WO2020117867A1 - Darinaparsine et composés d'acide rétinoïque pour le traitement de troubles associés à l'idh - Google Patents

Darinaparsine et composés d'acide rétinoïque pour le traitement de troubles associés à l'idh Download PDF

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WO2020117867A1
WO2020117867A1 PCT/US2019/064326 US2019064326W WO2020117867A1 WO 2020117867 A1 WO2020117867 A1 WO 2020117867A1 US 2019064326 W US2019064326 W US 2019064326W WO 2020117867 A1 WO2020117867 A1 WO 2020117867A1
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retinoic acid
leukemia
cells
darinaparsin
acid compound
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PCT/US2019/064326
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English (en)
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Pier Paolo Pandolfi
John Clohessy
Vera MUGONI
Ming Chen
Giulia CHELONI
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Beth Israel-Deaconess Medical Center, Inc.
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Publication of WO2020117867A1 publication Critical patent/WO2020117867A1/fr

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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
    • A61K31/28—Compounds containing heavy metals
    • A61K31/285—Arsenic compounds
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • Isocitrate dehydrogenase enzymes are key metabolic enzymes. Mutations to IDH1 and IDH2 have been identified as important early events in a variety of tumor types. For example, IDH enzymes are mutated in approximately 20% of human acute myeloid leukemias (AMLs). Pathogenic mutants of IDH enzymes have been identified that give rise to 2-hydroxyglutarate (2-HG), an oncometabolite that contributes to the oncogenic phenotype. Accordingly, 2-HG is a predictive biomarker in cancers having a pathogenic IDH1 or IDH2 allele.
  • 2-HG 2-hydroxyglutarate
  • IDH2-targeting Enasidenib AG-221
  • mIDH2 mutant form of IDH2
  • mIDH i.e., mIDHI and mIDH2
  • leukemias may adjust and evolve in response to treatment. Therefore, there is a need to identify common vulnerabilities in mIDH leukemias, and to develop rational therapies capable of preventing or overcoming resistance that arises in response to therapy.
  • the present invention is based on the discovery of common vulnerabilities in mIDH leukemia.
  • mIDH leukemia exhibits sensitivity to reactive oxygen species (ROS)-producing compounds, such as arsenic trioxide (ATO) and Darinaparsin.
  • ROS reactive oxygen species
  • ATO arsenic trioxide
  • ATRA retinoic acid compound-induced differentiation
  • the inventors have further shown that treatment of leukemia with the combination of a ROS promoting compound (e.g., arsenic trioxide and Darinaparsin) and a compound that promotes differentiation (e.g., a Pinl inhibitor, such as ATRA) provides a synergistic, powerful, and well-tolerated targeted therapy in both mouse and human models of AML.
  • a ROS promoting compound e.g., arsenic trioxide and Darinaparsin
  • a compound that promotes differentiation e.g., a Pinl inhibitor, such as ATRA
  • the present invention therefore features methods of treating cancer by contacting the cells of the cancer with a pharmaceutical compound.
  • the pharmaceutical compound can be Darinaparsin, a retinoic acid compound (e.g., ATRA), or a combination of Darinaparsin and a retinoic acid compound (e.g., Darinaparsin and ATRA).
  • the cancer can be a leukemia (e.g., mIDHl/mIDH2 leukemia) or a solid tumor (e.g., an IDH1- or IDH2-associated solid tumor).
  • a leukemia e.g., mIDHl/mIDH2 leukemia
  • a solid tumor e.g., an IDH1- or IDH2-associated solid tumor.
  • the solid tumors include, but are not limited to, glioma, paraganglioma, astroglioma, colorectal carcinoma, melanoma, cholangiocarcinoma, chondrosarcoma, thyroid carcinomas, prostate cancers, and non-small cell lung cancer.
  • the invention features a method of treating leukemia in a subject.
  • the method includes contacting the cells of said leukemia with (e.g., by administering to said subject) an effective amount of a pharmaceutical compound, the pharmaceutical compound being Darinaparsin, a retinoic acid compound, or a combination thereof, wherein one or more cells of the leukemia has a pathogenic IDH2 allele or has elevated 2- hydroxyglutarate (2-HG) levels; and wherein contacting said cells of said leukemia with said pharmaceutical compound treats said leukemia in said subject.
  • the invention features a method of treating leukemia in a subject, wherein one or more cells of the leukemia has a pathogenic IDH1 allele or has elevated 2-HG levels.
  • the method includes contacting the cells of said leukemia with (e.g., by administering to said subject) an effective amount of a pharmaceutical compound, the pharmaceutical compound being Darinaparsin, a retinoic acid compound, or a combination thereof, wherein contacting said cells of said leukemia with said pharmaceutical compound treats said leukemia in said subject.
  • Elevated 2-HG levels may be considered to include detection of any 2-HG at all.
  • a pathological level of 2-HG associated with an IDH1/IDH2 cancer may be determined by levels of 2-HG at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater than the 2-HG levels measured in a normal (e.g., wild-type and/or disease fee) subject, tissue, or cell.
  • a normal e.g., wild-type and/or disease fee
  • Elevated 2-HG levels can be determined via (i) the detection of D-2-HG in biological fluids detected by LC-MS/MS; (ii) the detection of total 2-HG (including both L-2-HG and D-2- HG) in the serum above 2mM; or (iii) the detection of the presence of mutant variants of IDH1/IDH2.
  • Darinaparsin and the retinoic acid compound results in the remission of the leukemia in the subject (e.g., the symptoms of the leukemia are reduced). In some embodiments, contacting said cells of said leukemia with the Darinaparsin and the retinoic acid compound results in the complete remission of the leukemia (e.g., all signs and symptoms of the leukemia are absent). In some embodiments, contacting said cells of said leukemia with the Darinaparsin and the retinoic acid compound cures the leukemia in the subject (e.g., all signs and symptoms of the leukemia are absent for 1 year or more, for 2 years or more, for 3 years or more, for 4 years or more, or for 5 years or more).
  • the Darinaparsin and the retinoic acid compound operate synergistically to treat said leukemia. In some embodiments, the Darinaparsin and the retinoic acid compound is more effective for treating said leukemia than the same quantities of either said Darinaparsin or said retinoic acid compound alone.
  • Darinaparsin and the retinoic acid compound increases the production of reactive oxygen species (ROS) in one or more cells of said leukemia, e.g., increases the production of ROS by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to production of ROS prior to treatment with the Darinaparsin and the retinoic acid compound.
  • contacting said cells of said leukemia with of the Darinaparsin and the retinoic acid compound increases the production of ROS by at least about 10%.
  • Darinaparsin and the retinoic acid compound promotes differentiation of one or more cells of said leukemia.
  • contacting said cells of said leukemia with of the combination of Darinaparsin and a retinoic acid compound is sufficient to inhibit and/or degrade Pinl in the subject.
  • this may include an increase in degradation of Pinl of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to Pinl activity prior to treatment with the Darinaparsin and the retinoic acid compound.
  • Darinaparsin and the retinoic acid compound increases the degradation of Pinl of at least about 10%.
  • this may include a reduction in Pinl activity of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to Pinl activity prior to treatment with the Darinaparsin and the retinoic acid compound.
  • the contacting said cells of said leukemia with of Darinaparsin and a retinoic acid compound is more effective for inhibiting and/or degrading Pinl in the subject than administration of the same quantities of either the Darinaparsin or the retinoic acid compound alone.
  • contacting said cells of said leukemia with the Darinaparsin and the retinoic acid compound is more effective for treating the leukemia than contacting said cells of said leukemia with of the same quantities of either the Darinaparsin or the retinoic acid compound alone.
  • the Darinaparsin and the retinoic acid compound may be administered concurrently (e.g., within about lmin, 2min, 5min, lOmin, 20min, 30min, or 60min) or separately.
  • the Darinaparsin may be administered either prior to or after the retinoic acid compound.
  • the invention features a method of treating leukemia in a subject.
  • the method includes contacting the cells of the leukemia with an effective amount of the Darinaparsin to said subject, wherein one or more cells of the leukemia have a pathogenic IDH1/IDH2 allele or have been previously determined to have elevated 2-hydroxy glutarate (2-HG) levels; and wherein contacting said cells of said leukemia with the Darinaparsin treats the leukemia in the subject.
  • Elevated 2-HG levels may be considered to include levels of 2-HG at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater than the 2-HG levels measured in a normal (e.g., wild-type and/or disease fee) subject, tissue, or cell.
  • a normal e.g., wild-type and/or disease fee
  • Darinaparsin increases the production of reactive oxygen species (ROS) in one or more cells of said leukemia (e.g., increases the production of ROS by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to production of ROS prior to treatment with the Darinaparsin).
  • ROS reactive oxygen species
  • Darinaparsin is sufficient to inhibit and/or degrade Pinl in the subject. In some embodiments, this may include an increase in degradation of Pinl of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to Pinl activity prior to treatment with the Darinaparsin.
  • this may include a reduction in Pinl activity of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to Pinl activity prior to treatment with the Darinaparsin.
  • contacting said cells of said leukemia with of the Darinaparsin increases the degradation of Pinl of at least about 5%.
  • the invention features a method of treating leukemia in a subject.
  • the method includes contacting the cells of the leukemia with an effective amount of a retinoic acid compound to the subject, wherein the subject has a pathogenic IDH1/IDH2 allele or wherein one or more cells of the leukemia have been previously determined to have elevated 2-hydroxyglutarate (2-HG) levels; and wherein contacting said cells of said leukemia with the retinoic acid compound treats the leukemia in the subject.
  • Elevated 2-HG levels may be considered to include levels of 2-HG at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater than the 2-HG levels measured in a normal (e.g., wild-type and/or disease fee) subject, tissue, or cell.
  • a normal e.g., wild-type and/or disease fee
  • contacting said cells of said leukemia with the retinoic acid compound promotes differentiation of one or more cells of the leukemia.
  • contacting said cells of said leukemia with the retinoic acid compound is sufficient to inhibit and/or degrade Pinl in the subject.
  • this may include an increase in degradation of Pinl of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to Pinl activity prior to treatment with retinoic acid compound.
  • this may include a reduction in Pinl activity of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to Pinl activity prior to treatment with the retinoic acid compound.
  • contacting said cells of said leukemia with of the retinoic acid compound increases the degradation of Pinl of at least about 5%.
  • the subject has elevated levels of Pinl activity (e.g., has previously been determined to have elevated levels of Pinl activity in one or more cells of the leukemia).
  • Levels of Pinl activity in the subject may be determined by measuring the levels of at least one Pinl marker, wherein elevated levels of the Pinl marker is indicative of elevated Pinl activity.
  • Non-limiting examples of Pinl markers include nucleic acid molecules (e.g., mRNA, DNA) that correspond to some or all of a Pinl gene, peptide sequences (e.g., amino acid sequences) that correspond to some or all of a Pinl protein, nucleic acid sequences which are homologous to Pinl gene sequences, peptide sequences which are homologous to Pinl peptide sequences, alteration of Pinl protein, antibodies to Pinl protein, substrates of Pinl protein, binding partners of Pinl protein, alteration of Pinl binding partners, and activity of Pinl.
  • nucleic acid molecules e.g., mRNA, DNA
  • peptide sequences e.g., amino acid sequences
  • alteration of a Pinl protein may include a post-translational modification (e.g., phosphorylation, acetylation, methylation, lipidation, or any other post-translational modification known in the art) of Pinl.
  • a Pinl marker is the level of Pin expression (e.g., Pinl protein expression levels and/or Pinl mRNA expression levels) in a subject.
  • Elevated levels of a Pinl marker include, for example, levels at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater than the marker levels measured in a normal (e.g., wild-type and/or disease fee) subject, tissue, or cell.
  • elevated levels of a Pinl marker include levels at least about 3% or greater than the marker levels measured in a normal subject, tissue, or cell.
  • the method further comprises contacting said cells of said leukemia with a compound that inhibits Pinl activity (e.g., in combination with the retinoic acid and the Darinaparsin).
  • a compound that inhibits Pinl activity e.g., in combination with the retinoic acid and the Darinaparsin.
  • the subject has decreased levels of lysine-specific demethylase (LSD1) activity.
  • LSD1 activity can be determined by methods known to one of skill in the art, including determining the levels of lysine histone methylation (e.g., H3K4me2 and/or H3K9me2).
  • Decreased levels of LSD 1 activity include, for example, a reduction of LSD1 activity of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater than the marker levels measured in a normal (e.g., wild- type and/or disease fee) subject, tissue, or cell.
  • decreased levels of LSD1 activity include a reduction of LSD1 activity of about 20% or greater than the marker levels measured in a normal subject, tissue, or cell.
