WO2020069187A1

WO2020069187A1 - Combination therapy for acute myeloid leukemia

Info

Publication number: WO2020069187A1
Application number: PCT/US2019/053250
Authority: WO
Inventors: Yang Shi; Abhinav Dhall; Barry M. ZEE; Jiexian MA
Original assignee: The Children's Medical Center Corporation
Priority date: 2018-09-28
Filing date: 2019-09-26
Publication date: 2020-04-02

Abstract

Described herein are drug combinations that show efficacy against non-APL acute myeloid leukemia (AML) with relatively little toxicity. Using differentiation therapies applied in the APL context, lysine demethylase 1 (LSD1) inhibitors are combined in therapy with differentiation agents, demonstrating enhanced differentiation and reduced proliferation.

Description

COMBINATION THERAPY FOR ACUTE MYELOID LEUKEMIA

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with Government support under Grant Nos. CA210104 and All 30737 awarded by the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The technology described herein relates to combination therapies for leukemia.

BACKGROUND

Acute myeloid leukemia (AML) is a deadly blood cancer that includes many distinct types. For a specific type of AML called APL, doctors prescribe a differentiation-based therapy that allows the majority of APL patients to live past five years of diagnosis with minimal toxic side effects.

Unfortunately, the most common types of AML are not APL, and for these patients no such differentiation-based therapy exists. Rather, relatively toxic chemotherapies are the standard approaches. If a differentiation-based therapy could be effective for non-APL AML, it would offer a strategy for treating patients that cannot otherwise tolerate standard chemotherapies. There is a need in the art for differentiation-based therapy for non-APL AMLs.

Described herein are drug combinations that show efficacy against AML cells in tissue culture with relatively little toxicity using multiple independent techniques. This includes combinations with (a) an LSD1 inhibitor with (b) a second agent comprising (i) an inhibitor of glycogen synthase kinase 3 (GSK-3) or (ii) an anthelmintic for the treatment of leukemia in a subject in need thereof. This combination is applicable to all subsets of AML excluding APL; the combination is effective in chemotherapy resistant, refractory or relapsed AML.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Preliminary studies with Pyrvinium and LSDli in HOXA9 and HOXB8 cell lines with GFP readout. The bar graphs in this figure show the synergistic effect of the combination of Pyrvinium (PP) with GSK-LSD1 (LSDli) in reducing the proliferation of AML cells. The figure shows the effect of increasing doses of PP (50 - 250 nM) in the presence and absence of 100 nM LSDli on mouse granulocyte monocyte progenitor (GMP) cells overexpressing the transcription factors HoxA9 or HoxB8 recapitulate AML disease. Black bars in the Alive columns show the percentage of cells alive at the end of 5 days of treatment with the combination of PP and LSDli drugs as measured through flow cytometry analysis (Intellicyt™ iQue). Black bars in the GFP+ columns show the percentage of cells expressing GFP protein (hence undergoing therapeutically relevant differentiation, see Scadden Cell 2016 paper) at the end of 5 days of treatment with the combination of PP and LSDli drugs as measured through flow cytometry analysis. Synergy was observed in the dose range of 100 nM LSD1 and 100 nM PP. The data represents an average of three independent in vitro treatment and flowcytometry analyses.

Figure 2. Dosage studies. The bar graphs in this figure show the synergistic effect of the combination of Pyrvinium (PP) with GSK-LSD1 (LSDli) in reducing the proliferation of cells in three different human cells lines of AML. The figure shows the effect of increasing doses of PP (5 - 25 nM) in the presence and absence of 50 nM LSDli on HL60, MOM13 and THP1 cells lines of AML. Fig 2A. Gray bars show the percentage of cells alive at the end of 5 days of treatment with the combination of PP and LSDli drugs as measured through flow cytometry analysis (Intellicyt™ iQue). Fig 2B. Gray bars show the percentage of cells expressing Cdl lb surface marker (hence undergoing therapeutically relevant differentiation) at the end of 5 days of treatment with the combination of PP and LSDli drugs as measured through flow cytometry analysis. The combination was highly effective in reducing the proliferation of leukemic cells in all three cell lines which represent different types and subtypes of human AML. The data represents an average of three independent in vitro treatment and flowcytometry analyses.

Figure 3. Additional dosage studies. The bar graphs in this figure show the synergistic effect of the combination of Pyrvinium (PP) with GSK-LSD1 (LSDli) in reducing the proliferation of cells in three different human cells lines of AML. The figure shows the effect of increasing doses of PP (5 - 25 nM) in the presence and absence of 100 nM LSDli on HL60, MOM13 and THP1 cells lines of AML. Fig 3A. Gray bars show the percentage of cells alive at the end of 5 days of treatment with the combination of PP and LSDli drugs as measured through flow cytometry analysis (Intellicyt™ iQue). Fig 3B. Gray bars show the percentage of cells expressing Cdl lb surface marker (hence undergoing therapeutically relevant differentiation) at the end of 5 days of treatment with the combination of PP and LSDli drugs as measured through flow cytometry analysis. The combination was highly effective in reducing the proliferation of leukemic cells in all three cell lines which represent different types and subtypes of human AML. The data represents an average of three independent in vitro treatment and flowcytometry analyses.

Figure 4. Preliminary studies with LY0290340 and LSDli in HOXA9 and HOXB8 cell lines with GFP readout. The bar graphs in this figure show the synergistic effect of the combination of Ly20903 l4 (Ly) with GSK-LSD1 (LSDli) in reducing the proliferation of AML cells. The figure shows the effect of increasing doses of Ly (50 - 250 nM) in the presence and absence of 100 nM LSDli on mouse granulocyte monocyte progenitor (GMP) cells overexpressing the transcription factors HoxA9 or HoxB8 recapitulate AML disease (see Scadden Cell 2016 paper). Black bars in the Alive columns show the percentage of cells alive at the end of 5 days of treatment with the combination of Ly and LSDli drugs as measured through flow cytometry analysis (Intellicyt™ iQue). Black bars in the GFP+ columns show the percentage of cells expressing GFP protein (hence undergoing therapeutically relevant differentiation, see Scadden Cell 2016 paper) at the end of 5 days of treatment with the combination of Ly and LSDli drugs as measured through flow cytometry analysis. Synergy was observed in the dose range of 100 nM LSD1 and 250 nM Ly. The data represents an average of three independent in vitro treatment and flowcytometry analyses.

