WO2021262874A1 - Utilisation d'un composé inhibiteur de dhodh en polythérapie anticancéreuse - Google Patents

Utilisation d'un composé inhibiteur de dhodh en polythérapie anticancéreuse Download PDF

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WO2021262874A1
WO2021262874A1 PCT/US2021/038727 US2021038727W WO2021262874A1 WO 2021262874 A1 WO2021262874 A1 WO 2021262874A1 US 2021038727 W US2021038727 W US 2021038727W WO 2021262874 A1 WO2021262874 A1 WO 2021262874A1
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cancer
inhibitor
formula
compound
cancer agent
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PCT/US2021/038727
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Gabrielle MCDONALD
Danielle ULANET
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Servier Pharmaceuticals , Llc
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    • 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
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4192—1,2,3-Triazoles
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • Dihydroorotate dehydrogenase is an enzyme that catalyzes one of the steps in the de novo pyrimidine nucleotide biosynthetic pathway. It catalyzes the only oxidation/reduction reaction in that pathway, which is the step of converting DHO (dihydroorotate) to orotate with the aid of flavin cofactor and an electron acceptor. Inhibitors of dihydroorotate dehydrogenase have been found to possess wider applications as chemotherapeutic agents.
  • the compound of Formula (I) was developed to treat conditions and disorders that would benefit from inhibition of DHODH, such as but not limited to solid cancers and hematological cancers.
  • Chkl inhibitors and DNA-PK inhibitors exert synergistic effects in reducing cell viability when used independently in combination with the compound of Formula (I).
  • the combination of the compound of Formula (I) with the Chkl inhibitors LY2606368 (prexasertib) or SCH900776 or the DNA-PK inhibitors M3814 or NU7441 synergistically inhibits the growth of a panel of AML and DLBCL cell lines. See Example 3 below.
  • this application provides a method for treating a cancer in a subject, comprising administering to the subject an effective amount of a first anti-cancer agent and an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is represented by Formula (I): or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor.
  • the pharmaceutically acceptable salt thereof is a tris(hydroxymethyl)aminomethane salt or a sodium salt.
  • this application provides the use of a first anti-cancer agent in the manufacture of a medicament for treating a cancer, wherein the medicament is used in combination with an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is represented by Formula (I): or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor.
  • the pharmaceutically acceptable salt thereof is a tris(hydroxymethyl)aminomethane salt or a sodium salt.
  • the application provides a first anti-cancer agent for use in treating a cancer in combination with a second anti-cancer agent, wherein the first anti-cancer agent is represented by Formula (I): or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor.
  • the pharmaceutically acceptable salt thereof is a tris(hydroxymethyl)aminomethane salt or a sodium salt.
  • the application provides a pharmaceutical composition comprising a first anti-cancer agent and a second anti-cancer agent, wherein the pharmaceutical composition additionally comprises an excipient, wherein the first anti-cancer agent is represented by Formula (I): or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor.
  • the pharmaceutically acceptable salt thereof is a tris(hydroxymethyl)aminomethane salt or a sodium salt.
  • Figure 1A and IB CRISPR screen implicates a role for DNA damage/repair pathways in response to the compound of Formula (I).
  • Figure 1A shows gene ontology (GO) analysis of significant hits from a genome wide CRISPR screen in A549 cells treated with the compound of Formula (I) compared to DMSO treated cells. GO terms associated with DNA repair or the DNA damage response are highlighted with boxes.
  • Figure B shows the log2 fold change depletion of sgRNAs targeting a given gene versus the p-value in A549 cells treated with the compound of Formula (I). Genes included in the highlighted GO terms in A) are indicated.
  • Figure 2 GO terms of proteins upregulated in A549 cells by treatment with the compound of Formula (I). GO analysis of proteins upregulated at least 1.5-fold in A549 cells treated with 10 mM of the compound of Formula (I) compared to DMSO. GO terms associated with DNA repair or the DNA damage response are highlighted with boxes.
  • Figure 3 GO terms of proteins upregulated in OCIFY 19 cells. GO analysis of proteins upregulated at least 1.5-fold in OCIFY 19 cells treated with 1 mM the compound of Formula (I) compared to DMSO. GO terms associated with DNA repair or the DNA damage response are highlighted with a box.