  • the method further comprises contacting said cells of said leukemia with a compound that inhibits LSD1 activity (e.g., reduced LSD1 activity by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to LSD1 activity prior to treatment).
  • a compound that inhibits LSD1 activity e.g., reduced LSD1 activity by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to LSD1 activity prior to treatment.
  • the method further comprises contacting said cells of said leukemia with an inhibitor of IDH1/IDH2.
  • the inhibitor of IDH1/IDH2 may reduce IDH1/IDH2 activity (e.g., as measured by a reduction in 2-HG levels) by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater relative to IDH1/IDH2 activity prior to treatment.
  • the inhibitor of IDH1/IDH2 may be specific for a pathogenic mutant form of IDH1/IDH2. When such an inhibitor targets a mutant form of IDH1/IDH2, it may reduce the aberrant activity of the enzyme by about 20%.
  • the pathogenic IDH1 allele is IDH1 R132C and the pathogenic IDH2 allele is IDH2 R140Q .
  • the method further includes contacting said cells of said leukemia with an effective amount of dexamethasone.
  • the invention features a kit for treating leukemia in a subject, wherein the kit includes: (a) an effective amount of Darinaparsin, (b) an effective amount of a retinoic acid compound, and (c) instructions for the use of the Darinaparsin in combination with the retinoic acid compound for treating the leukemia in the subject, wherein one or more cells of said leukemia has a pathogenic IDH1/IDH2 allele or wherein the subject has been previously determined to have elevated 2-hydroxyglutarate (2-HG) levels.
  • the kit includes: (a) an effective amount of Darinaparsin, (b) an effective amount of a retinoic acid compound, and (c) instructions for the use of the Darinaparsin in combination with the retinoic acid compound for treating the leukemia in the subject, wherein one or more cells of said leukemia has a pathogenic IDH1/IDH2 allele or wherein the subject has been previously determined to have elevated 2-hydroxyglutarate (2-HG) levels.
  • Elevated 2-HG levels may be considered to include levels of 2-HG at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater than the 2-HG levels measured in a normal (e.g., wild-type and/or disease fee) subject, tissue, or cell.
  • the kit further includes an effective amount of a compound that inhibits Pinl activity and instructions for the use of the compound that inhibits Pinl activity in combination with the Darinaparsin and the retinoic acid compound.
  • the kit further includes an effective amount of a compound that inhibits LSD1 activity and instructions for the use of the compound that inhibits LSD1 activity in combination with the Darinaparsin and said retinoic acid compound.
  • the kit further includes an effective amount of a compound that inhibits Pinl activity, an effective amount of a compound that inhibits LSD1 activity, and instructions for the use of the compound that inhibits Pinl activity and the compound that inhibits LSD1 activity in combination with the Darinaparsin and the retinoic acid compound.
  • the retinoic acid compound is administered in a low dose such as about 5 mg/kg body weight or less (e.g., about 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, or 5 mg/kg body weight or less), 1.5 ug/g body weight or less (e.g., about 0.1, 0.2, 0.5, 0.75, 1, or 1.5 ug/g body weight or less), less than about 25 mg/m 2 (e.g., less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg/m 2 ), or between 25 mg/m 2 and 45 mg/m 2 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mg/m 2 ).
  • a low dose such as about 5 mg/kg body weight or less (e.g., about 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, or 5 mg/kg
  • the low dose of the retinoic acid compound is a nontoxic dose of the retinoic acid compound. In some embodiments, the low dose of the retinoic acid compound is administered in combination with a low dose of retinoic acid. In some embodiments the low dose of the retinoic acid compound and the low dose of the retinoic acid are nontoxic.
  • a low dose of a retinoic acid compound related to in vivo treatments is 1.5 mg/g/day or lower. In some embodiments, a low dose of the retinoic acid compound comprises a dose of about 30 mg/m 2 body surface area or less. For example, a low dose of a retinoic acid compound related to treating a human with leukemia is 10-22.5 mg/m 2 (PO administration, BID). In some embodiments, a low dose of the retinoic acid compound comprises a dose of about 10 mg/m 2 body surface area or less.
  • the Darinaparsin is administered in a low dose, such as 2 mg/kg body weight or less (e.g., about 0.01, 0.02, 0.03, 0.032, 0.04, 0.05, 0.06, 0.07,
  • a low dose of the Darinaparsin is about 0.15, about 0.16, or about 0.032 mg/kg body weight. In some embodiments, the low dose of the Darinaparsin is a nontoxic dose of the Darinaparsin.
  • a low dose of Darinaparsin related to in vitro treatments of leukemia cells is 0.25 mM or lower.
  • a low dose of Darinaparsin related to in vivo treatments e.g., in mice treatments
  • a low dose of Darinaparsin comprises a dose of about 2.5 mg/kg body weight or less (e.g., a dose of between about 0.05 mg/kg body weight and about 2.5 mg/kg body weight).
  • a low dose of Darinaparsin related to treating a human with leukemia is 0.075-0.15 mg/kg (IV administration, QD).
  • a low dose of Darinaparsin comprises a dose of about 0.05 mg/kg body weight or less.
  • the low dose of the Darinaparsin is administered in combination with a low dose of a retinoic acid compound. In some embodiments the low dose of the Darinaparsin and the low dose of retinoic acid compound are nontoxic.
  • the retinoic acid compound is all-trans retinoic acid (ATRA), 13-cis-retinoic acid, retinol, retinyl acetate, retinal, or AC-55640, or is a compound structurally similar to retinoic acid.
  • the retinoic acid compound is a compound selected from Table 1 or Table 2.
  • the retinoic acid compound is ATRA.
  • the leukemia is acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the acute myeloid leukemia may be either relapsed acute myeloid leukemia or refractory acute myeloid leukemia.
  • the leukemia is an IDH1/IDH2 independent (e.g., resistant) leukemia.
  • An IDH2 independent leukemia includes, for example, a leukemia having a pathogenic IDH2 allele which has developed resistance to IDH2- targeting therapies (e.g., Enasidenib).
  • the leukemia described above can be acute promyelocytic leukemia (APL). In some embodiments, the leukemia is not acute promyelocytic leukemia APL.
  • one or more cells of said leukemia has a pathogenic IDH1/IDH2 allele and elevated 2-hydroxyglutarate (2-HG) levels.
  • one or more cells of the leukemia comprise a pathogenic IDH1 or IDH2 allele in combination with one or more other mutants.
  • the other mutants include, but are not limited to, FLT3, NPM1, DNMT3A, RUNX1, TET2, IDH2, CEBPA, TP53, IDH1, NRAS, RARA, PML, TTN, WT1, MYH11, BPIFC, CBFB, KIT, KRAS, KMT2A, PTPN11, MUC16, SMC1A, RUNX1T1, MPO, U2AF1, ABCA6, DMXL2, DNAH3, KLK3, FBX07, SMC3, MXRA5, MUC17, SF3B1, HPS3, PHF6, ASXL1, AHNAK, SENP6, MYCBP2, NF1, PCLO, CSMD3, LRP1B, MED 12, RAD21, AHNAK2, GDI2, PARP14
  • MLLT10 DNAH11, SLIT2, DNAH9, BRINP3, HMCN1, SYNE1, GABRG3, NPAS3, CSMD1, GATA2, ZCRB1, MTFR2, SLC43A1, SLA2, APOB, PRDM1, BTBD10, ABCA5, EP300, TNC, ADGRG4, BSN, IFT74, COL12A1, NUP98, CREBBP, EPG5, KIF1B, ABL1, SLC16A7, RALGPS2, CHL1, SCLT1, LRP2, VWA3B, FRYL, PRUNE2, PLCE1, GRIK4, HIVEP3, RYR3, ANK2, ATRNL1, VPS13B, GPR179, FCGBP, KIF15, and DNAH10.
  • Two exemplary mutants are NPMc+ and FLT3-ITD.
  • one or more cells of the leukemia comprise a pathogenic IDH1 allele in combination with NPMc+, FLT3-ITD, or both.
  • An exemplary pathogenic IDH1 allele is IDH1 R132C .
  • one or more cells of the leukemia comprise a pathogenic IDH2 allele in combination with NPMc+, FLT3-ITD, or both.
  • An exemplary pathogenic IDH2 allele is IDH2 R140Q .
  • the effective amount of the Darinaparsin is a low dose of about 2.5 mg/kg body weight or less
  • the effective amount of the retinoic acid compound is a low dose of about 30 mg/m 2 body surface area or less
  • the retinoic acid compound is selected from the group consisting of all-trans retinoic acid (ATRA), 13-cis-retinoic acid, retinol, retinyl acetate, retinal, and AC-55640.
  • the effective amount of the Darinaparsin is a low dose of between about 0.05 mg/kg body weight and about 2.5 mg/kg body weight
  • the effective amount of the retinoic acid compound is a low dose of about 10 mg/m 2 body surface area or less
  • the retinoic acid compound is ATRA.
  • one or more cells of the leukemia comprise a pathogenic IDH1 or IDH2 allele in combination with NPMc+, FLT3-ITD, or both, in which said pathogenic IDH1 allele is IDH1 R132C and said pathogenic IDH2 allele is IDH2 R140Q .
  • Fig. 1A is an image depicting the approach used to model in vivo independence from mIDH2 (IDH2R140Q) in AML.
  • Mouse model is based on serial transplantation of leukemia cells derived form Tg(M2rt-TA) transgenic mice and overexpressing HoxA9 and Meisla oncoproteins. Red boxes: leukemia. Blue boxes: healthy state.
  • Fig. IB is a series of images depicting Flow cytometry plots of bone marrow samples.
  • X- axis fluorescence Intensity (0 - 105), Y-axis, cell counts.
  • Fig. 1C is a graph depicting the percentage of Leukemic cells in the bone marrow ( BM) of 2 nd and 3 rd recipients maintained on DOX ON or DOX OFF diet. Quantitation is expressed as percentage of leukemic cells showing positivity for GFP and YFP fluorescence.
  • Fig. ID is a series of images showing May-Griinwald-Giemsa staining of peripheral blood (PB) smears at euthanization (6- 9 weeks after transplant).
  • PB peripheral blood
  • Fig. IE is a graph depicting the percentage of blasts in peripheral blood ( PB) in of 2 nd and 3 rd recipients maintained on DOX ON or DOX OFF diet.
  • Fig. IF is a graph depicting the LC- MS quantification of the 2-HG peripheral blood ( PB) in of 2 nd and 3 rd recipients maintained on DOX ON or DOX OFF diet.
  • Fig. 1G is a graph depicting real time quantitative PCR analyses for the expression of mIDH2 (IDH2 R140Q ) in leukemia cells.
  • Fig. 1H is a graph depicting real time quantitative PCR analyses for the expression of HoxA9 in leukemia cells.
  • Fig. II is a graph depicting real time quantitative PCR analyses for the expression of Meisl A in leukemia cells.
  • IDH2R140Q inhibitor IDHi
  • IDHi AGI-6780
  • ImM vehicle
  • Fig. IK is a series of images depicting representative magnification of methylcellulose colonies generated by blasts at 3rd plating.
  • Fig. 1L is a graph depicting the relative LC- MS quantification of the 2-HG from colonies pooled and extracted for metabolites at 3rd-plating.
  • Fig. 2A is an image depicting Hierarchical clustering of leukemia cells’ metabolites isolated from Dox+ cohorts. Rows: metabolites; columns: samples; color key indicates metabolite abundance level (blue: lowest; red: highest). Clustering generated by MetaboAnalyst’ s annotation tool.
  • Fig. 2B is an image depicting the Pathway enrichment analysis of altered metabolic pathways between sensitive (2nd RECIPIENT - derived cells) and resistant (3rd RECIPIENT - derived) leukemia cells.
  • Fig. 2C is an image depicting the bar chart showing metabolic pathways enriched in resistant leukemia cells isolated from 3rd RECIPIENTS.
  • Fig. 2D is a graph depicting Fold changes of Cysteine (CYS) and Glutathione (GSH) metabolites in resistant leukemia cells compared with sensitive leukemia cells.
  • CYS Cysteine
  • GSH Glutathione
  • Fig. 2E is a graph depicting Fold change of the NAD+/NADH and
  • NADP+/NADPH ratio in resistant leukemia cells compared with sensitive leukemia cells NAD+: nicotinamide adenine dinucleotide oxidized, NADP+: nicotinamide adenine dinucleotide phosphate oxidized, NADH: nicotinamide adenine dinucleotide reduced, NADPH: nicotinamide adenine dinucleotide phosphate reduced.
  • X- axis Fluorescence intensity levels;
  • Y-axis percentage of cell counts.
