Combination therapy with venetoclax and zotatifin for the treatment of cancer

Abstract

The invention features methods of treating a subject having a cancer (e.g., leukemia (e.g., acute myeloid leukemia), solid tumor, breast cancer, lung cancer, colorectal cancer, or pancreatic cancer) by administering venetoclax or a pharmaceutically acceptable salt thereof in combination with zotatifin or a pharmaceutically acceptable salt thereof.

Description

[0001] COMBINATION THERAPY WITH VENETOCLAX AND ZOTATIFIN FOR THE TREATMENT OF CANCER

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Patent Application Serial No. 63 / 559,748, filed on February 29, 2024. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

[0004] BACKGROUND OF THE INVENTION

[0005] This application relates to treating cancer.

[0006] Leukemias are cancers that start in cells that would normally develop into different types of blood cells. Most often, leukemia starts in early forms of white blood cells, but some leukemias start in other blood cell types. There are several types of leukemia, which are divided based mainly on whether the leukemia is acute (fast growing) or chronic (slower growing), and whether it starts in myeloid cells or lymphoid cells.

[0007] Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults, with a median age at diagnosis of 68 years. AML has other names, including acute myelocytic leukemia, acute myelogenous leukemia, acute granulocytic leukemia, and acute non-lymphocytic leukemia. The BCL2 inhibitor venetoclax is a common therapy for older patients with AML. However, due to the cellular heterogeneity of the disease, drug-resistant cell types often emerge and drive relapse, and the 5-year survival rate of AML patients is approximately 25%.

[0008] A first-in-class drug, zotatifin, inhibits the helicase activity of elF4A and curtails tumor growth in receptor tyrosine kinase-driven tumors. Zotatifin is currently being evaluated in phase 1 / 2 trials across multiple solid tumors. However, its effectiveness in blood cancers such as AML remains unexplored.

[0009] Accordingly, there exists a need for effective treatment options for cancer including leukemias such as AML.

[0010] SUMMARY OF THE INVENTION

[0011] In one aspect, the invention features a method for treating a subject having a cancer, the method including administering to the subject: a therapeutically effective amount of venetoclax, or a pharmaceutically acceptable salt thereof; and a therapeutically effective amount of zotatifin, or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the cancer is leukemia. In some embodiments, the leukemia is acute myeloid leukemia (AML). In some embodiments, the AML has mutations in FMS-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1 ), neuroblastoma RAS viral oncogene homolog (NRAS), or adenomatous polyposis coli (APC). In some embodiments, the subject previously received chemotherapy and is primary refractory. In some embodiments, the patient is resistant to venetoclax. In some embodiments, co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a synergistic effect. In some embodiments, co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a greater increase in progression-free survival (PFS) compared to administration of either agent alone. In some embodiments, co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a greater increase in duration of objective response (DOR) compared to administration of either agent alone.

[0013] In some embodiments, venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof are administered simultaneously.

[0014] In some embodiments, venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof are administered sequentially.

[0015] In some embodiments, venetoclax or a pharmaceutically acceptable salt thereof is administered orally as a tablet.

[0016] In some embodiments, zotatifin or a pharmaceutically acceptable salt thereof is administered intravenously, intraperitoneally, or subcutaneously.

[0017] In some embodiments, the subject is a human subject.

[0018] In another aspect, the invention features the use of a therapeutically effective amount of venetoclax or a pharmaceutically acceptable salt thereof in treating a patient who has been diagnosed with AML in combination with zotatifin or a pharmaceutically acceptable salt thereof.

[0019] In a further aspect, the invention features a method for treating a human subject having relapsed or refractory acute myeloid leukemia, comprising administering to the subject venetoclax in combination with zotatifin, wherein a dose of between about 50 mg to about 1500 mg of venetoclax is orally administered once daily to the subject.

[0020] In some embodiments, a dose of between about 0.001 mg / kg to about 0.1 mg / kg of zotatifin is intravenously administered once every week for 3 weeks to the subject.

[0021] In some embodiments, the AML of the subject includes an FLT3 mutation, NPM1 mutation, NRAS mutation, or APC mutation.

[0022] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0023] One advantage is that venetoclax and zotatifin, when administered together, are highly effective at lower doses than when either agent is administered alone, resulting in killing of cancer cells. Consequently, subjects receiving the combination of venetoclax and zotatifin may experience reduced toxicity from the combination therapy.

[0024] Definitions

[0025] As used herein, the term “about” refers to a value that is within 10% above or below the value being described.

[0026] The term “combination therapy” refers to a method of treatment including administering to a subject at least two therapeutic agents, optionally as one or more pharmaceutical compositions, as part of a therapeutic regimen. For example, a combination therapy may include administration of a single pharmaceutical composition including at least two therapeutic agents and one or more pharmaceutically acceptable carrier, excipient, diluent, or surfactant. A combination therapy may include administration of two or more pharmaceutical compositions, each composition including one or more therapeutic agent and one or more pharmaceutically acceptable carrier, excipient, diluent, or surfactant. The two or more agents may optionally be administered simultaneously (as a single or as separate compositions) or sequentially (as separate compositions). The effect of the combination of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof can be partially additive, wholly additive, or greater than additive (e.g., synergistic). In some embodiments, the effective amount of one or more of the therapeutic agents may be lower when used in a combination therapy than the therapeutic amount of the same therapeutic agent when it is used as a monotherapy, e.g., due to a synergistic effect of combining the two or more therapeutics.

[0027] As used herein, “duration of objective response” (DOR) is defined as the time from the first occurrence of a documented objective response to disease progression, or death from any cause, whichever occurs first.

