WO2021207598A1 - Combination treatment with an agonist of p53 and a second therapeutic agent - Google Patents

Combination treatment with an agonist of p53 and a second therapeutic agent Download PDF

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Publication number
WO2021207598A1
WO2021207598A1 PCT/US2021/026575 US2021026575W WO2021207598A1 WO 2021207598 A1 WO2021207598 A1 WO 2021207598A1 US 2021026575 W US2021026575 W US 2021026575W WO 2021207598 A1 WO2021207598 A1 WO 2021207598A1
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methyl
oxoquinuclidin
administered
lymphoma
compound
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PCT/US2021/026575
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French (fr)
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Eyal Attar
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Aprea Therapeutics, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • combination therapies using an agonist of p53 such as a p53 reactivator in combination with a second therapeutic agent, for example, an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1), for treating diseases and disorders.
  • a second therapeutic agent for example, an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1), for treating diseases and disorders.
  • a second therapeutic agent for example, an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinas
  • combination therapies using an agonist of p53 in combination with an inhibitor of Bruton’s tyrosine kinase for treating a disease or disorder such as lymphoma for treating a disease or disorder such as lymphoma.
  • combination therapies using an agonist of p53 in combination with an exportin 1 (XPO1) inhibitor for treating a disease or disorder such as a hyperproliferative malignancy for treating a disease or disorder such as cancer.
  • XPO1 exportin 1
  • combination therapies using an agonist of p53 in combination with an inhibitor of WEE1 for treating a disease or disorder such as cancer.
  • p53 halts the cell cycle and/or triggers apoptosis in response to various stress stimuli, including DNA damage, hypoxia, and oncogene activation (Ko, L. J. & Prives, C., Genes Dev.10, 1054-1072 (1996); Sherr, C. J., Genes Dev.12, 2984-2991 (1998)).
  • stress stimuli including DNA damage, hypoxia, and oncogene activation
  • Both p53-induced cell cycle arrest and apoptosis could be involved in p53-mediated tumor suppression.
  • a significant proportion of human tumors make a “mutant” p53 protein due to a TP53 mutation, making it highly desirable to restore the wild type p53 activity to yield growth suppression to tumors.
  • Tumor cells are particularly sensitive to p53 reactivation, supposedly for two main reasons.
  • Second, mutant p53 proteins tend to accumulate at high levels in tumor cells. Therefore, restoration of the wild type function to the abundant and presumably “activated” mutant p53 should trigger a massive apoptotic response in already sensitized tumor cells, whereas normal cells that harbor low or undetectable levels of p53 should not be affected.
  • Non-Hodgkin lymphomas are lymphoid malignant neoplasms with diverse biological and clinical behavior, variously derived from the clonal expansion of B cells, T cells, natural killer cells, or precursors of these cells.
  • Chronic lymphocytic leukemia (CLL) is one of the most common types of B-cell NHL, characterized by a progressive accumulation of functionally incompetent monoclonal lymphocytes (Siegel, R.L., et al., CA Cancer J Clin.70(1), 7-30 (2020)).
  • Mantle cell lymphoma is another type of mature B-cell NHL, which comprises about 7% of adult NHL in the United States with an incidence of approximately 4 to 8 cases per million persons per year (Harris, N.
  • Standard management for MCL includes chemoimmunotherapy (CIT) regimens, sometimes followed by consolidation with autologous stem cell transplantation in fit patients and/or maintenance rituximab, and most recently targeted agents such as Bruton’s tyrosine kinase (BTK) inhibitors have been found to be effective in the relapsed and/or refractory (R/R) MCL (Wang, M.
  • CIT chemoimmunotherapy
  • BTK tyrosine kinase
  • Therapeutically useful compounds have previously been generated based on showing mutant p53 dependent anti-proliferative activity in a cellular assay, including the compound PRIMA-1 (i.e., 2,2-bis(hydroxymethyl)quinuclidin-3-one) (disclosed in WO 02/24692), and its analogs (such as those disclosed in WO 03/070250). Nonetheless, there still remains a general need of effective combination therapies using these compounds in combination with a second therapeutic agent for treating cancer and other diseases or disorders. 2.
  • PRIMA-1 i.e., 2,2-bis(hydroxymethyl)quinuclidin-3-one
  • SUMMARY Provided herein are methods of treating solid tumor malignancy in a subject, comprising administering to the subject: (i) an agonist of p53; and (ii) an inhibitor of PD-1 mediated signaling.
  • methods of treating a lymphoma in a subject comprising administering to the subject: (i) a p53 reactivator; and (ii) a Bruton’s tyrosine kinase (BTK) inhibitor.
  • BTK tyrosine kinase
  • methods of treating a hyperproliferative malignancy in a subject comprising administering to the subject: (i) a p53 reactivator; and (ii) an inhibitor of Wee1-like protein kinase (WEE1).
  • the agonist of p53 is a compound that can give reactivation of a mutant p53 or a compound whose metabolite can give reactivation of a mutant p53.
  • the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W.
  • the agonist of p53 is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity.
  • the compound or metabolite promotes proper folding of mutant and wild-type p53 proteins.
  • the compound or metabolite is capable of shifting the equilibrium from unfolded towards a folded structure of wild-type or mutant p53, or wherein the compound or metabolite is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or metabolite is capable of reducing aggregation of the wild-type or mutant p53.
  • the compound or metabolite is capable of promoting a folded structure of wild-type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53.
  • the compound or metabolite is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promotes a folded conformation.
  • the p53 reactivator is a compound that can give reactivation of a mutant p53, or a degradation product thereof that can give reactivation of a mutant p53.
  • the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W.
  • the p53 reactivator is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity.
  • the compound or degradation product thereof promotes proper folding of mutant and wild-type p53 proteins.
  • the compound or degradation product thereof is capable of shifting the equilibrium from unfolded towards a folded structure of wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of reducing aggregation of the wild-type or mutant p53.
  • the compound or degradation product thereof is capable of promoting a folded structure of wild-type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53.
  • the compound or degradation product threreof is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promotes a folded conformation.
  • the compound is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one; 2,2-bis(hydroxymethyl)quinuclidin-3-one; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide;
  • the compound is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) having the following formula: , or a pharmaceutically acceptable salt thereof.
  • the compound is a compound selected from the group consisting of: 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; and 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide
  • the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2- yl)methyl)acetamide (Compound A), or a pharmaceutically acceptable salt thereof.
  • the compound is 2,2,2-trichloro-N-ethyl-N-((3- oxoquinuclidin-2-yl)methyl)acetamide (Compound B), or a pharmaceutically acceptable salt thereof.
  • the compound is 2,2,2-trichloro-N-((3-oxoquinuclidin-2- yl)methyl)acetamide (Compound C), or a pharmaceutically acceptable salt thereof.
  • the compound is N-ethyl-2,2,2-trifluoro-N-((3- oxoquinuclidin-2-yl)methyl)acetamide (Compound D), or a pharmaceutically acceptable salt thereof.
  • the compound is 2,2-difluoro-N-((3-oxoquinuclidin-2- yl)methyl)acetamide (Compound E), or a pharmaceutically acceptable salt thereof.
  • the inhibitor of PD-1 mediated signaling is an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, nivolumab, AMP-224, and AMP-514.
  • the anti-PD-1 antibody is pembrolizumab.
  • the agonist of p53 is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) and the inhibitor of PD-1 mediated signaling is pembrolizumab.
  • APR-246 is administered at a dose of about 4.5 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle.
  • APR-246 is administered at a dose of about 4.0 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle.
  • APR-246 is administered at a dose of about 3.5 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle.
  • APR-246 is administered on Days 1–4 and pembrolizumab is administered on Day 3 of the each 21-day cycle. [0045] In certain embodiments, APR-246 and pembrolizumab are administered for 1 to 20 cycles. [0046] In certain embodiments, the agonist of p53 is formulated in a first pharmaceutical composition and the inhibitor of PD-1 mediated signaling is formulated in a second pharmaceutical composition.
  • the solid tumor malignancy is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non- melanoma skin carcinoma, and a lung cancer.
  • the solid tumor malignancy is an advanced non-CNS- primary solid tumor.
  • the solid tumor malignancy is selected from the group consisting of gastric/gastroesophageal junction (GEJ) cancer, bladder/urothelial cancer, and non- small-cell lung cancer (NSCLC).
  • the BTK inhibitor is selected from the group consisting of ibrutinib, acalabrutinib, zanubrutinib, evobrutinib, dasatinib, spebrutinib, tirabrutinib, ABBV- 105, ONO-4059, LFM-A13, and HM71224, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • the BTK inhibitor is ibrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • the p53 reactivator is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) and the BTK inhibitor is ibrutinib.
  • APR-246 is administered at a dose of about 4.5 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle.
  • APR-246 is administered at a dose of about 4.0 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle.
  • APR-246 is administered at a dose of about 3.5 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle.
  • APR-246 is administered on Days 1–4 and ibrutinib is administered daily of each 28-day cycle.
  • APR-246 and ibrutinib are administered for 1 to 20 cycles.
  • the p53 reactivator is formulated in a first pharmaceutical composition and the BTK inhibitor is formulated in a second pharmaceutical composition.
  • the lymphoma is a Hodgkin lymphoma (HL) or a non- Hodgkin lymphoma (NHL).
  • the lymphoma is a non-Hodgkin lymphoma (NHL).
  • the non-Hodgkin lymphoma (NHL) is a mature (peripheral) B-cell neoplasm.
  • the non-Hodgkin lymphoma is selected from the group consisting of: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, diffuse large cell B-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma (plasmacytoma), extranodal marginal zone B-cell lymphoma, nodal marginal zone lymphoma, follicle center lymphoma, and Burkitt’s leukemia (Burkitt’s lymphoma).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • DLBCL diffuse large cell B-cell lymphoma
  • lymphoplasmacytic lymphoma splenic marginal zone B-cell lymphoma
  • hairy cell leukemia plasma cell myelo
  • the lymphoma is chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). [0064] In certain embodiments, the lymphoma is chronic lymphocytic leukemia (CLL). [0065] In certain embodiments, the lymphoma is mantle cell lymphoma (MCL). [0066] In certain embodiments, the non-Hodgkin lymphoma (NHL) is relapsed or refractory NHL. [0067] In certain embodiments, the non-Hodgkin lymphoma (NHL) is relapsed or refractory chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • the non-Hodgkin lymphoma is relapsed or refractory CLL.
  • the non-Hodgkin lymphoma is relapsed or refractory MCL.
  • the lymphoma comprises a cancer cell having mutant p53.
  • the subject has not been treated with any BTK inhibitor prior to the co-administration of the p53 reactivator and the BTK inhibitor.
  • the XPO1 inhibitor is selected from the group consisting of leptomycin A (LMA), leptomycin B (LMB), selinexor (KPT-330), Eltanexor (KPT-8602), KTP-185, KPT-249, KPT-251, KPT-276, verdinexor (KPT-335), piperlongumine, valtrate, felezonexor (CBS9106 or SL-801), and PKF050-638, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • LMA leptomycin A
  • LMB leptomycin B
  • KPT-330 selinexor
  • KPT-8602 Eltanexor
  • KTP-185 KPT-249
  • KPT-251, KPT-276 verdinexor
  • KPT-335 verdinexor
  • piperlongumine valtrate
  • felezonexor CBS9106 or SL-801
  • the XPO1 inhibitor is selinexor, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • the p53 reactivator is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) and the XPO1 inhibitor is selinexor.
  • APR-246 is administered at a daily dose of about 4.5 g/day and selinexor is administered at a dose of about 80 mg/day twice per week.
  • APR-246 is administered at a daily dose of about 4.0 g/day and selinexor is administered at a dose of about 80 mg/day twice per week.
  • APR-246 is administered at a daily dose of about 3.5 g/day and selinexor is administered at a dose of about 80 mg/day twice per week.
  • selinexor is administered on day 1 and day 3 of each week.
  • APR-246 and selinexor are administered for 1 to 20 cycles.
  • selinexor is administered at a reduced dose of about 100 mg once per week, about 80 mg once per week, or about 60 mg once per week.
  • the p53 reactivator is formulated in a first pharmaceutical composition and the XPO1 inhibitor is formulated in a second pharmaceutical composition.
  • the inhibitor of WEE1 is selected from the group consisting of 6-(2,6-Dichlorophenyl)-2-[4-[2-(diethylamino)ethoxy] anilino]-8-methylpyrido[2,3-d]pyrimidin- 7-one (PD0166285); 9-Hydroxy-4-phenylpyrrolo[3,4-c] carbazole-1,3(2H,6H)-dione (PD0407824); 4-(2-Chlorophenyl)-9-hydroxypyrrolo[3,4-c] carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor I); 6-Butyl-4-(2-chlorophenyl)-9-hydroxypyrrolo [3,4-c]carbazol
  • the inhibitor of WEE1 is MK-1775 having a formula of: (MK-1775), or a pharmaceutically acceptable salt thereof.
  • the p53 reactivator is formulated in a first pharmaceutical composition and the inhibitor of WEE1 is formulated in a second pharmaceutical composition.
  • the hyperproliferative malignancy is a hematological malignancy.
  • the hematological malignancy is leukemia, lymphoma, or myeloma.
  • the hematological malignancy is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymph
  • NHL non-Hodg
  • the hematologic malignancy is myeloma.
  • the myeloma is multiple myeloma (MM).
  • the hematologic malignancy is acute myeloid leukemia (AML).
  • the hematologic malignancy is chronic lymphocytic leukemia (CLL).
  • CLL chronic lymphocytic leukemia
  • the hematologic malignancy is myelodysplastic syndromes (MDS).
  • the hyperproliferative malignancy comprises a cancer cell having mutant p53.
  • the hyperproliferative malignancy is a solid tumor cancer.
  • the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, a lung cancer, a cervical cancer, a prostate cancer, a head and neck cancer, and a breast cancer.
  • the solid tumor cancer is breast cancer.
  • the solid tumor cancer is triple negative breast cancer. 3.
  • FIG.1 depicts the phase 1/2 clinical trials to determine the safety and preliminary efficacy of APR-246 in combination with pembrolizumab in subjects with solid tumor malignancies.
  • FIG.2 outlines the safety lead-in portion of the study, wherein subjects with advanced solid tumor malignancies receive pembrolizumab in combination with APR-246.
  • FIG.3 depicts the study scheme for the phase 1 and dose expansion study of APR- 246 in combination with ibrutinib in subjects with TP53-mutant relapsed and/or refractory non- Hodgkin lymphoma including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • DETAILED DESCRIPTION [00103] Described herein are combination treatments with an agonist of p53 such as a p53 reactivator (see Section 4.2.1) and a second therapeutic agent (see Sections 0 – 4.2.5) for treatment of diseases or disorders such as a cancer (see Section 4.4).
  • the second therapeutic agent provided herein can be an inhibitor of PD-1 mediated signaling such as an inhibitor of PD-1 (see Section 0).
  • the second therapeutic agent provided herein can be a Bruton’s tyrosine kinase (BTK) inhibitor (see Section 4.2.3).
  • the second therapeutic agent provided herein can be an exportin 1 (XPO1) inhibitor (see Section 4.2.4).
  • the second therapeutic agent provided herein can be an inhibitor of Wee1-like protein kinase (WEE1) (see Section 4.2.5). Manner of administration and dosing regimen are described in Section 4.4 herein.
  • the present disclosure is based, in part, on the surprising finding that the combination of an agonist of p53 such as a p53 reactivator and a second therapeutic agent produces synergistic effects in treating certain diseases and disorders such as a cancer.
  • the present disclosure is based, in part, on the surprising finding that the combination of a p53 reactivator and a BTK inhibitor produces synergistic effects in treating certain diseases and disorders (e.g., non-Hodgkin lymphoma).
  • the present disclosure is based, in part, on the surprising finding that the combination of a p53 reactivator and an XPO1 inhibitor produces synergistic effects in treating certain diseases and disorders (e.g., hyperproliferative malignancy).
  • the present disclosure is based, in part, on the surprising finding that the combination of a p53 reactivator and an inhibitor of WEE1 produces synergistic effects in treating certain diseases and disorders.
  • a subject is a mammal.
  • the subject is a human.
  • the subject is a mammal, e.g., a human, diagnosed with a disease or disorder.
  • the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.
  • administer or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.
  • the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or disorder resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder.
  • Treating includes both managing and ameliorating the disease.
  • the terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s).
  • a mutant p53 mediated disease or disorder refers to a disease or disorder that is caused or partially caused by mutation of the p53 gene (TP53).
  • TP53 p53 gene
  • a mutant p53 mediated cancer means the cancer that contains a cell having a mutant TP53.
  • alkyl unless otherwise stated, means an unbranched or branched, saturated or unsaturated (alkenyl or alkynyl) hydrocarbon radical.
  • Cx-Cy alkyl means a straight or branched chain hydrocarbon containing x to y carbon atoms.
  • C2-C6 alkyl means a straight or branched chain hydrocarbon containing 2 to 6 carbon atoms.
  • Representative examples of alkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n- hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n- decyl.
  • aryl means an aromatic group, such as phenyl or naphthyl.
  • cycloalkyl means a monocyclic or bicyclic, saturated or partially unsaturated (but not aromatic), hydrocarbon ring of three to ten carbon ring atoms. Cycloalkyl groups include fused and bridged bicyclic rings. For example, when fused, the cycloalkyl group may comprise two rings that share adjacent atoms (e.g., one covalent bond). When bridged, the cycloalkyl group may comprise two rings that share three or more atoms, separating the two bridgehead atoms by a bridge containing at least one atom.
  • cycloalkyl When a cycloalkyl group contains from x-y ring carbon atoms, it may be referred to herein as Cx-Cy cycloalkyl.
  • cycloalkyl is C3-C10 cycloalkyl, or is C5-C7 cycloalkyl, or is C5-C6 cycloalkyl, or is C3-C6 cycloalkyl, or is C3-C7 cycloalkyl.
  • cycloalkyl is C3-C8 cycloalkyl.
  • cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
  • heteroaryl means a mono-, bi-, or tricyclic heteroaromatic group containing one or more heteroatom(s) preferably selected from N, O and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl, thiophenetyl, pyranyl, carbazolyl, acridinyl, quinolinyl, benzimidazolyl, benzthiazolyl, purinyl, cinnolinyl and pteridinyl.
  • heteroatom(s) preferably selected from N, O and
  • non-aromatic heterocycle means a non-aromatic cyclic group containing one or more heteroatom(s) preferably selected from N, O and S, such as a pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, tetrahydrofuranyl or monosaccharide.
  • halogen means a fluorine, chlorine, bromine or iodine.
  • halo means a fluoro, chloro, bromo or iodo.
  • the term “substituted” means that the concerned groups are substituted with at least one functional group, such as hydroxyl, amine, sulfide, silyl, carboxylic acid, halogen, aryl, etc.
  • the term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • excipient means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material.
  • Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof.
  • encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents,
  • excipient can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.
  • excipients are pharmaceutically acceptable excipients.
  • Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • a pharmaceutically acceptable excipient is an aqueous pH buffered solution.
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time.
  • the term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days.
  • intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days.
  • the term “stereomerically pure” means a composition that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound.
  • a stereomerically pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the compound.
  • a stereomerically pure composition of a compound having two chiral centers will be substantially free of other diastereomers of the compound.
  • the depicted structure is to be accorded more weight.
  • the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of the structure.
  • the second therapeutic agent can be an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1).
  • BTK Bruton’s tyrosine kinase
  • XPO1 exportin 1
  • WEE1 Wee1-like protein kinase
  • Agents that can be used as agonists of p53 are set forth in Section 4.2.1. Agents that can be used as inhibitors of PD-1 mediated signaling such as inhibitors of PD-1 are set forth in Section 0. [00138]
  • agents that can be used as agonists of p53 such as a p53 reactivator and a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of certain diseases or disorders.
  • Agents that can be used as agonists of p53 such as p53 reactivators are set forth in Section 4.2.1.
  • Agents that can be used as BTK inhibitors are set forth in Section 4.2.3.
  • combination treatments with an agonist of p53 such as a p53 reactivator (see Section 4.2.1) and an exportin 1 (XPO1) inhibitor (see Section 4.2.4) for the treatment of certain diseases and disorders. Manners of administration and dosing regimen are described in Section 4.2.4.
  • combination treatments with a p53 reactivator see Section Error! Reference source not found.
  • an inhibitor of WEE1 see Section 4.2.5
  • the cancer can comprise cells that are mutant for p53 (see Section 4.4).
  • One therapeutic agent in the present combination therapies is an agonist of p53.
  • an agonist of p53 provided herein increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of p53 activity, e.g., in a cell such as an immune cell.
  • an agonist of p53 provided herein increases by about 50% of p53 activity, e.g., in a cell such as an immune cell.
  • the increased activity corresponds to an increased level of transcription of p53 target genes, which may be studied by quantifying mRNA increase.
  • An increased level of mRNA may be determined by methods known in the art.
  • mRNA is quantified by TaqMan analysis.
  • the p53 agonist provided herein increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of p53 activity, e.g., in a cell such as an immune cell.
  • the p53 agonist provided herein increases by about 50% of p53 activity, e.g., in a cell such as an immune cell.
  • the methods for measuring p53 activity and increase thereof are well known in the art.
  • the increased activity corresponds to an increased level of transcription of p53 target genes, which may be studied by quantifying mRNA increase.
  • An increased level of mRNA may be determined by methods known in the art.
  • mRNA is quantified by TaqMan analysis.
  • the agonist of p53 described herein is a p53 reactivator.
  • one therapeutic agent used in the combination therapies described herein is a p53 agonist such a p53 reactivator.
  • the p53 reactivator provided herein increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of p53 activity, e.g., in a cell such as an immune cell.
  • the p53 reactivator provided herein increases by about 50% of p53 activity, e.g., in a cell such as an immune cell.
  • the increased activity corresponds to an increased level of transcription of p53 target genes, which may be studied by quantifying mRNA increase.
  • An increased level of mRNA may be determined by methods known in the art.
  • mRNA is quantified by TaqMan analysis.
  • the agonist of p53 provided herein increases wild-type p53 activity.
  • the agonist of p53 provided herein is a p53 reactivator capable of reactivating a mutant p53.
  • the agonist of p53 provided herein is capable of increasing wild type p53 activity and capable of reactivating a mutant p53.
  • the p53 gene TP53 is a very common target for mutation in tumors. Around half of all human tumors carry mutation in TP53. p53 halts the cell cycle and/or triggers apoptosis in response to various stress stimuli, including DNA damage, hypoxia, and oncogene activation (Ko, L. J. & Prives, C., Genes Dev.10, 1054-1072 (1996); Sherr, C. J., Genes Dev.12, 2984- 2991 (1998)).
  • p53 Upon activation, p53 initiates the p53-dependent biological responses through transcriptional transactivation of specific target genes carrying p53 DNA binding motifs.
  • Analyses of a large number of mutant p53 genes in human tumors have revealed a strong selection for mutations that inactivate the DNA binding function of p53; most mutations in tumors are point mutations clustered in the part encoding the core domain of p53 (residues 94- 292) that harbors the DNA binding activity (Béroud, C. & Soussi, T., Nucl. Acids Res.26, 200- 204 (1998)).
  • Both p53-induced cell cycle arrest and apoptosis could be involved in p53-mediated tumor suppression.
  • autoimmune diseases In addition to hyperproliferative diseases, such as cancer, it is also known in the art that deficient p53 function is involved in a number of other disease states, e.g. autoimmune diseases and cardiac diseases.
  • human autoimmune diseases share the common feature of an imbalance between the production and destruction of various cell types including lymphocytes (SLE), synovial cells (RA), and fibroblasts (scleroderma). Genes including TP53 that regulates apoptosis are also expressed abnormally. According to the authors, specific therapies that induce apoptosis without incurring side effects should improve treatment of autoimmune disease.
  • TP53 codon 72 polymorphism contributes to a genetically determined variability in apoptotic susceptibility among old people, which has a potentially relevant role in the context of an age-related pathologic condition, such as myocardial ischaemia.
  • Okuda et al., Journal of Neuroimmunology, 135: 29-37 (2003) suggests that p53 may be involved in the regulatory process of experimental autoimmune encephalomyelitis (EAE) through the control of cytokine production and/or the apoptotic elimination of inflammatory cells.
  • EAE experimental autoimmune encephalomyelitis
  • EAE as a model for autoimmune inflammatory diseases of the central nervous system (CNS) is a widely used model for the human disease multiple sclerosis.
  • CNS central nervous system
  • p53 agonist e.g., a p53 reactivator
  • the p53 reactivator directly or indirectly targets a mutant p53 protein.
  • the p53 reactivator directly or indirectly targets a mutant p53 protein.
  • the mutant TP53 includes a missense mutation, which is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid.
  • Missense mutant p53 proteins can be broadly classified as DNA-contact mutants and structural mutants.
  • p53 DNA contact mutant contains mutations present on amino acids directly binding to DNA, such as in mutants carrying single amino acid changes R248Q, R248W, R273H, or R273C, where R248Q denotes that the wild-type residue arginine in position 248 has been replaced by a glutamine in the mutant.
  • the mutant TP53 includes a nonsense mutation.
  • a nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product.
  • Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP53 mutations in cancer.
  • the most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*.
  • the p53 reactivator provided herein reactivates or restores at least part of the wild-type p53 activity of a mutant p53, for example by promoting proper folding of the mutant p53 and restoring the normal p53 function of the mutant p53.
  • the p53 reactivator provided herein inhibits improper protein misfolding of the mutant p53 protein, and/or promotes proper protein folding of the mutant p53 by covalently binding to the mutant p53 protein.
  • the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) provided herein reacts as an electrophile with one or more thiols in the mutant p53.
  • the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) provided herein covalently binds to one or more thiols in the mutant p53.
  • the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) reacts with or binds to one or more thiols in the mutant p53 as an electrophile.
  • a metabolite or degradation product of the p53 reactivator reacts with or binds to one or more thiols in the mutant p53.
  • reversible or irreversible covalent bonds are formed between the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) and the mutant p53.
  • reversible covalent bonds are formed between the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) and the mutant p53.
  • irreversible covalent bonds are formed between the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) and the mutant p53.
  • the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) provided herein reacts with one or more thiols in the DNA binding domain of the mutant p53 to stabilize the mutant p53 conformation, thus restoring their transcriptional activities.
  • the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) reacts with one or more thiols of cysteine residues in the core domain of wild-type p53 protein and stabilizes wild-type p53 conformation. In other embodiments, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) reacts with one or more thiols of cysteine residues in the core domain of mutant p53 and restores the mutant p53 conformation to wild-type p53 like conformation.
  • the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) shifts the equilibrium from unfolded mutant p53 conformation towards a wild-type p53 like conformation.
  • the p53 reactivator provided herein inhibits improper protein misfolding and/or promotes proper protein folding by non-covalent binding to the mutant p53 protein.
  • Such p53 reactivators include chaperones that can non ⁇ covalently stabilize the mutant p53 protein.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof inhibits improper protein misfolding and/or promotes proper protein folding by covalent binding to the mutant p53 protein, for example, by electrophiles binding to one or more thiols in the mutant p53 DNA binding domain to stabilize a folded conformation, and restoring their transcriptional activities.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof binds to the thiol of cysteine residues in the core domain and stabilizes wild type p53 conformation.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof is shifting the equilibrium from unfolded towards a wild-type like p53 conformation.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof binds to thiol groups in the core domain and restores wild-type conformation.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof inhibits improper protein misfolding and/or promotes proper protein folding by non-covalent binding to the mutant p53 protein.
  • Such p53 reactivators include chaperones that can non ⁇ covalently stabilize mutant p53 structures.
  • the p53 reactivator provided herein reactivates mutant p53 by interfering with aggregation of misfolded p53 or reducing aggregation of mutant p53.
  • p53 misfolds or unfolds into an aggregation ⁇ prone stage that loses its DNA ⁇ binding capacity.
  • misfolded p53 may convert wild type p53 to a misfolded form and accelerate p53 aggregation.
  • the p53 reactivator provided herein may reactivate p53 by interfering with aggregation of misfolded p53.
  • the p53 reactivator provided herein or a degradation product or metabolite reduces non-folded or incorrectly folded mutant p53 that may otherwise aggregate, and thereby reducing aggregation.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof reactivates mutant p53 by interfering with aggregation of misfolded p53 or reducing aggregation of mutant p53. Sometimes, p53 misfolds or unfolds into an aggregation ⁇ prone stage that loses its DNA ⁇ binding capacity.
  • misfolded mutant p53 may cause accumulation of wild type p53 in a misfolded form and accelerate p53 aggregation.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof may reactivate p53 by interfering with aggregation of misfolded p53.
  • the p53 reactivator provided herein or a degradation product or metabolite reduces non-folded or incorrectly folded mutant p53 that may otherwise aggregate, and thereby reducing aggregation.
  • the mutant p53 comprises at least one amino acid replacement in the core domain of the mutant p53 (between residues 94 and 292) caused by a TP53 mutation.
  • the mutant p53 comprises replacement selected from the group consisting of V173A, S241F, R249S, R273H, R175H, R248Q, and Y220C. In some embodiments, the mutant p53 comprises one of the amino acid replacements of R175H and R273H.
  • the p53 reactivator provided herein can give reactivation of a mutant p53 protein. In certain embodiments, the p53 reactivator can result in reactivation of a mutant p53 protein. In certain embodiments, the p53 reactivator is transformed to a metabolite or a degradation product that reacts with a mutant p53 protein.
  • the p53 reactivator is transformed to the metabolite or the degradation product in vivo. In certain embodiments, the p53 reactivator is transformed to the metabolite or the degradation product in tumor tissue.
  • the p53 agonist provided herein e.g., a p53 reactivator
  • a degradation product or metabolite stabilizes wild type p53 protein in a situation where its production has been induced as part of a normal physiological process, and in this way enhancing the effect of said wild type p53 induction.
  • the p53 reactivator provided herein or a degradation product or metabolite stabilizes wild type p53 protein in a situation where its production has been induced as part of a normal physiological process, and in this way enhancing the effect of said wild type p53 induction.
  • the compound that can give reactivation of mutant p53 (the p53 reactivator) provided herein is a compound according to formula (I): wherein: R 1 is selected from the group consisting of H, —CH 2 —O—R 3 , —CH 2 —S—R 3 , and — CH 2 —NR 3 R 4 ; R 2 is selected from the group consisting of —CH 2 —O—R 3 , —CH 2 —S—R 3 , and — CH 2 —NR 3 R 4 ; each of R 3 and R 4 is independently selected from H; substituted or unsubstituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or C1-C10 alkyl; substituted or unsubstituted benzyl; substituted or unsubstituted mono- or bi-cyclic aryl; substituted or unsubstituted mono-, bi- or tri-cyclcycl
  • the agonist of p53 provided herein is a p53 reactivator having formula (I) wherein: R 1 is selected from H, —CH 2 —O—R 3 , —CH 2 —S—R 3 , and —CH 2 —NR 3 R 4 ; R 2 is selected from —CH 2 —O—R 3 , —CH 2 —S—R 3 , and —CH 2 —NR 3 R 4 ; R 3 and R 4 are the same or different and are independently selected from H; substituted or non- substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or C1-C10 alkyl; substituted or non-substituted benzyl; substituted or non-substituted mono- or bicyclic aryl; substituted or non-substituted mono-, bi- or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocycl
  • the pharmaceutically acceptable salt of the compound of formula (I) may be an acid addition salt of the compound formed with an inorganic acid or of an organic acid.
  • the pharmaceutically acceptable salt of the compound of formula (I) e.g. may be an acid addition salt of an inorganic mineral acid or of an organic acid.
  • R 1 is selected from H, — CH 2 —O—R 3 , —CH 2 —S—R 3 , and —CH 2 —NR 3 R 4 .
  • R 1 is selected from H, —CH 2 —O—R 3 , and —CH 2 —S—R 3 .
  • R 1 is selected from H and —CH 2 —O—R 3 . In other embodiments, R 1 is selected from —CH 2 —O—R 3 , and —CH 2 —S—R 3 . In some embodiments, R 1 is H. [00176] R 2 in formula (I) is selected from —CH 2 —O—R 3 , —CH 2 —S—R 3 , and —CH 2 — NR 3 R 4 . In some embodiments, R 2 is selected from —CH 2 —O—R 3 and —CH 2 —S—R 3 . In still other embodiments, R 2 is —CH 2 —O—R 3 .
  • R 1 is H, and R 2 is —CH 2 —O—R 3 , —CH 2 —S—R 3 or —CH 2 — NR 3 R 4 .
  • R 1 is H, and R 2 is —CH 2 —O—R 3 or —CH 2 —S—R 3 .
  • R 1 is H, and R 2 is —CH 2 —O—R 3 .
  • R 1 is selected from H, —CH 2 —O—R 3 and —CH 2 —S—R 3 ; and R 2 is selected from —CH 2 —O—R 3 and —CH 2 —S—R 3 .
  • R 1 is H; and R 2 is selected from —CH 2 —O—R 3 , —CH 2 —S—R 3 and —CH 2 —NR 3 R 4 ; e.g. from —CH 2 —O—R 3 and —CH 2 —S—R 3 , and in particular is — CH 2 —O—R 3 .
  • R 1 is selected from H and —CH 2 —O—R 3 ; and R 2 is —CH 2 — O—R 3 .
  • both R 1 and R 2 are —CH 2 —O—R 3 .
  • each R 3 is independently selected from H; substituted or unsubstituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl.
  • each R 3 is independently H or C1-C10 alkyl.
  • each R 3 is independently H or C1-C6 alkyl.
  • each R 3 is independently H or C1-C4 alkyl.
  • each R 3 is independently H or C1- C3 alkyl.
  • each R 3 is independently H or methyl.
  • each R 3 is independently selected from H; substituted or non- substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl.
  • each R 3 may be independently selected from H and C1-C10 alkyl, e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.
  • R 1 is H or —CH 2 —O—R 3
  • R 2 is —CH 2 —O—R 3
  • each R 3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl.
  • R 1 is H or —CH 2 —O—R 3
  • R 2 is —CH 2 —O—R 3
  • each R 3 is independently H or C1-C10 alkyl.
  • R 1 is H or —CH 2 —O—R 3
  • R 2 is —CH 2 —O—R 3
  • each R 3 is independently H or C1-C6 alkyl.
  • R 1 is H or —CH 2 —O—R 3
  • R 2 is —CH 2 —O—R 3
  • each R 3 is independently H or C1-C4 alkyl.
  • R 1 is H or —CH 2 —O—R 3
  • R 2 is —CH 2 —O—R 3
  • each R 3 is independently H or C1-C3 alkyl.
  • R 1 is H or —CH 2 —O—R 3
  • R 2 is —CH 2 —O—R 3
  • each R 3 is independently H or methyl.
  • R 1 is selected from H and —CH 2 —O—R 3
  • R 2 is —CH 2 — O—R 3
  • each R 3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl, in particular from H and C1-C10 alkyl, e.g.
  • R 1 and R 2 are both —CH 2 —O—R 3 , and each R 3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl.
  • R 1 and R 2 are both —CH 2 —O—R 3 , and each R 3 is independently H or C1-C10 alkyl.
  • R 1 and R 2 are both —CH 2 —O—R 3 , and each R 3 is independently H or C1-C6 alkyl. In yet another embodiment, R 1 and R 2 are both —CH 2 —O—R 3 , and each R 3 is independently H or C1- C4 alkyl. In yet another embodiment, R 1 and R 2 are both —CH 2 —O—R 3 , and each R 3 is independently H or C1-C3 alkyl. In a specific embodiment, R 1 and R 2 are both —CH 2 —O—R 3 , and each R 3 is independently H or methyl.
  • R 1 and R 2 are both —CH 2 —O—R 3 , and each R 3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl; in particular from H and C1-C10 alkyl; e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.
  • any C1-C10 alkyl e.g.
  • Any mono- or bicyclic aryl may be e.g. a monocyclic aryl, such as phenyl.
  • Any mono-, bi- or tricyclic C2-C10 heteroaryl may be e.g. a monocyclic or bicyclic C2-C5 heteroaryl, e.g.
  • Any mono-, bi- or tricyclic non-aromatic C2-C10 heterocyclyl may be e.g. a monocyclic or bicyclic C2-C5 heterocyclyl, e.g. a 5- or 6-membered monocyclic or 9- or 10-membered bicyclic C2-C5 heterocyclyl.
  • Any halogen may be selected from F, Cl, Br and I, preferably from F and Cl.
  • the substituted or unsubstituted C3-C12 cycloalkyl or C1-C10 alkyl is a non-substituted C3-C12 cycloalkyl or C1-C10 alkyl.
  • the substituted or unsubstituted benzyl is an unsubstituted benzyl.
  • the substituted or unsubstituted mono- or bi-cyclic aryl is an unsubstituted mono- or bi-cyclic aryl.
  • the substituted or unsubstituted mono-, bi- or tri-cyclic heteroaryl or non-aromatic heterocyclyl is an unsubstituted mono-, bi- or tri-cyclic heteroaryl or non- aromatic heterocyclyl.
  • each substituent independently is a C1-C10 alkyl, halo, halo-substituted C1-C10 alkyl, monocyclic heteroaryl, C1-C10 alkoxy, or amino group.
  • any substituted or non-substituted C3-C12 cycloalkyl or C1-C10 alkyl is non-substituted.
  • any substituted or non-substituted benzyl is non-substituted.
  • any substituted or non-substituted mono- or bicyclic aryl is non- substituted.
  • any substituted or non-substituted mono-, bi- or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl is non-substituted.
  • each substituent is selected from C1-C10 alkyl, e.g. C1-C6 alkyl, C1-C4 alkyl, or C1-C3 alkyl, such as methyl; halogen, e.g. Cl; halogen-substituted C1-C10 alkyl, e.g. trifluoromethyl; monocyclic C2-C5 heteroaryl, e.g. pyridyl; C1-C10 alkoxy, e.g. C1-C6 alkoxy, C1-C4 alkoxy, or C1-C3 alkoxy, such as methoxy; and amino.
  • C1-C10 alkyl e.g. C1-C6 alkyl, C1-C4 alkyl, or C1-C3 alkyl, such as methyl
  • halogen e.g. Cl
  • halogen-substituted C1-C10 alkyl e.g. trifluoromethyl
  • the number of substituents on each substituted group is 1, 2 or 3.
  • the compound provided herein is selected from those exemplified in the prior art documents referred to herein above, e.g. WO05/090341, WO04/084893, WO02/024692 and WO03/070250, which are incorporated herein in their entirety.
  • the compound of formula (I) is selected from 2-(hydroxymethyl)- 2-(methoxymethyl)quinuclidin-3-one (APR-246) and 2,2-bis(hydroxymethyl)quinuclidin-3-one, or a pharmaceutically acceptable salt thereof.
  • the compound of formula (I) is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) having the following formula: , or a pharmaceutically acceptable salt thereof.
  • the compound of formula (I) is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) or a pharmaceutically acceptable salt thereof.
  • the compound of formula (I) is 2,2- bis(hydroxymethyl)quinuclidin-3-one or a pharmaceutically acceptable salt thereof.
  • the agonist of p53 is a compound listed below.
  • the compound listed below is capable of reactivating mutatnt p53.
  • 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin)methyl)ace
  • the compound of formula (I) is selected from the group consisting of: 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin
  • the compound of formula (I) is a compound listed in Table 1 below, or a pharmaceutically acceptable salt thereof. Table 1.
  • the p53 reactivator is Compound A, or a pharmaceutically acceptable salt thereof.
  • the p53 reactivator is Compound B, or a pharmaceutically acceptable salt thereof.
  • the p53 reactivator is Compound C, or a pharmaceutically acceptable salt thereof.
  • the p53 reactivator is Compound D, or a pharmaceutically acceptable salt thereof.
  • the p53 reactivator is Compound E, or a pharmaceutically acceptable salt thereof.
  • the p53 reactivator provided herein is 2,2,2-trifluoro-N-((3- oxoquinuclidin-2-yl)methyl)acetamide having the following formula: (Compound A), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has reactivating activity of mutant p53.
  • the compound of Formula (I) has activating activity of wide type p53.
  • the agonist of p53 is a compound listed in the table below. In certain more specific embodiments, the compound listed in Table 2 below is capable of reactivating mutant p53.
  • the p53 reactivator provided herein is selected from the group consisting of the compounds in Table 2 below. Table 2.
  • the amino acid sequence of the compound is provided (N-terminus is myristoylated). b The amino acid sequence of the compound is provided. c The nucleic acid sequence of the compound is provided.
  • the inhibitor of PD-1 mediated signaling provided herein attenuates the interaction between the PD-1 receptor and PD-L1.
  • the inhibitor of PD-1 mediated signaling is an inhibitor of PD-1.
  • the inhibitor of PD-1 mediated signaling is an inhibitor of PD-L1.
  • the inhibitor of PD-1 is an anti-PD-1 antibody.
  • the inhibitor of PD-1 is an antisense oligonucleotide targeting PD-1 gene.
  • the inhibitor of PD-1 is a small molecule compound that can binds to PD-1 protein.
  • the inhibitor of PD-L1 is an anti-PD-L1 antibody. In some embodiments, the inhibitor of PD-L1 is an antisense oligonucleotide targeting PD-L1 gene. In other embodiments, the inhibitor of PD-L1 is a small molecule compound that can binds to PD- L1 protein.
  • the anti-PD-1 antibody provided herein is pembrolizumab (Merck); nivolumab (Bristol-Myers Squibb), cemiplimab (Regeneron), spartalizumab (Novartis), camrelizumab (Jiangsu HengRui Medicine), sintilimab (Innovent and Eli Lilly), tislelizumab (BeiGene), toripalimab (Junshi), nivolumab (Bristol-Myers Squibb), AMP-224 (GlaxoSmithKline), or AMP-514 (GlaxoSmithKline).
  • the anti-PD-1 antibody provided herein is pembrolizumab (KEYTRUDA ® ).
  • the heavy chain and light chain sequences of pembrolizumab are shown in Table 3. Table 3. Heavy chain (SEQ ID NO:5) Light chain (SEQ ID NO:6) 4.2.3 Bruton’s Tyrosine Kinase (BTK) Inhibitors
  • Another therapeutic agent used in the combination therapies described herein e.g., in Section 4.4.2
  • is a Bruton’s tyrosine kinase (BTK) inhibitor is a Bruton’s tyrosine kinase (BTK) inhibitor.
  • BTK tyrosine kinase
  • XLA X-linked agammaglobulinemia
  • BTK The suggested BTK’s role in B-cell development and immunoglobulin production combined with the nonlethality of XLA make BTK a promising therapeutic target (Herman, S., et al., Blood 117, 6287-6296 (2011)).
  • the therapeutic potential of BTK inhibition is supported by its effects on malignant cell lines in vitro. For example, in chronic lymphocytic leukemia (CLL), inhibition of BTK inhibits NF ⁇ B DNA binding, reduces cell migration, proliferation, and survival, and induces apoptosis (de Rooij, M., et al. Blood 119, 2590-2594 (2012); Cheng, S. et al.
  • CLL chronic lymphocytic leukemia
  • the BTK inhibitor used in the combination therapies methods described herein is selected from the group consisting of ibrutinib, acalabrutinib, zanubrutinib, evobrutinib, dasatinib, spebrutinib, tirabrutinib, ABBV-105, ONO-4059, LFM-A13, and HM71224, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • the BTK inhibitor is ibrutinib, or acalabrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. In one preferred embodiment, the BTK inhibitor is ibrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. In one embodiment, the BTK inhibitor is ibrutinib. In one embodiment, the BTK inhibitor is acalabrutinib.
  • Ibrutinib has a chemical name of 1-[(3R)-3-[4-amino-3-(4- phenoxyphenyl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one, having the following structure: . 4.2.4 Exportin 1 (XPO1) Inhibitors [00223] Another therapeutic agent used in the combination therapies described herein (e.g., in Section 4.4.3) is an exportin 1 (XPO1) inhibitor.
  • Exportin 1 (XPO1 or XPO-1), also known as chromosome region maintenance 1 (CRM1), is the main exporter of leucine-rich proteins form the nucleus through the neuclear pore complex (NPC) to the cytoplasm (Podar, K. et al., Exper Opinion on Pharmacotherapy 21(4), 399-4082 (2020); Wang A. Y. & Liu, H., Stem Cell Investigation, 6(6), 1-9 (2019)).
  • NPC neuclear pore complex
  • a GTPase Ran loaded with GTP binds cooperatively with XPO1 to translocate proteins including transcription factors, Tumor Suppressor Proteins (TSPs; e.g., p53, Rb, IkB, p73, BRCA1/2, etc.), cell-cyle regulators, and RNA molecules through the NPC to cytoplasm.
  • TSPs Tumor Suppressor Proteins
  • XPO1 mutation and/or overexpression have been reported in nearly all nalignancies and are associated with alleviated translocation of TSPs out of the nucleus, which leads to, e.g., inactivation of apoptosis, cell-cycle deregulations, and chemotherapeutic resistance (Podar, K. et al., Exper Opinion on Pharmacotherapy 21(4), 399-4082 (2020)).
  • XPO1 overexpression has been reported in several solid tumors and leukemias, including AML, and it is associated with worse outcome (Ranganathan, P., Blood, 125(17), 2689-2692 (2015)). Thus, XPO1 has become one of the therapeutic targets in cancer therapy.
  • Selinexor is a first-in-class small molecule XPO1 inhibitor that is developed for the treatment of cancer (Syed, Y. Y., Drugs, 79(13), 1485-1494 (2019)). Selinexor was approved by the U.S. Food and Drug Administration (FDA) for use in combination with the corticosteroid dexamethasone for the treatment of patients with relapsed refractory multiple myeloma who have received prior therapies and are resistant to several other forms of treatment (Selinexor Label, July 2019). [00226] In the presence of selinexor, XPO1 is inhibited and degraded, and is unable to export its cargo proteins.
  • FDA U.S. Food and Drug Administration
  • the XPO1 inhibitor used in the combination therapies methods described herein is selected from the group consisting of leptomycin A (LMA), leptomycin B (LMB), selinexor (KPT-330), Eltanexor (KPT-8602), KTP-185, KPT-249, KPT-251, KPT-276, verdinexor (KPT-335), piperlongumine, valtrate, felezonexor (CBS9106 or SL-801), and PKF050-638, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • LMA leptomycin A
  • LMB leptomycin B
  • KPT-330 selinexor
  • KPT-8602 Eltanexor
  • KTP-185 KPT-249
  • KPT-251, KPT-276 verdinexor
  • KPT-335 verdinexor
  • piperlongumine valtrate
  • felezonexor C
  • the XPO1 inhibitor is selinexor, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • the XPO1 inhibitor is selinexor.
  • Selinexor has a chemical name of (2Z)-3- ⁇ 3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl ⁇ -N′-pyrazin-2- ylprop-2-enehydrazide, having the following structure: .
  • WEE1-like protein kinase Another therapeutic agent used in the combination therapies described herein (e.g., in Section 4.4.4) is an inhibitor of Wee1-like protein kinase (WEE1).
  • WEE1 is a nuclear kinase belonging to the Ser/Thr family of protein kinases (see NCBI gene ID: 7465).
  • the inhibitor of WEE1 provided herein is a small- molecule inhibitor.
  • the inhibitor of WEE1 provided herein is a nucleic acid targeting WEE1 gene (e.g., siRNA, antisense oligonucleotide (ASOs), and miRNA).
  • the inhibitor of WEE1 provided herein is a polypeptide (e.g., an antibody or fragment thereof) capable of binding WEE1.
  • the inhibitor of WEE1 is 6-(2,6-dichlorophenyl)-2-[4-[2- (diethylamino)ethoxy]anilino]-8-methylpyrido[2,3-d]pyrimidin-7-one (PD0166285; CAS No. 185039-89-8) having a formula of:
  • the inhibitor of WEE1 is 9-hydroxy-4-phenylpyrrolo[3,4- c]carbazole-1,3(2H,6H)-dione (PD0407824; CAS No.622864-54-4) having a formula of: or a pharmaceutically acceptable salt thereof.
  • the inhibitor of WEE1 is 4-(2-chlorophenyl)-9- hydroxypyrrolo[3,4-c] carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor I; CAS No.622855-37-2) having a formula of: or a pharmaceutically acceptable salt thereof.
  • the inhibitor of WEE1 is 6-butyl-4-(2-chlorophenyl)-9- hydroxypyrrolo [3,4-c]carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor II; CAS No.622855-50-9) having a formula of:
  • the inhibitor of WEE1 is methyl 4-(4-((2-allyl-1-(6-(2- hydroxypropan-2-yl) pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d] pyrimidin-6- yl)amino)phenyl)piperazine-1-carboxylate (CJM061) having a formula of: or a pharmaceutically acceptable salt thereof.
  • the inhibitor of WEE1 is adavosertib (MK-1775 or AZD1775) having a formula of: or a pharmaceutically acceptable salt thereof.
  • compositions 4.3.1 Compositions Relating to Combination Therapies with Inhibitors of PD-1 Mediated Signaling
  • An agonist of p53 such as a p53 reactivator (e.g., APR-246) can be formulated with one or more pharmaceutically acceptable excipient.
  • an inhibitor of PD-1 mediated signaling such as an inhibitor of PD-1 (e.g., pembrolizumab) can be formulated in a pharmaceutical composition with one or more pharmaceutically acceptable excipients.
  • a combination therapy comprising a first pharmaceutical composition comprising a p53 reactivator provided herein and a first pharmaceutically acceptable excipient, and a second pharmaceutical composition comprising an inhibitor of PD-1 (e.g., pembrolizumab) provided herein and a second pharmaceutically acceptable excipient.
  • the first and the second pharmaceutically acceptable excipients can be the same or different.
  • a p53 reactivator provided herein and an inhibitor of PD-1 (e.g., pembrolizumab) provided herein are formulated together in a single pharmaceutical composition.
  • a pharmaceutical composition comprising a p53 reactivator provided herein, an inhibitor of PD-1 (e.g., pembrolizumab) provided herein, and one or more pharmaceutically acceptable excipient.
  • the p53 reactivator and/or the inhibitor of PD-1 (e.g., pembrolizumab) provided herein can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection (subcutaneous, intramuscular, intravenous, intraperitoneal, intraosseous, intracardiac, intraarticular, and intracavernous), sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal.
  • compositions may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, (7th ed.1999)).
  • effective concentrations of one or more compounds i.e., p53 reactivators or inhibitors of PD-1 (e.g., pembrolizumab) provided herein
  • pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient.
  • the concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein (e.g., cancer, including solid cancer and blood borne cancer).
  • a disease or disorder provided herein (e.g., cancer, including solid cancer and blood borne cancer).