  • Fig. 2G is a graph depicting the percentage of cells showing high levels of ROS (ROSHi) in sensitive (derived from 2nd RECIPIENTS) and resistant (derived from 3rd RECIPIENT) cells shown in (2F).
  • ROS ROS
  • Fig. 21 is a series of images depicting IHC for gH2A.C in mouse spleen sections and percentage of infiltrating blasts showing positivity for the gH2A.C marker.
  • Fig. 2J(a-e) a series of graphs showing the transcriptional rewiring of mIDH leukaemia at early (sensitive) stage, characterized by altered MAPK and ATRA associated pathways a. Bar chart showing the most significantly up-regulated KEGG pathways in HoxA9/Meisla/mIDH2 cells. The pathways are ranked on the basis of the gene set size indicating activation b-c.
  • GSE Gene Set Enrichment
  • GSE Gene Set Enrichment plots of Tretinoin related Signature in HoxA9/Meisla/mIDH2 vs HoxA9/Meisla.
  • d-e Gene Set Enrichment (GSE) plots of LSD1 Signature in HoxA9/Meisla/mIDH2 vs HoxA9/Meisla.
  • Fig. 3A is an image depicting the heat map of differentially expressed genes (P ⁇ 0.05) in sensitive and resistant samples or Hoxa9/Meisla leukemia, not primed by mIDH2.
  • the columns represent the samples and the rows represent the genes.
  • Gene expression is shown with pseudocolor scale (-3 to 3) with upregulated genes shown as a shade of blue and downregulated genes as a shade of red.
  • Fig. 3B is an image depicting the bar chart showing the most significantly up- regulated pathways (multiple test corrected p-value ⁇ 0.05) in resistant leukemia cells. The pathways are ranked on the basis of Z score indicating activation.
  • Fig. 3C is an image depicting Gene Set Enrichment (GSE) plots of MAPK
  • TNFcr TNFcr
  • Fig. 3D is an image depicting the Venn Plot showing shared genes between mouse mIDH2 leukemia and Schenken et ak; 2013 gene sets.
  • Fig. 3E is an image depicting the Heat map showing shared genes shown in (3D) are regulated concordantly in Resistant and Sensitive clone with respect to ATRA+TCP gene sets reported by Schenken et ak; 2013.
  • the figure legend ( right panel) describes representative images of leukemia cells at different stages of differentiation ( blasts, intermediate, differentiated)
  • Fig. 3G is a graph depicting relative LC-MS/MS quantitation of 2-HG in TF1 cells overexpressing IDH2R140Q or respective controls
  • Fig. 3H is a series of images and graphs depicting Cytospin images and quantitation of human TF1 cell line stably overexpressing the mutant variant R140Q of IDH2 (mIDH2) to the respective controls (CTRL) that have been treated with pharmacological concentrations of ATRA (10-6M) or vehicle (DMSO).
  • Fig. 3J is a series of images depicting different culture media color of TF1 cell line stably overexpressing mIDH2 or respective control (CTRL).
  • the asterisks indicate mIDH2-TFl leukemia cells treated with treated with ATRA (10-6M).
  • 1-4 flasks show cultured TF1 cell line (CTRL)
  • 5-8 flasks show cultured TF1 cell line overexpressing the mutant variant R140Q of IDH2 (mIDH2).
  • Fig. 3K(a-e) a series of graphs showing mouse HoxA9/Meisla/ mIDH2 leukaemia at early (sensitive) stage, marked by switching to One-Carbon metabolism and increased oxidative stress a.
  • Hierarchical clustering of leukaemia cells’ metabolites isolated from Dox-i- cohorts. Rows: metabolites; columns: samples; colour key indicates metabolite abundance level (blue: lowest; red: highest). Clustering generated by
  • MetaboAnalyst’ s annotation tool.
  • Pathway enrichment analysis shows altered metabolic pathways between HoxA9/Meisla/mIDH2 and respective HoxA9/Meisla control cells
  • c. LC-MS/MS fold change levels of precursors for One-Carbon metabolism isolated from HoxA9/Meisla/mIDH2 and respective HoxA9/Meisla control cells. Data are mean ⁇ SD.
  • Fig. 4A is an image depicting Western blot analysis for ATRA targets (Pinl), ATRA receptors (RARcr,RXRcr), and factors associated with ATRA - related differentiation pathway in hematopoietic cells (c/EBPer, c/EBRe).
  • Fig. 4B is an image depicting the Western blot analysis for factors associated with ATRA-related pathway in TF1 cell line, stably overexpressing the mIDH2) or the empty vector (CTRL).
  • Fig. 4C is an image depicting the Western blot analysis for factors associated with ATRA-related pathway in U937 cell line, stably overexpressing the mIDH2) or the empty vector (CTRL).
  • Fig. 4D is a graph depicting Percentage of the transferrin receptor (CD71+) positive TF1 cells overexpressing the mIDH2 or respective control (CTRL). Each cell line was treated with the Pinl Inhibitor (Juglone) or vehicle (DMSO) for 3 days.
  • Fig. 4E is a graph depicting the Colony forming assay of TF1 cells overexpressing the mIDH2 or respective control (CTRL) treated with Juglone or vehicle. Colonies were quantitated after 7 days from plating.
  • Fig. 4F is a graph depicting the quantification of the relative transcriptional expression levels of Hemoglobin (HBG) in TF1 cells silenced for Pinl and
  • Fig. 4G is a graph depicting the quantification of the relative transcriptional expression levels of KLF1 in TF1 cells silenced for Pinl and overexpressing the mIDH2 or respective controls (CTRL).
  • Fig. 4H is a graph depicting the quantification of the relative transcriptional expression levels of GATA2 in TF1 cells silenced for Pinl and overexpressing the mIDH2 or respective controls (CTRL).
  • Fig. 41 is a graph depicting the Fold Induction of CD71 in TF1 cells
  • Fig. 4J is a series of graphs depicting the Quantification of the relative transcriptional expression levels of Hemoglobin (HBG), Kruppel like factor 1 (KLF1), in TF1 cells silenced for Pinl and overexpressing the mIDH2 or respective controls (CTRL).
  • HBG Hemoglobin
  • KLF1 Kruppel like factor 1
  • Fig. 4K is an image depicting the scheme of ATO/ ATRA treatments performed in vivo on BL6J recipient mice transplanted with resistant leukemia
  • Fig. 4L is an image deciphering the percent of survival of recipient mice
  • Fig. 4M is an image deciphering the inhibition of LSD 1 contributes to increase intracellular levels of oxidative stress. Histogram reporting the intracellular oxidative stress of TF1 cells treated for 3 weeks with the LSD1 inhibitor (LSDli; GSK2879552, ImM) or DMSO (VHL) as respective control. Measurements were performed by flow cytometry analyses after incubation of TF1 cells with the CellROX molecular probe. Data are mean ⁇ SD.
  • Fig. 5A is a series of images and graphs depicting methylcellulose colony forming assay quantification of human primary AML blasts treated in vitro with pharmacological concentration of ATRA, ATO, a combination of both, or vehicles as control. Treatments were performed in either the presence of mIDH2 or absence of the mutation (CTRL).
  • Data are means ⁇ SD of duplicates.
  • Fig. 5B is an image depicting the Experimental approach to generate PDX for human AML harboring the mutation R140Qin the IDH2 gene
  • Fig. 5C is an image depicting the Scheme of the pharmacological therapy for PDX resembles therapeutic regimen of human APL patients. Treatment regimen was performed in cycles for a total of 65 days resembling APL0406 protocol (Lo-Coco et al; NEJM 2013).
  • Fig. 5E is a series of images depicting the cytospins of cells isolated from BM of PDX mice treated with ATRA and ATO.
  • Fig. 5F is a graph depicting the percentage of morphologically screened human cells isolated from BM of PDX generated by using IDH2R140Q AML.
  • Fig. 5G is an image depicting the scheme of leukemia evolution from mIDH dependent to mIDH2 independent states (e.g. acquisition of resistance to mIDH inhibition). Multiple alterations including genetic mutations, metabolic reprogramming, and transcriptional rewiring are acquired during progression and co-exist in the mIDH2 independent stage.
  • MapK/PI3K pathways upregulated pro-survival and proliferation programs
  • ATRA pro-differenting
  • sensitivity to ATO and ATRA is a vulnerability already present at an early stage in mIDH2 dependent leukemia, which is mantained in later stages of the disease progression.
  • Fig. 6B is a series of images depicting Haemotoxylin and Eosin (H&E) staining of internal organs (spleen, liver, and kidney) from 3rd RECIPIENTS showing infiltrating blasts.
  • H&E Haemotoxylin and Eosin
  • Enlarged images (40x) show perivascular accumulation of infiltrating blasts in spleen, liver, and kidney.
  • Fig. 6C is a series of images depicting H&E and immunohistochemistry (IHC) staining of kidney sections derived from 3 rd RECIPIENTS.
  • the IHC staining shows infiltrating blasts are positive for the antigen Ki-67, a cellular marker for proliferation.
  • Fig. 6D is a series of images depicting H&E staining of internal organs (spleen, liver, and kidney) from 2nd RECIPIENTS. High resolution images show absence or limited infiltrating blasts.
  • Fig. 6E is a series of images depicting H&E and IHC staining of kidney sections derived from 2 nd RECIPIENTS.
  • the IHC staining for the antigen Ki-67 is mostly negative across the examined tissue.
  • Fig. 6H is a graph depicting a methylcellulose colony forming assay of in vitro cultured blasts, derived from 2nd and 3rd RECIPIENTS.
  • mIDH2 IDH2R140Q.
  • Fig. 7 A is an image depicting the scheme of the experiment for metabolites’ analysis
  • Fig. 7B is an image depicting the One-Carbon metabolism pathway. The image is from Locasale J.W. Nature Reviews Cancerl3, 572-583 (2013).
  • Fig. 7C is a graph depicting Fold change of key metabolites in the Methionine pathway in resistant leukemia cells compared to sensitive cells.
  • MET Methionine
  • SAM S-adenosylmethionine
  • SAH Sadenosylhomomocysteine
  • hCYS homocysteine d
  • Fig. 7D is an image depicting the Representative scheme of the De-novo synthesis of Purine (Adenine and Guanine) and Pyrimidine (Thymine, Cytosine and Uracil).
  • Fig. 7E is an image depicting the Fold change of the precursors for Purine biosynthesis in resistant leukemia cells compared with sensitive leukemia cell.
  • IMP inosine monophosphate
  • AMP adenosine monophosphate
  • GMP guanosine
  • Fig. 7F is an image depicting the Fold change of the precursors for Pyrimidine biosynthesis in resistant leukemia cells compared with sensitive leukemia cells.
  • Orotate orotic acid
  • OMP orotidine monophoshate
  • UMP uridine monophosphate
  • UDP uridine diphosphate
  • Fig. 7G is a series of images and graphs depicting Flow cytometry plots and quantitation of the percentage of cells showing high levels of mitochondrial ROS (ROSHi) in blast cells overexpressing HoxA9/Meisl alone (HoxA9/Meisla mIDH2 OFF) or in combination with mutant IDH2 (HoxA9/Meisla mIDH2 ON).
  • ROSHi mitochondrial ROS
  • Fig. 8A is a scheme depicting samples processed for the Whole Exome
  • Fig. 8B is a pie-chart showing frequencies of transversion (2nd_07).
  • mIDH2 IDH2R140Q or transition mutations responsible for non-synonymous mutations in resistant genomes.
  • Pairs of nucleotide indicate different possible transversion (G>T, OA, A>C, T>G, G>C, C>G, A >T, T >A) and transition mutations targeting T or A (A>G, T>C) and C or G (G>A, OT).
  • Fig. 8C is a principal component analysis (PCA) plot showing clustering of WES samples using single nucleotide coverage of sensitive and resistant cells.
  • Fig. 8D is a venn diagram showing the number of mutated genes overlapping between resistant leukemia cells (3rd_08, 3rd_09, 3rd_ll). Mutations in Spl40 gene are shared between all analyzed resistant clones, but not found in the parental sensitive leukemia.
  • Fig. 8E is a series of circular plots of the expression of CNV and fusion genes for sensitive (2nd_07) and resistant (3rd_08, 3rd_09, 3rd_ll) leukemia cells.
  • Circular tracks from inside to outside Trackl: genome positions by chromosomes (black lines are cytobands) that are arranged circularly end to end.
  • Track 2 lines plot (red: positive; blue: negative) showing gene expression data represented as normalized read counts.
  • Track 3 Barplot showing segmented data (CNVs).
  • Track 4 Barplot with positive (gain: blue) and negative values (purple: loss) of called CNVs. Amplifications or deletion of relevant oncogenes such as Flt3, Dok genes, and Trp53 are highlighted.