Figure 5. Dosage studies. The bar graphs in this figure show the synergistic effect of the combination of Ly20903 l4 (Ly) with GSK-LSD1 (LSDli) in reducing the proliferation of cells in three different human cells lines of AML. The figure shows the effect of increasing doses of Ly (5 - 25 nM) in the presence and absence of 50 nM LSDli on HL60, MOM13 and THP1 cells lines of AML. Fig 2A. Gray bars show the percentage of cells alive at the end of 5 days of treatment with the combination of Ly and LSDli drugs as measured through flow cytometry analysis (Intellicyt™ iQue). Fig 2B. Gray bars show the percentage of cells expressing Cdl lb surface marker (hence undergoing therapeutically relevant differentiation) at the end of 5 days of treatment with the combination of Ly and LSDli drugs as measured through flow cytometry analysis. The combination was highly effective in reducing the proliferation of leukemic cells in all three cell lines which represent different types and subtypes of human AML. The data represents an average of three independent in vitro treatment and flowcytometry analyses. Figure 6. Combination studies with LY0290340 and LSDli in doxorubicin sensitive cell lines, day 3. This figure shows the effect of treatment of chemotherapy sensitive and chemotherapy resistant cells with a combination of 100 nM LSDli and 100 nM Ly. Fluorescence microscopy was used to detect ER-HOXA9 AML cells expressing GFP (hence undergoing differentiation) on Day 3 (D3) of drug treatment. (A) Top panel shows very few Doxorubicin sensitive/responsive cells as expressing GFP whereas Doxorubicin resistant cells (bottom panel) are highly responsive to a combination of LSDli and Ly treatment. (B) The percentage of apoptotic cells was measured using the Annexin V staining kit (Thermo Fisher Scientific) and the percentage of cells undergoing differentiation was measured by counting GFP+ cells using flow cytometry. Doxorubicin sensitive cells showed early upregulation of apoptosis (day 3) whereas Doxorubicin resistant cells showed lower rate of apoptosis and higher rate of differentiation.

Figure 7. Combination studies with LY0290340 and LSDli in doxorubicin sensitive cell lines, day 5. This figure shows the effect of treatment of chemotherapy sensitive and chemotherapy resistant cells with a combination of 100 nM LSDli and 100 nM Ly. Fluorescence microscopy was used to detect ER-HOXA9 AML cells expressing GFP (hence undergoing differentiation) on Day 5 (D5) of drug treatment. (A) Top panel shows very few Doxorubicin sensitive/responsive cells as expressing GFP whereas Doxorubicin resistant cells (bottom panel) are highly responsive to a combination of LSDli and Ly treatment. (B) The percentage of apoptotic cells was measured using the Annexin V staining kit (Thermo Fisher Scientific) and the percentage of cells undergoing differentiation was measured by counting GFP+ cells using flow cytometry. Doxorubicin resistant cells showed larger upregulation of apoptosis and differentiation when treated with a combination of LSDli and Ly compared to single agents.

Figure 8. Combination studies with LSDli (L), 6-mercaptopurine (M) and cerulenin (C) in HOXA9 cell line. These data show the in vivo efficacy of using LSDli and Ly as a combination therapy against AML. A) 100,000 HOXA9/MEIS1 overexpressing cells were injected in 8 weeks old (n=5) C57BL/6 within 24 hours of irradiating them with 3.5 Gy gamma irradiation. Leukemia burden was monitored by whole body live animal bioluminescence imaging using IVIS™. Treatment with 0.1 mg/kg body weight LSDli or 0.25 mg/kg body weight Ly or a combination of both was performed for 7 days starting day 9 post injection of leukemia forming cells through intraperitoneal injections. Treatment was stopped on day 15 and leukemic burden was continued to be measured. B) Leukemia burden as measured by bioluminescence in vehicle (DMSO) treated or LSDli and Ly combination treated mice. Combination therapy successfully reduced the rate of leukemia progression. Changing the doses of L, M, and C reveals that by 4 days of incubation, ER-HoxA9 cells have enhanced differentiation & reduced proliferation compared to no drug or single drugs as measured by flow cytometry. L = GSK-LSD1, M = 6-mercaptopurine, C = Cerulenin.

Figure 9. Wright-Giemsa staining of HOXA9, day 4. Morphology features based on nuclear lobulation, nucleusxytoplasmic ratio, cytoplasmic vacuoles, cytoplasmic color, and plasma membrane smoothness, are consistent with early myeloid differentiation upon LMC.

Figure 10. Combination studies with LSDli (L), 6-mercaptopurine (M) and cerulenin (C) in U937 cell line. Proliferation and differentiation of U937 cells is significantly reduced and enhanced respectively with the LMC combo compared to no drug or single drug alone. Morphology of U937 cells (next slide) suggests LMC combo induces early myeloid differentiation (less mature than ATRA incubation alone).

Figure 11. Wright-Giemsa staining of U937, day 4.

Figure 12. Schematic of GMP differentiation screen. (A) ER-HOXA9 cells are murine GMPs with a lysozyme-GFP (Lyz2-GFP) reporter that is activated upon myeloid maturation. Differentiation is induced by HOXA9 inactivation or, for the Inventors’ screen, in spite of HOXA9 nuclear localization. (B) Differentiation response of ER-HOXA9 to various drugs at 10 concentrations (dots) combined with GSK-LSD1 for 5 days. The two highlighted positive hits were GSK-LSD1 with cerulenin (LC) and GSK-LSD1 with 6- mercaptopurine (LM). (C) Structure of GSK-LSD1, 6-mercaptopurine, and cerulenin.

Figure 13. Differentiation response of LMC triple drug combination. ER-HOXA9 cells were incubated with DMSO (Veh), GSK-LSD1 (LSDli), 6-mercaptopurine (6MP), cerulenin (CER), and various double (LM and LC) and triple (LMC) combinations for various days. Viability (black circles, near top of panel) and differentiation (white circles) were measured by flow cytometry based on forward/side scatter parameters and Lyz2-GFP response respectively and reported as percentages of parent population. Circles represent averages of 3-6 technical replicates per condition, while error bars represent standard deviations. Figure 14. Dose response of double drug combos. ER-HOXA9 cells were incubated with GSK-LSD1 (LSDli) and 6-mercaptopurine (6MP) or GSK-LSD1 and cerulenin (CER) as double drug combos across multiple doses for 4 days. Differentiation as measured by Lyz2-GFP signal was assayed by flow cytometry. Figure 15. Dose response of triple drug combo. (A) ER-HOXA9 cells were incubated with GSK-LSD1 (L), 6-mercaptopurine (M), and cerulenin (C) across multiple doses for 4 days. Viability and differentiation as measured by Lyz2-GFP signal were assayed by flow cytometry. (B) ER-HOXA9 cells were incubated with TCP (T), M, and C across multiple doses for 4 days. Viability and differentiation was similarly measured as in A.

Figure 16. Morphological changes reflect LMC induction of differentiation. (A) ER- HOXA9 cells were treated with vehicle or drugs for 4 days and stained with Wright-Giemsa. Cells treated with LMC combination display morphologies consistent with murine neutrophils (middle row) and monocytes (bottom row). Bright-field images were acquired at 120^c magnification. Bar represents 10 microns. (B) Cells treated with vehicle or drugs were classified as immature progenitors or mature effectors in blinded fashion. Note some images in panel A are copied from Figure 9.

Figure 17. Proliferation response induced by LMC combo. Mouse and human cells (lymphoid lineage marked with *) treated with GSK-LSD1 (L), 6-mercaptopurine (M), and cerulenin (C) at two doses (+ and ++ differ by lO-fold concentration) for 4 days. Live cell number per treatment was normalized to cell number in vehicle for proliferation estimate, represented by circles of varying diameters.