  • FIGs 4A and 4B Combination of the compound of Formula (I) with either a Chkl or a DNA-PK inhibitor results in synergistic growth inhibition in a panel of AMF and DFBCF cell lines. Dose response matrix screening was carried out with the compound of Formula (I) in combination independently with a Chkl inhibitor (either FY2606368 (prexasertib) or SCH900776) and a DNA-PK inhibitor (either M3814 or NU7441).
  • Figure 6A shows synergy scores and Best Cl (combination index) values for each combination in each cell line.
  • Figure B provides a graphical depiction of the data shown above. The dotted lines represent the cutoff for likely combinatorial synergy using the different parameters (Cl ⁇ 0.7; synergy score >2.4).
  • Figures 5 A-C Differential activation of DNA damage response and combinatorial efficacy of Chkl inhibition.
  • Figure 5A shows Western blot of gH2AC levels in cells treated for the indicated times with 10 pM of the compound of Formula (I) or DMSO control.
  • Figure 5B shows a CTG growth assay on cells treated with varying concentrations of the compound of Formula (I) and prexasertib in matrix format for 48 hours in advanced RPMI containing 10% dialyzed FBS. Relative growth rates (compared to DMSO control) for each combination are depicted and color-coded as indicated by the bar to the right of each graph. Negative values indicate cell killing.
  • Figure 5C shows Western blot of gH2AC levels in cells treated with 10 mM the compound of Formula (I) and/or 100 nM prexasertib for 18 hours.
  • the invention is directed to a method for treating a cancer in a subject by administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a Chkl inhibitor, a Chk2 inhibitor, or a DNA-PK inhibitor.
  • the compound of Formula (I) acts agonistically with Chkl inhibitors, Chk2 inhibitors, or DNA-PK inhibitors against certain cancers.
  • the cancer being treated by the disclosed methods is responsive to inhibition of dihydroorotate dehydrogenase.
  • the cancer being treated by the disclosed methods is a hematological cancer.
  • the hematological cancer is selected from myeloma, lymphoma, and leukemia.
  • the hematological cancer is selected from acute myeloid leukemia, multiple myeloma, B-prolymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B cell lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, lymphocytic lymphoma cancer of the bladder, and T-cell lymphoma.
  • the cancer is myelodysplastic/myeloproliferative neoplasms.
  • the hematological cancer is selected from chemotherapy-resistant acute myeloid leukemia, cytarabine-resistant acute myeloid leukemia, acute monocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, diffuse mixed cell lymphoma, myelodysplastic syndrome, primary effusion lymphoma, erythroleukemia, chronic myeloid leukemia, chronic monocytic leukemia, double hit diffuse large B cell lymphoma, and triple hit diffuse large B cell lymphoma.
  • the hematological cancer is selected from angioimmunoblastic lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma, blastic NK-cell lymphoma, cutaneous T-cell lymphoma, lymphoblastic lymphoma, MALT lymphoma, mediastinal large B-cell lymphoma, nodal marginal zone B-cell lymphoma, small lymphocytic lymphoma, thyroid lymphoma, follicular lymphoma, Waldenstrom's macroglobulinemia, essential thrombocythemia, chronic idiopathic myelofibrosis, and polyeythemia rubra vera.
  • the hematological cancer is acute myeloid leukemia or diffuse large B-cell lymphoma.
  • the cancer being treated by the disclosed methods is a solid tumor.
  • the solid cancer is selected from lung cancer, breast cancer (e.g., triple negative breast cancer), melanoma, glioblastoma, prostate cancer, colon cancer, pancreatic cancer, bone cancer, cancer of the head or neck, skin cancer, cutaneous or intraocular malignant endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, 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, solid tumors of childhood, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), tumor
  • CNS central nervous system
  • the cancer is selected from biliary tract cancer or cancer of the ampulla of Vater, non-small cell lung cancer, broncho alveolar carcinoma, liver cancer, cancer of the ovary, cancer of the upper aerodigestive tract, and a cancer induced by asbestos.
  • the cancer is selected from biliary tract cancer, cancer of the ampulla of Vater, non- small cell lung cancer, bronchoalveolar carcinoma, liver cancer, cancer of the ovary, and cancer of the upper aerodigestive tract.
