WO2021228983A1 - A pharmaceutical composition comprising an arsenic compound, an inductor of type-1 ifn and a protein kinase inhibitor for treating cancer - Google Patents

A pharmaceutical composition comprising an arsenic compound, an inductor of type-1 ifn and a protein kinase inhibitor for treating cancer Download PDF

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WO2021228983A1
WO2021228983A1 PCT/EP2021/062701 EP2021062701W WO2021228983A1 WO 2021228983 A1 WO2021228983 A1 WO 2021228983A1 EP 2021062701 W EP2021062701 W EP 2021062701W WO 2021228983 A1 WO2021228983 A1 WO 2021228983A1
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malignant
carcinoma
cell
mice
leukemia
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PCT/EP2021/062701
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French (fr)
Inventor
Hugues De The
Laurent RENOU
Françoise PFLUMIO
Valérie LALLEMAND-BREITENBACH
Irina Naguibneva
Paola BALLERINI
Rima HADDAD
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives (Cea)
Centre National De La Recherche Scientifique (Cnrs)
College De France
Assistance Publique-Hôpitaux De Paris (Aphp)
Université de Paris
Université Paris-Saclay
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Publication of WO2021228983A1 publication Critical patent/WO2021228983A1/en

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    • 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
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and 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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/36—Arsenic; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention is in the field of oncology. More particularly, the invention relates to methods and compositions to treat cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein.
  • Promyelocytic leukemia (PML) expression is modified in numerous cancers and Promyelocytic leukemia-nuclear bodies (PML-NB) are disorganized in acute promyelocytic leukemia (APL) due to the promyelocytic leukemia - retinoic acid receptor alpha (PML-RARA) fusion (De The, Cancer Cell, 2017).
  • PML-NB Promyelocytic leukemia-nuclear bodies
  • APL acute promyelocytic leukemia
  • PML-RARA promyelocytic leukemia - retinoic acid receptor alpha
  • PML-RARA fusion protein is degraded by a combination of Arsenic trioxide (ATO) and Trans Retinoic Acid (ATRA) that results in the eradication of the APL leukemic clone and the long-term remission of APL patients (Lallemand-Breitenbach, JEM, 1999; de The, Nat Rev Cancer, 2018; Lo-Coco, N Engl J Med, 2013).
  • ATO Arsenic trioxide
  • ATRA Trans Retinoic Acid
  • T-ALL patient cells were found to be resistant to conventional chemotherapy. Because these cells contain a rearranged copy of Abll, the patient was shortly treated with Dasatinib, an inhibitor of tyrosine kinase (ITK, DASA) as it is done in patients with B-ALL carrying Bcr-Abl or other Abll rearrangements (Tanasi, Blood, 2019).
  • Dasatinib an inhibitor of tyrosine kinase (ITK, DASA) as it is done in patients with B-ALL carrying Bcr-Abl or other Abll rearrangements
  • MNC mononucleated cells
  • the invention relates to a method for treating a cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
  • PKI protein kinase inhibitor
  • M181Diagnosis A young 15 years-old boy was diagnosed with a T-ALL in May 2018.
  • the leukemia sample obtained from said patient was named M181Diagnosis (D).
  • Inventors have analyzed said leukemia cells and observed that a fusion between Ranbp2/Nup351 and Abl 1 was enriched in M181R.
  • the patient was already in advanced phase of disease development, the ITK treatment was not efficient enough and the boy died shortly after initiating the ITK treatment.
  • Inventors have decided to understand and to propose a new treatment to treat cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein or a protein interacting with the nuclear envelop.
  • DASA+ATO+PIPC treated mice had a significantly prolonged survival compared to the other treatment conditions (Fig 5H), showing major differences between results of treatment protocols.
  • ATO with inhibitors of TK represents a powerful way for eradication of T-ALL cells with high Abll activity and nuclear pore protein rearrangements.
  • the invention relates to a method for treating a cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
  • a method for treating a cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
  • PKI protein kinase inhibitor
  • treating refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • cancer refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.
  • cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the cancer is lymphoma.
  • lymphoma refers to a type of blood cancer that develops when lymphocytes grow out of control. Lymphomas can be grouped as Hodgkin lymphomas or non-Hodgkin lymphomas, depending on the types of cell they contain. T-cell lymphomas are non-Hodgkin lymphomas (NHL) that develop from T lymphocytes. NHL are diffuse large B-cell lymphoma (DLBCL, a high-grade B-cell lymphoma) and follicular lymphoma (a low-grade B-cell lymphoma). In children, the most common type is Burkitt lymphoma (a high-grade B-cell lymphoma).
  • the cancer is leukemia.
  • the cancer is acute lymphoblastic leukemia (ALL).
  • ALL acute lymphoblastic leukemia
  • Acute lymphoblastic leukemia has its general meaning in the art and refers to a cancer of the lymphoid lineage of blood cells characterized by the development of large numbers of immature lymphocytes.
  • Acute lymphoblastic leukemia include T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL).
  • the cancer is T-cell acute lymphoblastic leukemia (T-ALL).
  • T-ALL is a type of acute leukaemia meaning that it is aggressive and progresses quickly. It affects the lymphoid-cell-producing progenitor cells, in particular T lymphocytes as opposed to acute lymphoblastic leukaemia (ALL) which commonly affects B lymphocytes.
  • ALL acute lymphoblastic leukaemia
  • the cancer is B cell lymphomas.
  • the cancer is B cell leukemia.
  • the cancer is B-cell acute lymphoblastic leukemia (B-ALL).
  • the cancer is a solid cancer.
  • Solid cancer has various origins including breast, colon head and neck carcinomas.
  • the solid cancer is selected from the group consisting of but not limited to bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g.
  • osteoblastoma osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g.
  • ductal carcinoma in situ infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia
  • Castleman disease e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia
  • cervical cancer colorectal cancer
  • endometrial cancer e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell
  • esophagus cancer gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g.
  • choriocarcinoma chorioadenoma destruens
  • Hodgkin's disease non- Hodgkin's lymphoma, Kaposi's sarcoma
  • kidney cancer e.g. renal cell cancer
  • laryngeal and hypopharyngeal cancer liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma)
  • lung cancer e.g. small cell lung cancer, non-small cell lung cancer
  • mesothelioma plasmacytoma
  • nasal cavity and paranasal sinus cancer e.g.
  • esthesioneuroblastoma midline granuloma
  • nasopharyngeal cancer neuroblastoma
  • oral cavity and oropharyngeal cancer ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g.
  • thyroid cancer e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma
  • vaginal cancer e.g. vulvar cancer
  • uterine cancer e.g. uterine leiomyosarcoma
  • the cancer has a protein kinase gene fused to a gene coding for a nuclear-envelop protein.
  • protein kinase refers to enzyme family. Typically, protein kinase enzyme that modifies other proteins by chemically adding phosphate groups to them (phosphorylation).
  • protein kinase that modifies other proteins by chemically adding phosphate groups to them (phosphorylation).
  • protein kinase gene refers to gene encoding to protein kinase enzymes.
  • the protein kinase gene is Abll.
  • Abll refers to Abelson murine leukemia viral oncogene homolog 1. It is a proto-oncogene encoding a cytoplasmic and nuclear protein tyrosine kinase that has been implicated in processes of cell differentiation, cell division, cell adhesion, and stress response.
  • the naturally occurring human Abll gene has a nucleotide sequence as shown in Genbank Accession numbers NMNM_007313 and NM_005157.
  • the naturally occurring human Abll protein has an aminoacid sequence as shown in Genbank Accession numbers NP 005148 andNP_009297.
  • the murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM_001112703, NM_009594, NM_001283045, NM_001283046, NM_001283047 and NP_001106174, NP_001269974, NP_001269975, NP_001269976 and NP_033724).
  • the invention relates to a method for treating a cancer having ABL1 fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
  • a method for treating a cancer having ABL1 fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
  • PKI protein kinase inhibitor
  • the cancer is leukemia having ABL1 fused to a gene coding for a nuclear-envelop protein.
  • the cancer is acute lymphoblastic leukemia (ALL) having ABL1 fused to a gene coding for a nuclear-envelop protein.
  • ALL acute lymphoblastic leukemia
  • a gene coding for a nuclear-envelop protein also called as protein interacting with the nuclear envelop refers to DNA/RNA sequences coding for the entire or part of a protein that participates in nuclear-envelop architecture or takes part in protein complexes with important functions for the nuclear-envelop properties/roles in mammalian cells.
  • the gene coding for a nuclear-envelop protein is RANBP2.
  • RANBP2 refers to RAN binding protein 2 which is a protein in humans encoded by the RANBP2 gene. It is also known as nucleoporin 358 (NUP358).
  • the naturally occurring human RANBP2 gene has a nucleotide sequence as shown in Genbank Accession number NM 006267.
  • the naturally occurring human RANBP2 protein has an aminoacid sequence as shown in Genbank Accession number NP 006258.
  • the cancer has RANBP2-ABL1 fusion.
  • the gene coding for a nuclear-envelop protein is NUP214.
  • NUP214 refers to Nucleoporin 214 which is a protein in humans encoded by the NUP214 gene.
  • the naturally occurring human NUP214 gene has a nucleotide sequence as shown in Genbank Accession number NMJ305085.
  • the naturally occurring human RANBP2 protein has an aminoacid sequence as shown in Genbank Accession number NP_005076.
  • the cancer has NUP214-ABL1 fusion.
  • the invention relates to a method for treating a cancer having NUP214-ABL1 fusion or RANBP2-ABL1 fusion in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
  • a method for treating a cancer having NUP214-ABL1 fusion or RANBP2-ABL1 fusion in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
  • PKI protein kinase inhibitor
  • the cancer is leukemia having RANBP2-ABL1 fusion or NUP214-ABL1 fusion
  • the cancer is acute lymphoblastic leukemia having RANBP2- ABL1 fusion or NUP214-ABL1 fusion.
  • the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate.
  • the subject is a human afflicted with or susceptible to be afflicted with a cancer.
  • the subject suffers from T-ALL.
  • the subject suffers from B cell leukemia (B-ALL).
  • B-ALL B cell leukemia
  • the subject suffers from B cell lymphoma.
  • subject suffers from a solid cancer.
  • the subject has a protein kinase gene fused to a gene coding for a nuclear-envelop protein or a protein interacting with the nuclear envelop.
  • the subject has Ranbp2-Abll fusion.
  • the term “arsenic compound” is intended to include arsenic and any compound having the same biological properties as arsenic.
  • the expression "compound having the same biological properties as arsenic” is understood to mean any compound which, like arsenic, is an inhibitor of phosphatase and/or is capable of creating covalent adducts by binding with thiol groups.
  • the arsenic compound is selected from the group consisting of but not limited to arsenic, arsenic trioxide (As203 also called as ATO), arsenic hexoxide (AS4O6), melarsoprol and arsenic sulfur derivative.
  • the arsenic compound is ATO.
  • protein kinase inhibitor refers to any molecule that specifically blocks the action of one or more protein kinases. Protein kinase inhibitors are subdivided by the amino acids whose phosphorylation is inhibited. Most kinases act on both serine and threonine, the tyrosine kinases act on tyrosine, and a number (dual- specificity) kinases act on all three. As used herein, the term “serine/threonine kinase inhibitor” refers to a molecule that specifically blocks the action of one or more serine and/or threonine kinases.
  • the PKI is a TKI.
  • tyrosine kinase inhibitor refers to any compound, natural or synthetic molecule that specifically blocks the action of one or more tyrosine kinases. Typically, said inhibition results in a decreased phosphorylation of the tyrosine present on the intracellular domain of receptor tyrosine kinases (RTK) such as growth factor receptors.
  • RTK receptor tyrosine kinases
  • TKI may be multi-target tyrosine kinase inhibitor and may thus inhibit epidermal growth factor (EGF) receptor family (such as HER- 2); insulin-like growth factor (IGF) receptor family (such as IGF-1 receptor); platelet-derived growth factor (PDGF) receptor family, colony stimulating factor (CSF) receptor family (such as CSF-1 receptor); C- Kit receptor and vascular endothelial growth factor (VEGF) receptor family (such as VEGF- R1 (Flt-1) and VEGF-R2 (KDR/Flk-1)).