Figure 18. Differentiation and proliferation response induced by LMC combo. (A) U937 cells (human histiocytic sarcoma) were treated with vehicle or drugs for 4 days and stained with Wright-Giemsa. Cells treated with LMC combination display morphologies consistent with early macrophages. Bright-field images were acquired at 120 magnification. Bar represents 10 microns. (B) U937 treated with various doses of LSDli and 6MP for 4 days. Differentiation as measured by CDl lb expression was assayed by flow cytometry. (C) U937 treated with vehicle or triple drug combo for 4 days were assayed for expression changes in select genes. Increase in fold expression of CDKN1B and GADD45A is consistent with reduction in proliferation, while increase in ALOX5AP and LYZ1 is consistent with macrophage maturation. Note some images in panel A are copied from Figure 11.

Figure 19. Potential Mechanism of LMC combo (A) Total RNA was purified from ER-HOXA9 cells treated with LMC drug combo. Integrity was tested by agarose gel electrophoresis. (B) Total RNA was processed for gene expression by qRT-PCR or LC-MS analysis of 6MP incorporation as thioguanine. (C) Gene expression suggests activation of primary and secondary neutrophil granule components in LMC treated cells. (D) Standard curve of thioguanine MRM response when compound is diluted in water. (E) Elevated thioguanine levels in RNA was observed in LMC treated cells over 6MP treated cells.

Figure 20. Pharmacokinetic (PK) analysis of LMC combo (A) MRM transitions of LSDli, 6MP, and CER drugs measured for QQQ quantification. (B) Standard curve of MRM-based quantification of the three drugs when pooled together and diluted in water. (C) Stability of LMC drugs when pooled together into mouse plasma and incubated for various durations at 37°C.

Figure 21. Pharmacodynamic (PD) analysis of LM double combo (A) Irradiated mice were treated for 4 days with vehicle, single drugs, or double drug combos. Peripheral blood was collected by facial vein bleed for blood type differential assay of white blood cells (WBC), hemoglobin (HGB), hematocrit (HCT), and platelet (PLT) levels. (B) Differential blood assay of individual treated mice.

Figure 22. Drug response of double LM combo of HOXA9:MEISl cells (A) Wright- Giemsa stained HOXA9:MEISl cells treated with vehicle or double drug combo LM for 4 days suggests partial monocytic differentiation. (B) Flow cytometry measurement of LM drug combination in inducing differentiation across various doses of HOXA9:MEISl cells.

Figure 23. Drug response of double LM combo of HOXA9:MEISl leukemia (A) Irradiated mice (female age 8 weeks) were injected (i.v., tail vein) with 5-7.5E5 HOXA9:MEISl cells and treated with consecutive doses of 0.l25mg/kg GSK-LSD1 and 0.5- l.Omg/kg 6-MP for 9 total injections. Peripheral blood and bioluminescent imaging analysis were performed to monitor disease progression. (B) Bioluminescent imaging analysis (photons/sec) of vehicle and LM-treated mice with leukemia at indicated timepoints following leukemia cell injection. (C) Difference in weight of spleen and liver between vehicle (blue) and LM-treated (red) mice with leukemia at endpoint suggests difference in infiltration of leukemia to extramedullary hematopoietic sites.

Figure 24. Dose response of triple drug combo among ER-HOXA9 clones. (A) Clone# 2 of ER-HOXA9 cells incubated with GSK-LSD1 (L), 6-mercaptopurine (M), and cerulenin (C) across multiple doses for 4 days. Viability and differentiation as measured by Lyz2-GFP signal were assayed by flow cytometry. (B) Clone #3 of ER-HOXA9 cells incubated with three drugs for 4 days. (C) Clone #4 of ER-HOXA9 cells incubated with three drugs for 4 days.

Figure 25. Synergistic modeling of differentiation response of GSK-LSD1 drug combos in ER-HOXA9 cells. (A) 3D surface landscape of delta scores calculated based on the ZIP synergy model for the LSDli and 6MP (LM) double drug combo. Delta scores below for ZIP and other models are provided for the entire 5x5 concentration matrix or for a 3x3 matrix subset that contains the maximum point of synergy. Scores > 0 (red) denote synergy, while scores = (white) or < 0 (green) denote additivity or antagonism respectively. (B) 3D surface landscape of delta scores calculated based on the ZIP synergy model for the GSK- LSD1 and CER (LC) double drug combo. See (A) for similar descriptions. Plots and calculations generated by SynergyFinder web application.

Figure 26. Synergistic modeling of differentiation response of double drug combos in ER-HOXA9 cells. (A) 3D surface landscape of delta scores calculated based on the ZIP synergy model for the TCP and 6MP (TM) double drug combo. Delta scores below for ZIP and other models are provided as an average or maximum for the entire 6x5 concentration matrix. Scores > 0 denote synergy, while scores = or < 0 denote additivity or antagonism respectively. (B) 3D surface landscape of delta scores calculated based on the ZIP synergy model for the TCP and CER (TC) double drug combo. See A for similar descriptions. Plots and calculations generated by SynergyFinder web application.

Figure 27. Reversibility of triple drug combo for ER-HOXA9. ER-HOXA9 cells treated with various doses of GSK-LSD1 (L), 6MP (M), and CER (C) for 2 days and assayed 0 days (D2, W0) or 2 (D2, W2) or 4 (D2, W4) days after drug washing.

Figure 28. Cell cycle analysis of ER-HOXA9 cells treated with drugs. Hoechst- stained ER-HOXA9 cells treated with (A) Vehicle, (B) GSK-LSD1, (C) 6MP, (D) CER, and (E) LMC triple combo for 4 days were analyzed by flow cytometry to determine cell cycle phase distribution. Note accumulation of cells in G2/M with 6MP treatment.

Figure 29. Hypoxanthine rescue of 6MP induction of ER-HOXA9 cells. ER-HOXA9 cells were treated with GSK-LSD1, 6MP, and hypoxanthine for 4 days and assayed for differentiation response. Note reduction in percentage of differentiated cells from condition 5 (6MP only) to condition 19 (6MP with hypoxanthine). Bars and error bars represent average of 6 technical replicates and standard deviation respectively.

Figure 30. These data show the in vivo efficacy of using LSDli and Ly as a combination therapy against AML. (A) 100,000 HOXA9/MEIS1 overexpressing cells were injected in 8 weeks old (n=5) C57BL/6 within 24 hours of irradiating them with 3.5 Gy gamma irradiation. Leukemia burden was monitored by whole body live animal bioluminescence imaging using IVIS™. Treatment with 0.1 mg/kg body weight LSDli or 0.25 mg/kg body weight Ly or a combination of both was performed for 7 days starting day 9 post injection of leukemia forming cells through intraperitoneal injections. Treatment was stopped on day 15 and leukemic burden was continued to be measured. (B) Leukemia burden as measured by bioluminescence in vehicle (DMSO) treated or LSDli and Ly combination treated mice. Combination therapy successfully reduced the rate of leukemia progression.