  • the cancer is selected from triple negative breast cancer, melanoma, prostate cancer, and cancer of the esophagus.
  • the cancer is lung cancer.
  • the cancer is colon cancer.
  • the cancer being treated by the disclosed methods is a non- Hodgkin lymphoma or Hodgkin lymphoma.
  • the treated subject belongs to a subpopulation of non-Hodgkin lymphoma or Hodgkin lymphoma patients, where the non-Hodgkin lymphoma or Hodgkin lymphoma has progressed in spite of prior treatment, and for whom additional effective (curative or life-prolonging) standard therapy is not available.
  • the treated subject belongs to a subpopulation of resistant or refractory non- Hodgkin lymphoma or Hodgkin lymphoma.
  • the standard curative therapy is high-dose chemotherapy and autologous stem cell transplantation (HD-ASCT).
  • the subject's non-Hodgkin lymphoma or Hodgkin lymphoma has relapsed after HD-ASCT.
  • the treated subject belongs to a subpopulation of relapsed non- Hodgkin lymphoma or Hodgkin lymphoma (e.g., HD-ASCT-relapsed).
  • the subject is not eligible for HD-ASCT.
  • the subject has refused HD- ASCT.
  • the lymphoma being treated by the disclosed methods is a mature B-cell neoplasm, a mature T- and NK-cell neoplasm, a hodgkin lymphoma, or an Immunodeficiency-associated lymphoproliferative disorder.
  • the terms “subject” and “patient” may be used interchangeably, and mean a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like).
  • the subject is a human in need of treatment.
  • an “effective amount” of the compound of Formula (I), or a pharmaceutically acceptable salt is an amount sufficient to provide a therapeutic benefit in the treatment of a cancer or to delay or minimize one or more symptoms associated with the condition when combined with a second anti-cancer agent, such as a Chkl inhibitor, a Chk2 inhibitor, or a DNA- PK inhibitor.
  • a second anti-cancer agent such as a Chkl inhibitor, a Chk2 inhibitor, or a DNA- PK inhibitor.
  • An “effective amount” of the second anti-cancer agent, as described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition when combined with the compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • the term “therapeutically effective amount” and “effective amount” are used interchangeably.
  • an effective amount can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent.
  • an effective amount is an amount sufficient for eliciting therapeutic effects in the treatment of a cancer (including solid tumors and hematological cancers as further described herein).
  • the precise amount of the compound of Formula (I) (or pharmaceutically acceptable salt thereof), a Chkl inhibitor, a Chk2 inhibitor, and/or a DNA-PK inhibitor administered to a subject will depend on various factors, such as the given drug, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. For example, determination of an effective amount will also depend on the degree, severity and type of cell proliferation. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • an “effective amount” of any additional therapeutic agent(s) will depend on the type of drug used. Suitable dosages are known for approved therapeutic agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of the compound of Formula (I) or pharmaceutically acceptable salt thereof. In cases where no amount is expressly noted, an effective amount should be assumed. Non-limiting examples of an effective amount of the compound of Formula (I) or pharmaceutically acceptable salt thereof are provided herein below.
  • An effective amount of the compound of Formula (I), a pharmaceutically acceptable salt thereof, or a second anti-cancer agent is generally in the range from 0.1 pg to 100 mg/kg of body weight of the recipient (mammal) per day and particularly typically in the range from 1 to 10 mg/kg of body weight per day.
  • the actual amount per day for an adult mammal weighing 70 kg is usually between 70 and 700 mg, where this amount can be administered as an individual dose per day or usually in a series of part-doses (such as, for example, two, three, four, five or six) per day, so that the total daily dose is the same.
  • the effective dose of the compound of Formula (I) or pharmaceutically acceptable salt thereof to a subject can be 10 pg - 500 mg.
  • Effective amounts of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor are known to those skilled in the art.
  • treatment refers to reversing, alleviating, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment refers to reducing the size of a cancerous tumor or the number of cancer cells. Treatment may also be continued after symptoms have resolved, for example to reduce the likelihood of or delay their recurrence.
  • a “pharmaceutically acceptable salt” is a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound described herein.