  • Track 5 Track 5:
  • Interchromosomal translocations are plotted with lines connecting chromosome segments.
  • Fig. 9A is a scheme depicting the samples processed through RNA-Sequencing analysis to characterize the transcriptional changes associated with leukemia progression.
  • Resistant leukemia cells 3rd_09 and 3rd_13 were generated from the parental sensitive 2nd _9A.
  • Resistant leukemia cells 3rd_l l, 3rd_12, 3rd_08, and 3rd_10 were generated from the parental sensitive 2nd_7A.
  • mIDH2 IDH2R140Q.
  • Fig. 9B is a principal component analysis (PCA) plot showing the genes that are the sources with the majority of the variance in resistant leukemia cells, compared with sensitive leukemia cells.
  • the genes are represented by black dots.
  • Mpo myeloperoxidase
  • Lyz2 lysozyme 2
  • Rn45s 45s-pre-ribosomal RNA
  • Ngp neutrophilic granule protein
  • Psap prosaposin
  • Ftll ferritin light polypeptide 1
  • Zfp36 zinc finger protein 36
  • Hspa8 heat shock protein 8
  • Vim vimentin
  • Fosb FBJ osteosarcoma oncogene B
  • Fos FBJ osteosarcoma oncogene
  • Jun jun proto-oncogene.
  • Fig. 9C is a table showing the variance and associated statistical significance of major relevant genes for the Principal Component Analysis (PCA) plot shown in Fig. 9B.
  • Fig. 9D is an image showing the top altered networks in resistant cells generated by Ingenuity Pathways Analysis tool (www.ingenuity.com). The intensity of the node color indicates the degree of up-regulation (red) and down-regulation (green), while white nodes indicate non-modified genes that may be affected in a non-transcriptional manner. All networks shown were significantly affected, with a score >29.
  • Fig. 9E is a series of graph showing the quantification of intracellular flow staining for p-P44/42 (T201, Y204) and p-Akt (T308) in mouse leukemia cells.
  • Fig. 9F is a graph showing an increase in 2-HG levels in a human leukemia U937 cell line stably overexpressing the mutant variant R140Q of IDH2 (mIDH2) compared to a U937 control cell line (CTRL).
  • Fig. 9G is a series of images showing the representative cytospins images of blasts, intermediates, and differentiated cells used as reference for the screening.
  • Fig. 9H is a graph showing the quantification of human leukemia U937 cell line stably overexpressing the mutant variant R140Q of IDH2 (mIDH2) to the respective U937 controls (CTRL).
  • Fig. 91 is a graph showing the quantification of flow cytometry analysis for the percentage of CD 14 positive cells (CD14+).
  • Fig. 10A is a series of images depicting a western blot assay on TF1 cells overexpressing mutant IDH2 (TF1 IDH2R140Q) or respective control vector (TF1 CTRL) stably silenced for PIN1 expression (shPINl).
  • Non targeting (shSCR) was used as control for non-specific silencing effects.
  • Fig. 10B is a series of images depicting a western blot assay on TF1 cells overexpressing mutant IDH2 (TF1 IDH2R140Q) or respective control vector (TF1 CTRL) treated with the Pinl Inhibitor Juglone for 72h.
  • Fig. IOC is a series of images depicting a western blot assay on TF1 cells stably overexpressing mutant IDH2 (TF1 IDH2R140Q) and Pinl or respective control vector vectors (TF1 CTRL, CTRL).
  • Fig. 11A is a series of images depicting a methylcellulose colony forming assay and colony quantifications of mouse leukemia cells derived from 2 nd RECIPIENTS or leukemia cells overexpressing Hoxa9/Meisla.
  • Fig. 1 IB is a series of images depicting a methylcellulose colony forming assay and colony quantifications of mouse leukemia cells derived from 3 rd RECIPIENTS or leukemia cells overexpressing Hoxa9/Meisla.
  • Fig. llC is a scheme of the in vivo approach to evaluate combination arsenic trioxide (ATO) and ATRA treatment on mIDH2 leukemic cells in C57BL/6J mice.
  • ATO arsenic trioxide
  • Fig. 1 ID is a graph depicting Kaplan- Meier survival curves derived from the experiment shown in Fig. 11C.
  • Fig. 11E is a series of images depicting May-Grunwald-Giemsa staining of BM blasts sorted from recipients treated with ATO and ATRA as shown in Fig. 11C.
  • Fig. 1 IF is a series of images depicting H&E staining of lung tissues. Arrowheads indicate perivascular leukemia cells infiltrates.
  • Fig. 11G is a series of images depicting IHC of lung tissues for myeloperoxidase (MPO), a marker of myeloid cell differentiation. Arrowheads indicate strongly MPO positive cells in perivascular infiltrates.
  • MPO myeloperoxidase
  • Fig. 12A is a scheme of the in vivo experimental approach to evaluate combination ATO and ATRA treatment on mIDH2 U937 human leukemic cell lines.
  • Fig. 12B is a series of graphs depicting Kaplan- Meier survival curves of NSG mice transplanted with mIDH2-U937 or CTRL-U937 leukemia cells and treated with ATO, ATRA or a combination of both.
  • Fig. 12C is a series of graphs showing the percentage of leukemia cells in bone marrow (BM) of NSG mice. Data are means ⁇ SD of samples isolated from different mice.
  • Fig. 12D is a series of images showing morphological screening analysis of U937 cells isolated as GFP+ cells from NSG mice treated with retinoic acid (ATRA), Arsenic Trioxide (ATO), a combination of both, or respective vehicle solution. Arrowheads show cells displaying differentiated morphology
  • Fig. 12E is a series of graphs and images depicting cytospins of mIDH2-U937 or CTRL-U937 leukemia cells isolated as GFP+ cells from BM of NSG mice.
  • Fig. 12F is a series of flow cytometry plots showing CDl lb expression levels in mIDH2-U937 or CTRLU937 leukemia cells.
  • Fig. 12G is a series of graphs showing the quantification of flow cytometry analysis for the percentage of CDl lb positive cells (CDl lb+).
  • U937 cell line stably overexpressing the mutant variant R140Q of IDH2 (mIDH2) show about 30% reduced percentage of CD1 lb+ cells compared to the control (CTRL).
  • Fig. 12H is a series of graphs showing the quantification of spleen weight dissected from NSG mice transplanted with mIDH2-U937 or CTRL-U937 leukemia cells and treated with ATO, ATRA or a combination of both.
  • Fig. 13 a- 13k a series of graphs showing the sensitivity of mIDH2 leukaemia to ATRA and ATO.
  • Colonies were quantitated after 12 days from plating g, Colony forming assay of TF1 cells overexpressing the mIDH2 or respective control (CTRL) and treated with the PIN1 inhibitor (Juglone, 1 pM) or respective vehicle (VHL). Colonies were quantitated after 7 days from plating h, Fold induction of cell death (Annexin V/ 7AAD positive cells) in mouse leukaemia cells isolated from 2 nd or 3 rd recipients and in vitro treated with ATO (0.5 pM) for 96h.
  • Data are means ⁇ SD of duplicates k, Quantification of colony forming ability of mouse leukaemia cells harbouring mutations in IDH2 (IDH2 R140Q ) or IDH1 (IDH1 R132C ) in association with different genetic mutant backgrounds such as NPMc+ or FLT3ITD and treated with pharmacological concentrations of ATRA (1 mM), ATO (0.5 mM), a combination of both (ATRA+ATO), or vehicles (VHL) as control.
  • Non mutant IDH2 cells such as MLL-AF9 or NPMc+/ FLT3ITD were also included.
  • Fig. 14a-14h a series of graphs showing the combination of ATO and ATRA targeting mIDH2 human leukaemia cells a, Scheme of the in vivo experimental approach b, Kaplan- Meier survival curves of NSG mice transplanted with mIDH2-U937 or CTRL-U937 leukaemia cells and treated with ATO, ATRA or a combination of both c, Percentage of leukaemia cells in bone marrow (BM) of NSG mice. Data are means ⁇
  • U937 cell line stably overexpressing the mutant variant R140Q of IDH2 show about 30% reduced percentage of CDllb-i- cells compared to the control (CTRL).
  • DS differentiation syndrome
  • VEHICLE solutions Accumulation of human MPO positive cells is a major characteristic of DS.
  • Fig. 15h-15j a series of graphs showing the effect of ATO and ATRA
  • Fig. 16 a schematic depiction of ATO and ATRA targeting mIDH leukaemia.
  • Scheme of leukaemia evolution from mIDH dependent to mIDH2 independent states e.g. acquisition of resistance to mIDH inhibition.
  • Multiple alterations including metabolic reprogramming, and transcriptional rewiring are acquired during progression and co-exist in the mIDH2 independent stage.
  • PIN1, deregulated LSD1 signaling and high ROS levels as key factors of upregulated pro survival and proliferation programs (MAPK/PI3K pathways), alterations to functional ATRA signalling, and altered metabolic state (One-Carbon metabolism and increased nucleic acid synthesis).
  • mIDH2 IDH2 R140Q ; BM: bone marrow; ATRA: Retinoic acid; ATO: Arsenic Trioxide.
  • the term“about” refers to a value that is within 10% above or below the value being described.
  • any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.
  • Arsenic trioxide and“an arsenic trioxide compound” refer to a compound having the formula AS2O3 and derivatives thereof.
  • Arsenic trioxide generally has the following structure:
  • arsenic trioxide may include, for example, arsenic ores, such as, e.g., arsenopyrite (grey arsenic; FeAsS), realgar (also known as sandarach or red arsenic;
  • arsenic trioxide exhibits high toxicity in mammals, such as humans.
  • arsenic trioxide ingestion can result in severe side effects, including vomiting, abdominal pain, diarrhea, bleeding, convulsions, cardiovascular disorders, inflammation of the liver and kidneys, abnormal blood coagulation, hair loss, and death.
  • arsenic trioxide poisoning may rapidly lead to death. Chronic exposure to even low levels of arsenic trioxide can result in arsenicosis and skin cancer.
  • Arsenic trioxide is therefore desirably administered to a subject at low enough doses to minimize toxicity.
  • arsenic trioxide and derivatives thereof may be effective at treating leukemia.
  • administration of arsenic trioxide to a subject having leukemia may increase the production of reactive oxygen species in one or more leukemic cells of said subject.
  • organic arsenic compounds are converted to inorganic compounds when absorbed in a biological system (see, e.g., Frith, J. Military Vet. Health 21(4): 11-17, 2013).
  • Arsenic derivatives and uses thereof are described, for example, in Wax man et al. (Oncologist 6: 3-10, 2001).
  • Darinaparsin and“a Darinaparsin compound” refer to dimethylarsinic glutathione having the formula C12H22ASN3O6S and derivatives thereof.
  • Darinaparsin generally has the following structure:
  • exemplary retinoic acid compounds described herein include, without limitation, all-trans retinoic acid (ATRA), 13-cis retinoic acid (13cRA), and retinoic acid compounds, and derivatives thereof, e.g., as described herein. Examples of retinoic acid compounds include those shown in Tables 1 and 2 below.
  • retinoic acid compounds include any retinoic acid compounds, or derivatives thereof, known in the art, including those described in PCT Publication Nos. WO 2013/185055, WO 2015/143190, and WO 2016/145186, each of which is incorporated herein with respect to the compounds described therein.
  • the term“diterpene retinoic acid” encompasses any stereoisomer of retinoic acid (e.g., the retinoic acid may be in the all-trans configuration (ATRA) or one or more of the double bonds may be in the cis configuration, for example, 13cRA.
  • Derivatives of the diterpene retinoic acid include reduced forms such as retinal, retinol, and retinyl acetate.
  • each of Ar 1 and Ar 2 is, independently, optionally substituted aryl or an optionally substituted heteroaryl;
  • R 1 is H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group;
  • C1-C6 alkoxy represents a chemical substituent of formula -OR, where R is an optionally substituted C1-C6 alkyl group, unless otherwise specified.
  • the alkyl group can be substituted, e.g., the alkoxy group can have 1, 2, 3, 4, 5 or 6 substituent groups as defined herein.
  • alkyl As used herein, the term“alkyl,”“alkenyl” and“alkynyl” include straight-chain, branched-chain and cyclic monovalent substituents, as well as combinations of these, containing only C and H when unsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2 propenyl, 3 butynyl, and the like.
  • cycloalkyl represents a monovalent saturated or unsaturated non-aromatic cyclic alkyl group having between three to nine carbons (e.g., a C3-C9 cycloalkyl), unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl, and the like.
  • the cycloalkyl is a polycyclic (e.g., adamantyl).
  • Cycloalkyl groups may be unsubstituted or substituted with, e.g., 1, 2, 3, or 4 substituent groups as defined herein.