SUMMARY OF THE INVENTION

Described herein is a method of treating acute leukemia, including administering to a subject in need thereof, a therapeutically effective amount of a lysine demethylase 1 (LSD1) inhibitor, and at least one differentiation agent. In other embodiments, the LSD1 inhibitor is a cyclopropylamine. In other embodiments, the cyclopropylamine is GSK LSD1, tranylcypromine, RN-l, or Seclidemstat (SP-2577). In other embodiments, the at least one differentiation agent is selected from the group consisting of: a GSK-3 inhibitor, Wnt/b- Catenin inhibitor, an anthelmintic, a nucleotide analog and a fatty acid synthase inhibitor. In other embodiments, the GSK-3 inhibitor is LY2090314. In other embodiments, the anthelmintic is pyrvinium. In other embodiments, the nucleotide analog is 6-mercaptopurine. In other embodiments, the fatty acid synthase inhibitor is cerulenin. In other embodiments, the method includes two differentiation agents. In other embodiments, the method includes three differentiation agents. In other embodiments, the acute leukemia is non-acute promyelocytic leukemia (APL) acute myeloid leukemia. In other embodiments, the acute leukemia is acute promyelocytic leukemia (APL). In other embodiments, administering to a subject in need thereof includes one or more of: injection, intravenous, oral, enteral, topical and inhalation.

Described herein is a pharmaceutical composition, including a lysine demethylase 1 (LSD1) inhibitor, at least one differentiation agent, and a pharmaceutically acceptable carrier. In other embodiments, the LSD1 inhibitor is a cyclopropylamine. In other embodiments, the cyclopropylamine is GSK LSD1, tranylcypromine, RN-l, or Seclidemstat (SP-2577). In other embodiments, the at least one differentiation agent is selected from the group consisting of: a GSK-3 inhibitor, Wnt/p-Catenin inhibitor, an anthelmintic, a nucleotide analog and a fatty acid synthase inhibitor. In other embodiments, the GSK-3 inhibitor is LY2090314. In other embodiments, the anthelmintic is pyrvinium. In other embodiments, the nucleotide analog is 6-mercaptopurine. In other embodiments, the fatty acid synthase inhibitor is cerulenin. DETAILED DESCRIPTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al, Remington: The Science and Practice of Pharmacy 22^nd ed. , Pharmaceutical Press (September 15, 2012); Homyak et al, Introduction to Nanoscience and Nanotechnology , CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3^rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March’s Advanced Organic Chemistry Reactions, Mechanisms and Structure 7^th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3^rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2^nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7): 511 -9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al, Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6l62):323-7.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

Acute myeloid leukemia (AML) is a complex hematologic malignancy in need of better treatments. AML occurs in all age groups, but most frequently among the elderly. Currently, most mainstay therapeutic options for AMLs require cytotoxic chemotherapeutic regimens with or without subsequent bone marrow transplantation. Chemotherapy though is often not suitable for patients with lower physiologic reserves, especially the elderly.

An attractive and longstanding alternative with relatively milder side effects is differentiation therapy. At the forefront of this approach is all-trans retinoic acid (ATRA), which is essentially curative for patients with acute promyelocytic leukemia (APL). The promise of extending similar regimens to non-APL AML is largely unfulfilled despite inhibitors of mutant IDH1/2 showing potential. To address the lack of a tractable model for characterizing differentiation therapy, a murine granulocytic-macrophage precursor (GMP) cell line designated ER-HoxA9 recapitulates: 1) non-responsiveness of non-APL AML to ATRA and 2) differentiation blockade reinforced by overexpression of the transcription factor HoxA9 seen in approximately 70% of AML patients. Using this line and other tissue culture models, the Inventors found that inhibition of LSD1 (using the irreversible inhibitor GSK-LSD1 or LSDli) is able to induce myeloid differentiation and reduce proliferation albeit with low efficiency. Thus, the Inventors screened for additional drugs that enhanced the efficacy of LSDli in inducing differentiation of ER-HoxA9 cells.

The Inventors discovered and validated multiple drug candidates including: 1) LY2030914, 2) Pyrvinium palmoate, 3) 6-mercaptopurine, and 4) cerulenin that when co- treated with LSDli, induces differentiation and reduces proliferation of AML cells to a greater extent than when the drugs are used alone. Additionally, the Inventors compared these drug combinations between chemotherapeutics-sensitive and chemotherapeutics-resistant cells. The Inventors observe that these combos exert even stronger ability to induce differentiation and apoptosis in chemotherapy resistant cells. The Inventors believe that combinations of the aforementioned drugs together with LSDli represent promising tools to induce AML differentiation and are planning to test if these drug combinations show efficacy against AML cells in mouse animal disease models.

More specifically, differentiation arrest is a clinically relevant hallmark of acute myeloid leukemia (AML) as describe. An excess of proliferating and functionally impaired myeloid progenitors as well as a deficit of mature effectors broadly characterize these cancers. Consequently, normal hematopoietic and bone marrow functions are diminished. A longstanding attractive treatment option for AMLs is differentiated-based therapy, where the goal is to induce committed maturation of malignant cells. With the exception of select agents, such as all-trans retinoic acid (ATRA) for acute promyelocytic leukemia (APL) and enasidenib for mutant IDH2 AMLs, the promise of extending differentiation-based therapy broadly to AMLs remains largely unfulfilled. Rather most mainstay therapies rely on cytotoxic chemotherapy as a frontline measure to eliminate the majority of tumors followed by bone marrow transplantation to reconstitute the hematopoietic system and to induce graft- versus-host dependent elimination of residual cancer. Yet for elderly patients who have lower physiologic reserves, chemotherapies can be especially taxing and are commonly substituted with palliative care, resulting in 5-year survival rate for patients age 65-74 to remain relatively poor for men (7%) and women (9%). It is for these and other AML patients where non-ATRA approaches to differentiation-based therapy would be helpful.

An obstacle for investigating mechanisms to bypass the AML differentiation blockade is the lack of a tractable tissue culture system. Recently a murine granulocyte-monocyte progenitor (GMP) cell line was developed that recapitulates both ATRA non-responsiveness and blocked differentiation due to constitutive HOXA9 expression, a genetic feature observed in nearly half of AML patients. HOXA9 is a homeodomain-containing transcription factor that positively and negatively regulates proliferation-associated and differentiation-associated genes respectively. Using these GMPs the Inventors identified inhibition of LSD1 increases spontaneous differentiation in spite of HOXA9 activity. LSD1 (also annotated as KDM1A) is an FAD-dependent lysine demethylase that converts histone H3 lysine 4 mono- and di- methylation (H3K4mel and me2) to the unmodified state (H3K4un). Together with non lineage specific and lineage-specific binding partners such as RCOR1 and GFIlb respectively, LSD1 promotes a repressed chromatin environment. Inhibition blocks LSD1 demethylase activity and disrupts protein complex association, thereby tipping the balance for other enzymes to methylate H3K4. Recent findings suggest chromatin readers that recognize genomic regions decorated with these methyl post-translational modifications (PTMs) can promote a more accessible state for C/EBRa and PU.1 binding. The murine GMP findings suggest that epigenetic regulators may provide a pharmacologically attractive target to bypass the AML differentiation blockade.

However there exist major obstacles associated with epigenetic targeting drugs. The essential role of LSD 1 in normal myeloid differentiation means that inhibitors risk targeting non-malignant cells. Indeed, a recent AML Phase 1 clinical trial (NCT02177812) using an LSD1 inhibitor was terminated in part due to toxicity issues. In addition, the efficiency of differentiation is low; using the aforementioned GMP line, treatment with LSD1 inhibitor induces approximately 10% of live cells to differentiate. Thus, to improve the clinical potential of such drugs, the Inventors used the aforementioned GMP line to identify other compounds that could enhance the efficacy of inducing differentiation. The Inventors identified two compounds: 6-mercaptopurine (6MP) and cerulenin (CER), that together with the LSD1 inhibitor GSK-LSD1 (LSDli) as 2-plex and 3-plex combinations induce differentiation and reduce proliferation to a greater extent than the individual drugs when used alone. The Inventors propose these drugs target multiple pathways that converge on a metabolic and epigenetic state bypassing the AML differentiation blockade.

