WO2019109095A1 - Trioxyde d'arsenic et composés d'acide rétinoïque pour le traitement de troubles associés à l'idh2 - Google Patents

Trioxyde d'arsenic et composés d'acide rétinoïque pour le traitement de troubles associés à l'idh2 Download PDF

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WO2019109095A1
WO2019109095A1 PCT/US2018/063666 US2018063666W WO2019109095A1 WO 2019109095 A1 WO2019109095 A1 WO 2019109095A1 US 2018063666 W US2018063666 W US 2018063666W WO 2019109095 A1 WO2019109095 A1 WO 2019109095A1
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retinoic acid
leukemia
cells
acid compound
arsenic trioxide
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PCT/US2018/063666
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English (en)
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Pier Paolo Pandolfi
John Clohessy
Vera MUGONI
Ming Chen
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Beth Israel Deaconess Medical Center, Inc.
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Publication of WO2019109095A1 publication Critical patent/WO2019109095A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • Isocitrate dehydrogenase enzymes (IDH1 and IDH2) are key metabolic enzymes.
  • IDH2 IDH2 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-hydroxy glutarate (2-HG), an oncometabolite that contributes to the oncogenic phenotype. Accordingly, 2-HG is a predictive biomarker in cancers having a pathogenic IDH2 allele.
  • IDH2 -targeting Enasidenib AG-221
  • mIDH2 mutant form of IDH2
  • 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 of treating cancer by contacting the cells of the cancer with a pharmaceutical compound.
  • the pharmaceutical compound can be an arsenic trioxide compound (e.g., ATO), a retinoic acid compound (e.g., ATRA), or a combination of arsenic trioxide and a retinoic acid compound (ATO and ATRA).
  • the cancer can be a leukemia (e.g., mIDH2 leukemia) or a solid tumor (e.g., an IDH2- associated solid tumor).
  • a leukemia e.g., mIDH2 leukemia
  • a solid tumor e.g., an 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 arsenic trioxide, 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-hydroxy glutarate (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 IDH2 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 arsenic trioxide, 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 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 IDH2 gene.
  • contacting said cells of said leukemia with the arsenic trioxide 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 arsenic trioxide and the retinoic acid compound results in the complete remission of the leukemia (e.g., all signs and symptoms of the leukemia are absent).
  • contacting said cells of said leukemia with the arsenic trioxide 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 arsenic trioxide and the retinoic acid compound operate synergistically to treat said leukemia. In some embodiments, the arsenic trioxide and the retinoic acid compound is more effective for treating said leukemia than the same quantities of either said arsenic trioxide or said retinoic acid compound alone.
  • contacting said cells of said leukemia with of the arsenic trioxide 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 arsenic trioxide and the retinoic acid compound.
  • contacting said cells of said leukemia with of the arsenic trioxide and the retinoic acid compound increases the production of ROS by at least about 10%.
  • contacting said cells of said leukemia with of the arsenic trioxide 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 arsenic tri oxide and a retinoic acid compound 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 arsenic trioxide and the retinoic acid compound.
  • contacting said cells of said leukemia with of the arsenic trioxide 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
  • the contacting said cells of said leukemia with of arsenic trioxide 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 arsenic trioxide or the retinoic acid compound alone.
  • contacting said cells of said leukemia with the arsenic trioxide 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 arsenic trioxide or the retinoic acid compound alone.
  • arsenic trioxide and the retinoic acid compound may be administered concurrently (e.g., within about lmin, 2min, 5min, lOmin, 20min, 30min, or 60min) or separately.
  • the arsenic tri oxide 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 arsenic tri oxide to said subject, wherein one or more cells of the leukemia have a pathogenic 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 arsenic trioxide 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 arsenic trioxide 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 arsenic trioxide).
  • ROS reactive oxygen species
  • contacting said cells of said leukemia with the arsenic trioxide 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 arsenic tri oxide.
  • 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 arsenic trioxide.
  • contacting said cells of said leukemia with of the arsenic trioxide 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 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
  • 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 arsenic trioxide).
  • a compound that inhibits Pinl activity e.g., in combination with the retinoic acid and the arsenic trioxide.
  • 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 LSD1 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.
  • a normal e.g., wild-type and/or disease fee
  • decreased levels of LSD 1 activity include a reduction of LSD 1 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
  • the method further comprises contacting said cells of said leukemia with an inhibitor of IDH2.