  • the cycloalkyl group can be referred to as a“cycloalkenyl” group.
  • Exemplary cycloalkenyl groups include
  • the alkyl, alkenyl and alkynyl groups contain 1-12 carbons (e.g., Cl- C12 alkyl) or 2-12 carbons (e.g., C2-C12 alkenyl or C2-C12 alkynyl).
  • the alkyl groups are C1-C8, C1-C6, C1-C4, C1-C3, or C1-C2 alkyl groups; or C2-C8, C2-C6, C2-C4, or C2-C3 alkenyl or alkynyl groups.
  • any hydrogen atom on one of these groups can be replaced with a substituent as described herein.
  • aryl represents a mono- or bicyclic C6-C14 group with [4 n + 2] p electrons in conjugation and where n is 1, 2, or 3.
  • Aryl groups also include ring systems where the ring system having [4 n + 2] p electrons is fused to a non-aromatic cycloalkyl or a non-aromatic heterocyclyl.
  • Phenyl is an aryl group where n is 1.
  • Aryl groups may be unsubstituted or substituted with, e.g., 1, 2, 3, or 4 substituent groups as defined herein.
  • Still other exemplary aryl groups include, but are not limited to, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, and indenyl.
  • heteroaryl represents an aromatic (i.e., containing An+2 pi electrons within the ring system) 5- or 6-membered ring containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, as well as bicyclic, tricyclic, and tetracyclic groups in which any of the aromatic ring is fused to one, two, or three heterocyclic or carbocyclic rings (e.g., an aryl ring).
  • heteroaryls include, but are not limited to, furan, thiophene, pyrrole, thiadiazole (e.g., 1,2,3-thiadiazole or 1,2,4-thiadiazole), oxadiazole (e.g., 1,2,3-oxadiazole or 1,2,5-oxadiazole), oxazole, isoxazole, isothiazole, pyrazole, thiazole, triazole (e.g., 1,2,4-triazole or 1,2,3-triazole), pyridine, pyrimidine, pyrazine, pyrazine, triazine (e.g, 1,2,3-triazine 1,2,4-triazine, or 1,3,5-triazine), 1,2,4,5-tetrazine, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, and benzoxazolyl.
  • Heteroaryls may be unsubstituted or substituted with, e.g., 1, 2, 3, or 4 substituents groups as defined herein.
  • the term“heterocyclyl,” as used herein represents a non-aromatic 5-, 6- or 7- membered ring, unless otherwise specified, containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
  • Heterocyclyl groups may be unsubstituted or substituted with, e.g., 1, 2, 3, or 4 substituent groups as defined herein.
  • aryloxy refers to aromatic or heteroaromatic systems which are coupled to another residue through an oxygen atom.
  • O-aryl A typical example of an O-aryl is phenoxy.
  • thioaryloxy refers to aromatic or heteroaromatic systems which are coupled to another residue through a sulfur atom.
  • a halogen is selected from F, Cl, Br, and I, and more particularly it is fluoro or chloro.
  • each R or R’ is selected, independently, from H, Ci- 6 alkyl, C2-6 alkenyl, C2-6 alkynyl, aryl, or heteroaryl.
  • a substituent group e.g., alkyl, alkenyl, alkynyl, or aryl (including all heteroforms defined above) may itself optionally be substituted by additional substituents.
  • additional substituents e.g., alkyl, alkenyl, alkynyl, or aryl (including all heteroforms defined above
  • alkyl may optionally be substituted by the remaining substituents listed as substituents where this makes chemical sense, and where this does not undermine the size limit of alkyl per se; e.g., alkyl substituted by alkyl or by alkenyl would simply extend the upper limit of carbon atoms for these embodiments, and is not included.
  • Typical optional substituents on aromatic or heteroaromatic groups include independently halo, CN, N02, CF3, OCF3, COOR’, CONR’2, OR’, SR’, SOR’, S02R’, NR’2, NR’ (CO)R’ ,NR’ C(0)OR’ , NR’C(0)NR’2, NR’S02NR’2, or NR’S02R ⁇ wherein each R’ is independently H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and aryl (all as defined above); or the substituent may be an optionally substituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, O-aryl, O-heteroaryl and arylalkyl.
  • non-aromatic groups e.g., alkyl, alkenyl, and alkynyl groups
  • cancer refers to a proliferative disease in a subject (e.g., a human) having a pathogenic IDH1/IDH2 allele or having elevated 2- hydroxyglutarate (2-HG) levels.
  • a subject e.g., a human
  • IDHl/IDH2-associated leukemias IDHl/IDH2-associated solid tumors.
  • An IDHl/IDH2-associated leukemia can contain one or more mutants selected from FLT3, NPM1, DNMT3A, RUNX1, TET2, IDH2, CEBPA, TP53, IDH1, NRAS, RARA, PML, TTN, WT1, MYH11, BPIFC, CBFB, KIT, KRAS, KMT2A, PTPN11, MUC16, SMC1A, RUNX1T1, MPO, U2AF1, ABCA6, DMXL2, DNAH3, KLK3, FBX07, SMC3, MXRA5, MUC17, SF3B1, HPS3, PHF6, ASXL1, AHNAK, SENP6, MYCBP2, NF1, PCLO, CSMD3, LRP1B, MED 12, RAD21, AHNAK2, GDI2, PARP14, KLHL7, BCORL1, SPEN, BRWD1,UBR4, STAG2, LNX1, FREM2, MLLT10, DNAH11,
  • An IDHl/IDH2-associated solid tumor can be glioma, paraganglioma, astroglioma, colorectal carcinoma, melanoma, cholangiocarcinoma, chondrosarcoma, thyroid carcinomas, prostate cancers, or non-small cell lung cancer.
  • pathogenic IDH2 allele is meant to include any allele encoding an IDH2 protein having a mutation associated with increased cellular proliferation, associated with increased likelihood or severity of cancer, associated with increased likelihood or severity of leukemia, or associated with increased levels of the oncometabolite, 2-hydroxyglutarate (2-HG) compared with a wild-type cell, tissue, or subject.
  • Pathogenic alleles of IDH2 may encode, for example, any of the following mutations in the corresponding IDH2 protein: R140Q, R140W, R172K, R172M, R172G, or R172W.
  • the term“IDH2 inhibitor” is meant to include any compound that reduces the level of IDH2 activity or expression in a cell, tissue, or subject.
  • a reduction in IDH2 expression or activity may be measured by methods known to one of skill in the art, including the reduction in corresponding IDH2 mRNA or protein levels in a cell (e.g., a reduction of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater) or the reduction of 2-HG levels in a cell (e.g., a reduction of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater).
  • the IDH2 inhibitor may include the reduction in
  • Pinl activity refers to binding of the protein Pinl to a substrate (e.g., a substrate protein) and Pinl -catalyzed isomerization of the substrate.
  • Pinl generally acts as a peptidyl-prolyl isomerase (PPIase) that catalyzes prolyl isomerization of the substrate (e.g., conversion of a peptidyl-prolyl group on the substrate from a trans conformation to a cis conformation, or vice versa).
  • PPIase peptidyl-prolyl isomerase
  • “Elevated Pinl activity” or“elevated levels of Pinl activity,” as used herein, generally refer to an increase in Pinl-catalyzed isomerization of one or more Pinl substrates, for example, relative to a reference level of Pinl activity.
  • the reference level of Pinl activity is the level of Pinl activity in a wild-type cell (e.g., a wild-type cell of the same cell type as a cell of interest). In some embodiments, the reference level of Pinl activity is the level of Pinl activity in a wild-type subject (e.g., a subject not having leukemia), such that an increase in Pinl activity in a subject of interest relative to a wild-type subject indicates that the subject of interest has elevated Pinl activity. In some embodiments, alteration in Pinl activity can be assessed by determining the levels of a Pinl marker in a cell and/or a subject of interest, relative to a reference cell or subject (e.g., a wild-type cell or subject).
  • Elevated levels of Pinl activity include, for example, Pinl activity levels at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater than the activity level measured in a normal (e.g., wild-type and/or disease fee) subject, tissue, or cell.
  • a normal e.g., wild-type and/or disease fee
  • a decrease in Pinl activity includes a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% in the Pinl activity of a subject, tissue, or cell (e.g., after treatment by any of the
  • Pinl marker refers to a marker which is capable of being indicative of Pinl activity levels (e.g., in a sample obtained from a cell or subject of interest).
  • Non-limiting examples of Pinl markers include nucleic acid molecules (e.g., mRNA, DNA) that correspond to some or all of a Pinl gene, peptide sequences (e.g., amino acid sequences) that correspond to some or all of a Pinl protein, nucleic acid sequences which are homologous to Pinl gene sequences, peptide sequences which are homologous to Pinl peptide sequences, alteration of Pinl protein, antibodies to Pinl protein, substrates of Pinl protein, binding partners of Pinl protein, alteration of Pinl binding partners, and activity of Pinl.
  • nucleic acid molecules e.g., mRNA, DNA
  • peptide sequences e.g., amino acid sequences
  • alteration of a Pinl protein may include a post-translational modification (e.g., phosphorylation, acetylation, methylation, lipidation, or any other post- translational modification known in the art) of Pinl.
  • a Pinl marker is the level of Pin expression (e.g., Pinl protein expression levels and/or Pinl mRNA expression levels) in a subject.
  • elevated levels of a Pinl marker is meant a level of Pinl marker that is altered, which may, in some instances, indicate the presence of elevated Pinl activity.
  • Elevated levels of a Pinl marker include, for example, levels at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater than, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% less than the marker levels measured in a normal (e.g., wild-type and/or disease fee) subject, tissue, or cell.
  • a normal e.g., wild-type and/or disease fee
  • the terms“compound that inhibits LSD1” or“LSD1 inhibitor” are meant to include any compound that reduces the level of LSD1 activity or expression in a cell, tissue, or subject.
  • a reduction in LSD1 expression or activity may be measured by methods known to one of skill in the art, including the reduction in corresponding LSD1 mRNA or protein levels in a cell (e.g., a reduction of about 1%, 2%, 3%, 4%, 5%, 6%,
  • LSD 1- specific histone methylation such as H3K4me2 and/or H3K9me2 histone methylation (e.g., a reduction of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or greater).
  • “synergy” or“synergistic,” as used herein, refers to an improved effect when two agents are administered that is greater than the additive effects of each of the two agents when administered alone.
  • administration of an arsenic trioxide and a retinoic acid compound (e.g., ATRA) to a subject may result in a greater than additive effect on the subject than administration of either arsenic trioxide or the retinoic acid compound alone.
  • a“low dose” or“low dosage” is meant a dosage of at least 5% less (e.g., at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,
  • a low dosage of an agent formulated for oral administration may differ from a low dosage of the agent formulated for intravenous administration.
  • a low dosage of an agent may be selected to be a nontoxic dosage of the agent.
  • a low dosage may be selected as a dosage that minimizes particular side effects of an agent, but which may still retain some side effects.
  • a dosage may be selected that minimizes or eliminates side effects that can lead to significant mortality or severe illness among subjects while still permitting more tolerable side effects, such as headache.
  • a low dose of arsenic trioxide is a dose of about 2 mg/kg body weight or less (e.g., about 0.01, 0.02, 0.03, 0.032, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.25, 1.5, 1,75, or 2 mg/kg).
  • a low dose of arsenic trioxide is about 0.15, about 0.16, or about 0.032 mg/kg body weight. In other instances, a low dose of arsenic trioxide is a dose between about 0.5 mg/kg and about 12 mg/kg body weight (e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mg/kg). In some instances, a low dose of a retinoic acid compound is a dose of about 5 mg/kg body weight or less (e.g., about 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, or 5 mg/kg).
  • a low dose of a retinoic acid compound is a dose of about 25 mg/m 2 or less (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg/m 2 ). In other instances, the low dose of the retinoic acid compound is a dose of between 25 mg/m 2 and 45 mg/m 2 (e.g.,
  • A“nontoxic” dose of an agent is a dosage low enough to minimize or eliminate toxic side effects of the agent on the subject to which the agent is administered.
  • a nontoxic dosage may be achieved by reducing the quantity of the agent administered per dose and/or increasing the length of time between deliveries of individual doses.
  • the term“effective amount” or an amount“sufficient to” as used interchangeably herein, refers to a quantity of an agent that, when administered alone or with one or more additional therapeutic agents, induces a desired response or confers a therapeutic effect on the treated subject.
  • the desired response may be a therapeutic response.
  • the desired response is decreasing the signs or symptoms of a disorder described herein (e.g., leukemia).
  • the desired response is decreasing the risk of developing or decreasing the risk of recurrence of a disorder described herein (e.g., leukemia).