  • EGF epidermal growth factor
  • IGF insulin-like growth factor
  • PDGF platelet-derived growth factor
  • CSF colony stimulating factor
  • VEGF vascular endothelial growth factor
  • the tyrosine kinase inhibitor is a Bcr-Abl tyrosine kinase inhibitor.
  • the TKI may also be a multi-target tyrosine kinase inhibitor and may thus inhibit one or more tyrosine kinases, Bcr-Abl, but also c-Abl and the receptor tyrosine kinases PDGF-R, Fit3, VEGF-R, EGF-R, c-Kit, as well as combinations of two or more of these.
  • the Bcr-Abl TKI is selected from the group consisting of N- phenyl-2-pyrimidine-amine derivatives as described in EP0564409, pyrimidinylaminobenzamide derivatives as described in W02004005281, cyclic compounds as described in W00062778, bicyclic heteroaryl compounds as described in WO 2007075869, substituted 3-cyano quinoline derivatives as described in US6002008, 4-anilo-3- quinolinecarbonitrile derivatives as described in W0200504669 and amide derivatives as described in US7728131 and W02005063709.
  • the TKI is selected from the group consisting of but not limited to imatinib, nilotinib, dasatinib, ponatinib, bosutinib and bafetinib.
  • the TKI is Imatinib.
  • Imatinib sold under the brand name Gleevec® refers to (4-(4-methylpiperazin-l-yhnethyl)-N-[4-methyl-3-(4-pyridin-3-yl) pyrimidin-2-ylamino)phenyl-benzamide) also known as STI571 (Novartis; International Patent Publication No. WO 95/09852).
  • the TKI is nilotinib.
  • Nilotinib sold under the brand name TASIGNA® refers to 4-methyl-N-[3-(4-methyl-lH-imidazol-l-yl)- 5- (trifluoromethyl)phenyl]-3- [(4-pyridin-3-ylpyrimidin-2-yl) aminojbenzamide also known as AMN107(Novartis; International Patent Publication No. WO 2004/005281).
  • the TKI is dasatinib.
  • Dasatinib sold under the brand name SPRYCEL® refers to N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-l-piperazinyl]- 2-methyl-4-pyrimidinyl]amino]-5-thiazole carboxamide (also known as BMS-354825, Bristol- Myers Squibb) (International Patent Publication No. WO 2004/085388).
  • the term "inductor of type 1 IFNs” refers to any compound, natural or synthetic molecule that induces the expression and/or activity of type 1 IFN.
  • the induction of type 1 IFN gene involves recruitment of sequence-specific transcription factors that are activated by phosphorylation in response to signalling cascades during viral infections.
  • the inductor of type 1 IFNs is an interferon polypeptide.
  • interferon polypeptide or "IFN polypeptide” is intended to include any polypeptide defined as such in the literature, comprising for example any types of IFNs (type I and type II) and in particular, IFN-alpha, IFN-beta, IFN-omega and IFN-gamma.
  • interferon polypeptide as used herein, is also intended to encompass salts, functional derivatives, variants, muteins, fused proteins, analogs and active fragments thereof.
  • the polypeptide sequences for human interferon-alpha are deposited in database under accession numbers: AAA 52716, AAA 52724, and AAA 52713.
  • polypeptide sequences for human interferon-beta are deposited in database under accession numbers AAC41702, NP_002167, AAH 96152, AAH 96153, AAH 96150, AAH 96151, AAH 69314, and AAH 36040.
  • the polypeptide sequences for human interferon-gamma are deposited in database under accession numbers AAB 59534, AAM 28885, CAA 44325, AAK 95388, CAA 00226, AAP 20100, AAP 20098, AAK 53058, and NP-000610.
  • the inductor of type 1 IFNs is interferon-alpha (IFN-a).
  • IFN-a encompasses derivatives of IFN-a that are derivatized (e.g., are chemically modified relative to the naturally occurring peptide) to alter certain properties such as serum half-life.
  • IFN- a includes IFN- a derivatized with polyethylene glycol ("PEGylated IFN- a "), and the like. PEGylated IFN- a, and methods for making same, is discussed in, e.g., U.S. Pat. Nos. 5,382,657; 5,951,974; and 5,981,709.
  • PEGylated IFN-a encompasses conjugates of PEG and any of the above-described IFN- a molecules, including, but not limited to, PEG conjugated to interferon alpha-2a (Roferon, Hoffman La-Roche, Nutley, N. J.), interferon alpha- 2b (Intron, Schering-Plough, Madison, N.J.), interferon alpha-2c (Berofor Alpha, Boehringer Ingelheim, Ingelheim, Germany); and consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (Infergen®, InterMune, Inc., Brisbane, CA.).
  • the IFN-a has been modified with one or more polyethylene glycol moieties, i.e., pegylated.
  • pegylated-interferon peginterferon alfa-2a (40 kD) (Pegasys, Hoffmann-La Roche) and peginterferon alfa-2b (12 kD) (Peglntron, Merck), are commercially available, which differ in terms of their pharmacokinetic, viral kinetic, tolerability profiles, and hence, dosing.
  • Peginterferon alfa-2a (Pegasys) consists of interferon alfa-2a ( ⁇ 20 kD) covalently linked to a 40 kD branched polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG moiety is linked at a single site to the interferon alfa moiety via a stable amide bond to lysine.
  • Peginterferon alfa-2a has an approximate molecular weight of 60,000 daltons.
  • the biologic activity of peginterferon- alfa-2a derives from its interferon alfa-2a moiety which impacts both adaptive and innate immune responses against certain viruses.
  • the peginterferon alfa-2a Compared with the native interferon alfa-2a, the peginterferon alfa-2a has sustained absorption, delayed clear. Peginterferon alfa-2a is used as a fixed weekly dose. Peginterferon alfa-2a has a relatively constant absorption after injection and is distributed mostly in the blood and organs.
  • the inductor of type 1 IFNs is Polyinosinic:polycytidylic acid (usually abbreviated poly I:C or poly(TC); or PIPC).
  • the inductor of type 1 IFNs is Polyadenylic-polyuridylic acid (usually abbreviated poly(A:U).
  • the method according to the invention wherein the arsenic compound, the inductor of type 1 IFNs and the protein kinase inhibitor (PKI) are administered as a combined preparation.
  • PKI protein kinase inhibitor
  • the method according to the invention wherein the arsenic compound, the inductor of type 1 IFNs and the tyrosine kinase inhibitor (TKI) are administered as a combined preparation.
  • TKI tyrosine kinase inhibitor
  • the method according to the invention wherein the arsenic compound is ATO, the inductor of type 1 IFNs is PIPC and the TKI is DASA are administered as a combined preparation.
  • administering refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., ATO+ PIPC+ dasatinib) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • a disease, or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • the arsenic compound is administered orally.
  • the inductor of 1IFN and PKI are administered intravenously or subcutaneously.
  • the arsenic compound is administered orally.
  • the inductor of type 1 IFNs and TKI are administered intravenously or subcutaneously.
  • the terms “combined preparation”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication.
  • the combined therapy may be dual therapy or bi-therapy.
  • the arsenic compound, the inductor of type 1 IFNs and the protein kinase inhibitor (PKI) are administered simultaneously, separately or sequentially.
  • administration simultaneously refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time.
  • administration separately refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes.
  • administration sequentially refers to an administration of 2 active ingredients at different times, the administration route being identical or different.
  • a “therapeutically effective amount” is meant a sufficient amount of the arsenic compound, the inductor of type 1 IFNs and the protein kinase inhibitor (PKI) for use in a method for the treatment of cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
  • PKI protein kinase inhibitor
  • the invention refers to a pharmaceutical composition
  • a pharmaceutical composition comprising an arsenic compound, an inductor of type 1 IFNs and a tyrosine kinase inhibitor (TKI), as combined preparation.
  • TKI tyrosine kinase inhibitor
  • the invention refers to a pharmaceutical composition according to the invention, for use in the therapy.
  • the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of a cancer having ABL1 fused to a gene coding for a nuclear-envelop protein.
  • the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of ALL having Abll fused to a gene coding for a nuclear-envelop protein.
  • the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of ALL having ABL1-RANBP2 fusion or ABL1-NUP214 fusion.
  • the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of T- ALL having ABL1 fused to a gene coding for a nuclear-envelop protein.
  • the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of B cell leukaemia having ABL1 fused to a gene coding for a nuclear-envelop protein.
  • the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of a solid cancer having ABL1 fused to a gene coding for a nuclear-envelop protein.
  • An arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI) as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • PKI protein kinase inhibitor
  • “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
  • dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intrap eritoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the arsenic compound, the inductor of type 1 IFNs and the protein kinase inhibitor (PKI), as combined preparation is administered in combination with a classical treatment of cancer.
  • PKI protein kinase inhibitor
  • the invention also refers to a method for treating a cancer having ABL1 fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation and ii) classical treatment of cancer.
  • a method for treating a cancer having ABL1 fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation and ii) classical treatment of cancer.
  • PKI protein kinase inhibitor
  • the classical treatment refers to radiation therapy, immune checkpoint inhibitor or chemotherapeutic agent.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • chemotherapeutic agents include multkinase inhibitors such as sorafenib and sunitinib, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its thiotepa and
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolin
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • antihormonal agents that act to regulate or inhibit honnone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the term “radiation therapy” has its general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow.
  • One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy.
  • Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay.
  • the radiation therapy is external radiation therapy.
  • external radiation therapy examples include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction
  • immune checkpoint inhibitor refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins.
  • immune checkpoint protein has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules).
  • stimulatory checkpoint examples include CD27 CD28 CD40, CD 122, CD 137, 0X40, GITR, and ICOS.
  • inhibitory checkpoint molecules examples include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, PD-L1, LAG-3, TIM-3 and VISTA.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Protocol of the first round of treatment. 21 mice were treated during 15 days with a combination of DASA, ATO and PIPC. 1 mouse (group ATO+PIPC+DASA died at day 2 of treatment). After the end of the treatment, Blood and BM of mice were sampled to analyze leukemia expansion during treatment. A survival curve was started to test the effect of the treatment protocol on the viability of mice. - Days with treatment; X: days without treatment. Doses of drugs are given in Mat&Method section.
  • Figure 2 Results of the first experiment of treatment.
  • A-B Weight of mice during the treatment period.
  • C and D Levels of human CD45+hCD7+ leukemic cell infiltration in the blood (C) and the BM (D) of mice treated or not with the different drug combinations at the end of the treatment period. 5 mice/condition were used.
  • E Respective levels of human (hu)CD451owCD7high and CD45highCD71ow leukemic cells in the BM of mice at the end of the treatment period.
  • G Survival of mice in days after T- ALL transplant. Treatment period is shown in the graph. A total of 20 mice were followed up .**: p ⁇ 0.01; * ⁇ 0.05, Mann- Whitney non parametric test.
  • CD45highCD71ow are Ranbp2-Abll containing cells. Fish analysis of leukemic cells looking at Abll gene rearrangement. Shown are results obtained with 41 to 49 cells from patient cells before (black histogram) and after mouse BM engraftment. Dark grey histogram shows data from an untreated mouse recipient, whereas the two other histograms show data from cells sorted according to expression levels of huCD45 and huCD7 from an non treated mouse (mouse 1) and from a mouse treated with ATO+PIPC+DASA. No rearrangement of Abll was detected in huCD451oCD7high cells sorted from the BM of ATO+PIPC+DASA treated mouse (last column - empty - of the histogram).
  • Figure 4 Protocol of treatment extension. Mice were treated during 3 weeks with a combination of DAS A, ATO and PIPC after blood sampling. At the end of the treatment period, BM was sampled from all mice and 9 of them were sacrificed and their leukemic infiltration levels were measured in the blood, BM and spleen. Left-over mice were followed up during more than 100 days and survival was measured. - Days with treatment; X: days without treatment. Doses of drugs are similar to Figure 1.
  • Figure 5 Results of the treatment extension experiment. A-B. Weight of mice during treatment. C. Leukemic levels in the BM of all mice measured immediately after the treatment period. D. Leukemic levels in the BM and SPL of 9 mice sacrificed after treatment.
  • FIG. 6 In vitro treatments of human T-ALL cells.
  • A In vitro growth curve of M181R T-ALL cultured in presence or not of combination of ATO (50nM)+IFNg (20ng/mL) +DASA (ImM). Results are mean+/-SEM of technical triplicates.