  • Representative pharmaceutically acceptable, organic or inorganic base salts include, e.g., alkali metal salts (e.g., sodium), alkali earth salts (e.g., calcium), and ammonium salts (e.g., tris(hydroxymethyl)aminomethane, hydrabamine, and N-methylglucamine ammonium salt).
  • alkali metal salts e.g., sodium
  • alkali earth salts e.g., calcium
  • ammonium salts e.g., tris(hydroxymethyl)aminomethane, hydrabamine, and N-methylglucamine ammonium salt.
  • Representative pharmaceutically acceptable, organic or inorganic acid salts include, e.g., the acetate, amsonate (4,4-diaminostilbene-2, 2 -disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl
  • the invention is directed to a method for treating a cancer in a subject, comprising administering to the subject an effective amount of a first anti-cancer agent, with an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is represented by Formula (I): or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, Chk2 inhibitor, and a DNA-PK inhibitor.
  • the second anti-cancer agent is a Chkl or a Chk2 inhibitor.
  • a “Chkl inhibitor” refers to an inhibitor of checkpoint kinase 1 and a “Chk2 inhibitor” refers to an inhibitor of checkpoint kinase 2.
  • the second anti-cancer agent is a Chkl inhibitor or a dual Chkl/Chk2 inhibitor. In another embodiment, the second anti-cancer agent is a Chkl inhibitor.
  • Chkl inhibitor examples include SCH 900776 (MK-8776), LY2606368 (prexasertib), SRA737, LY2603618 (rabusertib), PF477736, CCT245737 ((R)-5-((4-((morpholin-2-ylmethyl)amino)-5- (trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile), GDC-0425, GDC-0575, UCN- 01 (7 -hydroxystaurosporine), PD-321852, SAR-020106, CCT-244747, S-024, S-144, D- 501036, V-158411, CHIR-124, and the like.
  • dual Chkl and Chk2 inhibitors examples include AZD7762, XL-844 (EXEL-9844), and the like.
  • the second anti-cancer agent is a Chk2 inhibitor or a dual Chkl/Chk2 inhibitor.
  • the second anti-cancer agent is a Chk2 inhibitor.
  • Chk2 inhibitor examples include PV1019 (NSC 744039) (7-nitro-lH-indole-2-carboxylic acid ⁇ 4-[l-(guanidinohydrazone)- ethyl]-phenyl ⁇ -amide), CCT241533, VRX0466617, C3742, ABI (2-arylbenzimidazole), hymenialdisine, debromohymenialdisine (DBH), indoloazepine, and NCS 109555 (4,4'- diacetyldiphenylurea-bis(guanylhydrazone), and the like.
  • the Chkl inhibitor is selected from SCH 900776, LY2606368 (prexasertib), 3RA737, LY2603618 (rabuseitib), PF477736, CCT245737, ( DC-0425, GDC- 0575. UCN-01, PD-321852, SAR-020106, CCT-244747, S-024. S-144 D-501Q36, V-158411, CHIR-124, AZD7762, and XL-844.
  • the Chkl inhibitor is SRA737 or LY2603618 (rabuseitib).
  • the Chkl inhibitor is LY2606368 (prexasertib) or SCH900776 (MK-8776) whose structures are shown below:
  • the dual Chkl/2 inhibitor is AZD7762 or XL-844.
  • the Chk2 inhibitor is selected from PV1019, CCT241533, VRX0466617, C3742, ABI, hymenialdisine, debromohymenialdisine, indoloazepine, and NCS 109555.
  • the method is for treating a hematological cancer in a subject, comprising administering to the subject an effective amount of a first anti-cancer agent, with an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is the compound of Formula (I) or a pharmaceutically acceptable salt thereof; wherein the second anti cancer agent is LY2606368 (prexasertib) or SCH900776 (MK-8776); and wherein the hematological cancer is acute myeloid leukemia or diffuse large B-cell lymphoma.
  • the method is for treating a solid tumor in a subject, comprising administering to the subject an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is the compound of Formula (I) or a pharmaceutically acceptable salt thereof; wherein the second anti-cancer agent is LY2606368 (prexasertib); and wherein the solid tumor is colon cancer or lung cancer.