  • the inhibitor of IDH2 may reduce 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 IDH2 activity prior to treatment.
  • the inhibitor of IDH2 may be specific for a pathogenic mutant form of IDH2. When such an inhibitor targets a mutant form of IDH2, it may reduce the aberrant activity of the enzyme by about 20%.
  • 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 arsenic tri oxide, (b) an effective amount of a retinoic acid compound, and (c) instructions for the use of the arsenic trioxide 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 IDH2 allele or wherein the subject has been previously determined to have elevated 2 -hydroxy glutarate (2-HG) levels.
  • the kit includes: (a) an effective amount of arsenic tri oxide, (b) an effective amount of a retinoic acid compound, and (c) instructions for the use of the arsenic trioxide 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 IDH2 allele or wherein the subject has been previously determined to have elevated 2 -hydroxy glutarate (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.
  • a normal e.g., wild-type and/or disease fee
  • 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 arsenic trioxide 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 arsenic trioxide 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 arsenic tri oxide 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 arsenic trioxide. In some embodiments the low dose of the retinoic acid compound and the low dose of the arsenic trioxide are nontoxic.
  • a low dose of a retinoic acid compound related to in vivo treatments is 1.5 pg/g/day or lower.
  • a low dose of the retinoic acid compound comprises a dose of about 30 mg/m 2 body surface area or less.
  • 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).
  • a low dose of the retinoic acid compound comprises a dose of about 10 mg/m 2 body surface area or less.
  • the arsenic trioxide 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, 0.08,
  • a low dose of arsenic trioxide is about 0.15, about 0.16, or about 0.032 mg/kg body weight. In some embodiments, the low dose of the arsenic trioxide is a nontoxic dose of the arsenic trioxide.
  • a low dose of arsenic trioxide related to in vitro treatments of leukemia cells is 0.25 mM or lower. In some embodiments, a low dose of arsenic trioxide related to in vivo treatments (e.g., in mice treatments) is 2.5 pg/g/day or lower. In some embodiments, a low dose of arsenic tri oxide 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). For example, a low dose of arsenic trioxide related to treating a human with leukemia is 0.075-0.15 mg/kg (IV administration, QD). In some embodiments, a low dose of arsenic tri oxide comprises a dose of about 0.05 mg/kg body weight or less.
  • the low dose of arsenic trioxide is administered in combination with a low dose of a retinoic acid compound. In some embodiments the low dose of arsenic trioxide and the low dose of retinoic acid compound are nontoxic.
  • the retinoic acid compound is all-trans retinoic acid (ATRA), l3-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 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 IDH2 allele and elevated 2-hydroxy glutarate (2-HG) levels.
  • one or more cells of the leukemias comprise a pathogenic 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,
  • 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 arsenic tri oxide 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), l3-cis-retinoic acid, retinol, retinyl acetate, retinal, and AC-55640.
  • the effective amount of arsenic tri oxide 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 IDH2 allele in combination with NPMc+, FLT3-ITD, or both, in which 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 from Tg(M2rt-TA) transgenic mice and overexpressing HoxA9 and Meisla oncoproteins. Red boxes: leukemia. Blue boxes: healthy state.
  • Fig. 1B 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. 1D 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. 1E 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. 1F 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 MeislA in leukemia cells.
  • IDH2R140Q inhibitor IDHi
  • IDHi AGI-6780
  • ImM vehicle
  • Fig. 1K 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.
  • vehicle DMSO
  • IDHi AGI-6780
  • ImM ImM
  • 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
  • GSE Gene Set Enrichment
  • 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, TNFa, and ATRA related signature in resistant (3rd RECIPIENT) vs sensitive (2nd RECIPIENT) leukemia cells (left panel) and the associated Heat maps (right panels).
  • GSE Gene Set Enrichment
  • Fig. 3D is an image depicting the Venn Plot showing shared genes between mouse mIDH2 leukemia and Schenken et al; 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 al; 2013.
  • Fig. 3F is a series of graphs and images depicting the quantifications of morphological screening of murine leukemia cells isolated from the bone marrow of sensitive (2 nd
  • 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. 31 is a graph depicting Cell proliferation assay on TF1 cell line, stably
  • 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
  • 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+ 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
  • d. LC-MS/MS fold change levels of precursors for pyrimidine metabolism. OMP: orotidine -5’-monophosphate, UDP: uridine diphosphate.