  • the active compound is in an amount sufficient to exert a therapeutically useful effect on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems and then extrapolated therefrom for dosages for humans.
  • concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. [00241] It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated.
  • compositions should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • the p53 reactivators e.g, APR-246
  • inhibitors of PD-1 e.g., pembrolizumab
  • pharmaceutically acceptable salts thereof may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions.
  • liposomal suspensions including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art. 4.3.2 Compositions Relating to Combination Therapies with Bruton’s Tyrosine Kinase Inhibitors [00243]
  • the agonist of p53 such as the p53 reactivator provided herein can be formulated in a pharmaceutical composition that comprises a p53 reactivator provided herein and a pharmaceutically acceptable excipient.
  • an inhibitor of Bruton’s tyrosine kinase provided herein can be formulated in a pharmaceutical composition that comprises an inhibitor of Bruton’s tyrosine kinase (BTK) provided herein and a pharmaceutically acceptable excipient.
  • a combination therapy comprising administering a first pharmaceutical composition comprising a p53 reactivator and a first pharmaceutically acceptable excipient, and administering a second pharmaceutical composition comprising a BTK inhibitor and a second pharmaceutically acceptable excipient.
  • a p53 reactivator provided herein and a BTK inhibitor provided herein are formulated together in a pharmaceutical composition.
  • compositions comprising a p53 reactivator provided herein, a BTK inhibitor provided herein, and a pharmaceutically acceptable excipient.
  • the p53 reactivator and/or the BTK inhibitor provided herein can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection, sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal.
  • the compounds described above may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, (7th ed.1999)).
  • compositions effective concentrations of one or more compounds (i.e., p53 reactivator or BTK inhibitor provided herein) or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient.
  • concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein (e.g., non-Hodgkin lymphoma).
  • the active compound is in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems described herein and then extrapolated therefrom for dosages for humans.
  • concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • concentrations and dosage values may also vary with the severity of the condition to be alleviated.
  • compositions should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • the p53 reactivators e.g., APR-246
  • BTK inhibitor e.g., ibrutinib
  • pharmaceutically acceptable salts thereof may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions.
  • liposomal suspensions including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art. 4.3.3 Compositions Relating to Combination Therapies with XPO1 Inhibitors [00249]
  • the agonist of p53 such as the p53 reactivator provided herein can be formulated in a pharmaceutical composition that comprises a p53 reactivator provided herein and a pharmaceutically acceptable excipient.
  • an exportin 1 (XPO1) inhibitor provided herein can be formulated in a pharmaceutical composition that comprises the XPO1 inhibitor and a pharmaceutically acceptable excipient.
  • a combination therapy comprising administering a first pharmaceutical composition comprising a p53 reactivator and a first pharmaceutically acceptable excipient, and administering a second pharmaceutical composition comprising an XPO1 inhibitor and a second pharmaceutically acceptable excipient.
  • a p53 reactivator provided herein e.g., APR-246
  • an XPO1 inhibitor provided herein e.g., selinexor
  • compositions comprising a p53 reactivator provided herein (e.g., APR-246), an XPO1 inhibitor provided herein (e.g., selinexor), and a pharmaceutically acceptable excipient.
  • a p53 reactivator e.g., APR-246
  • an XPO1 inhibitor e.g., selinexor
  • a pharmaceutically acceptable excipient e.g., a pharmaceutically acceptable excipient.
  • the p53 reactivator and/or the XPO1 inhibitor provided herein can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection, infusion, sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal.
  • compositions may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, (7th ed.1999)).
  • effective concentrations of one or more compounds i.e., p53 reactivator or XPO1 inhibitor provided herein
  • pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient.
  • concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein.
  • the active compound is in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems described herein and then extrapolated therefrom for dosages for humans.
  • concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data.
  • concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • the p53 reactivators e.g., APR-246
  • the XPO1 inhibitor e.g., selinexor
  • pharmaceutically acceptable salts thereof may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated.
  • liposomal suspensions including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art. 4.3.4 Compositions Relating to Combination Therapies with WEE1 Inhibitors [00255]
  • a p53 reactivator e.g., APR-246 can be formulated with one or more pharmaceutically acceptable excipient.
  • an inhibitor of WEE1 can be formulated in a pharmaceutical composition with one or more pharmaceutically acceptable excipients.
  • a combination therapy comprising a first pharmaceutical composition comprising a p53 reactivator provided herein and a first pharmaceutically acceptable excipient, and a second pharmaceutical composition comprising an inhibitor of WEE1 (e.g., MK-1775) provided herein and a second pharmaceutically acceptable excipient.
  • the first and the second pharmaceutically acceptable excipients can be the same or different.
  • a p53 reactivator provided herein and an inhibitor of WEE1 (e.g., MK-1775) provided herein are formulated together in a single pharmaceutical composition.
  • a pharmaceutical composition comprising a p53 reactivator provided herein, an inhibitor of WEE1 (e.g., MK-1775) provided herein, and one or more pharmaceutically acceptable excipient.
  • the p53 reactivator and/or the inhibitor of WEE1 can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection (subcutaneous, intramuscular, intravenous, intraperitoneal, intraosseous, intracardiac, intraarticular, and intracavernous), sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal.
  • the compounds described above may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, (7th ed.1999)).
  • compositions effective concentrations of one or more compounds (i.e., p53 reactivators or inhibitors of WEE1 (e.g., MK-1775) provided herein) or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient.
  • concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein (e.g., cancer, including solid cancer and blood borne cancer).
  • the active compound is in an amount sufficient to exert a therapeutically useful effect on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems and then extrapolated therefrom for dosages for humans.
  • concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • concentrations and dosage values may also vary with the severity of the condition to be alleviated.
  • compositions should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • the p53 reactivators e.g, APR-246
  • inhibitors of WEE1 e.g., MK-1775
  • pharmaceutically acceptable salts thereof may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions.
  • liposomal suspensions including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art. 4.3.5 A Liquid Composition Comprising an Agonist of P53 [00261]
  • the agonist of p53 such as a p53 reactivator provided here (e.g., APR-246 or Compound A) is formulated in an aqueous solution, as described in US patent no.9,061,016, which is incorporated herein by reference, and as described in more detail below.
  • the formulation comprising the p53 reactivator is a stock solution and preferably is a pharmaceutical formulation in the form of a concentrated stock solution.
  • the formulation preferably is sterile, and this may be achieved by known sterilization methods such as filtration, allowing for long term storage.
  • the formulation preferably is sterile, and this may be achieved by known sterilization methods such as filtration, allowing for long term storage essentially without any deterioration of the p53 reactivator, e.g. by a chemical reaction of degradation, and essentially without formation of degradation products.
  • the formulation provided herein can be used, e.g.
  • the formulation provided herein is an aqueous solution of the p53 reactivator provided herein (e.g., APR-246 or Compound A), wherein the p53 reactivator is present at a concentration within a range of about 10 mg/mL to about 250 mg/mL, a range of about 50 mg/mL to about 200 mg/mL, or a range of about 75 mg/mL to about 150 mg/mL of the formulation.
  • the formulation may be diluted prior to use, e.g., administration to a patient.
  • the dilution factor depends on the concentration of the p53 reactivator in the formulation and the required amount of the compound needed, e.g., to meet the therapeutically effective dose.
  • the final diluted product in case of parenteral administration, has a pH within the range of about pH 4 to about pH 6.
  • the final diluted product for parenteral administration has a pH within the range of about pH 4.2 to about pH 5.5.
  • the liquid formulation may contain sodium chloride at a concentration of between 0% and 3%, a concentration of between 0.5% and 1.5%, or a concentration of between 0.8% and 1% weight by volume of the formulation.
  • the p53 reactivator e.g., APR-246 or Compound A
  • the liquid formulation in the form of an acid addition salt with one or several different pharmaceutically acceptable acids.
  • the pharmaceutically acceptable acid may be a mineral acid, e.g., selected from the group consisting of hydrochloric acid, hydrogen bromide, hydrogen iodide, sulphuric acid, nitric acid, phosphoric acid and the like.
  • the pharmaceutically acceptable acid may be an organic acid, e.g., a sulfonic or carboxylic acid, particularly an alkyl or aryl sulfonic acid or an alkyl or aryl carboxylic acid, such as selected from the group consisting of methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, acetic acid, tartaric acid, maleic acid, citric acid, benzoic acid, salicylic acid, ascorbic acid and the like.
  • the composition provided herein contains a pH regulating agent.
  • pH regulating agent means at least one pharmaceutically acceptable organic or inorganic (mineral) acid, or at least one pharmaceutically acceptable acid buffer or a mixture of any of these.
  • the pH regulating agent may be any such acid or buffer, or a mixture of acids or buffers, or a mixture of acid(s) and buffer(s). Examples of useful acids and buffers are as indicated herein.
  • the composition may contain at least one pharmaceutically acceptable acid.
  • the acid may be an inorganic mineral acid, e.g., selected from the group consisting of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid, phosphoric acid or the like, or an organic acid, e.g., selected from the group consisting of acetic acid, succinic acid, tartaric acid, maleic acid, ascorbic acid, citric acid, glutamic acid, benzoic acid, ascorbic acid, methanesulfonic acid, ethanesulfonic acid and the like. It is contemplated that the composition may contain one or several acids, selected from inorganic and organic acids. In one embodiment, the required pH of the formulation is achieved by addition of hydrochloric acid.
  • the composition provided herein also may comprise at least one pharmaceutically acceptable buffer, particularly selected from the group of citric buffer, acetate buffer, phosphate buffer and the like, separately or as a mixture thereof, as well as in combination with any pharmaceutically acceptable acid, as defined herein, e.g., hydrochloric acid.
  • the liquid composition provided herein is aqueous, which means that it contains water. However, it is contemplated that the aqueous solution and the aqueous phase used to prepare the composition also may contain other pharmaceutically acceptable liquids as a solvent phase, e.g., polyethylene glycol (PEG) and alcohols, e.g., ethanol.
  • PEG polyethylene glycol
  • alcohols e.g., ethanol
  • the aqueous phase mainly comprises water as a solvent.
  • the solvent phase is comprised of from 50 to 100% water, at least 80% water, at least 90% water, at least 95% water, at least 98% water or 100% water.
  • the composition described herein is provided as a stable stock solution, particularly as a concentrated stock solution for long term storage at a temperature range of 2-8° C., in a container, for example, a sealed and sterilized container.
  • the composition may comprise a stable aqueous WFI (water for injection) solution of the p53 reactivator optionally as an acid addition salt, in particular a hydrochloride addition salt, in a concentration of at about 10 mg/mL to about 250 mg/mL, at about 50 mg/mL to about 200 mg/mL, or at about 75 mg/mL to about 150 mg/mL, and a pH regulating agent in such an amount as to provide a pH in the solution in a range of between pH 3.0 and pH 5.0, between pH 3.2 and pH 4.7, between pH 3.5 and pH 4.5, or between pH 3.8 and pH 4.2, e.g., approximately 4.0.
  • a stable aqueous WFI water for injection solution of the p53 reactivator optionally as an acid addition salt, in particular a hydrochloride addition salt, in a concentration of at about 10 mg/mL to about 250 mg/mL, at about 50 mg/mL to about 200 mg/mL, or at about 75 mg/
  • the pH of the stock solution may have a lower limit selected from a pH of about 3.0, or about 3.2, e.g. about 3.4, such as about 3.6 or about 3.8, and an upper limit of about 5.0, or about 4.7, or about 4.5, or about 4.2, e.g. about 4.0.
  • Other components also may be added to or present in the aqueous phase, such as pharmaceutically acceptable inorganic salts, e.g., NaCl, preservatives, or further pharmaceutically acceptable compounds, e.g., further therapeutically active ingredients, such as cytostatics, particularly cisplatin, daunorubicin, cerubidine, cytarabine and fludarabine.
  • NaCl is added to the aqueous phase in an amount so as to provide a final liquid composition as defined herein above, containing NaCl at a concentration of between 0% and 3%, between 0.5% and 1.5%, or between 0.8% and 1% weight by volume of the formulation.
  • the composition is a sterile formulation. In this case, sterilization of the composition may be accomplished by passing the formulation, e.g., a formulated stock solution, through a sterile filter with a nominal pore size of 0.2 ⁇ m into a cleaned and sterilized container.
  • the composition may be provided as a ready-to-use injection solution, wherein a liquid formulation, e.g., a stock solution, is brought to the desired volume by addition of one or more pharmaceutically acceptable solvents, such as selected from the group consisting of WFI, a glucose solution, electrolyte solution containing amino acids, lipids, vitamins, and other minerals, Ringer's solution, Hartmann's solution, or a sodium chloride solution in the form of an isotonic, hypotonic or hypertonic solution.
  • a pharmaceutically acceptable solvents such as selected from the group consisting of WFI, a glucose solution, electrolyte solution containing amino acids, lipids, vitamins, and other minerals, Ringer's solution, Hartmann's solution, or a sodium chloride solution in the form of an isotonic, hypotonic or hypertonic solution.
  • a pharmaceutically acceptable solvents such as selected from the group consisting of WFI, a glucose solution, electrolyte solution containing amino acids, lipids, vitamins, and
  • the p53 reactivator is APR-246, which is formulated in liquid formulation, which comprises at least one pH regulating agent in an amount such as to provide a pH in the aqueous solution of from about 3.0 to about 5.0.
  • APR-246 is present in the aqueous solution at a concentration of from 10 mg/mL to 250 mg/mL.
  • the aqueous solution comprises NaCl at a concentration of between 0% to 3% weight by volume.
  • the p53 reactivator is Compound A.
  • the agonist of p53 such as the p53 reactivator provided here (e.g., APR-246 or Compound A) is formulated in a composition for oral administration.
  • the oral dosage form is a solid form.
  • Pharmaceutical compositions that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art.
  • Typical oral dosage forms are prepared by combining the active ingredients in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques.
  • Excipients can take a wide variety of forms depending on the form of preparation desired for administration. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.
  • tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy.
  • compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.
  • a tablet can be prepared by compression or molding.
  • Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free- flowing form such as powder or granules, optionally mixed with an excipient.
  • Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • excipients that can be used in oral dosage forms provided herein include, but are not limited to, binders, fillers, disintegrants, and lubricants.
  • Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.
  • Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105, and mixtures thereof.
  • Suitable anhydrous or low moisture excipients or additives include AVICEL- PH-103TM and Starch 1500 LM.
  • fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
  • Disintegrants are used in compositions to provide tablets that disintegrate when exposed to an aqueous environment. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art.
  • Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof. [00288] Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions.
  • Lubricants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil, zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.
  • Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, and mixtures thereof. 4.3.7 A Lyophilized Composition Comprising an Inhibitor of PD-1 Mediated Signaling
  • the inhibitor of PD-1 mediated signaling such as an inhibitor of PD-1 (e.g., pembrolizumab) provided herein is supplied in a sterile form for reconstitution as a suspension for subcutaneous injection or reconstitution as a solution with further dilution for intravenous infusion.
  • the inhibitor of PD-1 e.g., pembrolizumab
  • the inhibitor of PD-1 is formulated for parenteral administration.
  • compositions for parenteral administration include intravenous, subcutaneous, and intramuscular administrations.
  • Compositions for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, sterile suspensions ready for injection, and sterile emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • the unit dose parenteral preparations can be packaged in an ampoule, a vial or a syringe with a needle.
  • pharmaceutically acceptable excipients used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances.
  • the inhibitor of PD-1 e.g., pembrolizumab
  • Lyophilized powders can be reconstituted for administration as solutions, emulsions, and other mixtures.
  • the sterile, lyophilized powder can be prepared by dissolving an inhibitor of PD-1 (e.g., pembrolizumab) provided herein, or a pharmaceutically acceptable salt thereof, in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose.
  • the solvent may also contain a buffer, such as citrate, phosphate, or other buffers known to those of skill in the art.
  • the lyophilized formulations are suitable for reconstitution with a suitable diluent to the appropriate concentration prior to administration.
  • the lyophilized formulation is suitable for reconstitution with an aqueous solution for intravenous administrations.
  • the lyophilized formulation provided herein is suitable for reconstitution with water.
  • the lyophilized formulations comprise the inhibitor of PD-1 (e.g., pembrolizumab) provided herein, a buffer and a bulking agent.
  • a lyophilized formulation comprises an inhibitor of PD-1 (e.g., pembrolizumab) provided herein, in about 0.1 to about 60% based on the total weight of the lyophilized formulation.
  • the lyophilized formulation can be reconstituted for parenteral administration to a patient using any pharmaceutically acceptable diluent.
  • Such diluents include, but are not limited to Sterile Water for Injection (SWFI), Dextrose 5% in Water (D5W), or a cosolvent system.
  • a p53 agonist such as a p53 reactivator (see Section 4.2.1) in combination with a second therapeutic agent, for example, an inhibitor of PD-1 mediated signaling (see Section 4.2.3), a Bruton’s tyrosine kinase (BTK) inhibitor (see Section 4.2.3), an exportin 1 (XPO1) inhibitor (see Section 4.2.4), or an inhibitor of Wee1-like protein kinase (WEE1) (see Section 4.2.5), for treating a disease or disorder.
  • a p53 agonist such as a p53 reactivator (see Section 4.2.1) in combination with a second therapeutic agent, for example, an inhibitor of PD-1 mediated signaling (see Section 4.2.3), a Bruton’s tyrosine kinase (BTK) inhibitor (see
  • such combination therapies relate to methods comprising administering an effective amount of the p53 agonist such as a p53 reactivator and the second therapeutic agent to a subject.
  • the subject is a human.
  • combination therapies using a compound of Formula (I) in combination with a second therapeutic agent, for example, an inhibitor of PD-1 mediated signaling (see Section 4.2.3), a Bruton’s tyrosine kinase (BTK) inhibitor (see Section 4.2.3), an exportin 1 (XPO1) inhibitor (see Section 4.2.4), or an inhibitor of Wee1-like protein kinase (WEE1) (see Section 4.2.5), for treating a disease or disorder, wherein the compound of Formula (I) shows synergism with the second therapeutic agent provided herein for treating the disease or disorder.
  • a second therapeutic agent for example, an inhibitor of PD-1 mediated signaling (see Section 4.2.3), a Bruton’s tyrosine kinase (BTK) inhibitor (see Section 4.2.3), an
  • such combination therapies relate to methods comprising administering an effective amount of the compound of Formula (I) that shows synergism and the second therapeutic agent to a subject.
  • the subject is a human.
  • 4.4.1 Combination Therapies with Inhibitors of PD-1 Mediated Signaling [00301]
  • a composition or a combination of compositions for use in the prevention and/or treatment of a disease or condition comprising the agonist of p53 such as a p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 mediated signaling such as an inhibitor of PD-1 (e.g., pembrolizumab).
  • Such uses relate to a method comprising administering an effective amount of the p53 reactivator and the inhibitor of PD-1 (e.g., pembrolizumab) to a subject.
  • the subject is a human.
  • Provided herein are methods of preventing and/or treating a disease or disorder (e.g., a solid tumor malignancy) in a subject comprising administering to a subject an agonist of p53 and an inhibitor of PD-1 mediated signaling.
  • a disease or disorder e.g., a solid tumor malignancy
  • methods of preventing a disease or disorder in a subject comprising administering to a subject an agonist of p53 and an inhibitor of PD-1 mediated signaling.
  • a disease or disorder e.g. a solid tumor malignancy
  • methods of treating a disease or disorder comprising administering to a subject an agonist of p53 and an inhibitor of PD-1 mediated signaling.
  • a pharmaceutically effective amount of the agonist of p53 is administered.
  • the agonist of p53 and the inhibitor of PD-1 mediated signaling are concomitantly administered.
  • the co-administration of the agonist of p53 and the inhibitor of PD-1 mediated signaling is pharmaceutically effective to treat the disease or disorder (e.g., a solid tumor malignancy).
  • a disease or disorder e.g., a solid tumor malignancy
  • methods of treating a disease or disorder comprising administering to a subject a compound of Formula (I) and an inhibitor of PD-1 mediated signaling, wherein the compound of Formula (I) shows synergism with the inhibitor of PD-1 mediated signaling for treating the disease or disorder.
  • a pharmaceutically effective amount of the compound of Formula (I) is administered.
  • the co-administration of the compound of Formula (I) and the inhibitor of PD-1 mediated signaling is pharmaceutically effective to treat the disease or disorder (e.g., a solid tumor malignancy).
  • the compound that has synergism with the inhibitor of PD-1 mediated signaling is APR-246. In another specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is Compound A. In another specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is Compound B. In another specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is Compound C. In another specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is Compound D. In another specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is Compound E.
  • the disease or disorder is a neoplastic disease.
  • the disease or disorder is a cancer.
  • the disease or disorder is a solid tumor malignancy.
  • the neoplastic, cancerous, or malignant cell in a subject to be treated with a method provided herein comprises a mutation in TP53.
  • provided herein is a method to treat or prevent a solid tumor cancer with a mutation in the p53 gene.
  • the cancer to be treated with a method provided herein comprises cancer cells that carry a mutation in TP53.
  • the mutation in TP53 is R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, V173A, S241F, R249S or a combination thereof.
  • the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation.
  • the mutant TP53 includes a nonsense mutation.
  • a nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product.
  • Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP53 mutations in cancer.
  • the most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*.
  • the cancer to be treated does not comprise any cells carrying a mutation in the TP53 gene.
  • the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis.
  • diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct
  • the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small- cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer.
  • the disease or disorder is a solid tumor cancer.
  • the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer.
  • the solid tumor malignancy is an advanced non-CNS-primary solid tumor.
  • the solid tumor malignancy is selected from the group consisting of gastric/gastroesophageal junction (GEJ) cancer, bladder/urothelial cancer, and non- small-cell lung cancer (NSCLC).
  • GEJ gastric/gastroesophageal junction
  • NSCLC non- small-cell lung cancer
  • the dose administered to a subject in the context of the present disclosure should be sufficient to effect a therapeutic response.
  • dosage will depend upon a variety of factors including the potency of the specific compound, the age, condition and body weight of the patient, as well as the stage/severity of the disease.
  • the dose will also be determined by the route (administration form) timing and frequency of administration.
  • the p53 reactivator e.g., APR-246
  • the inhibitor of PD-1 e.g., pembrolizumab
  • the p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 are administered together in the same pharmaceutical composition.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 are administered simultaneously.
  • the term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 (e.g., pembrolizumab) are not administered simultaneously, and instead the two compounds are administered at different times.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 (e.g., pembrolizumab) are administered at least once during a dosing period.
  • a dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once.
  • a dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.
  • a dosing period is a dosing cycle.
  • the prophylactic or therapeutic agent can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time).
  • the agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity.
  • Stable disease or lack is determined by methods known in the art such as evaluation of patient symptoms, physical examination, and visualization of the tumor that has been imaged using X- ray, CAT, PET, MRI scan, or other commonly accepted evaluation modalities.
  • the prophylactic or therapeutic agent can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID).
  • the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug).
  • the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time.
  • the term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days.
  • the term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days.
  • the frequency of administration is in the range of about a daily dose to about a monthly dose.
  • administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks.
  • the compound is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks.
  • APR-246 is administered at a dose of less than 150 mg/kg.
  • APR-246 is administered at a dose of less than 100 mg/kg. In some embodiments, APR-246 is administered at a dose of between 100 mg/kg and 25 mg/kg. In other embodiments, APR-246 is administered at dose of less than 75 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 65 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 50 mg/kg. [00320] In other embodiments, APR-246 is administered at a fixed dose within the interval 2.7-7.5 g. In some embodiments, the fixed dose of APR-246 is no more than 4.5 g.
  • the fixed dose of APR-246 is no more than 4.0 g. In some embodiments, the fixed dose of APR-246 is no more than 3.5 g. [00321] In some embodiments, APR-246 is administered at about 4.5 g/day for 4 days in combination with pembrolizumab at the dose of about 200 mg once of each 21-day cycle. In some specific embodiments, APR-246 is administered at about 4.5 g/day on Days 1–4 in combination with pembrolizumab at the dose of about 200 mg on Day 3 of each 21-day cycle. In some embodiments, APR-246 and pembrolizumab are administered for 1 to 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 1 cycle.
  • APR-246 and pembrolizumab are administered for 2 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 3 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 4 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 5 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 6 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 7 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 8 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 9 cycles.
  • APR-246 and pembrolizumab are administered for 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for more than 10 cycles. [00322] In some embodiments, APR-246 is administered at about 4.0 g/day for 4 days in combination with pembrolizumab at the dose of about 200 mg once of each 21-day cycle. In some specific embodiments, APR-246 is administered at about 4.0 g/day on Days 1–4 in combination with pembrolizumab at the dose of about 200 mg on Day 3 of each 21-day cycle. In some embodiments, APR-246 and pembrolizumab are administered for 1 to 10 cycles.
  • APR-246 and pembrolizumab are administered for 1 cycle. In some embodiments, APR-246 and pembrolizumab are administered for 2 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 3 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 4 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 5 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 6 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 7 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 8 cycles.
  • APR-246 and pembrolizumab are administered for 9 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for more than 10 cycles. [00323] In some embodiments, APR-246 is administered at about 3.5 g/day for 4 days in combination with pembrolizumab at the dose of about 200 mg once of each 21-day cycle. In some specific embodiments, APR-246 is administered at about 3.5 g/day on Days 1–4 in combination with pembrolizumab at the dose of about 200 mg on Day 3 of each 21-day cycle.
  • APR-246 and pembrolizumab are administered for 1 to 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 1 cycle. In some embodiments, APR-246 and pembrolizumab are administered for 2 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 3 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 4 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 5 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 6 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 7 cycles.
  • APR-246 and pembrolizumab are administered for 8 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 9 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for more than 10 cycles.
  • Exemplary Embodiments [00324] Embodiment A1. A method of treating solid tumor malignancy in a subject, comprising administering to the subject: (i) an agonist of p53; and (ii) an inhibitor of PD-1 mediated signaling. [00325] Embodiment A2.
  • Embodiment A1 wherein the agonist of p53 is a compound that can give reactivation of a mutant p53 or a compound whose metabolite can give reactivation of a mutant p53.
  • Embodiment A3 The method of Embodiment A2, wherein the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W.
  • Embodiment A4 Embodiment A4.
  • Embodiment A2 wherein the agonist of p53 is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity.
  • Embodiment A5. The method of Embodiment A2, wherein the compound or metabolite promotes proper folding of mutant and wild-type p53 proteins.
  • Embodiment A6 Embodiment A6.
  • Embodiment A2 wherein the compound or metabolite is capable of shifting the equilibrium from unfolded towards a folded structure of wild-type or mutant p53, or wherein the compound or metabolite is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or metabolite is capable of reducing aggregation of the wild-type or mutant p53.
  • Embodiment A7 The method of Embodiment A2, wherein the compound or metabolite is capable of promoting a folded structure of wild-type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53.
  • Embodiment A8 The method of Embodiment A7, wherein the compound or metabolite is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promote a folded conformation.
  • the compound that can give reactivation of mutant p53 is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one; 2,2-bis(hydroxymethyl)quinuclidin-3-one; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((((3-oxoquinu
  • Embodiment A10 The method of Embodiment A9, wherein the compound is 2- (hydroxymethyl)-2- (methoxymethyl) quinuclidin-3-one having the following formula: (APR-246), or a pharmaceutically acceptable salt thereof.
  • Embodiment A11 The method of Embodiment A9, wherein the compound is 2,2,2- trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide having the following formula: (Compound A), or a pharmaceutically acceptable salt thereof.
  • Embodiment A13 The method of Embodiment A12, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab; nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, nivolumab, AMP-224, and AMP-514.
  • Embodiment A14 Embodiment A14.
  • Embodiment A13 wherein the anti-PD-1 antibody is pembrolizumab.
  • Embodiment A15 The method of Embodiment A1, wherein the agonist of p53 is APR-246 and the inhibitor of PD-1 mediated signaling is pembrolizumab.
  • Embodiment A16 The method of Embodiment A15, wherein APR-246 is administered at a dose of about 4.5 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle.
  • Embodiment A17 Embodiment A17.
  • Embodiment A15 wherein APR-246 is administered at a dose of about 4.0 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle.
  • Embodiment A18 The method of Embodiment A15, wherein APR-246 is administered at a dose of about 3.5 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle.
  • Embodiment A19 The method of any one of Embodiments A15 to A18, wherein APR-246 is administered on Days 1–4 and pembrolizumab is administered on Day 3 of the each 21-day cycle.
  • Embodiment A20 The method of any one of Embodiments A16 to A19, wherein APR-246 and pembrolizumab are administered for 1 to 20 cycles.
  • Embodiment A21 The method of any one of Embodiments A1 to A20, wherein the agonist of p53 is formulated in a first pharmaceutical composition and the inhibitor of PD-1 mediated signaling is formulated in a second pharmaceutical composition.
  • Embodiment A22 Embodiment A22.
  • Embodiment A23 The method of any one of Embodiments A1 to A20, wherein the solid tumor malignancy is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer.
  • Embodiment A23 Embodiment A23.
  • Embodiment A24 The method of any one of Embodiments A1 to A20, wherein the solid tumor malignancy is selected from the group consisting of gastric/gastroesophageal junction (GEJ) cancer, bladder/urothelial cancer, and non-small-cell lung cancer (NSCLC).
  • GEJ gastric/gastroesophageal junction
  • NSCLC non-small-cell lung cancer
  • combination therapies using a p53 reactivator (such as the p53 reactivator described in Section 4.2.1) in combination with a BTK inhibitor (such as the BTK inhibitor described in Section 4.2.3) for treating a disease or disorder (such as lymphoma, e.g., a non-Hodgkin lymphoma).
  • a p53 reactivator such as the p53 reactivator described in Section 4.2.1
  • a BTK inhibitor such as the BTK inhibitor described in Section 4.2.3
  • a disease or disorder such as lymphoma, e.g., a non-Hodgkin lymphoma.
  • a disease or disorder e.g., lymphoma
  • a p53 reactivator a compound that can give reactivation of a mutant p53
  • BTK Bruton’s tyrosine kinase
  • a pharmaceutically effective amount of the p53 reactivator is administered.
  • the p53 reactivator and the BTK inhibitor are concomitantly administered.
  • the co-administration of the compound that can give reactivation of a mutant p53 (the p53 reactivator) and the BTK inhibitor is pharmaceutically effective to treat the disease or disorder (e.g., non-Hodgkin lymphoma).
  • a disease or disorder e.g., lymphoma
  • methods of treating a disease or disorder (e.g., lymphoma) in a subject comprising administering to a subject a compound of Formula (I) and a Bruton’s tyrosine kinase (BTK) inhibitor, wherein the compound of Formula (I) shows synergism with the Bruton’s tyrosine kinase (BTK) inhibitor for treating the disease or disorder.
  • a pharmaceutically effective amount of the compound of Formula (I) is administered.
  • the co-administration of the compound of Formula (I) and the BTK inhibitor is pharmaceutically effective to treat the disease or disorder (e.g., non- Hodgkin lymphoma).
  • the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is APR-246.
  • the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is Compound A.
  • the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is Compound B.
  • the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is Compound C.
  • the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is Compound D.
  • the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is Compound E.
  • the subject is a human.
  • the subject is a subject diagnosed with lymphoma. In some embodiments, the subject is a subject diagnosed with non-Hodgkin lymphoma (NHL). In some embodiments, the subject is a subject diagnosed with mature (peripheral) B-cell neoplasm. In some preferred embodiments, the subject is a subject diagnosed with chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). In one embodiment, the subject is a subject diagnosed with chronic lymphocytic leukemia (CLL). In another embodiment, the subject is a subject diagnosed with cell lymphoma (MCL).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • the subject is a subject diagnosed with relapsed and/or refractory (R/R) CLL or relapsed and/or refractory (R/R) MCL.
  • the subject is a subject diagnosed with R/R CLL.
  • the subject is subject diagnosed with R/R MCL.
  • the subject is diagnosed to have TP53 mutation.
  • the subject carries TP53 mutation.
  • the subject has mutant p53 protein.
  • the subject is a subject diagnosed with a TP53 mutant lymphoma.
  • the subject is a subject diagnosed with a TP53 mutant non- Hodgkin lymphoma (NHL). In some embodiments, the subject is a subject diagnosed with a TP53 mutant mature (peripheral) B-cell neoplasm. In some preferred embodiments, the subject is a subject diagnosed with a TP53 mutant chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). In one embodiment, the subject is a subject diagnosed with a TP53 mutant chronic lymphocytic leukemia (CLL). In another embodiment, the subject is a subject diagnosed with a TP53 mutant mantle cell lymphoma (MCL).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • the subject is a subject diagnosed with a TP53 mutant R/R CLL or a TP53 mutant R/R MCL. In yet another embodiment, the subject is a subject diagnosed with a TP53 mutant R/R CLL. In yet another embodiment, the subject is a subject diagnosed with a TP53 mutant R/R MCL. [00353] In some embodiments, the methods described herein further comprises determining by gene sequencing if the subject has TP53 mutation. [00354] In some embodiments, the disease or disorder is a mutant p53 mediated cancer (including, e.g., hematological tumors with mutations in the p53 gene). In other embodiments, the disease or disorder is not a mutant p53 mediated cancer.
  • the disease or disorder is a disease characterized by apoptotic dysfunction and/or overexpression of a Bruton’s tyrosine kinase.
  • the disease or disorder is mutant p53 mediated and characterized by apoptotic dysfunction and/or overexpression of a Bruton’s tyrosine kinase.
  • the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma. In some embodiments, the disease or disorder is a lymphoma.
  • the disease or disorder is Hodgkin's lymphoma or non- Hodgkin's lymphoma (NHL). In some embodiments, the disease or disorder is a Hodgkin lymphoma. In some embodiments, the disease or disorder is a non-Hodgkin lymphoma (NHL). In some embodiments, the disease or disorder is a mature (peripheral) B-cell neoplasm.
  • the disease or disorder a non-Hodgkin lymphoma (NHL) selected from the group consisting of: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, diffuse large cell B-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma (plasmacytoma), extranodal marginal zone B-cell lymphoma, nodal marginal zone lymphoma, follicle center lymphoma, and Burkitt’s leukemia (Burkitt’s lymphoma).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • B-cell prolymphocytic leukemia diffuse large cell B-cell lymphoma
  • LLBCL diffuse large cell B-cell lymphoma
  • the disease or disorder is chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). In some preferred embodiments, the disease or disorder is chronic lymphocytic leukemia (CLL). In some preferred embodiments, the disease or disorder is mantle cell lymphoma (MCL). [00358] In some embodiments, the disease or disorder is a TP53 mutant lymphoma. In some embodiments, the disease or disorder is a TP53 mutant non-Hodgkin lymphoma (NHL). In some embodiments, the disease or disorder is a TP53 mutant mature (peripheral) B-cell neoplasm.
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • the disease or disorder is a TP53 mutant chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL).
  • the disease or disorder is a TP53 mutant chronic lymphocytic leukemia (CLL).
  • the disease or disorder is a TP53 mutant mantle cell lymphoma (MCL).
  • the disease or disorder is a TP53 mutant R/R CLL or a TP53 mutant R/R MCL.
  • the disease or disorder is a TP53 mutant R/R CLL.
  • the disease or disorder is a TP53 mutant R/R MCL.
  • the mutation in TP53 is R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, V173A, S241F, R249S or a combination thereof.
  • the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation.
  • the mutant TP53 includes a nonsense mutation.
  • a nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product.
  • Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP53 mutations in cancer.
  • the most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*.
  • the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis.
  • diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct
  • the disease or disorder is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (
  • the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer.
  • the disease or disorder is a solid tumor cancer.
  • the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer.
  • the amount of the p53 reactivator or the BTK inhibitor provided herein, or a pharmaceutical composition that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some embodiments, should be decided according to the judgment of the practitioner and each patient’s circumstances. Effective doses may also be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the p53 reactivator is administered to the subject as part a composition.
  • the composition is a pharmaceutical composition described in Section 4.3.2.
  • the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) can be formulated in different pharmaceutical compositions and administered separately to the subject in need thereof. In other embodiments, the p53 reactivator (e.g., APR- 246) and the BTK inhibitor (e.g., ibrutinib) are administered together in the same pharmaceutical composition. [00367] In some embodiments, the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) are administered simultaneously.
  • the term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours.
  • the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) are not administered simultaneously, and instead the two compounds are administered at different times.
  • the BTK inhibitor e.g., ibrutinib
  • the BTK inhibitor is administered before or concurrently with the administration of the p53 reactivator (e.g., APR-246).
  • the BTK inhibitor e.g., ibrutinib
  • the p53 reactivator e.g., APR-246
  • the subject has been previously treated with the BTK inhibitor (e.g., ibrutinib) prior to the administration of the p53 reactivator (e.g., APR-246).
  • the subject has been previously treated with the p53 reactivator (e.g., APR-246) prior to the administration of the BTK inhibitor (e.g., ibrutinib).
  • the subject has not been previously treated with any BTK inhibitor (e.g., ibrutinib, acalabrutinib, zanubrutinib, etc.) prior to the co-administration of the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib).
  • the co-administration of the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) is concomitant administration.
  • the co-administration of the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) is pharmaceutically effective to treat lymphoma.
  • the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) are administered at least once during a dosing period.
  • a dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once.
  • a dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.
  • a dosing period is a dosing cycle.
  • the prophylactic or therapeutic agent can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time).
  • the agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity.
  • the prophylactic or therapeutic agent can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID).
  • the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug).
  • the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time.
  • the term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days.
  • the term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days.
  • the frequency of administration is in the range of about a daily dose to about a monthly dose.
  • administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks.
  • the compound is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks.
  • APR-246 is administered at a dose of less than about 150 mg/kg.
  • APR-246 is administered at a dose of less than about 100 mg/kg. In some embodiments, APR-246 is administered at a dose of between about 100 mg/kg and about 25 mg/kg. In other embodiments, APR-246 is administered at dose of less than about 75 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than about 65 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than about 50 mg/kg. [00382] In other embodiments, APR-246 is administered at a fixed dose within the interval of 2.7-7.5 g. In some embodiments, the fixed dose of APR-246 is no more than about 4.5 g.
  • the fixed dose of APR-246 is no more than about 4.0 g. In some embodiments, the fixed dose of APR-246 is no more than about 3.5 g. In one embodiments, APR-246 is administered at a dose of about 4.5 g/day, about 4.0 g/day, or about 3.5 g/day. In one specific embodiment, APR-246 is administered at a dose of about 4.5 g/day. In another specific embodiment, APR-246 is administered at a dose of about 4.0 g/day. In yet another specific embodiment, APR-246 is administered at a dose of about 3.5 g/day.
  • APR-246 is administered in a multiple-step administration to avoid high plasma concentration and/or to minimize the risk of adverse events.
  • APR-246 is administered in a 2-step administration consisting of a first loading dose and a subsequent maintenance dose.
  • the first loading dose is not equal to the subsequent maintenance dose.
  • the first loading dose is not equal to the subsequent maintenance dose.
  • the first loading dose is about 1.5 g, about 1.33 g, or about 1.16 g.
  • the subsequent maintenance dose is about 3.0 g, about 2.67 g, or about 2.34 g.
  • APR-246 is administered 1, 2, 3, 4, 5, 6, 7, or 8 times of each cycle of 14 days, 21 days, every 28 days, every 35 days, or every 42 days. In preferred embodiments, APR-246 is administered for 4 days in each treatment cycle. In preferred embodiments, APR-246 is administered for 4 days in each treatment cycle of 28 days. In some embodiments, APR-246 is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles. [00385] In a specific embodiment, APR-246 is administered once daily for 4 consecutive days in each treatment cycle of 28 days. [00386] In some preferred embodiments, APR-246 is administered intravenously.
  • the BTK inhibitor described herein is administered at a dose between 1 mg/day to 1000 mg/day.
  • the BTK inhibitor is administered at a dose of about 100 mg/day, about 200 mg/day, about 300 mg/day, about 400 mg/day, about 500 mg/day, about 600 mg/day, about 700 mg/day, about 800 mg/day, about 900 mg/day, or about 1000 mg/day.
  • the BTK inhibitor is administered at a dose of about 420 mg/day or about 560 mg/day. In one embodiment, the BTK inhibitor is administered at a dose of about 420 mg/day.
  • the BTK inhibitor is administered at a dose of about 560 mg/day.
  • ibrutinib is administered at a dose of about 420 mg or about 560 mg.
  • the BTK inhibitor e.g., ibrutinib
  • the BTK inhibitor is administered daily.
  • ibrutinib is administered daily for each treatment cycle of 28 days.
  • the BTK inhibitor e.g., ibrutinib
  • the BTK inhibitor is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles.
  • the BTK inhibitor is administered orally.
  • combination treatment of the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) show synergistic effects in treating lymphoma.
  • APR-246 is administered at a dose of about 4.5 g/day for 4 days in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. In some specific embodiments, APR-246 is administered at a dose about 4.5 g/day on Days 1–4 in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle.
  • APR-246 and ibrutinib are administered for 1 to 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for 1 cycle. In some embodiments, APR-246 and ibrutinib are administered for 2 cycles. In some embodiments, APR-246 and ibrutinib are administered for 3 cycles. In some embodiments, APR-246 and ibrutinib are administered for 4 cycles. In some embodiments, APR-246 and ibrutinib are administered for 5 cycles. In some embodiments, APR-246 and ibrutinib are administered for 6 cycles. In some embodiments, APR-246 and ibrutinib are administered for 7 cycles.
  • APR-246 and ibrutinib are administered for 8 cycles. In some embodiments, APR-246 and ibrutinib are administered for 9 cycles. In some embodiments, APR-246 and ibrutinib are administered for 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for more than 10 cycles. [00393] In some embodiments, APR-246 is administered at a dose of about 4.0 g/day for 4 days in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle.
  • APR-246 is administered at a dose about 4.5 g/day on Days 1–4 in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle.
  • APR-246 and ibrutinib are administered for 1 to 10 cycles.
  • APR-246 and ibrutinib are administered for 1 cycle.
  • APR-246 and ibrutinib are administered for 2 cycles.
  • APR-246 and ibrutinib are administered for 3 cycles.
  • APR-246 and ibrutinib are administered for 4 cycles.
  • APR-246 and ibrutinib are administered for 5 cycles. In some embodiments, APR-246 and ibrutinib are administered for 6 cycles. In some embodiments, APR-246 and ibrutinib are administered for 7 cycles. In some embodiments, APR-246 and ibrutinib are administered for 8 cycles. In some embodiments, APR-246 and ibrutinib are administered for 9 cycles. In some embodiments, APR-246 and ibrutinib are administered for 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for more than 10 cycles.
  • APR-246 is administered at a dose of about 3.5 g/day for 4 days in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. In some specific embodiments, APR-246 is administered at a dose about 4.5 g/day on Days 1–4 in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. In some embodiments, APR-246 and ibrutinib are administered for 1 to 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for 1 cycle.
  • APR-246 and ibrutinib are administered for 2 cycles. In some embodiments, APR-246 and ibrutinib are administered for 3 cycles. In some embodiments, APR-246 and ibrutinib are administered for 4 cycles. In some embodiments, APR-246 and ibrutinib are administered for 5 cycles. In some embodiments, APR-246 and ibrutinib are administered for 6 cycles. In some embodiments, APR-246 and ibrutinib are administered for 7 cycles. In some embodiments, APR-246 and ibrutinib are administered for 8 cycles. In some embodiments, APR-246 and ibrutinib are administered for 9 cycles.
  • APR-246 and ibrutinib are administered for 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for more than 10 cycles. [00395] In certain embodiments, the methods described herein further comprises determining by gene sequencing if the subject has TP53 mutation. In certain embodiments, a next generation sequencing (NGS) method is used for the gene sequencing. In certain embodiments, the gene sequencing data are interpreted to determine if the subject has or likely has TP53 mutation.
  • NGS next generation sequencing
  • a disease or disorder described herein e.g., lymphoma
  • combination therapies of the p53 reactivator and the Bruton’s tyrosine kinase (BTK) inhibitor provided herein for use in treating chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • compositions or a combination of pharmaceutical compositions of the p53 reactivator and the Bruton’s tyrosine kinase (BTK) inhibitor provided herein for use in treating a disease or disorder (e.g., lymphoma).
  • a disease or disorder e.g., lymphoma
  • pharmaceutical compositions or a combination of pharmaceutical compositions of the p53 reactivator and the Bruton’s tyrosine kinase (BTK) inhibitor provided herein for use in treating chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • Embodiment B1 A method of treating a lymphoma in a subject, comprising administering to the subject: (i) a p53 reactivator; and (ii) a Bruton’s tyrosine kinase (BTK) inhibitor.
  • BTK tyrosine kinase
  • Embodiment B1 wherein the p53 reactivator is a compound that can give reactivation of a mutant p53, or a degradation product thereof that can give reactivation of a mutant p53.
  • Embodiment B3 The method of Embodiment B2, wherein the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W.
  • Embodiment B4 Embodiment B4.
  • Embodiment B5 The method of any one of Embodiments B2-B4, wherein the compound or degradation product thereof promotes proper folding of mutant and wild-type p53 proteins.
  • Embodiment B7 The method of any one of Embodiments B2-B4, wherein the compound or degradation product thereof is capable of promoting a folded structure of wild- type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53.
  • Embodiment B8 The method of any one of Embodiments B2-B6, wherein the compound or degradation product threreof is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promote a folded conformation.
  • Embodiment B9 The method of any one of Embodiments B2-B8, wherein the compound that can give reactivation of mutant p53 is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one; 2,2-bis(hydroxymethyl)quinuclidin-3-one; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-3-one
  • Embodiment B10 The method of Embodiment B9, wherein the compound is 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one (APR-246) having the following formula: , or a pharmaceutically acceptable salt thereof.
  • APR-246 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one
  • Embodiment B9 wherein the compound is a compound selected from the group consisting of: 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; and 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, or a pharmaceutically acceptable salt thereof.
  • Embodiment B12 The method of Embodiment B11, wherein the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound A), or a pharmaceutically acceptable salt thereof.
  • Embodiment B13 The method of Embodiment B11, wherein the compound is 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound B), or a pharmaceutically acceptable salt thereof.
  • Embodiment B14 Embodiment B14.
  • Embodiment B11 wherein the compound is 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound C), or a pharmaceutically acceptable salt thereof.
  • Embodiment B15 The method of Embodiment B11, wherein the compound is N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound D), or a pharmaceutically acceptable salt thereof.
  • Embodiment B16 Embodiment B16.
  • Embodiment B11 wherein the compound is 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound E), or a pharmaceutically acceptable salt thereof.
  • Compound E 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide
  • Embodiments B1-B16 wherein the BTK inhibitor is selected from the group consisting of ibrutinib, acalabrutinib, zanubrutinib, evobrutinib, dasatinib, spebrutinib, tirabrutinib, ABBV-105, ONO-4059, LFM-A13, and HM71224, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • the BTK inhibitor is selected from the group consisting of ibrutinib, acalabrutinib, zanubrutinib, evobrutinib, dasatinib, spebrutinib, tirabrutinib, ABBV-105, ONO-4059, LFM-A13, and HM71224, or a stereoisomer, mixture of stereoisomers, tautomer, isotopo
  • Embodiment B16 wherein the BTK inhibitor is ibrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • Embodiment B19 The method of any one of Embodiments B1-B10 and B17- B18, wherein the p53 reactivator is APR-246 and the BTK inhibitor is ibrutinib.
  • Embodiment B20 Embodiment B20.
  • Embodiment B19 wherein APR-246 is administered at a dose of about 4.5 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle.
  • Embodiment B21 The method of Embodiment B19, wherein APR-246 is administered at a dose of about 4.0 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle.
  • Embodiment B22 Embodiment B22.
  • Embodiment B19 wherein APR-246 is administered at a dose of about 3.5 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle.
  • Embodiment B23 The method of any one of Embodiments B20-B22, wherein APR-246 is administered on Days 1–4 and ibrutinib is administered daily of each 28-day cycle.
  • Embodiment B24 The method of any one of Embodiments B20-B23, wherein APR-246 and ibrutinib are administered for 1 to 20 cycles.
  • Embodiment B25 Embodiment B25.
  • Embodiment B26 The method of any one of Embodiments B1-B25, wherein the lymphoma is a Hodgkin lymphoma (HL) or a non-Hodgkin lymphoma (NHL).
  • Embodiment B27 The method of Embodiment B26, wherein the lymphoma is a non-Hodgkin lymphoma (NHL).
  • Embodiment B28 The method of any one of Embodiments B1-B24, wherein the compound that can give reactivation of the mutant p53 is formulated in a first pharmaceutical composition and the BTK inhibitor is formulated in a second pharmaceutical composition.
  • Embodiment B27 wherein the non-Hodgkin lymphoma (NHL) is a mature (peripheral) B-cell neoplasm.
  • Embodiment B29 The method of Embodiment B27, wherein the non-Hodgkin lymphoma (NHL) is selected from the group consisting of: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, diffuse large cell B-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma (plasmacytoma), extranodal marginal zone B-cell lymphoma, nodal marginal zone lymphoma, follicle center lymphoma, and Burkitt’s leukemia (Burkitt’s lymphoma).
  • CLL chronic lymphocytic leukemia
  • Embodiment B30 The method of any one of Embodiments B1-B29, wherein the lymphoma is chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL).
  • Embodiment B31 The method of Embodiment B30, wherein the lymphoma is chronic lymphocytic leukemia (CLL).
  • Embodiment B32 The method of Embodiment B30, wherein the lymphoma is mantle cell lymphoma (MCL).
  • Embodiment B33 Embodiment B33.
  • Embodiment B27 wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory NHL.
  • Embodiment B34 The method of Embodiment B33, wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL).
  • Embodiment B35 The method of Embodiment B34, wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory CLL.
  • Embodiment B36 Embodiment B36.
  • Embodiment B34 wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory MCL.
  • Embodiment B37 The method of any one of Embodiments B1-B36, wherein the lymphoma comprises a cancer cell having mutant p53.
  • Embodiment B38 The method of any one of Embodiments B1-B37, wherein further comprises determining by gene sequencing if the subject has TP53 mutation.
  • Embodiment B39 Embodiment B39.
  • a disease or disorder e.g., hyperproliferative malignancy
  • methods of preventing and/or treating a disease or disorder comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an exportin 1 (XPO1) inhibitor.
  • methods of preventing a disease or disorder in a subject comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an exportin 1 (XPO1) inhibitor.
  • methods of treating a disease or disorder e.g.
  • hematological malignancy comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an exportin 1 (XPO1) inhibitor.
  • a pharmaceutically effective amount of the p53 reactivator is administered.
  • the p53 reactivator and the XPO1 inhibitor are concomitantly administered.