  • B Cell numbers recovered after 72 hours of culture of Ml 81R T-ALL in presence of ImM ATO, lpMDASA and 20ng/mL IFNg.
  • C Cell cycle analysis measured in M181R cells cultured as described in (B).
  • D Apoptosis levels in cells from (B).
  • E In vitro growth of M106 T-ALL in the same conditions as in (A).
  • Figure 7 Analysis of Ranbp2-Abll phosphorylation and PML protein levels in M181R T-ALL. Quantification of RANBP2-ABL1, STAT5 and CRKL phosphorylation levels in M181R T-ALL after in vitro treatment with combination of DASA (ImM), ATO (1 mM) and IFNg (20ng/mL) during 48 hours. Results are from western blot analysis.
  • Figure 8 Establishment of patient-derived xenograft of RANBP2-ABL1 B cell acute lymphoblastic leukemia injected in primary immune-deficient mice.
  • A. Shows engraftment levels of human (hCD45+ cells) B-ALL with RANBP2-ABL1 in primary immune- deficient mice.
  • B. Shows that the recovered cells are of B cell origin (CD 19+) and part of them are CD34+.
  • Figure 9 Results of treatments of immune-deficient mice injected with RANBP2- ABL1 B cell acute lymphoblastic leukemia.
  • Human leukemic cells from primary mice were transplanted in a large cohort of secondary recipient mice. Mice were treated 6 weeks later with different combination of drugs. Treatment lasted 3 weeks.
  • BM was then sampled from all mice and tested for human leukemic cells levels. A few mice (1-3/groups) were euthanasied after treatment to measure leukemia infiltration in spleens. The rest of mice were kept for survival.
  • A Shows the results of human leukemic levels in BM samplings the day after the end of treatment.
  • B-D Shows the results of human leukemic levels in BM samplings the day after the end of treatment.
  • A, D show the number of human CD45+ leukemic cells in the BM and SPL of treated mice.
  • B shows the spleen weight of the same mice after treatment.
  • C show leukemia levels in spleen of mice injected with a NUP214-ABL1 T- ALL#1 treated with different drug combinations.
  • E, F shows leukemia levels in blood (E) and BM (F) of a second NUP214- ABL1 T-ALL (T-ALL#2) after treatment with the indicated drugs.
  • FIG. 11 Results of treatments of a T cell acute lymphoblastic leukemia that does not harbor any ABL1 gene rearrangement.
  • Immune deficient mice were injected with 5000 leukemic cells from PDX of this T-ALL. 3 weeks later, mice were treated with different drug combinations during 3 weeks. At the end of treatment, blood and BM were sampled on all live mice and human leukemia levels were measured. 2-5 mice were euthanasied to measure leukemia infiltration in BM and SPL. Left-over mice were kept for survival.
  • A, B, E show leukemia levels in the Blood (A), the BM (B) and spleen (E) of mice injected with a T-ALL with no ABLl rearrangement and treated with different drug combinations.
  • C, F show the number of human CD45+ leukemic cells in the BM (C) and in the SPL (F) of treated mice.
  • D shows the spleen weight of the mice euthanasied after treatment.
  • G show survival curves of treated mice.
  • Human T-ALL 250xl0 3 cells were cultured in 500pL of alpha-minimum essential medium (a-MEM, Gibco) containing 10% of FCS, 1% of Penicilline/Stretomycine/L- Glutamine (Gibco), recombinant human stem cell factor (hSCF ; 50 ng/mL), Fms-related tyrosine kinase 3 ligand (Flt3-L ; 20 ng/mL) (both from Miltenyi Biotech), Interleukin- 7 (IL-7, 10 ng/mL ; Preprotech) and Insulin (20 nM ; Sigma-Aldrich) in 24-well plates and incubated at 37°C in 21% of O2.
  • a-MEM alpha-minimum essential medium
  • FCS 1% of Penicilline/Stretomycine/L- Glutamine
  • hSCF recombinant human stem cell factor
  • Cells were harvested at indicated time points, numbered and further processed as to measure apoptosis, cell cycle and proteins were extracted to measure expression, phosphorylation and sumoylation levels. When necessary, equal numbers of leukemic cells were replated in medium freshly renewed every other day.
  • T-ALL cultures were set with Dasatinib (0.1-ImM, SPRY CEL, Bristol-Myers Squibb), and/or Arsenic Tri-Oxyde (ATO, 0.1-ImM, SIGMA) and/or g-Interferon (IFNy, 20ng/mL, Myltenyi-Biotech) during 1 to 12 days.
  • ATO Arsenic Tri-Oxyde
  • IFNy g-Interferon
  • Human T-ALL CD45 + /CD7 + cells were harvested at different time points and live and dead cells were numbered using an automated cytometer (Guava Easycyte, Bioscience/Merk, Guyancourt, France). When necessary, equal number of live cells were reseeded in fresh medium with drugs and tested during additional days.
  • NOD/scid/IL-2Ry null mice (NSG, originally from The Jackson Laboratory, Bar Harbor, USA) and NSG mice bearing a kit gene mutation (NSG-W41, kindly provided by C Waskow, Dresden, Germany) were produced in specific pathogen-free animal facilities (CEA, Fontenay-aux-Roses, France).
  • Transplantation of human leukemic cells into ID mice was done using retroorbital intravenous injection under isoflurane anesthesia. The number of transplanted leukemic cells was 0.25 to lxlO 6 cells/mouse. Human leukemia development was followed by sampling bone marrow (BM) and blood cells.
  • BM bone marrow
  • BM-infiltrating leukemic cell levels were determined by labeling BM cells with anti-hCD45, hCD7, hCD4, hCD8, hCD3, TCRab antibodies (from eBioscience/Thermofisher, Miltenyi Bitotech and Biolegend/Ozyme) and analyses were done using a FACS-Canto or a LSR-II apparatus (Becton Dickinson). All animal experimentation was done after approval of a local ethical committee (project A19-023), and with the authorization number APAFIS#21276-2019070116158626 from the French Ministere de FEnseignement Superieur et de lalich.
  • Drug treatments in vivo Treatments started in mice with leukemic burden being >3% (up to 90%) hCD45+CD7+ cells in the blood as detected by flow cytometry in individual tested mice (see figures for details).
  • DASA (20mg/Kg, diluted in 80mM citric acid, pH2.1) was delivered using per os administration.
  • ATO (5mg/Kg, diluted in TBS, pH7) and PolyTPolyC (PIPC, 20mg/Kg diluted in PBS, SIGMA) were injected using intraperitoneal route. The time scale of drug delivery is indicated for every experiment performed. Mice were weighed every day of treatments, in order to ensure they were not losing too much weight and also to adjust the delivered drug doses.
  • Absolute number of leukemic cells, the proportion of apoptotic cells, the level of cell cycle progression and phosphorylation of different factors were measured by flow cytometry using FACS Canto and LSRII cytometers (Becton Dickinson). Data analysis was carried out with FlowJo software. Cells were stained with fluorescein (FITC)-, phycoerythrin (PE)-, PE- cyanin7 (PC7)- or allophycocyanin (APC)-conjugated mouse monoclonal antibodies specific for human markers. Absolute number of cells was quantified by determining the number of hCD45 + /hCD7 + cells in a fraction of the culture multiplied by the total volume of the culture well.
  • FITC fluorescein
  • PE phycoerythrin
  • PC7 PE- cyanin7
  • APC allophycocyanin
  • Annexin V staining by selecting cells and debris and gating on hCD45 + /hCD7 + cells.
  • cells were labeled with anti-CD45 and anti-CD7 antibodies, washed with Annexin V Binding Buffer (IX) (ref 556454, BD Biosciences) before being stained with Annexin V (ref 640941, BD Biosciences) during 15 minutes at room temperature.
  • Annexin V ref 640941, BD Biosciences
  • cells were stained with anti-CD45-APC and anti-CD7-PC7 antibodies, permeabilized using Cytofix/Cytoperm (554722, BD Biosciences) during 15 minutes minimum at 4°C, washed with Perm/Wash Buffer (554723, BD Biosciences) and labeled with anti-Ki67 antibodies (556027, BD Biosciences) at 4°C during 45 minutes and Hoechst 33342 (H3570, Life Technologies) 10 minutes before the end of incubation. Cells were washed with Perm/Wash Buffer and resuspended in PBS.
  • Phosphorylation ABLl protein targets (STAT5, CRKL) was measured using antibodies directed against phospho-STAT5 and phospho-CRKL.
  • Cells were stained with anti-CD45 antibodies, fixed with BD Cytofix Fixation Buffer (10 min at 37°C), then permeabilized in BD Phosflow Perm buffer III (30 min on ice), washed with Perm/Wash Buffer and labeled with anti-Phospho-STAT5 (Alexa Fluor 488 anti-STAT5 (pY694) Ref: 612598) or anti-Phospho- CRKL (Alexa Fluor 647 mouse anti-CRKL (pY207) Ref: 560790), both from BD Phosflow antibodies at 4°C during 30 minutes, washed with Perm/Wash Buffer and resuspended in PBS.
  • Cell sorting was performed using an Influx cell sorting cytometer (BD Influx system; BD Bioscience). Transduced cells were sorted based on the
  • Cell protein extracts were prepared according standard procedure with lysis buffer containing 50 mM Tris pH8.0, 300 mM NaCl, 10% glycerol, ImM EDTA, ImM EGTA, 1% NP-40, 0.5% DOC, 0,1%SDS supplemented with a protease inhibitor mix (cOmplete TM , Mini Protease Inhibitor Cocktail, Ref: 11836153001) and phosphatase inhibitor cocktails 1 and 2 (Ref: P2850 and P5726), all from Sigma-Aldrich.
  • a protease inhibitor mix cOmplete TM , Mini Protease Inhibitor Cocktail, Ref: 11836153001
  • phosphatase inhibitor cocktails 1 and 2 Ref: P2850 and P5726
  • Proteins were separated by 4-12% SDS PAGE (NUPAGE 4-12% BT GEL, Ref: 10247002 Fisher Scientific), transferred onto nitrocellulose membrane, blocked in 5% non-fat milk in TBST and immunoblotted in 5% BSA- TBST solution overnight.
  • PathScan-Bcr/Abl Western Multiplex Detection Cocktail containing Phospho-c-Abll (Tyr245) (73E5), Phospho-Stat5 (Tyr694) (D47E7), Phospho- CRKL (Tyr 207) and Rabl 1 (D4F5)XP rabbit antibodies , (Ref: 5300; Cell Signalling) at 1 :250 dilution.
  • Leukemic cells were grown in complete medium in presence of cytokines for 24 hours before adding Colcemid (50-75 pL/10mL culture medium) during 45 minutes at 37°C. Cells were then submitted to a hypotonic shock in presence of KCL 0.075M at 37°C during 30 minutes. Before the end of the incubation period, a fixation mixture of methanol and acetic acid (ratio 3:1) was prepared. Cells were pre-fixed with the fixation mixture added and gently mixed with the hypotonic chock medium. Cells were immediately spin down during 5’ at 1500rpm. Pellets of cells were resuspended in lOmL of fixation mixture and kept during 15’ at room temperature.
  • Denaturation was done at 75°C during two minutes.
  • the nuclei pictures were automatically detected on the fully motorized ZEISS Axio Imager.Z2 light microscope. It is equipped with a motorized stage high-resolution CCD camera. A total of at least 50 interphase nuclei were scored, using Metafer software (MetaSystems).
  • M181Diagnosis D
  • Most of Ml 8 ID leukemic cells were positive for CD45, CD 7, CD5, CD8, CD44 and partially CD4 (47% and 55% in Blood and BM) and CD la (19% and 27%).
  • Albeit intensive chemotherapy treatment was given, the boy relapsed later during the year.
  • M181R The leukemic cells from relapse (hereafter called M181R) were phenotypically different as they were CD45+CD7+CD5+CD44+, mostly (>75%) CD8+ but negative for CD4, sCD3, sTCRab and CD la.
  • M181R patient cells were found to be resistant to conventional chemotherapy. Because these cells contain a rearranged copy of Abll, the patient was shortly treated with Dasatinib, an inhibitor of tyrosine kinase (ITK, DASA) as it is done in patients with B-ALL carrying Bcr- Abl or other Abll rearrangements (Tanasi, Blood, 2019). As a consequence, the blast cells decreased dramatically to 26000 MNC/pL (54% blast cells), but re-increased to 77000 MNC/pL (80% blast cells) in a few days after DASA was stopped, indicating an important sensitivity of the leukemic cells to ITK.