  • a second anti-cancer agent wherein the first anti-cancer agent is the compound of Formula (I) or a pharmaceutically acceptable salt thereof; wherein the second anti-cancer agent is LY2606368 (prexasertib); and wherein the solid tumor is colon cancer or lung cancer.
  • the second anti-cancer agent is a DNA-PK inhibitor.
  • a “DNA-PK inhibitor” refers to an inhibitor of the DNA-dependent protein kinase.
  • Examples of a DNA-PK inhibitor include nedisertib (M3814/MSC2490484A) ((5)-[2-chloro-4- fluoro-5-(7-morpholinoquinazolin-4-yl)phenyl]-(6-methoxypyridazin-3-yl)methanol), NU7441 (KU-57788) (2-N- morpholino-8-dibcnzothiophcnyl-chromcn-4-onc), AZD7648 (7-methyl-2-[(7- methyl [ 1 ,2,4] triazolo [1,5- aj yridin- 6 -yl)amino] - 9 -(tetrahydro- 2H-pyran- 4 - yl) - 7
  • the DNA-PK inhibitor is selected from nedisertib, NU7441, AZD7648, VX-984, IC60211, SU-11752, CC-115, avadomide, wortmannin, vanillins, LY2094002, KU-0060648, NU7026, NU7059, and NU7427.
  • the DNA- PK inhibitor is CC-115 or avadomide.
  • the DNA-PK inhibitor is nedisertib or NU7441 (KU-57788) whose structures are shown below:
  • the method is for treating a hematological cancer in a subject, comprising administering to the subject an effective amount of a first anti-cancer agent, with an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is the compound of Formula (I) or a pharmaceutically acceptable salt thereof; wherein the second anti cancer agent is nedisertib (M3814) or NU7441 (KU-57788); and wherein the hematological cancer is acute myeloid leukemia or diffuse large B-cell lymphoma.
  • the first anti-cancer agent and the second anti-cancer agent can be administered simultaneously in the same or different formulations. In an alternative embodiment, the first anti-cancer agent and the second anti-cancer agent can be administered sequentially, i.e., at diferent points in time. [0036] In some embodiments, the first anti-cancer agent is a pharmaceutically acceptable salt of the compound of Formula (I). In one embodiment, the first anti-cancer agent is the tris(hydroxymethyl)aminomethane (“Tris”) salt of the compound of Formula (I). In another embodiment, the first anti-cancer agent is the sodium salt of the compound of Formula (I).
  • Tris tris(hydroxymethyl)aminomethane
  • the compound of Formula (I) or a pharmaceutically acceptable salt thereof is a solid state or crystalline form of the compound of Formula (I), as described in PCT/US2019/067897, filed December 20, 2019. The entire teachings of PCT/US2019/067897 are incorporated herein by reference.
  • the compound of Formula (I) is Form A, B, C, or D of the crystalline form of the compound of Formula (I).
  • the compound of Formula (I) is Form A, B, or C of the crystalline form of the Tris salt of the compound of Formula (I).
  • the compound of Formula (I) or the Tris salt thereof is in amorphous form.
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) once a week in one or more 28- day or 4- week cycles.
  • a pharmaceutically acceptable salt thereof e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for a period of 2 to 5 days each week, with 1 cycle of therapy defined as 4 consecutive weeks of treatment [0040]
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for a period of 2 to 5 consecutive days followed by a 2 to 5 day break each week, in one or more 28-day or 4-week cycle.
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for 2 consecutive days followed by a 5-day break each week, in one or more 28-day or 4-week cycles.
  • a pharmaceutically acceptable salt thereof e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g ., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for 3 consecutive days followed by a 4-day break each week, in one or more 28-day or 4-week cycles.
  • a pharmaceutically acceptable salt thereof e.g ., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for 4 consecutive days followed by a 3-day break each week, in one or more 28-day or 4-week cycles.
  • a pharmaceutically acceptable salt thereof e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for 5 consecutive days followed by a 2-day break each week, in one or more 28-day or 4-week cycles.
  • a pharmaceutically acceptable salt thereof e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for a period of 2 to 5 consecutive days followed by a 2 to 5 day break each week, in one or more 28-day or 4-week cycle, where the number of consecutive days of treatment is increased in at least one of the weeks of the 28-day or 4-week cycle, in one or more 28-day or 4-week cycles.