  • 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/EBPs).
  • 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 overexpressing the mIDH2 or respective controls (CTRL).
  • HBG Hemoglobin
  • 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 overexpressing mutant IDH2 alone (mIDH2) or in combination with PIN1 (mIDH2 PIN1++) and induced to differentiation after treatment with the IDH inhibitor AG-221 or respective vehicle ( DMSO).
  • 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. 4M is an image deciphering the inhibition of LSD1 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 Rl40Qin 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 Haemotoxyiin and Eosin (H&E) staining of internal organs (spleen, liver, and kidney) from 3rd RECIPIENTS showing infiltrating blasts.
  • H&E Haemotoxyiin 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.
  • Fig. 7A 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
  • AMP adenosine monophosphate
  • GMP guanosine monophosphate
  • 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 Sequencing (WES) analysis. Genomic DNA was isolated from resistant leukemia cells (3rd_08, 3rd_09, 3rd_l l) or their common parental sensitive cells (2nd_07).
  • 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, C>A, A>C, T>G, G>C, OG, 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_l l). 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_l 1) 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 Interchromosomal translocations (fusion genes) 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_l3 were generated from the parental sensitive 2nd _9A.
  • Resistant leukemia cells 3rd_l 1, 3rd_l2, 3rd_08, and 3rd_l0 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 myeloperoxidase
  • Rn45s 45s-pre-ribosomal RNA
  • Ngp neutrophilic granule protein
  • Psap
  • prosaposin Fill : 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.
  • PCA Principal Component Analysis
  • 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 CD14 positive cells (CD14+).
  • Fig. 10A is a series of images depicting a western blot assay on TF1 cells
  • Fig. 10B is a series of images depicting a western blot assay on TF1 cells
  • Fig. 10C 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. 11B 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. 11C is a scheme of the in vivo approach to evaluate combination arsenic tri oxide (ATO) and ATRA treatment on mIDH2 leukemic cells in C57BL/6J mice.
  • ATO arsenic tri oxide
  • Fig. 11D 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. 11F 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 CD1 lb positive cells (CD1 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 fromNSG mice transplanted with mIDH2-U937 or CTRL-U937 leukemia cells and treated with ATO, ATRA or a combination of both.
  • Fig. l3a-l3k 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. i, Fold induction of cell death (Annexin V/7AAD positive cells) in TF1 leukaemia cells
  • IDHl 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. l4a-l4h 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.
  • BM bone marrow
  • Data are means ⁇ SD of samples isolated from different mice d, 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 e, Cytospins showing mIDH2-U937 or CTRL - U937 leukaemia cells isolated as GFP+ cells from BM of NSG mice. Arrowheads show cells displaying differentiated morphology f, Flow cytometry plots showing CDl lb expression levels in mIDH2-U937 or CTRL-U937 leukaemia cells g, Quantification of flow cytometry analysis for the percentage of CD1 lb positive cells (CD1 lb+).
  • U937 cell line stably overexpressing the mutant variant R140Q of IDH2 show about 30% reduced percentage of CD1 lb+ cells compared to the control (CTRL).
  • Fig. l5a-l5g a series of graphs showing combined ATRA and ATO treatment eradicating mIDH2 leukaemia a, Methylcellulose colony forming assay quantification of human primary mIDH2 AML blasts treated in vitro with pharmacological concentrations of ATRA (1 mM), ATO (0.5 mM), a combination of both ATRA and ATO, or vehicles (VHL) as control. Treatments were performed in either the presence of mIDH2 or absence of the mutation (CTRL). Data are means ⁇ SD of duplicates b and d, Kaplan-Meyer curve showing the percentage of disease free survival related to ATO/ ATRA pre-clinical study on mIDH2 PDX.
  • N 2 different mIDH2 AML were isolated from bone marrow at diagnosis. Data analysed by Log-Rank (Mentel-Cox) test. #DS: mice involved by differentiation syndrome (DS) (aka. ATRA syndrome). c and e, Representative images of cytospins showing cells isolated from BM of PDX mice treated with ATRA and ATO. n.d: not detected as mice were not euthanized f-g,
  • Fig. l5h-l5j a series of graphs showing the effect of ATO and ATRA combination in limiting leukemia aggressiveness in vivo in a pre-clinical study on PDX generated with AML harboring wild-type IDH1 and IDH2 genes h.