  • ITK tyrosine kinase
  • RANBP2 is a nucleoporin, also called NUP358, localized in the nuclear pore complex, with other proteins such as UBC9 and SENP2.
  • Ranbp2 has an activity of E3 SUMOl ligase on different substrates, including SP100, MDM2, PML, etc. that is important for their location in the nuclear bodies (Pichler, CELL, 2002; Miyauchi, JBC, 2002; Sakin, JBC, 2015).
  • PML expression is modified in numerous cancers and PML NB are disorganized in Acute promyelocytic leukemia (APL) due to the PML-RARA fusion (H. de The. Cancer Cell 2017).
  • APL Acute promyelocytic leukemia
  • PML-RARA fusion protein is degraded by a combination of ATO and Trans Retinoic Acid (ATRA) that results in the eradication of the APL leukemic clone and the long term remission of APL patients (Lallemand-Breitenbach, JEM, 1999; de The, Nat Rev Cancer, 2018; Lo-Coco, N Engl J Med, 2013).
  • ATRA Trans Retinoic Acid
  • type 1 IFNs potentiate ATO effect on NB formation.
  • We hypothesized that combined treatment of M181R with ITK, ATO and IFNs would target ABLl function and RANBP2/SUM01/PML pathway, resulting in efficient killing of leukemic cells.
  • mice were transplanted with M181R blood MNC (10 6 /mouse) using IV route (data not shown).
  • 3 male mice were analyzed 4 weeks later that had less than 1% leukemic cells in their BM (data not shown).
  • 2 females were sacrificed 3 additional weeks later and they exhibited 40.5% and 45.6% leukemic cells in their BM whereas their spleen (SPL) contained >70% infiltration (data not shown).
  • SPL spleen
  • Protocol included periods of on and off treatment during 15 days (Figure 1), due to weight loss of mice, especially when ATO, PIPC and DASA were combined ( Figure 2A-B).
  • Figure 1 due to weight loss of mice, especially when ATO, PIPC and DASA were combined.
  • Figure 2A-B At the end of treatment, BM and blood were sampled from all treated mice to measure the leukemic cell infiltration, and mice survival was followed up (Fig2C-G).
  • CD45highCD71ow leukemic cells from M181R patient sample are enriched in Ranbp2-Abll fusion
  • mice were injected with xenografted cells from the BM of a primary NSG-W41 mouse transplanted with M181R patient cells from the sham treated CTL group. 5 weeks later mice were bled and leukemia infiltration was found to be very high (>80% for most of the mice) (data not shown). Treatments were initiated the following day using the same drug doses than before and lasted 3 weeks with adaptation of ATO+PIPC frequency of delivery based on the weight loss of mice (Fig4).
  • BM were sampled from all live mice, a total of 9 mice were euthanasied to measure leukemia multi-sites infiltration and a survival curve was initiated with the remaining treated mice (Fig 5C-H).
  • Fig5H most CTL mice succumbed before the end of the treatment and only one was left, but sick, and thus was analyzed one day post-treatment (Fig5H).
  • the results of additional CTL mice are combined with the results of the other treatment conditions, albeit they were sick and had to be analyzed before the other groups (Fig5C and 5E).
  • the results show that all drug treatment conditions had an effect, either on the leukemic cell numbers and/or infiltration levels in the BM and the SPL (Fig8B-G).
  • TTL time to leukemia
  • T-ALL (named M106) was also sensitive to the drug treatments in vitro, especially to the combination of drugs, indicating a possible more general effect on other T-ALL samples (Fig6E), that we need to investigate further in vitro and in vivo by treating mice engrafted with other T-ALL and B-ALL, having fusion proteins implicating Ranbp2 and/or Abll, such as B-ALL with Ranbp2-Abll, T-ALL with Nup214-Abll, lymphomas with Ranbp2-Alk, etc...
  • DAS A and ATO are efficiently targeting respectively Abll phosphorylation and PML degradation in M181R T-ALL
  • mice injected with RANBP2-ABL1 B cell acute lymphoblastic leukemia Treatment of mice injected with RANBP2-ABL1 B cell acute lymphoblastic leukemia
  • mice injected with NUP214-ABL1 T cell acute lymphoblastic leukemia We then tested the DASA+ATO+PIPC treatment in mice injected with a NUP214-
  • ABL1 T cell acute lymphoblastic leukemia The combinations DASA+ATO+PIPC seems more effective than other combinations in human T-ALL with NUP214-ABL1 in decreasing leukemic cell levels in the BM and the spleen of immune-deficient mice (Fig. 10A-F). Then, in order to assay the specificities of the drug combination effects, we tested the

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Abstract

The present invention relates to a method for treating a cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein or a protein interacting with the nuclear envelop in a subject comprising administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a tyrosine kinase inhibitor (TKI), as combined preparation. Indeed, inventors have validated the present invention with in vivo and in vitro models. Treatments started in mice with leukemic burden being >3% (up to 90%) hCD45+CD7+ cells in the blood as detected by flow cytometry in individual tested mice. DASA (20mg/Kg, diluted in 80mM citric acid, pH2.1) was delivered using per os administration. ATO (5mg/Kg, diluted in TBS, pH7) and PolyI:PolyC (PIPC, 20mg/Kg diluted in PBS, SIGMA) were injected using intraperitoneal route. The time scale of drug delivery is indicated for every experiment performed. Mice were weighed every day of treatments, in order to ensure they were not losing too much weight and also to adjust the delivered drug doses. DASA+ATO+PIPC treated mice had a significantly prolonged survival compared to the other treatment conditions, showing major differences between results of treatment protocols. ATO combined with IFN-type 1 inducers and inhibitors of TK represents a powerful way for eradication of T-ALL cells that implicate high ABL1 activity and nuclear pore protein lesions.

Description

WO 2021/228983 _ j _ PCT/EP2021/062701
METHODS AND PHARMACEUTICAL COMPOSITIONS FOR TREATING
CANCERS
FIELD OF THE INVENTION:
The invention is in the field of oncology. More particularly, the invention relates to methods and compositions to treat cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein.
BACKGROUND OF THE INVENTION:
Promyelocytic leukemia (PML) expression is modified in numerous cancers and Promyelocytic leukemia-nuclear bodies (PML-NB) are disorganized in acute promyelocytic leukemia (APL) due to the promyelocytic leukemia - retinoic acid receptor alpha (PML-RARA) fusion (De The, Cancer Cell, 2017). PML-RARA fusion protein is degraded by a combination of Arsenic trioxide (ATO) and Trans Retinoic Acid (ATRA) that results in the eradication of the APL leukemic clone and the long-term remission of APL patients (Lallemand-Breitenbach, JEM, 1999; de The, Nat Rev Cancer, 2018; Lo-Coco, N Engl J Med, 2013).
Ml 81 Relapse (R) T-ALL patient cells were found to be resistant to conventional chemotherapy. Because these cells contain a rearranged copy of Abll, the patient was shortly treated with Dasatinib, an inhibitor of tyrosine kinase (ITK, DASA) as it is done in patients with B-ALL carrying Bcr-Abl or other Abll rearrangements (Tanasi, Blood, 2019). As a consequence, the number of mononucleated cells (MNC) decreased dramatically to 26000 MNC/pL (among which 54% were leukemic blast cells), but re-increased to 77000 MNC/pL (80% blast cells) in a few days after DASA was stopped, indicating a sensitivity of the leukemic cells to ITK. However, because the patient was already in advanced phase of disease development, this treatment was not efficient enough and the boy died shortly after initiating the ITK treatment.
Accordingly, there is a need to find new therapeutic tools to treat cancer patients with similar genetic lesions.
SUMMARY OF THE INVENTION:
The invention relates to a method for treating a cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation. In particular, the invention is defined by claims.
DETAILED DESCRIPTION OF THE INVENTION:
A young 15 years-old boy was diagnosed with a T-ALL in May 2018. The leukemia sample obtained from said patient was named M181Diagnosis (D). Inventors have analyzed said leukemia cells and observed that a fusion between Ranbp2/Nup351 and Abl 1 was enriched in M181R. Unfortunately, the patient was already in advanced phase of disease development, the ITK treatment was not efficient enough and the boy died shortly after initiating the ITK treatment. Inventors have decided to understand and to propose a new treatment to treat cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein or a protein interacting with the nuclear envelop.
Indeed, inventors have validated their proposal with in vivo models. Treatments started in mice with leukemic burden being >3% (up to 90%) hCD45+CD7+ cells in the blood as detected by flow cytometry in individual tested mice. DASA (20mg/Kg, diluted in 80mM citric acid, pH2.1) was delivered using per os administration. ATO (5mg/Kg, diluted in TBS, pH7) and PolyTPolyC (PIPC, 20mg/Kg diluted in PBS, SIGMA) were injected using intraperitoneal route. The time scale of drug delivery is indicated for every experiment performed. Mice were weighed every day of treatments, in order to ensure they were not losing too much weight and also to adjust the delivered drug doses.
DASA+ATO+PIPC treated mice had a significantly prolonged survival compared to the other treatment conditions (Fig 5H), showing major differences between results of treatment protocols. ATO with inhibitors of TK represents a powerful way for eradication of T-ALL cells with high Abll activity and nuclear pore protein rearrangements.
Accordingly, the invention relates to a method for treating a cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
As used herein, the terms “cancer”, “tumor”, “cancerous” or malignant” in a subject refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
In a particular embodiment, the cancer is lymphoma. As used herein, the term “lymphoma” refers to a type of blood cancer that develops when lymphocytes grow out of control. Lymphomas can be grouped as Hodgkin lymphomas or non-Hodgkin lymphomas, depending on the types of cell they contain. T-cell lymphomas are non-Hodgkin lymphomas (NHL) that develop from T lymphocytes. NHL are diffuse large B-cell lymphoma (DLBCL, a high-grade B-cell lymphoma) and follicular lymphoma (a low-grade B-cell lymphoma). In children, the most common type is Burkitt lymphoma (a high-grade B-cell lymphoma).
In a particular embodiment, the cancer is leukemia.
In a particular embodiment, the cancer is acute lymphoblastic leukemia (ALL).
As used herein, the term “acute lymphoblastic leukemia” has its general meaning in the art and refers to a cancer of the lymphoid lineage of blood cells characterized by the development of large numbers of immature lymphocytes. Acute lymphoblastic leukemia include T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL).
In a particular embodiment, the cancer is T-cell acute lymphoblastic leukemia (T-ALL). T-ALL is a type of acute leukaemia meaning that it is aggressive and progresses quickly. It affects the lymphoid-cell-producing progenitor cells, in particular T lymphocytes as opposed to acute lymphoblastic leukaemia (ALL) which commonly affects B lymphocytes.
In another embodiment, the cancer is B cell lymphomas.
In another embodiment, the cancer is B cell leukemia.
In a particular embodiment, the cancer is B-cell acute lymphoblastic leukemia (B-ALL).
In another embodiment, the cancer is a solid cancer. Solid cancer has various origins including breast, colon head and neck carcinomas. Typically, the solid cancer is selected from the group consisting of but not limited to bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non- Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).
In the context of the invention, the cancer has a protein kinase gene fused to a gene coding for a nuclear-envelop protein.
As used herein, the term “protein kinase” refers to enzyme family. Typically, protein kinase enzyme that modifies other proteins by chemically adding phosphate groups to them (phosphorylation). Different types of protein kinase exist in the art: i) Tyrosine-specific protein kinases: said enzymes phosphorylate tyrosine amino acid residues; ii) Receptor tyrosine kinases: said kinases consist of a transmembrane receptor with a tyrosine kinase domain protruding into the cytoplasm; iii) Histidine-specific protein kinases; iv) serin-threonin protein kinases. As used herein, the term “protein kinase gene” refers to gene encoding to protein kinase enzymes. In the context of the invention, the protein kinase gene is Abll. As used herein, the term “Abll” refers to Abelson murine leukemia viral oncogene homolog 1. It is a proto-oncogene encoding a cytoplasmic and nuclear protein tyrosine kinase that has been implicated in processes of cell differentiation, cell division, cell adhesion, and stress response. The naturally occurring human Abll gene has a nucleotide sequence as shown in Genbank Accession numbers NMNM_007313 and NM_005157. The naturally occurring human Abll protein has an aminoacid sequence as shown in Genbank Accession numbers NP 005148 andNP_009297. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM_001112703, NM_009594, NM_001283045, NM_001283046, NM_001283047 and NP_001106174, NP_001269974, NP_001269975, NP_001269976 and NP_033724).