  • a pharmaceutically acceptable salt thereof e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) for a period of 2 to 5 consecutive days followed by a 2 to 5 day break each week, in one or more 28-day or 4-week cycle, where the number of consecutive days of treatment is decreased in at least one of the weeks of the 28-day or 4-week cycle, in one or more 28-day or 4-week cycles.
  • a pharmaceutically acceptable salt thereof e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof is administered to the subject for 2 consecutive days followed by a 5-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is increased in at least one of the weeks of the 28-day or 4-week cycle.
  • the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof is administered to the subject for 2 consecutive days followed by a 5-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is decreased in at least one of the weeks of a 28-day or 4-week cycle.
  • the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt is administered to the subject for 3 consecutive days followed by a 4-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is increased in at least one of the weeks of a 28-day or 4-week cycle.
  • the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof is administered to the subject for 3 consecutive days followed by a 4-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is decreased in at least one of the weeks of a 28-day or 4-week cycle.
  • the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof is administered to the subject for 4 consecutive days followed by a 3-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is increased in at least one of the weeks of the 28-day or 4-week cycle.
  • the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof is administered to the subject for 4 consecutive days followed by a 3-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is decreased in at least one of the weeks of a 28-day or 4-week cycle.
  • the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof is administered to the subject for 5 consecutive days followed by a 2-day break for the first week of a 28-day or 4-week cycle, where the number of consecutive days of treatment is increased in at least one of the weeks of the 28-day or 4-week cycle.
  • the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof e.g ., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • a pharmaceutically acceptable salt thereof e.g ., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) orally.
  • the effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • the effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) once daily (QD).
  • the dosing regimen for the cancer treatment method described in the foregoing paragraphs comprises administering an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., the Tris salt of the compound of Formula (I) and the sodium salt of the compound of Formula (I)) twice daily (BID).
  • compositions [0057] The same dosing regimens described above also apply to the second anti-cancer agent if it is administered concurrently with the first anti-cancer agent (i.e., the compound of Formula (I), or a pharmaceutically acceptable salt thereof) either in the same pharmaceutical formulation or in separate pharmaceutical formulations. If the second anti-cancer agent is not administered concurrently with the first anti-cancer agent, those skilled in the art will be able to determine suitable dosing regimens using routine experimentation.
  • first anti-cancer agent i.e., the compound of Formula (I), or a pharmaceutically acceptable salt thereof
  • the invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a first anti-cancer agent and a second anti-cancer agent, wherein the pharmaceutical composition additionally comprises an excipient, wherein the first anti cancer agent is represented by Formula (I): or a pharmaceutically acceptable salt thereof; and wherein the second anti-cancer agent is selected from the group consisting of a Chkl inhibitor, a Chk2 inhibitor, and a DNA-PK inhibitor.
  • the second anti-cancer agent is a Chkl inhibitor or a dual Chkl/Chk2 inhibitor. In another embodiment, the second anti-cancer agent is a Chkl inhibitor. In one embodiment, the Chkl inhibitor is selected from 8CI I 900776, LY2606368 (prexasertib), 3RA737, LY2603618 (rabusertib), PE-477736, CCT245737, GDC-0425, GDC- 0575. UCN-01, PD-321852, SAR-020106, CCT-244747, S-024. S-144. D-501036, V-158411, CHIR-124, AZD7762, and XL-844.
  • the Chkl inhibitor is SRA737 or LY2603618 (rabusertib). In another embodiment, the Chkl inhibitor is LY2606368 (prexasertib) or SCH900776 (MK-8776).
  • the second anti-cancer agent is a dual Chkl/2 inhibitor. In another embodiment, the dual Chkl/2 inhibitor is AZD7762 or XL- 844. In one embodiment, the second anti-cancer agent is a Chk2 inhibitor.
  • the Chk2 inhibitor is selected from PV1019, CCT241533, VRX0466617, C3742, ABI, hymenialdisine, debromohymenialdisine, indoloazepine, and NCS 109555.
  • the second anti-cancer agent is a DNA-PK inhibitor.