  • Scheme of the experiment NSG mice were transplanted with human AML blasts harboring mutations in WT1 and KIT genes and chromosomal alterations (invl6), but wild-type IDH1 and IDH2 genes i.
  • Scheme of the ATO and ATRA treatments j Survival curve shows no significant survival extension for mice treated with ATO, ATRA, or combination of both ATRA and ATO, as compared with the respective VHL treated mice.
  • 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 LSI I 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.
  • 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 AS 2 O 3 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; AsS), orpiment (yellow arsenic; AS 2 S 3 ), and arsenolite, an oxidation product of arsenic sulphides (white arsenic; AS 2 O 3 ).
  • 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 tri oxide 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 Waxman et al. ( Oncologist 6: 3-10, 2001).
  • retinoic acid examples include, without limitation, all-trans retinoic acid (ATRA), 13- cis retinoic acid (l3cRA), and retinoic acid compounds, and derivatives thereof, e.g., as described herein.
  • ATRA all-trans retinoic acid
  • l3cRA 13- cis retinoic acid
  • retinoic acid compounds examples include those shown in Tables 1 and 2 below. Table 1.
  • 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, l3cRA.
  • 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;
  • a pharmaceutical compound refers to a pharmaceutical product that fits a unique need of a patient. It can be a single therapeutic agent or a combination of multiple therapeutic agents.
  • a pharmaceutical compound used in this invention can be arsenic trioxide, a retinoic acid compound described above, or a combination thereof.
  • 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.l.]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 cyclopentenyl, cyclohexenyl, and the like.
  • the alkyl, alkenyl and alkynyl groups contain 1-12 carbons (e.g., C1-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 G r r group with 14n + 2] p electrons in conjugation and where n is 1, 2, or 3.
  • Aryl groups also include ring systems where the ring system having 14n + 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, l,2-dihydronaphthyl, l,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, and indenyl.
  • heteroaryl represents an aromatic (i.e., containing 4n+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 l,2,4-thiadiazole), oxadiazole (e.g., l,2,3-oxadiazole or l,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, l,2,3-triazine l,2,4-triazine, or l,3,5-triazine), l,2,4,5-tetrazine, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazo
  • heterocyclyl 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.
  • 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.
  • 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.
  • 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 IDH2 allele or having elevated 2 -hydroxy glutarate (2-HG) levels.
  • a subject e.g., a human
  • IDH2-associated leukemias IDH2-associated solid tumors.
  • An 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, BRWDl,UBR4, STAG2, LNX1, FREM2, MLLT10, DNAH11, SLIT2,
  • An 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- hydroxy glutarate (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.
  • 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 be specific for a pathogenic mutant form of IDH2 (e.g., Enasidenib).
  • “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” 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 compositions of methods described herein).
  • 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 LSD 1 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 LSD 1 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 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%,
  • “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 tri oxide 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%, 94%, 95%, 96%, 97%, 98%, or 99% less) than the standard recommended dosage or lowest standard recommended dosage of a particular agent, e.g., a therapeutic agent formulated for a given route of administration for treatment of any human disease or condition.
  • 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). In certain instances, a low dose of arsenic trioxide is about 0.15, about 0.16, or about 0.032 mg/kg body weight.
  • 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).
  • 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., 25, 26, 27, 28, 29, 30, 31, 32,
  • 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.
  • the term“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 predisposition toward a disorder.
  • 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
  • the treatment at least partially
  • 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 tri oxide 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 -hydroxy glutarate (2-HG), an oncometabolite that contributes to the oncogenic phenotype. Accordingly, 2-HG is a predictive biomarker in cancers having a pathogenic IDH2 allele.
  • AMLs human acute myeloid leukemias
  • the methods described herein are used to treat a subject having a disorder associated with a mutation in the IDH2 enzyme (mIDH2), thereby giving rise to a pathogenic phenotype (e.g., leukemia).
  • 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-hydroxy glutarate (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-hydroxy glutarate (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 compound, 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 tri oxide 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 tri oxide 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), l3-cis retinoic acid (l3cRA), and retinoic acid compounds, and derivatives thereof, e.g., as described herein. Retinoic acid compounds of the invention may be ay 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
  • 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). Combination Therapies
  • arsenic tri oxide 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 arsenic tri oxide 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, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine,
  • 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.
  • Pinl is overexpressed in some human cancer samples and the levels of Pinl are correlated with the aggressiveness of tumors.