Described herein is a method of treating leukemia, including administering to a subject in need thereof, a therapeutically effective amount of a demethylase inhibitor, and at least one differentiation agent. In other embodiments, the methods treats acute leukemia, including administering to a subject in need thereof, a therapeutically effective amount of a flavin-dependent demethylase inhibitor, and at least one differentiation agent. In other embodiments, the method treats acute leukemia, including administering to a subject in need thereof, a therapeutically effective amount of a lysine demethylase 1 (LSD1) inhibitor, and at least one differentiation agent. In other embodiments, the LSD1 inhibitor is a cyclopropylamine. In other embodiments, LSD1 inhibitors include MAO inhibitors such as argyline, tranylcypromine and phenelzine, baicalin, resveratrol and geranylgeranoic acid (GGA), peptide-based, polyamine-based, metal complex inhibitors, among others. In other embodiments, the cyclopropylamine is GSK LSD1, tranylcypromine, RN-l, or Seclidemstat (SP-2577). In other embodiments, the at least one differentiation agent is selected from the group consisting of: a GSK-3 inhibitor, Wnt/p-Catenin inhibitor, an anthelmintic, a nucleotide analog and a fatty acid synthase inhibitor. In other embodiments, the GSK-3 inhibitor include a metal ion such as lithium ion, valproic acid, iodotubercidin, naproxen and cromolyn, famotidine, curcumin, olanzapine, CHIR99021 and pyrimidine derivatives. In other embodiments, the GSK-3 inhibitor is LY2090314. In other embodiments, the anthelmintic is pyrvinium. In other embodiments, the nucleotide analog is 6-mercaptopurine. In other embodiments, the fatty acid synthase inhibitor is a HMG-CoA synthetase activity inhibitor. In other embodiments, the fatty acid synthase inhibitor is cerulenin. In other embodiments, the method includes two differentiation agents. In other embodiments, the method includes three differentiation agents. In other embodiments, the acute leukemia is non-acute promyelocytic leukemia (APL) acute myeloid leukemia. In other embodiments, the acute leukemia is acute promyelocytic leukemia (APL). In other embodiments, administering to a subject in need thereof includes one or more of: injection, intravenous, oral, enteral, topical and inhalation. In other embodiments, the differentiation agent is capable of inducing differentiation in myeloblasts. In other embodiments, the administration of the demethylase inhibitor, and at least one differentiation agent is simultaneous. In other embodiments, the administration of the demethylase inhibitor, and at least one differentiation agent is sequential. In other embodiments, the method of treatment is primary therapy. In other embodiments, the method of treatment is adjuvant to standard therapy.

Described herein is a pharmaceutical composition, including a demethylase inhibitor, at least one differentiation agent, and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition includes a lysine demethylase 1 (LSD1) inhibitor, at least one differentiation agent, and a pharmaceutically acceptable carrier. In other embodiments, the LSD1 inhibitor is a cyclopropylamine. In other embodiments, LSD1 inhibitors include MAO inhibitors such as argyline, tranylcypromine and phenelzine, baicalin, resveratrol and geranylgeranoic acid (GGA), peptide-based, polyamine-based, metal complex inhibitors, among others. In other embodiments, the cyclopropylamine is GSK LSD1, tranylcypromine, RN-l, or Seclidemstat (SP-2577). In other embodiments, the at least one differentiation agent is selected from the group consisting of: a GSK-3 inhibitor, Wnt/b- Catenin inhibitor, an anthelmintic, a nucleotide analog and a fatty acid synthase inhibitor. In other embodiments, the GSK-3 inhibitor includes a metal ion such as lithium ion, valproic acid, iodotubercidin, naproxen and cromolyn, famotidine, curcumin, olanzapine, CHIR99021 and pyrimidine derivatives. In other embodiments, the GSK-3 inhibitor is LY2090314. In other embodiments, the anthelmintic is pyrvinium. In other embodiments, the nucleotide analog is 6-mercaptopurine. In other embodiments, the fatty acid synthase inhibitor is a HMG-CoA synthetase activity inhibitor. In other embodiments, the fatty acid synthase inhibitor is cerulenin. In other embodiments, the pharmaceutical composition includes two differentiation agents. In other embodiments, the pharmaceutical composition includes three differentiation agents.

Example 1

Drug pathway

LY2090314 or LY is a potent GSK-3 inhibitor for GSK-3a/p, it selectively inhibits the activity of GSK-3 by interrupting ATP binding. LY2090314 is able to stabilize 13- catenin, some clinical trial about LY2090314 are on the way (NCT01214603).

Pyrvinium is an anthelmintic effective for pinworms, which can inhibit androgen receptor as well as Wnt/l3-Catenin. Example 2

Cell lines

ER-HoxA9 and ER-Hoxb8 are myeloid progenitor cell lines, which over-express HOXA9 and HoxB8 gene that exist in 70%-80% AML subtypes. These cells are also transfected with GFP-Lysozyme gene, if differentiation happens, the Inventors can detect GFP expression by flow cytometry or fluorescent microscopy.

The Inventors discovered Pyrvinium and LY2090314, each combined with LSD1 inhibitor (GSK-LSD1), have a strong effect to induce differentiation. In Figs. 1-5, black bars that show the combined effect of LSD1 with Pyrvinium and LY2090314 in reducing the number of Alive cells and Increasing the percentage of GFP (a readout for differentiation).

Example 3

The Inventors tested the potential Lsdl inhibitor and LY2090314 (combo) in chemotherapeutics sensitive and resistant cells (incubation for several days, checked on d3 and d5), The Inventors found this combo have synergy with chemotherapeutics to induce differentiation and apoptosis, especially in resistant cell lines, the combo induce stronger differentiation. Results are shown in Figs. 6-7.

Example 4

Drug pathways part 2

6-Mercaptopurine or M is a synthetic nucleotide analog developed by Gertrude Elion (Nobel 1988) and her team. M is in active clinical use for treating acute lymphoid leukemia and other diseases (trade name Purinethol), where in the former, the drug is given daily up to years to maintain remission (such regimen contrasts with other chemotherapeutic agents like cytarabine). Recent studies find potential efficacy of M for acute myeloid leukemia. Regarding mechanism, M resembles guanine and is incorporated into genomic DNA as deoxythioguanine (dTG). Nonenzymatic methylation of dTG generates a modified form that preferentially basepairs with thymine instead of cytosine and activates the mismatch repair pathway. This sequence of events may explain the latency of M efficacy in cells and in animals.