  • An effective amount of an agent may desirably provide a therapeutic effect without causing substantial toxicity in the subject.
  • an effective amount of a composition administered to a human subject will vary depending upon a number of factors associated with that subject, for example, the overall health of the subject, the condition to be treated, and/or the severity of the condition.
  • An effective amount of a composition can be determined by varying the dosage of the product and measuring the resulting therapeutic response. The effective amount can be dependent, for example, on the source applied, the subject being treated, the severity and type of the condition being treated, and the manner of administration.
  • “treat”,“treating”, or“treatment” refers to application or administration of a pharmaceutical compound by any route, e.g., orally, topically, or by inhalation to a subject with the purpose to cure, alleviate, relieve, alter, remedy, improve, or affect the disease, the symptom, or the predisposition.
  • the compound can be administered alone or in combination with one or more additional compounds. Treatments may be sequential, with a compound being administered before or after the administration of other agents. Alternatively, compounds may be administered concurrently.
  • the subject e.g., a patient, can be one having a disorder (e.g., a leukemia), a symptom of a disorder, or a disorder (e.g., a leukemia), a symptom of a disorder, or a
  • Treatment is not limited to curing or complete healing, but can result in one or more of alleviating, relieving, altering, partially remedying, ameliorating, improving or affecting the disorder, reducing one or more symptoms of the disorder or the predisposition toward the disorder.
  • the treatment at least partially) alleviates or relieves symptoms related to a fibrotic disease.
  • the treatment at least partially alleviates or relieves symptoms related to an inflammatory disease.
  • the treatment reduces at least one symptom of the disorder or delays onset of at least one symptom of the disorder. The effect is beyond what is seen in the absence of treatment.
  • Treatment of leukemia may be considered to include administration of any of the compounds described herein resulting in the remission of the leukemia in the subject (e.g., the symptoms of the leukemia are reduced). Treatment of the leukemia may further be considered to include administration of any of the compounds described herein resulting in the complete remission of the leukemia (e.g., all signs and symptoms of the leukemia are absent). Treatment of the leukemia may further be considered to include administration of any of the compounds described herein, wherein the treatment cures the leukemia in the subject (e.g., all signs and symptoms of the leukemia are absent for 1 year or more, for 2 years or more, for 3 years or more, for 4 years or more, or for 5 years or more).
  • subject refers to any organism or portion thereof to be administered a composition as described herein (e.g., arsenic trioxide, a retinoic acid compound, and combinations or derivatives thereof).
  • a subject may be an animal, such as a mammal (e.g., a human, mouse, rat, rabbit, dog, cat, goat, pig, and horse).
  • the subject is human.
  • administering may also be considered to include contacting.
  • a compound is administered to a cell, it may be considered to be equivalent to contacting the cell with the compound.
  • the term“contacting one or more cells” with a compound of the invention is meant to include administering a compound of the invention to a subject, such that the compound contacts one or more cells of the subject.
  • the present invention is based on the discovery of common vulnerabilities in mIDH2 leukemia.
  • mIDH2 leukemia exhibits sensitivity to reactive oxygen species (ROS)-producing compounds, such as arsenic trioxide (ATO).
  • ROS reactive oxygen species
  • ATO arsenic trioxide
  • mIDH2 leukemia exhibits sensitivity to retinoic acid compound- induced differentiation (e.g., ATRA-induce differentiation).
  • a ROS-promoting compound e.g., arsenic trioxide
  • a compound that promotes differentiation e.g., a Pinl inhibitor, such as ATRA
  • the present invention therefore features methods, compositions, and kits relating to the treatment of mIDH2 leukemia by administering an arsenic trioxide compound (e.g., ATO), a retinoic acid compound (e.g., ATRA), or a combination of an arsenic trioxide and a retinoic acid compound (ATO and ATRA).
  • an arsenic trioxide compound e.g., ATO
  • a retinoic acid compound e.g., ATRA
  • ATO and ATRA retinoic acid compound
  • Isocitrate dehydrogenases are enzymes (IDH enzymes) are metabolic enzymes that catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate.
  • the protein encoded by the IDH2 gene is the NADP(+) -dependent isocitrate dehydrogenase found in the mitochondria. It plays a role in intermediary metabolism and energy production.
  • IDH enzymes have been identified as important early events in a variety of tumor types. For example, IDH enzymes are mutated in approximately 20% of human acute myeloid leukemias (AMLs). Pathogenic mutants of IDH2 enzymes have been identified that give rise to 2-hydroxyglutarate (2-HG), an oncometabolite that contributes to the oncogenic phenotype. Accordingly, 2-HG is a predictive biomarker in cancers having a pathogenic IDH2 allele. In some embodiments of the invention, the methods described herein are used to treat a subject having a disorder associated with a mutation in the IDH2 enzyme
  • the mIDH2 disorder is associated with a pathogenic IDH2 allele, wherein a pathogenic IDH2 allele is meant to include any allele encoding an IDH2 protein having a mutation associated with increased cellular proliferation, associated with increased likelihood or severity of cancer, associated with increased likelihood or severity of leukemia, or associated with increased levels of the oncometabolite, 2-hydroxyglutarate (2-HG) compared with a wild-type cell, tissue, or subject.
  • Pathogenic alleles of IDH2 may encode, for example, any of the following mutations in the corresponding IDH2 protein: R140Q, R140W, R172K, R172M, R172G, or R172W.
  • the methods described herein are used to a subject having a leukemia, wherein the leukemia is associated with a mutation in the IDH2 enzyme.
  • the subject having a leukemia has a pathogenic IDH2 allele and/or has elevated 2-hydroxyglutarate (2-HG) levels (e.g., in one or more cells of the leukemia).
  • the leukemia is an IDH2 sensitive leukemia (e.g., a leukemia that is responsive to IDH2 inhibitors).
  • the leukemia is an IDH2 independent or resistant leukemia (e.g., leukemia resistant to known IDH2 inhibitors, such as, Enasidenib).
  • the present invention features methods of treating leukemia (e.g., mIDH2- associated leukemia) using arsenic trioxide and/or a retinoic acid compounds, and derivatives thereof.
  • leukemia e.g., mIDH2- associated leukemia
  • subject is treated with arsenic trioxide in combination with a retinoic acid compound (e.g., as described herein).
  • Arsenic trioxide generally has the following structure:
  • Arsenic trioxide exhibits high toxicity in subjects of the invention, including mammals (e.g., humans).
  • subjects of the invention including mammals (e.g., humans).
  • arsenic trioxide ingestion can result in severe side effects, including vomiting, abdominal pain, diarrhea, bleeding, convulsions, cardiovascular disorders, inflammation of the liver and kidneys, abnormal blood coagulation, hair loss, and death.
  • Chronic exposure to even low levels of arsenic trioxide can result in arsenicosis and skin cancer.
  • Arsenic trioxide is therefore desirably administered to a subject at low enough doses to minimize toxicity.
  • Arsenic trioxide and derivatives thereof may be effective at increasing the production of reactive oxygen species in a cell, tissue, or subject.
  • Arsenic trioxide and derivatives thereof may also be effective in reducing Pinl activity in a cell, tissue, or subject.
  • arsenic trioxide may operate synergistically with a retinoic acid compound to treat a disorder described herein.
  • the combination of arsenic trioxide and the retinoic acid compound are administered in amounts that result in minimal toxicity.
  • Retinoic acid compounds are generally derivatives of the diterpene retinoic acid (e.g., as described herein). Retinoic acid compounds may be effective in promoting differentiation in a cell (e.g., a leukemic cell). Retinoic acid compounds may also be effective in reducing Pinl activity in a cell, tissue, or subject. Exemplary retinoic acid compounds of the invention include all-trans retinoic acid (ATRA), 13-cis retinoic acid (13cRA), and retinoic acid compounds, and derivatives thereof, e.g., as described herein. Retinoic acid compounds of the invention may be a y compound selected from Table 1 or Table 2. In some instances, a retinoic acid compound is administered in combination with arsenic trioxide. In certain instances, the combination of arsenic trioxide and the retinoic acid compound are administered in amounts that result in minimal toxicity.
  • ATRA all-trans retinoic acid
  • 13cRA 13
  • Certain embodiments of the invention feature a deuterated retinoic acid compound that is made by replacing some or all hydrogen with deuterium using state of the art techniques (e.g., as described herein and at www.concertpharma.com).
  • arsenic trioxide and/or retinoic acid compound(s) of the invention may be further combined with additional therapeutic agents for treatment of any of the disorders described herein (e.g., leukemia).
  • such compounds may act synergistically with arsenic trioxide and/or a retinoic acid compound to treat the disorder (e.g., leukemia). Additionally, co administration with arsenic trioxide and/or a retinoic acid compound may result in the efficacy of the additional therapeutic agent at lower and safer doses (e.g., at least 5% less, for example, at least 10%, 20%, 50%, 80%, 90%, or even 95% less) than when the additional therapeutic agent is administered alone.
  • the additional therapeutic agent e.g., leukemia
  • the arsenic trioxide and/or retinoic acid compounds may be combined with anti-proliferative and other anti-cancer compounds (e.g., anti- angiogenic compounds) for treating a disorder (e.g., leukemia).
  • anti-proliferative agents that can be used in combination with a retinoic acid compound include, without limitation, microtubule inhibitors, topoisomerase inhibitors, platins, alkylating agents, and anti metabolites.
  • anti-proliferative agents that are useful in the methods and compositions of the invention include, without limitation, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine,
  • Pinl is an enzyme that catalyzed the cis-trans isomerization of phosphorylated Ser/Thr-Pro motifs and which has been shown to be involved in an increasing number of diseases. Elevated Pinl activity has been associated with the development and progression of cancer. For example, Pinl is overexpressed in some human cancer samples and the levels of Pinl are correlated with the aggressiveness of tumors. Moreover, inhibition of Pinl by various approaches, including the Pinl inhibitor, Pinl antisense polynucleotides, or genetic depletion, kills human and yeast dividing cells by inducing premature mitotic entry and apoptosis.
  • Pinl upon phosphorylation, Pinl latches onto phosphoproteins and twists the peptide bond next to the proline, which regulates the function of phosphoproteins and participates in controlling the timing of mitotic progression.
  • Pinl has been shown to regulate the expression and/or activity of a diverse array of proteins associated with cancer progression.
  • known Pinl substrates include, without limitation, Her2, PKM2, FAK, Raf-1, AKT, b-catenin, c-Myc, p53, and numerous other proteins known to play roles in cancer progression.
  • the arsenic trioxide and/or retinoic acid compounds may be combined with a compound that inhibits Pinl activity or expression.
  • the arsenic trioxide and/or retinoic acid compounds may be combined with a compound known to interact with other proteins implicated in Pinl signaling pathways (see, e.g., the targets and compounds in Table 3).
  • Table 3 Exemplary Additional Therapeutic Agents
  • Lysine-specific histone demethylase 1 is a flavin-dependent monoamine oxidase, which can demethylate mono- and di-methylated lysines, specifically H3K4 and H3K9).
  • the LSD1 enzyme has roles critical in embryogenesis and tissue-specific differentiation, as well as oocyte growth.
  • the inventors have also identified a shared set of ATRA responsive genes previously reported to be regulated by inhibition of the demethylase LSD1 and associated with ATRA sensitivity.
  • the arsenic trioxide and/or retinoic acid compounds may be combined with a compound that inhibits LSD1 activity or expression.
  • Treatment may be performed alone or in conjunction with another therapy and may be provided at home, the doctor’s office, a clinic, a hospital’s outpatient department, or a hospital. Treatment optionally begins at a hospital so that the doctor can observe the therapy’s effects closely and make any adjustments that are needed, or it may begin on an outpatient basis.
  • the duration of the therapy depends on the type of disease or disorder being treated, the age and condition of the patient, the stage and type of the patient’s disease, and how the patient responds to the treatment.
  • Routes of administration for the various embodiments include, but are not limited to, topical, transdermal, nasal, and systemic administration (such as, intravenous, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ophthalmic, otic, or oral administration).
  • systemic administration refers to all nondermal routes of administration, and specifically excludes topical and transdermal routes of administration ⁇
  • each component of the combination can be controlled independently.
  • one or more of the compounds may be administered three times per day, while another compound or compounds may be administered once per day.
  • one compound may be administered earlier and another compound may be administered later.
  • Combination therapy may be given in on-and-off cycles that include rest periods so that the patient’s body has a chance to recover from any as yet unforeseen side effects.
  • the compounds may also be formulated together such that one administration delivers both compounds.
  • Each compound of the combination may be formulated in a variety of ways that are known in the art.
  • a plurality of therapeutic agents e.g., arsenic trioxide, a retinoic acid compound, and/or an additional therapeutic agent, as described herein
  • multiple agents are formulated together for the simultaneous or near simultaneous administration of the agents.