  • 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-l, AKT, b-catenin, c-Myc, p53, and numerous other proteins known to play roles in cancer progression.
  • the arsenic tri oxide 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).
  • 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
  • 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
  • agents e.g., arsenic trioxide, a retinoic acid compound, and/or an additional therapeutic agent, as described herein
  • co-formulated compositions can include the drugs together in the same pill, ointment, cream, foam, capsule, liquid, etc. It is to be understood that, when referring to the formulation of combinations of the invention, 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.
  • Certain embodiments of the invention feature formulations of arsenic tri oxide 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-l+, Lin-) cells as reported in Quek et al., Nat.
  • 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-l+
  • BD FACSAria ll 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 (I 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 m ⁇ 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 Female NSG (NOD.CgPrkdc scld I12' glml 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.
  • mice engrafted with human AML blasts were purchased from Jackson
  • mice 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.
  • ATO 2.5 pg/g
  • ATRA 1.5 pg/g
  • RA Retinoic acid
  • APL As203 treatment in transgenic models of acute promyelocytic leukemia
  • the Hoxa9/Meisla/IDH2Rl40Q murine primary leukaemia cells were cultured and expanded in RPMI media supplemented with murine SCF (2.4 ng/mL), FLT3 Ligand
  • Methylcellulose colony assay Methylcellulose colony assay.
  • methylcellulose medium was supplemented with Doxycycline (Gibco), Arsenic Tri oxide (Sigma), Tretinoin (Sigma), AGI-6780 or AG-221 (Cayman Chemical), Juglone (Cayman Chemical) or respective vehicle solutions.
  • Doxycycline Gibco
  • Arsenic Tri oxide Sigma
  • Tretinoin Sigma
  • AGI-6780 or AG-221
  • Juglone Cayman Chemical
  • Single colonies were picked up 1- 2 weeks after culture and then processed by cytospin, assayed for cell viability, flow cytometry analysis, and/ or harvested for nucleic acids extraction.
  • 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 CCL incubator. Colonies were counted as units of at least 30 cells were generated after plating (2 - 4 weeks). Differentiation Assay.
  • Human TF1 cells were pre-treated for 12 days with AG-221 (1 mM) or vehicle and washed with PBS to remove residual GM-CSF. Cells were then induced to differentiate using EPO (2 unit/ml, StemCell Technologies) and Hemin (10 pM, Sigma). Induction continued for 12 days and the cell pellets were collected and subjected to real-time qPCR to detect fatal haemoglobin (HBG) and KLF-l gene expression or flow cytometry to quantify CD71.
  • EPO unit/ml
  • Hemin 10 pM, Sigma
  • Cell Proliferation and viability Assay Cells (TF1 or U937, 8-10 *l0 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 1 ml of 2%FBS in PBS IX.
  • 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 al, Nat Rev Cancer 2013, 13:227-232.
  • 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 al, Nat Rev Cancer 2013, 13:227-232.
  • the cells were fixed in 4% PFA at 37°C, 5% C02 for 10 minutes and permeabilized with 90% ice- cold methanol and processed as reported in Pandolfi et al., Cancer Cell 2017, 32:552-560.
  • Flow cytometry data were collected on a J LSR II (VF415) or LSR J-Fortessa (JF415) flow cytometer (Becton Dickinson). Resulting profiles were further processed and analysed using the FlowLogic or FloJo software. FACS Cell sorting was performed on a JF Aria II SORP or Sony SH800z Cell Sorter, with the support of the Dana-Farber Cancer Institute Flow Cytometry Core Facility.
  • Tissues were fixed in 4% paraformaldehyde overnight, paraffin embedded, and then sectioned at 5pm. 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% H 2 0 2 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-l4520) or Anti-Myeloperoxidase antibody (1 :50, abeam #ab9535), or Phospho-Histone H2A.X (1 :200, Cell Signalling #97l8S) 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.
  • Stained sections were counterstained using hematoxylin and dehydrated before they were sealed with a coverslip with Richard- Allan Scientific® CytosealTM XYL 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. RNA-Seq / Gene expression analysis.
  • 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 (v0.6.l) 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,
  • TndelRealigner (www.broadinstitute.org/gatk/) was used to realign reads near indels from the Mouse Genomes Project (v5).