  • the co-administration of the compound that can give reactivation of a mutant p53 (the p53 reactivator) and the XPO1 inhibitor is pharmaceutically effective to treat the disease or disorder (e.g., hyperproliferative malignancy).
  • a disease or disorder e.g., hyperproliferative malignancy
  • methods of treating a disease or disorder comprising administering to a subject a compound of Formula (I) and an exportin 1 (XPO1) inhibitor, wherein the compound of Formula (I) shows synergism with the exportin 1 (XPO1) inhibitor for treating the disease or disorder.
  • a pharmaceutically effective amount of the compound of Formula (I) is administered.
  • the co-administration of the compound of Formula (I) and the exportin 1 (XPO1) inhibitor is pharmaceutically effective to treat the disease or disorder (e.g., hyperproliferative malignancy).
  • the compound that has synergism with the XPO1 inhibitor is APR-246. In another specific embodiment, the compound that has synergism with the XPO1 inhibitor is Compound A. In another specific embodiment, the compound that has synergism with the XPO1 inhibitor is Compound B. In another specific embodiment, the compound that has synergism with the XPO1 inhibitor is Compound C. In another specific embodiment, the compound that has synergism with the XPO1 inhibitor is Compound D. In another specific embodiment, the compound that has synergism with the XPO1 inhibitor is Compound E. [00441] In some embodiments, the subject is a human.
  • the subject is a subject diagnosed with hyperproliferative malignancy, e.g., a hematological malignancy.
  • the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis.
  • diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct
  • the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small- cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer.
  • the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma.
  • the cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphom
  • NHL non-Hodgkin's lymph
  • the disease or disorder is myeloma.
  • the disease or disorder is myelodysplastic syndromes (MDS).
  • the disease or disorder is acute myeloid leukemia (AML).
  • the disease or disorder is chronic lymphocytic leukemia (CLL).
  • the myeloma is multiple myeloma (MM).
  • the disease or disorder is a solid tumor cancer.
  • the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer.
  • the subject is diagnosed to have TP53 mutation.
  • the subject carries TP53 mutation.
  • the subject has mutant p53 protein.
  • the methods described herein further comprises determining by gene sequencing if the subject has TP53 mutation.
  • the disease or disorder is a mutant p53 mediated cancer (including, e.g., hematological tumors with mutations in the p53 gene). In other embodiments, the disease or disorder is not a mutant p53 mediated cancer.
  • the mutation in TP53 is R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, V173A, or S241F.
  • the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation.
  • the mutant TP53 includes a nonsense mutation.
  • a nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP53 mutations in cancer. The most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*.
  • the disease or disorder is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DL), lymphocytic lympho
  • the amount of the p53 reactivator or the XPO1 inhibitor provided herein, or a pharmaceutical composition that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some embodiments, should be decided according to the judgment of the practitioner and each patient’s circumstances. Effective doses may also be extrapolated from dose-response curves derived from in vitro or animal model test systems. [00452] In some embodiments, the p53 reactivator is administered to the subject as part a composition.
  • the composition is a pharmaceutical composition described in Section 4.3.3.
  • the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) can be formulated in different pharmaceutical compositions and administered separately to the subject in need thereof.
  • the p53 reactivator (e.g., APR- 246) and the XPO1 inhibitor (e.g., selinexor) are administered together in the same pharmaceutical composition.
  • the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) are administered simultaneously.
  • the term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours.
  • the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) are not administered simultaneously, and instead the two compounds are administered at different times.
  • the XPO1 inhibitor e.g., selinexor
  • the XPO1 inhibitor is administered before or concurrently with the administration of the p53 reactivator (e.g., APR-246).
  • the XPO1 inhibitor (e.g., selinexor) is administered after the administration of the p53 reactivator (e.g., APR-246).
  • the subject has been previously treated with the XPO1 inhibitor (e.g., selinexor) prior to the administration of the p53 reactivator (e.g., APR-246).
  • the subject has been previously treated with the p53 reactivator (e.g., APR-246) prior to the administration of the XPO1 inhibitor (e.g., selinexor).
  • the subject has not been previously treated with any XPO1 inhibitor (e.g., selinexor) prior to the co-administration of the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor).
  • the co-administration of the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) is concomitant administration.
  • the co-administration of the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) is pharmaceutically effective to treat hyperproliferative malignancy, e.g., a hematological malignancy.
  • the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) are administered at least once during a dosing period.
  • a dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once.
  • a dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.
  • a dosing period is a dosing cycle.
  • the prophylactic or therapeutic agent can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time).
  • the agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity. Stable disease or lack is determined by methods known in the art such as evaluation of patient symptoms, physical examination, and visualization of the tumor that has been imaged using X- ray, CAT, PET, MRI scan, or other commonly accepted evaluation modalities.
  • the prophylactic or therapeutic agent can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID).
  • the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug).
  • the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time.
  • the term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days.
  • the term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals.
  • intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days.
  • the frequency of administration is in the range of about a daily dose to about a monthly dose.
  • administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks.
  • the compound is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks.
  • APR-246 is administered at a dose of less than about 150 mg/kg. In some embodiments, APR-246 is administered at a dose of less than about 100 mg/kg. In some embodiments, APR-246 is administered at a dose of between about 100 mg/kg and about 25 mg/kg. In other embodiments, APR-246 is administered at dose of less than about 75 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than about 65 mg/kg.
  • the APR-246 is administered at a dose of less than about 50 mg/kg. [00469] In other embodiments, APR-246 is administered at a fixed dose within the interval of 2.7-7.5 g. In some embodiments, the fixed dose of APR-246 is no more than about 4.5 g. In some embodiments, the fixed dose of APR-246 is no more than about 4.0 g. In some embodiments, the fixed dose of APR-246 is no more than about 3.5 g. In one embodiments, APR-246 is administered at a dose of about 4.5 g/day, about 4.0 g/day, or about 3.5 g/day. In one specific embodiment, APR-246 is administered at a dose of about 4.5 g/day.
  • APR-246 is administered at a dose of about 4.0 g/day. In yet another specific embodiment, APR-246 is administered at a dose of about 3.5 g/day. [00470] In some embodiments, APR-246 is administered in a multiple-step administration to avoid high plasma concentration and/or to minimize the risk of adverse events. In some embodiments, APR-246 is administered in a 2-step administration consisting of a first loading dose and a subsequent maintenance dose. In some preferred embodiments, the first loading dose is not equal to the subsequent maintenance dose. In other embodiments, the first loading dose is not equal to the subsequent maintenance dose. In some embodiments, the first loading dose is about 1.5 g, about 1.33 g, or about 1.16 g.
  • the subsequent maintenance dose is about 3.0 g, about 2.67 g, or about 2.34 g.
  • APR-246 is administered 1, 2, 3, 4, 5, 6, 7, or 8 times of each cycle of 14 days, 21 days, every 28 days, every 35 days, or every 42 days. In preferred embodiments, APR-246 is administered for 4 days in each treatment cycle. In preferred embodiments, APR-246 is administered for 4 days in each treatment cycle of 28 days. In some embodiments, APR-246 is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles. [00472] In a specific embodiment, APR-246 is administered once daily for 4 consecutive days in each treatment cycle of 28 days.
  • APR-246 is administered intravenously.
  • the XPO1 inhibitor described herein e.g., selinexor
  • the XPO1 inhibitor described herein is administered at a dose between 1 mg/day to 1000 mg/day.
  • the XPO1 inhibitor is administered at a dose between about 10 mg/day and about 20 mg/day, between about 20 mg/day and 30 mg/day, between about 30 mg/day and 40 mg/day, between about 40 mg/day and about 50 mg/day, between about 50 mg/day and 60 mg/day, between about 60 mg/day and 70 mg/day, between about 70 mg/day and 80 mg/day, between about 80 mg/day and 90 mg/day, or between about 90 mg/day and about 100 mg/day.
  • the XPO1 inhibitor is administered at a dose of about 80 mg/day. In one embodiment, the XPO1 inhibitor is administered at a dose of about 100 mg/day.
  • the XPO1 inhibitor is administered at a dose of about 60 mg/day. In one specific embodiment, the XPO1 inhibitor is administered at a dose of about 80 mg/day and administered twice per week. In another specific embodiment, the XPO1 inhibitor is administered at a dose of about 100 mg/day and administered once per week. In another specific embodiment, the XPO1 inhibitor is administered at a dose of about 80 mg/day and administered once per week. In another specific embodiment, the XPO1 inhibitor is administered at a dose of about 60 mg/day and administered once per week. [00475] In some preferred embodiments, selinexor is administered at a daily dose of about 80 mg twice per week.
  • the XPO1 inhibitor is administered at a daily dose of about 80 mg on day 1 and day 3 per week.
  • the XPO1 inhibitor e.g., selinexor
  • the XPO1 inhibitor is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles.
  • the XPO1 inhibitor e.g., selinexor
  • the XPO1 inhibitor is administered orally.
  • combination treatment of the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) show synergistic effects in treating hyperproliferative malignancy, e.g., a hematological malignancy.
  • APR-246 is administered at a dose of about 4.5 g/day in combination with selinexor administered at a dose of about 80 mg/day twice per week in each 28-day cycle.
  • APR-246 and selinexor are administered for 1 to 10 cycles.
  • APR-246 and selinexor are administered for 1 cycle.
  • APR-246 and selinexor are administered for 2 cycles.
  • APR-246 and selinexor are administered for 3 cycles.
  • APR-246 and selinexor are administered for 4 cycles.
  • APR-246 and selinexor are administered for 5 cycles.
  • APR-246 and selinexor are administered for 6 cycles. In some embodiments, APR-246 and selinexor are administered for 7 cycles. In some embodiments, APR-246 and selinexor are administered for 8 cycles. In some embodiments, APR-246 and selinexor are administered for 9 cycles. In some embodiments, APR-246 and selinexor are administered for 10 cycles. In some embodiments, APR-246 and selinexor are administered for more than 10 cycles. [00480] In some embodiments, APR-246 is administered at a dose of about 4.0 g/day in combination with selinexor administered at a dose of about 80 mg/day twice per week in each 28-day cycle.
  • APR-246 and selinexor are administered for 1 to 10 cycles. In some embodiments, APR-246 and selinexor are administered for 1 cycle. In some embodiments, APR-246 and selinexor are administered for 2 cycles. In some embodiments, APR-246 and selinexor are administered for 3 cycles. In some embodiments, APR-246 and selinexor are administered for 4 cycles. In some embodiments, APR-246 and selinexor are administered for 5 cycles. In some embodiments, APR-246 and selinexor are administered for 6 cycles. In some embodiments, APR-246 and selinexor are administered for 7 cycles. In some embodiments, APR-246 and selinexor are administered for 8 cycles.
  • APR-246 and selinexor are administered for 9 cycles. In some embodiments, APR-246 and selinexor are administered for 10 cycles. In some embodiments, APR-246 and selinexor are administered for more than 10 cycles. [00481] In some embodiments, APR-246 is administered at a dose of about 3.5 g/day in combination with selinexor administered at a dose of about 80 mg/day twice per week in each 28-day cycle. In some embodiments, APR-246 and selinexor are administered for 1 to 10 cycles. In some embodiments, APR-246 and selinexor are administered for 1 cycle. In some embodiments, APR-246 and selinexor are administered for 2 cycles.
  • APR-246 and selinexor are administered for 3 cycles. In some embodiments, APR-246 and selinexor are administered for 4 cycles. In some embodiments, APR-246 and selinexor are administered for 5 cycles. In some embodiments, APR-246 and selinexor are administered for 6 cycles. In some embodiments, APR-246 and selinexor are administered for 7 cycles. In some embodiments, APR-246 and selinexor are administered for 8 cycles. In some embodiments, APR-246 and selinexor are administered for 9 cycles. In some embodiments, APR-246 and selinexor are administered for 10 cycles. In some embodiments, APR-246 and selinexor are administered for more than 10 cycles.
  • the methods described herein further comprises determining by gene sequencing if the subject has TP53 mutation.
  • a next generation sequencing (NGS) method is used for the gene sequencing.
  • the gene sequencing data are interpreted to determine if the subject has or likely has TP53 mutation.
  • combination therapies of the p53 reactivator and the exportin 1 (XPO1) inhibitor provided herein for use in treating a disease or disorder described herein (e.g., hyperproliferative malignancy, e.g., a hematological malignancy).
  • combination therapies of the p53 reactivator and the exportin 1 (XPO1) inhibitor provided herein for use in treating hematological malignancy are also provided herein.
  • pharmaceutical compositions or a combination of pharmaceutical compositions of the p53 reactivator and the exportin 1 (XPO1) inhibitor provided herein for use in treating hematological malignancy Exemplary Embodiments [00485] Embodiment C1.
  • a method of treating a hyperproliferative malignancy in a subject comprising administering to the subject: (i) a p53 reactivator; and (ii) an exportin 1 (XPO1) inhibitor.
  • a p53 reactivator is a compound that can give reactivation of a mutant p53, or a degradation product thereof that can give reactivation of a mutant p53.
  • Embodiment C2 wherein the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W.
  • Embodiment C4 The method of any one of Embodiments C1 and C2, wherein the p53 reactivator is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity.
  • Embodiment C6 The method of any one of Embodiments C2-C4, wherein the compound or degradation product thereof is capable of shifting the equilibrium from unfolded towards a folded structure of wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of reducing aggregation of the wild-type or mutant p53.
  • Embodiment C7 Embodiment C7.
  • Embodiment C8 The method of any one of Embodiments C2-C6, wherein the compound or degradation product threreof is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promote a folded conformation.
  • Embodiment C9 The method of any one of Embodiments C2-C8, wherein the compound that can give reactivation of mutant p53 is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one; 2,2-bis(hydroxymethyl)quinuclidin-3-one; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-3-one
  • Embodiment C10 The method of Embodiment C9, wherein the compound is 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one (APR-246) having the following formula: , or a pharmaceutically acceptable salt thereof.
  • APR-246 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one
  • Embodiment C9 wherein the compound is a compound selected from the group consisting of: 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; and 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, or a pharmaceutically acceptable salt thereof.
  • Embodiment C12 The method of Embodiment C11, wherein the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound A), or a pharmaceutically acceptable salt thereof.
  • Embodiment C13 The method of Embodiment C11, wherein the compound is 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound B), or a pharmaceutically acceptable salt thereof.
  • Embodiment C14 Embodiment C14.
  • Embodiment C11 wherein the compound is 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound C), or a pharmaceutically acceptable salt thereof.
  • Embodiment C15 The method of Embodiment C11, wherein the compound is N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound D), or a pharmaceutically acceptable salt thereof.
  • Embodiment C16 Embodiment C16.
  • Embodiment C11 wherein the compound is 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound E), or a pharmaceutically acceptable salt thereof.
  • Compound E 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide
  • Embodiments C1-C16 wherein the XPO1 inhibitor is selected from the group consisting of leptomycin A (LMA), leptomycin B (LMB), selinexor (KPT-330), Eltanexor (KPT-8602), KTP-185, KPT-249, KPT-251, KPT-276, verdinexor (KPT-335), piperlongumine, valtrate, felezonexor (CBS9106 or SL-801), and PKF050-638, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • LMA leptomycin A
  • LMB leptomycin B
  • KPT-330 selinexor
  • Eltanexor KPT-8602
  • KTP-185 KPT-249
  • KPT-251, KPT-276 verdinexor
  • KPT-335 verdinexor
  • piperlongumine valtrate
  • Embodiment 17 wherein the XPO1 inhibitor is selinexor, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
  • Embodiment C19 The method of any one of Embodiments C1-C10 and C17- C18, wherein the p53 reactivator is APR-246 and the XPO1 inhibitor is selinexor.
  • Embodiment C20 The method of Embodiment C19, wherein APR-246 is administered at a daily dose of about 4.5 g/day and selinexor is administered at a dose of about 80 mg/day twice per week.
  • Embodiment C21 The method of Embodiment C19, wherein APR-246 is administered at a daily dose of about 4.0 g/day and selinexor is administered at a dose of about 80 mg/day twice per week.
  • Embodiment C22 The method of Embodiment C19, wherein APR-246 is administered at a daily dose of about 3.5 g/day and selinexor is administered at a dose of about 80 mg/day twice per week.
  • Embodiment C23 The method of any one of Embodiments C20-C22, wherein selinexor is administered on day 1 and day 3 of each week.
  • Embodiment C24 The method of any one of Embodiments C20-C22, wherein selinexor is administered on day 1 and day 3 of each week.
  • Embodiment C19 wherein selinexor is administered at a reduced dose of about 100 mg once per week, about 80 mg once per week, or about 60 mg once per week.
  • Embodiment C25 The method of any one of Embodiments C1-C24, wherein the compound that can give reactivation of the mutant p53 is formulated in a first pharmaceutical composition and the XPO1 inhibitor is formulated in a second pharmaceutical composition.
  • Embodiment C26 The method of any one of Embodiments C1-C25, wherein the hyperproliferative malignancy is a hematological malignancy.
  • Embodiment C27 Embodiment C27.
  • Embodiment C26 wherein the hematological malignancy is leukemia, lymphoma, or myeloma.
  • Embodiment C28 The method of Embodiment C26, wherein the hematological malignancy is selected from the group consisting of: Hodgkin's lymphoma, non- Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lympho
  • NHL non- Ho
  • Embodiment C29 The method of Embodiment C26, wherein the hematologic malignancy is myeloma.
  • Embodiment C30 The method of Embodiment C29, wherein the myeloma is multiple myeloma (MM).
  • Embodiment C31 The method of Embodiment C26, wherein the hematologic malignancy is acute myeloid leukemia (AML).
  • Embodiment C32 The method of Embodiment C26, wherein the hematologic malignancy is chronic lymphocytic leukemia (CLL).
  • Embodiment C33 The method of Embodiment C26, wherein the hematologic malignancy is chronic lymphocytic leukemia (CLL).
  • Embodiment C34 The method of any one of Embodiments C1-C33, wherein further comprises determining by gene sequencing if the subject has TP53 mutation.
  • Embodiment C35 The method of any one of Embodiments C1-C34, wherein the subject is not treated with any XPO1 inhibitor prior to the co-administration of the p53 reactivator and the XPO1 inhibitor.
  • compositions for use in the prevention and/or treatment of a disease or condition comprising the p53 reactivator provided herein (e.g., the p53 reactivator described in Section 4.2.1 such as APR-246) and the inhibitor of WEE1 provided herein (e.g., the inhibitors of WEE1 described in Section 4.2.5 such as MK-1775).
  • a disease or condition e.g., a hyperproliferative malignancy
  • WEE1 e.g., the p53 reactivator described in Section 4.2.1 such as APR-246
  • the inhibitor of WEE1 provided herein e.g., the inhibitors of WEE1 described in Section 4.2.5 such as MK-1775.
  • the subject is a human.
  • a disease or disorder e.g., hyperproliferative malignancy
  • methods of preventing and/or treating a disease or disorder comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an inhibitor of WEE1.
  • methods of preventing a disease or disorder in a subject comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an inhibitor of WEE1.
  • methods of treating a disease or disorder e.g.
  • hyperproliferative malignancy comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an inhibitor of WEE1.
  • a pharmaceutically effective amount of the p53 reactivator is administered.
  • the p53 reactivator and the inhibitor of WEE1 are concomitantly administered.
  • the co- administration of the compound that can give reactivation of a mutant p53 (the p53 reactivator) and the inhibitor of WEE1 is pharmaceutically effective to treat the disease or disorder (e.g., hyperproliferative malignancy).
  • a disease or disorder e.g., a hyperproliferative malignancy
  • methods of treating a disease or disorder comprising administering to a subject a compound of Formula (I) and an inhibitor of WEE1, wherein the compound of Formula (I) shows synergism with the inhibitor of WEE1 for treating the disease or disorder.
  • a pharmaceutically effective amount of the compound of Formula (I) is administered.
  • the co-administration of the compound of Formula (I) and the inhibitor of WEE1 is pharmaceutically effective to treat the disease or disorder (e.g., hyperproliferative malignancy).
  • the compound that has synergism with the inhibitor of WEE1 is APR-246.
  • the compound that has synergism with the inhibitor of WEE1 is Compound A. In another specific embodiment, the compound that has synergism with the inhibitor of WEE1 is Compound B. In another specific embodiment, the compound that has synergism with the inhibitor of WEE1 is Compound C. In another specific embodiment, the compound that has synergism with the inhibitor of WEE1 is Compound D. In another specific embodiment, the compound that has synergism with the inhibitor of WEE1 is Compound E. [00523] In some embodiments, the disease or disorder is a neoplastic disease. In certain embodiments, the disease or disorder is a cancer. In certain embodiments, the disease or disorder is a hematogical malignancy.
  • the neoplastic, cancerous, or malignant cell in a subject to be treated with a method provided herein comprises a mutation in TP53.
  • provided herein is a method to treat or prevent a hematological cancer with a mutation in the p53 gene.
  • the cancer to be treated with a method provided herein comprises cancer cells that carry a mutation in TP53.
  • the mutation in TP53 is R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, V173A, S241F, R249S or a combination thereof.
  • the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation.
  • the mutant TP53 includes a nonsense mutation.
  • a nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP53 mutations in cancer. The most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*.
  • the cancer to be treated does not comprise any cells carrying a mutation in the TP53 gene.
  • the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis.
  • diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct
  • the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small- cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer.
  • the disease or disorder is a tumor of the hematopoietic or lymphoid tissue.
  • the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma.
  • the cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myel
  • NHL non
  • the disease or disorder is myelodysplastic syndromes (MDS).
  • the disease or disorder is acute myeloid leukemia (AML).
  • the disease or disorder is chronic lymphocytic leukemia (CLL).
  • the disease or disorder is multiple myeloma (MM).
  • the disease or disorder is lymphoma.
  • the disease or disorder is a solid tumor cancer.
  • the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, a lung cancer, a cervical cancer, a prostate cancer, a head and neck cancer, and a breast cancer.
  • the solid tumor cancer is breast cancer.
  • the solid tumor cancer is selected from the group consisting of breast cancer, cervical cancer, prostate cancer, and head and neck cancer. In certain embodiments, the solid tumor cancer is triple negative breast cancer.
  • the amount of a prophylactic or therapeutic agent (the p53 reactivator and the inhibitor of WEE1 (e.g., MK-1775) provided herein), or a composition provided herein that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some embodiments, should be decided according to the judgment of the practitioner and each patient’s circumstances.
  • the dose administered to a subject in the context of the present disclosure should be sufficient to effect a therapeutic response.
  • dosage will depend upon a variety of factors including the potency of the specific compound, the age, condition and body weight of the patient, as well as the stage/severity of the disease.
  • the dose will also be determined by the route (administration form) timing and frequency of administration.
  • the p53 reactivator e.g., APR-246
  • the inhibitor of WEE1 e.g., MK-1775
  • the p53 reactivator (e.g., APR-246) and the inhibitor of WEE1 (e.g., MK-1775) are administered together in the same pharmaceutical composition.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of WEE1 (e.g., MK-1775) are administered simultaneously.
  • the term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of WEE1 (e.g., MK-1775) are not administered simultaneously, and instead the two compounds are administered at different times.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of WEE1 (e.g., MK-1775) are administered at least once during a dosing period.
  • a dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once.
  • a dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.
  • a dosing period is a dosing cycle.
  • the prophylactic or therapeutic agent can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time).
  • the agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity.
  • the prophylactic or therapeutic agent can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID).
  • the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug).
  • the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time.
  • the term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days.
  • the term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days.
  • the frequency of administration is in the range of about a daily dose to about a monthly dose.
  • administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks.
  • the compound is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks.
  • APR-246 is administered at a dose of less than 150 mg/kg.
  • APR-246 is administered at a dose of less than 100 mg/kg. In some embodiments, APR-246 is administered at a dose of between 100 mg/kg and 25 mg/kg. In other embodiments, APR-246 is administered at dose of less than 75 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 65 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 50 mg/kg. [00541] In other embodiments, APR-246 is administered at a fixed dose within the interval 2.7-7.5 g. In some embodiments, the fixed dose of APR-246 is no more than 4.5 g.
  • the fixed dose of APR-246 is no more than 3.7 g.
  • APR- 246 is administered at a fixed dose during a period of about 3 to 7 hours.
  • APR-246 is administered at a fixed dose during a period of about 6 or 4 hours.
  • Exemplary Embodiments [00542] Embodiment D1. A method of treating cancer in a subject, comprising administering to the subject: (i) a compound that can activate a mutant p53; and (ii) an inhibitor of Wee1-like protein kinase (WEE1). [00543] Embodiment D2.
  • Embodiment D1 wherein the cancer in the subject comprises a cell that carries a mutation in TP53.
  • Embodiment D3 The method of Embodiment D2, wherein the mutation in TP53 is selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W, or a combination thereof.
  • Embodiment D4 The method of Embodiment D2 or D3, wherein the compound that can activate the mutant p53 promotes proper folding of the mutant p53 and restores at least part of a normal p53 function.
  • Embodiment D2 or D3 wherein the compound that can activate of the mutant p53 can result in a shift of the equilibrium from unfolded towards a wild-type like p53 conformation.
  • Embodiment D6 The method of Embodiment D2 or D3, wherein the compound that can activate the mutant p53 interferes with aggregation of misfolded mutant p53 or reduces aggregation of the mutant p53.
  • Embodiment D7 The method of Embodiment D2 or D3, wherein the compound that can activate the mutant p53 or its metabolite or degradation product thereof can restore a p53 wild type function by covalent binding to the mutant p53.
  • Embodiment D8 The method of Embodiment D7, wherein the compound that can activate the mutant p53 can bind to thiol groups in the core domain of the mutant p53 and restore wild-type conformation.
  • Embodiment D9. The method of any one of Embodiments D1-D8, wherein the compound reactivates a mutant p53.
  • Embodiment D11 The method of Embodiment D10, wherein the compound is 2- (hydroxymethyl)-2- (methoxymethyl) quinuclidin-3-one (APR-246) having the following formula: , or a pharmaceutically acceptable salt thereof.
  • APR-246 2- (hydroxymethyl)-2- (methoxymethyl) quinuclidin-3-one
  • Embodiment D10 wherein the compound is a compound selected from the group consisting of: 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; and 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, or a pharmaceutically acceptable salt thereof.
  • Embodiment D13 The method of Embodiment D12, wherein the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound A), or a pharmaceutically acceptable salt thereof.
  • Embodiment D14 The method of Embodiment D12, wherein the compound is 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound B), or a pharmaceutically acceptable salt thereof.
  • Embodiment D15 Embodiment D15.
  • Embodiment D12 wherein the compound is 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound C), or a pharmaceutically acceptable salt thereof.
  • Embodiment D16 The method of Embodiment D12, wherein the compound is N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound D), or a pharmaceutically acceptable salt thereof.
  • Embodiment D17 Embodiment D17.
  • Embodiment D12 wherein the compound is 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound E), or a pharmaceutically acceptable salt thereof.
  • Compound E 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide
  • any one of Embodiments D1-D17, wherein the inhibitor of WEE1 is selected from the group consisting of: 6-(2,6-Dichlorophenyl)-2-[4-[2-(diethylamino)ethoxy] anilino]-8-methylpyrido[2,3- d]pyrimidin-7-one (PD0166285); 9-Hydroxy-4-phenylpyrrolo[3,4-c] carbazole-1,3(2H,6H)-dione (PD0407824); 4-(2-Chlorophenyl)-9-hydroxypyrrolo[3,4-c] carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor I); 6-Butyl-4-(2-chlorophenyl)-9-hydroxypyrrolo [3,4-c]carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor II); 2-Allyl-1-(
  • Embodiment D19 The method of any one of Embodiments D1-D17, wherein the inhibitor of WEE1 is MK-1775 having a formula of: (MK-1775), or a pharmaceutically acceptable salt thereof.
  • Embodiment D20 The method of any one of Embodiments D1-D19, wherein the p53 reactivator is formulated in a first pharmaceutical composition and the inhibitor of WEE1 is formulated in a second pharmaceutical composition.
  • Embodiment D21 The method of any one of Embodiments D1-D20, wherein the cancer is a hematological malignancy.
  • Embodiment D22 The method of any one of Embodiments D1-D20, wherein the cancer is a hematological malignancy.
  • Embodiment D21 wherein the hematological malignancy is leukemia, lymphoma, or myeloma.
  • Embodiment D23 The method of any one of Embodiments D1-D22, wherein the cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymph
  • NHL non-Hod
  • Embodiment D24 The method of Embodiment D23, wherein the cancer is myelodysplastic syndromes (MDS).
  • Embodiment D25 The method of Embodiment D23, wherein the cancer is acute myeloid leukemia (AML).
  • Embodiment D26 The method of Embodiment D23, wherein the cancer is chronic lymphocytic leukemia (CLL).
  • Embodiment D27 The method of Embodiment D23, wherein the cancer is multiple myeloma (MM).
  • Embodiment D28 The method of Embodiment D23, wherein the cancer is multiple myeloma (MM).
  • Embodiment D29 The method of Embodiment D28, wherein the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, a lung cancer, a cervical cancer, a prostate cancer, a head and neck cancer, and a breast cancer.
  • the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma
  • Embodiment D30 The method of Embodiment D28, wherein the solid tumor cancer is breast cancer.
  • Embodiment D31 The method of Embodiment D28, wherein the solid tumor cancer is triple negative breast cancer.
  • assays for Testing and Demonstrating Synergistic Effects of an Activator of P53 and a Second Therapeutic Agent are also provided herein, for example, an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1).
  • an agonist of p53 such as a p53 reactivator and a second therapeutic agent
  • a second therapeutic agent for example, an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1).
  • a compound e.g., a compound of Formula (I) provided herein
  • a second therapeutic agent for example, an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1).
  • the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is a compound of Formula (I).
  • the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is APR-246. In another specific embodiment, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is Compound A. In another specific embodiment, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is Compound B. In another specific embodiment, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is Compound C. In another specific embodiment, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is Compound D.
  • the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is Compound E.
  • the compound has synergism with the second therapeutic compound disclosed herein activates p53 in immune cells.
  • the compound has synergism with the second therapeutic compound disclosed herein activates a mutant p53 in immune cells.
  • the compound has synergism with the second therapeutic compound disclosed herein activates normal p53 in immune cells.
  • provided herein are assays for demonstrating the effects of the combination treatment with an agonist of p53 and an inhibitor of PD-1 mediated signalling such as an inhibitor of PD-1.
  • assays for demonstrating the effects of the combination treatment with an agonist of p53 such as a p53 reactivator and a BTK inhibitor.
  • assays for demonstrating the effects of the combination treatment with an agonist of p53 such as a p53 reactivator and an XPO1 inhibitor (e.g., selinexor).
  • XPO1 inhibitor e.g., selinexor
  • a model system for a cancer may be treated with a p53 agonist such as p53 reactivator, a second therapeutic agent, or a combination of the p53 agonist or reactivator and the second therapeutic agent, and the effects of the combination treatment are analyzed and compared to the monotherapies.
  • a p53 agonist such as p53 reactivator, a second therapeutic agent, or a combination of the p53 agonist or reactivator and the second therapeutic agent
  • the effects of the combination treatment are analyzed and compared to the monotherapies.
  • the model system for a solid tumor malignancy may be treated with a p53 reactivator, a BTK inhibitor, or a combination of the p53 reactivator and the BTK inhibitor, and the effects of the combination treatment are analyzed and compared to the monotherapies.
  • the model system for a solid tumor malignancy may be treated with a p53 reactivator, an XPO1 inhibitor, or a combination of the p53 reactivator and the XPO1 inhibitor, and the effects of the combination treatment are analyzed and compared to the monotherapies.
  • the model system for a hematological malignancy may be treated with a p53 reactivator, a WEE1 inhibitor, or a combination of the p53 reactivator and the WEE1 inhibitor, and the effects of the combination treatment are analyzed and compared to the monotherapies.
  • a p53 agonist such as p53 reactivator and a second therapeutic agent can then be analyzed using well-known analytical tools.
  • synergistic effects of a p53 reactivator and an inhibitor of PD-1 can then be analyzed using well-known analytical tools.
  • synergistic effects of a p53 reactivator and a BTK inhibitor can also be analyzed using well-known analytical tools.
  • synergistic effects of a p53 reactivator and an XPO1 inhibitor can then be analyzed using well-known analytical tools.
  • synergistic Effects of a p53 reactivator and a WEE1 inhibitor can then be analyzed using well-known analytical tools.
  • the analytical tool is COMBENEFIT. Combenefit (Di Veroli.
  • Bioinformatics, 32(18), 2016, 2866–2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files.
  • Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413–2415).
  • synergistic effects of an exemplary activator of p53 in combination with an exemplary second therapeutic agent (e.g., an inhibitor of PD-1 mediated signaling, a BTK inhibitor, an XPO1 inhibitor, or a WEE1 inhibitor) were tested in one or more cell lines, to assess both single agent activity and evaluate additive, synergistic or antagonistic interactions across to a range of doses.
  • an exemplary second therapeutic agent e.g., an inhibitor of PD-1 mediated signaling, a BTK inhibitor, an XPO1 inhibitor, or a WEE1 inhibitor
  • any synergistic effects in various combinational doses can be determined according to the Highest Single Agent (HSA), Bliss and Loewe models. Synergistic effects can be determined if additive model Combination Index values obtained in these cell lines are found to indicate synergism.
  • the CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells.
  • the homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium.
  • the homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present.
  • the amount of ATP is directly proportional to the number of cells present in culture.
  • IC 50 Dose response Determinations by XLFIT
  • the serial dilution of compounds can be performed in cell culture medium based on a 1.5- or 2-fold dilution factor.
  • the serially diluted compounds can be added to the cells and incubated for 72 h followed by cell viability test using the CTG assay.
  • the current viability values can be calculated and IC 50 values were determined by XLfit software version 5.5.0.5 (a Microsoft Excel-based plug-in to perform regression, curve fitting and statistical analysis).
  • XLfit software version 5.5.0.5 a Microsoft Excel-based plug-in to perform regression, curve fitting and statistical analysis.
  • Analysis of Combination by COMBENEFIT [00594] Combenefit (Di Veroli.
  • the experimental dose–response surface that delineates combination effects in concentration space is first read by the Combenefit software as a matrix of % of the control value across concentrations. Single agent effects are extracted from this data and fitted with a dose response curve. Based on the two single agent dose response curves, a model-based combination dose–response surface is derived. This surface provides a “reference” dose–response surface for a non-synergistic (additive/independent) combination, whose characteristics are determined by the selected model (i.e., Loewe, Bliss or HSA). The experimental combination dose response surface is then compared to the model-generated one, resulting in a synergy distribution in concentration space.
  • the selected model i.e., Loewe, Bliss or HSA
  • the graphical outputs consist of: (i) the single agent dose response data and its fitting; (ii) the combination dose response (four different displays); (iii) the model generated reference combination dose response, i.e., the prediction of effect if the drugs are not synergistic (three different displays); (iv) the resulting synergy distribution (three different displays); and (v) a graphic mapping the synergy distribution onto the dose–response surface.
  • This synergy distribution can be further summarized via various metrics (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866-2868) as follows: • SYN_MAX - the maximum level of synergy observed.
  • SYN_SUM the sum of synergy observed in concentration logarithmic space. For instance, an integrated synergy of 50 is equivalent to an extra synergistic effect of 50%, which is spread over a square of 1 log x 1 log in the 2-d log-concentration space.
  • SYN_SUM_WEIGHTED The integrated weighted synergy incorporates a weight based on the dose response which bias the total score towards synergy achieving highest effect. Hence, a synergy of 50% leading to a combined full effect (100%) will have more weight than if the corresponding effect was only 20 or 30%.
  • SYN_AVERAGE_C1 Concentration value of drug 1 where synergy appears to be localized.
  • a “combination index” (CI) is then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability.
  • the p53 agonist has synergism with the inhibitor of PD-1 mediated signaling (e.g., an inhibitor of PD-1 such an anti-PD-1 antibody) in activating immune cells.
  • the p53 agonist disclosed herein activates normal p53 in immune cells.
  • any synergistic effects in activating or enhancing immune cells can be determined according to in vitro functional assays well known to those persons of ordinary skills in the art (Wang et al., Cancer Immunol. Res.2:846-856 (2014)).
  • the synergistic effects to promote T-cell responses can be evaluated in vitro, e.g., using human T cells.
  • enhancement of IFN ⁇ release or enhanced T-cell proliferation can be measured to indicate the activation and stimulation of immune cells.
  • PD-L1 expression and/or upregulation can be measured to indicate the activation and stimulation of immune cells.
  • IFN ⁇ secretion and T-cell proliferation are measured to indicate the activation and stimulation of immune cells (e.g., T cells).
  • Dendritic cells DC are generated by culturing monocytes isolated from PBMCs using a monocyte purification kit in vitro for 7 days with 500 U/mL interleukin-4 (IL-4) and 250 U/mL GM-CSF.
  • IL-4 interleukin-4
  • CD4 + T cells (1 x 10 5 ) and allogeneic DCs (1 x 10 4 ) are co-cultured with or without dose titrations of a PD-1 mediated signaling inhibitor, which is added at the initiation of the assay.
  • IFN ⁇ secretion in culture supernatants is analyzed by ELISA and cells are labeled with 3 H-thymidine for an additional 18 hours to measure T-cell proliferation.
  • Assay of Staphylococcal Enterotoxin B Stimulation of PBMCs [00605] In this assay, IL-2 levels are measured to indicate the activation and stimulation of immune cells (e.g., T cells) in the presence of staphylococcal enterotoxin B (SEB).
  • SEB staphylococcal enterotoxin B
  • PBMCs from healthy human donors are cultured for 3 days with a PD-1 mediated signaling inhibitor or an isotype control at the initiation of the assay together with serial dilutions of staphylococcal enterotoxin B (SEB).
  • SEB staphylococcal enterotoxin B
  • IL-2 levels in culture supernatants are measured by ELISA analysis.
  • Cytomegalovirus (CMV)-Restimulation Assay [00606] In a cytomegalovirus (CMV)-restimulation assay, antigen-specific recall response in vitro is measured.
  • CD4 + CD25 + regulatory T cells and CD4 + CD25- responder T cells are purified from PBMCs (CD4 + CD25 + Treg isolation kit; Miltenyi Biotec).
  • Tregs 5 x 10 4
  • 1 x 10 5 responder T cells 1 x 10 5 responder T cells
  • 2 x 10 4 monocyte-derived DCs 2 x 10 4 monocyte-derived DCs
  • IFN ⁇ production is assessed in supernatants, and cells are labeled with 3 H-thymidine for an additional 18 hours for proliferation analysis.
  • Restoration of T cell proliferation and IFN ⁇ release by the alloreactive T cells is measured to indicate the activation and stimulation of responder T cells, which are suppressed by Tregs.
  • the invention is generally disclosed herein using affirmative language to describe the numerous embodiments.
  • the invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis.
  • particular subject matter such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis.
  • Example 1 A Phase 1/2 Study of APR-246 in Combination with Pembrolizumab in Subjects with Solid Tumor Malignancies
  • This clinical trial is a phase 1/2, open-label, study to determine the safety and preliminary efficacy of APR-246 in combination with pembrolizumab in subjects with solid tumor malignancies including gastric/gastroesophageal junction (GEJ) cancer, bladder/urothelial cancer, and non-small-cell lung cancer (NSCLC).
  • the study includes a safety lead-in portion comprised of subjects with advanced non-CNS-primary solid tumors, followed by an expansion portion with separate cohorts for subjects with gastric/GEJ cancer, urothelial cancer and non- small-cell lung cancer (see FIG.1).
  • Dose-limiting toxicity is assessed after three subjects are enrolled in each respective cohort and the last enrolled subject completes the 3-week safety assessment period (i.e., one cycle of combination regimen).
  • a subject that discontinues therapy during Cycle 1 without DLT is considered evaluable for the purpose of safety only if at least 75% of scheduled doses of APR- 246 are administered in the first cycle.
  • R2D recommended phase 2 dose
  • the study continues enrollment at Dose Level -1 (4.0 g/day of APR-246). If ⁇ 1 subject out of 6 experiences DLT at this dose level, the dose level (4.0 g/day of APR-246) is deemed the RP2D for that cohort. If ⁇ 2 subjects out of the total 3 – 6 subjects at that dose level experience DLT, the study continues enrollment at Dose Level -2 (3.5 g/day of APR-246). If ⁇ 1 subject out of 6 experiences DLT at this dose level, the dose level (3.5 g/day of APR-246) is deemed the RP2D for that cohort.
  • phase 2 portion begins once the recommended phase 2 dose (RP2D) of APR-246 in combination with pembrolizumab is determined and assess the antitumor activity of this combination. Up to 100 subjects are enrolled in three cohorts according to their underlying disease, as outlined in FIG.1.
  • Example 2 A Phase 1 and Dose Expansion Study of APR-246 in Combination with Ibrutinib in Subject with TP53-Mutant Relapsed and/or Refractory Non-Hodgkin Lymphoma (NHL) [00616]
  • This example relates a Phase 1, open-label, dose-finding and cohort expansion study to determine the preliminary safety, tolerability, and pharmacokinetic (PK) profile of APR-246 in combination with ibrutinib therapy in subjects with TP53-mutant NHL, including relapsed and/or refractory (R/R) CLL and R/R MCL.
  • the study includes a safety lead-in dose de- escalation portion in subjects with R/R CLL.
  • ibrutinib In safety lead-in cohort, treatment consists of APR-246 at 4.5 g/day on days 1–4 of each 28-day cycle administered concurrently with ibrutinib at standard dose in R/R CLL of 420 mg PO daily. [00619]
  • the safety lead-in cohort initially enrolls 3 subjects. Subjects are assessed for dose limiting toxicity (DLT) related to APR-246 with ibrutinib combination therapy after the first 3 subjects are enrolled in respective cohorts and the last enrolled subject has completed the 4-week safety assessment period (i.e., one cycle of combination regimen).
  • DLT dose limiting toxicity
  • the does de-escalation is as follows: • The safety lead-in cohort initially enrolls 3 subjects • If ⁇ 1 subject out of 3 experiences a dose limiting toxicity (DLT), 3 additional subjects are recruited and treated at the same dose level (4.5 g/day of APR-246 on Days 1–4 of each 28-day cycle). • If ⁇ 2 subjects out of 3–6 subjects in a cohort experience a DLT, the study continues enrollment of 3 additional subjects at Dose Level -1 (4.0 g/day of APR- 246 on Days 1–4 of each 28-day cycle). • If >1 subject out of 3 experience DLT, no additional subjects is recruited and treated at the same dose level.
  • DLT dose limiting toxicity
  • Dose Modification Expansion Study begins after the preliminary safety, tolerability, or PK profile of APR-246 in combination with ibrutinib has been established. Expansion proceeds with APR-246 in combination with ibrutinib in subjects with R/R CLL and R/R MCL.
  • Expansion Cohort 1 subjects with TP53-mutant R/R CLL (N ⁇ 20); and (2) Expansion Cohort 2: subjects with TP53-mutant R/R MCL (N ⁇ 40).
  • Expansion Cohort 2 subjects with TP53-mutant R/R MCL (N ⁇ 40).
  • subjects with R/R CLL receive APR-246 at the RP2D identified in safety lead-in study of APR-246 with ibrutinib based on safety, PK, and/or preliminary efficacy data in the Safety lead-in cohort.
  • the DRT makes recommendations to continue treating MCL patients at the daily ibrutinib dose of 560 mg and APR-2464.5 g daily on Days 1-4 or if a lower dose of APR-246 should be evaluated in MCL patients enrolled in the expansion cohort with ibrutinib.
  • imaging may be omitted and MRD assessment is completed using flow cytometry and/or molecular techniques.
  • subjects may continue treatment as long as toxicity remains acceptable and the subject has not withdrawn consent.
  • Further study is designed based on an integrated assessment of safety, tolerability, and preliminary evidence of clinical activity. The objective of the expansion portion is to gain additional safety tolerability, PK, and preliminary efficacy data regarding the combination therapy of APR-246 with ibrutinib.
  • Secondary endpoints of the study include: (1) pharmacokinetic parameters: Cmax (maximum concentration), AUC (area under the curve), V d and clearance (CL) of APR-246 and Cmax (maximum concentration), Tmax (time of maximum concentration), and AUC (area under the curve) of ibrutinib; (2) complete remission (CR) rate, defined as the proportion of subjects who achieve CR as per disease-specific response criteria; (3) overall response rate (ORR), defined as the proportion of subjects achieving a response, as per disease-specific response criteria; (4) Duration of response (DOR), defined as a time from documentation of tumor response to disease progression or death as a result of any cause; (5) progression-free survival (PFS), defined as the time from the first study dose date to the date of first documentation of confirmed disease progression or death (whichever occurs first).
  • Cmax maximum concentration
  • AUC area under the curve
  • the exploratory endpoints include: (1) Exploratory analyses of molecular markers for response predication and monitoring may include, but are not limited to: TP53 VAF by Next- generation sequencing (NGS), mutations in other genes by NGS, RNA expression; and (2) the exposure response relationship for safety and efficacy of APR-246 when combined with ibrutinib therapy. 5.2.3 Inclusion Criteria [00632] Subjects must meet the inclusion criteria to be eligible to be enrolled, e.g., among other criteria, the subject must have at least one TP53 mutation that is not benign or likely benign based on local testing. [00633] In the detailed inclusion criteria, the subject: 1.
  • R/R CLL has documented histologic diagnosis of R/R CLL or R/R MCL by WHO criteria with at least two prior systemic therapies. In R/R CLL, response and PD are assessed by iwCLL 2018 criteria; in R/R MCL, response and PD are assessed by Lugano criteria. 3. has most recent regimen that did not include BTK inhibitor therapy (e.g., ibrutinib, acalabrutinib, zanubrutinib, experimental BTK inhibitor) 4.
  • BTK inhibitor therapy e.g., ibrutinib, acalabrutinib, zanubrutinib, experimental BTK inhibitor
  • cytopenias a-c
  • CLL/MCL patients may proceed with enrollment after discussion with the Medical Monitor. Cytopenias may not be due to evidence of MDS or hypoplastic BM.
  • Cytopenias may not be due to evidence of MDS or hypoplastic BM. 6.
  • Subjects who are enrolled should use an effective form of contraception for up to 30 days after the last dose of APR-246 or up to 4 months after the last dose of ibrutinib, whichever time period is longer. 5.2.4 Treatment Administration [00634] Study treatment is administered on an outpatient basis. No investigational or commercial agents or therapies other than those described herein is administered with the intent to treat the subject’s disease. Administration of APR-246 [00635] 2-(Hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one (APR-246) is administered as a 6-hour IV infusion daily on days 1–4 of each 28-day cycle. The APR-246 starting dose is fixed at 4.5 g.
  • APR-246 is administered in a 2-step infusion: • Step 1: Loading dose of 1.5 g for the first 45 minutes ( ⁇ 2 min); and • Step 2: Maintenance dose of 3 g over 5 hours 15 minutes ( ⁇ 30 min). [00636] The dose of APR-246 may be reduced per the safety lead-in dose de-escalation procedure, or treatment interrupted if the subject develops AEs. [00637] APR-246 vials are stored at 2 – 8 o C (35.6 – 46.4 o F). At the pharmacies and at the study centers, the prepared APR-246 study product (diluted in sodium chloride solution) are stored at not more than 25 oC. The infusion is completed within 24 hours from the time of preparation.
  • Ibrutinib is administered at standard dose of 420 mg for R/R CLL and 560 mg for R/R MCL orally daily together with a meal on each day of a 28-day cycle. On Day 1 of Cycle 1, ibrutinib is taken 1 hour prior to administration of the APR-246 infusion.
  • Subjects may remain on study treatment to the end of the trial while deriving clinical benefit, unless unacceptable toxicity, progression, death or subject withdrawal.
  • a DLT is defined as any of the TEAEs defined by the National Cancer Institute’s Common Terminology Criteria for Adverse Events, version 5.0 (NCI-CTCAE v5.0), and for hematologic AEs in R/R CLL/MCL patients, iwCLL criteria for CLL for hematologic toxicity, occurring during the first 28 days of study drug, unless the AE is clearly unrelated to study drug (related to the patient’s disease under study, their medical condition or concomitant medications, or clearly attributable to ibrutinib).
  • DLT definitions are: • Any ⁇ Grade 3 non-hematologic toxicity except for: • First occurrence of Grade 3 electrolyte abnormalities and/or creatinine clearance decrease resolving to Grade 2 (or baseline if baseline is ⁇ Grade 2) within 48 hours with supportive treatment. • Grade 3 fatigue, nausea, vomiting, diarrhea or other manageable constitutional symptom that is responsive to supportive therapy. • Grade 3 infection responding to appropriate antimicrobial therapy. • Any neurologic toxicity of grade 4, or grade 3 that does not return to ⁇ grade 1 or baseline within 7 days.
  • Any ⁇ Grade 3 hematologic toxicity are considered a DLT except for: • Grade 3 neutropenia without fever • Grade 4 neutropenia without fever lasting 8 days or less • Grade 3 thrombocytopenia that does not result in bleeding or transfusion • Grade 3/4 lymphopenia/lymphocytosis • Grade 3/4 WBC decreased • Grade 3/4 WBC increased [00643] Any toxicity, regardless of the NCI-CTCAE v5.0 grade, resulting in discontinuation, dose reduction or treatment with less than 75% of planned doses of APR-246 study drug, are reviewed by the DRT, and a considered a DLT if determined that the toxicity is clearly related to study drug, unless reversible CNS-related effects previously described for APR-246 or related to the patient’s underlying disease, other medical condition or concomitant medications, or clearly attributable to ibrutinib.
  • R2D The Recommended Phase 2 Dose (RP2D) of APR-246 is defined as the dose at which less than 2 out of 6 subjects experience DLT during the 4-week safety assessment period after administration of APR-246 in combination with ibrutinib. Up to 10 additional patients may be enrolled at the RP2D to confirm the confidence at that dose level.
  • DTR Data Review Team
  • Sample Size A total of approximately 120 evaluable patients are included in the study.
  • two cohorts enroll subjects with TP53-mutant R/R CLL in a 3+3 dose de-escalation design.
  • a maximal of 6 x 6 x 6 (18) DLT evaluable patients are included in this portion of the study by allowing 2 APR-246 dose reductions for the 2 cohorts.
  • An additional up to 10 patients are enrolled at the RP2D to confirm the safety at that dose level.
  • 20 patients with R/R CLL and 40 patients with R/R MCL are included to further investigate the safety profile at RP2D and efficacy effects.
  • Safety population Subjects are evaluable for safety if they receive at least one dose of APR-246 with ibrutinib.