  • co-formulated compositions can include the drugs together in the same pill, ointment, cream, foam, capsule, liquid, etc.
  • the formulation technology employed is also useful for the formulation of the individual agents of the combination, as well as other combinations of the invention.
  • the pharmacokinetic profiles for each agent can be suitably matched.
  • Certain embodiments of the invention feature formulations of arsenic trioxide and/or a retinoic acid compound for, e.g., controlled or extended release.
  • Many strategies can be pursued to obtain controlled and/or extended release in which the rate of release outweighs the rate of metabolism of the therapeutic compound.
  • controlled release can be obtained by the appropriate selection of formulation parameters and ingredients (e.g., appropriate controlled release compositions and coatings). Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes.
  • the release mechanism can be controlled such that the arsenic trioxide and/or retinoic acid compound is released at period intervals, the release could be simultaneous, or a delayed release of one of the agents of the combination can be affected, when the early release of one particular agent is preferred over the other.
  • kits that contain, e.g., a plurality of pills (e.g., two pills or three pills), a pill and a powder, a suppository and a liquid in a vial, two topical creams, ointments, foams etc.
  • the kit can include optional components that aid in the administration of the unit dose to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc.
  • the unit dose kit can contain instructions for preparation and administration of the compositions.
  • the kit may be manufactured as a single use unit dose for one patient, multiple uses for a particular patient (at a constant dose or in which the individual compounds may vary in potency as therapy progresses), or the kit may contain multiple doses suitable for administration to multiple patients (“bulk packaging”).
  • the kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.
  • the Hoxa9-Meisl- IDH2R140Q model of murine leukaemia cells was generated by the retroviral transduction of KSL (c-Kit+, Sca-1+, Lin-) cells as reported in Quek et a , Nat. Med., 2018, 24: 1167-1177.
  • the generation of murine leukaemia cells showing independence by the mutant IDH2 was casually generated after three serial bone marrow transplantations of murine leukaemia cells.
  • Serial bone marrow transplantation experiments were started by harvesting leukemic bone marrow cells from a single donor mouse and transplanting 5-10 * 10 4 cells into 8-10 recipients. The recipients were age- matched, female C57BL/6J (6-8 weeks old).
  • hematopoietic stem cells LSK were isolated from the bone marrow of a single Tg (M2rt/TA, IDH2R140Q) donor (3-5 months old) maintained on doxycycline diet.
  • Tg M2rt/TA, IDH2R140Q
  • PBS medium containing bone marrow extracts was filtered and purified from red blood cells by incubation on ACK Lysing Buffer (Gibco).
  • Lineage negative cells were immunostained using Lineage Cell Detection Cocktail- Biotin, mouse (Miltenyi Biotec) and streptavidin - APC-Cy7.
  • LSK cells Lineage-, cKit+, Sca-1+
  • BD FACSAria II high speed cell sorters Becton Dickinson
  • IL3 25ng/ml
  • IL6 25ng/ml
  • TPO 50 ng/ml
  • SCF 50ng/ml
  • FLT3Ligand 50ng/ml
  • Sorted LSK were transduced by spinoculation with Hoxa9-IRES-GFP and Meisla-IRES-YFP retroviruses and maintained in culture for no more than 72 hours before transplantation on a primary host (1 st RECIPIENT, C57BL/6J females).
  • Age-matched C57BL/6J recipient female mice (age 8-10 weeks, Jackson Laboratories) were injected intravenously (tail vein) or retro-orbitally with 5-10 x 10 5 leukaemia cells in a volume of 200 pi of sterile PBS. All recipient animals received 650 cGy of radiation 24 hours prior to injection of leukemic cells.
  • Leukemic bone marrow cells harvested from 2 nd and 3 rd RECIPIENTS were ex vivo expanded in RPMI media (Gibco) supplemented with murine SCF, FLT3 Ligand, IL-3, and IL-6 (2.5 ng/mL) for subsequent studies.
  • Intravenous Xenograft Leukaemia model Intravenous Xenograft Leukaemia model.
  • mice 6-8 weeks of age Female NSG (NOD.CgPrkde scld I12 lslni 1 W
  • Cells 25 x 10 4 ) were introduced intravenously by tail vein injection.
  • Mice were treated with ATRA (lOmg/mouse) by subcutaneous implantation of ATRA pellets (5mg/pellet /21 days release), Arsenic trioxide (2.5 mg/Kg/day), or respective vehicle solutions via intraperitoneal injection. The experiment, as well as all treatments, were stopped after 21 days following the exhaustion of ATRA pellets.
  • AML patient - derived xenograft (PDX) model AML patient - derived xenograft (PDX) model.
  • NSG mice engrafted with human AML blasts were purchased from Jackson Laboratories (NOD scid gamma, NOD -scid IL2Rg nu11 , NOD3 scid IL2Rgamma nu11 ).
  • Female NSG mice (age: 4-5 weeks old) were irradiated (400 cGy) 24 hours prior to the retro-orbital transplantation of sorted human AML blasts. Engraftment and disease progression was detected as evidence of human CD45- positive cells in the peripheral blood. Mice were randomly divided into groups before starting of the treatments.
  • Treatment regimen was performed in cycles for a total of 65 days resembling APL0406 protocol (Lo-Coco et al; NEJM 2013). Accordingly, each cycle (65-71 days) was as follows: 15-21 days combination of ATO and ATRA, 10 days no therapy administered (off period) followed by two rounds of single drug administration: 10 days of ATO therapy before 10 days of ATRA therapy. Before the starting of a new cycle, no therapy was administered for 10 days.
  • ATO 2.5 pg/g
  • ATRA 1.5 pg/g
  • the Hoxa9/Meisla/IDH2R140Q murine primary leukaemia cells were cultured and expanded in RPMI media supplemented with murine SCF (2.4 ng/mL), FLT3 Ligand (5ng/mL), IL-3 (2 ng / mL), and IL6 (2.5 ng/mL) (PeproTech).
  • murine SCF 2.4 ng/mL
  • FLT3 Ligand 5ng/mL
  • IL-3 2 ng / mL
  • IL6 2.5 ng/mL
  • doxycycline Sigma
  • Conditioned media (1:2) was used for serial re-plating.
  • Methylcellulose colony assay Methylcellulose colony assay.
  • Methocult M3434 medium MethoCult H4434 Classic (Stem Cell Technologies). According to specific conditions, methylcellulose medium was supplemented with Doxycycline (Gibco), Arsenic Trioxide (Sigma), Tretinoin (Sigma), AGI-6780 or AG-221 (Cay
  • Mouse leukaemia cells were isolated from bone marrow as GFP+ cells and plated at 5000 cells / dish. Then, 2500 cells/dish were used for serial plating. Human AML blasts (1.5 -2.5 *104 cells/dish) were plated in 35-mm Petri dishes and incubated in a humidified CO2 incubator. Colonies were counted as units of at least 30 cells were generated after plating (2 - 4 weeks).
  • KLF1, PIN1, ITGAM, LILRA5, RARA, PRAM1 and GAPDH gene expression were measured using the Taqman assay (Invitrogen) according to manufacturer’s instruction. 2HG treatments.
  • Human TF1 cells TF-1 cells were treated with vehicle (0.1% EtoH) or 0.1 mM (2R)-Octyl-2-HG and harvested at indicated time for Western blot analyses.
  • Cell Proliferation and viability Assay Cells (TF1 or U937, 8-10 *10 3 cells) were plated in complete medium on multiwell plates and treated with ATRA and/or ATO for 4 days. Cells were assayed every 24h for cell viability or proliferation by using CellTiter Glo, MTS Assay (Promega) according with manufacturer protocols.
  • mice were euthanized and single-cell suspensions from the bone marrow were generated by bone crushing in PBS supplemented with 2% FBS.
  • Cell suspensions were passed through IOOmM cell strainers, centrifuged, and then re- suspended in 1-2 ml ACK red cell lysis buffer (GIBCO). Red blood cells were lysed on ice for lmin. Cell suspension were then washed in 2%FBS/PBS, centrifuged, and then re-suspended in 1ml of 2%FBS in PBS1X.
  • Cells were subsequently processed for GFP+/YFP+ sorting, KLS isolation, or intracellular flow staining. Staining with specific antibodies (1:100) or Annexin V/ 7AAD was performed for 15 - 30 minutes at room temperature in the dark, following the protocol reported in Carracedo et ak, Nat Rev Cancer 2013, 13:227-232. For intracellular phosphoflow cytometry on mouse leukaemia blasts, the cells were fixed in 4% PFA at
  • Human AML blasts (2-5*10 4 cells) were fixed in 4%PFA for 5 minutes at 37 °C, washed twice with cold PBS and permeabilized with 90% methanol ( ice-cold). Pellets were washed twice in ice cold PBS and resuspended in Staining buffer (0.5%BSA in PBS1X) and incubated (at 4 °C) with anti - PIN1 antibody ( Rb mAh to PIN1, ab76309, abeam) or respective IgG control (Rb IgG monoclonal isotype control, abl72730, abeam). Cells were washed twice and incubated with Donkey anti - rabbit Alexa Fluor 647 for 1 hour at room temperature. Pellets were washed three times before flow analysis.
  • Tissues were fixed in 4% paraformaldehyde overnight, paraffin embedded, and then sectioned at 5 pm. After deparaffinization and rehydration, antigen retrieval was performed in a pressure cooker with sodium citrate buffer at 95 °C for 25 minutes. Sections were incubated in a 0.3% H2O2 solution in lx PBS, and then a 10% serum solution in lx PBS for 30 minutes each solution was used to block endogenous peroxidase and background from the secondary antibody, respectively.
  • the sections were stained with the primary antibodies: Ki67 (1:200, Thermo Fisher Scientific, #MA5-14520) or Anti-Myeloperoxidase antibody (1:50, abeam #ab9535), or Phospho- Histone H2A.X (1:200, Cell Signalling #9718S) and incubated in a biotinylated secondary antibody in lx PBS (1:500-1:1000) at room temperature for 30 minutes.
  • the Vectastain ABC Elite kit was used to enhance specific staining, and the staining was visualized using a 3’-diaminobenzidine (DAB) substrate.
  • DAB 3’-diaminobenzidine
  • Blocking was performed for one hour using a 5% bovine serum albumin solution in lx PBS.
  • the sections were stained with the P-Histone H2A.X primary antibody (1:200) in a 2% bovine serum albumin and lx PBS solution at 4 °C overnight, and then incubated with a goat anti-rabbit IgG Alexa Fluor 546 conjugate (1:1000) in PBS IX at room temperature for 1 hour before staining with DAPI (1: 1000) in lx PBS for 10 minutes.
  • Cells were washed with lx PBS and milli-Q water before being sealed with a coverslip with Fluorescence Mounting Medium.
  • RNA derived from resistant and sensitive leukaemia cells was subjected to next- generation sequencing (NGS) to generate deep coverage RNASeq data.
  • NGS next- generation sequencing
  • Sequencing libraries of Poly A selected mRNA were generated from the double- stranded cDNA, using the Illumina TruSeq kit according to the manufacturer's protocol. Library quality control was checked using the Agilent DNA High Sensitivity Chip and qRT-PCR. High quality libraries were sequenced on an Illumina HiSeq 4000. To achieve comprehensive coverage for each sample, we generated about 30-35 million single end reads.
  • the raw sequencing data was processed to remove any adaptors, PCR primers, and low quality transcripts using FASTQC and fastx. These provided a very comprehensive estimate of sample quality on the basis of read quality, read length, GC content, uncalled based, ratio of bases called, sequence duplication, adaptors, and PCR primer contamination.
  • These high quality, cleaned reads were aligned against the mouse transcriptome (mmlO) using bowtiel with parameters: p 12 -q -n 2 -m 1 -S -best.
  • Gene expression measurement was performed from aligned reads by counting the unique reads using htseq-count (vO.6.1) with parameters: -a 10 -m intersection_strict.
  • the read count based gene expression data was normalized and analyzed using the“DESEq2 R package”.
  • the differentially expressed genes were identified on the basis of FDR value and fold change. Genes were considered significantly differentially expressed if the multiple hypothesis test-t corrected p-value was ⁇ 0.05.
  • the resulting gene expression matrix was subsequently subjected to gene set enrichment analysis (F-GSEA, https://bioconductor.org/packages/release/bioc/html/fgsea.html) and then visualized through the R pheatmap package. WES sequencing and read alignment.