Thus, the invention relates to a method for treating a cancer having ABL1 fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
In some embodiment, the cancer is leukemia having ABL1 fused to a gene coding for a nuclear-envelop protein.
In some embodiment, the cancer is acute lymphoblastic leukemia (ALL) having ABL1 fused to a gene coding for a nuclear-envelop protein.
As used herein, the term “a gene coding for a nuclear-envelop protein” also called as protein interacting with the nuclear envelop refers to DNA/RNA sequences coding for the entire or part of a protein that participates in nuclear-envelop architecture or takes part in protein complexes with important functions for the nuclear-envelop properties/roles in mammalian cells.
In particular embodiment, the gene coding for a nuclear-envelop protein is RANBP2.
As used herein, the term RANBP2 refers to RAN binding protein 2 which is a protein in humans encoded by the RANBP2 gene. It is also known as nucleoporin 358 (NUP358). The naturally occurring human RANBP2 gene has a nucleotide sequence as shown in Genbank Accession number NM 006267. The naturally occurring human RANBP2 protein has an aminoacid sequence as shown in Genbank Accession number NP 006258.
In some embodiment, the cancer has RANBP2-ABL1 fusion.
In particular embodiment, the gene coding for a nuclear-envelop protein is NUP214. As used herein, the term NUP214 refers to Nucleoporin 214 which is a protein in humans encoded by the NUP214 gene. The naturally occurring human NUP214 gene has a nucleotide sequence as shown in Genbank Accession number NMJ305085. The naturally occurring human RANBP2 protein has an aminoacid sequence as shown in Genbank Accession number NP_005076.
In some embodiment, the cancer has NUP214-ABL1 fusion.
Thus, the invention relates to a method for treating a cancer having NUP214-ABL1 fusion or RANBP2-ABL1 fusion in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
In some embodiment, the cancer is leukemia having RANBP2-ABL1 fusion or NUP214-ABL1 fusion
In some embodiment, the cancer is acute lymphoblastic leukemia having RANBP2- ABL1 fusion or NUP214-ABL1 fusion.
As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with a cancer. In a particular embodiment, the subject suffers from T-ALL. In a particular embodiment, the subject suffers from B cell leukemia (B-ALL). In a particular embodiment, the subject suffers from B cell lymphoma. In another embodiment, subject suffers from a solid cancer. In a further embodiment, the subject has a protein kinase gene fused to a gene coding for a nuclear-envelop protein or a protein interacting with the nuclear envelop. Typically, in the context of the invention, the subject has Ranbp2-Abll fusion.
As used herein, the term “arsenic compound” is intended to include arsenic and any compound having the same biological properties as arsenic. The expression "compound having the same biological properties as arsenic" is understood to mean any compound which, like arsenic, is an inhibitor of phosphatase and/or is capable of creating covalent adducts by binding with thiol groups. In some embodiments, the arsenic compound is selected from the group consisting of but not limited to arsenic, arsenic trioxide (As203 also called as ATO), arsenic hexoxide (AS4O6), melarsoprol and arsenic sulfur derivative. In a particular embodiment, the arsenic compound is ATO.
As used herein, the term “protein kinase inhibitor” refers to any molecule that specifically blocks the action of one or more protein kinases. Protein kinase inhibitors are subdivided by the amino acids whose phosphorylation is inhibited. Most kinases act on both serine and threonine, the tyrosine kinases act on tyrosine, and a number (dual- specificity) kinases act on all three. As used herein, the term "serine/threonine kinase inhibitor" refers to a molecule that specifically blocks the action of one or more serine and/or threonine kinases.
In a further embodiment, the PKI is a TKI. As used herein, the term "tyrosine kinase inhibitor" (TKI) refers to any compound, natural or synthetic molecule that specifically blocks the action of one or more tyrosine kinases. Typically, said inhibition results in a decreased phosphorylation of the tyrosine present on the intracellular domain of receptor tyrosine kinases (RTK) such as growth factor receptors. Accordingly, TKI may be multi-target tyrosine kinase inhibitor and may thus inhibit epidermal growth factor (EGF) receptor family (such as HER- 2); insulin-like growth factor (IGF) receptor family (such as IGF-1 receptor); platelet-derived growth factor (PDGF) receptor family, colony stimulating factor (CSF) receptor family (such as CSF-1 receptor); C- Kit receptor and vascular endothelial growth factor (VEGF) receptor family (such as VEGF- R1 (Flt-1) and VEGF-R2 (KDR/Flk-1)).
In a particular embodiment, the tyrosine kinase inhibitor is a Bcr-Abl tyrosine kinase inhibitor. In another embodiment, the TKI may also be a multi-target tyrosine kinase inhibitor and may thus inhibit one or more tyrosine kinases, Bcr-Abl, but also c-Abl and the receptor tyrosine kinases PDGF-R, Fit3, VEGF-R, EGF-R, c-Kit, as well as combinations of two or more of these. In one embodiment, the Bcr-Abl TKI is selected from the group consisting of N- phenyl-2-pyrimidine-amine derivatives as described in EP0564409, pyrimidinylaminobenzamide derivatives as described in W02004005281, cyclic compounds as described in W00062778, bicyclic heteroaryl compounds as described in WO 2007075869, substituted 3-cyano quinoline derivatives as described in US6002008, 4-anilo-3- quinolinecarbonitrile derivatives as described in W0200504669 and amide derivatives as described in US7728131 and W02005063709.
In a particular embodiment, the TKI is selected from the group consisting of but not limited to imatinib, nilotinib, dasatinib, ponatinib, bosutinib and bafetinib.
In a particular embodiment, the TKI is Imatinib. Imatinib sold under the brand name Gleevec® refers to (4-(4-methylpiperazin-l-yhnethyl)-N-[4-methyl-3-(4-pyridin-3-yl) pyrimidin-2-ylamino)phenyl-benzamide) also known as STI571 (Novartis; International Patent Publication No. WO 95/09852).
In another embodiment, the TKI is nilotinib. Nilotinib sold under the brand name TASIGNA® refers to 4-methyl-N-[3-(4-methyl-lH-imidazol-l-yl)- 5- (trifluoromethyl)phenyl]-3- [(4-pyridin-3-ylpyrimidin-2-yl) aminojbenzamide also known as AMN107(Novartis; International Patent Publication No. WO 2004/005281).
In another embodiment, the TKI is dasatinib. Dasatinib sold under the brand name SPRYCEL® refers to N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-l-piperazinyl]- 2-methyl-4-pyrimidinyl]amino]-5-thiazole carboxamide (also known as BMS-354825, Bristol- Myers Squibb) (International Patent Publication No. WO 2004/085388).
As used herein, the term "inductor of type 1 IFNs” refers to any compound, natural or synthetic molecule that induces the expression and/or activity of type 1 IFN. Typically, the induction of type 1 IFN gene involves recruitment of sequence-specific transcription factors that are activated by phosphorylation in response to signalling cascades during viral infections. In a particular embodiment, the inductor of type 1 IFNs is an interferon polypeptide.
As used herein, the term "interferon polypeptide" or "IFN polypeptide" is intended to include any polypeptide defined as such in the literature, comprising for example any types of IFNs (type I and type II) and in particular, IFN-alpha, IFN-beta, IFN-omega and IFN-gamma. The term interferon polypeptide, as used herein, is also intended to encompass salts, functional derivatives, variants, muteins, fused proteins, analogs and active fragments thereof. The polypeptide sequences for human interferon-alpha are deposited in database under accession numbers: AAA 52716, AAA 52724, and AAA 52713. The polypeptide sequences for human interferon-beta are deposited in database under accession numbers AAC41702, NP_002167, AAH 96152, AAH 96153, AAH 96150, AAH 96151, AAH 69314, and AAH 36040. The polypeptide sequences for human interferon-gamma are deposited in database under accession numbers AAB 59534, AAM 28885, CAA 44325, AAK 95388, CAA 00226, AAP 20100, AAP 20098, AAK 53058, and NP-000610.
In some embodiments, the inductor of type 1 IFNs is interferon-alpha (IFN-a). The term "IFN-a" encompasses derivatives of IFN-a that are derivatized (e.g., are chemically modified relative to the naturally occurring peptide) to alter certain properties such as serum half-life. As such, the term "IFN- a " includes IFN- a derivatized with polyethylene glycol ("PEGylated IFN- a "), and the like. PEGylated IFN- a, and methods for making same, is discussed in, e.g., U.S. Pat. Nos. 5,382,657; 5,951,974; and 5,981,709. PEGylated IFN-a encompasses conjugates of PEG and any of the above-described IFN- a molecules, including, but not limited to, PEG conjugated to interferon alpha-2a (Roferon, Hoffman La-Roche, Nutley, N. J.), interferon alpha- 2b (Intron, Schering-Plough, Madison, N.J.), interferon alpha-2c (Berofor Alpha, Boehringer Ingelheim, Ingelheim, Germany); and consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (Infergen®, InterMune, Inc., Brisbane, CA.). Thus, in some embodiments, the IFN-a has been modified with one or more polyethylene glycol moieties, i.e., pegylated. Two forms of pegylated-interferon, peginterferon alfa-2a (40 kD) (Pegasys, Hoffmann-La Roche) and peginterferon alfa-2b (12 kD) (Peglntron, Merck), are commercially available, which differ in terms of their pharmacokinetic, viral kinetic, tolerability profiles, and hence, dosing. In particular, Peginterferon alfa-2a (Pegasys) consists of interferon alfa-2a (~20 kD) covalently linked to a 40 kD branched polyethylene glycol (PEG). The PEG moiety is linked at a single site to the interferon alfa moiety via a stable amide bond to lysine. Peginterferon alfa-2a has an approximate molecular weight of 60,000 daltons. The biologic activity of peginterferon- alfa-2a derives from its interferon alfa-2a moiety which impacts both adaptive and innate immune responses against certain viruses. Compared with the native interferon alfa-2a, the peginterferon alfa-2a has sustained absorption, delayed clear. Peginterferon alfa-2a is used as a fixed weekly dose. Peginterferon alfa-2a has a relatively constant absorption after injection and is distributed mostly in the blood and organs.
In a particular embodiment, the inductor of type 1 IFNs is Polyinosinic:polycytidylic acid (usually abbreviated poly I:C or poly(TC); or PIPC).
In a particular embodiment, the inductor of type 1 IFNs is Polyadenylic-polyuridylic acid (usually abbreviated poly(A:U).
In a particular embodiment, the method according to the invention, wherein the arsenic compound, the inductor of type 1 IFNs and the protein kinase inhibitor (PKI) are administered as a combined preparation.
In a particular embodiment, the method according to the invention, wherein the arsenic compound, the inductor of type 1 IFNs and the tyrosine kinase inhibitor (TKI) are administered as a combined preparation.
In a particular embodiment, the method according to the invention, wherein the arsenic compound is ATO, the inductor of type 1 IFNs is PIPC and the TKI is DASA are administered as a combined preparation.
As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., ATO+ PIPC+ dasatinib) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.
In a particular embodiment, the arsenic compound is administered orally. In another embodiment, the inductor of 1IFN and PKI are administered intravenously or subcutaneously.
In a particular embodiment, the arsenic compound is administered orally. In another embodiment, the inductor of type 1 IFNs and TKI are administered intravenously or subcutaneously.
As used herein, the terms “combined preparation”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy. In the context of the invention, the arsenic compound, the inductor of type 1 IFNs and the protein kinase inhibitor (PKI) are administered simultaneously, separately or sequentially.
As used herein, the term "administration simultaneously" refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term "administration separately" refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term "administration sequentially" refers to an administration of 2 active ingredients at different times, the administration route being identical or different.