  • the safety population is the primary analysis population used for all analyses such as patient disposition, patient demographics, exposure, safety parameters, and efficacy parameters.
  • the safety population is the primary analysis population for efficacy.
  • Efficacy evaluable (EE) population All subjects who complete at least one treatment cycle of APR-246 and ibrutinib and who have at least one post treatment clinical response assessment. Subjects who fail to complete one treatment cycle is also considered EE if they show clear evidence of clinically significant disease progression.
  • the EE population is the secondary analysis population for efficacy.
  • PK population Subjects is evaluable for PK if at least one sample for PK evaluation has been obtained Safety Analysis
  • Safety data are summarized for the safety population. These data include adverse events and laboratory parameters. AE terms are coded using the Medical Dictionary for Drug Regulatory Activities (MedDRA)®, version 22.0 or higher. AEs are summarized by System Organ Class (SOC), preferred term, severity, and relationship to treatment. Serious adverse events (SAEs), deaths, and adverse events (AEs) leading to early discontinuation of study treatment are summarized. Laboratory parameters are summarized by maximum NCI-CTCAE version 5.0 severity grade and also by change from pre-treatment to scheduled time points using descriptive statistics. Laboratory parameter listings include the normal ranges for each parameter.
  • DOR Duration of response
  • CR complete remission
  • OS Overall survival
  • PFS Progression-free survival
  • APR-246 and ibrutinib are summarized using descriptive statistics (mean, standard deviation, CV% mean, geometric mean, CV% geometric mean) and compared with historical control data.
  • Concentrations of APR-246 and ibrutinib are determined, and pharmacokinetic parameters (e.g., C max , T max , AUC, V d and CL) are derived using popPK or non-compartmental methods.
  • APR-246 AUC and Cmax are then be tested for association with signs of efficacy and safety.
  • a PK/PD model is developed to evaluate the exposure- response relationship between APR-246 plasma exposure and outcome measures.
  • Demographic and clinical data ethnicity, current age, body weight, sex, disease status, etc. are utilized to assess intersubject variability in the PK and PK/PD relationships.
  • Exploratory analyses [00663] Descriptive statistics/results from exploratory analyses of molecular markers for response predication and monitoring are prepared and may include but are not limited to: TP53 VAF by Next-generation sequencing (NGS), mutations in other genes by NGS, RNA expression.
  • NGS Next-generation sequencing
  • TP53 Sequencing -- Variant Interpretation Algorithm Inclusion of patients in the study is based on TP53 sequencing performed in a laboratory at each participating site according to established local routines. A study-specific variant interpretation algorithm is used to discriminate between eligible and non-eligible TP53 sequence variants. [00665] In order to select patients with high unmet medical need due to poor prognosis, the study enrolls patients that have any TP53 mutation which is not pre-defined as “benign” or “likely benign.” Eligible TP53 mutations herein includes, for example, variants classified as pathogenic, likely pathogenic and variant of uncertain significance (VUS) in a specified database (UMD-TP53).
  • VUS pathogenic, likely pathogenic and variant of uncertain significance
  • TP53 sequence variants Patients harboring at least one such TP53 sequence variant are eligible for inclusion, while patients who only have variant(s) classified as benign or likely benign are not eligible. Thus, also TP53 VUS are eligible, avoiding exclusion of patients with possible pathogenic TP53 variants presently classified as VUS.
  • exemplary p53 reactivator i.e., APR-246
  • BTK inhibitor i.e., ibrutinib
  • a matrix combinational dose response screening of APR-246 and ibrutinib was performed similarly in 3 AML cell lines with different TP53 and FLT3 status and one MDS/AML cell line to assess both single agent activity and evaluate additive, synergistic or antagonistic interactions across to a range of doses.
  • KBM3 ISCOVE’s modified Dulbecco's medium (Sigma Cat. no. I3390), 20% FBS (Sigma Cat. no. F084) and 1% L-glutamine (Sigma, Cat.no. G7513).
  • ISCOVE modified Dulbecco's medium
  • MV4-11 ISCOVE’s modified Dulbecco's medium (Sigma Cat. no.
  • a given pair of drugs was combined as a series of 10 dilution (dilution factor 2) concentrations of Ibrutinib and 7 dilution concentrations of APR-246 (dilution factor 1.5), which resulted in a 10 ⁇ 7 dose matrix according to "Repl1" Excel Combenefit template, and incubated for 72 h at 37 °C.
  • Day 3 [00676] Plates were taken from the incubator to reach room temperature approx.30 min. 100 ⁇ L CellTiter-Glo was added to all wells (except edge wells). Plates were then shaken on a plate shaker for 3 minutes at RT thereafter the plates were set for 10 min at RT to allow cell lysis to appear.
  • the luminescence was measured using the PerkinElmer Victorx4 instrument using the built-in program according to Instruction 11. Combenefit 2.021 was used for data analysis.
  • CCG CellTiter-Glo viability
  • the CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells. The homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium.
  • the homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present.
  • the amount of ATP is directly proportional to the number of cells present in culture.
  • Dose Response determinations by XLFIT [00678]
  • IC50 determinations the serial dilution of compounds was performed in cell culture medium based on 1.5- or 2-fold dilution factor. The serially diluted compounds were added to the cells and incubated for 72 h followed by cell viability test using the CTG assay. For each concentration, the current viability values were calculated and IC50 values were determined by XLfit software version 5.5.0.5 (a Microsoft Excel-based plug-in to perform regression, curve fitting and statistical analysis).
  • Combenefit (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866–2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files.
  • Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods.
  • Loewe the synergy score quantifies the excess over the highest single drug response.
  • Loewe model the synergy score quantifies the excess over the expected response if the two drugs are the same compound.
  • the expected response is a multiplicative effect as if the two drugs act independently (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413–2415).
  • the experimental dose–response surface that delineates combination effects in concentration space is first read by the Combenefit software as a matrix of % of the control value across concentrations. Single agent effects are extracted from this data and fitted with a dose response curve. Based on the two single agent dose response curves, a model-based combination dose–response surface is derived.
  • This surface provides a ‘reference’ dose–response surface for a non-synergistic (additive/independent) combination, whose characteristics are determined by the selected model (i.e., Loewe, Bliss or HSA).
  • the experimental combination dose response surface is then compared to the model-generated one, resulting in a synergy distribution in concentration space.
  • the graphical outputs consist of: (i) the single agent dose response data and its fitting; (ii) the combination dose response (four different displays); (iii) the model generated reference combination dose response, i.e., the prediction of effect if the drugs are not synergistic (three different displays); (iv) the resulting synergy distribution (three different displays); and (v) a graphic mapping the synergy distribution onto the dose–response surface.
  • This synergy distribution can be further summarized via various metrics (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866-2868) as follows: • SYN_MAX - the maximum level of synergy observed.
  • SYN_SUM the sum of synergy observed in concentration logarithmic space. For instance, an integrated synergy of 50 is equivalent to an extra synergistic effect of 50%, which is spread over a square of 1 log x 1 log in the 2-d log-concentration space.
  • SYN_SUM_WEIGHTED The integrated weighted synergy incorporates a weight based on the dose response which bias the total score towards synergy achieving highest effect. Hence, a synergy of 50% leading to a combined full effect (100%) will have more weight than if the corresponding effect was only 20 or 30%.
  • SYN_AVERAGE_C1 Concentration value of drug 1 where synergy appears to be localized.
  • a “combination index” (CI) is then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability.
  • Additive model Combination Index values corresponding to synergy and strong synergy were also obtained in these cell lines.
  • the synergy was apparent in APR-246 concentrations ranging from 1.3 to 6.6 ⁇ M for MOLM-13, 2 to 2.5 ⁇ M for MV4-11 and 2.9 to 4.4 ⁇ M for KBM3 cell line.
  • Ibrutinib concentrations with synergy ranged from 0.08 to 10 ⁇ M for KBM3, 0.04 to 1.25 ⁇ M for MV4-11 and MOLM-13.
  • the FLT3 wild type and TP53 mutated cell line SKM-1 showed an antagonistic effect in the majority of cases.
  • KBM3 and MOLM-13 are TP53 wild type, whereas MV4-11(R248W heterozygous) and the MDS/AML cell line SKM-1 (R248Q, homo/hemizygous) are TP53 mutated.
  • MV4-11(R248W heterozygous) and the MDS/AML cell line SKM-1 (R248Q, homo/hemizygous) are TP53 mutated.
  • exemplary p53 reactivator i.e., APR-246
  • an exemplary XPO1 inhibitor i.e., selinexor
  • a matrix combinational dose response screening of APR-246 and selinexor was performed similarly in the two AML cell lines with different TP53 status and one MDS/AML cell line to assess both single agent activity and evaluate additive, synergistic or antagonistic interactions across to a range of doses.
  • MOLM-13 Highest Single Agent
  • Bliss and Loewe models Additive model Combination Index values corresponding to synergy and strong synergy were also obtained in these cell lines.
  • a given pair of drugs e.g., APR-246 and selinexor
  • 10 dilution dilution factor 2
  • 7 dilution concentrations of APR-246 dilution factor 1.5
  • Day 3 Plates was taken from the incubator to reach room temperature approx.30min. 100 ⁇ L CellTiter-Glo was added to all wells (except edge wells). Plates were then shaken on a plate shaker for 3 minutes at room temperature.
  • the CellTiter-Glo viability (CTG) assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells.
  • the homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium.
  • the homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present.
  • the amount of ATP is directly proportional to the number of cells present in culture.
  • Dose response determinations by XLFIT [00704] For IC 50 determinations, the serial dilution of compounds was performed in cell culture medium based on a 1.5- or 2-fold dilution factor. The serially diluted compounds were added to the cells and incubated for 72 h followed by cell viability test using the CTG assay. For each concentration, the current viability values were calculated and IC 50 values were determined by XLfit software version 5.5.0.5 (a Microsoft Excel-based plug-in to perform regression, curve fitting and statistical analysis).
  • Combenefit (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866–2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files.
  • Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods.
  • Loewe the synergy score quantifies the excess over the highest single drug response.
  • Loewe model the synergy score quantifies the excess over the expected response if the two drugs are the same compound.
  • the expected response is a multiplicative effect as if the two drugs act independently (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413–2415).
  • the experimental dose–response surface that delineates combination effects in concentration space is first read by the Combenefit software as a matrix of % of the control value across concentrations. Single agent effects are extracted from this data and fitted with a dose response curve. Based on the two single agent dose response curves, a model-based combination dose–response surface is derived.
  • This surface provides a “reference” dose–response surface for a non-synergistic (additive/independent) combination, whose characteristics are determined by the selected model (i.e., Loewe, Bliss or HSA).
  • the experimental combination dose response surface is then compared to the model-generated one, resulting in a synergy distribution in concentration space.
  • the graphical outputs consist of: (i) the single agent dose response data and its fitting; (ii) the combination dose response (four different displays); (iii) the model generated reference combination dose response, i.e., the prediction of effect if the drugs are not synergistic (three different displays); (iv) the resulting synergy distribution (three different displays); and (v) a graphic mapping the synergy distribution onto the dose–response surface.
  • This synergy distribution can be further summarized via various metrics (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866-2868) as follows: • SYN_MAX - the maximum level of synergy observed.
  • SYN_SUM the sum of synergy observed in concentration logarithmic space. For instance, an integrated synergy of 50 is equivalent to an extra synergistic effect of 50%, which is spread over a square of 1 log x 1 log in the 2-d log-concentration space.
  • SYN_SUM_WEIGHTED The integrated weighted synergy incorporates a weight based on the dose response which bias the total score towards synergy achieving highest effect. Hence, a synergy of 50% leading to a combined full effect (100%) will have more weight than if the corresponding effect was only 20 or 30%.
  • SYN_AVERAGE_C1 Concentration value of drug 1 where synergy appears to be localized.
  • a “combination index” (CI) is then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability.
  • a matrix combinational dose response screening of APR-246 and Selinexor was performed in two AML cell lines with different TP53 status (MOLM-13 carries wild type TP53 and HL-60 has a large deletion of TP53) and one MDS/AML cell line to assess both single agent activity and evaluate additive, synergistic or antagonistic interactions across to a range of doses.
  • both APR-246 and selinexor showed dose-dependent cytotoxic activity with IC 50 values ranging between 1.3-8.1 ⁇ M for APR-246 and 0.05-0.14 ⁇ M for selinexor. IC 50 values are summarized in Table 11 below. Table 11.
  • Combination index for SKM-1 Table 15 Combination index for HL-60
  • IC 50 0.05 ⁇ M
  • SKM-1 0.9-0.14 ⁇ M
  • Additive model Combination Index values corresponding to synergy and strong synergy were also obtained in these cell lines.
  • the synergy was apparent in APR-246 concentrations ranging from 1.3 to 6.7 ⁇ M for MOLM-13, 1 to 1.5 ⁇ M for SKM-1 and 1.9 to 4.4 ⁇ M for HL-60 cell line. Selinexor concentrations with synergy ranged from 0.005 to 0.16 ⁇ M for MOLM-13, 0.04 to 2.5 ⁇ M for SKM-1 and 0.16 to 10 ⁇ M for HL-60.
  • Example 5 Synergistic Effects of APR-246 in Combination with Selinexor in Multiple Myeloma Cell Lines
  • synergistic effects of an exemplary p53 reactivator i.e., APR-246 in combination with an exemplary XPO1 inhibitor (i.e., selinexor) are tested in three human multiple myeloma cell lines MOLP-8 with wild type TP53, RPMI-8226, TP53 mutated (Homozygous, 853G>A, E285K), and OPM-2 TP53 mutated (Homozygous, c.524G>A p.R175H).
  • a matrix combinational dose response screening of APR-246 and selinexor are performed similarly to in the AML cell lines in Example 1, with three myeloma cell lines having different TP53 status, to evaluate additive, synergistic or antagonistic interactions across a range of doses.
  • ISCOVE modified Dulbecco's medium (Sigma Cat. No. I3390), 20% FBS (Sigma Cat. no. F084) and 1% L-glutamine (Sigma, Cat. No. G7513).
  • RPMI-8226 RPMI (R8758, Sigma Aldrich) with 2 mM L-glutamine + 10% FBS (F0804, Sigma Aldrich), heat inactivated at 56 °C for 60 min for MOLM-13: RPMI (R8758, Sigma Aldrich) + 10% FBS (F0804, Sigma Aldrich), heat inactivated at 56 °C for 60 min.
  • MOLM-13 RPMI (R8758, Sigma Aldrich) + 10% FBS (F0804, Sigma Aldrich), heat inactivated at 56 °C for 60 min.
  • MOLP-8 cells are seeded out at ca.5 x 10 5 cells/mL in a 12-well plate and split 1:2 every 2-3 days, viability decreases after thawing.
  • RPMI-8226 cells are seeded out at ca.5 x 10 5 cells/mL in a 6-well plate and split 1:2 every 2-3 days.
  • OPM2 cells are seeded out at ca.1 x 10 6 cells/mL in a 12-well plate; maintained at 0.3-0.7 x 10 6 cells/mL; split 1:2 every 2-3 days; viability decreases after thawing.
  • Cell Assays [00725] Day 1 [00726] For each cell line one well of 12 well plate (65% confluent) cells are transferred to a 15 mL tube and centrifuged at 1,000 RPM for 5 min. The supernatant is discarded, and the cell pellet is resuspended in 3 mL medium.
  • the cells are counted in the TC20 automated cell counter.
  • a 6 mL cell culture medium is prepared with approximately 24 x 10 4 cells/mL (corresponding to 12,000 cells when 50 ⁇ L is seeded out). Cells are seeded in 96- well plates in column 1-12, row B-H.
  • APR-246 and Selinexor are combined as a series of 10 dilutions, (dilution factor 2) concentrations of Selinexor and 7 dilution concentrations of APR-246 (dilution factor 1.5), resulting in a 10 ⁇ 7 dose matrix, and incubated for 72 h at 37 °C.
  • Dose response determinations by XLFIT For IC 50 determinations, serial dilutions of compounds are performed in cell culture medium based on a 1.5- or 2-fold dilution factor. The serially diluted compounds are added to the cells and incubated for 72 h, followed by cell viability test using the CTG assay. The IC 50 values are determined as described in Example 1. Analysis of combination by COMBENEFIT [00733] The synergy is evaluated by several methods, according to Example 1, including Combenefit analyses using the standard Loewe, Bliss and HSA methods, the Additive method, and by calculation of the combination index.
  • MDA-MB-231 For MDA-MB-231, DMEM with Glutamax (31966-021, Gibco) +10% FBS (F0804, Sigma Aldrich). Equipment & Software • PerkinElmer Multimode Plate Reader Victorx4 • PerkinElmer 2030 Software 4.0 • Combenefit 2,021 software • XLFIT 5.5.5.0. Cell Assays [00738] Day 1 [00739] A 75 cm 2 flask (100% confluent) MDA-MB-231 was trypsinized and suspended in 6 ml cell culture medium and a 75 cm 2 flask (100% confluent) SKM-1 was transferred to 15 mL tubes. Cells were centrifuged at 1,000 rpm for 5 min.
  • a given pair of drugs was combined as a series of 7-10 dilution (dilution factor 2) concentrations of MK-1775 and 7 or 9 dilution concentrations of APR-246 (dilution factor 1.5), which resulted in a 10 ⁇ 7 dose matrix according to "Repl1" Excel Combenefit template, and incubated for 72 h at 37 °C.
  • Day 3 [00743] Plates were taken from the incubator to reach room temperature approx.30 min.
  • 100 ⁇ L CellTiter-Glo was added to all wells (except edge wells).
  • the CellTiter-Glo viability (CTG) assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells.
  • the homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium.
  • the homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present.
  • the amount of ATP is directly proportional to the number of cells present in culture.
  • Dose response determinations by XLFIT [00746]
  • the serial dilution of compounds was performed in cell culture medium based on 1.5- or 2-fold dilution factor. The serially diluted compounds were added to the cells and incubated for 72 h followed by cell viability test using the CTG assay.
  • Combenefit (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866–2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files. Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods. In the HSA model, the synergy score quantifies the excess over the highest single drug response.
  • the synergy score quantifies the excess over the expected response if the two drugs are the same compound.
  • the expected response is a multiplicative effect as if the two drugs act independently (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413–2415).
  • the experimental dose–response surface that delineates combination effects in concentration space was first read by the Combenefit software as a matrix of % of the control value across concentrations. Single agent effects were extracted from this data and fitted with a dose response curve. Based on the two single agent dose response curves, a model-based combination dose–response surface was derived.
  • This surface provided a “reference” dose– response surface for a non-synergistic (additive/independent) combination, whose characteristics are determined by the selected model (i.e., Loewe, Bliss or HSA).
  • the experimental combination dose response surface was then compared to the model-generated one, resulting in a synergy distribution in concentration space.
  • the graphical outputs consist of: (i) the single agent dose response data and its fitting; (ii) the combination dose response (four different displays); (iii) the model generated reference combination dose response, i.e., the prediction of effect if the drugs are not synergistic (three different displays); (iv) the resulting synergy distribution (three different displays); and (v) a graphic mapping the synergy distribution onto the dose–response surface. [00750] This synergy distribution were further summarized via various metrics (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866-2868) as follows: • SYN_MAX - the maximum level of synergy observed.
  • SYN_SUM the sum of synergy observed in concentration logarithmic space. For instance, an integrated synergy of 50 is equivalent to an extra synergistic effect of 50%, which is spread over a square of 1 log x 1 log in the 2-d log-concentration space.
  • SYN_SUM_WEIGHTED The integrated weighted synergy incorporates a weight based on the dose response which bias the total score towards synergy achieving highest effect. Hence, a synergy of 50% leading to a combined full effect (100%) will have more weight than if the corresponding effect was only 20 or 30%.
  • SYN_AVERAGE_C1 Concentration value of drug 1 where synergy appears to be localized.
  • a “combination index” (CI) was then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability.
  • a matrix combinational dose response screening of APR-246 and MK-1775 was performed in one MDS/AML cell line and one breast cancer cell line with mutant TP53 to assess both single agent activity and to evaluate additive, synergistic or antagonistic interactions across to a range of doses.
  • both APR-246 and MK-1775 showed dose-dependent cytotoxic activity with IC 50 values ranging between 1.5-65 ⁇ M for APR-246 and 0.2 ⁇ M for MK-1775.
  • IC 50 values are summarized in Table 16. Table 16.
  • IC 50 values ( ⁇ M) for APR-246 (mean ⁇ SD, n 2 or 3) and MK-1775.
  • the synergy was apparent in APR-246 concentrations ranging from 50 to 75 ⁇ M for MDA-MB- 231 cell line and 1 to 1.7 ⁇ M for SKM-1 cell line.
  • MK-1775 concentrations with synergy ranged from 0.14 to 0.16 ⁇ M for SKM-1 cell line and 0.1 to 1 ⁇ M for MDA-MB-231 cell line.
  • the Loewe model an antagonistic effect appeared in some dose combinations.
  • the Loewe, Bliss and HSA use different mathematical and/ or probabilistic theories for representing synergy effects while combining different drugs.
  • the Bliss independence model is expected to hold true for non-interacting drugs that elicit their responses independently, by targeting separate pathways.

Abstract

Provided herein are methods of treating a disease or disorder in a subject using a combination therapy of a p53 agonist such as a p53 reactivator and a second therapeutic agent such as an inhibitor of PD-1 mediated signaling, a Bruton's tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1).

Description

COMBINATION TREATMENT WITH AN AGONIST OF P53 AND A SECOND THERAPEUTIC AGENT CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.63/008,519, filed April 10, 2020, U.S. Provisional Application No.63/028,478, filed May 21, 2020, U.S. Provisional Application No.63/038,651, filed June 12, 2020, and U.S. Provisional Application No.63/083,471, filed September 25, 2020, the content of each of which is incorporated by reference herein in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0002] This application incorporates by reference in its entirety the Computer Readable Form (CRF) of a Sequence Listing in ASCII text format submitted herewith. The Sequence Listing text file submitted herewith, entitled 14599-020-228_SEQ_LISTING.TXT, was created on April 8, 2021, and is 7,285 bytes in size. FIELD [0003] Provided herein are combination therapies using an agonist of p53 such as a p53 reactivator in combination with a second therapeutic agent, for example, an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1), for treating diseases and disorders. [0004] In one aspect, provided herein are combination therapies using an agonist of p53 in combination with an inhibitor of PD-1 mediated signaling for treating a disease or disorder such as a solid tumor malignancy. In another aspect, provided herein are combination therapies using an agonist of p53 in combination with an inhibitor of Bruton’s tyrosine kinase for treating a disease or disorder such as lymphoma. In yet another aspect, provided herein are combination therapies using an agonist of p53 in combination with an exportin 1 (XPO1) inhibitor for treating a disease or disorder such as a hyperproliferative malignancy. In yet another aspect, provided herein are combination therapies using an agonist of p53 in combination with an inhibitor of WEE1 for treating a disease or disorder such as cancer. 1. BACKGROUND [0005] p53 plays a critical role as a tumor suppressor and its gene TP53 is a common target for mutations in tumors. p53 halts the cell cycle and/or triggers apoptosis in response to various stress stimuli, including DNA damage, hypoxia, and oncogene activation (Ko, L. J. & Prives, C., Genes Dev.10, 1054-1072 (1996); Sherr, C. J., Genes Dev.12, 2984-2991 (1998)). [0006] Both p53-induced cell cycle arrest and apoptosis could be involved in p53-mediated tumor suppression. A significant proportion of human tumors make a “mutant” p53 protein due to a TP53 mutation, making it highly desirable to restore the wild type p53 activity to yield growth suppression to tumors. Tumor cells are particularly sensitive to p53 reactivation, supposedly for two main reasons. First, tumor cells are sensitized to apoptosis due to oncogene activation (reviewed in Evan, G. & Littlewood, T., Science.281, 1317-1322 (1998)). Second, mutant p53 proteins tend to accumulate at high levels in tumor cells. Therefore, restoration of the wild type function to the abundant and presumably “activated” mutant p53 should trigger a massive apoptotic response in already sensitized tumor cells, whereas normal cells that harbor low or undetectable levels of p53 should not be affected. [0007] In normal cells p53 is found at very low levels due to negative feedback regulation of the level, unless undergoing developmental or maturation processes involving p53. [0008] Non-Hodgkin lymphomas (NHL) are lymphoid malignant neoplasms with diverse biological and clinical behavior, variously derived from the clonal expansion of B cells, T cells, natural killer cells, or precursors of these cells. Chronic lymphocytic leukemia (CLL) is one of the most common types of B-cell NHL, characterized by a progressive accumulation of functionally incompetent monoclonal lymphocytes (Siegel, R.L., et al., CA Cancer J Clin.70(1), 7-30 (2020)). The incidence rates among men and women in the United States are approximately 6.75 and 3.65 cases per 100,000 population per year, respectively (Yamamoto, J.F., et al., Cancer Causes Control.19(4), 379-390 (2008); Fitzmaurice, C., et al., JAMA Oncol. 3(4), 524-548, (2017)). [0009] Mantle cell lymphoma (MCL) is another type of mature B-cell NHL, which comprises about 7% of adult NHL in the United States with an incidence of approximately 4 to 8 cases per million persons per year (Harris, N. L., et al., Blood 84(5), 1361-1392 (1994); Armitage, J.O., et al., J Clin Oncol.16(8), 2780-2795 (1998); Zhou, Y., et al., Cancer 113(4), 791-798 (2008)). Standard management for MCL includes chemoimmunotherapy (CIT) regimens, sometimes followed by consolidation with autologous stem cell transplantation in fit patients and/or maintenance rituximab, and most recently targeted agents such as Bruton’s tyrosine kinase (BTK) inhibitors have been found to be effective in the relapsed and/or refractory (R/R) MCL (Wang, M. L., et al., N. Engl. J. Med.369(6), 507-516 (2013)). [0010] Therapeutically useful compounds have previously been generated based on showing mutant p53 dependent anti-proliferative activity in a cellular assay, including the compound PRIMA-1 (i.e., 2,2-bis(hydroxymethyl)quinuclidin-3-one) (disclosed in WO 02/24692), and its analogs (such as those disclosed in WO 03/070250). Nonetheless, there still remains a general need of effective combination therapies using these compounds in combination with a second therapeutic agent for treating cancer and other diseases or disorders. 2. SUMMARY [0011] Provided herein are methods of treating solid tumor malignancy in a subject, comprising administering to the subject: (i) an agonist of p53; and (ii) an inhibitor of PD-1 mediated signaling. [0012] Also provided herein are methods of treating a lymphoma in a subject, comprising administering to the subject: (i) a p53 reactivator; and (ii) a Bruton’s tyrosine kinase (BTK) inhibitor. [0013] Also provided herein are methods of treating a hyperproliferative malignancy in a subject, comprising administering to the subject: (i) a p53 reactivator; and (ii) an exportin 1 (XPO1) inhibitor. [0014] Also provided herein are methods of treating a hyperproliferative malignancy in a subject, comprising administering to the subject: (i) a p53 reactivator; and (ii) an inhibitor of Wee1-like protein kinase (WEE1). [0015] In certain embodiments, the agonist of p53 is a compound that can give reactivation of a mutant p53 or a compound whose metabolite can give reactivation of a mutant p53. [0016] In certain embodiments, the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W. [0017] In certain embodiments, the agonist of p53 is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity. [0018] In certain embodiments, the compound or metabolite promotes proper folding of mutant and wild-type p53 proteins. [0019] In certain embodiments, the compound or metabolite is capable of shifting the equilibrium from unfolded towards a folded structure of wild-type or mutant p53, or wherein the compound or metabolite is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or metabolite is capable of reducing aggregation of the wild-type or mutant p53. [0020] In certain embodiments, the compound or metabolite is capable of promoting a folded structure of wild-type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53. [0021] In certain embodiments, the compound or metabolite is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promotes a folded conformation. [0022] In certain embodiments, the p53 reactivator is a compound that can give reactivation of a mutant p53, or a degradation product thereof that can give reactivation of a mutant p53. [0023] In certain embodiments, the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W. [0024] In certain embodiments, the p53 reactivator is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity. [0025] In certain embodiments, the compound or degradation product thereof promotes proper folding of mutant and wild-type p53 proteins. [0026] In certain embodiments, the compound or degradation product thereof is capable of shifting the equilibrium from unfolded towards a folded structure of wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of reducing aggregation of the wild-type or mutant p53. [0027] In certain embodiments, the compound or degradation product thereof is capable of promoting a folded structure of wild-type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53. [0028] In certain embodiments, the compound or degradation product threreof is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promotes a folded conformation. [0029] In certain embodiments, the compound is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one; 2,2-bis(hydroxymethyl)quinuclidin-3-one; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; 2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide; N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide; N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide; 1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide; 1-methyl-N-((3-oxoquinuclidin-2-yl)methyl)cyclopropane-1-sulfonamide; N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide; 1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; 5-methyl-1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; tert-butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine-1(2H)- carboxylate; 5-methyl-1,3-bis((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; N-methyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((3-chloro-1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; N,N-dimethyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carbonitrile; and 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; or a pharmaceutically acceptable salt thereof. [0030] In certain embodiments, the compound is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) having the following formula:
Figure imgf000008_0001
, or a pharmaceutically acceptable salt thereof. [0031] In certain embodiments, the compound is a compound selected from the group consisting of: 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; and 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, or a pharmaceutically acceptable salt thereof. [0032] In certain embodiments, the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2- yl)methyl)acetamide (Compound A), or a pharmaceutically acceptable salt thereof. [0033] In certain embodiments, the compound is 2,2,2-trichloro-N-ethyl-N-((3- oxoquinuclidin-2-yl)methyl)acetamide (Compound B), or a pharmaceutically acceptable salt thereof. [0034] In certain embodiments, the compound is 2,2,2-trichloro-N-((3-oxoquinuclidin-2- yl)methyl)acetamide (Compound C), or a pharmaceutically acceptable salt thereof. [0035] In certain embodiments, the compound is N-ethyl-2,2,2-trifluoro-N-((3- oxoquinuclidin-2-yl)methyl)acetamide (Compound D), or a pharmaceutically acceptable salt thereof. [0036] In certain embodiments, the compound is 2,2-difluoro-N-((3-oxoquinuclidin-2- yl)methyl)acetamide (Compound E), or a pharmaceutically acceptable salt thereof. [0037] In certain embodiments, the inhibitor of PD-1 mediated signaling is an anti-PD-1 antibody or an anti-PD-L1 antibody. [0038] In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, nivolumab, AMP-224, and AMP-514. [0039] In certain embodiments, the anti-PD-1 antibody is pembrolizumab. [0040] In certain embodiments, the agonist of p53 is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) and the inhibitor of PD-1 mediated signaling is pembrolizumab. [0041] In certain embodiments, APR-246 is administered at a dose of about 4.5 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle. [0042] In certain embodiments, APR-246 is administered at a dose of about 4.0 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle. [0043] In certain embodiments, APR-246 is administered at a dose of about 3.5 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle. [0044] In certain embodiments, APR-246 is administered on Days 1–4 and pembrolizumab is administered on Day 3 of the each 21-day cycle. [0045] In certain embodiments, APR-246 and pembrolizumab are administered for 1 to 20 cycles. [0046] In certain embodiments, the agonist of p53 is formulated in a first pharmaceutical composition and the inhibitor of PD-1 mediated signaling is formulated in a second pharmaceutical composition. [0047] In certain embodiments, the solid tumor malignancy is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non- melanoma skin carcinoma, and a lung cancer. [0048] In certain embodiments, the solid tumor malignancy is an advanced non-CNS- primary solid tumor. [0049] In certain embodiments, the solid tumor malignancy is selected from the group consisting of gastric/gastroesophageal junction (GEJ) cancer, bladder/urothelial cancer, and non- small-cell lung cancer (NSCLC). [0050] In certain embodiments, the BTK inhibitor is selected from the group consisting of ibrutinib, acalabrutinib, zanubrutinib, evobrutinib, dasatinib, spebrutinib, tirabrutinib, ABBV- 105, ONO-4059, LFM-A13, and HM71224, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. [0051] In certain embodiments, the BTK inhibitor is ibrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. [0052] In certain embodiments, the p53 reactivator is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) and the BTK inhibitor is ibrutinib. [0053] In certain embodiments, APR-246 is administered at a dose of about 4.5 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. [0054] In certain embodiments, APR-246 is administered at a dose of about 4.0 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. [0055] In certain embodiments, APR-246 is administered at a dose of about 3.5 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. [0056] In certain embodiments, APR-246 is administered on Days 1–4 and ibrutinib is administered daily of each 28-day cycle. [0057] In certain embodiments, APR-246 and ibrutinib are administered for 1 to 20 cycles. [0058] In certain embodiments, the p53 reactivator is formulated in a first pharmaceutical composition and the BTK inhibitor is formulated in a second pharmaceutical composition. [0059] In certain embodiments, the lymphoma is a Hodgkin lymphoma (HL) or a non- Hodgkin lymphoma (NHL). [0060] In certain embodiments, the lymphoma is a non-Hodgkin lymphoma (NHL). [0061] In certain embodiments, the non-Hodgkin lymphoma (NHL) is a mature (peripheral) B-cell neoplasm. [0062] In certain embodiments, the non-Hodgkin lymphoma (NHL) is selected from the group consisting of: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, diffuse large cell B-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma (plasmacytoma), extranodal marginal zone B-cell lymphoma, nodal marginal zone lymphoma, follicle center lymphoma, and Burkitt’s leukemia (Burkitt’s lymphoma). [0063] In certain embodiments, the lymphoma is chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). [0064] In certain embodiments, the lymphoma is chronic lymphocytic leukemia (CLL). [0065] In certain embodiments, the lymphoma is mantle cell lymphoma (MCL). [0066] In certain embodiments, the non-Hodgkin lymphoma (NHL) is relapsed or refractory NHL. [0067] In certain embodiments, the non-Hodgkin lymphoma (NHL) is relapsed or refractory chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). [0068] In certain embodiments, the non-Hodgkin lymphoma (NHL) is relapsed or refractory CLL. [0069] In certain embodiments, the non-Hodgkin lymphoma (NHL) is relapsed or refractory MCL. [0070] In certain embodiments, the lymphoma comprises a cancer cell having mutant p53. [0071] In certain embodiments, further comprises determining by gene sequencing if the subject has TP53 mutation. [0072] In certain embodiments, the subject has not been treated with any BTK inhibitor prior to the co-administration of the p53 reactivator and the BTK inhibitor. [0073] In certain embodiments, the XPO1 inhibitor is selected from the group consisting of leptomycin A (LMA), leptomycin B (LMB), selinexor (KPT-330), Eltanexor (KPT-8602), KTP-185, KPT-249, KPT-251, KPT-276, verdinexor (KPT-335), piperlongumine, valtrate, felezonexor (CBS9106 or SL-801), and PKF050-638, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. [0074] In certain embodiments, the XPO1 inhibitor is selinexor, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. [0075] In certain embodiments, the p53 reactivator is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) and the XPO1 inhibitor is selinexor. [0076] In certain embodiments, APR-246 is administered at a daily dose of about 4.5 g/day and selinexor is administered at a dose of about 80 mg/day twice per week. [0077] In certain embodiments, APR-246 is administered at a daily dose of about 4.0 g/day and selinexor is administered at a dose of about 80 mg/day twice per week. [0078] In certain embodiments, APR-246 is administered at a daily dose of about 3.5 g/day and selinexor is administered at a dose of about 80 mg/day twice per week. [0079] In certain embodiments, selinexor is administered on day 1 and day 3 of each week. [0080] In certain embodiments, APR-246 and selinexor are administered for 1 to 20 cycles. [0081] In certain embodiments, selinexor is administered at a reduced dose of about 100 mg once per week, about 80 mg once per week, or about 60 mg once per week. [0082] In certain embodiments, the p53 reactivator is formulated in a first pharmaceutical composition and the XPO1 inhibitor is formulated in a second pharmaceutical composition. [0083] In certain embodiments, the inhibitor of WEE1 is selected from the group consisting of 6-(2,6-Dichlorophenyl)-2-[4-[2-(diethylamino)ethoxy] anilino]-8-methylpyrido[2,3-d]pyrimidin- 7-one (PD0166285); 9-Hydroxy-4-phenylpyrrolo[3,4-c] carbazole-1,3(2H,6H)-dione (PD0407824); 4-(2-Chlorophenyl)-9-hydroxypyrrolo[3,4-c] carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor I); 6-Butyl-4-(2-chlorophenyl)-9-hydroxypyrrolo [3,4-c]carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor II); 2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((4-(4-methylpiperazin-1-yl)phenyl) amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Adavosertib (MK-1775 or AZD1775)); and Methyl 4-(4-((2-allyl-1-(6-(2-hydroxypropan-2-yl) pyridin-2-yl)-3-oxo-2,3-dihydro-1H- pyrazolo[3,4-d] pyrimidin-6-yl)amino)phenyl)piperazine-1-carboxylate (CJM061). [0084] In certain embodiments, the inhibitor of WEE1 is MK-1775 having a formula of:
Figure imgf000013_0001
(MK-1775), or a pharmaceutically acceptable salt thereof. [0085] In certain embodiments, the p53 reactivator is formulated in a first pharmaceutical composition and the inhibitor of WEE1 is formulated in a second pharmaceutical composition. [0086] In certain embodiments, the hyperproliferative malignancy is a hematological malignancy. [0087] In certain embodiments, the hematological malignancy is leukemia, lymphoma, or myeloma. [0088] In certain embodiments, the hematological malignancy is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. [0089] In certain embodiments, the hematologic malignancy is myeloma. [0090] In certain embodiments, the myeloma is multiple myeloma (MM). [0091] In certain embodiments, the hematologic malignancy is acute myeloid leukemia (AML). [0092] In certain embodiments, the hematologic malignancy is chronic lymphocytic leukemia (CLL). [0093] In certain embodiments, the hematologic malignancy is myelodysplastic syndromes (MDS). [0094] In certain embodiments, the hyperproliferative malignancy comprises a cancer cell having mutant p53. [0095] In certain embodiments, further comprises determining by gene sequencing if the subject has TP53 mutation. [0096] In certain embodiments, the hyperproliferative malignancy is a solid tumor cancer. [0097] In certain embodiments, the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, a lung cancer, a cervical cancer, a prostate cancer, a head and neck cancer, and a breast cancer. [0098] In certain embodiments, the solid tumor cancer is breast cancer. [0099] In certain embodiments, the solid tumor cancer is triple negative breast cancer. 3. BRIEF DESCRIPTION OF THE FIGURES [00100] FIG.1 depicts the phase 1/2 clinical trials to determine the safety and preliminary efficacy of APR-246 in combination with pembrolizumab in subjects with solid tumor malignancies. [00101] FIG.2 outlines the safety lead-in portion of the study, wherein subjects with advanced solid tumor malignancies receive pembrolizumab in combination with APR-246. [00102] FIG.3 depicts the study scheme for the phase 1 and dose expansion study of APR- 246 in combination with ibrutinib in subjects with TP53-mutant relapsed and/or refractory non- Hodgkin lymphoma including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). 4. DETAILED DESCRIPTION [00103] Described herein are combination treatments with an agonist of p53 such as a p53 reactivator (see Section 4.2.1) and a second therapeutic agent (see Sections 0 – 4.2.5) for treatment of diseases or disorders such as a cancer (see Section 4.4). In some embodiments, the second therapeutic agent provided herein can be an inhibitor of PD-1 mediated signaling such as an inhibitor of PD-1 (see Section 0). In some embodiments, the second therapeutic agent provided herein can be a Bruton’s tyrosine kinase (BTK) inhibitor (see Section 4.2.3). In some embodiments, the second therapeutic agent provided herein can be an exportin 1 (XPO1) inhibitor (see Section 4.2.4). In some embodiments, the second therapeutic agent provided herein can be an inhibitor of Wee1-like protein kinase (WEE1) (see Section 4.2.5). Manner of administration and dosing regimen are described in Section 4.4 herein. [00104] The present disclosure is based, in part, on the surprising finding that the combination of an agonist of p53 such as a p53 reactivator and a second therapeutic agent produces synergistic effects in treating certain diseases and disorders such as a cancer. [00105] For example, the present disclosure is based, in part, on the surprising finding that the combination of a p53 reactivator and a BTK inhibitor produces synergistic effects in treating certain diseases and disorders (e.g., non-Hodgkin lymphoma). [00106] As another example, the present disclosure is based, in part, on the surprising finding that the combination of a p53 reactivator and an XPO1 inhibitor produces synergistic effects in treating certain diseases and disorders (e.g., hyperproliferative malignancy). [00107] As another example, the present disclosure is based, in part, on the surprising finding that the combination of a p53 reactivator and an inhibitor of WEE1 produces synergistic effects in treating certain diseases and disorders. 4.1 Definitions [00108] Techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed using conventional methodology by those skilled in the art. Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control. [00109] The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of a therapeutic compound, a combination of therapeutic compounds or pharmaceutical compositions thereof provided herein, which is sufficient to result in the desired outcome. [00110] The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal. In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder. [00111] “Administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art. [00112] As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or disorder resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder. The term “treating” includes both managing and ameliorating the disease. [00113] The terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s). [00114] The term “a mutant p53 mediated disease or disorder” as used herein refers to a disease or disorder that is caused or partially caused by mutation of the p53 gene (TP53). For example, a mutant p53 mediated cancer means the cancer that contains a cell having a mutant TP53. [00115] As used herein, the term “alkyl” unless otherwise stated, means an unbranched or branched, saturated or unsaturated (alkenyl or alkynyl) hydrocarbon radical. The term “Cx-Cy alkyl” means a straight or branched chain hydrocarbon containing x to y carbon atoms. For example, “C2-C6 alkyl” means a straight or branched chain hydrocarbon containing 2 to 6 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n- hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n- decyl. [00116] As used herein, the term “aryl” means an aromatic group, such as phenyl or naphthyl. [00117] As used herein, the term “cycloalkyl” means a monocyclic or bicyclic, saturated or partially unsaturated (but not aromatic), hydrocarbon ring of three to ten carbon ring atoms. Cycloalkyl groups include fused and bridged bicyclic rings. For example, when fused, the cycloalkyl group may comprise two rings that share adjacent atoms (e.g., one covalent bond). When bridged, the cycloalkyl group may comprise two rings that share three or more atoms, separating the two bridgehead atoms by a bridge containing at least one atom. When a cycloalkyl group contains from x-y ring carbon atoms, it may be referred to herein as Cx-Cy cycloalkyl. In certain embodiments, cycloalkyl is C3-C10 cycloalkyl, or is C5-C7 cycloalkyl, or is C5-C6 cycloalkyl, or is C3-C6 cycloalkyl, or is C3-C7 cycloalkyl. In certain embodiments, cycloalkyl is C3-C8 cycloalkyl. In certain embodiments, cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. [00118] As used herein, the term “heteroaryl” means a mono-, bi-, or tricyclic heteroaromatic group containing one or more heteroatom(s) preferably selected from N, O and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl, thiophenetyl, pyranyl, carbazolyl, acridinyl, quinolinyl, benzimidazolyl, benzthiazolyl, purinyl, cinnolinyl and pteridinyl. [00119] As used herein, the term “non-aromatic heterocycle” means a non-aromatic cyclic group containing one or more heteroatom(s) preferably selected from N, O and S, such as a pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, tetrahydrofuranyl or monosaccharide. [00120] As used herein, the term “halogen” means a fluorine, chlorine, bromine or iodine. [00121] As used herein, the term “halo” means a fluoro, chloro, bromo or iodo. [00122] As used herein, and unless specified otherwise, the term “substituted” means that the concerned groups are substituted with at least one functional group, such as hydroxyl, amine, sulfide, silyl, carboxylic acid, halogen, aryl, etc. [00123] As used herein, the term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans. [00124] As used herein, “excipient” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof. The term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle. [00125] In some embodiments, excipients are pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences (18th ed.1990). [00126] In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. In some embodiments, a pharmaceutically acceptable excipient is an aqueous pH buffered solution. [00127] The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range. [00128] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise. [00129] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of” otherwise analogous embodiments described in terms of “consisting of” are also provided. [00130] The term “between” as used in a phrase as such “between A and B” or “between A- B” refers to a range including both A and B. [00131] As used herein, numerical values are often presented in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly indicates otherwise. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. [00132] The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). [00133] As used herein, the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time. The term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days. The term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days. [00134] As used herein and unless otherwise indicated, the term “stereomerically pure” means a composition that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound. For example, a stereomerically pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure composition of a compound having two chiral centers will be substantially free of other diastereomers of the compound. [00135] It should be noted that if there is a discrepancy between a depicted structure and a name given that structure, the depicted structure is to be accorded more weight. In addition, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of the structure. 4.2 Agents Used in the Combination Therapies [00136] Provided herein are methods and compositions relating to the combination therapies of an agonist of p53 such as a p53 reactivator and a second therapeutic agent. For example, the second therapeutic agent can be an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1). [00137] In one aspect, provided herein are methods and compositions relating to the combination of an agonist of p53 and an inhibitor of PD-1 mediated signaling such as an inhibitor of PD-1. Agents that can be used as agonists of p53 are set forth in Section 4.2.1. Agents that can be used as inhibitors of PD-1 mediated signaling such as inhibitors of PD-1 are set forth in Section 0. [00138] In another aspect, also provided herein are methods and compositions relating to the combination of an agonist of p53 such as a p53 reactivator and a Bruton’s tyrosine kinase (BTK) inhibitor for treatment of certain diseases or disorders. Agents that can be used as agonists of p53 such as p53 reactivators are set forth in Section 4.2.1. Agents that can be used as BTK inhibitors are set forth in Section 4.2.3. [00139] In yet another aspect, also provided herein are combination treatments with an agonist of p53 such as a p53 reactivator (see Section 4.2.1) and an exportin 1 (XPO1) inhibitor (see Section 4.2.4) for the treatment of certain diseases and disorders. Manners of administration and dosing regimen are described in Section 4.2.4. [00140] In yet another aspect, also provided herein are combination treatments with a p53 reactivator (see Section Error! Reference source not found.) and an inhibitor of WEE1 (see Section 4.2.5) for treatment of certain diseases and disorders (see Section Error! Reference source not found.4.4.4). The cancer can comprise cells that are mutant for p53 (see Section 4.4). Manner of administration and coordination of treatment regimen is described and exemplified in Section 4.4.4. 4.2.1 Agonists of p53 [00141] One therapeutic agent in the present combination therapies is an agonist of p53. In some embodiments, an agonist of p53 provided herein increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of p53 activity, e.g., in a cell such as an immune cell. In a specific embodiment, an agonist of p53 provided herein increases by about 50% of p53 activity, e.g., in a cell such as an immune cell. The methods for measuring p53 activity and increase thereof are well known in the art. For example, in some embodiments, the increased activity corresponds to an increased level of transcription of p53 target genes, which may be studied by quantifying mRNA increase. An increased level of mRNA may be determined by methods known in the art. In some embodiments, mRNA is quantified by TaqMan analysis. [00142] In some embodiments, the p53 agonist provided herein increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of p53 activity, e.g., in a cell such as an immune cell. In a specific embodiment, the p53 agonist provided herein increases by about 50% of p53 activity, e.g., in a cell such as an immune cell. The methods for measuring p53 activity and increase thereof are well known in the art. For example, in some embodiments, the increased activity corresponds to an increased level of transcription of p53 target genes, which may be studied by quantifying mRNA increase. An increased level of mRNA may be determined by methods known in the art. In some embodiments, mRNA is quantified by TaqMan analysis. [00143] In certain embodiments, the agonist of p53 described herein is a p53 reactivator. [00144] In certain embodiments, one therapeutic agent used in the combination therapies described herein (e.g., in Section 4.4) is a p53 agonist such a p53 reactivator. In some embodiments, the p53 reactivator provided herein increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of p53 activity, e.g., in a cell such as an immune cell. In a specific embodiment, the p53 reactivator provided herein increases by about 50% of p53 activity, e.g., in a cell such as an immune cell. The methods for measuring p53 activity and increase thereof are well known in the art. For example, in some embodiments, the increased activity corresponds to an increased level of transcription of p53 target genes, which may be studied by quantifying mRNA increase. An increased level of mRNA may be determined by methods known in the art. In some embodiments, mRNA is quantified by TaqMan analysis. [00145] In some embodiments, the agonist of p53 provided herein increases wild-type p53 activity. In some embodiments, the agonist of p53 provided herein is a p53 reactivator capable of reactivating a mutant p53. In some embodiments, the agonist of p53 provided herein is capable of increasing wild type p53 activity and capable of reactivating a mutant p53. [00146] The p53 gene TP53 is a very common target for mutation in tumors. Around half of all human tumors carry mutation in TP53. p53 halts the cell cycle and/or triggers apoptosis in response to various stress stimuli, including DNA damage, hypoxia, and oncogene activation (Ko, L. J. & Prives, C., Genes Dev.10, 1054-1072 (1996); Sherr, C. J., Genes Dev.12, 2984- 2991 (1998)). Upon activation, p53 initiates the p53-dependent biological responses through transcriptional transactivation of specific target genes carrying p53 DNA binding motifs. [00147] Analyses of a large number of mutant p53 genes in human tumors have revealed a strong selection for mutations that inactivate the DNA binding function of p53; most mutations in tumors are point mutations clustered in the part encoding the core domain of p53 (residues 94- 292) that harbors the DNA binding activity (Béroud, C. & Soussi, T., Nucl. Acids Res.26, 200- 204 (1998)). [00148] Both p53-induced cell cycle arrest and apoptosis could be involved in p53-mediated tumor suppression. While p53-induced cell cycle arrest could conceivably be reversed in different ways, p53-induced cell death would have advantage of being irreversible. There is indeed evidence from animal in vivo models (Symonds et al., Cell 78, 703-711 (1994)) and human tumors (Bardeesy et al., Cancer Res 55, 215-219 (1995)) indicating that p53-dependent apoptosis plays a major role in the elimination of emerging tumors, particularly in response to oncogenic signaling. Moreover, the ability of p53 to induce apoptosis often determines the efficacy of cancer therapy (Lowe et al., Science 266, 807-10 (1994)). [00149] In addition to hyperproliferative diseases, such as cancer, it is also known in the art that deficient p53 function is involved in a number of other disease states, e.g. autoimmune diseases and cardiac diseases. [00150] For example, as shown in Mountz et al., Immunology, 6: 27-37 (1994), human autoimmune diseases share the common feature of an imbalance between the production and destruction of various cell types including lymphocytes (SLE), synovial cells (RA), and fibroblasts (scleroderma). Genes including TP53 that regulates apoptosis are also expressed abnormally. According to the authors, specific therapies that induce apoptosis without incurring side effects should improve treatment of autoimmune disease. [00151] For another example, Bonafe et al., Cell Death and Differentiation, 11: 962-973 (2004) suggests that TP53 codon 72 polymorphism contributes to a genetically determined variability in apoptotic susceptibility among old people, which has a potentially relevant role in the context of an age-related pathologic condition, such as myocardial ischaemia. [00152] Okuda et al., Journal of Neuroimmunology, 135: 29-37 (2003) suggests that p53 may be involved in the regulatory process of experimental autoimmune encephalomyelitis (EAE) through the control of cytokine production and/or the apoptotic elimination of inflammatory cells. EAE as a model for autoimmune inflammatory diseases of the central nervous system (CNS) is a widely used model for the human disease multiple sclerosis. [00153] These results suggest that pharmacological restoration of p53 function would be beneficial in a number of disorders and diseases. [00154] In some embodiments of the combination therapies provided herein, the p53 agonist (e.g., a p53 reactivator) directly or indirectly targets a mutant p53 protein. [00155] In some embodiments of the combination therapies provided herein, the p53 reactivator directly or indirectly targets a mutant p53 protein. [00156] In some embodiments, the mutant TP53 includes a missense mutation, which is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. Missense mutant p53 proteins can be broadly classified as DNA-contact mutants and structural mutants. p53 DNA contact mutant contains mutations present on amino acids directly binding to DNA, such as in mutants carrying single amino acid changes R248Q, R248W, R273H, or R273C, where R248Q denotes that the wild-type residue arginine in position 248 has been replaced by a glutamine in the mutant. p53 structural mutants have an amino acid replacement that alters the overall architecture and/or stability to abolish its DNA-binding ability, as reported in mutants carrying the R175H, Y220C, G245S, R249S, or R282W replacements. [00157] In other embodiments, the mutant TP53 includes a nonsense mutation. A nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP53 mutations in cancer. The most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*. [00158] In some embodiments, the p53 reactivator provided herein reactivates or restores at least part of the wild-type p53 activity of a mutant p53, for example by promoting proper folding of the mutant p53 and restoring the normal p53 function of the mutant p53. [00159] In certain embodiments, the p53 reactivator provided herein inhibits improper protein misfolding of the mutant p53 protein, and/or promotes proper protein folding of the mutant p53 by covalently binding to the mutant p53 protein. [00160] In certain embodiment, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) provided herein reacts as an electrophile with one or more thiols in the mutant p53. In certain embodiment, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) provided herein covalently binds to one or more thiols in the mutant p53. In certain embodiment, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) reacts with or binds to one or more thiols in the mutant p53 as an electrophile. In certain embodiments, a metabolite or degradation product of the p53 reactivator) reacts with or binds to one or more thiols in the mutant p53. In certain embodiments, reversible or irreversible covalent bonds are formed between the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) and the mutant p53. In one embodiment, reversible covalent bonds are formed between the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) and the mutant p53. In another embodiment, irreversible covalent bonds are formed between the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) and the mutant p53. [00161] In certain embodiments, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) provided herein reacts with one or more thiols in the DNA binding domain of the mutant p53 to stabilize the mutant p53 conformation, thus restoring their transcriptional activities. In some embodiments, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) reacts with one or more thiols of cysteine residues in the core domain of wild-type p53 protein and stabilizes wild-type p53 conformation. In other embodiments, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) reacts with one or more thiols of cysteine residues in the core domain of mutant p53 and restores the mutant p53 conformation to wild-type p53 like conformation. In yet other embodiments, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) shifts the equilibrium from unfolded mutant p53 conformation towards a wild-type p53 like conformation. [00162] In certain embodiments, the p53 reactivator provided herein inhibits improper protein misfolding and/or promotes proper protein folding by non-covalent binding to the mutant p53 protein. Such p53 reactivators include chaperones that can non‐covalently stabilize the mutant p53 protein. [00163] In some more specific embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof inhibits improper protein misfolding and/or promotes proper protein folding by covalent binding to the mutant p53 protein, for example, by electrophiles binding to one or more thiols in the mutant p53 DNA binding domain to stabilize a folded conformation, and restoring their transcriptional activities. In some embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof binds to the thiol of cysteine residues in the core domain and stabilizes wild type p53 conformation. In other embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof is shifting the equilibrium from unfolded towards a wild-type like p53 conformation. In yet other embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof binds to thiol groups in the core domain and restores wild-type conformation. [00164] In other more specific embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof inhibits improper protein misfolding and/or promotes proper protein folding by non-covalent binding to the mutant p53 protein. Such p53 reactivators include chaperones that can non‐covalently stabilize mutant p53 structures. [00165] In certain embodiments, the p53 reactivator provided herein reactivates mutant p53 by interfering with aggregation of misfolded p53 or reducing aggregation of mutant p53. Sometimes, p53 misfolds or unfolds into an aggregation‐prone stage that loses its DNA‐binding capacity. Similarly, misfolded p53 may convert wild type p53 to a misfolded form and accelerate p53 aggregation. Thus, in some embodiments, the p53 reactivator provided herein may reactivate p53 by interfering with aggregation of misfolded p53. In other embodiments, the p53 reactivator provided herein or a degradation product or metabolite reduces non-folded or incorrectly folded mutant p53 that may otherwise aggregate, and thereby reducing aggregation. [00166] In other embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof reactivates mutant p53 by interfering with aggregation of misfolded p53 or reducing aggregation of mutant p53. Sometimes, p53 misfolds or unfolds into an aggregation‐ prone stage that loses its DNA‐binding capacity. Similarly, misfolded mutant p53 may cause accumulation of wild type p53 in a misfolded form and accelerate p53 aggregation. Thus, in some embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof may reactivate p53 by interfering with aggregation of misfolded p53. In other embodiments, the p53 reactivator provided herein or a degradation product or metabolite reduces non-folded or incorrectly folded mutant p53 that may otherwise aggregate, and thereby reducing aggregation. Increased melting temperature correlates with shifting the equilibrium towards the folded structure and may be studied by for example Circular Dicroism (CD) or Differential Scanning Fluorimetry (DSF). Aggregation of the unfolded protein chain is in competition with folding and aggregation may be studied by intrinsic fluorescence measurements or light scattering, and the actual aggregates may be investigated by atomic force microscopy. [00167] In some embodiments, the mutant p53 comprises at least one amino acid replacement in the core domain of the mutant p53 (between residues 94 and 292) caused by a TP53 mutation. In some embodiments, the mutant p53 comprises replacement selected from the group consisting of V173A, S241F, R249S, R273H, R175H, R248Q, and Y220C. In some embodiments, the mutant p53 comprises one of the amino acid replacements of R175H and R273H. [00168] In certain embodiments, the p53 reactivator provided herein can give reactivation of a mutant p53 protein. In certain embodiments, the p53 reactivator can result in reactivation of a mutant p53 protein. In certain embodiments, the p53 reactivator is transformed to a metabolite or a degradation product that reacts with a mutant p53 protein. In certain embodiments, the p53 reactivator is transformed to the metabolite or the degradation product in vivo. In certain embodiments, the p53 reactivator is transformed to the metabolite or the degradation product in tumor tissue. [00169] Without being bound by any theory, in some embodiments, the p53 agonist provided herein (e.g., a p53 reactivator) or a degradation product or metabolite stabilizes wild type p53 protein in a situation where its production has been induced as part of a normal physiological process, and in this way enhancing the effect of said wild type p53 induction. Without being bound by any theory, in some embodiments, the p53 reactivator provided herein or a degradation product or metabolite stabilizes wild type p53 protein in a situation where its production has been induced as part of a normal physiological process, and in this way enhancing the effect of said wild type p53 induction. [00170] In certain embodiments, the compound that can give reactivation of mutant p53 (the p53 reactivator) provided herein is a compound according to formula (I):