  • SNPs and indels were identified using the SAMtools mpileup function and variants were called using VarScan suite (v.2, varscan.sourceforge.net). Variants were filtered using an in-house Perl script, to account for SNVs and indels not included in the control sample (wild type), for mapping quality, number of mismatches, read depth, and known variants (Mouse genome project, mgp v.3). Finally, variants were annotated using Annovar (http://annovar.openbioinformatics.org, v.20150322)
  • Copy number variations were called using the binary segmentation algorithm implemented in the“DNACopy R package”.
  • the default parameters and segmented data were merged suing mergeSegments.pl from VarScan suite.
  • mIDH2 R140Q mIDH2 dependent AML model was used to predict mechanisms promoting resistance to mIDH2 inhibitors in patients.
  • mIDH2 dependency was manipulated in a model of mouse bone marrow (BM) cells overexpressing mIDH2 transduced with Hoxa9-GFP and Meisla-IRES-YFP retroviruses transplanted in a primary host in the presence or absence of doxycycline to induce mIDH2 expression.
  • BM mouse bone marrow
  • Meisla-IRES-YFP retroviruses transplanted in a primary host in the presence or absence of doxycycline to induce mIDH2 expression.
  • Dependence on mIDH2 in secondary transplanted disease was confirmed (Figs.
  • Example 2 De novo resistance is associated with a distinct metabolic switch to one- carbon metabolism and altered redox balance
  • mIDH2 Since the expression of mIDH2 directly impacts cellular metabolism, polar metabolites were extracted from sensitive and resistant AML cells from both 2 nd and 3 rd transplanted mice (Fig. 7A). Comparison of metabolite abundance established multiple metabolic processes altered between the mIDH2 leukemic states (Fig. 2A and 2B). Resistant cells were significantly enriched in glycerophospholipid, pyrimidine, purine, cysteine and methionine metabolism pathways (Fig. 2B). These pathways can be fueled by one-carbon metabolism, which consists of both the folate and the methionine cycles (Figs. 7B and 1C).
  • one-carbon metabolism is a main cellular engine contributing to nucleotide metabolism and global methylation, as well as cellular redox status. Similar to recent findings for purine metabolism in cancer, a general increase in purine precursors in 3 rd compared to 2 nd recipient AMLs was observed (Figs. 7D-F).
  • Glutathione metabolism was the most significant and highly enriched metabolic pathway in resistant AML cells (Fig. 2C). 3 rd recipient AML cells demonstrated high levels of glutathione and cysteine, glutathione’s major precursor (Fig. 2D), suggesting that these cells may have an altered redox balance. Indeed, altered NADPH/NADP+ and NADH/NAD+ ratios confirm this oxidative stress (Fig. 2E), and excessive ROS levels were visualized using MitoSOXTM Red staining (Figs. 2F-G), especially relative to Hoxa9/Meisla driven leukemias lacking mIDH2 (Fig. 7G).
  • HoxA9/Meisla/mIDH2 leukemia at early (sensitive) stage were marked by the same features of late (resistant) leukemia. See Figs. 2J(a-e) and 3K(a-e). This evidenced that the mutant IDH leukemia at early stage show the same characteristics/features of the late stage leukemia, when using ATO combined with ATRA, providing a rational for the utilization of ATO and ATRA not only in mutant IDH2 leukemia at late sage (when cells became resistant to the inhibitor), but also at early stage (when cells are sensitive to the inhibitor).
  • Fig. 8A To determine if such genotoxic stress may contribute to AML evolution through targeted mutation whole exome sequencing (WES) of a 2 nd recipient and three of its derived 3 rd recipient‘daughter’ leukemias was performed (Fig. 8A).
  • WES targeted mutation whole exome sequencing
  • Fig. 8B Non-synonymous base substitutions and both G>A:C>T and A>G:T>C transitions accounted for two- thirds of the single base substitutions (SBS) observed, as is frequently found in cancers without biased substitution
  • PCA principal component analysis
  • translocations involve known hematopoietic genes including Rael ( Rael;Pappa t(4;2)), Meisl ( EeahMeis P, t(l 1 ; 10)), and both Ppplrl3b (Asppl) and Cadps2 (Ppplrl 3b:Cadps2 t(6;12)) (Fig. 8E).
  • Rael Rael;Pappa t(4;2)
  • Meisl EeahMeis P, t(l 1 ; 10)
  • Ppplrl3b Asppl
  • Cadps2 Ppplrl 3b:Cadps2 t(6;12)
  • RNAseq analysis was performed to create a global picture of leukemia progression from a state of mIDH2 dependence to independence (Fig. 9A).
  • Primary leukemias derived from Hoxa9;Meisl overexpression without mIDH2 was used for comparison.
  • PCA analysis identified distinct clusters of 2 nd and 3 rd recipient leukemias (Fig. 9B). Differential gene expression patterns highlighted clear differences between sensitive and resistant leukemias (Fig. 3A), suggestive of common pathways leading to independence from mIDH2.
  • a gene-centric PCA identified genes associated with the Jun/Fos AP-1 transcription factor family (Jim, Fos, Fosb), granulopoiesis and myeloid differentiation ( My a, Lyz2, Ngp ), and inflammation ( Zfp36 ) as differential (Fig. 9C). Altered expression status of myeloid granule components also suggested that 2 nd and 3 rd recipient leukemias may represent different stages of myeloid development (Fig. 9C).
  • GSEA Gene set enrichment analysis identified similar pathways altered in resistant AMLs, including enrichment of Kras, MAPK, TNF signaling and response to Tretinoin (aka. all-trans retinoic acid; ATRA) (Fig. 3C), while an interactive network of all differentially regulated genes highlighted the central role of Erk/MAPK signaling in defining the signatures associated with resistant disease (Fig. 8C) consistent with previously published data, suggesting that the present model recapitulates mechanisms relevant to human AML. Indeed, cancer signaling pathway activation was confirmed by flow cytometry analysis (Fig. 8E).
  • ATRA has been recently established as a specific inhibitor of the PIN 1 proto-oncogene.
  • PIN 1 has been reported to be upregulated by C/EBPa-p30, and can promote activation of MAPK. Similarly, it promotes PI3K activity and regulates AKT’s stability and phosphorylation.
  • ATRA is a specific inhibitor of PIN1, it is also important to note that PIN1 itself can be a negative regulator of ATRA, and there is an expanding role for PIN 1 as a negative regulator of hematopoietic differentiation.
  • PIN 1 may contribute to proliferative signaling (MAPK and PI3K dependent), increased survival to ROS-mediated apoptosis, and the ATRA mediated differentiation block observed.
  • a marked upregulation of Pin 1 protein in both Dox ON and OFF mIDH2 leukemias at late stages of progression Fig. 4A, Lanes 5-8. This suggested that 3 rd recipient leukemias may be sensitized to ATRA and that targeting PIN 1 may relieve both the oncogenic signaling and the differentiation block in these cells.
  • ATRA-sensitive retinoic acid receptors were upregulated in mIDH2 2 nd recipient AML, as well as increased C/EBRa and C/EBRe (Fig. 4A, Lanes 3 and 4).
  • PIN1 itself, the negative regulator of the pathway, was also concomitantly induced (Fig. 4A, Lanes 3 and 4).
  • validated Rara/Rxra responsive genes were not transcriptionally activated, suggesting that RARs are transcriptionally inactive.
  • mIDH2 overexpressing TF- 1 and U937 cell lines were used to understand if the same was true for human leukemia. Indeed, both TF- 1 (Fig. 4B) and U937 (Fig. 4C) lines demonstrated upregulation of PIN1 in the presence of mIDH2, along with retinoic acid receptors and C/EBRa. Inhibition of PIN1 by using a specific inhibitor (Juglone) clearly demonstrated PIN 1 to be required for blocking differentiation driven by erythropoietin (EPO) (Fig. 4D) and reduced their clonogenic ability (Figs. 4E, 10A, and 10B).
  • EPO erythropoietin
  • a mIDH2 independent (e.g, resistant) mouse AML was transplanted into BL6 (Fig. 11C) and placed mice on a treatment regime as outlined in Fig. 4K. Both single agent and combination treatments demonstrated efficacy as measured by increase in mean survival of mice treated (Fig. 11D). Strikingly, a number of mice treated with the ATO/ ATRA combination demonstrated a strong differentiation response in the leukemic blasts associated with an excessive infiltration of mature myeloid cells in the lung (Fig. 11E-G). Thus, steroid dexamethasone was also administered. As outlined in Figs. 4K-L, dexamethasone treatment successfully overcame the differentiation syndrome, and enabled extensive survival benefits upon treatment with the ATO/ ATRA combination.
  • a panel of human primary AMLs was treated with ATO/ATRA to evaluate their potential for clinical efficacy, as measured by colony formation in methylcellulose.
  • AML harboring mIDH2 showed a significantly reduced number of colonies upon treatment, and strong synergy was observed in combination treatments (Fig. 5A).
  • NSG mice transplanted with human primary mIDH2 positive AML (Fig. 5B) were subjected to the treatment protocol as outlined in Fig. 5C to evaluate in vivo efficacy. To minimize onset of DS a metronomic dosing schedule was utilized.
  • mIDH2- overexpression in TF1 cells inhibited the induction of the differentiation marker CD71 upon differentiation driven by erythropoietin (EPO), while specific targeting of PIN 1 with Juglone restored the upregulation of this marker. Also similarly, these findings were mimicked by genetic targeting of PIN 1 in mIDH2- overexpressing TF1 cells, with up-regulation of the CD71 and CD44 markers detected by flow cytometry and the differentiation associated HGB and KLF1 genes
  • the therapeutic opportunity afforded by ROS induction was evaluated in this model through the use of arsenic trioxide, hypothesizing that the pro oxidant activity of ATO could cause genotoxic stress and cell death.
  • the murine AML model demonstrated increased apoptosis in both a sensitive mIDH2 dependent and resistant mIDH2 independent setting (Fig. 13h), while the expression of mIDH2 in TF1 cells conferred sensitivity to induction of apoptosis upon treatment with ATO that was not observed in control infected cells (Fig. 13i).
  • Example 9 ATRA and ATO combination for treatment of mIDH AML
  • Fig. 14a 21 days after initiating treatment mice engrafted with mIDH2- overexpressing U937 treated with the ATO/ATRA combination remained alive, while all control mice had succumbed to disease (Fig. 14b).
  • Bone marrow (BM) analysis demonstrated a significant decrease in the percentage of human leukemic blasts, and increased differentiation, in both the ATRA and ATO/ATRA treated cohorts of mice transplanted with mIDH2-overexpressing cells (Fig. 14c-14e).
  • mIDH2 expressing U937 cells treated with the combination therapy demonstrated the mature myeloid CDllb marker amongst all the cohorts analysed (Fig. 14f and 14g).
  • Reduced spleen weights were observed in mice transplanted with mIDH2-expressing U937 cells treated with ATO/ATRA, demonstrating the power of this treatment combination to impact the severity of disease (Fig. 14h).
  • NSG mice transplanted with human primary mIDH2 positive AML were subjected to the treatment protocol, and to minimize onset of differentiation syndrome (DS), we utilized a metronomic dosing schedule, as for APL patients’ treatment protocols.
  • DS onset of differentiation syndrome
  • the ATRA/ ATO combination selectively targets the PINl-PML-RARa oncogenic node, while in mIDH-leukemia, the ATRA/ ATO combination treatment selectively targets the oncogenic mIDH-PINl-RARa node.
  • mIDH-leukemia a cohort of primary human AML cells that harbor either mIDHI or mIDH2
  • Our analysis points to the ability of these oncogenes to sensitize to the effects of ATRA plus ATO.
  • the combination of these agents with specific mIDH targeting agents may in turn potentiate and extend the efficacy of these inhibitors.

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Abstract

La présente invention concerne des méthodes et des kits se rapportant au traitement de la leucémie mIDH1, la leucémie mIDH2 et de tumeurs solides associées à mIDH par l'administration de darinaparsine, d'un composé d'acide rétinoïque ou d'une association d'un composé d'arsenic et d'un composé d'acide rétinoïque.
PCT/US2019/064326 2018-12-03 2019-12-03 Darinaparsine et composés d'acide rétinoïque pour le traitement de troubles associés à l'idh WO2020117867A1 (fr)

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Citations (2)

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WO2017146794A1 (fr) * 2016-02-26 2017-08-31 Celgene Corporation Inhibiteurs d'idh2 pour le traitement de tumeurs solides et malignes hématologiques

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US20040146575A1 (en) * 1997-11-10 2004-07-29 Memorial Sloan-Kettering Cancer Center Process for producing arsenic trioxide formulations and methods for treating cancer using arsenic trioxide or melarsoprol
WO2017146794A1 (fr) * 2016-02-26 2017-08-31 Celgene Corporation Inhibiteurs d'idh2 pour le traitement de tumeurs solides et malignes hématologiques

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