By a "therapeutically effective amount" is meant a sufficient amount of the arsenic compound, the inductor of type 1 IFNs and the protein kinase inhibitor (PKI) for use in a method for the treatment of cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
A further aspect, the invention relates to a pharmaceutical composition comprising an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
In a particular embodiment, the invention refers to a pharmaceutical composition comprising an arsenic compound, an inductor of type 1 IFNs and a tyrosine kinase inhibitor (TKI), as combined preparation.
In a particular embodiment, the invention refers to a pharmaceutical composition according to the invention, for use in the therapy.
In a particular embodiment, the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of a cancer having ABL1 fused to a gene coding for a nuclear-envelop protein.
In a particular embodiment, the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of ALL having Abll fused to a gene coding for a nuclear-envelop protein.
In a particular embodiment, the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of ALL having ABL1-RANBP2 fusion or ABL1-NUP214 fusion.
In a particular embodiment, the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of T- ALL having ABL1 fused to a gene coding for a nuclear-envelop protein.
In a particular embodiment, the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of B cell leukaemia having ABL1 fused to a gene coding for a nuclear-envelop protein.
In a particular embodiment, the invention refers to a pharmaceutical composition according to the invention, for use in the treatment of a solid cancer having ABL1 fused to a gene coding for a nuclear-envelop protein.
In a particular embodiment, the pharmaceutical composition according to the invention wherein the the arsenic compound is ATO, the inductor of type 1 IFNs is PIPC and the TKI is dasatinib. An arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI) as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intrap eritoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
In one embodiment, the arsenic compound, the inductor of type 1 IFNs and the protein kinase inhibitor (PKI), as combined preparation, is administered in combination with a classical treatment of cancer.
Thus, the invention also refers to a method for treating a cancer having ABL1 fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation and ii) classical treatment of cancer.
In a particular embodiment, the classical treatment refers to radiation therapy, immune checkpoint inhibitor or chemotherapeutic agent.
As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include multkinase inhibitors such as sorafenib and sunitinib, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxombicin and deoxydoxorubicin), epimbicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
As used herein, the term “radiation therapy” has its general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.
As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins.
As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules).
Examples of stimulatory checkpoint include CD27 CD28 CD40, CD 122, CD 137, 0X40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, PD-L1, LAG-3, TIM-3 and VISTA. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1: Protocol of the first round of treatment. 21 mice were treated during 15 days with a combination of DASA, ATO and PIPC. 1 mouse (group ATO+PIPC+DASA died at day 2 of treatment). After the end of the treatment, Blood and BM of mice were sampled to analyze leukemia expansion during treatment. A survival curve was started to test the effect of the treatment protocol on the viability of mice. - Days with treatment; X: days without treatment. Doses of drugs are given in Mat&Method section.
Figure 2: Results of the first experiment of treatment. A-B. Weight of mice during the treatment period. C and D. Levels of human CD45+hCD7+ leukemic cell infiltration in the blood (C) and the BM (D) of mice treated or not with the different drug combinations at the end of the treatment period. 5 mice/condition were used. E. Respective levels of human (hu)CD451owCD7high and CD45highCD71ow leukemic cells in the BM of mice at the end of the treatment period. F. Levels of huCD45highCD71ow. G. Survival of mice in days after T- ALL transplant. Treatment period is shown in the graph. A total of 20 mice were followed up .**: p<0.01; *<0.05, Mann- Whitney non parametric test.
Figure 3: CD45highCD71ow are Ranbp2-Abll containing cells. Fish analysis of leukemic cells looking at Abll gene rearrangement. Shown are results obtained with 41 to 49 cells from patient cells before (black histogram) and after mouse BM engraftment. Dark grey histogram shows data from an untreated mouse recipient, whereas the two other histograms show data from cells sorted according to expression levels of huCD45 and huCD7 from an non treated mouse (mouse 1) and from a mouse treated with ATO+PIPC+DASA. No rearrangement of Abll was detected in huCD451oCD7high cells sorted from the BM of ATO+PIPC+DASA treated mouse (last column - empty - of the histogram).
Figure 4: Protocol of treatment extension. Mice were treated during 3 weeks with a combination of DAS A, ATO and PIPC after blood sampling. At the end of the treatment period, BM was sampled from all mice and 9 of them were sacrificed and their leukemic infiltration levels were measured in the blood, BM and spleen. Left-over mice were followed up during more than 100 days and survival was measured. - Days with treatment; X: days without treatment. Doses of drugs are similar to Figure 1. Figure 5: Results of the treatment extension experiment. A-B. Weight of mice during treatment. C. Leukemic levels in the BM of all mice measured immediately after the treatment period. D. Leukemic levels in the BM and SPL of 9 mice sacrificed after treatment. E-G analysis of the weight (E), the cell numbers (F) and the leukemic cell levels (G) in the SPL of 9 mice sacrificed after treatment. In the sham CTL group, data from 6 additional mice are shown that were analyzed before ending the treatment as they were sick. H. Survival curve of the mice. C and E: One-way ANOVA test, ***, p=0.001; ****, p<0.0001; H, Mantel-Cox test, ***, p < 0.0005.
Figure 6: In vitro treatments of human T-ALL cells. A. In vitro growth curve of M181R T-ALL cultured in presence or not of combination of ATO (50nM)+IFNg (20ng/mL) +DASA (ImM). Results are mean+/-SEM of technical triplicates. B. Cell numbers recovered after 72 hours of culture of Ml 81R T-ALL in presence of ImM ATO, lpMDASA and 20ng/mL IFNg. C. Cell cycle analysis measured in M181R cells cultured as described in (B). D. Apoptosis levels in cells from (B). E. In vitro growth of M106 T-ALL in the same conditions as in (A).
Figure 7: Analysis of Ranbp2-Abll phosphorylation and PML protein levels in M181R T-ALL. Quantification of RANBP2-ABL1, STAT5 and CRKL phosphorylation levels in M181R T-ALL after in vitro treatment with combination of DASA (ImM), ATO (1 mM) and IFNg (20ng/mL) during 48 hours. Results are from western blot analysis.
Figure 8: Establishment of patient-derived xenograft of RANBP2-ABL1 B cell acute lymphoblastic leukemia injected in primary immune-deficient mice. A. Shows engraftment levels of human (hCD45+ cells) B-ALL with RANBP2-ABL1 in primary immune- deficient mice. B. Shows that the recovered cells are of B cell origin (CD 19+) and part of them are CD34+.
Figure 9: Results of treatments of immune-deficient mice injected with RANBP2- ABL1 B cell acute lymphoblastic leukemia. Human leukemic cells from primary mice were transplanted in a large cohort of secondary recipient mice. Mice were treated 6 weeks later with different combination of drugs. Treatment lasted 3 weeks. BM was then sampled from all mice and tested for human leukemic cells levels. A few mice (1-3/groups) were euthanasied after treatment to measure leukemia infiltration in spleens. The rest of mice were kept for survival. A. Shows the results of human leukemic levels in BM samplings the day after the end of treatment. B-D. Shows weight of and leukemic infiltration in spleens (SPL) of mice analyzed the day after the end of treatment. E. Survival curve of the mice treated with different DASA/ATO/PIPC combinations. Figure 10: Results of treatments of immune-deficient mice injected with NUP214- ABL1 T cell acute lymphoblastic leukemia. Immune deficient mice were injected with 10exp6 leukemic cells from primary PDX. 4 months later, mice were treated with different drug combination during 3-4 weeks. At the end of treatment, blood and BM were sampled on all live mice and human leukemia levels were measured. 2-5 mice of T-ALL#1 were euthanasied to measure leukemia infiltration in BM and SPL. A, D show the number of human CD45+ leukemic cells in the BM and SPL of treated mice. B shows the spleen weight of the same mice after treatment. C show leukemia levels in spleen of mice injected with a NUP214-ABL1 T- ALL#1 treated with different drug combinations. E, F shows leukemia levels in blood (E) and BM (F) of a second NUP214- ABL1 T-ALL (T-ALL#2) after treatment with the indicated drugs.
Figure 11: Results of treatments of a T cell acute lymphoblastic leukemia that does not harbor any ABL1 gene rearrangement. Immune deficient mice were injected with 5000 leukemic cells from PDX of this T-ALL. 3 weeks later, mice were treated with different drug combinations during 3 weeks. At the end of treatment, blood and BM were sampled on all live mice and human leukemia levels were measured. 2-5 mice were euthanasied to measure leukemia infiltration in BM and SPL. Left-over mice were kept for survival. A, B, E show leukemia levels in the Blood (A), the BM (B) and spleen (E) of mice injected with a T-ALL with no ABLl rearrangement and treated with different drug combinations. C, F show the number of human CD45+ leukemic cells in the BM (C) and in the SPL (F) of treated mice. D shows the spleen weight of the mice euthanasied after treatment. G show survival curves of treated mice.
EXAMPLE:
Material & Methods
Human T-ALL
Human T-ALL cell samples were obtained with the informed consent of patients in accordance with the Declaration of Helsinki and the Ethic regulations. Blood and/or bone marrow samples were collected at diagnosis and relapse at Hopital A. Trousseau and at Hopital Saint Louis, in Paris, France and processed either by the hospital teams or in the laboratory of F Pflumio in Fontenay-aux-Roses. Basically blood and BM cells were ficolled, numbered and characterized using flow cytometry with anti-human CD antibodies. When fresh sample was available, part of the leukemic cells was injected in immune-deficient (ID) mice and part was frozen in liquid nitrogen in Foetal Calf Serum (FCS) containing 10% of Dimethylsufoxide (DMSO) (Sigma-Aldrich). When only frozen samples were available, leukemic cells were thawed, carefully washed, numbered and thereafter transplanted in ID mice or used for other purposes (culture, biochemistry etc)
Culture conditions
Human T-ALL (250xl03 cells) were cultured in 500pL of alpha-minimum essential medium (a-MEM, Gibco) containing 10% of FCS, 1% of Penicilline/Stretomycine/L- Glutamine (Gibco), recombinant human stem cell factor (hSCF ; 50 ng/mL), Fms-related tyrosine kinase 3 ligand (Flt3-L ; 20 ng/mL) (both from Miltenyi Biotech), Interleukin- 7 (IL-7, 10 ng/mL ; Preprotech) and Insulin (20 nM ; Sigma-Aldrich) in 24-well plates and incubated at 37°C in 21% of O2. Cells were harvested at indicated time points, numbered and further processed as to measure apoptosis, cell cycle and proteins were extracted to measure expression, phosphorylation and sumoylation levels. When necessary, equal numbers of leukemic cells were replated in medium freshly renewed every other day.
Drug treatments in vitro
T-ALL cultures were set with Dasatinib (0.1-ImM, SPRY CEL, Bristol-Myers Squibb), and/or Arsenic Tri-Oxyde (ATO, 0.1-ImM, SIGMA) and/or g-Interferon (IFNy, 20ng/mL, Myltenyi-Biotech) during 1 to 12 days. Human T-ALL CD45+/CD7+ cells were harvested at different time points and live and dead cells were numbered using an automated cytometer (Guava Easycyte, Bioscience/Merk, Guyancourt, France). When necessary, equal number of live cells were reseeded in fresh medium with drugs and tested during additional days.
Mice and leukemic cell transplantation
NOD/scid/IL-2Ry null mice (NSG, originally from The Jackson Laboratory, Bar Harbor, USA) and NSG mice bearing a kit gene mutation (NSG-W41, kindly provided by C Waskow, Dresden, Germany) were produced in specific pathogen-free animal facilities (CEA, Fontenay-aux-Roses, France). Transplantation of human leukemic cells into ID mice was done using retroorbital intravenous injection under isoflurane anesthesia. The number of transplanted leukemic cells was 0.25 to lxlO6 cells/mouse. Human leukemia development was followed by sampling bone marrow (BM) and blood cells. BM-infiltrating leukemic cell levels were determined by labeling BM cells with anti-hCD45, hCD7, hCD4, hCD8, hCD3, TCRab antibodies (from eBioscience/Thermofisher, Miltenyi Bitotech and Biolegend/Ozyme) and analyses were done using a FACS-Canto or a LSR-II apparatus (Becton Dickinson). All animal experimentation was done after approval of a local ethical committee (project A19-023), and with the authorization number APAFIS#21276-2019070116158626 from the French Ministere de FEnseignement Superieur et de la Recherche.