  • the DNA-PK inhibitor is selected from nedisertib, NU7441, AZD7648, VX-984, IC60211, SU-11752, CC-115, avadomide, wortmannin, vanillins, LY2094002, KU-0060648, NU7026, NU7059, and NU7427.
  • the DNA-PK inhibitor is CC-115 or avadomide. In another embodiment, the DNA-PK inhibitor is nedisertib or NU7441 (KU- 57788). [0060] In some embodiments, the first anti-cancer agent is a pharmaceutically acceptable salt of the compound of Formula (I). In one embodiment, the first anti-cancer agent is the tris(hydroxymethyl)aminomethane salt of the compound of Formula (I). In another embodiment, the first anti-cancer agent is the sodium salt of the compound of Formula (I).
  • the first anti-cancer agent and/or second anti-cancer agent may be formulated together with a pharmaceutically acceptable carrier, adjuvant, or vehicle into pharmaceutical compositions prior to being administered to a subject.
  • pharmaceutically acceptable carrier, adjuvant, or vehicle refers to a carrier, adjuvant, or vehicle that may be administered to a subject, together with the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a second anti-cancer agent and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
  • a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof, and/or a second anti-cancer agent
  • the second anti-cancer agent optionally is part of the same formulation for the first anti-cancer agent (i.e., the compound of Formula (I), or a pharmaceutically acceptable salt thereof), if it is administered concurrently with the first anti-cancer agent, or, alternatively, the second anti-cancer agent can be administered separately.
  • Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d- a- tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene
  • Cyclodextrins such as a-, b-, and g-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-P-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a second anti-cancer agent.
  • the pharmaceutical compositions may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection.
  • the pharmaceutical compositions may contain any conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles.
  • the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • suitable vehicles and solvents that may be employed are mannitol, water, Ringer’s solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions.
  • surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • the pharmaceutical compositions may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
  • carriers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried com starch.
  • aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
  • compositions may also be administered in the form of suppositories for rectal administration.
  • These compositions can be prepared by mixing the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a second anti cancer agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
  • the pharmaceutical compositions may be administered topically to the skin.
  • the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier.
  • Carriers for topical administration of the compounds of one aspect of this application include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents.
  • suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions of one aspect of this application may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topical transdermal patches are also included in one aspect of this application.
  • compositions may be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • the amount of active ingredient that may be combined with one or more pharmaceutical excipients to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration.
  • a typical preparation will contain from about 5% to about 95% active compound (w/w).
  • such preparations contain from about 20% to about 80% active compound.
  • the pharmaceutical composition comprises between about 10 mg to about 1500 mg of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (based on the weight of the free form of the compound of Formula (I), apart from the weight of any conformer, salt former, water of hydration, solvent of solvation and the like).
  • the pharmaceutical composition comprises between about 90 mg and about 240 mg; between about 100 mg and about 240 mg; between about 10 mg and about 500 mg; or between about 10 mg and about 1000 mg of the compound of Formula (I) or a pharmaceutically acceptable salt.
  • the pharmaceutical composition comprises about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 175 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 225 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 275 mg, about 280 mg, about 290 mg, or about 300 mg of the compound of Formula (I) or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition comprises about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, or about 1500 mg.
  • the pharmaceutical composition is in the form of an orally acceptable dosage form, such as for example a capsule, and comprises about 50 mg, about 75 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 175 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 225 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 275 mg, about 280 mg, about 290 mg, or about 300 mg of the Tris salt of the compound of Formula
  • the compound of Formula (I) and formulations thereof can be prepared according to methods described in WO 2014/128669, WO 2019/164794, PCT/US2019/067897, and US Patent No. 9,630,932, the contents of which are incorporated herein by reference.
  • a CRISPR depletion screen and proteomics analyses were carried out to evaluate in an unbiased manner factors contributing to response to DHODH inhibition and pointed to DNA damage response and DNA double-strand break (DSB) repair pathways as likely adaptive mechanisms.
  • co-treatment with Chkl or DNA-PK inhibitors resulted in synergistic effects with the compound of Formula (I).
  • Cell lines were purchased from ATCC or DSMZ and cultured in vendor-recommended media or Gibco advanced RPMI 1640 supplemented with 10% dialyzed FBS, 2mM glutamine and 25mM HEPES where indicated.