  • the BAM files were then re-sorted and quality scores were recalibrated using GATK‘Table Recalibration’ and PCR duplicates were marked using Picard‘MarkDuplicates’.
  • 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
  • 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 doxy cy dine to induce mIDH2 expression.
  • BM mouse bone marrow
  • Example 2 De novo resistance is associated with a distinct metabolic switch to one- carbon metabolism and altered redox balance
  • mIDH2 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 7C).
  • 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).
  • Fig. 8 A 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. 8 A). Non-synonymous base substitutions and both G>A:OT 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 (Fig. 8B). Additionally, principal component analysis (PCA) established that these leukemias did not appear to cluster (Fig. 8C).
  • SBS single base substitutions
  • PCA principal component analysis
  • the translocation involving Ppplr 13b and Cadps2 was of particular interest since loss of function for both of these genes has been implicated in hematopoietic stem cell maintenance.
  • the Ppplr 13b gene is also implicated in leukemia development, coordinating tumor suppression with the TP 53. While the Ppprl3b:Cadps2 translocation was only identified in two out of three of the 3 rd recipient AMLs (3rd_08 and 3rd_l 1), the third leukemia showed genomic loss of Trp53, suggesting that targeting of Trp53 may contribute to progression.
  • Example 4 Aberrant ATRA signaling characterizes resistance to mIDH2 withdrawal
  • 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-l transcription factor family ( Jun , Fos, Fosb ), granulopoiesis and myeloid differentiation ( Myo , 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).
  • pharmacological and sub-pharmacological doses of ATRA demonstrated profound differentiation effects on mIDH2 expressing AMLs (Fig. 3F).
  • TF1 cell lines overexpressing the mIDH2 allele and increased 2-HG levels were generated (Fig. 3G).
  • Treatment of mIDH2 overexpressing TF1 with ATRA (10 6 M) resulted in increased differentiation as compared to controls (Fig. 3H), with a concomitant decrease in cell growth (Figs. 31 and 3Jj).
  • LSD 1 a concomitant decrease in cell growth
  • ATRA has been recently established as a specific inhibitor of the PIN1 proto-oncogene.
  • PIN1 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 PIN1 as a negative regulator of hematopoietic differentiation.
  • PIN1 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 Pint 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 PIN1 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-l and U937 cell lines were used to understand if the same was true for human leukemia. Indeed, both TF-l (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 PIN1 to be required for blocking differentiation driven by erythropoietin (EPO) (Fig. 4D) and reduced their clonogenic ability (Figs. 4E, 10A, and 10B).
  • EPO erythropoietin
  • TCGA Cancer Genome Atlas
  • Example 6 ATRA and ATO combination for treatment of mIDH2 AMT.
  • 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.
  • Example 7 Differentiation therapy for the treatment of human mIDH2 AMT.
  • 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. Importantly, a notable survival advantage for both ATO and ATRA treated mice was observed, while combination treated mice appeared to demonstrate a durable remission in most cases (Fig. 5D). Interestingly, the single agent ATO and ATRA treated cohorts demonstrated a significant increase in differentiating blasts in the bone marrow as evaluated by morphology (Figs. 5E and 5F).
  • ATRA can inhibit the enzymatic activity of its negative regulator PIN1 through direct binding, as reported in Wei et al, Nat. Med. 2015, 21 :457-466.
  • PIN1 negative regulator
  • the ability of ATRA was evaluated to promote differentiation of leukemic blasts in a murine model of AML. Indeed, both pharmacological and sub-pharmacological doses of ATRA demonstrated striking differentiation effects on murine mIDH2 expressing AMLs, both in the mIDH2 dependent and independent phases of disease evolution (Fig. l3a).
  • mIDH2- overexpression in TF1 cells inhibited the induction of the differentiation marker CD71 upon differentiation driven by erythropoietin (EPO), while specific targeting of PIN1 with Juglone restored the upregulation of this marker.
  • EPO erythropoietin
  • 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. l3h), 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).
  • mIDH2-expressing AML cells demonstrated enhanced sensitivity to the combination as measured by reduced colony forming capacity.
  • 2 nd recipient leukaemias demonstrated sensitivity to both single agents and a synergistic response to combination (Fig. l3j).
  • Late stage disease i.e. 3 rd recipient, mIDH2- independent leukaemias in both the presence and absence of mIDH2 demonstrated a similar pattern of responses, showing sensitivity to single agents and synergistic effect of combination treatments, indicating efficacy in both mIDH2 dependent and independent disease and consistent with the hypothesis based on the AML RNAseq data (Fig. 4c-f).