Figure imgf000027_0001
wherein: R1 is selected from the group consisting of H, —CH2—O—R3, —CH2—S—R3, and — CH2—NR3R4; R2 is selected from the group consisting of —CH2—O—R3, —CH2—S—R3, and — CH2—NR3R4; each of R3 and R4 is independently selected from H; substituted or unsubstituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or C1-C10 alkyl; substituted or unsubstituted benzyl; substituted or unsubstituted mono- or bi-cyclic aryl; substituted or unsubstituted mono-, bi- or tri-cyclic C3-C10 heteroaryl or non-aromatic C3-C10 heterocyclyl containing one or more heteroatoms independently selected from N, O and S; or R3 and R4 in —CH2—NR3R4 together with the nitrogen atom to which they are attached, form a substituted or unsubstituted non-aromatic C3-C10 mono- or bi-cyclic heterocyclyl containing one or more heteroatoms independently selected from N, O and S and optionally comprising one or more keto substitute groups; wherein the substituents of the substituted groups are independently selected from unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or C1-C10 alkyl; halogen; halo-substituted C1-C10 alkyl, mono- or bicyclic aryl; mono-, bi- or tricyclic C3-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl containing one or more heteroatoms independently selected from N, O and S; C1-C10 alkoxy; amino; and C1- C10 alkylamino; or a pharmaceutically acceptable salt thereof. [00171] In some embodiments, the agonist of p53 provided herein is a p53 reactivator having formula (I)
Figure imgf000028_0001
wherein: R1 is selected from H, —CH2—O—R3, —CH2—S—R3, and —CH2—NR3R4; R2 is selected from —CH2—O—R3, —CH2—S—R3, and —CH2—NR3R4; R3 and R4 are the same or different and are independently selected from H; substituted or non- substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or C1-C10 alkyl; substituted or non-substituted benzyl; substituted or non-substituted mono- or bicyclic aryl; substituted or non-substituted mono-, bi- or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl containing one or several heteroatoms independently selected from N, O and S; or R3 and R4 in —CH2—NR3R4 are bonded together and form, together with the nitrogen atom to which they are bonded, a substituted or non-substituted non-aromatic C2-C10 mono- or bicyclic heterocyclyl optionally containing one or several further heteroatoms independently selected from N, O and S and optionally comprising one or several cyclic keto groups; wherein the substituents of the substituted groups are independently selected from unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or C1-C10 alkyl; halogen; halogen- substituted C1-C10 alkyl, mono- or bicyclic aryl; mono-, bi- or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl containing one or several heteroatoms independently selected from N, O and S; C1-C10 alkoxy; amino; and C1-C10 alkylamino; or a pharmaceutically acceptable salts thereof. [00172] The pharmaceutically acceptable salt of the compound of formula (I) may be an acid addition salt of the compound formed with an inorganic acid or of an organic acid. [00173] The pharmaceutically acceptable salt of the compound of formula (I) e.g. may be an acid addition salt of an inorganic mineral acid or of an organic acid. [00174] In some embodiments of the compound of formula (I), R1 is selected from H, — CH2—O—R3, —CH2—S—R3, and —CH2—NR3R4. [00175] In some embodiments, R1 is selected from H, —CH2—O—R3, and —CH2—S—R3. In some embodiments, R1 is selected from H and —CH2—O—R3. In other embodiments, R1 is selected from —CH2—O—R3, and —CH2—S—R3. In some embodiments, R1 is H. [00176] R2 in formula (I) is selected from —CH2—O—R3, —CH2—S—R3, and —CH2— NR3R4. In some embodiments, R2 is selected from —CH2—O—R3 and —CH2—S—R3. In still other embodiments, R2 is —CH2—O—R3. [00177] In one embodiment, R1 is H, and R2 is —CH2—O—R3, —CH2—S—R3 or —CH2— NR3R4. In another embodiment, R1 is H, and R2 is —CH2—O—R3 or —CH2—S—R3. In yet another embodiment, R1 is H, and R2 is —CH2—O—R3. [00178] In one embodiment, R1 is selected from H, —CH2—O—R3 and —CH2—S—R3; and R2is selected from —CH2—O—R3 and —CH2—S—R3. [00179] In one embodiment, R1 is H; and R2 is selected from —CH2—O—R3, —CH2—S—R3 and —CH2—NR3R4; e.g. from —CH2—O—R3 and —CH2—S—R3, and in particular is — CH2—O—R3. [00180] In one embodiment, R1 is selected from H and —CH2—O—R3; and R2 is —CH2— O—R3. [00181] In one embodiment, both R1 and R2 are —CH2—O—R3. [00182] In one embodiment, each R3 is independently selected from H; substituted or unsubstituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl. In one embodiment, each R3 is independently H or C1-C10 alkyl. In another embodiment, each R3 is independently H or C1-C6 alkyl. In yet another embodiment, each R3 is independently H or C1-C4 alkyl. In yet another embodiment, each R3 is independently H or C1- C3 alkyl. In a specific embodiment, each R3 is independently H or methyl. [00183] In one embodiment, each R3 is independently selected from H; substituted or non- substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl. For example, each R3 may be independently selected from H and C1-C10 alkyl, e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl. [00184] In one embodiment, R1 is H or —CH2—O—R3, R2 is —CH2—O—R3, and each R3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl. In another embodiment, R1 is H or —CH2—O—R3, R2 is —CH2—O—R3, and each R3 is independently H or C1-C10 alkyl. In yet another embodiment, R1 is H or —CH2—O—R3, R2 is —CH2—O—R3, and each R3 is independently H or C1-C6 alkyl. In yet another embodiment, R1 is H or —CH2—O—R3, R2 is —CH2—O—R3, and each R3 is independently H or C1-C4 alkyl. In yet another embodiment, R1 is H or —CH2—O—R3, R2 is —CH2—O—R3, and each R3 is independently H or C1-C3 alkyl. In a specific embodiment, R1 is H or —CH2—O—R3, R2 is —CH2—O—R3, and each R3 is independently H or methyl. [00185] In one embodiment, R1 is selected from H and —CH2—O—R3, and R2 is —CH2— O—R3, and each R3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl, in particular from H and C1-C10 alkyl, e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl. [00186] In one embodiment, R1 and R2 are both —CH2—O—R3, and each R3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl. In another embodiment, R1 and R2 are both —CH2—O—R3, and each R3 is independently H or C1-C10 alkyl. In yet another embodiment, R1 and R2 are both —CH2—O—R3, and each R3 is independently H or C1-C6 alkyl. In yet another embodiment, R1 and R2 are both —CH2—O—R3, and each R3 is independently H or C1- C4 alkyl. In yet another embodiment, R1 and R2 are both —CH2—O—R3, and each R3 is independently H or C1-C3 alkyl. In a specific embodiment, R1 and R2 are both —CH2—O—R3, and each R3 is independently H or methyl. [00187] In one embodiment, R1 and R2 are both —CH2—O—R3, and each R3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl; in particular from H and C1-C10 alkyl; e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl. [00188] In a compound of formula (I), as defined herein above, any C1-C10 alkyl e.g. may be a C1-C6 alkyl, or a C1-C4 alkyl, e.g. methyl, ethyl, propyl or butyl. Any C3-C12 cycloalkyl may be e.g. a C3-C8 cycloalkyl, or a C3-C6 cycloalkyl. Any mono- or bicyclic aryl may be e.g. a monocyclic aryl, such as phenyl. Any mono-, bi- or tricyclic C2-C10 heteroaryl may be e.g. a monocyclic or bicyclic C2-C5 heteroaryl, e.g. a 5- or 6-membered monocyclic or a 9-membered bicyclic C2-C5 heteroaryl. Any mono-, bi- or tricyclic non-aromatic C2-C10 heterocyclyl may be e.g. a monocyclic or bicyclic C2-C5 heterocyclyl, e.g. a 5- or 6-membered monocyclic or 9- or 10-membered bicyclic C2-C5 heterocyclyl. Any halogen may be selected from F, Cl, Br and I, preferably from F and Cl. Any heterocycle, aromatic or not, containing one or several heteroatoms independently selected from N, O and S, e.g. may contain 1-5 heteroatoms, e.g. independently selected from N and O. [00189] In one embodiment of the compound of formula (I) as defined herein above, the substituted or unsubstituted C3-C12 cycloalkyl or C1-C10 alkyl is a non-substituted C3-C12 cycloalkyl or C1-C10 alkyl. [00190] In one embodiment, the substituted or unsubstituted benzyl is an unsubstituted benzyl. [00191] In one embodiment, the substituted or unsubstituted mono- or bi-cyclic aryl is an unsubstituted mono- or bi-cyclic aryl. [00192] In one embodiment, the substituted or unsubstituted mono-, bi- or tri-cyclic heteroaryl or non-aromatic heterocyclyl is an unsubstituted mono-, bi- or tri-cyclic heteroaryl or non- aromatic heterocyclyl. [00193] In one embodiment, when any of the above groups is substituted, each substituent independently is a C1-C10 alkyl, halo, halo-substituted C1-C10 alkyl, monocyclic heteroaryl, C1-C10 alkoxy, or amino group. [00194] In one embodiment, in a compound of formula (I) as defined herein above, any substituted or non-substituted C3-C12 cycloalkyl or C1-C10 alkyl is non-substituted. [00195] In one embodiment, any substituted or non-substituted benzyl is non-substituted. [00196] In one embodiment, any substituted or non-substituted mono- or bicyclic aryl is non- substituted. [00197] In one embodiment, any substituted or non-substituted mono-, bi- or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl is non-substituted. [00198] In one embodiment, when any of the above groups is substituted, each substituent is selected from C1-C10 alkyl, e.g. C1-C6 alkyl, C1-C4 alkyl, or C1-C3 alkyl, such as methyl; halogen, e.g. Cl; halogen-substituted C1-C10 alkyl, e.g. trifluoromethyl; monocyclic C2-C5 heteroaryl, e.g. pyridyl; C1-C10 alkoxy, e.g. C1-C6 alkoxy, C1-C4 alkoxy, or C1-C3 alkoxy, such as methoxy; and amino. [00199] In one embodiment, when any of the above groups is substituted, the number of substituents on each substituted group is 1, 2 or 3. [00200] In another embodiment, the compound provided herein is selected from those exemplified in the prior art documents referred to herein above, e.g. WO05/090341, WO04/084893, WO02/024692 and WO03/070250, which are incorporated herein in their entirety. [00201] In one embodiment, the compound of formula (I) is selected from 2-(hydroxymethyl)- 2-(methoxymethyl)quinuclidin-3-one (APR-246) and 2,2-bis(hydroxymethyl)quinuclidin-3-one, or a pharmaceutically acceptable salt thereof. [00202] In one embodiment, the compound of formula (I) is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) having the following formula:
Figure imgf000032_0001
, or a pharmaceutically acceptable salt thereof. [00203] In one embodiment, the compound of formula (I) is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) or a pharmaceutically acceptable salt thereof. [00204] In another embodiment, the compound of formula (I) is 2,2- bis(hydroxymethyl)quinuclidin-3-one or a pharmaceutically acceptable salt thereof. [00205] In yet other embodiments, the agonist of p53 is a compound listed below. In certain more specific embodiments, the compound listed below is capable of reactivating mutatnt p53. 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; 2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide; N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide; N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide; 1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide; 1-methyl-N-((3-oxoquinuclidin-2-yl)methyl)cyclopropane-1-sulfonamide; N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide; 1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; 5-methyl-1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; tert-butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine- 1(2H)-carboxylate; 5-methyl-1,3-bis((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; N-methyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((3-chloro-1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; N,N-dimethyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carbonitrile; and 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide, or a pharmaceutically acceptable salt thereof. [00206] In yet another embodiments, the compound of formula (I) is selected from the group consisting of: 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; 2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide; N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide; N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide; 1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide; 1-methyl-N-((3-oxoquinuclidin-2-yl)methyl)cyclopropane-1-sulfonamide; N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide; 1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; 5-methyl-1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; tert-butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine- 1(2H)-carboxylate; 5-methyl-1,3-bis((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; N-methyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((3-chloro-1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; N,N-dimethyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carbonitrile; and 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide, or a pharmaceutically acceptable salt thereof. [00207] In yet another embodiment, the compound of formula (I) is a compound listed in Table 1 below, or a pharmaceutically acceptable salt thereof. Table 1.
Figure imgf000035_0001
[00208] In yet another embodiment, the p53 reactivator is Compound A, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the p53 reactivator is Compound B, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the p53 reactivator is Compound C, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the p53 reactivator is Compound D, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the p53 reactivator is Compound E, or a pharmaceutically acceptable salt thereof. [00209] In a specific embodiment, the p53 reactivator provided herein is 2,2,2-trifluoro-N-((3- oxoquinuclidin-2-yl)methyl)acetamide having the following formula:
Figure imgf000036_0001
(Compound A), or a pharmaceutically acceptable salt thereof. [00210] In some embodiments, the compound of Formula (I) has reactivating activity of mutant p53. In other embodiments, the compound of Formula (I) has activating activity of wide type p53. [00211] In some embodiments, the agonist of p53 is a compound listed in the table below. In certain more specific embodiments, the compound listed in Table 2 below is capable of reactivating mutant p53. [00212] In some embodiments, the p53 reactivator provided herein is selected from the group consisting of the compounds in Table 2 below. Table 2.
Figure imgf000036_0002
Figure imgf000037_0001
Figure imgf000038_0001
a The amino acid sequence of the compound is provided (N-terminus is myristoylated). b The amino acid sequence of the compound is provided. c The nucleic acid sequence of the compound is provided.
Figure imgf000039_0001
4.2.2 Inhibitors of PD-1 Mediated Signaling [00213] In certain embodiments, the inhibitor of PD-1 mediated signaling provided herein attenuates the interaction between the PD-1 receptor and PD-L1. In some embodiments, the inhibitor of PD-1 mediated signaling is an inhibitor of PD-1. In other embodiments, the inhibitor of PD-1 mediated signaling is an inhibitor of PD-L1. [00214] In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody. In some embodiments, the inhibitor of PD-1 is an antisense oligonucleotide targeting PD-1 gene. In other embodiments, the inhibitor of PD-1 is a small molecule compound that can binds to PD-1 protein. [00215] In some embodiments, the inhibitor of PD-L1 is an anti-PD-L1 antibody. In some embodiments, the inhibitor of PD-L1 is an antisense oligonucleotide targeting PD-L1 gene. In other embodiments, the inhibitor of PD-L1 is a small molecule compound that can binds to PD- L1 protein. [00216] In some embodiments, the anti-PD-1 antibody provided herein is pembrolizumab (Merck); nivolumab (Bristol-Myers Squibb), cemiplimab (Regeneron), spartalizumab (Novartis), camrelizumab (Jiangsu HengRui Medicine), sintilimab (Innovent and Eli Lilly), tislelizumab (BeiGene), toripalimab (Junshi), nivolumab (Bristol-Myers Squibb), AMP-224 (GlaxoSmithKline), or AMP-514 (GlaxoSmithKline). [00217] In a specific embodiment, the anti-PD-1 antibody provided herein is pembrolizumab (KEYTRUDA®). [00218] The heavy chain and light chain sequences of pembrolizumab are shown in Table 3. Table 3. Heavy chain
Figure imgf000040_0001
(SEQ ID NO:5) Light chain
Figure imgf000040_0002
(SEQ ID NO:6) 4.2.3 Bruton’s Tyrosine Kinase (BTK) Inhibitors [00219] Another therapeutic agent used in the combination therapies described herein (e.g., in Section 4.4.2) is a Bruton’s tyrosine kinase (BTK) inhibitor. [00220] Bruton’s tyrosine kinase (BTK), a nonreceptor tyrosine kinase member of the Tec kinase family, plays a significant role in B-cell development and is a unique therapeutic target in B-cell malignancies such as chronic lymphocytic leukemia and mantle cell lymphoma (Aalipour, A. & Advani, R. H., Thera. Adv. Hematol.5(4), 121-133 (2014)). Loss of function mutations in BTK result in X-linked agammaglobulinemia (XLA), which is characterized by low peripheral blood B cells, low levels of Ig, and recurring infections. The suggested BTK’s role in B-cell development and immunoglobulin production combined with the nonlethality of XLA make BTK a promising therapeutic target (Herman, S., et al., Blood 117, 6287-6296 (2011)). [00221] The therapeutic potential of BTK inhibition is supported by its effects on malignant cell lines in vitro. For example, in chronic lymphocytic leukemia (CLL), inhibition of BTK inhibits NFκB DNA binding, reduces cell migration, proliferation, and survival, and induces apoptosis (de Rooij, M., et al. Blood 119, 2590-2594 (2012); Cheng, S. et al. Leukemia 28, 649- 657 (2014); Herman, S., et al., Blood 117, 6287-6296 (2011)). For another example, in mantle cell lymphoma (MCL), inhibition of BTK induces apoptosis and decreases levels of the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 (Cinar, M., et al. Leuk. Res.37, 1271-1277 (2013)). Moreover, siRNA-mediated knockdown of BTK in MCL cells showed significant reduction of phospho-STAT3 and inhibition of the NFκB pathway, thereby reducing MCL growth and cellular migration (Ou, Z., et al. Blood 122, 3079 (2013)). [00222] In some embodiment, the BTK inhibitor used in the combination therapies methods described herein is selected from the group consisting of ibrutinib, acalabrutinib, zanubrutinib, evobrutinib, dasatinib, spebrutinib, tirabrutinib, ABBV-105, ONO-4059, LFM-A13, and HM71224, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. In one embodiment, the BTK inhibitor is ibrutinib, or acalabrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. In one preferred embodiment, the BTK inhibitor is ibrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. In one embodiment, the BTK inhibitor is ibrutinib. In one embodiment, the BTK inhibitor is acalabrutinib. Ibrutinib has a chemical name of 1-[(3R)-3-[4-amino-3-(4- phenoxyphenyl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one, having the following structure:
Figure imgf000041_0001
. 4.2.4 Exportin 1 (XPO1) Inhibitors [00223] Another therapeutic agent used in the combination therapies described herein (e.g., in Section 4.4.3) is an exportin 1 (XPO1) inhibitor. [00224] Exportin 1 (XPO1 or XPO-1), also known as chromosome region maintenance 1 (CRM1), is the main exporter of leucine-rich proteins form the nucleus through the neuclear pore complex (NPC) to the cytoplasm (Podar, K. et al., Exper Opinion on Pharmacotherapy 21(4), 399-4082 (2020); Wang A. Y. & Liu, H., Stem Cell Investigation, 6(6), 1-9 (2019)). A GTPase Ran loaded with GTP binds cooperatively with XPO1 to translocate proteins including transcription factors, Tumor Suppressor Proteins (TSPs; e.g., p53, Rb, IkB, p73, BRCA1/2, etc.), cell-cyle regulators, and RNA molecules through the NPC to cytoplasm. During cancer initiation or progression, malignant cells seem to develop the ability to export key nuclear proteins that affect treatment outcome. During cancer XPO1 mutation and/or overexpression have been reported in nearly all nalignancies and are associated with alleviated translocation of TSPs out of the nucleus, which leads to, e.g., inactivation of apoptosis, cell-cycle deregulations, and chemotherapeutic resistance (Podar, K. et al., Exper Opinion on Pharmacotherapy 21(4), 399-4082 (2020)). XPO1 overexpression has been reported in several solid tumors and leukemias, including AML, and it is associated with worse outcome (Ranganathan, P., Blood, 125(17), 2689-2692 (2015)). Thus, XPO1 has become one of the therapeutic targets in cancer therapy. [00225] Selinexor (XPOVIO™) is a first-in-class small molecule XPO1 inhibitor that is developed for the treatment of cancer (Syed, Y. Y., Drugs, 79(13), 1485-1494 (2019)). Selinexor was approved by the U.S. Food and Drug Administration (FDA) for use in combination with the corticosteroid dexamethasone for the treatment of patients with relapsed refractory multiple myeloma who have received prior therapies and are resistant to several other forms of treatment (Selinexor Label, July 2019). [00226] In the presence of selinexor, XPO1 is inhibited and degraded, and is unable to export its cargo proteins. This leads to nuclear accumulation of important TSPs, including p53, p21, p27, and FOXO, resulting in cell-cycle arrest, apoptosis, antiproliferation, and other antitumor activities (Conforti, F. et al., Clin. Cancer Res., 21(20), 4508-4513 (2015)). [00227] In some embodiment, the XPO1 inhibitor used in the combination therapies methods described herein is selected from the group consisting of leptomycin A (LMA), leptomycin B (LMB), selinexor (KPT-330), Eltanexor (KPT-8602), KTP-185, KPT-249, KPT-251, KPT-276, verdinexor (KPT-335), piperlongumine, valtrate, felezonexor (CBS9106 or SL-801), and PKF050-638, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. In one preferred embodiment, the XPO1 inhibitor is selinexor, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. In one embodiment, the XPO1 inhibitor is selinexor. Selinexor has a chemical name of (2Z)-3-{3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl}-N′-pyrazin-2- ylprop-2-enehydrazide, having the following structure:
Figure imgf000043_0001
. 4.2.5 Inhibitors of Wee1-like protein kinase (WEE1) [00228] Another therapeutic agent used in the combination therapies described herein (e.g., in Section 4.4.4) is an inhibitor of Wee1-like protein kinase (WEE1). [00229] The inhibitors of WEE1 provided herein include any compound that reduces the activity and/or any biological function of WEE1. WEE1 is a nuclear kinase belonging to the Ser/Thr family of protein kinases (see NCBI gene ID: 7465). [00230] In certain embodiments, the inhibitor of WEE1 provided herein is a small- molecule inhibitor. In other embodiments, the inhibitor of WEE1 provided herein is a nucleic acid targeting WEE1 gene (e.g., siRNA, antisense oligonucleotide (ASOs), and miRNA). In yet other embodiments, the inhibitor of WEE1 provided herein is a polypeptide (e.g., an antibody or fragment thereof) capable of binding WEE1. [00231] In some embodiments, the inhibitor of WEE1 is 6-(2,6-dichlorophenyl)-2-[4-[2- (diethylamino)ethoxy]anilino]-8-methylpyrido[2,3-d]pyrimidin-7-one (PD0166285; CAS No. 185039-89-8) having a formula of:
Figure imgf000044_0001
or a pharmaceutically acceptable salt thereof. [00232] In some embodiments, the inhibitor of WEE1 is 9-hydroxy-4-phenylpyrrolo[3,4- c]carbazole-1,3(2H,6H)-dione (PD0407824; CAS No.622864-54-4) having a formula of:
Figure imgf000044_0002
or a pharmaceutically acceptable salt thereof. [00233] In other embodiments, the inhibitor of WEE1 is 4-(2-chlorophenyl)-9- hydroxypyrrolo[3,4-c] carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor I; CAS No.622855-37-2) having a formula of:
Figure imgf000044_0003
or a pharmaceutically acceptable salt thereof. [00234] In yet other embodiments, the inhibitor of WEE1 is 6-butyl-4-(2-chlorophenyl)-9- hydroxypyrrolo [3,4-c]carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor II; CAS No.622855-50-9) having a formula of:
Figure imgf000045_0001
or a pharmaceutically acceptable salt thereof. [00235] In yet other embodiments, the inhibitor of WEE1 is methyl 4-(4-((2-allyl-1-(6-(2- hydroxypropan-2-yl) pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d] pyrimidin-6- yl)amino)phenyl)piperazine-1-carboxylate (CJM061) having a formula of:
Figure imgf000045_0002
or a pharmaceutically acceptable salt thereof. [00236] In yet other embodiments, the inhibitor of WEE1 is adavosertib (MK-1775 or AZD1775) having a formula of:
Figure imgf000045_0003
or a pharmaceutically acceptable salt thereof. 4.3 Pharmaceutical Compositions 4.3.1 Compositions Relating to Combination Therapies with Inhibitors of PD-1 Mediated Signaling [00237] An agonist of p53 such as a p53 reactivator (e.g., APR-246) can be formulated with one or more pharmaceutically acceptable excipient. Similarly, an inhibitor of PD-1 mediated signaling such as an inhibitor of PD-1 (e.g., pembrolizumab) can be formulated in a pharmaceutical composition with one or more pharmaceutically acceptable excipients. In some embodiments, provided herein is a combination therapy comprising a first pharmaceutical composition comprising a p53 reactivator provided herein and a first pharmaceutically acceptable excipient, and a second pharmaceutical composition comprising an inhibitor of PD-1 (e.g., pembrolizumab) provided herein and a second pharmaceutically acceptable excipient. The first and the second pharmaceutically acceptable excipients can be the same or different. In some embodiments, a p53 reactivator provided herein and an inhibitor of PD-1 (e.g., pembrolizumab) provided herein are formulated together in a single pharmaceutical composition. In other embodiments, provided herein is a pharmaceutical composition comprising a p53 reactivator provided herein, an inhibitor of PD-1 (e.g., pembrolizumab) provided herein, and one or more pharmaceutically acceptable excipient. [00238] The p53 reactivator and/or the inhibitor of PD-1 (e.g., pembrolizumab) provided herein can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection (subcutaneous, intramuscular, intravenous, intraperitoneal, intraosseous, intracardiac, intraarticular, and intracavernous), sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal. The compounds described above may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, (7th ed.1999)). [00239] In the compositions, effective concentrations of one or more compounds (i.e., p53 reactivators or inhibitors of PD-1 (e.g., pembrolizumab) provided herein) or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient. In certain embodiments, the concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein (e.g., cancer, including solid cancer and blood borne cancer). [00240] The active compound is in an amount sufficient to exert a therapeutically useful effect on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems and then extrapolated therefrom for dosages for humans. The concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. [00241] It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. [00242] The p53 reactivators (e.g, APR-246) or inhibitors of PD-1 (e.g., pembrolizumab) provided herein, or pharmaceutically acceptable salts thereof, may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art. 4.3.2 Compositions Relating to Combination Therapies with Bruton’s Tyrosine Kinase Inhibitors [00243] The agonist of p53 such as the p53 reactivator provided herein can be formulated in a pharmaceutical composition that comprises a p53 reactivator provided herein and a pharmaceutically acceptable excipient. Similarly, an inhibitor of Bruton’s tyrosine kinase (BTK) provided herein can be formulated in a pharmaceutical composition that comprises an inhibitor of Bruton’s tyrosine kinase (BTK) provided herein and a pharmaceutically acceptable excipient. In certain embodiments, provided herein is a combination therapy comprising administering a first pharmaceutical composition comprising a p53 reactivator and a first pharmaceutically acceptable excipient, and administering a second pharmaceutical composition comprising a BTK inhibitor and a second pharmaceutically acceptable excipient. In other embodiments, a p53 reactivator provided herein and a BTK inhibitor provided herein are formulated together in a pharmaceutical composition. In other embodiments, provided herein are pharmaceutical compositions comprising a p53 reactivator provided herein, a BTK inhibitor provided herein, and a pharmaceutically acceptable excipient. [00244] The p53 reactivator and/or the BTK inhibitor provided herein can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection, sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal. The compounds described above may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, (7th ed.1999)). [00245] In the compositions, effective concentrations of one or more compounds (i.e., p53 reactivator or BTK inhibitor provided herein) or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient. In certain embodiments, the concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein (e.g., non-Hodgkin lymphoma). [00246] The active compound is in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems described herein and then extrapolated therefrom for dosages for humans. The concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. [00247] It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. [00248] The p53 reactivators (e.g., APR-246) or the BTK inhibitor (e.g., ibrutinib) provided herein, or pharmaceutically acceptable salts thereof, may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art. 4.3.3 Compositions Relating to Combination Therapies with XPO1 Inhibitors [00249] The agonist of p53 such as the p53 reactivator provided herein can be formulated in a pharmaceutical composition that comprises a p53 reactivator provided herein and a pharmaceutically acceptable excipient. Similarly, an exportin 1 (XPO1) inhibitor provided herein (e.g., selinexor) can be formulated in a pharmaceutical composition that comprises the XPO1 inhibitor and a pharmaceutically acceptable excipient. In certain embodiments, provided herein is a combination therapy comprising administering a first pharmaceutical composition comprising a p53 reactivator and a first pharmaceutically acceptable excipient, and administering a second pharmaceutical composition comprising an XPO1 inhibitor and a second pharmaceutically acceptable excipient. In other embodiments, a p53 reactivator provided herein (e.g., APR-246) and an XPO1 inhibitor provided herein (e.g., selinexor) are formulated together in a pharmaceutical composition. In other embodiments, provided herein are pharmaceutical compositions comprising a p53 reactivator provided herein (e.g., APR-246), an XPO1 inhibitor provided herein (e.g., selinexor), and a pharmaceutically acceptable excipient. [00250] The p53 reactivator and/or the XPO1 inhibitor provided herein can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection, infusion, sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal. The compounds described above may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, (7th ed.1999)). [00251] In the compositions, effective concentrations of one or more compounds (i.e., p53 reactivator or XPO1 inhibitor provided herein) or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient. In certain embodiments, the concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein. [00252] The active compound is in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems described herein and then extrapolated therefrom for dosages for humans. The concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. [00253] It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. [00254] The p53 reactivators (e.g., APR-246) or the XPO1 inhibitor (e.g., selinexor) provided herein, or pharmaceutically acceptable salts thereof, may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art. 4.3.4 Compositions Relating to Combination Therapies with WEE1 Inhibitors [00255] A p53 reactivator (e.g., APR-246) can be formulated with one or more pharmaceutically acceptable excipient. Similarly, an inhibitor of WEE1 (e.g., MK-1775) can be formulated in a pharmaceutical composition with one or more pharmaceutically acceptable excipients. In some embodiments, provided herein is a combination therapy comprising a first pharmaceutical composition comprising a p53 reactivator provided herein and a first pharmaceutically acceptable excipient, and a second pharmaceutical composition comprising an inhibitor of WEE1 (e.g., MK-1775) provided herein and a second pharmaceutically acceptable excipient. The first and the second pharmaceutically acceptable excipients can be the same or different. In some embodiments, a p53 reactivator provided herein and an inhibitor of WEE1 (e.g., MK-1775) provided herein are formulated together in a single pharmaceutical composition. In other embodiments, provided herein is a pharmaceutical composition comprising a p53 reactivator provided herein, an inhibitor of WEE1 (e.g., MK-1775) provided herein, and one or more pharmaceutically acceptable excipient. [00256] The p53 reactivator and/or the inhibitor of WEE1 (e.g., MK-1775) provided herein can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection (subcutaneous, intramuscular, intravenous, intraperitoneal, intraosseous, intracardiac, intraarticular, and intracavernous), sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal. The compounds described above may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, (7th ed.1999)). [00257] In the compositions, effective concentrations of one or more compounds (i.e., p53 reactivators or inhibitors of WEE1 (e.g., MK-1775) provided herein) or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient. In certain embodiments, the concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein (e.g., cancer, including solid cancer and blood borne cancer). [00258] The active compound is in an amount sufficient to exert a therapeutically useful effect on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems and then extrapolated therefrom for dosages for humans. The concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. [00259] It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. [00260] The p53 reactivators (e.g, APR-246) or inhibitors of WEE1 (e.g., MK-1775) provided herein, or pharmaceutically acceptable salts thereof, may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art. 4.3.5 A Liquid Composition Comprising an Agonist of P53 [00261] In some specific embodiments, the agonist of p53 such as a p53 reactivator provided here (e.g., APR-246 or Compound A) is formulated in an aqueous solution, as described in US patent no.9,061,016, which is incorporated herein by reference, and as described in more detail below. [00262] In one embodiment, the formulation comprising the p53 reactivator is a stock solution and preferably is a pharmaceutical formulation in the form of a concentrated stock solution. The formulation preferably is sterile, and this may be achieved by known sterilization methods such as filtration, allowing for long term storage. [00263] In one embodiment, the formulation preferably is sterile, and this may be achieved by known sterilization methods such as filtration, allowing for long term storage essentially without any deterioration of the p53 reactivator, e.g. by a chemical reaction of degradation, and essentially without formation of degradation products. [00264] The formulation provided herein can be used, e.g. for administration to a patient in need thereof by direct injection or preferentially diluted with appropriate injectable solutions for i.v. infusion. [00265] In one embodiment, the formulation provided herein is an aqueous solution of the p53 reactivator provided herein (e.g., APR-246 or Compound A), wherein the p53 reactivator is present at a concentration within a range of about 10 mg/mL to about 250 mg/mL, a range of about 50 mg/mL to about 200 mg/mL, or a range of about 75 mg/mL to about 150 mg/mL of the formulation. [00266] The formulation may be diluted prior to use, e.g., administration to a patient. The dilution factor depends on the concentration of the p53 reactivator in the formulation and the required amount of the compound needed, e.g., to meet the therapeutically effective dose. In some embodiments, in case of parenteral administration, the final diluted product has a pH within the range of about pH 4 to about pH 6. In some embodiments, the final diluted product for parenteral administration has a pH within the range of about pH 4.2 to about pH 5.5. [00267] The liquid formulation may contain sodium chloride at a concentration of between 0% and 3%, a concentration of between 0.5% and 1.5%, or a concentration of between 0.8% and 1% weight by volume of the formulation. [00268] In one embodiment, the p53 reactivator (e.g., APR-246 or Compound A) is present in the liquid formulation in the form of an acid addition salt with one or several different pharmaceutically acceptable acids. The pharmaceutically acceptable acid may be a mineral acid, e.g., selected from the group consisting of hydrochloric acid, hydrogen bromide, hydrogen iodide, sulphuric acid, nitric acid, phosphoric acid and the like. As an alternative, the pharmaceutically acceptable acid may be an organic acid, e.g., a sulfonic or carboxylic acid, particularly an alkyl or aryl sulfonic acid or an alkyl or aryl carboxylic acid, such as selected from the group consisting of methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, acetic acid, tartaric acid, maleic acid, citric acid, benzoic acid, salicylic acid, ascorbic acid and the like. [00269] In some embodiments, to be at the required pH, the composition provided herein contains a pH regulating agent. The term “pH regulating agent,” as used herein, means at least one pharmaceutically acceptable organic or inorganic (mineral) acid, or at least one pharmaceutically acceptable acid buffer or a mixture of any of these. Thus, the pH regulating agent may be any such acid or buffer, or a mixture of acids or buffers, or a mixture of acid(s) and buffer(s). Examples of useful acids and buffers are as indicated herein. [00270] For example, the composition may contain at least one pharmaceutically acceptable acid. The acid may be an inorganic mineral acid, e.g., selected from the group consisting of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid, phosphoric acid or the like, or an organic acid, e.g., selected from the group consisting of acetic acid, succinic acid, tartaric acid, maleic acid, ascorbic acid, citric acid, glutamic acid, benzoic acid, ascorbic acid, methanesulfonic acid, ethanesulfonic acid and the like. It is contemplated that the composition may contain one or several acids, selected from inorganic and organic acids. In one embodiment, the required pH of the formulation is achieved by addition of hydrochloric acid. [00271] The composition provided herein also may comprise at least one pharmaceutically acceptable buffer, particularly selected from the group of citric buffer, acetate buffer, phosphate buffer and the like, separately or as a mixture thereof, as well as in combination with any pharmaceutically acceptable acid, as defined herein, e.g., hydrochloric acid. [00272] The liquid composition provided herein is aqueous, which means that it contains water. However, it is contemplated that the aqueous solution and the aqueous phase used to prepare the composition also may contain other pharmaceutically acceptable liquids as a solvent phase, e.g., polyethylene glycol (PEG) and alcohols, e.g., ethanol. In some embodiments, the aqueous phase mainly comprises water as a solvent. For example, the solvent phase is comprised of from 50 to 100% water, at least 80% water, at least 90% water, at least 95% water, at least 98% water or 100% water. [00273] In one embodiment, the composition described herein is provided as a stable stock solution, particularly as a concentrated stock solution for long term storage at a temperature range of 2-8° C., in a container, for example, a sealed and sterilized container. For example, the composition may comprise a stable aqueous WFI (water for injection) solution of the p53 reactivator optionally as an acid addition salt, in particular a hydrochloride addition salt, in a concentration of at about 10 mg/mL to about 250 mg/mL, at about 50 mg/mL to about 200 mg/mL, or at about 75 mg/mL to about 150 mg/mL, and a pH regulating agent in such an amount as to provide a pH in the solution in a range of between pH 3.0 and pH 5.0, between pH 3.2 and pH 4.7, between pH 3.5 and pH 4.5, or between pH 3.8 and pH 4.2, e.g., approximately 4.0. For example, the pH of the stock solution may have a lower limit selected from a pH of about 3.0, or about 3.2, e.g. about 3.4, such as about 3.6 or about 3.8, and an upper limit of about 5.0, or about 4.7, or about 4.5, or about 4.2, e.g. about 4.0. [00274] Other components also may be added to or present in the aqueous phase, such as pharmaceutically acceptable inorganic salts, e.g., NaCl, preservatives, or further pharmaceutically acceptable compounds, e.g., further therapeutically active ingredients, such as cytostatics, particularly cisplatin, daunorubicin, cerubidine, cytarabine and fludarabine. [00275] In one embodiment, NaCl is added to the aqueous phase in an amount so as to provide a final liquid composition as defined herein above, containing NaCl at a concentration of between 0% and 3%, between 0.5% and 1.5%, or between 0.8% and 1% weight by volume of the formulation. [00276] In one embodiment, the composition is a sterile formulation. In this case, sterilization of the composition may be accomplished by passing the formulation, e.g., a formulated stock solution, through a sterile filter with a nominal pore size of 0.2 μm into a cleaned and sterilized container. [00277] The composition may be provided as a ready-to-use injection solution, wherein a liquid formulation, e.g., a stock solution, is brought to the desired volume by addition of one or more pharmaceutically acceptable solvents, such as selected from the group consisting of WFI, a glucose solution, electrolyte solution containing amino acids, lipids, vitamins, and other minerals, Ringer's solution, Hartmann's solution, or a sodium chloride solution in the form of an isotonic, hypotonic or hypertonic solution. An example of such pharmaceutically acceptable solution is Baxter Viaflo 9 mg/ml. [00278] In a specific embodiment, the p53 reactivator is APR-246, which is formulated in liquid formulation, which comprises at least one pH regulating agent in an amount such as to provide a pH in the aqueous solution of from about 3.0 to about 5.0. In some embodiments, APR-246 is present in the aqueous solution at a concentration of from 10 mg/mL to 250 mg/mL. In some embodiments, the aqueous solution comprises NaCl at a concentration of between 0% to 3% weight by volume. In another specific embodiment, the p53 reactivator is Compound A. 4.3.6 An Oral Dosage Form Comprising an Agonist of P53 [00279] In some specific embodiments, the agonist of p53 such as the p53 reactivator provided here (e.g., APR-246 or Compound A) is formulated in a composition for oral administration. In a specific embodiment, the oral dosage form is a solid form. [00280] Pharmaceutical compositions that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. [00281] Typical oral dosage forms are prepared by combining the active ingredients in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents. [00282] If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary. [00283] For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free- flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. [00284] Examples of excipients that can be used in oral dosage forms provided herein include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof. [00285] Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105, and mixtures thereof. Suitable anhydrous or low moisture excipients or additives include AVICEL- PH-103™ and Starch 1500 LM. [00286] Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. [00287] Disintegrants are used in compositions to provide tablets that disintegrate when exposed to an aqueous environment. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof. [00288] Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms. [00289] Lubricants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil, zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, and mixtures thereof. 4.3.7 A Lyophilized Composition Comprising an Inhibitor of PD-1 Mediated Signaling [00290] In some embodiments, the inhibitor of PD-1 mediated signaling such as an inhibitor of PD-1 (e.g., pembrolizumab) provided herein is supplied in a sterile form for reconstitution as a suspension for subcutaneous injection or reconstitution as a solution with further dilution for intravenous infusion. [00291] In some embodiments, the inhibitor of PD-1 (e.g., pembrolizumab) is formulated for parenteral administration. Parenteral administration of the compositions includes intravenous, subcutaneous, and intramuscular administrations. Compositions for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, sterile suspensions ready for injection, and sterile emulsions. The solutions may be either aqueous or nonaqueous. The unit dose parenteral preparations can be packaged in an ampoule, a vial or a syringe with a needle. [00292] As described above, pharmaceutically acceptable excipients used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances. [00293] In a specific embodiment, the inhibitor of PD-1 (e.g., pembrolizumab) is formulated as lyophilized powder for reconstitution for parenteral administration. Lyophilized powders can be reconstituted for administration as solutions, emulsions, and other mixtures. [00294] The sterile, lyophilized powder can be prepared by dissolving an inhibitor of PD-1 (e.g., pembrolizumab) provided herein, or a pharmaceutically acceptable salt thereof, in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose. The solvent may also contain a buffer, such as citrate, phosphate, or other buffers known to those of skill in the art. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. Generally, the resulting solution will be apportioned into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the inhibitor of PD-1 (e.g., pembrolizumab). The lyophilized powder can be stored under appropriate conditions, such as at about 4 oC to room temperature. [00295] In one aspect, the lyophilized formulations are suitable for reconstitution with a suitable diluent to the appropriate concentration prior to administration. In some embodiments, the lyophilized formulation is suitable for reconstitution with an aqueous solution for intravenous administrations. In certain embodiments, the lyophilized formulation provided herein is suitable for reconstitution with water. [00296] In certain embodiment, the lyophilized formulations comprise the inhibitor of PD-1 (e.g., pembrolizumab) provided herein, a buffer and a bulking agent. [00297] In certain embodiments, a lyophilized formulation comprises an inhibitor of PD-1 (e.g., pembrolizumab) provided herein, in about 0.1 to about 60% based on the total weight of the lyophilized formulation. [00298] The lyophilized formulation can be reconstituted for parenteral administration to a patient using any pharmaceutically acceptable diluent. Such diluents include, but are not limited to Sterile Water for Injection (SWFI), Dextrose 5% in Water (D5W), or a cosolvent system. 4.4 Combination Therapies [00299] Provided herein are combination therapies using a p53 agonist such as a p53 reactivator (see Section 4.2.1) in combination with a second therapeutic agent, for example, an inhibitor of PD-1 mediated signaling (see Section 4.2.3), a Bruton’s tyrosine kinase (BTK) inhibitor (see Section 4.2.3), an exportin 1 (XPO1) inhibitor (see Section 4.2.4), or an inhibitor of Wee1-like protein kinase (WEE1) (see Section 4.2.5), for treating a disease or disorder. In some embodiments, such combination therapies relate to methods comprising administering an effective amount of the p53 agonist such as a p53 reactivator and the second therapeutic agent to a subject. In one embodiment, the subject is a human. [00300] Provided herein are combination therapies using a compound of Formula (I) (see Section 4.2.1) in combination with a second therapeutic agent, for example, an inhibitor of PD-1 mediated signaling (see Section 4.2.3), a Bruton’s tyrosine kinase (BTK) inhibitor (see Section 4.2.3), an exportin 1 (XPO1) inhibitor (see Section 4.2.4), or an inhibitor of Wee1-like protein kinase (WEE1) (see Section 4.2.5), for treating a disease or disorder, wherein the compound of Formula (I) shows synergism with the second therapeutic agent provided herein for treating the disease or disorder. In some embodiments, such combination therapies relate to methods comprising administering an effective amount of the compound of Formula (I) that shows synergism and the second therapeutic agent to a subject. In one embodiment, the subject is a human. 4.4.1 Combination Therapies with Inhibitors of PD-1 Mediated Signaling [00301] In one aspect, provided herein is a composition or a combination of compositions for use in the prevention and/or treatment of a disease or condition comprising the agonist of p53 such as a p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 mediated signaling such as an inhibitor of PD-1 (e.g., pembrolizumab). Such uses relate to a method comprising administering an effective amount of the p53 reactivator and the inhibitor of PD-1 (e.g., pembrolizumab) to a subject. In one embodiment, the subject is a human. [00302] Provided herein are methods of preventing and/or treating a disease or disorder (e.g., a solid tumor malignancy) in a subject comprising administering to a subject an agonist of p53 and an inhibitor of PD-1 mediated signaling. In certain embodiments, provided herein are methods of preventing a disease or disorder in a subject, comprising administering to a subject an agonist of p53 and an inhibitor of PD-1 mediated signaling. In certain embodiments, provided herein are methods of treating a disease or disorder (e.g. a solid tumor malignancy), comprising administering to a subject an agonist of p53 and an inhibitor of PD-1 mediated signaling. In certain embodiments, a pharmaceutically effective amount of the agonist of p53 is administered. In certain embodiments, the agonist of p53 and the inhibitor of PD-1 mediated signaling are concomitantly administered. In certain embodiments, the co-administration of the agonist of p53 and the inhibitor of PD-1 mediated signaling is pharmaceutically effective to treat the disease or disorder (e.g., a solid tumor malignancy). [00303] Provided herein are methods of treating a disease or disorder (e.g., a solid tumor malignancy) in a subject comprising administering to a subject a compound of Formula (I) and an inhibitor of PD-1 mediated signaling, wherein the compound of Formula (I) shows synergism with the inhibitor of PD-1 mediated signaling for treating the disease or disorder. In certain embodiments, a pharmaceutically effective amount of the compound of Formula (I) is administered. In certain embodiments, the co-administration of the compound of Formula (I) and the inhibitor of PD-1 mediated signaling is pharmaceutically effective to treat the disease or disorder (e.g., a solid tumor malignancy). In one specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is APR-246. In another specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is Compound A. In another specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is Compound B. In another specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is Compound C. In another specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is Compound D. In another specific embodiment, the compound that has synergism with the inhibitor of PD-1 mediated signaling is Compound E. [00304] In some embodiments, the disease or disorder is a neoplastic disease. In certain embodiments, the disease or disorder is a cancer. In certain embodiments, the disease or disorder is a solid tumor malignancy. In certain embodiments, the neoplastic, cancerous, or malignant cell in a subject to be treated with a method provided herein comprises a mutation in TP53. In a specific embodiment, provided herein is a method to treat or prevent a solid tumor cancer with a mutation in the p53 gene. In certain embodiments, the cancer to be treated with a method provided herein comprises cancer cells that carry a mutation in TP53. In some embodiments, the mutation in TP53 is R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, V173A, S241F, R249S or a combination thereof. In some embodiments, the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation. In other embodiments, the mutant TP53 includes a nonsense mutation. A nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP53 mutations in cancer. The most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*. [00305] In other embodiments, the cancer to be treated does not comprise any cells carrying a mutation in the TP53 gene. [00306] In some embodiments, the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis. Examples of such diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct) cancer, primary or secondary central nervous system tumor, primary or secondary brain tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer, cancer of the kidney and/or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system, primary central nervous system lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma or a combination thereof. [00307] In some embodiments, the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small- cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer. [00308] In other embodiments, the disease or disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer. [00309] In some embodiments, the solid tumor malignancy is an advanced non-CNS-primary solid tumor. In some embodiments, the solid tumor malignancy is selected from the group consisting of gastric/gastroesophageal junction (GEJ) cancer, bladder/urothelial cancer, and non- small-cell lung cancer (NSCLC). [00310] The amount of a prophylactic or therapeutic agent (the p53 reactivator and the inhibitor of PD-1 (e.g., pembrolizumab) provided herein), or a composition provided herein that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some embodiments, should be decided according to the judgment of the practitioner and each patient’s circumstances. [00311] The dose administered to a subject in the context of the present disclosure should be sufficient to effect a therapeutic response. One skilled in the art will recognize that dosage will depend upon a variety of factors including the potency of the specific compound, the age, condition and body weight of the patient, as well as the stage/severity of the disease. The dose will also be determined by the route (administration form) timing and frequency of administration. [00312] The p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 (e.g., pembrolizumab) can be formulated in different pharmaceutical compositions and administered separately to the subject in need thereof. Alternatively, the p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 (e.g., pembrolizumab) are administered together in the same pharmaceutical composition. [00313] In some embodiments, the p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 (e.g., pembrolizumab) are administered simultaneously. The term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours. [00314] In some embodiments, the p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 (e.g., pembrolizumab) are not administered simultaneously, and instead the two compounds are administered at different times. In some embodiments, the p53 reactivator (e.g., APR-246) and the inhibitor of PD-1 (e.g., pembrolizumab) are administered at least once during a dosing period. A dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once. A dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In certain embodiments, a dosing period is a dosing cycle. [00315] The prophylactic or therapeutic agent (the p53 reactivator and/or the inhibitor of PD-1 provided herein) can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time). The agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity. Stable disease or lack is determined by methods known in the art such as evaluation of patient symptoms, physical examination, and visualization of the tumor that has been imaged using X- ray, CAT, PET, MRI scan, or other commonly accepted evaluation modalities. [00316] The prophylactic or therapeutic agent (the p53 reactivator and/or the inhibitor of PD-1 provided herein) can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID). In addition, the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug). As used herein, the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time. The term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days. The term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days. [00317] In some embodiments, the frequency of administration is in the range of about a daily dose to about a monthly dose. In certain embodiments, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks. [00318] In certain embodiments, the compound is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks. [00319] In some embodiments, APR-246 is administered at a dose of less than 150 mg/kg. In some embodiments, APR-246 is administered at a dose of less than 100 mg/kg. In some embodiments, APR-246 is administered at a dose of between 100 mg/kg and 25 mg/kg. In other embodiments, APR-246 is administered at dose of less than 75 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 65 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 50 mg/kg. [00320] In other embodiments, APR-246 is administered at a fixed dose within the interval 2.7-7.5 g. In some embodiments, the fixed dose of APR-246 is no more than 4.5 g. In some embodiments, the fixed dose of APR-246 is no more than 4.0 g. In some embodiments, the fixed dose of APR-246 is no more than 3.5 g. [00321] In some embodiments, APR-246 is administered at about 4.5 g/day for 4 days in combination with pembrolizumab at the dose of about 200 mg once of each 21-day cycle. In some specific embodiments, APR-246 is administered at about 4.5 g/day on Days 1–4 in combination with pembrolizumab at the dose of about 200 mg on Day 3 of each 21-day cycle. In some embodiments, APR-246 and pembrolizumab are administered for 1 to 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 1 cycle. In some embodiments, APR-246 and pembrolizumab are administered for 2 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 3 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 4 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 5 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 6 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 7 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 8 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 9 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for more than 10 cycles. [00322] In some embodiments, APR-246 is administered at about 4.0 g/day for 4 days in combination with pembrolizumab at the dose of about 200 mg once of each 21-day cycle. In some specific embodiments, APR-246 is administered at about 4.0 g/day on Days 1–4 in combination with pembrolizumab at the dose of about 200 mg on Day 3 of each 21-day cycle. In some embodiments, APR-246 and pembrolizumab are administered for 1 to 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 1 cycle. In some embodiments, APR-246 and pembrolizumab are administered for 2 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 3 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 4 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 5 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 6 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 7 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 8 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 9 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for more than 10 cycles. [00323] In some embodiments, APR-246 is administered at about 3.5 g/day for 4 days in combination with pembrolizumab at the dose of about 200 mg once of each 21-day cycle. In some specific embodiments, APR-246 is administered at about 3.5 g/day on Days 1–4 in combination with pembrolizumab at the dose of about 200 mg on Day 3 of each 21-day cycle. In some embodiments, APR-246 and pembrolizumab are administered for 1 to 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 1 cycle. In some embodiments, APR-246 and pembrolizumab are administered for 2 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 3 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 4 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 5 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 6 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 7 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 8 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 9 cycles. In some embodiments, APR-246 and pembrolizumab are administered for 10 cycles. In some embodiments, APR-246 and pembrolizumab are administered for more than 10 cycles. Exemplary Embodiments [00324] Embodiment A1. A method of treating solid tumor malignancy in a subject, comprising administering to the subject: (i) an agonist of p53; and (ii) an inhibitor of PD-1 mediated signaling. [00325] Embodiment A2. The method of Embodiment A1, wherein the agonist of p53 is a compound that can give reactivation of a mutant p53 or a compound whose metabolite can give reactivation of a mutant p53. [00326] Embodiment A3. The method of Embodiment A2, wherein the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W. [00327] Embodiment A4. The method of Embodiment A2, wherein the agonist of p53 is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity. [00328] Embodiment A5. The method of Embodiment A2, wherein the compound or metabolite promotes proper folding of mutant and wild-type p53 proteins. [00329] Embodiment A6. The method of Embodiment A2, wherein the compound or metabolite is capable of shifting the equilibrium from unfolded towards a folded structure of wild-type or mutant p53, or wherein the compound or metabolite is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or metabolite is capable of reducing aggregation of the wild-type or mutant p53. [00330] Embodiment A7. The method of Embodiment A2, wherein the compound or metabolite is capable of promoting a folded structure of wild-type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53. [00331] Embodiment A8. The method of Embodiment A7, wherein the compound or metabolite is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promote a folded conformation. [00332] Embodiment A9. The method of Embodiment A2, wherein the compound that can give reactivation of mutant p53 is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one; 2,2-bis(hydroxymethyl)quinuclidin-3-one; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; 2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide; N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide; N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide; 1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide; 1-methyl-N-((3-oxoquinuclidin-2-yl)methyl)cyclopropane-1-sulfonamide; N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide; 1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; 5-methyl-1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; tert-butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine- 1(2H)-carboxylate; 5-methyl-1,3-bis((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; N-methyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((3-chloro-1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; N,N-dimethyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carbonitrile; and 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide, or a pharmaceutically acceptable salt thereof. [00333] Embodiment A10. The method of Embodiment A9, wherein the compound is 2- (hydroxymethyl)-2- (methoxymethyl) quinuclidin-3-one having the following formula:
Figure imgf000069_0001
(APR-246), or a pharmaceutically acceptable salt thereof. [00334] Embodiment A11. The method of Embodiment A9, wherein the compound is 2,2,2- trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide having the following formula:
Figure imgf000069_0002
(Compound A), or a pharmaceutically acceptable salt thereof. [00335] Embodiment A12. The method of any one of Embodiments A1 to A11, wherein the inhibitor of PD-1 mediated signaling is an anti-PD-1 antibody or an anti-PD-L1 antibody. [00336] Embodiment A13. The method of Embodiment A12, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab; nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, nivolumab, AMP-224, and AMP-514. [00337] Embodiment A14. The method of Embodiment A13, wherein the anti-PD-1 antibody is pembrolizumab. [00338] Embodiment A15. The method of Embodiment A1, wherein the agonist of p53 is APR-246 and the inhibitor of PD-1 mediated signaling is pembrolizumab. [00339] Embodiment A16. The method of Embodiment A15, wherein APR-246 is administered at a dose of about 4.5 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle. [00340] Embodiment A17. The method of Embodiment A15, wherein APR-246 is administered at a dose of about 4.0 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle. [00341] Embodiment A18. The method of Embodiment A15, wherein APR-246 is administered at a dose of about 3.5 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle. [00342] Embodiment A19. The method of any one of Embodiments A15 to A18, wherein APR-246 is administered on Days 1–4 and pembrolizumab is administered on Day 3 of the each 21-day cycle. [00343] Embodiment A20. The method of any one of Embodiments A16 to A19, wherein APR-246 and pembrolizumab are administered for 1 to 20 cycles. [00344] Embodiment A21. The method of any one of Embodiments A1 to A20, wherein the agonist of p53 is formulated in a first pharmaceutical composition and the inhibitor of PD-1 mediated signaling is formulated in a second pharmaceutical composition. [00345] Embodiment A22. The method of any one of Embodiments A1 to A20, wherein the solid tumor malignancy is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer. [00346] Embodiment A23. The method of any one of Embodiments A1 to A20, wherein the solid tumor malignancy is an advanced non-CNS-primary solid tumor. [00347] Embodiment A24. The method of any one of Embodiments A1 to A20, wherein the solid tumor malignancy is selected from the group consisting of gastric/gastroesophageal junction (GEJ) cancer, bladder/urothelial cancer, and non-small-cell lung cancer (NSCLC). 4.4.2 Combination Therapies with Bruton’s Tyrosine Kinase Inhibitors [00348] In another aspect, also provided herein are combination therapies using a p53 reactivator (such as the p53 reactivator described in Section 4.2.1) in combination with a BTK inhibitor (such as the BTK inhibitor described in Section 4.2.3) for treating a disease or disorder (such as lymphoma, e.g., a non-Hodgkin lymphoma). [00349] Provided herein are methods of treating a disease or disorder (e.g., lymphoma) in a subject comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and a Bruton’s tyrosine kinase (BTK) inhibitor. In certain embodiments, a pharmaceutically effective amount of the p53 reactivator is administered. In certain embodiments, the p53 reactivator and the BTK inhibitor are concomitantly administered. In certain embodiments, the co-administration of the compound that can give reactivation of a mutant p53 (the p53 reactivator) and the BTK inhibitor is pharmaceutically effective to treat the disease or disorder (e.g., non-Hodgkin lymphoma). [00350] Provided herein are methods of treating a disease or disorder (e.g., lymphoma) in a subject comprising administering to a subject a compound of Formula (I) and a Bruton’s tyrosine kinase (BTK) inhibitor, wherein the compound of Formula (I) shows synergism with the Bruton’s tyrosine kinase (BTK) inhibitor for treating the disease or disorder. In certain embodiments, a pharmaceutically effective amount of the compound of Formula (I) is administered. In certain embodiments, the co-administration of the compound of Formula (I) and the BTK inhibitor is pharmaceutically effective to treat the disease or disorder (e.g., non- Hodgkin lymphoma). In one specific embodiment, the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is APR-246. In another specific embodiment, the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is Compound A. In another specific embodiment, the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is Compound B. In another specific embodiment, the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is Compound C. In another specific embodiment, the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is Compound D. In another specific embodiment, the compound that has synergism with the Bruton’s tyrosine kinase (BTK) inhibitor is Compound E. [00351] In some embodiments, the subject is a human. In some embodiments, the subject is a subject diagnosed with lymphoma. In some embodiments, the subject is a subject diagnosed with non-Hodgkin lymphoma (NHL). In some embodiments, the subject is a subject diagnosed with mature (peripheral) B-cell neoplasm. In some preferred embodiments, the subject is a subject diagnosed with chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). In one embodiment, the subject is a subject diagnosed with chronic lymphocytic leukemia (CLL). In another embodiment, the subject is a subject diagnosed with cell lymphoma (MCL). In yet another embodiment, the subject is a subject diagnosed with relapsed and/or refractory (R/R) CLL or relapsed and/or refractory (R/R) MCL. In yet another embodiment, the subject is a subject diagnosed with R/R CLL. In yet another embodiment, the subject is subject diagnosed with R/R MCL. [00352] In some embodiments, the subject is diagnosed to have TP53 mutation. In some embodiments, the subject carries TP53 mutation. In some embodiments, the subject has mutant p53 protein. In some embodiments, the subject is a subject diagnosed with a TP53 mutant lymphoma. In some embodiments, the subject is a subject diagnosed with a TP53 mutant non- Hodgkin lymphoma (NHL). In some embodiments, the subject is a subject diagnosed with a TP53 mutant mature (peripheral) B-cell neoplasm. In some preferred embodiments, the subject is a subject diagnosed with a TP53 mutant chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). In one embodiment, the subject is a subject diagnosed with a TP53 mutant chronic lymphocytic leukemia (CLL). In another embodiment, the subject is a subject diagnosed with a TP53 mutant mantle cell lymphoma (MCL). In yet another embodiment, the subject is a subject diagnosed with a TP53 mutant R/R CLL or a TP53 mutant R/R MCL. In yet another embodiment, the subject is a subject diagnosed with a TP53 mutant R/R CLL. In yet another embodiment, the subject is a subject diagnosed with a TP53 mutant R/R MCL. [00353] In some embodiments, the methods described herein further comprises determining by gene sequencing if the subject has TP53 mutation. [00354] In some embodiments, the disease or disorder is a mutant p53 mediated cancer (including, e.g., hematological tumors with mutations in the p53 gene). In other embodiments, the disease or disorder is not a mutant p53 mediated cancer. [00355] In some embodiments, the disease or disorder is a disease characterized by apoptotic dysfunction and/or overexpression of a Bruton’s tyrosine kinase. [00356] In some embodiments, the disease or disorder is mutant p53 mediated and characterized by apoptotic dysfunction and/or overexpression of a Bruton’s tyrosine kinase. [00357] In some embodiments, the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma. In some embodiments, the disease or disorder is a lymphoma. In some embodiments, the disease or disorder is Hodgkin's lymphoma or non- Hodgkin's lymphoma (NHL). In some embodiments, the disease or disorder is a Hodgkin lymphoma. In some embodiments, the disease or disorder is a non-Hodgkin lymphoma (NHL). In some embodiments, the disease or disorder is a mature (peripheral) B-cell neoplasm. In some embodiments, the disease or disorder a non-Hodgkin lymphoma (NHL) selected from the group consisting of: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, diffuse large cell B-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma (plasmacytoma), extranodal marginal zone B-cell lymphoma, nodal marginal zone lymphoma, follicle center lymphoma, and Burkitt’s leukemia (Burkitt’s lymphoma). In some preferred embodiments, the disease or disorder is chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). In some preferred embodiments, the disease or disorder is chronic lymphocytic leukemia (CLL). In some preferred embodiments, the disease or disorder is mantle cell lymphoma (MCL). [00358] In some embodiments, the disease or disorder is a TP53 mutant lymphoma. In some embodiments, the disease or disorder is a TP53 mutant non-Hodgkin lymphoma (NHL). In some embodiments, the disease or disorder is a TP53 mutant mature (peripheral) B-cell neoplasm. In some preferred embodiments, the disease or disorder is a TP53 mutant chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). In one embodiment, the disease or disorder is a TP53 mutant chronic lymphocytic leukemia (CLL). In another embodiment, the disease or disorder is a TP53 mutant mantle cell lymphoma (MCL). In yet another embodiment, the disease or disorder is a TP53 mutant R/R CLL or a TP53 mutant R/R MCL. In yet another embodiment, the disease or disorder is a TP53 mutant R/R CLL. In yet another embodiment, the disease or disorder is a TP53 mutant R/R MCL. [00359] In some embodiments, the mutation in TP53 is R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, V173A, S241F, R249S or a combination thereof. In some embodiments, the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation. In other embodiments, the mutant TP53 includes a nonsense mutation. A nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP53 mutations in cancer. The most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*. [00360] In some embodiments, the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis. Examples of such diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct) cancer, primary or secondary central nervous system tumor, primary or secondary brain tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer, cancer of the kidney and/or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system, primary central nervous system lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma, neuroblastoma, and retinoblastoma. [00361] In some embodiments, the disease or disorder is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. [00362] In some embodiments, the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer. [00363] In other embodiments, the disease or disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer. [00364] The amount of the p53 reactivator or the BTK inhibitor provided herein, or a pharmaceutical composition that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some embodiments, should be decided according to the judgment of the practitioner and each patient’s circumstances. Effective doses may also be extrapolated from dose-response curves derived from in vitro or animal model test systems. [00365] In some embodiments, the p53 reactivator is administered to the subject as part a composition. In some embodiments, the composition is a pharmaceutical composition described in Section 4.3.2. [00366] In some embodiments, the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) can be formulated in different pharmaceutical compositions and administered separately to the subject in need thereof. In other embodiments, the p53 reactivator (e.g., APR- 246) and the BTK inhibitor (e.g., ibrutinib) are administered together in the same pharmaceutical composition. [00367] In some embodiments, the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) are administered simultaneously. The term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours. [00368] In some embodiments, the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) are not administered simultaneously, and instead the two compounds are administered at different times. [00369] In certain embodiments, the BTK inhibitor (e.g., ibrutinib) is administered before or concurrently with the administration of the p53 reactivator (e.g., APR-246). [00370] In certain embodiments, the BTK inhibitor (e.g., ibrutinib) is administered after the administration of the p53 reactivator (e.g., APR-246). [00371] In certain embodiments, the subject has been previously treated with the BTK inhibitor (e.g., ibrutinib) prior to the administration of the p53 reactivator (e.g., APR-246). [00372] In certain embodiments, the subject has been previously treated with the p53 reactivator (e.g., APR-246) prior to the administration of the BTK inhibitor (e.g., ibrutinib). [00373] In certain embodiments, the subject has not been previously treated with any BTK inhibitor (e.g., ibrutinib, acalabrutinib, zanubrutinib, etc.) prior to the co-administration of the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib). [00374] In certain embodiments, the co-administration of the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) is concomitant administration. [00375] In certain embodiments, the co-administration of the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) is pharmaceutically effective to treat lymphoma. [00376] In some embodiments, the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) are administered at least once during a dosing period. A dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once. A dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In certain embodiments, a dosing period is a dosing cycle. [00377] The prophylactic or therapeutic agent (the p53 reactivator and/or the BTK inhibitor provided herein) can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time). The agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity. Stable disease or lack is determined by methods known in the art such as evaluation of patient symptoms, physical examination, and visualization of the tumor that has been imaged using X- ray, CAT, PET, MRI scan, or other commonly accepted evaluation modalities. [00378] The prophylactic or therapeutic agent (the p53 reactivator and/or the BTK inhibitor provided herein) can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID). In addition, the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug). As used herein, the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time. The term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days. The term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days. [00379] In some embodiments, the frequency of administration is in the range of about a daily dose to about a monthly dose. In certain embodiments, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks. [00380] In certain embodiments, the compound is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks. [00381] In some embodiments, APR-246 is administered at a dose of less than about 150 mg/kg. In some embodiments, APR-246 is administered at a dose of less than about 100 mg/kg. In some embodiments, APR-246 is administered at a dose of between about 100 mg/kg and about 25 mg/kg. In other embodiments, APR-246 is administered at dose of less than about 75 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than about 65 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than about 50 mg/kg. [00382] In other embodiments, APR-246 is administered at a fixed dose within the interval of 2.7-7.5 g. In some embodiments, the fixed dose of APR-246 is no more than about 4.5 g. In some embodiments, the fixed dose of APR-246 is no more than about 4.0 g. In some embodiments, the fixed dose of APR-246 is no more than about 3.5 g. In one embodiments, APR-246 is administered at a dose of about 4.5 g/day, about 4.0 g/day, or about 3.5 g/day. In one specific embodiment, APR-246 is administered at a dose of about 4.5 g/day. In another specific embodiment, APR-246 is administered at a dose of about 4.0 g/day. In yet another specific embodiment, APR-246 is administered at a dose of about 3.5 g/day. [00383] In some embodiments, APR-246 is administered in a multiple-step administration to avoid high plasma concentration and/or to minimize the risk of adverse events. In some embodiments, APR-246 is administered in a 2-step administration consisting of a first loading dose and a subsequent maintenance dose. In some preferred embodiments, the first loading dose is not equal to the subsequent maintenance dose. In other embodiments, the first loading dose is not equal to the subsequent maintenance dose. In some embodiments, the first loading dose is about 1.5 g, about 1.33 g, or about 1.16 g. In some embodiments, the subsequent maintenance dose is about 3.0 g, about 2.67 g, or about 2.34 g. [00384] In some embodiment, APR-246 is administered 1, 2, 3, 4, 5, 6, 7, or 8 times of each cycle of 14 days, 21 days, every 28 days, every 35 days, or every 42 days. In preferred embodiments, APR-246 is administered for 4 days in each treatment cycle. In preferred embodiments, APR-246 is administered for 4 days in each treatment cycle of 28 days. In some embodiments, APR-246 is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles. [00385] In a specific embodiment, APR-246 is administered once daily for 4 consecutive days in each treatment cycle of 28 days. [00386] In some preferred embodiments, APR-246 is administered intravenously. [00387] In some embodiments, the BTK inhibitor described herein (e.g., ibrutinib) is administered at a dose between 1 mg/day to 1000 mg/day. In some embodiments, the BTK inhibitor is administered at a dose of about 100 mg/day, about 200 mg/day, about 300 mg/day, about 400 mg/day, about 500 mg/day, about 600 mg/day, about 700 mg/day, about 800 mg/day, about 900 mg/day, or about 1000 mg/day. In some embodiments, the BTK inhibitor is administered at a dose of about 420 mg/day or about 560 mg/day. In one embodiment, the BTK inhibitor is administered at a dose of about 420 mg/day. In another embodiment, the BTK inhibitor is administered at a dose of about 560 mg/day. [00388] In some preferred embodiments, ibrutinib is administered at a dose of about 420 mg or about 560 mg. In one preferred embodiment, the BTK inhibitor (e.g., ibrutinib) is administered daily. In one embodiment, ibrutinib is administered daily for each treatment cycle of 28 days. [00389] In some embodiments, the BTK inhibitor (e.g., ibrutinib) is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles. [00390] In some preferred embodiments, the BTK inhibitor (e.g., ibrutinib) is administered orally. [00391] In certain embodiments, combination treatment of the p53 reactivator (e.g., APR-246) and the BTK inhibitor (e.g., ibrutinib) show synergistic effects in treating lymphoma. [00392] In some embodiments, APR-246 is administered at a dose of about 4.5 g/day for 4 days in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. In some specific embodiments, APR-246 is administered at a dose about 4.5 g/day on Days 1–4 in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. In some embodiments, APR-246 and ibrutinib are administered for 1 to 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for 1 cycle. In some embodiments, APR-246 and ibrutinib are administered for 2 cycles. In some embodiments, APR-246 and ibrutinib are administered for 3 cycles. In some embodiments, APR-246 and ibrutinib are administered for 4 cycles. In some embodiments, APR-246 and ibrutinib are administered for 5 cycles. In some embodiments, APR-246 and ibrutinib are administered for 6 cycles. In some embodiments, APR-246 and ibrutinib are administered for 7 cycles. In some embodiments, APR-246 and ibrutinib are administered for 8 cycles. In some embodiments, APR-246 and ibrutinib are administered for 9 cycles. In some embodiments, APR-246 and ibrutinib are administered for 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for more than 10 cycles. [00393] In some embodiments, APR-246 is administered at a dose of about 4.0 g/day for 4 days in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. In some specific embodiments, APR-246 is administered at a dose about 4.5 g/day on Days 1–4 in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. In some embodiments, APR-246 and ibrutinib are administered for 1 to 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for 1 cycle. In some embodiments, APR-246 and ibrutinib are administered for 2 cycles. In some embodiments, APR-246 and ibrutinib are administered for 3 cycles. In some embodiments, APR-246 and ibrutinib are administered for 4 cycles. In some embodiments, APR-246 and ibrutinib are administered for 5 cycles. In some embodiments, APR-246 and ibrutinib are administered for 6 cycles. In some embodiments, APR-246 and ibrutinib are administered for 7 cycles. In some embodiments, APR-246 and ibrutinib are administered for 8 cycles. In some embodiments, APR-246 and ibrutinib are administered for 9 cycles. In some embodiments, APR-246 and ibrutinib are administered for 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for more than 10 cycles. [00394] In some embodiments, APR-246 is administered at a dose of about 3.5 g/day for 4 days in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. In some specific embodiments, APR-246 is administered at a dose about 4.5 g/day on Days 1–4 in combination with ibrutinib administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. In some embodiments, APR-246 and ibrutinib are administered for 1 to 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for 1 cycle. In some embodiments, APR-246 and ibrutinib are administered for 2 cycles. In some embodiments, APR-246 and ibrutinib are administered for 3 cycles. In some embodiments, APR-246 and ibrutinib are administered for 4 cycles. In some embodiments, APR-246 and ibrutinib are administered for 5 cycles. In some embodiments, APR-246 and ibrutinib are administered for 6 cycles. In some embodiments, APR-246 and ibrutinib are administered for 7 cycles. In some embodiments, APR-246 and ibrutinib are administered for 8 cycles. In some embodiments, APR-246 and ibrutinib are administered for 9 cycles. In some embodiments, APR-246 and ibrutinib are administered for 10 cycles. In some embodiments, APR-246 and ibrutinib are administered for more than 10 cycles. [00395] In certain embodiments, the methods described herein further comprises determining by gene sequencing if the subject has TP53 mutation. In certain embodiments, a next generation sequencing (NGS) method is used for the gene sequencing. In certain embodiments, the gene sequencing data are interpreted to determine if the subject has or likely has TP53 mutation. [00396] In another aspect, also provided herein are combination therapies of the p53 reactivator and the Bruton’s tyrosine kinase (BTK) inhibitor provided herein for use in treating a disease or disorder described herein (e.g., lymphoma). In some embodiments, also provided herein are combination therapies of the p53 reactivator and the Bruton’s tyrosine kinase (BTK) inhibitor provided herein for use in treating chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). [00397] In another aspect, also provided herein are pharmaceutical compositions or a combination of pharmaceutical compositions of the p53 reactivator and the Bruton’s tyrosine kinase (BTK) inhibitor provided herein for use in treating a disease or disorder (e.g., lymphoma). In some embodiments, also provided herein are pharmaceutical compositions or a combination of pharmaceutical compositions of the p53 reactivator and the Bruton’s tyrosine kinase (BTK) inhibitor provided herein for use in treating chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). [00398] In another aspect, also provided herein are method of treating lymphoma in a subject, comprising administering to the subject a therapeutically effective amount of a compound that can give reactivation of a mutant p53, wherein the lymphoma does not comprise a cancer cell having mutant p53 or the lymphoma comprises a cancer cell having wild-type p53. Exemplary Embodiments [00399] Embodiment B1. A method of treating a lymphoma in a subject, comprising administering to the subject: (i) a p53 reactivator; and (ii) a Bruton’s tyrosine kinase (BTK) inhibitor. [00400] Embodiment B2. The method of Embodiment B1, wherein the p53 reactivator is a compound that can give reactivation of a mutant p53, or a degradation product thereof that can give reactivation of a mutant p53. [00401] Embodiment B3. The method of Embodiment B2, wherein the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W. [00402] Embodiment B4. The method of any one of Embodiments B2 and B3, wherein the p53 reactivator is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity. [00403] Embodiment B5. The method of any one of Embodiments B2-B4, wherein the compound or degradation product thereof promotes proper folding of mutant and wild-type p53 proteins. [00404] Embodiment B6. The method of any one of Embodiments B2-B4, wherein the compound or degradation product thereof is capable of shifting the equilibrium from unfolded towards a folded structure of wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of reducing aggregation of the wild-type or mutant p53. [00405] Embodiment B7. The method of any one of Embodiments B2-B4, wherein the compound or degradation product thereof is capable of promoting a folded structure of wild- type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53. [00406] Embodiment B8. The method of any one of Embodiments B2-B6, wherein the compound or degradation product threreof is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promote a folded conformation. [00407] Embodiment B9 The method of any one of Embodiments B2-B8, wherein the compound that can give reactivation of mutant p53 is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one; 2,2-bis(hydroxymethyl)quinuclidin-3-one; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; 2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide; N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide; N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide; 1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide; 1-methyl-N-((3-oxoquinuclidin-2-yl)methyl)cyclopropane-1-sulfonamide; N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide; 1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; 5-methyl-1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; tert-butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine- 1(2H)-carboxylate; 5-methyl-1,3-bis((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; N-methyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((3-chloro-1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; N,N-dimethyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carbonitrile; and 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; or a pharmaceutically acceptable salt thereof. [00408] Embodiment B10. The method of Embodiment B9, wherein the compound is 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one (APR-246) having the following formula:
Figure imgf000083_0001
, or a pharmaceutically acceptable salt thereof. [00409] Embodiment B11. The method of Embodiment B9, wherein the compound is a compound selected from the group consisting of: 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; and 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, or a pharmaceutically acceptable salt thereof. [00410] Embodiment B12. The method of Embodiment B11, wherein the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound A), or a pharmaceutically acceptable salt thereof. [00411] Embodiment B13. The method of Embodiment B11, wherein the compound is 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound B), or a pharmaceutically acceptable salt thereof. [00412] Embodiment B14. The method of Embodiment B11, wherein the compound is 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound C), or a pharmaceutically acceptable salt thereof. [00413] Embodiment B15. The method of Embodiment B11, wherein the compound is N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound D), or a pharmaceutically acceptable salt thereof. [00414] Embodiment B16. The method of Embodiment B11, wherein the compound is 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound E), or a pharmaceutically acceptable salt thereof. [00415] Embodiment B17. The method of any one of Embodiments B1-B16, wherein the BTK inhibitor is selected from the group consisting of ibrutinib, acalabrutinib, zanubrutinib, evobrutinib, dasatinib, spebrutinib, tirabrutinib, ABBV-105, ONO-4059, LFM-A13, and HM71224, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. [00416] Embodiment B18. The method of Embodiment B16, wherein the BTK inhibitor is ibrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. [00417] Embodiment B19. The method of any one of Embodiments B1-B10 and B17- B18, wherein the p53 reactivator is APR-246 and the BTK inhibitor is ibrutinib. [00418] Embodiment B20. The method of Embodiment B19, wherein APR-246 is administered at a dose of about 4.5 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. [00419] Embodiment B21. The method of Embodiment B19, wherein APR-246 is administered at a dose of about 4.0 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. [00420] Embodiment B22. The method of Embodiment B19, wherein APR-246 is administered at a dose of about 3.5 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28-day cycle. [00421] Embodiment B23. The method of any one of Embodiments B20-B22, wherein APR-246 is administered on Days 1–4 and ibrutinib is administered daily of each 28-day cycle. [00422] Embodiment B24. The method of any one of Embodiments B20-B23, wherein APR-246 and ibrutinib are administered for 1 to 20 cycles. [00423] Embodiment B25. The method of any one of Embodiments B1-B24, wherein the compound that can give reactivation of the mutant p53 is formulated in a first pharmaceutical composition and the BTK inhibitor is formulated in a second pharmaceutical composition. [00424] Embodiment B26. The method of any one of Embodiments B1-B25, wherein the lymphoma is a Hodgkin lymphoma (HL) or a non-Hodgkin lymphoma (NHL). [00425] Embodiment B27. The method of Embodiment B26, wherein the lymphoma is a non-Hodgkin lymphoma (NHL). [00426] Embodiment B28. The method of Embodiment B27, wherein the non-Hodgkin lymphoma (NHL) is a mature (peripheral) B-cell neoplasm. [00427] Embodiment B29. The method of Embodiment B27, wherein the non-Hodgkin lymphoma (NHL) is selected from the group consisting of: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, diffuse large cell B-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma (plasmacytoma), extranodal marginal zone B-cell lymphoma, nodal marginal zone lymphoma, follicle center lymphoma, and Burkitt’s leukemia (Burkitt’s lymphoma). [00428] Embodiment B30. The method of any one of Embodiments B1-B29, wherein the lymphoma is chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). [00429] Embodiment B31. The method of Embodiment B30, wherein the lymphoma is chronic lymphocytic leukemia (CLL). [00430] Embodiment B32. The method of Embodiment B30, wherein the lymphoma is mantle cell lymphoma (MCL). [00431] Embodiment B33. The method of Embodiment B27, wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory NHL. [00432] Embodiment B34. The method of Embodiment B33, wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). [00433] Embodiment B35. The method of Embodiment B34, wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory CLL. [00434] Embodiment B36. The method of Embodiment B34, wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory MCL. [00435] Embodiment B37. The method of any one of Embodiments B1-B36, wherein the lymphoma comprises a cancer cell having mutant p53. [00436] Embodiment B38. The method of any one of Embodiments B1-B37, wherein further comprises determining by gene sequencing if the subject has TP53 mutation. [00437] Embodiment B39. The method of any one of Embodiments B1-B38, wherein the subject is not treated with any BTK inhibitor prior to the co-administration of the p53 reactivator and the BTK inhibitor. 4.4.3 Combination Therapies with Exportin 1 (XPO1) Inhibitors [00438] In yet another aspect, also provided herein are combination therapies using a p53 reactivator (such as the p53 reactivator described in Section 4.2.1) in combination with an XPO1 inhibitor (such as the XPO1 inhibitor described in Section 4.2.4) for treating a disease or disorder (such as hyperproliferative malignancy, e.g., a hematological malignancy). [00439] Provided herein are methods of preventing and/or treating a disease or disorder (e.g., hyperproliferative malignancy) in a subject comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an exportin 1 (XPO1) inhibitor. In certain embodiments, provided herein are methods of preventing a disease or disorder in a subject, comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an exportin 1 (XPO1) inhibitor. In certain embodiments, provided herein are methods of treating a disease or disorder (e.g. hematological malignancy), comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an exportin 1 (XPO1) inhibitor. In certain embodiments, a pharmaceutically effective amount of the p53 reactivator is administered. In certain embodiments, the p53 reactivator and the XPO1 inhibitor are concomitantly administered. In certain embodiments, the co-administration of the compound that can give reactivation of a mutant p53 (the p53 reactivator) and the XPO1 inhibitor is pharmaceutically effective to treat the disease or disorder (e.g., hyperproliferative malignancy). [00440] Provided herein are methods of treating a disease or disorder (e.g., hyperproliferative malignancy) in a subject comprising administering to a subject a compound of Formula (I) and an exportin 1 (XPO1) inhibitor, wherein the compound of Formula (I) shows synergism with the exportin 1 (XPO1) inhibitor for treating the disease or disorder. In certain embodiments, a pharmaceutically effective amount of the compound of Formula (I) is administered. In certain embodiments, the co-administration of the compound of Formula (I) and the exportin 1 (XPO1) inhibitor is pharmaceutically effective to treat the disease or disorder (e.g., hyperproliferative malignancy). In one specific embodiment, the compound that has synergism with the XPO1 inhibitor is APR-246. In another specific embodiment, the compound that has synergism with the XPO1 inhibitor is Compound A. In another specific embodiment, the compound that has synergism with the XPO1 inhibitor is Compound B. In another specific embodiment, the compound that has synergism with the XPO1 inhibitor is Compound C. In another specific embodiment, the compound that has synergism with the XPO1 inhibitor is Compound D. In another specific embodiment, the compound that has synergism with the XPO1 inhibitor is Compound E. [00441] In some embodiments, the subject is a human. In some embodiments, the subject is a subject diagnosed with hyperproliferative malignancy, e.g., a hematological malignancy. [00442] In some embodiments, the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis. Examples of such diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct) cancer, primary or secondary central nervous system tumor, primary or secondary brain tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer, cancer of the kidney and/or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system, primary central nervous system lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma or a combination thereof. [00443] In some embodiments, the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small- cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer. [00444] In some embodiments, the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma. In some embodiments, the cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. In a specific embodiment, the disease or disorder is myeloma. In a specific embodiment, the disease or disorder is myelodysplastic syndromes (MDS). In another specific embodiment, the disease or disorder is acute myeloid leukemia (AML). In another specific embodiment, the disease or disorder is chronic lymphocytic leukemia (CLL). In yet another specific embodiment, the myeloma is multiple myeloma (MM). [00445] In other embodiments, the disease or disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer. [00446] In some embodiments, the subject is diagnosed to have TP53 mutation. In some embodiments, the subject carries TP53 mutation. In some embodiments, the subject has mutant p53 protein. [00447] In some embodiments, the methods described herein further comprises determining by gene sequencing if the subject has TP53 mutation. [00448] In some embodiments, the disease or disorder is a mutant p53 mediated cancer (including, e.g., hematological tumors with mutations in the p53 gene). In other embodiments, the disease or disorder is not a mutant p53 mediated cancer. [00449] In some embodiments, the mutation in TP53 is R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, V173A, or S241F. In some embodiments, the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation. In other embodiments, the mutant TP53 includes a nonsense mutation. A nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP53 mutations in cancer. The most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*. [00450] In some embodiments, the disease or disorder is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. [00451] The amount of the p53 reactivator or the XPO1 inhibitor provided herein, or a pharmaceutical composition that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some embodiments, should be decided according to the judgment of the practitioner and each patient’s circumstances. Effective doses may also be extrapolated from dose-response curves derived from in vitro or animal model test systems. [00452] In some embodiments, the p53 reactivator is administered to the subject as part a composition. In some embodiments, the composition is a pharmaceutical composition described in Section 4.3.3. [00453] In some embodiments, the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) can be formulated in different pharmaceutical compositions and administered separately to the subject in need thereof. In other embodiments, the p53 reactivator (e.g., APR- 246) and the XPO1 inhibitor (e.g., selinexor) are administered together in the same pharmaceutical composition. [00454] In some embodiments, the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) are administered simultaneously. The term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours. [00455] In some embodiments, the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) are not administered simultaneously, and instead the two compounds are administered at different times. [00456] In certain embodiments, the XPO1 inhibitor (e.g., selinexor) is administered before or concurrently with the administration of the p53 reactivator (e.g., APR-246). [00457] In certain embodiments, the XPO1 inhibitor (e.g., selinexor) is administered after the administration of the p53 reactivator (e.g., APR-246). [00458] In certain embodiments, the subject has been previously treated with the XPO1 inhibitor (e.g., selinexor) prior to the administration of the p53 reactivator (e.g., APR-246). [00459] In certain embodiments, the subject has been previously treated with the p53 reactivator (e.g., APR-246) prior to the administration of the XPO1 inhibitor (e.g., selinexor). [00460] In certain embodiments, the subject has not been previously treated with any XPO1 inhibitor (e.g., selinexor) prior to the co-administration of the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor). [00461] In certain embodiments, the co-administration of the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) is concomitant administration. [00462] In certain embodiments, the co-administration of the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) is pharmaceutically effective to treat hyperproliferative malignancy, e.g., a hematological malignancy. [00463] In some embodiments, the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) are administered at least once during a dosing period. A dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once. A dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In certain embodiments, a dosing period is a dosing cycle. [00464] The prophylactic or therapeutic agent (the p53 reactivator and/or the XPO1 inhibitor provided herein) can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time). The agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity. Stable disease or lack is determined by methods known in the art such as evaluation of patient symptoms, physical examination, and visualization of the tumor that has been imaged using X- ray, CAT, PET, MRI scan, or other commonly accepted evaluation modalities. [00465] The prophylactic or therapeutic agent (the p53 reactivator and/or the XPO1 inhibitor provided herein) can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID). In addition, the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug). As used herein, the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time. The term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days. The term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days. [00466] In some embodiments, the frequency of administration is in the range of about a daily dose to about a monthly dose. In certain embodiments, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks. [00467] In certain embodiments, the compound is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks. [00468] In some embodiments, APR-246 is administered at a dose of less than about 150 mg/kg. In some embodiments, APR-246 is administered at a dose of less than about 100 mg/kg. In some embodiments, APR-246 is administered at a dose of between about 100 mg/kg and about 25 mg/kg. In other embodiments, APR-246 is administered at dose of less than about 75 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than about 65 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than about 50 mg/kg. [00469] In other embodiments, APR-246 is administered at a fixed dose within the interval of 2.7-7.5 g. In some embodiments, the fixed dose of APR-246 is no more than about 4.5 g. In some embodiments, the fixed dose of APR-246 is no more than about 4.0 g. In some embodiments, the fixed dose of APR-246 is no more than about 3.5 g. In one embodiments, APR-246 is administered at a dose of about 4.5 g/day, about 4.0 g/day, or about 3.5 g/day. In one specific embodiment, APR-246 is administered at a dose of about 4.5 g/day. In another specific embodiment, APR-246 is administered at a dose of about 4.0 g/day. In yet another specific embodiment, APR-246 is administered at a dose of about 3.5 g/day. [00470] In some embodiments, APR-246 is administered in a multiple-step administration to avoid high plasma concentration and/or to minimize the risk of adverse events. In some embodiments, APR-246 is administered in a 2-step administration consisting of a first loading dose and a subsequent maintenance dose. In some preferred embodiments, the first loading dose is not equal to the subsequent maintenance dose. In other embodiments, the first loading dose is not equal to the subsequent maintenance dose. In some embodiments, the first loading dose is about 1.5 g, about 1.33 g, or about 1.16 g. In some embodiments, the subsequent maintenance dose is about 3.0 g, about 2.67 g, or about 2.34 g. [00471] In some embodiment, APR-246 is administered 1, 2, 3, 4, 5, 6, 7, or 8 times of each cycle of 14 days, 21 days, every 28 days, every 35 days, or every 42 days. In preferred embodiments, APR-246 is administered for 4 days in each treatment cycle. In preferred embodiments, APR-246 is administered for 4 days in each treatment cycle of 28 days. In some embodiments, APR-246 is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles. [00472] In a specific embodiment, APR-246 is administered once daily for 4 consecutive days in each treatment cycle of 28 days. [00473] In some preferred embodiments, APR-246 is administered intravenously. [00474] In some embodiments, the XPO1 inhibitor described herein (e.g., selinexor) is administered at a dose between 1 mg/day to 1000 mg/day. In some embodiments, the XPO1 inhibitor is administered at a dose between about 10 mg/day and about 20 mg/day, between about 20 mg/day and 30 mg/day, between about 30 mg/day and 40 mg/day, between about 40 mg/day and about 50 mg/day, between about 50 mg/day and 60 mg/day, between about 60 mg/day and 70 mg/day, between about 70 mg/day and 80 mg/day, between about 80 mg/day and 90 mg/day, or between about 90 mg/day and about 100 mg/day. In some embodiments, the XPO1 inhibitor is administered at a dose of about 80 mg/day. In one embodiment, the XPO1 inhibitor is administered at a dose of about 100 mg/day. In another embodiment, the XPO1 inhibitor is administered at a dose of about 60 mg/day. In one specific embodiment, the XPO1 inhibitor is administered at a dose of about 80 mg/day and administered twice per week. In another specific embodiment, the XPO1 inhibitor is administered at a dose of about 100 mg/day and administered once per week. In another specific embodiment, the XPO1 inhibitor is administered at a dose of about 80 mg/day and administered once per week. In another specific embodiment, the XPO1 inhibitor is administered at a dose of about 60 mg/day and administered once per week. [00475] In some preferred embodiments, selinexor is administered at a daily dose of about 80 mg twice per week. In one preferred embodiment, the XPO1 inhibitor is administered at a daily dose of about 80 mg on day 1 and day 3 per week. [00476] In some embodiments, the XPO1 inhibitor (e.g., selinexor) is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles. [00477] In some preferred embodiments, the XPO1 inhibitor (e.g., selinexor) is administered orally. [00478] In certain embodiments, combination treatment of the p53 reactivator (e.g., APR-246) and the XPO1 inhibitor (e.g., selinexor) show synergistic effects in treating hyperproliferative malignancy, e.g., a hematological malignancy. [00479] In some embodiments, APR-246 is administered at a dose of about 4.5 g/day in combination with selinexor administered at a dose of about 80 mg/day twice per week in each 28-day cycle. In some embodiments, APR-246 and selinexor are administered for 1 to 10 cycles. In some embodiments, APR-246 and selinexor are administered for 1 cycle. In some embodiments, APR-246 and selinexor are administered for 2 cycles. In some embodiments, APR-246 and selinexor are administered for 3 cycles. In some embodiments, APR-246 and selinexor are administered for 4 cycles. In some embodiments, APR-246 and selinexor are administered for 5 cycles. In some embodiments, APR-246 and selinexor are administered for 6 cycles. In some embodiments, APR-246 and selinexor are administered for 7 cycles. In some embodiments, APR-246 and selinexor are administered for 8 cycles. In some embodiments, APR-246 and selinexor are administered for 9 cycles. In some embodiments, APR-246 and selinexor are administered for 10 cycles. In some embodiments, APR-246 and selinexor are administered for more than 10 cycles. [00480] In some embodiments, APR-246 is administered at a dose of about 4.0 g/day in combination with selinexor administered at a dose of about 80 mg/day twice per week in each 28-day cycle. In some embodiments, APR-246 and selinexor are administered for 1 to 10 cycles. In some embodiments, APR-246 and selinexor are administered for 1 cycle. In some embodiments, APR-246 and selinexor are administered for 2 cycles. In some embodiments, APR-246 and selinexor are administered for 3 cycles. In some embodiments, APR-246 and selinexor are administered for 4 cycles. In some embodiments, APR-246 and selinexor are administered for 5 cycles. In some embodiments, APR-246 and selinexor are administered for 6 cycles. In some embodiments, APR-246 and selinexor are administered for 7 cycles. In some embodiments, APR-246 and selinexor are administered for 8 cycles. In some embodiments, APR-246 and selinexor are administered for 9 cycles. In some embodiments, APR-246 and selinexor are administered for 10 cycles. In some embodiments, APR-246 and selinexor are administered for more than 10 cycles. [00481] In some embodiments, APR-246 is administered at a dose of about 3.5 g/day in combination with selinexor administered at a dose of about 80 mg/day twice per week in each 28-day cycle. In some embodiments, APR-246 and selinexor are administered for 1 to 10 cycles. In some embodiments, APR-246 and selinexor are administered for 1 cycle. In some embodiments, APR-246 and selinexor are administered for 2 cycles. In some embodiments, APR-246 and selinexor are administered for 3 cycles. In some embodiments, APR-246 and selinexor are administered for 4 cycles. In some embodiments, APR-246 and selinexor are administered for 5 cycles. In some embodiments, APR-246 and selinexor are administered for 6 cycles. In some embodiments, APR-246 and selinexor are administered for 7 cycles. In some embodiments, APR-246 and selinexor are administered for 8 cycles. In some embodiments, APR-246 and selinexor are administered for 9 cycles. In some embodiments, APR-246 and selinexor are administered for 10 cycles. In some embodiments, APR-246 and selinexor are administered for more than 10 cycles. [00482] In certain embodiments, the methods described herein further comprises determining by gene sequencing if the subject has TP53 mutation. In certain embodiments, a next generation sequencing (NGS) method is used for the gene sequencing. In certain embodiments, the gene sequencing data are interpreted to determine if the subject has or likely has TP53 mutation. [00483] In another aspect, also provided herein are combination therapies of the p53 reactivator and the exportin 1 (XPO1) inhibitor provided herein for use in treating a disease or disorder described herein (e.g., hyperproliferative malignancy, e.g., a hematological malignancy). In some embodiments, also provided herein are combination therapies of the p53 reactivator and the exportin 1 (XPO1) inhibitor provided herein for use in treating hematological malignancy. [00484] In another aspect, also provided herein are pharmaceutical compositions or a combination of pharmaceutical compositions of the p53 reactivator and the exportin 1 (XPO1) inhibitor provided herein for use in treating a disease or disorder (e.g., hyperproliferative malignancy). In some embodiments, also provided herein are pharmaceutical compositions or a combination of pharmaceutical compositions of the p53 reactivator and the exportin 1 (XPO1) inhibitor provided herein for use in treating hematological malignancy. Exemplary Embodiments [00485] Embodiment C1. A method of treating a hyperproliferative malignancy in a subject, comprising administering to the subject: (i) a p53 reactivator; and (ii) an exportin 1 (XPO1) inhibitor. [00486] Embodiment C2. The method of Embodiment C1, wherein the p53 reactivator is a compound that can give reactivation of a mutant p53, or a degradation product thereof that can give reactivation of a mutant p53. [00487] Embodiment C3. The method of Embodiment C2, wherein the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W. [00488] Embodiment C4. The method of any one of Embodiments C1 and C2, wherein the p53 reactivator is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity. [00489] Embodiment C5. The method of any one of Embodiments C2-C4, wherein the compound or degradation product thereof promotes proper folding of mutant and wild-type p53 proteins. [00490] Embodiment C6. The method of any one of Embodiments C2-C4, wherein the compound or degradation product thereof is capable of shifting the equilibrium from unfolded towards a folded structure of wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of reducing aggregation of the wild-type or mutant p53. [00491] Embodiment C7. The method of any one of Embodiments C2-C4, wherein the compound or degradation product thereof is capable of promoting a folded structure of wild- type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53. [00492] Embodiment C8. The method of any one of Embodiments C2-C6, wherein the compound or degradation product threreof is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promote a folded conformation. [00493] Embodiment C9 The method of any one of Embodiments C2-C8, wherein the compound that can give reactivation of mutant p53 is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one; 2,2-bis(hydroxymethyl)quinuclidin-3-one; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; 2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide; N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide; N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide; 1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide; 1-methyl-N-((3-oxoquinuclidin-2-yl)methyl)cyclopropane-1-sulfonamide; N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide; 1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; 5-methyl-1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; tert-butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine- 1(2H)-carboxylate; 5-methyl-1,3-bis((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; N-methyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((3-chloro-1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; N,N-dimethyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carbonitrile; and 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; or a pharmaceutically acceptable salt thereof. [00494] Embodiment C10. The method of Embodiment C9, wherein the compound is 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one (APR-246) having the following formula:
Figure imgf000098_0001