Drug treatments in vivo Treatments started in mice with leukemic burden being >3% (up to 90%) hCD45+CD7+ cells in the blood as detected by flow cytometry in individual tested mice (see figures for details). DASA (20mg/Kg, diluted in 80mM citric acid, pH2.1) was delivered using per os administration. ATO (5mg/Kg, diluted in TBS, pH7) and PolyTPolyC (PIPC, 20mg/Kg diluted in PBS, SIGMA) were injected using intraperitoneal route. The time scale of drug delivery is indicated for every experiment performed. Mice were weighed every day of treatments, in order to ensure they were not losing too much weight and also to adjust the delivered drug doses.
Flow cytometry
Absolute number of leukemic cells, the proportion of apoptotic cells, the level of cell cycle progression and phosphorylation of different factors were measured by flow cytometry using FACS Canto and LSRII cytometers (Becton Dickinson). Data analysis was carried out with FlowJo software. Cells were stained with fluorescein (FITC)-, phycoerythrin (PE)-, PE- cyanin7 (PC7)- or allophycocyanin (APC)-conjugated mouse monoclonal antibodies specific for human markers. Absolute number of cells was quantified by determining the number of hCD45+/hCD7+ cells in a fraction of the culture multiplied by the total volume of the culture well.
Cell apoptosis analysis was done using Annexin V staining, by selecting cells and debris and gating on hCD45+/hCD7+ cells. In short, cells were labeled with anti-CD45 and anti-CD7 antibodies, washed with Annexin V Binding Buffer (IX) (ref 556454, BD Biosciences) before being stained with Annexin V (ref 640941, BD Biosciences) during 15 minutes at room temperature. Cells were washed and resuspended in Annexin V Binding Buffer.
For cell cycle, cells were stained with anti-CD45-APC and anti-CD7-PC7 antibodies, permeabilized using Cytofix/Cytoperm (554722, BD Biosciences) during 15 minutes minimum at 4°C, washed with Perm/Wash Buffer (554723, BD Biosciences) and labeled with anti-Ki67 antibodies (556027, BD Biosciences) at 4°C during 45 minutes and Hoechst 33342 (H3570, Life Technologies) 10 minutes before the end of incubation. Cells were washed with Perm/Wash Buffer and resuspended in PBS.
Phosphorylation ABLl protein targets (STAT5, CRKL) was measured using antibodies directed against phospho-STAT5 and phospho-CRKL. Cells were stained with anti-CD45 antibodies, fixed with BD Cytofix Fixation Buffer (10 min at 37°C), then permeabilized in BD Phosflow Perm buffer III (30 min on ice), washed with Perm/Wash Buffer and labeled with anti-Phospho-STAT5 (Alexa Fluor 488 anti-STAT5 (pY694) Ref: 612598) or anti-Phospho- CRKL (Alexa Fluor 647 mouse anti-CRKL (pY207) Ref: 560790), both from BD Phosflow antibodies at 4°C during 30 minutes, washed with Perm/Wash Buffer and resuspended in PBS. Cell sorting was performed using an Influx cell sorting cytometer (BD Influx system; BD Bioscience). Transduced cells were sorted based on the expression levels of hCD45 and hCD7.
Western blot analysis
Cell protein extracts were prepared according standard procedure with lysis buffer containing 50 mM Tris pH8.0, 300 mM NaCl, 10% glycerol, ImM EDTA, ImM EGTA, 1% NP-40, 0.5% DOC, 0,1%SDS supplemented with a protease inhibitor mix (cOmplete™, Mini Protease Inhibitor Cocktail, Ref: 11836153001) and phosphatase inhibitor cocktails 1 and 2 (Ref: P2850 and P5726), all from Sigma-Aldrich. Proteins were separated by 4-12% SDS PAGE (NUPAGE 4-12% BT GEL, Ref: 10247002 Fisher Scientific), transferred onto nitrocellulose membrane, blocked in 5% non-fat milk in TBST and immunoblotted in 5% BSA- TBST solution overnight. We used PathScan-Bcr/Abl Western Multiplex Detection Cocktail containing Phospho-c-Abll (Tyr245) (73E5), Phospho-Stat5 (Tyr694) (D47E7), Phospho- CRKL (Tyr 207) and Rabl 1 (D4F5)XP rabbit antibodies , (Ref: 5300; Cell Signalling) at 1 :250 dilution. After washing 4x5min in TBST, membrane was incubated with secondary Goat anti rabbit IgG IR800 conjugate (Ref: R-05060-250 Diagenomics) (1:15000), lh at RT and visualised by Odyssey CLX Imaging System. To detect PML, we used rabbit anti-PML (...) at 1:1000 dilution.
FISH analysis
Leukemic cells were grown in complete medium in presence of cytokines for 24 hours before adding Colcemid (50-75 pL/10mL culture medium) during 45 minutes at 37°C. Cells were then submitted to a hypotonic shock in presence of KCL 0.075M at 37°C during 30 minutes. Before the end of the incubation period, a fixation mixture of methanol and acetic acid (ratio 3:1) was prepared. Cells were pre-fixed with the fixation mixture added and gently mixed with the hypotonic chock medium. Cells were immediately spin down during 5’ at 1500rpm. Pellets of cells were resuspended in lOmL of fixation mixture and kept during 15’ at room temperature. Cells were spun down 5’ at 1500rpm, supernatant was discarded and lOmL fixation mixture was added. Fixation was left 15’ at 4°C before to be spun down again and either directly spread on slides or kept several days at 4°C before being deposited on slides. We performed a FISH analysis on nuclei preparations, using the LM02/ABL1 Break Apart FISH probe (Empire Genomics, Ref. EG-LMO2-ABL1-20-GRGOAQRE). This reagent combines two break apart probes, targeting ABLl and LM02 genes, labeled with two different sets of fluorochroms, Aqua-Green and Red-Gold, respectively. The protocol of hybridation was followed according to the manufacturer’s instructions. Denaturation was done at 75°C during two minutes. The nuclei pictures were automatically detected on the fully motorized ZEISS Axio Imager.Z2 light microscope. It is equipped with a motorized stage high-resolution CCD camera. A total of at least 50 interphase nuclei were scored, using Metafer software (MetaSystems).
Results
T-ALL leukemia with Ranbp2-Abll translocation
A young 15 years-old boy was diagnosed with a T-ALL in May 2018. The leukemia sample was named M181Diagnosis (D). Most of Ml 8 ID leukemic cells were positive for CD45, CD 7, CD5, CD8, CD44 and partially CD4 (47% and 55% in Blood and BM) and CD la (19% and 27%). A minority (3-4%) of mononucleated cells expressed surface TCRab and CD3, although we cannot exclude these were normal mature T cells (data not shown). Albeit intensive chemotherapy treatment was given, the boy relapsed later during the year. The leukemic cells from relapse (hereafter called M181R) were phenotypically different as they were CD45+CD7+CD5+CD44+, mostly (>75%) CD8+ but negative for CD4, sCD3, sTCRab and CD la. Genetic analysis indicated shared gene abnormalities between Ml 8 ID and M181R leukemic samples, including a t(ll ;14)tpl3 ;qll) translocation showing the rearrangement of Lmo2 with TCRD, the deletion of CDKN2A/2B and Notchl and I17r mutations, that are mutations typically found in T-ALL (reviewed in Girardi, Blood, 2017). Interestingly, a fusion between Ranbp2/Nup351 and Abll was enriched in M181R (data not shown). Looking back into Ml 8 ID revealed that this cell population represented a minority (14%) of diagnosed leukemic cells. We sequenced the cDNA of the fusion transcript Ranbp2/Abll. Exactly like described in B-ALL by the group of C Mullighan (Roberts, Nat Med, 2008), the protein fusion between RANBP2 and ABLl in this T-ALL retains the leucin-zipper domain but loses the SUMO E3 ligase and the RAN binding domains of RANBP2 whereas the tyrosine kinase domain of ABLl is conserved (data not shown). The concomitant presence of Lmo2 rearrangement and Ranbp2-Abll fusion in the same cells is unexpected and rather a unique feature of this peculiar T-ALL. Indeed, Ranbp2-Abll is normally the primary oncogenic event in B-ALL where it is associated with other gene abnormalities, such as Ikaros or Pax5 deletions . In T-ALL this is the first description of a fusion between those two genes, Abll being rather rearranged with other partners such as Nup214 in 6% of T-ALL (Girardi, Blood, 2017). Another difference lays in the fact that Nup214-Abll fusion is always associated with TLX1 or TLX3 and not with LM02 expression, as it is the case for Ml 81 T-ALL here (Graux, Nat Genet, 2004).
Development of xenograft mouse models for M181 T-ALL Because patient leukemic samples may represent interesting tools for drug screening, we transplanted M181R and Ml 8 ID cells into immune-deficient NSG and NSG-W41 mice. Both pathologic cells spread into the mouse body and reached high leukemia infiltration in the Blood, BM and SPL (data not shown). Liver was found spotted with white colonies, indicating leukemic cells were also present in this vital organ. We previously classified T-ALL patient samples into short, long and no Time-To-Leukemia samples (TTL) depending on the length of time required for leukemic cells to reach over 20% leukemia in the recipient BM (Poglio, Leukemia, 2015). According to this work, the leukemia Ml 8 ID and M181R fell in the category of short TTL since leukemia infiltration was over 70% CD45+/CD7+/CD5+ cells in the mouse BM at 7-8 weeks after transplant (data not shown).
Drug treatments of ID mice engrafted with M181D and M181R leukemic cells
M181R patient cells were found to be resistant to conventional chemotherapy. Because these cells contain a rearranged copy of Abll, the patient was shortly treated with Dasatinib, an inhibitor of tyrosine kinase (ITK, DASA) as it is done in patients with B-ALL carrying Bcr- Abl or other Abll rearrangements (Tanasi, Blood, 2019). As a consequence, the blast cells decreased dramatically to 26000 MNC/pL (54% blast cells), but re-increased to 77000 MNC/pL (80% blast cells) in a few days after DASA was stopped, indicating an important sensitivity of the leukemic cells to ITK. However because the patient was already in advanced phase of disease development, this treatment was not efficient enough and the boy died shortly after initiating the ITK treatment. Nevertheless, as patient responded to ITK, we decided this result could be used in new therapy design as an efficient drug and we wished to associate ITK with additional molecules.
In order to increase ITK efficiency, we tested the combination of DASA with ATO and PIPC, the latter being an inducer of typel Interferons (IFNs). ATO and IFNs were chosen to target RANBP2 activity. Indeed, RANBP2 is a nucleoporin, also called NUP358, localized in the nuclear pore complex, with other proteins such as UBC9 and SENP2. Ranbp2 has an activity of E3 SUMOl ligase on different substrates, including SP100, MDM2, PML, etc. that is important for their location in the nuclear bodies (Pichler, CELL, 2002; Miyauchi, JBC, 2002; Sakin, JBC, 2015). Sumoylation of PML is implicated in the efficient functions of nuclear bodies (NBs), including induction of senescence (Lallemand-Breitenbach, Curr Op Cell Biol, 2018). PML expression is modified in numerous cancers and PML NB are disorganized in Acute promyelocytic leukemia (APL) due to the PML-RARA fusion (H. de The. Cancer Cell 2017). PML-RARA fusion protein is degraded by a combination of ATO and Trans Retinoic Acid (ATRA) that results in the eradication of the APL leukemic clone and the long term remission of APL patients (Lallemand-Breitenbach, JEM, 1999; de The, Nat Rev Cancer, 2018; Lo-Coco, N Engl J Med, 2013). In vivo, type 1 IFNs potentiate ATO effect on NB formation. We hypothesized that combined treatment of M181R with ITK, ATO and IFNs would target ABLl function and RANBP2/SUM01/PML pathway, resulting in efficient killing of leukemic cells.