Cerulenin or C is a natural fungal product initially purified by Satoshi Omura (Nobel 2015) and his team. C is not currently in clinical use. Regarding mechanism, most studies report C inhibits the fatty acid synthase complex, thereby reducing the generation of palmitate. As palmitate is a building block for phospholipid synthesis, a secondary effect of C is lower phospholipid synthesis and reduced cell proliferation. The immense demand for fatty acids by cancer cells (e.g. to generate plasma membrane) may explain the much shorter latency of response to C than to M in the Inventors’ cell culture experiments.

Example 5

Observations

• Changing the doses of L, M, and C reveals that by 4 days of incubation, ER-HoxA9 cells have enhanced differentiation & reduced proliferation compared to no drug or single drugs as measured by flow cytometry. L = GSK-LSD1, M = 6-mercaptopurine, C = Cerulenin. Results are shown in Fig. 8.

• Morphology features based on nuclear lobulation, nucleus: cytoplas ic ratio, cytoplasmic vacuoles, cytoplasmic color, and plasma membrane smoothness, are consistent with early myeloid differentiation upon LMC. Results are shown in Fig. 9.

• Proliferation and differentiation of U937 cells is significantly reduced and enhanced respectively with the LMC combo compared to no drug or single drug alone. Results are shown in Fig. 10.

• Morphology of E1937 cells (next slide) suggests LMC combo induces early myeloid differentiation (less mature than ATRA incubation alone). Results are shown in Fig. 11

Example 6

Tissue culture

ER-HOXA9 GMP cells were a gift from Dr. David Sykes (Massachusetts General Hospital). For passaging, ER-HOXA9 cells were grown in RPMI-1640 (Invitrogen) media containing 10% volume heat inactivated fetal bovine serum (FBS, Gemini Bioproducts), 2% volume conditioned media from CHO cells that constitutively secrete stem cell factor (SCF), and 0.5 mM beta-estradiol. For all tissue culture experiments, cells were grown in a 37°C humidified incubator with 5% CO2.

For drug plating, 2.5>< l0³ (2,500) cells were seeded onto flat-bottom 96-well plastic plates (Genesee Scientific) in 200 pL of media. Drugs or vehicle at specified concentrations were added to wells such that the total vehicle concentration was the same for all wells (generally 0.2% volume DMSO). Example 7

Flow cytometry

On day of flow analysis, each well was re-suspended before transferring 100 pL of the cell suspension into round-bottom 96-well plastic plates (Greiner Bio-One) and centrifuging the round-bottom plate at 30-40 g’s for 1 minute. Approximately 70 pL of supernatant from the round-bottom plate was removed from each well, leaving the cell pellet concentrated in 30 pL media. If antibody was used for detection, approximately 10 pL of 4x concentrated antibody diluted in staining buffer (phosphate buffered saline with 2mM EDTA and 2mg/ml glucose) was added to each well for 40 pL volume.

Plates were analyzed on an iQue Screener Plus flow cytometer (Intellicyt) where wells were shake at 2200-2400 rpm and sipped for approximately 3-5 seconds with pump set at standard rate of 29 rpm. Live cells were gated by forward-scatter (FSC) and side-scatter (SSC) properties (i.e. high FSC, low SSC). Live singlets were gated by FSC height versus area and SSC height versus area properties. Finally LYZ-GFP dim cells were gated on BL1 channel (excitation: 488nm, emission bandwidth: 530/30nm); the gate was established such that vehicle only wells had approximately 1% GFPdim positive events of total live singlets. CDl lb positive cells were gated on RL1 channel (excitation: 640nm, emission bandwidth: 675/30nm); the gate was established such that negative control wells stained with APC- conjugated isotype control had less than 1% APC positive events of total live events.

Example 8

Wright-Giemsa staining

ER-HOXA9 cells in media were centrifuged with a cytospin (ThermoFisher) onto non-coated glass slides (VWR) at 800 rpm for 2 minutes at low acceleration. U937 cells were centrifuged by cytospin at 500 rpm (approximately 28 g’s) for 2 minutes. Slides were air dried for at least 15 minutes before staining in 100% volume buffered Wright-Giemsa (ThermoFisher) for 2 minutes, followed by staining in 20%:80% volume Wright- Giemsa:PBS (phosphate buffered saline, pH = 7.2) for 12 minutes. Slides were washed in reverse osmosis water for 5-10 times. Washed slides were air-dried at least 3 hours and glass coverslips were fixed on samples with DPX mountant (Fluka).

Example 9

Results The ER-HOXA9 line originates from GMPs isolated from the bone marrow of a transgenic mouse, where the endogenous lysozyme (LYZ2) promoter drives GFP expression (Lyz-GFP). During myeloid differentiation lysozyme expression is activated. These GMPs were transduced with a virus coding for a constitutively expressed ER-HOXA9 transgene. The estrogen receptor (ER) domain allows for posttranslational control of the fusion protein such that addition of estradiol maintains transgene nuclear localization (Figure 12A). Thus, while ER-HOXA9 cells are functionally not leukemias, these cells represent a leukemia-like differentiation arrest at the early GMP stage when cultured with estradiol to maintain proliferative potential and with SCF to maintain progenitor potential.

In the presence of estradiol, approximately 1% of live ER-HOXA9 cells are positive for Lyz-GFP (Figure 24). The Inventors attribute this percentage to spontaneous differentiation within the culture. When the Inventors treat cells with the LSD1 inhibitor GSK-LSD1, the Inventors find GSK-LSD1 induces myeloid differentiation in approximately 10% of live cells. The Inventors’ findings are consistent with previous reports describing a role of LSD1 in maintaining the AML differentiation blockade. GSK-LSD1 and similarly structured compounds such as tranylcypromine (TCP) covalently modify the cofactor FAD and irreversibly inhibit demethylase activity. The Inventors used GSK-LSD1 as the Inventors’ primary compound for inhibiting LSD1.

Example 10

Compound Screen

To identify drugs that, when combined with GSK-LSD1, induce myeloid differentiation to a greater extent than with GSK-LSD1 alone, the Inventors screened a chemical library comprised of drugs targeting lipid and nucleic acid metabolism. The Inventors screened cells in the presence of estradiol, and thus in spite of HOXA9 activation (Figure 12A). The Inventors reasoned the most promising compounds would be those that induced differentiation without significant cytotoxicity. Most compounds in the library did not significantly induce differentiation (Figure 12B). Among the compounds that induced differentiation at above 15% and maintained viability at above 60%, the two most promising compounds were 6-mercaptopurine (6MP or M) and cerulenin (CER or C) (Figure 12C). Interestingly two functionally related compounds, thioguanine and C75, that target nucleic acid and lipid metabolism respectively also induced differentiation of ER-HOXA9 cells. The Inventors decided to focus on the three compounds: GSK-LSD1, 6MP, and CER as a novel combinatorial strategy to induce myeloid differentiation.

Example 11

Time-dependency, dose dependency, stability of the differentiation response

To determine time-dependency of the differentiation response, the Inventors treated ER-HOXA9 cells with various combinations of GSK-LSD1, 6MP, and CER at a fixed concentration for one to four days (Figure 13). The Inventors find that the double combination of GSK-LSD1 and CER (LC) induces a measurable increase in differentiation as early as 1-2 days of treatment, while the double combination of GSK-LSD1 and 6MP (LM) induces a differentiation response at a later point at 3-4 days of treatment. When the Inventors combined the three drugs together as a triple combination (LMC), the Inventors observe a greater percentage of cells undergoing differentiation particularly at 3-4 days of treatment. At all time points, cell viability remained above 50%. Thus, the three drugs as double (LM and LC) and triple (LMC) combinations induce rapid differentiation of ER- HOXA9 cells.