  • a genome-wide CRISPR depletion screen was performed in A549 cells in the presence or absence of the compound of Formula (I) to identify specific genes and pathways limiting response to the compound of Formula (I).
  • Uridine was provided in the medium at a concentration of 5 mM, resulting in alternate periods of uridine availability and depletion over the course of the culture period.
  • a VSV-G pseudotype lentiviral plasmid library containing sgRNAs was generated using the Cellecta Three-Module Human Genome -Wide CRIPSR construct library with the pRSG16-U6-sg-HTS6C-UbiC-TagRFP-2A-Puro plasmid.
  • a lentiviral plasmid encoding stable Cas9 from Cellecta (pR-CMV-Cas9-2A-Blast) was introduced into A549 cells.
  • the sgRNA library was introduced into the Cas9-expressing cells at a multiplicity of infection of 0.3 and selected under antibiotics for 48 hours.
  • sgRNAs were amplified by PCR using primers specific for the regions of the plasmid flanking the sgRNA (FwdU6-l: 5’- CAAGGCTGTTAGAGAGATAATTGG-3’ R2: 5 ’ -CGACAAC AACGC AGAAATTTTGAAT - 3’). The PCR product was sequenced using the Hiseq4000 sequencing platform with 80-100 million single-end 50 base pair reads.
  • Cells were cultured at le6/mL (OCILY 19) or 5e5/mL (A549) in standard culture media containing ImM the compound of Formula (I) or DMSO for 24 (OCILY 19) or 28 (A549) hours. Cells were collected, washed in PBS and snap frozen. Cells were treated for 28 hours under culture conditions that resulted in depletion of uridine from the medium within 16 hours; thus, the cells experienced a period of uridine availability followed by a period of uridine depletion (comparable to the conditions in the CRISPR screen).
  • Each 10- plex contained the same pooled sample labelled with TMT-131 and all relative abundance data were normalized to this channel to compare relative protein abundance across 10-plexes.
  • GO analysis of proteins with at least a 1.5-fold increase in expression in the presence of the compound of Formula (I) compared to DMSO identified DNA damage response and repair pathways as upregulated in response to the compound of Formula (I) in A549 cells ( Figure 2). Any proteins that decreased in the DMSO-treated cells by 25% or more compared to TO were excluded from the analysis.
  • EC X ECy where for a particular data point is the ratio of the X compound’s measured concentration to its effective concentration at the chosen effect level.
  • the Cl is a rough estimate of how much drug was needed in combination relative to the single agent doses required to achieve the chosen effect level. Cl values 0.5-0.7 are typical for in vitro measurements of current clinical combinations.
  • the Cl error (oCI) is calculated using standard error propagation through the Cl calculation based on the isobologram errors. Best Cl is the Cl with the largest signal-to-noise ratio level (1-CI)/ oCI.
  • Proteins were transferred to a nitrocellulose membrane using the iBlot 2 dry transfer system, blocked in Odyssey TBS blocking buffer (LI-COR), and incubated with the indicated antibodies overnight (Cell Signaling Technologies) followed by a 1 hour incubation with the appropriate secondary antibody (LI-COR) and imaged on the LI-COR Odessey CLx system.
  • LI-COR Odyssey TBS blocking buffer
  • CTG assays were performed using CTG as a readout of viable cells.
  • cells were plated in 96-well tissue culture plates (Coming) at either 10,000 cells/well for cell lines of hematologic origin or 2000 cells/well for solid tumor-derived cell lines. Cells were incubated with Formula (I) or DMSO control in combination with LY2606368 (prexasertib) for 96 hours. CTG readings were performed at TO and at T96 using a Molecular Devices SpectraMAX Paradigm plate reader.

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Abstract

L'invention concerne une méthode de traitement du cancer faisant appel à un premier agent anti-cancéreux en association avec un inhibiteur de la Chk1, un inhibiteur de la Chk2, ou un inhibiteur d'ADN-PK, le premier agent anticancéreux étant représenté par la formule (I) :
PCT/US2021/038727 2020-06-24 2021-06-23 Utilisation d'un composé inhibiteur de dhodh en polythérapie anticancéreuse WO2021262874A1 (fr)

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