In order to test our hypothesis, 26 (5 females and 20 males) NSG-W41 mice were transplanted with M181R blood MNC (106 /mouse) using IV route (data not shown). 3 male mice were analyzed 4 weeks later that had less than 1% leukemic cells in their BM (data not shown). 2 females were sacrificed 3 additional weeks later and they exhibited 40.5% and 45.6% leukemic cells in their BM whereas their spleen (SPL) contained >70% infiltration (data not shown). Thus treatment started a week later and doses of DASA, ATO and IFNs (DASA (20mg/Kg, diluted in 80mM citric acid, pH2.1) was delivered using per os administration. ATO (5mg/Kg, diluted in TBS, pH7) and PolyTPolyC (PIPC, 20mg/Kg diluted in PBS, SIGMA) were based on previous works (Schubert, Oncotarget, 2017; Lallemand-Breitenbach, JEM, 1999; Sahin, Nat Comm, 2014). Protocol included periods of on and off treatment during 15 days (Figure 1), due to weight loss of mice, especially when ATO, PIPC and DASA were combined (Figure 2A-B). At the end of treatment, BM and blood were sampled from all treated mice to measure the leukemic cell infiltration, and mice survival was followed up (Fig2C-G). As shown in Figure 2C, blood infiltration was up to 100% leukemic cells in sham treated control mice (CTL) and in ATO+PIPC treated mice whereas DASA treatment alone or in combination with ATO and PIPC diminished leukemic cell load reaching significance for the whole drug combination. When looking in the BM, leukemic cell levels were not significantly decreased, whatever drug was used in the mice although 3/5 mice treated with DASA+ ATO+PIPC had lower leukemic cell infiltration (Fig 2D). Close look at CD45 and CD7 expression levels on the leukemic cells indicated the presence of two major cell populations in 2/5 mice receiving DASA alone or in combination with ATO+PIPC. One cell population had high CD7 and low CD45 expression and the other one had high CD45 and low CD7 expression (data not shown). The CD451owCD7high leukemic cells were absent or infrequent in the BM of CTL and ATO+PIPC treated mice (Fig 2E) indicative of a selection process when DASA was included in the treatment. Plotting only the % of BM cells with CD45highCD71ow cells for all treatment conditions revealed that DASA+ATO+PIPC treatment efficiently and significantly decreased the levels of this cell population infiltration in the BM compared to CTL and ATO+PIPC conditions. Even though a tendency to decrease more this cell population was observed with DASA+ATO+PIPC compared to DASA alone, it did not reached significance (Fig2F). Survival of mice was not significantly improved in drug treated mice compared to CTL even though DASA and DASA+ATO+PIPC treated mice had a few days longer life span
(Figure 2G).
CD45highCD71ow leukemic cells from M181R patient sample are enriched in Ranbp2-Abll fusion
On the basis of the previous treatment results, we wished to test to which extent CD45highCD71ow and CD451owCD7high cells from treated mice were enriched in Ranbp2- Abll fusion. To answer this question, we sorted both cell populations from a mouse that had been treated with DASA+ATO+PIPC (data not shown) and compared the levels of Ranbp2- Abll fusion in both cell samples using Fish analysis with a probe targeting Abll gene sequence. We also tested BM cells from a sham treated mouse CTL and included a fluorescent probe targeting Lmo2 rearrangement to ensure Lmo2 rearrangement and Ranbp2-Abll fusion were also in the same cells after mouse engraftment. The results of careful analysis indicated that Abll is rearranged in sorted CD45highCD71ow leukemic cells and in leukemic cells from the tested untreated CTL mouse (that were mainly CD45highCD71ow), but it was not detected in sorted CD451owCD7high leukemic cells (Fig3). Interestingly engraftment into mouse BM seemed to enrich in Abll rearranged cells since higher levels of rearranged Abll were detected in mouse BM compared to M181R blood cells, indicative of a selection process in the mouse BM. Further analysis of such xenografted cells confirmed the presence of both Lmo2 and Abll rearrangements in the same cells (data not shown) although additional cells containing only Lmo2 (13%) or only Abll (7%) were also detected in the tested sample.
Altogether these results were supporting the idea that DASA combined to ATO and PIPC was efficiently targeting the leukemic cells containing Ranbp2-Abll fusion.
Extending the treatment period considerably enhanced DASA+ATO+PIPC treatment efficiency
Based on the first treatment experiment, we wished to test whether extending the treatment period, and thus the number of doses of drugs provided to the mice would improve drug combination efficiency. A total of 40 mice were injected with xenografted cells from the BM of a primary NSG-W41 mouse transplanted with M181R patient cells from the sham treated CTL group. 5 weeks later mice were bled and leukemia infiltration was found to be very high (>80% for most of the mice) (data not shown). Treatments were initiated the following day using the same drug doses than before and lasted 3 weeks with adaptation of ATO+PIPC frequency of delivery based on the weight loss of mice (Fig4). At the end of the treatment period, BM were sampled from all live mice, a total of 9 mice were euthanasied to measure leukemia multi-sites infiltration and a survival curve was initiated with the remaining treated mice (Fig 5C-H). Of note, most CTL mice succumbed before the end of the treatment and only one was left, but sick, and thus was analyzed one day post-treatment (Fig5H). The results of additional CTL mice are combined with the results of the other treatment conditions, albeit they were sick and had to be analyzed before the other groups (Fig5C and 5E). The results show that all drug treatment conditions had an effect, either on the leukemic cell numbers and/or infiltration levels in the BM and the SPL (Fig8B-G). However the strongest effects were obtained when all drugs were combined. An extremely low leukemic cell load was detected in BM (0.01%) (Fig5C-D) and SPL (<0.01%) (Fig5E-G) of treated mice (3 logs difference from the DASA treated group). The SPL weight was 10 times less than in CTL and ATO+PIPC treated mice (Fig5F). The survival curve-is showing a strong difference between DASA treated and untreated groups (Fig5H). Moreover, combining DASA+ATO+PIPC allowed higher survival than DASA alone with respectively 7/7 vs 0/7 mice being alive 30 days after the end of treatment, the 2 last mice of the DASA treatment group being euthanasied at day 27 (Fig5H). Pictures of remaining mice at day 25 show that DASA treated mice (3/7 at that time) were very static indicating they were sick whereas DASA+ATO+PIPC treated mice were still vital and healthy (data not shown), showing major differences between results of treatment protocols.
In vitro growth of M181 T-ALL
In order to have easy access to M181R T-ALL growth, we tested leukemic cell expansion in vitro. We have previously shown that human T-ALL require stromal cell co cultures to efficiently and durably expand ex-vivo (Armstrong, Blood, 2009). We also found that short “time to leukemia” (TTL) T-ALL readily grow in liquid culture (Poglio, Leukemia, 2015). Here interestingly, we observed that M181R did not require the presence of stromal supporting cells for ex-vivo expansion (Fig6A). Treatment of M181R cells from the BM of a CTL untreated mouse with ATO (50nM) +IFNg (20ng/mL), DASA (ImM) or the three drugs together indicated that DASA exerted the most pronounced effect on leukemic cell growth over time, the combination of drugs being as efficient as DASA alone (Fig6A). However this might be a question of ATO concentration since adding more (ATO: ImM) during the culture in combination with IFNy (20ng/mL) +/- DASA (ImM) decreased leukemic cell growth (Fig6B), in relation to decreased cell cycle progression (Fig 6C) and increased apoptosis (Fig6D). This part of the project requires more work in culture but already offers ideal tools to investigate the molecular mechanisms.
Interestingly enough, another T-ALL (named M106) was also sensitive to the drug treatments in vitro, especially to the combination of drugs, indicating a possible more general effect on other T-ALL samples (Fig6E), that we need to investigate further in vitro and in vivo by treating mice engrafted with other T-ALL and B-ALL, having fusion proteins implicating Ranbp2 and/or Abll, such as B-ALL with Ranbp2-Abll, T-ALL with Nup214-Abll, lymphomas with Ranbp2-Alk, etc...
DAS A and ATO are efficiently targeting respectively Abll phosphorylation and PML degradation in M181R T-ALL
In order to understand the molecular mechanisms underlying the synergistic effects of DASA, ATO and IFNs, we investigated the levels of phosphorylation of Ranbp2-ABL1 and some targets of Abll pathway, as well as PML expression. Measures were performed in M181R cells (patient cells and cells recovered from mice) treated in vitro (data not shown) and in vivo (data not shown). We observed a constantly elevated level of Ranbp-Abll protein phosphorylation, as well as phosphorylation of STAT5 and CRKL, downstream effectors of Abll pathway in the leukemic cells, whatever their origin (patient and xenograft), indicating that Ranbp2-Abll fusion protein is constitutively activated. Phosphorylation of these proteins was strongly inhibited by Glivec and DASA, two known inhibitors of tyrosine kinase, as shown by western blot (data not shown) and flow cytometry (data not shown) when M181R cells were treated in cultures and during growth in ID mice. We also found that ATO strongly diminished PML expression in M181R (data not shown).
All these results show efficient targeting of the ABLl and PML pathways in M181R cells respectively by ITK and ATO. Thus the combination of ATO with inhibitors of TK represents a powerful way for eradication of T-ALL cells and more generally pathological cells that implicate high Abll activity and nuclear pore protein lesions.
Treatment of mice injected with RANBP2-ABL1 B cell acute lymphoblastic leukemia
In order to test DASA+ATO+PIPC treatment on mice model, we injected human RANBP2-ABL1 B cell acute lymphoblastic leukemia in primary immune-deficient mice. We observed a high engraftment levels of human B-ALL with RANBP2-ABL1 in primary immune- deficient mice (Fig. 8A and 8B).
We then tested the DASA+ATO+PIPC treatment in the secondary mice injected with RANBP2-ABL1 B cell acute lymphoblastic leukemia. We demonstrated that combining DASA+ATO+PIPC is more effective than other combinations in human B-AL with RANBP2- ABLl in decreasing leukemic cell levels in the BM of immune-deficient mice (Fig. 9A). This combination appeared also more effective in decreasing leukemic cell levels in the spleen of mice (Fig. 9B-D). The mice treated with DASA+ATO+PIPC survive longer than the mice treated with other combinations or not treated (Fig. 9E).
Treatment of mice injected with NUP214-ABL1 T cell acute lymphoblastic leukemia We then tested the DASA+ATO+PIPC treatment in mice injected with a NUP214-
ABL1 T cell acute lymphoblastic leukemia. The combinations DASA+ATO+PIPC seems more effective than other combinations in human T-ALL with NUP214-ABL1 in decreasing leukemic cell levels in the BM and the spleen of immune-deficient mice (Fig. 10A-F). Then, in order to assay the specificities of the drug combination effects, we tested the
DASA+ATO+PIPC treatment in mice injected with a T-ALL with no ABL1 rearrangement. The results indicate some effects of combining DASA+ATO+PIPC in the blood of mice injected with this leukemia (used as control) (Fig. 11A). Nevertheless, the three combined drugs did not affect leukemic cell levels in the BM and in the spleens of the mice (Fig. 11B-F). Moreover, the mice treated with DASA+ATO+PIPC did not survive longer than the mice treated with other combinations (Fig. 11G). These results reveal the specificity of the drug combination effects for the leukemia with a nuclear envelop protein- ABLl fusions, such as NUP214-ABL1 or RANBP2- ABL 1. REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:
1. A method for treating a cancer having a protein kinase gene fused to a gene coding for a nuclear-envelop protein in a subject comprising a step of administering to the subject a therapeutically effective of an arsenic compound, an inductor of type 1 IFNs and a protein kinase inhibitor (PKI), as combined preparation.
2. The method according to claim 1, wherein the cancer has ABL1 fused to a gene coding for a nuclear-envelop protein.
3. The method according to claim 1, wherein, the cancer has RANBP2-ABL1 fusion or NUP214-ABL1 fusion.
4. The method according to claims 1 to 3 wherein, the cancer is selected from the group consisting of but not limited to neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non- Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
5. The method according to claim 4, wherein the cancer is acute lymphoblastic leukemia (ALL).
6. The method according to claims 1 to 5, wherein the inductor of type-1 IFNs is poly- inosinic:polycytidinic acid (Poly(LC).
7. The method according to claims 1 to 6, wherein PKI is a tyrosine kinase inhibitor (TKI).
8. The method according to claims 1 to 7, wherein the TKI is selected from the group consisting of but not limited to imatinib, nilotinib, dasatinib, ponatinib, bosutinib and bafetinib.
9. The method according to claims 1 to 8, wherein the arsenic compound is selected from the group consisting of arsenic, arsenic trioxide (As203 or ATO), melarsoprol and arsenic sulfur derivative.
10. The method according to claims 1 to 9 wherein said combined preparation is suitable for a separately, sequentially or simultaneous use.
11. A pharmaceutical composition comprising an arsenic compound, an inductor of type-1 IFNs and a protein kinase inhibitor (PKI), as a combined preparation.
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