To assay dose-dependency of the differentiation response, the Inventors treated ER- HOXA9 cells with various concentrations of GSK-LSD1, 6MP, and CER as double combinations (LM or LC) for 4 days (Figure 14). The Inventors find that both double combinations display enhanced differentiation with increasing concentration of drugs. Modeling the response with Bliss, Highest Single Agent (HAS), and Zero Interaction Potentcy (ZIP) models suggests that both LM and LC synergistically induce differentiation of ER-HOXA9 cells (Figure 25). The Inventors next determined the dose-dependency of the differentiation response at various concentrations of the three drugs as triple combination (LMC) (Figure 15A). Consistent with the double combination data, the Inventors find a narrow range of CER concentration induces a differentiation response when combined with LSDli or 6MP. The Inventors observe a synergistic and additive response when CER and 6MP respectively are paired with a different LSD1 inhibitor TCP (T) (Figure 15B) (Figure 26). These results suggest that LSD1 inhibition by different drugs (GSK-LSD1 or TCP) induces differentiation that can be enhanced by CER and 6MP co-treatment.

To assay stability of the differentiation response induced by the three drugs, the Inventors treated ER-HOXA9 cells with drugs at various concentration and combinations for 2 days (Figure 27). The Inventors then washed cells with drug-free media and cultured cells in absence of drugs for 2 or 4 additional days. Despite a high initial differentiation response of ER-HOXA9 cells to the GSK-LSD1 and CER double combination (conditions 14 and 15, Figure 27), the overall percentage of differentiated cells drops after 2 and 4 days of washout. In contrast, the double GSK-LSD1 and 6MP (LM, condition 11) and triple combination (LMC, condition 27) produces a more durable differentiation response even after 4 days of drug washout. This result suggests that differentiation response induce by the triple LMC combination is durable for a short term and that the stability of differentiation is dependent on 6MP treatment.

Example 12

Lysozyme activation

To confirm the differentiation response as measured by lysozyme activation with the flow cytometer, the Inventors stained ER-HOXA9 cells treated with the various drugs with Wright-Giemsa (Figure 16A) and imaged with brightfield microscopy. In vehicle treated cells, the Inventors observed morphologies consistent with GMPs, namely small and uniform -10 micron diameter, large nuclearxytoplasmic ratio, and basophilic cytoplasm. When cells are treated with the drug combinations, the Inventors observed morphologies consistent with mouse neutrophils and monocytes, namely a larger -15 micron diameter with pseudopod-like extensions, smaller nuclearxytoplasmic ratio, ring-shaped and indented nucleus, less basophilic cytoplasm, and presence of granules. Blinded counted of cells as either non- differentiated or differentiated cells reveals larger number of differentiated cells in the triple drug combination treatment group (Figure 16B). Thus, the morphological changes induced by LMC treatment are consistent with myeloid differentiation.

Example 13

Drug combination sensitivity to proliferation, differentiation To assay the broad sensitivity of LMC combination with respect to proliferation, the Inventors treated various human and mouse myeloid and lymphoid cells with combinations of GSK-LSD1, 6MP, and CER for 4 days (Figure 6). Proliferation was determined by counting the number of live cells using flow cytometry and normalizing the counts to number of cells of the respective vehicle treatment group. The Inventors find that generally, most lines do not proliferate slower with CER or GSK-LSD1 as single agents. In contrast, most lines grow slower with 6MP at the concentrations tested. Additionally, most lines tested show reduced growth with the triple LMC combination. To confirm the proliferation response, the Inventors measured DNA content for ER-HOXA9 cells treated with the drugs alone or as triple combination (Figure 28). Consistent with previous reports, the Inventors find 6MP alone or as a triple combination leads to accumulation of cells at the G2/M phase in cell cycle. No significant change in cell cycle distribution was observed with LSDli or CER treatment after 4 days. Thus, the LMC combination reduces the proliferation of cells in a largely 6MP-dependent manner.

To investigate the sensitivity of the drugs more closely with respect to differentiation, the Inventors focused on EG937 cells treated with vehicle or the triple LMC drug combination. EG937 cells originated from a histiocytic sarcoma. The Inventors find that the morphology of vehicle-treated EG937 cells is consistent with human monocytes, while the morphology of a fraction of LMC-treated EG937 cells is consistent with macrophages (Figure 18A). Maturation from monocyte to macrophage was confirmed by flow cytometry, where the double LM combination was sufficient to induce increased CDl lb expression (Figure 18B). Finally, the Inventors determined that LMC-treated U937 cells up-regulate expression of several gene markers for mature macrophages using qRT-PCR (Figure 18C). Thus, the drug combinations appear to induce myeloid differentiation in not only mouse but also human cell lines.

Example 14

Synergistic mechanisms

To explore potential mechanisms of how LMC induce differentiation in a synergistic manner, the Inventors assayed the metabolism and incorporation of 6MP as 6-thioguanosine into total RNA. The Inventors treated ER-HOXA9 cells with vehicle, drugs as single agents, and drugs as a triple combination for 4 days. After treatment, the Inventors isolated intact total RNA from cells (Figure 19A). The Inventors processed the samples for liquid chromatography-mass spectrometry (LC-MS) as well as qRT-PCR (Figure 19B). After confirming induction of genes associated with primary and secondary neutrophil granules (Figure 19C), the Inventors find that 6MP incorporation into total RNA was elevated with the triple LMC treated cells over 6MP-only treated cells (Figure 19E). While it is unclear how 6MP incorporation into total RNA relates to differentiation context, reports from other groups suggest that following 6MP incorporation into genomic DNA, spontaneous methylation of the sulfhydryl-group leads to preferential base-pairing with deoxythymidine instead of deoxycytosine. It is possible that a similar methylation event at the RNA level could lead to preferential base-pairing with uridine rather than cytidine, leading to defects in translation fidelity and efficiency that promote differentiation response.

Example 15

Specificity, stability and toxicity

To determine the specificity of the 6MP response, the Inventors treated ER-HOXA9 cells with GSK-LSD1, 6MP, and hypoxanthine (Figure 29). Hypoxanthine is a purine similar to 6MP but has an oxygen atom in place of the sulfur atom, and thus is metabolized via the salvage pathway into the normal guanosine and adenosine, rather than to thioguanosine. Hypoxanthine alone does induce differentiation. Interestingly, hypoxanthine is able to compete with 6MP in reducing the percentage of differentiated cells (compare condition 5 and 19 in Figure 29). To a lesser extent, hypoxanthine is able to attenuate the differentiation response of cells to the double LM combo. These results suggest that the response induced by 6MP depend on the same pathways targeted by hypoxanthine, namely the nucleotide salvage pathway.