  • ATRA/ ATO combination was not due simply to oncogenic interaction with HOXA9 and Meisla, we evaluated the ability of ATRA and ATO to effectively target mIDH AMLs derived from a number of distinct genetic contexts.
  • Each of the AMLs harbouring mIDH demonstrated extraordinar sensitivity to the ATRA/ ATO combination as compared to AML derived from MLL-AF9 overexpression with wild type IDH alleles, as negative controls.
  • NPMc+/FLT3-ITD alone were used as positive control as this genotype was previously shown to confer sensitivity to ATRA/ ATO.
  • mIDH conferred superior sensitivity even when compared with this genetic make-up (Fig. l3k).
  • IDH2 mutation 3 harbouring an IDH1 mutation, and seven AMLs, with wild type IDH, with ATO/ ATRA to evaluate their potential for clinical efficacy, as measured by colony formation in methylcellulose.
  • AML harbouring mIDH2 showed significantly reduced number of colonies upon treatment (Fig. l5a), and strong synergy was observed in combination treatments (Fig. l5a).
  • colonies arising from single agent ATRA or ATO treatment gave primarily blast-like morphology, while the combination treatment resulted in almost complete loss of this morphology.
  • mice treated with ATRA/ ATO died not as a result of frank AML, but rather succumbed to differentiation syndrome (DS) characterized by infiltration of lungs with mature myeloid cells demonstrating strong myeloperoxidase (MPO) expression, a feature also associated with APL patients treated with ATRA.
  • DS differentiation syndrome
  • MPO myeloperoxidase
  • 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 PIN1 -PML-RARa oncogenic node, while in mIDH-leukemia, the ATRA/ ATO combination treatment selectively targets the oncogenic mIDH-PINl-RARa node.
  • the ATRA/ ATO combination treatment selectively targets the oncogenic mIDH-PINl-RARa node.

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Abstract

La présente invention concerne des méthodes et des kits se rapportant au traitement du cancer par l'administration de trioxyde d'arsenic, d'un composé d'acide rétinoïque ou d'une association de trioxyde d'arsenic et d'un composé d'acide rétinoïque.
PCT/US2018/063666 2017-12-01 2018-12-03 Trioxyde d'arsenic et composés d'acide rétinoïque pour le traitement de troubles associés à l'idh2 WO2019109095A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20080119559A1 (en) * 2005-03-23 2008-05-22 Florida Atlantic University Treatment or prevention of cancer and precancerous disorders
US20140371176A1 (en) * 2011-03-25 2014-12-18 Glaxosmithkline Intellectual Property (No.2) Limited Cyclopropylamines as lsd1 inhibitors
US20160159771A1 (en) * 2013-07-11 2016-06-09 Agios Pharmaceuticals, Inc. Therapeutically active compounds and their methods of use
US20160222106A1 (en) * 2014-12-04 2016-08-04 Janssen Biotech, Inc. Anti-CD38 Antibodies for Treatment of Acute Myeloid Leukemia
US20170202799A1 (en) * 2014-07-17 2017-07-20 Beth Israel Deaconess Medical Center, Inc. Atra for modulating pin1 activity and stability
US20170246174A1 (en) * 2016-02-26 2017-08-31 Celgene Corporation Methods of treatment of malignancies

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20080119559A1 (en) * 2005-03-23 2008-05-22 Florida Atlantic University Treatment or prevention of cancer and precancerous disorders
US20140371176A1 (en) * 2011-03-25 2014-12-18 Glaxosmithkline Intellectual Property (No.2) Limited Cyclopropylamines as lsd1 inhibitors
US20160159771A1 (en) * 2013-07-11 2016-06-09 Agios Pharmaceuticals, Inc. Therapeutically active compounds and their methods of use
US20170202799A1 (en) * 2014-07-17 2017-07-20 Beth Israel Deaconess Medical Center, Inc. Atra for modulating pin1 activity and stability
US20160222106A1 (en) * 2014-12-04 2016-08-04 Janssen Biotech, Inc. Anti-CD38 Antibodies for Treatment of Acute Myeloid Leukemia
US20170246174A1 (en) * 2016-02-26 2017-08-31 Celgene Corporation Methods of treatment of malignancies

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