, or a pharmaceutically acceptable salt thereof. [00495] Embodiment C11. The method of Embodiment C9, wherein the compound is a compound selected from the group consisting of: 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; and 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, or a pharmaceutically acceptable salt thereof. [00496] Embodiment C12. The method of Embodiment C11, wherein the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound A), or a pharmaceutically acceptable salt thereof. [00497] Embodiment C13. The method of Embodiment C11, wherein the compound is 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound B), or a pharmaceutically acceptable salt thereof. [00498] Embodiment C14. The method of Embodiment C11, wherein the compound is 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound C), or a pharmaceutically acceptable salt thereof. [00499] Embodiment C15. The method of Embodiment C11, wherein the compound is N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound D), or a pharmaceutically acceptable salt thereof. [00500] Embodiment C16. The method of Embodiment C11, wherein the compound is 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound E), or a pharmaceutically acceptable salt thereof. [00501] Embodiment C17. The method of any one of Embodiments C1-C16, wherein the XPO1 inhibitor is selected from the group consisting of leptomycin A (LMA), leptomycin B (LMB), selinexor (KPT-330), Eltanexor (KPT-8602), KTP-185, KPT-249, KPT-251, KPT-276, verdinexor (KPT-335), piperlongumine, valtrate, felezonexor (CBS9106 or SL-801), and PKF050-638, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. [00502] Embodiment C18. The method of Embodiment 17, wherein the XPO1 inhibitor is selinexor, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. [00503] Embodiment C19. The method of any one of Embodiments C1-C10 and C17- C18, wherein the p53 reactivator is APR-246 and the XPO1 inhibitor is selinexor. [00504] Embodiment C20. The method of Embodiment C19, wherein APR-246 is administered at a daily dose of about 4.5 g/day and selinexor is administered at a dose of about 80 mg/day twice per week. [00505] Embodiment C21. The method of Embodiment C19, wherein APR-246 is administered at a daily dose of about 4.0 g/day and selinexor is administered at a dose of about 80 mg/day twice per week. [00506] Embodiment C22. The method of Embodiment C19, wherein APR-246 is administered at a daily dose of about 3.5 g/day and selinexor is administered at a dose of about 80 mg/day twice per week. [00507] Embodiment C23. The method of any one of Embodiments C20-C22, wherein selinexor is administered on day 1 and day 3 of each week. [00508] Embodiment C24. The method of Embodiment C19, wherein selinexor is administered at a reduced dose of about 100 mg once per week, about 80 mg once per week, or about 60 mg once per week. [00509] Embodiment C25. The method of any one of Embodiments C1-C24, wherein the compound that can give reactivation of the mutant p53 is formulated in a first pharmaceutical composition and the XPO1 inhibitor is formulated in a second pharmaceutical composition. [00510] Embodiment C26. The method of any one of Embodiments C1-C25, wherein the hyperproliferative malignancy is a hematological malignancy. [00511] Embodiment C27. The method of Embodiment C26, wherein the hematological malignancy is leukemia, lymphoma, or myeloma. [00512] Embodiment C28. The method of Embodiment C26, wherein the hematological malignancy is selected from the group consisting of: Hodgkin's lymphoma, non- Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. [00513] Embodiment C29. The method of Embodiment C26, wherein the hematologic malignancy is myeloma. [00514] Embodiment C30. The method of Embodiment C29, wherein the myeloma is multiple myeloma (MM). [00515] Embodiment C31. The method of Embodiment C26, wherein the hematologic malignancy is acute myeloid leukemia (AML). [00516] Embodiment C32. The method of Embodiment C26, wherein the hematologic malignancy is chronic lymphocytic leukemia (CLL). [00517] Embodiment C33. The method of any one of Embodiments C1-C32, wherein the hyperproliferative malignancy comprises a cancer cell having mutant p53. [00518] Embodiment C34. The method of any one of Embodiments C1-C33, wherein further comprises determining by gene sequencing if the subject has TP53 mutation. [00519] Embodiment C35. The method of any one of Embodiments C1-C34, wherein the subject is not treated with any XPO1 inhibitor prior to the co-administration of the p53 reactivator and the XPO1 inhibitor. 4.4.4 Combination Therapies with Inhibitors of WEE1 [00520] In yet another aspect, also provided herein is a composition or a combination of compositions for use in the prevention and/or treatment of a disease or condition (e.g., a hyperproliferative malignancy) comprising the p53 reactivator provided herein (e.g., the p53 reactivator described in Section 4.2.1 such as APR-246) and the inhibitor of WEE1 provided herein (e.g., the inhibitors of WEE1 described in Section 4.2.5 such as MK-1775). Such uses relate to a method comprising administering an effective amount of the p53 reactivator and the inhibitor of WEE1 (e.g., MK-1775) to a subject. In one embodiment, the subject is a human. [00521] Provided herein are methods of preventing and/or treating a disease or disorder (e.g., hyperproliferative malignancy) in a subject comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an inhibitor of WEE1. In certain embodiments, provided herein are methods of preventing a disease or disorder in a subject, comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an inhibitor of WEE1. In certain embodiments, provided herein are methods of treating a disease or disorder (e.g. hyperproliferative malignancy), comprising administering to a subject a p53 reactivator (a compound that can give reactivation of a mutant p53) and an inhibitor of WEE1. In certain embodiments, a pharmaceutically effective amount of the p53 reactivator is administered. In certain embodiments, the p53 reactivator and the inhibitor of WEE1 are concomitantly administered. In certain embodiments, the co- administration of the compound that can give reactivation of a mutant p53 (the p53 reactivator) and the inhibitor of WEE1 is pharmaceutically effective to treat the disease or disorder (e.g., hyperproliferative malignancy). [00522] Provided herein are methods of treating a disease or disorder (e.g., a hyperproliferative malignancy) in a subject comprising administering to a subject a compound of Formula (I) and an inhibitor of WEE1, wherein the compound of Formula (I) shows synergism with the inhibitor of WEE1 for treating the disease or disorder. In certain embodiments, a pharmaceutically effective amount of the compound of Formula (I) is administered. In certain embodiments, the co-administration of the compound of Formula (I) and the inhibitor of WEE1 is pharmaceutically effective to treat the disease or disorder (e.g., hyperproliferative malignancy). In one specific embodiment, the compound that has synergism with the inhibitor of WEE1 is APR-246. In another specific embodiment, the compound that has synergism with the inhibitor of WEE1 is Compound A. In another specific embodiment, the compound that has synergism with the inhibitor of WEE1 is Compound B. In another specific embodiment, the compound that has synergism with the inhibitor of WEE1 is Compound C. In another specific embodiment, the compound that has synergism with the inhibitor of WEE1 is Compound D. In another specific embodiment, the compound that has synergism with the inhibitor of WEE1 is Compound E. [00523] In some embodiments, the disease or disorder is a neoplastic disease. In certain embodiments, the disease or disorder is a cancer. In certain embodiments, the disease or disorder is a hematogical malignancy. In certain embodiments, the neoplastic, cancerous, or malignant cell in a subject to be treated with a method provided herein comprises a mutation in TP53. In a specific embodiment, provided herein is a method to treat or prevent a hematological cancer with a mutation in the p53 gene. In certain embodiments, the cancer to be treated with a method provided herein comprises cancer cells that carry a mutation in TP53. In some embodiments, the mutation in TP53 is R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, V173A, S241F, R249S or a combination thereof. In some embodiments, the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation. In other embodiments, the mutant TP53 includes a nonsense mutation. A nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP53 mutations in cancer. The most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*. [00524] In other embodiments, the cancer to be treated does not comprise any cells carrying a mutation in the TP53 gene. [00525] In some embodiments, the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis. Examples of such diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct) cancer, primary or secondary central nervous system tumor, primary or secondary brain tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer, cancer of the kidney and/or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system, primary central nervous system lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma or a combination thereof. [00526] In some embodiments, the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small- cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer. [00527] In some embodiments, the disease or disorder is a tumor of the hematopoietic or lymphoid tissue. [00528] In some embodiments, the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma. In some embodiments, the cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. In a specific embodiment, the disease or disorder is myelodysplastic syndromes (MDS). In another specific embodiment, the disease or disorder is acute myeloid leukemia (AML). In another specific embodiment, the disease or disorder is chronic lymphocytic leukemia (CLL). In yet another specific embodiment, the disease or disorder is multiple myeloma (MM). In some embodiments, the disease or disorder is lymphoma. [00529] In other embodiments, the disease or disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, a lung cancer, a cervical cancer, a prostate cancer, a head and neck cancer, and a breast cancer. In certain embodiments, the solid tumor cancer is breast cancer. [00530] In some embodiments, the solid tumor cancer is selected from the group consisting of breast cancer, cervical cancer, prostate cancer, and head and neck cancer. In certain embodiments, the solid tumor cancer is triple negative breast cancer. [00531] The amount of a prophylactic or therapeutic agent (the p53 reactivator and the inhibitor of WEE1 (e.g., MK-1775) provided herein), or a composition provided herein that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some embodiments, should be decided according to the judgment of the practitioner and each patient’s circumstances. [00532] The dose administered to a subject in the context of the present disclosure should be sufficient to effect a therapeutic response. One skilled in the art will recognize that dosage will depend upon a variety of factors including the potency of the specific compound, the age, condition and body weight of the patient, as well as the stage/severity of the disease. The dose will also be determined by the route (administration form) timing and frequency of administration. [00533] The p53 reactivator (e.g., APR-246) and the inhibitor of WEE1 (e.g., MK-1775) can be formulated in different pharmaceutical compositions and administered separately to the subject in need thereof. Alternatively, the p53 reactivator (e.g., APR-246) and the inhibitor of WEE1 (e.g., MK-1775) are administered together in the same pharmaceutical composition. [00534] In some embodiments, the p53 reactivator (e.g., APR-246) and the inhibitor of WEE1 (e.g., MK-1775) are administered simultaneously. The term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours. [00535] In some embodiments, the p53 reactivator (e.g., APR-246) and the inhibitor of WEE1 (e.g., MK-1775) are not administered simultaneously, and instead the two compounds are administered at different times. In some embodiments, the p53 reactivator (e.g., APR-246) and the inhibitor of WEE1 (e.g., MK-1775) are administered at least once during a dosing period. A dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once. A dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In certain embodiments, a dosing period is a dosing cycle. [00536] The prophylactic or therapeutic agent (the p53 reactivator and/or the WEE1 inhibitor provided herein) can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time). The agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity. Stable disease or lack is determined by methods known in the art such as evaluation of patient symptoms, physical examination, and visualization of the tumor that has been imaged using X- ray, CAT, PET, MRI scan, or other commonly accepted evaluation modalities. [00537] The prophylactic or therapeutic agent (the p53 reactivator and/or the WEE1 inhibitor provided herein) can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID). In addition, the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug). As used herein, the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time. The term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days. The term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days. [00538] In some embodiments, the frequency of administration is in the range of about a daily dose to about a monthly dose. In certain embodiments, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks. [00539] In certain embodiments, the compound is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks. [00540] In some embodiments, APR-246 is administered at a dose of less than 150 mg/kg. In some embodiments, APR-246 is administered at a dose of less than 100 mg/kg. In some embodiments, APR-246 is administered at a dose of between 100 mg/kg and 25 mg/kg. In other embodiments, APR-246 is administered at dose of less than 75 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 65 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 50 mg/kg. [00541] In other embodiments, APR-246 is administered at a fixed dose within the interval 2.7-7.5 g. In some embodiments, the fixed dose of APR-246 is no more than 4.5 g. In some embodiments, the fixed dose of APR-246 is no more than 3.7 g. In some embodiments, APR- 246 is administered at a fixed dose during a period of about 3 to 7 hours. In some embodiments, APR-246 is administered at a fixed dose during a period of about 6 or 4 hours. Exemplary Embodiments [00542] Embodiment D1. A method of treating cancer in a subject, comprising administering to the subject: (i) a compound that can activate a mutant p53; and (ii) an inhibitor of Wee1-like protein kinase (WEE1). [00543] Embodiment D2. The method of Embodiment D1, wherein the cancer in the subject comprises a cell that carries a mutation in TP53. [00544] Embodiment D3. The method of Embodiment D2, wherein the mutation in TP53 is selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W, or a combination thereof. [00545] Embodiment D4. The method of Embodiment D2 or D3, wherein the compound that can activate the mutant p53 promotes proper folding of the mutant p53 and restores at least part of a normal p53 function. [00546] Embodiment D5. The method of Embodiment D2 or D3, wherein the compound that can activate of the mutant p53 can result in a shift of the equilibrium from unfolded towards a wild-type like p53 conformation. [00547] Embodiment D6. The method of Embodiment D2 or D3, wherein the compound that can activate the mutant p53 interferes with aggregation of misfolded mutant p53 or reduces aggregation of the mutant p53. [00548] Embodiment D7. The method of Embodiment D2 or D3, wherein the compound that can activate the mutant p53 or its metabolite or degradation product thereof can restore a p53 wild type function by covalent binding to the mutant p53. [00549] Embodiment D8. The method of Embodiment D7, wherein the compound that can activate the mutant p53 can bind to thiol groups in the core domain of the mutant p53 and restore wild-type conformation. [00550] Embodiment D9. The method of any one of Embodiments D1-D8, wherein the compound reactivates a mutant p53. [00551] Embodiment D10. The method of any one of Embodiments D1-D9, wherein the compound that can activate the mutant p53 is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one; 2,2-bis(hydroxymethyl)quinuclidin-3-one; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; 2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide; N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide; N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide; 1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide; 1-methyl-N-((3-oxoquinuclidin-2-yl)methyl)cyclopropane-1-sulfonamide; N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide; 1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; 5-methyl-1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; tert-butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine- 1(2H)-carboxylate; 5-methyl-1,3-bis((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; N-methyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((3-chloro-1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; N,N-dimethyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carbonitrile; and 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide, or a pharmaceutically acceptable salt thereof. [00552] Embodiment D11. The method of Embodiment D10, wherein the compound is 2- (hydroxymethyl)-2- (methoxymethyl) quinuclidin-3-one (APR-246) having the following formula:
Figure imgf000109_0001
, or a pharmaceutically acceptable salt thereof. [00553] Embodiment D12. The method of Embodiment D10, wherein the compound is a compound selected from the group consisting of: 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; and 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, or a pharmaceutically acceptable salt thereof. [00554] Embodiment D13. The method of Embodiment D12, wherein the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound A), or a pharmaceutically acceptable salt thereof. [00555] Embodiment D14. The method of Embodiment D12, wherein the compound is 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound B), or a pharmaceutically acceptable salt thereof. [00556] Embodiment D15. The method of Embodiment D12, wherein the compound is 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound C), or a pharmaceutically acceptable salt thereof. [00557] Embodiment D16. The method of Embodiment D12, wherein the compound is N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound D), or a pharmaceutically acceptable salt thereof. [00558] Embodiment D17. The method of Embodiment D12, wherein the compound is 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide (Compound E), or a pharmaceutically acceptable salt thereof. [00559] Embodiment D18. The method of any one of Embodiments D1-D17, wherein the inhibitor of WEE1 is selected from the group consisting of: 6-(2,6-Dichlorophenyl)-2-[4-[2-(diethylamino)ethoxy] anilino]-8-methylpyrido[2,3- d]pyrimidin-7-one (PD0166285); 9-Hydroxy-4-phenylpyrrolo[3,4-c] carbazole-1,3(2H,6H)-dione (PD0407824); 4-(2-Chlorophenyl)-9-hydroxypyrrolo[3,4-c] carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor I); 6-Butyl-4-(2-chlorophenyl)-9-hydroxypyrrolo [3,4-c]carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor II); 2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((4-(4-methylpiperazin-1-yl)phenyl) amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Adavosertib (MK-1775 or AZD1775)); and Methyl 4-(4-((2-allyl-1-(6-(2-hydroxypropan-2-yl) pyridin-2-yl)-3-oxo-2,3-dihydro-1H- pyrazolo[3,4-d] pyrimidin-6-yl)amino)phenyl)piperazine-1-carboxylate (CJM061). [00560] Embodiment D19. The method of any one of Embodiments D1-D17, wherein the inhibitor of WEE1 is MK-1775 having a formula of:
Figure imgf000111_0001
(MK-1775), or a pharmaceutically acceptable salt thereof. [00561] Embodiment D20. The method of any one of Embodiments D1-D19, wherein the p53 reactivator is formulated in a first pharmaceutical composition and the inhibitor of WEE1 is formulated in a second pharmaceutical composition. [00562] Embodiment D21. The method of any one of Embodiments D1-D20, wherein the cancer is a hematological malignancy. [00563] Embodiment D22. The method of Embodiment D21, wherein the hematological malignancy is leukemia, lymphoma, or myeloma. [00564] Embodiment D23. The method of any one of Embodiments D1-D22, wherein the cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. [00565] Embodiment D24. The method of Embodiment D23, wherein the cancer is myelodysplastic syndromes (MDS). [00566] Embodiment D25. The method of Embodiment D23, wherein the cancer is acute myeloid leukemia (AML). [00567] Embodiment D26. The method of Embodiment D23, wherein the cancer is chronic lymphocytic leukemia (CLL). [00568] Embodiment D27. The method of Embodiment D23, wherein the cancer is multiple myeloma (MM). [00569] Embodiment D28. The method of any one of Embodiments D1-D20, wherein the cancer is a solid tumor cancer. [00570] Embodiment D29. The method of Embodiment D28, wherein the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, a lung cancer, a cervical cancer, a prostate cancer, a head and neck cancer, and a breast cancer. [00571] Embodiment D30. The method of Embodiment D28, wherein the solid tumor cancer is breast cancer. [00572] Embodiment D31. The method of Embodiment D28, wherein the solid tumor cancer is triple negative breast cancer. 4.5 Assays for Testing and Demonstrating Synergistic Effects of an Activator of P53 and a Second Therapeutic Agent [00573] Also provided herein are assays for demonstrating the effects of the combination treatment with an agonist of p53 such as a p53 reactivator and a second therapeutic agent, for example, an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1). [00574] Also provided herein are assays for demonstrating the synergistic effects of the combination treatment with a compound (e.g., a compound of Formula (I) provided herein) that activates p53 and a second therapeutic agent, for example, an inhibitor of PD-1 mediated signaling, a Bruton’s tyrosine kinase (BTK) inhibitor, an exportin 1 (XPO1) inhibitor, or an inhibitor of Wee1-like protein kinase (WEE1). In certain embodiments, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is a compound of Formula (I). In one specific embodiment, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is APR-246. In another specific embodiment, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is Compound A. In another specific embodiment, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is Compound B. In another specific embodiment, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is Compound C. In another specific embodiment, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is Compound D. In another specific embodiment, the compound that activates p53 and has synergism with the second therapeutic compound disclosed herein is Compound E. [00575] In certain embodiments, the compound has synergism with the second therapeutic compound disclosed herein activates p53 in immune cells. In other embodiments, the compound has synergism with the second therapeutic compound disclosed herein activates a mutant p53 in immune cells. In other embodiments, the compound has synergism with the second therapeutic compound disclosed herein activates normal p53 in immune cells. [00576] In one embodiment, provided herein are assays for demonstrating the effects of the combination treatment with an agonist of p53 and an inhibitor of PD-1 mediated signalling such as an inhibitor of PD-1. [00577] In another embodiment, also provided herein are assays for demonstrating the effects of the combination treatment with an agonist of p53 such as a p53 reactivator and a BTK inhibitor. [00578] In yet another embodiment, also provided herein are assays for demonstrating the effects of the combination treatment with an agonist of p53 such as a p53 reactivator and an XPO1 inhibitor (e.g., selinexor). [00579] In yet another embodiment, also provided herein are assays for demonstrating the effects of the combination treatment with a p53 reactivator and a WEE1 inhibitor. [00580] A model system for a cancer may be treated with a p53 agonist such as p53 reactivator, a second therapeutic agent, or a combination of the p53 agonist or reactivator and the second therapeutic agent, and the effects of the combination treatment are analyzed and compared to the monotherapies. [00581] In one embodiment, the model system for a solid tumor malignancy may be treated with a p53 reactivator, an inhibitor of PD-1, or a combination of the p53 reactivator and the inhibitor of PD-1, and the effects of the combination treatment are analyzed and compared to the monotherapies. [00582] In another embodiment, the model system for a solid tumor malignancy may be treated with a p53 reactivator, a BTK inhibitor, or a combination of the p53 reactivator and the BTK inhibitor, and the effects of the combination treatment are analyzed and compared to the monotherapies. [00583] In yet another embodiment, the model system for a solid tumor malignancy may be treated with a p53 reactivator, an XPO1 inhibitor, or a combination of the p53 reactivator and the XPO1 inhibitor, and the effects of the combination treatment are analyzed and compared to the monotherapies. [00584] In yet another embodiment, the model system for a hematological malignancy (e.g., an in vivo cancer model such as a mouse model) may be treated with a p53 reactivator, a WEE1 inhibitor, or a combination of the p53 reactivator and the WEE1 inhibitor, and the effects of the combination treatment are analyzed and compared to the monotherapies. [00585] Synergistic effects of a p53 agonist such as p53 reactivator and a second therapeutic agent can then be analyzed using well-known analytical tools. [00586] In one embodiment, synergistic effects of a p53 reactivator and an inhibitor of PD-1 can then be analyzed using well-known analytical tools. [00587] In another embodiment, synergistic effects of a p53 reactivator and a BTK inhibitor can also be analyzed using well-known analytical tools. [00588] In yet another embodiment, synergistic effects of a p53 reactivator and an XPO1 inhibitor can then be analyzed using well-known analytical tools. [00589] In yet another embodiment, synergistic Effects of a p53 reactivator and a WEE1 inhibitor can then be analyzed using well-known analytical tools. [00590] In one embodiment, the analytical tool is COMBENEFIT. Combenefit (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866–2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files. Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413–2415). [00591] In some embodiments, synergistic effects of an exemplary activator of p53 (e.g., APR-246) in combination with an exemplary second therapeutic agent (e.g., an inhibitor of PD-1 mediated signaling, a BTK inhibitor, an XPO1 inhibitor, or a WEE1 inhibitor) were tested in one or more cell lines, to assess both single agent activity and evaluate additive, synergistic or antagonistic interactions across to a range of doses. In one embodiment, any synergistic effects in various combinational doses can be determined according to the Highest Single Agent (HSA), Bliss and Loewe models. Synergistic effects can be determined if additive model Combination Index values obtained in these cell lines are found to indicate synergism. CellTiter-Glo viability (CTG) assay [00592] The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells. The homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. The homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. Dose response Determinations by XLFIT [00593] For IC50 determinations, the serial dilution of compounds can be performed in cell culture medium based on a 1.5- or 2-fold dilution factor. The serially diluted compounds can be added to the cells and incubated for 72 h followed by cell viability test using the CTG assay. For each concentration, the current viability values can be calculated and IC50 values were determined by XLfit software version 5.5.0.5 (a Microsoft Excel-based plug-in to perform regression, curve fitting and statistical analysis). Analysis of Combination by COMBENEFIT [00594] Combenefit (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866–2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files. Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods. In the HSA model, the synergy score quantifies the excess over the highest single drug response. In the Loewe model, the synergy score quantifies the excess over the expected response if the two drugs are the same compound. In the Bliss model, the expected response is a multiplicative effect as if the two drugs act independently (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413–2415). [00595] The experimental dose–response surface that delineates combination effects in concentration space is first read by the Combenefit software as a matrix of % of the control value across concentrations. Single agent effects are extracted from this data and fitted with a dose response curve. Based on the two single agent dose response curves, a model-based combination dose–response surface is derived. This surface provides a “reference” dose–response surface for a non-synergistic (additive/independent) combination, whose characteristics are determined by the selected model (i.e., Loewe, Bliss or HSA). The experimental combination dose response surface is then compared to the model-generated one, resulting in a synergy distribution in concentration space. [00596] The graphical outputs consist of: (i) the single agent dose response data and its fitting; (ii) the combination dose response (four different displays); (iii) the model generated reference combination dose response, i.e., the prediction of effect if the drugs are not synergistic (three different displays); (iv) the resulting synergy distribution (three different displays); and (v) a graphic mapping the synergy distribution onto the dose–response surface. [00597] This synergy distribution can be further summarized via various metrics (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866-2868) as follows: • SYN_MAX - the maximum level of synergy observed. • SYN_SUM - the sum of synergy observed in concentration logarithmic space. For instance, an integrated synergy of 50 is equivalent to an extra synergistic effect of 50%, which is spread over a square of 1 log x 1 log in the 2-d log-concentration space. • SYN_SUM_WEIGHTED - The integrated weighted synergy incorporates a weight based on the dose response which bias the total score towards synergy achieving highest effect. Hence, a synergy of 50% leading to a combined full effect (100%) will have more weight than if the corresponding effect was only 20 or 30%. • SYN_AVERAGE_C1 - Concentration value of drug 1 where synergy appears to be localized. • SYN_AVERAGE_C2 - Concentration value of drug 2 where synergy appears to be localized. [00598] Combination effects are also analyzed using the Additive model (see Valeriote et al., Cancer Chemother Rep.1975, 59:895–900; Lepri et al., Hematol Oncol.1991, 9:79–86; and Jonsson E et al., Eur J Clin Pharmacol.1998, 54:509–14.) In samples with two co-incubated substances, a predicted cell viability (%) is calculated according to the following formula: Predicted cell viability (%) = cell viability of substance 1 (%) x cell viability of substance 2 (%) x 0.01. [00599] A “combination index” (CI) is then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability. The following classifications are used in this example: CI > 1.2 sub-additive effect CI = 0.8 - 1.2 additive effect CI < 0.8 synergistic effect CI < 0.5 strong synergistic effect [00600] If the measured cell viability for a combination of two substances is higher than the cell viability for one or both substances, the effect is considered antagonistic. If the predicted viability is very low, the quote “measured viability / predicted viability” may give false CI values. Thus, a lower limit of < 5% of the predicted viability may be set. 4.6 Assays for Testing Synergistic Effects of an Activator of P53 and an Inhibitor of PD-1 Mediated Signaling [00601] Also provided herein are assays for demonstrating the effects of the combination treatment with an agonist of p53 and an inhibitor of PD-1 mediated signaling. [00602] In certain embodiments, the p53 agonist has synergism with the inhibitor of PD-1 mediated signaling (e.g., an inhibitor of PD-1 such an anti-PD-1 antibody) in activating immune cells. In certain embodiments, the p53 agonist disclosed herein activates normal p53 in immune cells. [00603] In one embodiment, any synergistic effects in activating or enhancing immune cells can be determined according to in vitro functional assays well known to those persons of ordinary skills in the art (Wang et al., Cancer Immunol. Res.2:846-856 (2014)). The synergistic effects to promote T-cell responses can be evaluated in vitro, e.g., using human T cells. In some embodiments of such in vitro assays, enhancement of IFNγ release or enhanced T-cell proliferation can be measured to indicate the activation and stimulation of immune cells. In other embodiments of such in vitro assays, PD-L1 expression and/or upregulation can be measured to indicate the activation and stimulation of immune cells. Mixed Lymphocyte Reaction Assay [00604] In this assay, IFNγ secretion and T-cell proliferation are measured to indicate the activation and stimulation of immune cells (e.g., T cells). Dendritic cells (DC) are generated by culturing monocytes isolated from PBMCs using a monocyte purification kit in vitro for 7 days with 500 U/mL interleukin-4 (IL-4) and 250 U/mL GM-CSF. CD4+ T cells (1 x 105) and allogeneic DCs (1 x 104) are co-cultured with or without dose titrations of a PD-1 mediated signaling inhibitor, which is added at the initiation of the assay. After 5 days, IFNγ secretion in culture supernatants is analyzed by ELISA and cells are labeled with 3H-thymidine for an additional 18 hours to measure T-cell proliferation. Assay of Staphylococcal Enterotoxin B Stimulation of PBMCs [00605] In this assay, IL-2 levels are measured to indicate the activation and stimulation of immune cells (e.g., T cells) in the presence of staphylococcal enterotoxin B (SEB). PBMCs from healthy human donors (N = 18) are cultured for 3 days with a PD-1 mediated signaling inhibitor or an isotype control at the initiation of the assay together with serial dilutions of staphylococcal enterotoxin B (SEB). IL-2 levels in culture supernatants are measured by ELISA analysis. Cytomegalovirus (CMV)-Restimulation Assay [00606] In a cytomegalovirus (CMV)-restimulation assay, antigen-specific recall response in vitro is measured. In this assay, 2 x 105 PBMCs from a CMV-positive donor are stimulated using lysate of CMV-infected cells, with serial dilutions of PD-1 mediated signaling inhibitor, which is added at the initiation of the assay. After 4 days, supernatants are assayed for IFNγ. Suppression Assay with Regulatory T Cells [00607] CD4+CD25+ regulatory T cells and CD4+CD25- responder T cells are purified from PBMCs (CD4+CD25+ Treg isolation kit; Miltenyi Biotec). In an allogeneic mixed lymphocyte reaction (MLR) assay, Tregs (5 x 104) are co-cultured with 1 x 105 responder T cells and 2 x 104 monocyte-derived DCs, with a PD-1 mediated signaling inhibitor. After 5 days, IFNγ production is assessed in supernatants, and cells are labeled with 3H-thymidine for an additional 18 hours for proliferation analysis. Restoration of T cell proliferation and IFNγ release by the alloreactive T cells is measured to indicate the activation and stimulation of responder T cells, which are suppressed by Tregs. [00608] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include, aspects that are not expressly included in the invention are nevertheless disclosed herein. [00609] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of invention described in the claims. 5. EXAMPLES [00610] The following is a description of various methods and materials used in the studies, and are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for. 5.1 Example 1: A Phase 1/2 Study of APR-246 in Combination with Pembrolizumab in Subjects with Solid Tumor Malignancies [00611] This clinical trial is a phase 1/2, open-label, study to determine the safety and preliminary efficacy of APR-246 in combination with pembrolizumab in subjects with solid tumor malignancies including gastric/gastroesophageal junction (GEJ) cancer, bladder/urothelial cancer, and non-small-cell lung cancer (NSCLC). The study includes a safety lead-in portion comprised of subjects with advanced non-CNS-primary solid tumors, followed by an expansion portion with separate cohorts for subjects with gastric/GEJ cancer, urothelial cancer and non- small-cell lung cancer (see FIG.1). [00612] During the safety lead-in portion of the study, subjects with advanced solid tumor malignancies receive pembrolizumab in combination with APR-246, as outlined in FIG.2. [00613] Dosing begins at the full dose of both drugs. Lower dose levels of APR-246 are used as necessary depending on observed toxicity. [00614] The initial cohort of subjects receive APR-246 at 4.5 g/day administered as an intravenous (IV) infusion on Days 1–4 in combination with pembrolizumab at the dose of 200 mg on Day 3 of each 21-day cycle. This cohort enrolls up to a maximum of 6 subjects. Dose-limiting toxicity (DLT) is assessed after three subjects are enrolled in each respective cohort and the last enrolled subject completes the 3-week safety assessment period (i.e., one cycle of combination regimen). A subject that discontinues therapy during Cycle 1 without DLT is considered evaluable for the purpose of safety only if at least 75% of scheduled doses of APR- 246 are administered in the first cycle. At the first dose level of 4.5 g/day of APR-246, if ≤ 1 subject out of 3 experiences a DLT, 3 additional subjects are enrolled. If ≤ 1 subject out of 6 experiences DLT, the dose level (4.5 g/day of APR-246) is deemed the recommended phase 2 dose (RP2D) for that cohort. If ≥ 2 subjects out of the total 3 – 6 subjects in the cohort experience DLT, the study continues enrollment at Dose Level -1 (4.0 g/day of APR-246). If ≤ 1 subject out of 6 experiences DLT at this dose level, the dose level (4.0 g/day of APR-246) is deemed the RP2D for that cohort. If ≥ 2 subjects out of the total 3 – 6 subjects at that dose level experience DLT, the study continues enrollment at Dose Level -2 (3.5 g/day of APR-246). If ≤ 1 subject out of 6 experiences DLT at this dose level, the dose level (3.5 g/day of APR-246) is deemed the RP2D for that cohort. If ≥ 2 subjects out of the total 3 – 6 subjects at this dose level experience DLT, the trial is halted and the Data Review Team (DRT) considers dosing modifications. [00615] The phase 2 portion begins once the recommended phase 2 dose (RP2D) of APR-246 in combination with pembrolizumab is determined and assess the antitumor activity of this combination. Up to 100 subjects are enrolled in three cohorts according to their underlying disease, as outlined in FIG.1. 5.2 Example 2: A Phase 1 and Dose Expansion Study of APR-246 in Combination with Ibrutinib in Subject with TP53-Mutant Relapsed and/or Refractory Non-Hodgkin Lymphoma (NHL) [00616] This example relates a Phase 1, open-label, dose-finding and cohort expansion study to determine the preliminary safety, tolerability, and pharmacokinetic (PK) profile of APR-246 in combination with ibrutinib therapy in subjects with TP53-mutant NHL, including relapsed and/or refractory (R/R) CLL and R/R MCL. The study includes a safety lead-in dose de- escalation portion in subjects with R/R CLL. Once the safety, tolerability, and PK of APR-246 in combination with ibrutinib is established, an expansion portion proceeds with APR-246 in combination with ibrutinib therapy in subjects with R/R CLL and R/R MCL. The study design is described in FIG.3. 5.2.1 Study Design Safety Lead-in Study [00617] In the safety lead-in portion of the study to determine the Recommended Phase 2 dose (RP2D), a safety lead-in cohort enrolls subjects with TP53-mutant CLL in a 3 + 3 dose de- escalation design: APR-246 + ibrutinib (N ≤ 28), in subjects with no prior Bruton's tyrosine kinase (BTK) inhibitor therapy (ex. ibrutinib). [00618] In safety lead-in cohort, treatment consists of APR-246 at 4.5 g/day on days 1–4 of each 28-day cycle administered concurrently with ibrutinib at standard dose in R/R CLL of 420 mg PO daily. [00619] The safety lead-in cohort initially enrolls 3 subjects. Subjects are assessed for dose limiting toxicity (DLT) related to APR-246 with ibrutinib combination therapy after the first 3 subjects are enrolled in respective cohorts and the last enrolled subject has completed the 4-week safety assessment period (i.e., one cycle of combination regimen). [00620] The does de-escalation is as follows: • The safety lead-in cohort initially enrolls 3 subjects • If ≤1 subject out of 3 experiences a dose limiting toxicity (DLT), 3 additional subjects are recruited and treated at the same dose level (4.5 g/day of APR-246 on Days 1–4 of each 28-day cycle). • If ≥ 2 subjects out of 3–6 subjects in a cohort experience a DLT, the study continues enrollment of 3 additional subjects at Dose Level -1 (4.0 g/day of APR- 246 on Days 1–4 of each 28-day cycle). • If >1 subject out of 3 experience DLT, no additional subjects is recruited and treated at the same dose level. If ≤1 subject out of 3 experiences DLT at the reduced dose of 4.0 g/day of APR-246 in the first 28 days of Cycle 1, 3 additional subjects are recruited and treated at the same dose level. • If ≥ 2 subjects out of the total 3–6 subjects in the cohort of Dose Level-1 experience DLT, the study continues enrollment of 3 additional subjects at Dose Level-2 (3.5 g/day of APR-246 on Days 1–4 of each 28-day cycle). • If ≤1 subject out of 3 experiences a DLT, 3 additional subjects are recruited and treated at the same dose level (3.5 g/day of APR-246 on Days 1–4 of each 28-day cycle). • If ≥ 2 subjects of the total 3-6 subjects in the cohort experience DLT, the study is temporarily discontinue enrollment and the Data Review Team consider further enrollment and possible dose/schedule adjustments. [00621] Dose modification is summarized in Table 4 below. Table 4. Dose Modification
Figure imgf000122_0001
Figure imgf000123_0001
Expansion Study [00622] The expansion portion of the study begins after the preliminary safety, tolerability, or PK profile of APR-246 in combination with ibrutinib has been established. Expansion proceeds with APR-246 in combination with ibrutinib in subjects with R/R CLL and R/R MCL. In expansion, up to 60 subjects are enrolled and stratified into one of two cohorts: (1) Expansion Cohort 1: subjects with TP53-mutant R/R CLL (N ≤ 20); and (2) Expansion Cohort 2: subjects with TP53-mutant R/R MCL (N ≤ 40). [00623] In Expansion Cohort 1, subjects with R/R CLL receive APR-246 at the RP2D identified in safety lead-in study of APR-246 with ibrutinib based on safety, PK, and/or preliminary efficacy data in the Safety lead-in cohort. [00624] In Expansion Cohort 2, subjects with R/R MCL receive APR-246 at the RP2D identified in safety lead-in study of APR-246 with ibrutinib based on safety, PK, and/or preliminary efficacy data in the Safety lead-in cohort. [00625] If ibrutinib is selected for combination with the RP2D of APR-246 in the expansion phase for patients with MCL, the DRT will review safety data after the first 5 patients with MCL have been treated to determine if the RP2D of APR-246 is safe and well tolerated in combination with ibrutinib administered at 560 mg daily. Based on safety, tolerability, and available PK data, the DRT makes recommendations to continue treating MCL patients at the daily ibrutinib dose of 560 mg and APR-2464.5 g daily on Days 1-4 or if a lower dose of APR-246 should be evaluated in MCL patients enrolled in the expansion cohort with ibrutinib. [00626] Responses of CLL subjects are assessed according to disease specific response criteria for R/R CLL, e.g., the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) 2018 guidelines for response assessment of CLL via blood, bone marrow (BM), and imaging (computed tomography [CT]), as appropriate, on Day 1 of Cycles 4, 7, 13, 19, and 25 (Hallek, M., et al., Blood 131(25), 2745-2760 (2018)). Responses and PD of CLL subjects are assessed according to Revised Criteria for Response Assessment of Hodgkin’s and Non- Hodgkin’s Lymphoma (Lugano Criteria) via blood, bone marrow (BM), and imaging (positron emission tomography [PET]/CT), as appropriate, on Day 1 of Cycles 4, 7, 13, 19, and 25 (van Heertum, R. L., et al., Drug. Des. Devel. Ther.11, 1719-1728 (2017)). If IV contrast is contraindicated, CT without contrast can be used. For patients with bone marrow (BM) disease, BM assessments are done at Day 1 of Cycle 7, 13 and 25 months and if required to confirm complete response. For patients who achieve complete remission, imaging may be omitted and MRD assessment is completed using flow cytometry and/or molecular techniques. [00627] In each portion of the study, subjects may continue treatment as long as toxicity remains acceptable and the subject has not withdrawn consent. [00628] Further study is designed based on an integrated assessment of safety, tolerability, and preliminary evidence of clinical activity. The objective of the expansion portion is to gain additional safety tolerability, PK, and preliminary efficacy data regarding the combination therapy of APR-246 with ibrutinib. 5.2.2 Study Endpoints [00629] Primary endpoints of the study are as follows: (1) Occurrence of dose limiting toxicity (DLT), classified and graded according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events (NCI-CTCAE, version 5.0); (2) Frequency of treatment-emergent adverse events (TEAEs), and serious adverse events (SAEs) related to APR- 246 in combination with ibrutinib therapy; and (3) The highest dose of APR-246 with acceptable toxicity (the Recommended Phase 2 dose (RP2D) of APR-246) (the dose producing ≤ 20% of DLT). [00630] Secondary endpoints of the study include: (1) pharmacokinetic parameters: Cmax (maximum concentration), AUC (area under the curve), Vd and clearance (CL) of APR-246 and Cmax (maximum concentration), Tmax (time of maximum concentration), and AUC (area under the curve) of ibrutinib; (2) complete remission (CR) rate, defined as the proportion of subjects who achieve CR as per disease-specific response criteria; (3) overall response rate (ORR), defined as the proportion of subjects achieving a response, as per disease-specific response criteria; (4) Duration of response (DOR), defined as a time from documentation of tumor response to disease progression or death as a result of any cause; (5) progression-free survival (PFS), defined as the time from the first study dose date to the date of first documentation of confirmed disease progression or death (whichever occurs first). [00631] The exploratory endpoints include: (1) Exploratory analyses of molecular markers for response predication and monitoring may include, but are not limited to: TP53 VAF by Next- generation sequencing (NGS), mutations in other genes by NGS, RNA expression; and (2) the exposure response relationship for safety and efficacy of APR-246 when combined with ibrutinib therapy. 5.2.3 Inclusion Criteria [00632] Subjects must meet the inclusion criteria to be eligible to be enrolled, e.g., among other criteria, the subject must have at least one TP53 mutation that is not benign or likely benign based on local testing. [00633] In the detailed inclusion criteria, the subject: 1. Is able to understand and is willing and able to comply with the study requirements and to provide written informed consent. 2. has documented histologic diagnosis of R/R CLL or R/R MCL by WHO criteria with at least two prior systemic therapies. In R/R CLL, response and PD are assessed by iwCLL 2018 criteria; in R/R MCL, response and PD are assessed by Lugano criteria. 3. has most recent regimen that did not include BTK inhibitor therapy (e.g., ibrutinib, acalabrutinib, zanubrutinib, experimental BTK inhibitor) 4. Has Prothrombin time (or international normalized ratio) and partial thromboplastin time not to exceed 1.2 times of the institution’s normal range (patients with an elevated prothrombin time and known lupus anticoagulant may be eligible for participation after consulting the Medical Monitor). 5. Has adequate bone marrow function independent of growth factor or transfusion support, per local laboratory reference range at screening as follows: a. platelet count ≥ 75000/mm3; b. ANC ≥ 1000/mm3 unless cytopenia is clearly due to marrow involvement from CLL or MC; c. total hemoglobin ≥ 9 g/dL (without transfusion support within 2 weeks of screening); d. If any of the above-mentioned cytopenias (a-c) are present due to significant BM involvement (requiring transfusion or G-CSF support) CLL/MCL patients may proceed with enrollment after discussion with the Medical Monitor. Cytopenias may not be due to evidence of MDS or hypoplastic BM. 6. Has adequate organ function as defined by the following laboratory values: a. Creatinine clearance ≥ 30 mL/min (by Cockcroft-Gault or MDRD method); b. Total serum bilirubin ≤ 1.5 × ULN unless due to Gilbert’s syndrome, NHL organ involvement, controlled immune hemolysis or considered an effect of regular blood transfusions; c. ALT and AST ≤ 3 × ULN, unless due to NHL organ involvement. 7. Age ≥18 years at the time of signing the informed consent form. 8. At least one TP53 mutation which is not benign or likely benign (refer to Lab Manual) based on local testing. 9. ECOG performance status of 0, 1 or 2. 10. Projected life expectancy of ≥ 12 weeks. 11. Female patients must be surgically sterile, postmenopausal (for at least 1 year), or have negative results for a pregnancy test performed at screening, on a serum sample obtained within 7 days prior to initiation of study treatment. 12. Women of childbearing potential and men with female partners of childbearing potential must be willing to use an effective form of contraception. a. Subjects who are enrolled should use an effective form of contraception for up to 30 days after the last dose of APR-246 or up to 4 months after the last dose of ibrutinib, whichever time period is longer. 5.2.4 Treatment Administration [00634] Study treatment is administered on an outpatient basis. No investigational or commercial agents or therapies other than those described herein is administered with the intent to treat the subject’s disease. Administration of APR-246 [00635] 2-(Hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one (APR-246) is administered as a 6-hour IV infusion daily on days 1–4 of each 28-day cycle. The APR-246 starting dose is fixed at 4.5 g. APR-246 is administered in a 2-step infusion: • Step 1: Loading dose of 1.5 g for the first 45 minutes (± 2 min); and • Step 2: Maintenance dose of 3 g over 5 hours 15 minutes (± 30 min). [00636] The dose of APR-246 may be reduced per the safety lead-in dose de-escalation procedure, or treatment interrupted if the subject develops AEs. [00637] APR-246 vials are stored at 2 – 8 oC (35.6 – 46.4 oF). At the pharmacies and at the study centers, the prepared APR-246 study product (diluted in sodium chloride solution) are stored at not more than 25 ºC. The infusion is completed within 24 hours from the time of preparation. [00638] Detailed instructions on vial concentration, preparation, and dispensing can be found in the Pharmacy Binder. The infusion timing, including start/stop times and the time of rate change are recorded. Administration of Ibrutinib [00639] Ibrutinib is administered at standard dose of 420 mg for R/R CLL and 560 mg for R/R MCL orally daily together with a meal on each day of a 28-day cycle. On Day 1 of Cycle 1, ibrutinib is taken 1 hour prior to administration of the APR-246 infusion. [00640] Subjects may remain on study treatment to the end of the trial while deriving clinical benefit, unless unacceptable toxicity, progression, death or subject withdrawal. Subjects may remain on study treatment after disease progression if they are continuing to derive clinical benefit in the opinion of the investigator. Dose Limiting Toxicity [00641] A DLT is defined as any of the TEAEs defined by the National Cancer Institute’s Common Terminology Criteria for Adverse Events, version 5.0 (NCI-CTCAE v5.0), and for hematologic AEs in R/R CLL/MCL patients, iwCLL criteria for CLL for hematologic toxicity, occurring during the first 28 days of study drug, unless the AE is clearly unrelated to study drug (related to the patient’s disease under study, their medical condition or concomitant medications, or clearly attributable to ibrutinib). [00642] DLT definitions are: • Any ≥ Grade 3 non-hematologic toxicity except for: • First occurrence of Grade 3 electrolyte abnormalities and/or creatinine clearance decrease resolving to Grade 2 (or baseline if baseline is ≥ Grade 2) within 48 hours with supportive treatment. • Grade 3 fatigue, nausea, vomiting, diarrhea or other manageable constitutional symptom that is responsive to supportive therapy. • Grade 3 infection responding to appropriate antimicrobial therapy. • Any neurologic toxicity of grade 4, or grade 3 that does not return to ≤
Figure imgf000128_0001
grade 1 or baseline within 7 days. • Any ≥ Grade 3 hematologic toxicity are considered a DLT except for: • Grade 3 neutropenia without fever • Grade 4 neutropenia without fever lasting 8 days or less • Grade 3 thrombocytopenia that does not result in bleeding or transfusion • Grade 3/4 lymphopenia/lymphocytosis • Grade 3/4 WBC decreased • Grade 3/4 WBC increased [00643] Any toxicity, regardless of the NCI-CTCAE v5.0 grade, resulting in discontinuation, dose reduction or treatment with less than 75% of planned doses of APR-246 study drug, are reviewed by the DRT, and a considered a DLT if determined that the toxicity is clearly related to study drug, unless reversible CNS-related effects previously described for APR-246 or related to the patient’s underlying disease, other medical condition or concomitant medications, or clearly attributable to ibrutinib. [00644] G-CSF support for the management of neutropenia is allowed including during the DLT period. Recommended Phase 2 Dose [00645] The Recommended Phase 2 Dose (RP2D) of APR-246 is defined as the dose at which less than 2 out of 6 subjects experience DLT during the 4-week safety assessment period after administration of APR-246 in combination with ibrutinib. Up to 10 additional patients may be enrolled at the RP2D to confirm the confidence at that dose level. [00646] Data Review Team (DRT) consisting of the Medical Monitor, Site Principal Investigators, and other clinical research personnel that the Sponsor may deem appropriate, hold Data Review Meetings (DRMs) on an interim basis at a frequency dependent on study accrual. At these meetings, the DRT review AEs and DLTs and make recommendations regarding the RP2D. In the expansion portion of the study, the DRT evaluate safety and tolerability after 5 subjects have completed 1 cycle of treatment in each cohort. All accumulated safety data are discussed during DRMs. 5.2.5 Statistics [00647] Demographic data and disease-related characteristics are summarized using descriptive statistics (count and percent, mean, median, standard deviation, minimum, maximum). Continuous variables are presented by n, mean, median, standard deviation and range (minimum and maximum), and categorical variables are presented by count and percentage of subjects as appropriate. Data are presented by each dose cohort in safety lead-in dose-finding portion and by each treatment arm and dose cohort in the expansion portion. All subject data, efficacy and safety data are summarized. Sample Size [00648] A total of approximately 120 evaluable patients are included in the study. In the Safety Lead-In portion of the study, two cohorts enroll subjects with TP53-mutant R/R CLL in a 3+3 dose de-escalation design. A maximal of 6 x 6 x 6 (18) DLT evaluable patients are included in this portion of the study by allowing 2 APR-246 dose reductions for the 2 cohorts. An additional up to 10 patients are enrolled at the RP2D to confirm the safety at that dose level. [00649] In the Expansion cohort, 20 patients with R/R CLL and 40 patients with R/R MCL are included to further investigate the safety profile at RP2D and efficacy effects. Analysis Populations [00650] Safety population: Subjects are evaluable for safety if they receive at least one dose of APR-246 with ibrutinib. The safety population is the primary analysis population used for all analyses such as patient disposition, patient demographics, exposure, safety parameters, and efficacy parameters. The safety population is the primary analysis population for efficacy. [00651] Efficacy evaluable (EE) population: All subjects who complete at least one treatment cycle of APR-246 and ibrutinib and who have at least one post treatment clinical response assessment. Subjects who fail to complete one treatment cycle is also considered EE if they show clear evidence of clinically significant disease progression. The EE population is the secondary analysis population for efficacy. [00652] Pharmacokinetic (PK) population: Subjects is evaluable for PK if at least one sample for PK evaluation has been obtained Safety Analysis [00653] Safety data are summarized for the safety population. These data include adverse events and laboratory parameters. AE terms are coded using the Medical Dictionary for Drug Regulatory Activities (MedDRA)®, version 22.0 or higher. AEs are summarized by System Organ Class (SOC), preferred term, severity, and relationship to treatment. Serious adverse events (SAEs), deaths, and adverse events (AEs) leading to early discontinuation of study treatment are summarized. Laboratory parameters are summarized by maximum NCI-CTCAE version 5.0 severity grade and also by change from pre-treatment to scheduled time points using descriptive statistics. Laboratory parameter listings include the normal ranges for each parameter. Each value is classified as falling above, below, or within the normal range. [00654] Only AEs related to study screening procedures are collected from the time of signing informed consent, throughout study enrollment, and up to 30 days after last dose. Data summaries include only treatment-emergent adverse events (TEAEs), defined as events occurring at the start of APR-246 infusion on Day 1, Cycle 1 up to and including 30 days after last dose of study treatment. Efficacy [00655] Overall response rate (ORR), defined as the proportion of subjects achieving complete remission (CR) or partial remission (PR) measure per Lugano criteria (see, e.g., van Heertum, R. L., et al, Drug. Des. Devel. Ther.11, 1719-1728 (2017)) for NHL (MCL), or iwCLL 2018 criteria for patients with CLL (see, e.g., Hallek, M., et al., Blood 131(25), 2745-2760 (2018)). Both Overall response rate and complete remission with exact 95% CI are summarized by cohort. [00656] Duration of response (DOR) is defined as the time from the date when criteria for response are met to the date of progressive disease (PD) or death due to any cause, whichever occurs first. Subjects alive with no progressive disease (PD) has their DOR censored at the date of the last clinical assessment. The duration of complete remission (CR) are summarized in each treatment arm by providing the median DOR together with associated 95% CI, using Kaplan- Meier methodology. DOR endpoints are not formally compared between treatment arms. [00657] Overall response is summarized in number (%) of subjects in each category of responses and ORR is analyzed by using the similar method as CR rate. [00658] Survival data are collected at treatment and follow-up periods. Subjects are followed until death. Overall survival (OS) is defined as the number of days from the first day of treatment to the date of death. Kaplan-Meier methodology is utilized. [00659] Progression-free survival (PFS) is defined as the time from the first day of treatment to disease progression or death from MDS, whichever occurs first. If neither event occurs, PFS is censored at the date of the last clinical assessment. Kaplan-Meier methodology is utilized. Pharmacokinetic Analysis [00660] The pharmacokinetics of APR-246 and ibrutinib are summarized using descriptive statistics (mean, standard deviation, CV% mean, geometric mean, CV% geometric mean) and compared with historical control data. [00661] Concentrations of APR-246 and ibrutinib are determined, and pharmacokinetic parameters (e.g., Cmax, Tmax, AUC, Vd and CL) are derived using popPK or non-compartmental methods. [00662] APR-246 AUC and Cmax are then be tested for association with signs of efficacy and safety. If an observable trend exists, a PK/PD model is developed to evaluate the exposure- response relationship between APR-246 plasma exposure and outcome measures. Demographic and clinical data (ethnicity, current age, body weight, sex, disease status, etc.) are utilized to assess intersubject variability in the PK and PK/PD relationships. Exploratory analyses [00663] Descriptive statistics/results from exploratory analyses of molecular markers for response predication and monitoring are prepared and may include but are not limited to: TP53 VAF by Next-generation sequencing (NGS), mutations in other genes by NGS, RNA expression. 5.2.6 TP53 Sequencing -- Variant Interpretation Algorithm [00664] Inclusion of patients in the study is based on TP53 sequencing performed in a laboratory at each participating site according to established local routines. A study-specific variant interpretation algorithm is used to discriminate between eligible and non-eligible TP53 sequence variants. [00665] In order to select patients with high unmet medical need due to poor prognosis, the study enrolls patients that have any TP53 mutation which is not pre-defined as “benign” or “likely benign.” Eligible TP53 mutations herein includes, for example, variants classified as pathogenic, likely pathogenic and variant of uncertain significance (VUS) in a specified database (UMD-TP53). Patients harboring at least one such TP53 sequence variant are eligible for inclusion, while patients who only have variant(s) classified as benign or likely benign are not eligible. Thus, also TP53 VUS are eligible, avoiding exclusion of patients with possible pathogenic TP53 variants presently classified as VUS. 5.3 Example 3: Synergistic Effects of APR-246 in Combination with Ibrutinib [00666] In this example, synergistic effects of an exemplary p53 reactivator (i.e., APR-246) in combination with an exemplary BTK inhibitor (i.e., ibrutinib) were tested in AML cell lines (MV4-11, MOLM-13, and KBM-3) and MDS/AML cell line SKM-1. A matrix combinational dose response screening of APR-246 and ibrutinib was performed similarly in 3 AML cell lines with different TP53 and FLT3 status and one MDS/AML cell line to assess both single agent activity and evaluate additive, synergistic or antagonistic interactions across to a range of doses. 5.3.1 Materials and Methods Materials [00667] For cell lines KBM3: ISCOVE’s modified Dulbecco's medium (Sigma Cat. no. I3390), 20% FBS (Sigma Cat. no. F084) and 1% L-glutamine (Sigma, Cat.no. G7513). [00668] For MV4-11: ISCOVE’s modified Dulbecco's medium (Sigma Cat. no. I3390), 10% FBS (Sigma Cat. no. F084) and 1% L-glutamine (Sigma, Cat.no. G7513). [00669] For MOLM-13: RPMI (R8758, Sigma Aldrich) + 10% FBS (F0804, Sigma Aldrich), heat inactivated at 56 °C for 60 min. [00670] For SKM-1: RPMI (R8758, Sigma Aldrich) + 20% FBS (F0804, Sigma Aldrich), heat inactivated at 56 °C for 60 min. Cell Assays [00671] Day 1 [00672] 75 cm2 flask (100% confluent) cells were transferred to a 15 mL tube and centrifuged at 1000 RPM for 5 min. The supernatant was discarded, and the cell pellet resuspended in 6 mL medium. The cells were counted in the TC20 automated cell counter. [00673] For MOLM-13, 10 mL cell culture medium was prepared with 90,000 cells/mL (corresponding to 4,500 cells when 50 μL is seeded out) and for other cell lines 60,000 cells/mL (corresponding to 3,000 cells when 50 μL is seeded out). Cells were seeded in 96-well plates in column 2-12, row B-G, for blank, 100 μL cell culture medium was added. [00674] A given pair of drugs was combined as a series of 10 dilution (dilution factor 2) concentrations of Ibrutinib and 7 dilution concentrations of APR-246 (dilution factor 1.5), which resulted in a 10 × 7 dose matrix according to "Repl1" Excel Combenefit template, and incubated for 72 h at 37 °C. [00675] Day 3 [00676] Plates were taken from the incubator to reach room temperature approx.30 min. 100 μL CellTiter-Glo was added to all wells (except edge wells). Plates were then shaken on a plate shaker for 3 minutes at RT thereafter the plates were set for 10 min at RT to allow cell lysis to appear. The luminescence was measured using the PerkinElmer Victorx4 instrument using the built-in program according to Instruction 11. Combenefit 2.021 was used for data analysis. CellTiter-Glo viability (CTG) assay [00677] The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells. The homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. The homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. Dose Response determinations by XLFIT [00678] For IC50 determinations, the serial dilution of compounds was performed in cell culture medium based on 1.5- or 2-fold dilution factor. The serially diluted compounds were added to the cells and incubated for 72 h followed by cell viability test using the CTG assay. For each concentration, the current viability values were calculated and IC50 values were determined by XLfit software version 5.5.0.5 (a Microsoft Excel-based plug-in to perform regression, curve fitting and statistical analysis). Analysis of combination by COMBENEFIT [00679] Combenefit (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866–2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files. Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods. In the HSA model, the synergy score quantifies the excess over the highest single drug response. In the Loewe model, the synergy score quantifies the excess over the expected response if the two drugs are the same compound. In the Bliss model, the expected response is a multiplicative effect as if the two drugs act independently (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413–2415). [00680] The experimental dose–response surface that delineates combination effects in concentration space is first read by the Combenefit software as a matrix of % of the control value across concentrations. Single agent effects are extracted from this data and fitted with a dose response curve. Based on the two single agent dose response curves, a model-based combination dose–response surface is derived. This surface provides a ‘reference’ dose–response surface for a non-synergistic (additive/independent) combination, whose characteristics are determined by the selected model (i.e., Loewe, Bliss or HSA). The experimental combination dose response surface is then compared to the model-generated one, resulting in a synergy distribution in concentration space. [00681] The graphical outputs consist of: (i) the single agent dose response data and its fitting; (ii) the combination dose response (four different displays); (iii) the model generated reference combination dose response, i.e., the prediction of effect if the drugs are not synergistic (three different displays); (iv) the resulting synergy distribution (three different displays); and (v) a graphic mapping the synergy distribution onto the dose–response surface. [00682] This synergy distribution can be further summarized via various metrics (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866-2868) as follows: • SYN_MAX - the maximum level of synergy observed. • SYN_SUM - the sum of synergy observed in concentration logarithmic space. For instance, an integrated synergy of 50 is equivalent to an extra synergistic effect of 50%, which is spread over a square of 1 log x 1 log in the 2-d log-concentration space. • SYN_SUM_WEIGHTED - The integrated weighted synergy incorporates a weight based on the dose response which bias the total score towards synergy achieving highest effect. Hence, a synergy of 50% leading to a combined full effect (100%) will have more weight than if the corresponding effect was only 20 or 30%. • SYN_AVERAGE_C1 - Concentration value of drug 1 where synergy appears to be localized. • SYN_AVERAGE_C2 - Concentration value of drug 2 where synergy appears to be localized. Analysis of combinations using the Additive model [00683] Combination effects were also analyzed using the Additive model (see Valeriote et al., Cancer Chemother Rep.1975, 59:895–900; Lepri et al., Hematol Oncol.1991, 9:79–86; and Jonsson E et al., Eur J Clin Pharmacol.1998, 54:509–14.) In samples with two co-incubated substances, a predicted cell viability (%) is calculated according to the following formula: Predicted cell viability (%) = cell viability of substance 1 (%) x cell viability of substance 2 (%) x 0.01. [00684] A “combination index” (CI) is then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability. The following classifications are used in this example: • CI > 1.2 sub-additive effect • CI = 0.8 - 1.2 additive effect • CI < 0.8 synergistic effect • CI < 0.5 strong synergistic effect [00685] If the measured cell viability for a combination of two substances is higher than the cell viability for one or both substances, the effect is considered antagonistic. If the predicted viability is very low, the quote “measured viability / predicted viability” may give false CI values. Thus, a lower limit of < 5% of the predicted viability was set. 5.3.2 Results [00686] As a single agent, both APR-246 and Ibrutinib showed dose-dependent cytotoxic activity with IC50 values ranging between 1.3-7.3 µM for APR-246 and 0.1- 8.6 µM for Ibrutinib. IC50 values are summarized in Table 5 below. Table 5. IC50 values (µM) for APR-246 (mean ± SD, n=2 or 3) and Ibrutinib.