To assay the stability of the drugs for animal studies, the Inventors developed and optimized pharmacokinetic (PK) assays to quantify the drugs (Figure 20A). To the Inventors’ knowledge, no PK assay was published for CER in the primary literature, although the Inventors found reports of an MRM transition for CER in an online technical report. The Inventors were able to confirm all three MRM transitions using the drugs as individual standards. The LC-MS methods were able to linearly quantify GSK-LSD1, 6MP, and CER across several logs of concentration (Figure 20B). The Inventors incubated the three drugs together in mouse plasma for several timepoints and performed relative quantification of the drugs. The Inventors find in order of increasing plasma stability: CER, 6MP, and GSK- LSD1, with CER having an approximate half-life of 57 minutes (Figure 20C). It is possible that CER is metabolized and converted in mouse plasma to another metabolite of a different mass that would not be detected by the MRM method. However, in light of these results and the earlier finding that the GSK-LSD1 and CER double combination (LC) is effective at a narrow dose range of CER, the Inventors focused on the LSDli and 6MP double combination (LM) for in vivo animal work.

To assay the toxicity of the drugs for animal studies, the Inventors performed pharmacodynamic (PD) assays to measure hematological indices in mice using a Sysmex instrument (Figure 21A). ETsing irradiated non-leukemic female C57BL6/J mice, the Inventors performed 4 sequential injections of GSK-LSD1 and 6MP via the intra-peritoneal route. Comparing the hematological indices before and after injection, the Inventors find that GSK-LSD1 injections at 0.5mg/kg reduce platelet counts (Figure 21B). Conversely, 6MP injections at 3.5mg/kg lead to no significant reduction in white blood cell, red blood cell, or platelet counts. Finally, when the Inventors injected both GSK-LSD1 and 6MP at the aforementioned concentrations together, the Inventors observe both platelet and white blood cell reduction. No mice in the treatment regimens had a weight reduction below 10% of the initial starting mass or displayed behavioral endpoints (e.g. lack of curiosity). Thus, these results suggest that while GSK-LSD1 and 6MP (LM) induce limited hematological toxicity, the combo is otherwise well tolerated in non-leukemic animals.

Example 16

Efficacy

The Inventors next proceeded to testing the efficacy of GSK-LSD1 and 6MP in a mouse HOXA9:MEISl leukemia model. These murine cells are genetically similar to ER- HOXA9 cells (i.e. constitutive HOXA9 expression) and similarly proliferate as myeloid progenitors. Interestingly, the morphology of HOXA9:MEISl cells (Figure 22A) suggest that these cells are at a more mature stage than ER-HOXA9 cells (see Figure 16A). Treating HOXA9:MEISl cells with GSK-LSD1 and 6MP double combination (LM) reveals morphology changes consistent with mouse monocytes. To identify the optimal GSK-LSD1 and 6MP concentration ranges for inducing CDl lb expression, the Inventors treated HOXA9:MEISl cells with various drug concentrations. The Inventors find that HOXA9:MEISl cells display a synergistic differential response at intermediate concentrations of LSDli and 6MP (Figure 22B). These data suggest an optimal GSK-LSD1 and 6MP concentration window to use for in vivo animal experiments.

To test the relevance of the GSK-LSD1 and 6MP double combination in reducing AML progression, the Inventors performed intravenous injection (i.v.) of HOXA9:MEISl cells into irradiated female C57BL6/J mice via tail vein (Figure 23A). HOXA9:MEISl cells were cultured with only SCF and no estradiol, and were confirmed to be actively doubling in the days prior to injection. Disease progression and drug toxicity were monitored non- invasively with both peripheral blood analysis of hematological indices and bioluminescent imaging (BLI). After confirming engraftment (approximately 1 week following injection), the Inventors began drug treatments with low doses of GSK-LSD1 and 6MP as two separate pulses. The Inventors find the concentration and duration of drug treatments was sufficient to avoid the hematological toxicity previously observed (Figure 22B). The Inventors observed a short-term reduction in disease burden, as measured by BLI quantification (Figure 23B), following the first pulse of treatment. However, in spite of a second pulse of drug treatment, BLI quantification reveals similar disease progression rates and time-to-endpoints between vehicle and combo treated mice. The Inventors sacrificed all mice and confirmed elevated white blood cell counts in peripheral blood. Interestingly, when the Inventors compared the masses of liver and spleen in mice, the Inventors find a trend where organ weights from combo treated mice were elevated above normal physiological ranges yet below organ weights from vehicle treated mice (Figure 23C). The reduced organ masses suggest less infiltration of the leukemia cells into these tissues. However, much larger sample sizes and further optimization of the GSK-LSD1 and 6MP combo regimen are needed to extend the time-to-endpoint of treated mice over vehicle mice.

The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments. Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, therapeutic agents used in combination for treatment of acute myeloid leukemia, dosing regimens and schedules associated with such treatment, methods of administration, among others. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term“about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

In some embodiments, the terms“a” and“an” and“the” and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.

Claims

nil CLAIMS

1. A method of treating acute leukemia, comprising:

administering to a subject in need thereof, a therapeutically effective amount of: a lysine demethylase 1 (LSD1) inhibitor, and

at least one differentiation agent.

2. The method of claim 1, wherein the LSD1 inhibitor is a cyclopropylamine.

3. The method of claim 2, wherein the cyclopropylamine is GSK LSD1, tranylcypromine, RN-l, or Seclidemstat (SP-2577).

4. The method of claim 1, wherein the at least one differentiation agent is selected from the group consisting of: a GSK-3 inhibitor, Wnt/p-Catenin inhibitor, an anthelmintic, a nucleotide analog and a fatty acid synthase inhibitor.

5. The method of claim 4, wherein the GSK-3 inhibitor is LY2090314.

6. The method of claim 4, wherein the anthelmintic is pyrvinium.

7. The method of claim 4, wherein the nucleotide analog is 6-mercaptopurine.

8. The method of claim 4, wherein the fatty acid synthase inhibitor is cerulenin.

9. The method of claim 1, wherein the method comprises two differentiation agents.

10. The method of claim 1, wherein the method comprises three differentiation agents.

11. The method of claim 1, wherein the acute leukemia is non-acute promyelocytic leukemia (APL) acute myeloid leukemia.

12. The method of claim 1, wherein the acute leukemia is acute promyelocytic leukemia (APL).

13. The method of claim 1, wherein administering to a subject in need thereof comprises one or more of: injection, intravenous, oral, enteral, topical and inhalation.

14. A pharmaceutical composition, comprising:

a lysine demethylase 1 (LSD1) inhibitor,

at least one differentiation agent, and

a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 14, wherein the LSD1 inhibitor is a

cyclopropylamine.

16. The pharmaceutical composition of claim 15, wherein the cyclopropylamine is GSK LSD1, tranylcypromine, RN-l, or Seclidemstat (SP-2577).

17. The pharmaceutical composition of claim 14, wherein the at least one differentiation agent is selected from the group consisting of: a GSK-3 inhibitor, Wnt/p-Catenin inhibitor, an anthelmintic, a nucleotide analog and a fatty acid synthase inhibitor.

18. The pharmaceutical composition of claim 17, wherein the GSK-3 inhibitor is

LY2090314.

19. The pharmaceutical composition of claim 17, wherein the anthelmintic is pyrvinium.

20. The pharmaceutical composition of claim 17, wherein the nucleotide analog is 6- mercaptopurine.

21. The pharmaceutical composition of claim 17, wherein the fatty acid synthase inhibitor is cerulenin.