Figure imgf000135_0001
[00687] Synergy scores were calculated with the Combenefit software using three mathematical models (Bliss, Loewe, HSA) as described above. In addition, the Additive model was used to calculate a combination index for all combinations. Maximum synergy values and synergy sum for each model from Combenefit are summarized in Table 6 below. Table 6. Maximum synergy values and synergy sum for each model
Figure imgf000136_0001
[00688] Combination Index values for the individual cell lines with various doses of APR-246 and ibrutinib are shown in Tables 7-10 below (N.C = not calculated).
Table 7. Combination index for MOLM-13
Figure imgf000137_0001
Table 8. Combination index for MV4-11
Figure imgf000137_0002
Table 9. Combination index for KBM-3
Figure imgf000137_0003
Table 10. Combination index for SKM-1
Figure imgf000138_0001
[00689] In MV4-11 and MOLM-13 cell lines, ibrutinib showed higher cytotoxicity (IC50= 0.1-0.6 µM) compared to KBM-3 and SKM-1 (IC500 = 5.6 - 8.9 µM). [00690] Thus, in MOLM-13, MV4-11, and KBM3, the combination of APR-246 and ibrutinib showed synergy using HSA, Bliss, Loewe and Additive reference models. Additive model Combination Index values corresponding to synergy and strong synergy were also obtained in these cell lines. The synergy was apparent in APR-246 concentrations ranging from 1.3 to 6.6 µM for MOLM-13, 2 to 2.5 µM for MV4-11 and 2.9 to 4.4 µM for KBM3 cell line. Ibrutinib concentrations with synergy ranged from 0.08 to 10 µM for KBM3, 0.04 to 1.25 µM for MV4-11 and MOLM-13. [00691] In contrast, the FLT3 wild type and TP53 mutated cell line SKM-1 showed an antagonistic effect in the majority of cases. KBM3 and MOLM-13 are TP53 wild type, whereas MV4-11(R248W heterozygous) and the MDS/AML cell line SKM-1 (R248Q, homo/hemizygous) are TP53 mutated. 5.4 Example 4: Synergistic Effects of APR-246 in Combination with Selinexor [00692] In this example, synergistic effects of an exemplary p53 reactivator (i.e., APR-246) in combination with an exemplary XPO1 inhibitor (i.e., selinexor) were tested in two AML cell lines (MOLM-13 with wild type TP53, and HL-60 with null TP53) and MDS/AML cell line SKM-1. A matrix combinational dose response screening of APR-246 and selinexor was performed similarly in the two AML cell lines with different TP53 status and one MDS/AML cell line to assess both single agent activity and evaluate additive, synergistic or antagonistic interactions across to a range of doses. [00693] In the MOLM-13, SKM-1 and HL-60 cell lines tested, the combination of APR-246 and selinexor was found to be synergistic in the majority of combinational doses according to the Highest Single Agent (HSA), Bliss and Loewe models. Additive model Combination Index values corresponding to synergy and strong synergy were also obtained in these cell lines. 5.4.1 Materials and Methods Materials [00694] For cell lines HL-60: ISCOVE’s modified Dulbecco's medium (Sigma Cat. No. I3390), 20% FBS (Sigma Cat. no. F084) and 1% L-glutamine (Sigma, Cat. No. G7513). [00695] For MOLM-13: RPMI (R8758, Sigma Aldrich) + 10% FBS (F0804, Sigma Aldrich), heat inactivated at 56 °C for 60 min. [00696] For SKM-1: RPMI (R8758, Sigma Aldrich) + 20% FBS (F0804, Sigma Aldrich), heat inactivated at 56 °C for 60 min. Cell Assays [00697] Day 1 [00698] 75 cm2 flask (100% confluent) cells were transferred to a 15 mL tube and centrifuged at 1,000 RPM for 5 min. The supernatant was discarded, and the cell pellet resuspended in 6 mL medium. The cells were counted in the TC20 automated cell counter. [00699] For MOLM-13, 6 mL cell culture medium was prepared with 9,000 cells/mL (corresponding to 4,500 cells when 50 μL is seeded out) and for other cell lines 60,000 cells/ml (corresponding to 3,000 cells when 50 μL is seeded out). Cells were seeded in 96-well plates in column 2-12, row B-G. [00700] A given pair of drugs (e.g., APR-246 and selinexor) was combined as a series of 10 dilution (dilution factor 2) concentrations of selinexor and 7 dilution concentrations of APR-246 (dilution factor 1.5), which resulted in a 10 × 7 dose matrix according to "Repl1" Excel Combenefit template, and incubated for 72 h at 37 °C. [00701] Day 3 [00702] Plates was taken from the incubator to reach room temperature approx.30min. 100 μL CellTiter-Glo was added to all wells (except edge wells). Plates were then shaken on a plate shaker for 3 minutes at room temperature. Thereafter, the plates were set for 10 min at room temperature to allow cell lysis to appear. The luminescence was measured using the PerkinElmer Victorx4 instrument using the built-in program according to Instruction 11. Combenefit 2.021 was used for data analysis. CellTiter-Glo viability (CTG) assay [00703] The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells. The homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. The homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. Dose response determinations by XLFIT [00704] For IC50 determinations, the serial dilution of compounds was performed in cell culture medium based on a 1.5- or 2-fold dilution factor. The serially diluted compounds were added to the cells and incubated for 72 h followed by cell viability test using the CTG assay. For each concentration, the current viability values were calculated and IC50 values were determined by XLfit software version 5.5.0.5 (a Microsoft Excel-based plug-in to perform regression, curve fitting and statistical analysis). Analysis of combination by COMBENEFIT [00705] Combenefit (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866–2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files. Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods. In the HSA model, the synergy score quantifies the excess over the highest single drug response. In the Loewe model, the synergy score quantifies the excess over the expected response if the two drugs are the same compound. In the Bliss model, the expected response is a multiplicative effect as if the two drugs act independently (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413–2415). [00706] The experimental dose–response surface that delineates combination effects in concentration space is first read by the Combenefit software as a matrix of % of the control value across concentrations. Single agent effects are extracted from this data and fitted with a dose response curve. Based on the two single agent dose response curves, a model-based combination dose–response surface is derived. This surface provides a “reference” dose–response surface for a non-synergistic (additive/independent) combination, whose characteristics are determined by the selected model (i.e., Loewe, Bliss or HSA). The experimental combination dose response surface is then compared to the model-generated one, resulting in a synergy distribution in concentration space. [00707] The graphical outputs consist of: (i) the single agent dose response data and its fitting; (ii) the combination dose response (four different displays); (iii) the model generated reference combination dose response, i.e., the prediction of effect if the drugs are not synergistic (three different displays); (iv) the resulting synergy distribution (three different displays); and (v) a graphic mapping the synergy distribution onto the dose–response surface. [00708] This synergy distribution can be further summarized via various metrics (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866-2868) as follows: • SYN_MAX - the maximum level of synergy observed. • SYN_SUM - the sum of synergy observed in concentration logarithmic space. For instance, an integrated synergy of 50 is equivalent to an extra synergistic effect of 50%, which is spread over a square of 1 log x 1 log in the 2-d log-concentration space. • SYN_SUM_WEIGHTED - The integrated weighted synergy incorporates a weight based on the dose response which bias the total score towards synergy achieving highest effect. Hence, a synergy of 50% leading to a combined full effect (100%) will have more weight than if the corresponding effect was only 20 or 30%. • SYN_AVERAGE_C1 - Concentration value of drug 1 where synergy appears to be localized. • SYN_AVERAGE_C2 - Concentration value of drug 2 where synergy appears to be localized. Analysis of combinations using the additive model [00709] Combination effects were also analyzed using the Additive model (see Valeriote et al., Cancer Chemother Rep.1975, 59:895–900; Lepri et al., Hematol Oncol.1991, 9:79–86; and Jonsson E et al., Eur J Clin Pharmacol.1998, 54:509–14.) In samples with two co-incubated substances, a predicted cell viability (%) is calculated according to the following formula: Predicted cell viability (%) = cell viability of substance 1 (%) x cell viability of substance 2 (%) x 0.01. [00710] A “combination index” (CI) is then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability. The following classifications are used in this example: • CI > 1.2 sub-additive effect • CI = 0.8 - 1.2 additive effect • CI < 0.8 synergistic effect • CI < 0.5 strong synergistic effect [00711] If the measured cell viability for a combination of two substances is higher than the cell viability for one or both substances, the effect is considered antagonistic. If the predicted viability is very low, the quote “measured viability / predicted viability” may give false CI values. Thus, a lower limit of < 5% of the predicted viability was set. 5.4.2 Results [00712] A matrix combinational dose response screening of APR-246 and Selinexor was performed in two AML cell lines with different TP53 status (MOLM-13 carries wild type TP53 and HL-60 has a large deletion of TP53) and one MDS/AML cell line to assess both single agent activity and evaluate additive, synergistic or antagonistic interactions across to a range of doses. [00713] As a single agent, both APR-246 and selinexor showed dose-dependent cytotoxic activity with IC50 values ranging between 1.3-8.1 µM for APR-246 and 0.05-0.14 µM for selinexor. IC50 values are summarized in Table 11 below. Table 11. IC50 values (µM) for APR-246 (mean ± SD, n=2) and Selinexor.
Figure imgf000142_0001
[00714] Synergy scores were calculated with the Combenefit software using three mathematical models (Bliss, Loewe, HSA) as described above. In addition, the Additive model was used to calculate a combination index for all combinations. Maximum synergy values and synergy sum for each model from Combenefit are summarized in Table 12 below. Table 12. Maximum synergy values and synergy sum for each model
Figure imgf000143_0001
[00715] Combination Index values for the individual cell lines with various doses of APR-246 and selinexor are shown in Tables 13-15 below (N.C = not calculated). Table 13. Combination index for MOLM-13
Figure imgf000143_0002
Table 14. Combination index for SKM-1
Figure imgf000143_0003
Table 15. Combination index for HL-60
Figure imgf000144_0001
[00716] In MOLM-13 cell line, Selinexor showed higher cytotoxicity (IC50= 0.05 µM) compared to HL-60 and SKM-1 (IC50 = 0.9-0.14 µM). [00717] In cell lines MOLM-13, SKM-1 and HL-60, the combination of APR-246 and selinexor showed synergy according to the HSA, Bliss and Loewe reference models. Additive model Combination Index values corresponding to synergy and strong synergy were also obtained in these cell lines. The synergy was apparent in APR-246 concentrations ranging from 1.3 to 6.7 µM for MOLM-13, 1 to 1.5 µM for SKM-1 and 1.9 to 4.4 µM for HL-60 cell line. Selinexor concentrations with synergy ranged from 0.005 to 0.16 µM for MOLM-13, 0.04 to 2.5 µM for SKM-1 and 0.16 to 10 µM for HL-60. 5.5 Example 5: Synergistic Effects of APR-246 in Combination with Selinexor in Multiple Myeloma Cell Lines [00718] In this example, synergistic effects of an exemplary p53 reactivator (i.e., APR-246) in combination with an exemplary XPO1 inhibitor (i.e., selinexor) are tested in three human multiple myeloma cell lines MOLP-8 with wild type TP53, RPMI-8226, TP53 mutated (Homozygous, 853G>A, E285K), and OPM-2 TP53 mutated (Homozygous, c.524G>A p.R175H). [00719] A matrix combinational dose response screening of APR-246 and selinexor are performed similarly to in the AML cell lines in Example 1, with three myeloma cell lines having different TP53 status, to evaluate additive, synergistic or antagonistic interactions across a range of doses. 5.5.1 Materials and Methods Materials [00720] For cell lines HL-60: ISCOVE’s modified Dulbecco's medium (Sigma Cat. No. I3390), 20% FBS (Sigma Cat. no. F084) and 1% L-glutamine (Sigma, Cat. No. G7513). [00721] For cell line RPMI-8226: RPMI (R8758, Sigma Aldrich) with 2 mM L-glutamine + 10% FBS (F0804, Sigma Aldrich), heat inactivated at 56 °C for 60 min for MOLM-13: RPMI (R8758, Sigma Aldrich) + 10% FBS (F0804, Sigma Aldrich), heat inactivated at 56 °C for 60 min. Cell Culture Method [00722] MOLP-8 cells are seeded out at ca.5 x 105 cells/mL in a 12-well plate and split 1:2 every 2-3 days, viability decreases after thawing. [00723] RPMI-8226 cells are seeded out at ca.5 x 105 cells/mL in a 6-well plate and split 1:2 every 2-3 days. [00724] OPM2 cells are seeded out at ca.1 x 106 cells/mL in a 12-well plate; maintained at 0.3-0.7 x 106 cells/mL; split 1:2 every 2-3 days; viability decreases after thawing. Cell Assays [00725] Day 1 [00726] For each cell line one well of 12 well plate (65% confluent) cells are transferred to a 15 mL tube and centrifuged at 1,000 RPM for 5 min. The supernatant is discarded, and the cell pellet is resuspended in 3 mL medium. The cells are counted in the TC20 automated cell counter. [00727] For each cell line, a 6 mL cell culture medium is prepared with approximately 24 x 104 cells/mL (corresponding to 12,000 cells when 50 μL is seeded out). Cells are seeded in 96- well plates in column 1-12, row B-H. [00728] APR-246 and Selinexor are combined as a series of 10 dilutions, (dilution factor 2) concentrations of Selinexor and 7 dilution concentrations of APR-246 (dilution factor 1.5), resulting in a 10 × 7 dose matrix, and incubated for 72 h at 37 °C. [00729] Day 3 [00730] Plates are taken from the incubator to reach room temperature for approximately 30 min. 100 μL CellTiter-Glo is added to all wells (except edge wells). Plates are then shaken on a plate shaker for 3 minutes at room temperature. Thereafter, the plates are set for 10 min at room temperature to allow cell lysis to appear. The luminescence is measured using the PerkinElmer Victorx4 instrument using the built-in program according to Instruction 11. Combenefit 2.021 is used for data analysis. CellTiter-Glo viability (CTG) assay [00731] The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method described in Example 1. Dose response determinations by XLFIT [00732] For IC50 determinations, serial dilutions of compounds are performed in cell culture medium based on a 1.5- or 2-fold dilution factor. The serially diluted compounds are added to the cells and incubated for 72 h, followed by cell viability test using the CTG assay. The IC50 values are determined as described in Example 1. Analysis of combination by COMBENEFIT [00733] The synergy is evaluated by several methods, according to Example 1, including Combenefit analyses using the standard Loewe, Bliss and HSA methods, the Additive method, and by calculation of the combination index. 5.6 Example 6: Synergistic Effects of APR-246 in Combination with Wee1 inhibitor MK-1775 [00734] In this example, synergistic effects of an exemplary p53 reactivator (i.e., APR-246) in combination with an exemplary Wee1 inhibitor (i.e., MK-1775) were tested in one MDS/AML cell lines with mutant p53 (SKM-1 (R248Q homo/hemizygous)) and one breast cancer cell line with mutant p53 (MDA-MB-231 (R280K)). [00735] In SKM-1 and MDA-MB-231 the combination of APR-246 and MK-1775 showed synergy according to the Highest Single Agent (HSA) and Bliss models. Additive model Combination Index values corresponding to synergy and strong synergy were also obtained in these cell lines. In contrast, by the Loewe model an antagonistic effect appeared in some dose combinations. 5.6.1 Materials and Methods Materials [00736] For SKM-1: RPMI (R8758, Sigma Aldrich) + 20% FBS (F0804, Sigma Aldrich), heat inactivated at 56 °C for 60 min. [00737] For MDA-MB-231, DMEM with Glutamax (31966-021, Gibco) +10% FBS (F0804, Sigma Aldrich). Equipment & Software • PerkinElmer Multimode Plate Reader Victorx4 • PerkinElmer 2030 Software 4.0 • Combenefit 2,021 software • XLFIT 5.5.5.0. Cell Assays [00738] Day 1 [00739] A 75 cm2 flask (100% confluent) MDA-MB-231 was trypsinized and suspended in 6 ml cell culture medium and a 75 cm2 flask (100% confluent) SKM-1 was transferred to 15 mL tubes. Cells were centrifuged at 1,000 rpm for 5 min. Then the supernatant was discarded, and the cell pellet was resuspended in 6 mL media. The cells were counted in the TC20 automated cell counter (EXP-18-GY8902). [00740] 6 mL cell culture medium was prepared for each cell with 60,000 cells/mL (corresponding to 3,000 cells when 50 μL is seeded out). Cells were seeded in 96-well plates in column 1-12, row B-H. For MDA-MB-231, overnight incubation before treatment required. [00741] A given pair of drugs was combined as a series of 7-10 dilution (dilution factor 2) concentrations of MK-1775 and 7 or 9 dilution concentrations of APR-246 (dilution factor 1.5), which resulted in a 10 × 7 dose matrix according to "Repl1" Excel Combenefit template, and incubated for 72 h at 37 °C. [00742] Day 3 [00743] Plates were taken from the incubator to reach room temperature approx.30 min. [00744] 100 μL CellTiter-Glo was added to all wells (except edge wells). Plates were then shaken on a plate shaker for 3 minutes at room temperature thereafter the plates were set for 10 min at room temperature to allow cell lysis to appear. The luminescence was measured using the PerkinElmer Victorx4 instrument using the built-in program according to Instruction 11. Combenefit 2.021 was used for data analysis. CellTiter-Glo viability (CTG) assay [00745] The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells. The homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. The homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. Dose response determinations by XLFIT [00746] For IC50 determinations, the serial dilution of compounds was performed in cell culture medium based on 1.5- or 2-fold dilution factor. The serially diluted compounds were added to the cells and incubated for 72 h followed by cell viability test using the CTG assay. For each concentration, the current viability values were calculated and IC50 values were determined by XLfit software version 5.5.0.5 (a Microsoft Excel-based plug-in to perform regression, curve fitting and statistical analysis). Analysis of combination by COMBENEFIT [00747] Combenefit (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866–2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files. Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods. In the HSA model, the synergy score quantifies the excess over the highest single drug response. In the Loewe model, the synergy score quantifies the excess over the expected response if the two drugs are the same compound. In the Bliss model, the expected response is a multiplicative effect as if the two drugs act independently (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413–2415). [00748] The experimental dose–response surface that delineates combination effects in concentration space was first read by the Combenefit software as a matrix of % of the control value across concentrations. Single agent effects were extracted from this data and fitted with a dose response curve. Based on the two single agent dose response curves, a model-based combination dose–response surface was derived. This surface provided a “reference” dose– response surface for a non-synergistic (additive/independent) combination, whose characteristics are determined by the selected model (i.e., Loewe, Bliss or HSA). The experimental combination dose response surface was then compared to the model-generated one, resulting in a synergy distribution in concentration space. [00749] The graphical outputs consist of: (i) the single agent dose response data and its fitting; (ii) the combination dose response (four different displays); (iii) the model generated reference combination dose response, i.e., the prediction of effect if the drugs are not synergistic (three different displays); (iv) the resulting synergy distribution (three different displays); and (v) a graphic mapping the synergy distribution onto the dose–response surface. [00750] This synergy distribution were further summarized via various metrics (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866-2868) as follows: • SYN_MAX - the maximum level of synergy observed. • SYN_SUM - the sum of synergy observed in concentration logarithmic space. For instance, an integrated synergy of 50 is equivalent to an extra synergistic effect of 50%, which is spread over a square of 1 log x 1 log in the 2-d log-concentration space. • SYN_SUM_WEIGHTED - The integrated weighted synergy incorporates a weight based on the dose response which bias the total score towards synergy achieving highest effect. Hence, a synergy of 50% leading to a combined full effect (100%) will have more weight than if the corresponding effect was only 20 or 30%. • SYN_AVERAGE_C1 - Concentration value of drug 1 where synergy appears to be localized. • SYN_AVERAGE_C2 - Concentration value of drug 2 where synergy appears to be localized. Analysis of combinations using the Additive Model [00751] Combination effects were also analyzed using the Additive model (see Valeriote et al., Cancer Chemother Rep.1975, 59:895–900; Lepri et al., Hematol Oncol.1991, 9:79–86; and Jonsson E et al., Eur J Clin Pharmacol.1998, 54:509–14.) In samples with two co-incubated substances, a predicted cell viability (%) was calculated according to the following formula: Predicted cell viability (%) = cell viability of substance 1 (%) x cell viability of substance 2 (%) x 0.01. [00752] A “combination index” (CI) was then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability. The following classifications were used in this example: • CI > 1.2 sub-additive effect • CI = 0.8 - 1.2 additive effect • CI < 0.8 synergistic effect • CI < 0.5 strong synergistic effect [00753] If the measured cell viability for a combination of two substances is higher than the cell viability for one or both substances, the effect is considered antagonistic. If the predicted viability is very low, the quote “measured viability / predicted viability” may give false CI values. Thus, a lower limit of < 5% of the predicted viability was set. 5.6.2 Results [00754] A matrix combinational dose response screening of APR-246 and MK-1775 was performed in one MDS/AML cell line and one breast cancer cell line with mutant TP53 to assess both single agent activity and to evaluate additive, synergistic or antagonistic interactions across to a range of doses. [00755] As a single agent, both APR-246 and MK-1775 showed dose-dependent cytotoxic activity with IC50 values ranging between 1.5-65 µM for APR-246 and 0.2 µM for MK-1775. IC50 values are summarized in Table 16. Table 16. IC50 values (µM) for APR-246 (mean ± SD, n=2 or 3) and MK-1775.
Figure imgf000150_0001
[00756] Synergy scores were calculated with the Combenefit software using three mathematical models (Bliss, Loewe, HSA) as described above. In addition, the Additive model was used to calculate a combination index for all combinations. Maximum synergy values and synergy sum for each model are summarized in Table 17. Table 17. Maximum synergy values and synergy sum for each model
Figure imgf000150_0002
[00757] Combination Index values for various doses of APR-246 and MK-1775 in SKM-1 cell line are shown in Tables 18-20 below (N.C = not calculated). Table 18. Combination index for SKM-1 Cell Line
Figure imgf000151_0001
Table 19. Combination index for SKM-1 Cell Line
Figure imgf000151_0002
Table 20. Combination index for SKM-1 Cell Line
Figure imgf000151_0003
[00758] Combination Index values for various doses of APR-246 and MK-1775 in MDA- MB-231 cell line are shown in Tables 21-23 below (N.C = not calculated). Table 21. Combination index for MDA-MB-231 Cell Line
Figure imgf000152_0001
Table 22. Combination index for MDA-MB-231 Cell Line
Figure imgf000152_0002
Table 23. Combination index for MDA-MB-231 Cell Line
Figure imgf000152_0003
[00759] In SKM-1 and MDA-MB-231 cell lines, the combination of APR-246 and MK-1775 showed synergy according to the HAS and Bliss reference models. Additive model Combination Index values corresponding to synergy and strong synergy were also obtained in these cell lines. The synergy was apparent in APR-246 concentrations ranging from 50 to 75 µM for MDA-MB- 231 cell line and 1 to 1.7 µM for SKM-1 cell line. MK-1775 concentrations with synergy ranged from 0.14 to 0.16 µM for SKM-1 cell line and 0.1 to 1 µM for MDA-MB-231 cell line. In contrast, by the Loewe model an antagonistic effect appeared in some dose combinations. [00760] The Loewe, Bliss and HSA use different mathematical and/ or probabilistic theories for representing synergy effects while combining different drugs. The Bliss independence model is expected to hold true for non-interacting drugs that elicit their responses independently, by targeting separate pathways. Loewe additivity, in contrast, is more compatible with the case where the drugs have similar modes of action on the same targets or pathways (Tang 2015). The HSA model assumes that the expected combination effect equals to the higher individual drug effect at the dose in the combination. [00761] From the foregoing, it will be appreciated that, although specific embodiments have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of what is provided herein. All of the references referred to above are incorporated herein by reference in their entireties.

Claims

WHAT IS CLAIMED: 1. A method of treating solid tumor malignancy in a subject, comprising administering to the subject: (i) an agonist of p53; and (ii) an inhibitor of PD-1 mediated signaling.
2. A method of treating a lymphoma in a subject, comprising administering to the subject: (i) a p53 reactivator; and (ii) a Bruton’s tyrosine kinase (BTK) inhibitor.
3. A method of treating a hyperproliferative malignancy in a subject, comprising administering to the subject: (i) a p53 reactivator; and (ii) an exportin 1 (XPO1) inhibitor.
4. A method of treating a hyperproliferative malignancy in a subject, comprising administering to the subject: (i) a p53 reactivator; and (ii) an inhibitor of Wee1-like protein kinase (WEE1).
5. The method of claim 1, wherein the agonist of p53 is a compound that can give reactivation of a mutant p53 or a compound whose metabolite can give reactivation of a mutant p53.
6. The method of claim 5, wherein the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W.
7. The method of claim 5, wherein the agonist of p53 is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity.
8. The method of claim 5, wherein the compound or metabolite promotes proper folding of mutant and wild-type p53 proteins.
9. The method of claim 5, wherein the compound or metabolite is capable of shifting the equilibrium from unfolded towards a folded structure of wild-type or mutant p53, or wherein the compound or metabolite is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or metabolite is capable of reducing aggregation of the wild-type or mutant p53.
10. The method of claim 5, wherein the compound or metabolite is capable of promoting a folded structure of wild-type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53.
11. The method of claim 10, wherein the compound or metabolite is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promotes a folded conformation.
12. The method of of any one of claims 2-4, wherein the p53 reactivator is a compound that can give reactivation of a mutant p53, or a degradation product thereof that can give reactivation of a mutant p53.
13. The method of claim 12, wherein the mutant p53 comprises a mutation selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, and R282W.
14. The method of claim 12, wherein the p53 reactivator is capable of reactivating the mutant p53 to restore at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of wild type p53 activity.
15. The method of any one of claims 12-14, wherein the compound or degradation product thereof promotes proper folding of mutant and wild-type p53 proteins.
16. The method of any one of claims 12-14, wherein the compound or degradation product thereof is capable of shifting the equilibrium from unfolded towards a folded structure of wild- type or mutant p53, or wherein the compound or degradation product thereof is capable of interfering with aggregation of misfolded wild-type or mutant p53, or wherein the compound or degradation product thereof is capable of reducing aggregation of the wild-type or mutant p53.
17. The method of any one of claims 12-14, wherein the compound or degradation product thereof is capable of promoting a folded structure of wild-type or mutant p53, and/or is capable of restoring or enhancing wild type function by covalent binding to the wild-type or mutant p53.
18. The method of any one of claims 12-14, wherein the compound or degradation product threreof is capable of binding to thiol groups in the core domain of wild-type or mutant p53 and promotes a folded conformation.
19. The method of any one of claims 5-18, wherein the compound is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one; 2,2-bis(hydroxymethyl)quinuclidin-3-one; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; 2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide; N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide; N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide; 1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide; 1-methyl-N-((3-oxoquinuclidin-2-yl)methyl)cyclopropane-1-sulfonamide; N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide; 1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; 5-methyl-1-((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; tert-butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine- 1(2H)-carboxylate; 5-methyl-1,3-bis((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; N-methyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((3-chloro-1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; N,N-dimethyl-1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; 2-((1H-1,2,4-triazol-1-yl)methyl)quinuclidin-3-one; 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carbonitrile; and 1-((3-oxoquinuclidin-2-yl)methyl)-1H-1,2,4-triazole-3-carboxamide; or a pharmaceutically acceptable salt thereof.
20. The method of claim 19, wherein the compound is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) having the following formula:
Figure imgf000157_0001
, or a pharmaceutically acceptable salt thereof.
21. The method of claim 19, wherein the compound is a compound selected from the group consisting of: 2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; 2,2,2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; and 2,2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, or a pharmaceutically acceptable salt thereof.
22. The method of claim 21, wherein the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin- 2-yl)methyl)acetamide (Compound A), or a pharmaceutically acceptable salt thereof.
23. The method of claim 21, wherein the compound is 2,2,2-trichloro-N-ethyl-N-((3- oxoquinuclidin-2-yl)methyl)acetamide (Compound B), or a pharmaceutically acceptable salt thereof.
24. The method of claim 21, wherein the compound is 2,2,2-trichloro-N-((3-oxoquinuclidin- 2-yl)methyl)acetamide (Compound C), or a pharmaceutically acceptable salt thereof.
25. The method of claim 21, wherein the compound is N-ethyl-2,2,2-trifluoro-N-((3- oxoquinuclidin-2-yl)methyl)acetamide (Compound D), or a pharmaceutically acceptable salt thereof.
26. The method of claim 21, wherein the compound is 2,2-difluoro-N-((3-oxoquinuclidin-2- yl)methyl)acetamide (Compound E), or a pharmaceutically acceptable salt thereof.
27. The method of any one of claims 1, 5-11, and 19-26, wherein the inhibitor of PD-1 mediated signaling is an anti-PD-1 antibody or an anti-PD-L1 antibody.
28. The method of claim 27, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, nivolumab, AMP-224, and AMP-514.
29. The method of claim 28, wherein the anti-PD-1 antibody is pembrolizumab.
30. The method of any one of claims 1, 5-11, 19 and 20, wherein the agonist of p53 is 2- (hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one (APR-246) and the inhibitor of PD-1 mediated signaling is pembrolizumab.
31. The method of claim 30, wherein APR-246 is administered at a dose of about 4.5 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle.
32. The method of claim 30, wherein APR-246 is administered at a dose of about 4.0 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle.
33. The method of claim 30, wherein APR-246 is administered at a dose of about 3.5 g/day for 4 days and pembrolizumab is administered at a dose of about 200 mg once in each 21-day cycle.
34. The method of any one of claims 30-33, wherein APR-246 is administered on Days 1–4 and pembrolizumab is administered on Day 3 of the each 21-day cycle.
35. The method of any one of claims 30-34, wherein APR-246 and pembrolizumab are administered for 1 to 20 cycles.
36. The method of any one of claims 1, 5-11, and 19-35, wherein the agonist of p53 is formulated in a first pharmaceutical composition and the inhibitor of PD-1 mediated signaling is formulated in a second pharmaceutical composition.
37. The method of any one of claims 1, 5-11, and 19-35, wherein the solid tumor malignancy is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer.
38. The method of any one of claims 1, 5-11, and 19-35, wherein the solid tumor malignancy is an advanced non-CNS-primary solid tumor.
39. The method of any one of claims 1, 5-11, and 19-35, wherein the solid tumor malignancy is selected from the group consisting of gastric/gastroesophageal junction (GEJ) cancer, bladder/urothelial cancer, and non-small-cell lung cancer (NSCLC).
40. The method of any one of claims 2 and 12-26, wherein the BTK inhibitor is selected from the group consisting of ibrutinib, acalabrutinib, zanubrutinib, evobrutinib, dasatinib, spebrutinib, tirabrutinib, ABBV-105, ONO-4059, LFM-A13, and HM71224, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
41. The method of claim 40, wherein the BTK inhibitor is ibrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
42. The method of any one of claims 2, 12-20, 40 and 41, wherein the p53 reactivator is 2- (hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one (APR-246) and the BTK inhibitor is ibrutinib.
43. The method of claim 42, wherein APR-246 is administered at a dose of about 4.5 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28- day cycle.
44. The method of claim 42, wherein APR-246 is administered at a dose of about 4.0 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28- day cycle.
45. The method of claim 42, wherein APR-246 is administered at a dose of about 3.5 g/day for 4 days and ibrutinib is administered daily at a dose of about 420 mg or 560 mg in each 28- day cycle.
46. The method of any one of claims 42-45, wherein APR-246 is administered on Days 1–4 and ibrutinib is administered daily of each 28-day cycle.
47. The method of any one of claims 42-46, wherein APR-246 and ibrutinib are administered for 1 to 20 cycles.
48. The method of any one of claims 2, 12-26, and 40-47, wherein the p53 reactivator is formulated in a first pharmaceutical composition and the BTK inhibitor is formulated in a second pharmaceutical composition.
49. The method of any one of claims 2, 12-26, and 40-48, wherein the lymphoma is a Hodgkin lymphoma (HL) or a non-Hodgkin lymphoma (NHL).
50. The method of claim 49, wherein the lymphoma is a non-Hodgkin lymphoma (NHL).
51. The method of claim 50, wherein the non-Hodgkin lymphoma (NHL) is a mature (peripheral) B-cell neoplasm.
52. The method of claim 50, wherein the non-Hodgkin lymphoma (NHL) is selected from the group consisting of: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, diffuse large cell B-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma (plasmacytoma), extranodal marginal zone B-cell lymphoma, nodal marginal zone lymphoma, follicle center lymphoma, and Burkitt’s leukemia (Burkitt’s lymphoma).
53. The method of any one of claims 2, 12-26, and 40-48, wherein the lymphoma is chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL).
54. The method of claim 53, wherein the lymphoma is chronic lymphocytic leukemia (CLL).
55. The method of claim 53, wherein the lymphoma is mantle cell lymphoma (MCL).
56. The method of claim 50, wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory NHL.
57. The method of claim 56, wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL).
58. The method of claim 57, wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory CLL.
59. The method of claim 57, wherein the non-Hodgkin lymphoma (NHL) is relapsed or refractory MCL.
60. The method of any one of claims 2, 12-26, and 40-59, wherein the lymphoma comprises a cancer cell having mutant p53.
61. The method of any one of claims 2, 12-26, and 40-60, wherein further comprises determining by gene sequencing if the subject has TP53 mutation.
62. The method of any one of claims 2, 12-26, and 40-61, wherein the subject has not been treated with any BTK inhibitor prior to the co-administration of the p53 reactivator and the BTK inhibitor.
63. The method of any one of claims 3 and 12-26, wherein the XPO1 inhibitor is selected from the group consisting of leptomycin A (LMA), leptomycin B (LMB), selinexor (KPT-330), Eltanexor (KPT-8602), KTP-185, KPT-249, KPT-251, KPT-276, verdinexor (KPT-335), piperlongumine, valtrate, felezonexor (CBS9106 or SL-801), and PKF050-638, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
64. The method of claim 63, wherein the XPO1 inhibitor is selinexor, or a stereoisomer, mixture of stereoisomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof.
65. The method of any one of claims 3, 12-20, and 63 and 64, wherein the p53 reactivator is 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one (APR-246) and the XPO1 inhibitor is selinexor.
66. The method of claim 65, wherein APR-246 is administered at a daily dose of about 4.5 g/day and selinexor is administered at a dose of about 80 mg/day twice per week.
67. The method of claim 65, wherein APR-246 is administered at a daily dose of about 4.0 g/day and selinexor is administered at a dose of about 80 mg/day twice per week.
68. The method of claim 65, wherein APR-246 is administered at a daily dose of about 3.5 g/day and selinexor is administered at a dose of about 80 mg/day twice per week.
69. The method of any one of claims 65-68, wherein selinexor is administered on day 1 and day 3 of each week.
70. The method of any one of claims 65-69, wherein APR-246 and selinexor are administered for 1 to 20 cycles.
71. The method of claim 65, wherein selinexor is administered at a reduced dose of about 100 mg once per week, about 80 mg once per week, or about 60 mg once per week.
72. The method of any one of claims 3, 12-26, and 63-71, wherein the p53 reactivator is formulated in a first pharmaceutical composition and the XPO1 inhibitor is formulated in a second pharmaceutical composition.
73. The method of any one of claims 4 and 12-26, wherein the inhibitor of WEE1 is selected from the group consisting of 6-(2,6-Dichlorophenyl)-2-[4-[2-(diethylamino)ethoxy] anilino]-8-methylpyrido[2,3-d]pyrimidin- 7-one (PD0166285); 9-Hydroxy-4-phenylpyrrolo[3,4-c] carbazole-1,3(2H,6H)-dione (PD0407824); 4-(2-Chlorophenyl)-9-hydroxypyrrolo[3,4-c] carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor I); 6-Butyl-4-(2-chlorophenyl)-9-hydroxypyrrolo [3,4-c]carbazole-1,3-(2H,6H)-dione (WEE1 Inhibitor II); 2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((4-(4-methylpiperazin-1-yl)phenyl) amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Adavosertib (MK-1775 or AZD1775)); and Methyl 4-(4-((2-allyl-1-(6-(2-hydroxypropan-2-yl) pyridin-2-yl)-3-oxo-2,3-dihydro-1H- pyrazolo[3,4-d] pyrimidin-6-yl)amino)phenyl)piperazine-1-carboxylate (CJM061).
74. The method of claim 73, wherein the inhibitor of WEE1 is MK-1775 having a formula of:
Figure imgf000164_0001
(MK-1775), or a pharmaceutically acceptable salt thereof.
75. The method of claim 73 or 74, wherein the p53 reactivator is formulated in a first pharmaceutical composition and the inhibitor of WEE1 is formulated in a second pharmaceutical composition.
76. The method of any one of claims 3-4, 12-26, and 63-75, wherein the hyperproliferative malignancy is a hematological malignancy.
77. The method of claim 76, wherein the hematological malignancy is leukemia, lymphoma, or myeloma.
78. The method of claim 76, wherein the hematological malignancy is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia.
79. The method of claim 76, wherein the hematologic malignancy is myeloma.
80. The method of claim 79, wherein the myeloma is multiple myeloma (MM).
81. The method of claim 76, wherein the hematologic malignancy is acute myeloid leukemia (AML).
82. The method of claim 76, wherein the hematologic malignancy is chronic lymphocytic leukemia (CLL).
83. The method of claim 76, wherein the hematologic malignancy is myelodysplastic syndromes (MDS).
84. The method of any one of claims 3-4, 12-26 and 63-83, wherein the hyperproliferative malignancy comprises a cancer cell having mutant p53.
85. The method of any one of claims 3-4, 12-26 and 63-84, wherein further comprises determining by gene sequencing if the subject has TP53 mutation.
86. The method of any one of claims 3-4, 12-26 and 63-75, wherein the hyperproliferative malignancy is a solid tumor cancer.
87. The method of claim 86, wherein the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non- melanoma skin carcinoma, a lung cancer, a cervical cancer, a prostate cancer, a head and neck cancer, and a breast cancer.
88. The method of claim 86, wherein the solid tumor cancer is breast cancer.
89. The method of claim 86, wherein the solid tumor cancer is triple negative breast cancer.
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