[0028] As used herein, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of the compound of any of the compounds described herein. For example, pharmaceutically acceptable salts of any of the compounds described herein include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1 -19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable organic acid. As used herein, “disorder” is used in this disclosure to mean, and is used interchangeably with, the terms condition, disease, or illness, unless otherwise indicated.

[0029] As used herein, the term “primary refractory” refers to a subject who has previously received induction therapy for a disease and has not achieved a complete remission.

[0030] As used herein, “progression-free survival” (PFS) refers to the length of time during and after treatment during which the disease being treated (e.g., cancer, e.g., leukemia, e.g., AML) does not get worse. Progression-free survival may include the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease.

[0031] As used herein, the term “resistant to treatment” refers to a treatment of a disorder with a therapeutic agent, where the therapeutic agent is ineffective or where the therapeutic agent was previously effective and has become less effective over time. Resistance to treatment includes acquired resistance to treatment, which refers to a decrease in the efficacy of a treatment over a period of time where the subject is being administered the therapeutic agent. Acquired resistance to treatment may result from the acquisition of a mutation in a target protein, or in multiple proteins and genetic loci, that renders the treatment ineffective or less effective. Accordingly, resistance to treatment may persist even after cessation of administration of the therapeutic agent.

[0032] As used herein, the terms “subject” and “patient” are interchangeable and refer to a subject (e.g., a mammalian subject, e.g., a human subject) that receives treatment or diagnosis for a disorder as described herein.

[0033] As used herein, “therapeutically effective amount” refers to an amount of venetoclax or a pharmaceutically acceptable salt thereof or zotatifin or a pharmaceutically acceptable salt thereof sufficient to treat a cancer (e.g., leukemia, e.g., acute myeloid leukemia) as described herein in a subject (e.g., a human).

[0034] As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a disorder. The disorder may be a cancer, such as a leukemia (e.g., acute myeloid leukemia). Treatment may be administered to a subject who does not exhibit signs of the disease and / or to a subject who exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. For any term present in the art which is identical to any term expressly defined in this disclosure, the term's definition presented in this disclosure will control in all respects. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are described herein.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0038] FIGS. 1 A-1 E are a series of graphs showing EIF4A1 is highly expressed and can be targeted in primitive AML cell states. FIG. 1 A is a Sina plot showing expression of EIF4A1 in individual cells analyzed by single-cell RNA-sequencing on 5 healthy and 16 AML BM samples. P-values were calculated using the Wilcoxon test with Bonferroni correction. Healthy samples include HSC, Prog, and GMP. AML samples include HSC-like, Prog-like, and GMP-like. FIG. 1 B is a heatmap showing expression of elF4F complex members across 43 AML cell lines analyzed by bulk RNA-seq. FIG. 1 C is a scatter plot showing the reduction in AML cell viability after Zotatifin treatment (2.5 pM, five days, x-axis) and EIF4A1 expression (y-axis). Higher expression is associated with lower cell survival. FIG. 1 D is a series of line graphs showing half-maximal inhibitory concentration (IC50) curves for five AML cell lines treated for 24 or 72 hours with serial 10-fold titrated doses of Zotatifin from 0.1 pM to 10 pM. Results are representative of 2-4 independent experiments. Error bars represent mean±SD of n=3 technical replicates. For FIG. 1 D, shown, from top to bottom at the zotatifin concentration of 10000 nM, are the normalized viability observed for U937, HL-60, OCI-AML3, MOLM-13, and MV4-11 . FIG. 1 E is a bar plot showing the percent viability after treating primary BM cells from healthy donors and AML patients with Zotatifin (100 nM, 24 hours). Viability of flow cytometry-gated CD34+ HSPCs from healthy BM samples and aberrant CD34+CD33+ cells from AML BM samples was normalized to DMSO treatment (dotted line at 100%). Each bar represents a biological replicate (n = 8 healthy donors, n = 11 for AML patients) and data is shown as mean ± SD of n = 3 technical replicates. P-value was calculated by unpaired t-test. HSC: hematopoietic stem cell, Prog: progenitor, GMP: granulocyte-macrophage progenitor, HSPC: hematopoietic stem and progenitor cells, BM: bone marrow.

[0039] FIG. 2 is a heatmap showing expression of translation initiation factors in healthy and AML cell types by single-cell RNA-sequencing. Heatmap shows normalized expression of genes related to translation initiation in indicated cell types. Heatmaps were generated using published single-cell RNA- sequencing data on 16 AML and 5 healthy BM samples (van Galen et al., Cell. 2019;176:1265-81 .e24). Rows are ordered by the gene with the highest expression in HSC-like, Prog-like, and GMP-like AML cells. HSC: hematopoietic stem cell, Prog: progenitor, GMP: granulocyte-macrophage progenitor, ProMono: promonocyte, Mono: monocyte, eDC: classical dendritic cell.

[0040] FIGS. 3A-3I are a series of graphs and images showing that zotatifin downregulates AKT, metabolic and cell cycle pathways, upregulates inflammatory and apoptotic pathways, and decreases STAT-5, and MCL-1 in vitro. FIG. 3A is a diagram illustrates the principles of ribosome profiling. Ribo-seq captures ribosome-protected mRNA fragments (RPFs), while RNA-seq measures the whole transcriptome. The ratio of RPFs to total mRNA is used to calculate translation efficiency (TE). Created with Biorender. FIG. 3B is a series of metagene plots for the relative densities of RPFs across all transcripts following 4-hour treatment of MOLM-13 cells with control (DMSO, black) or Zotatifin at 100 nM (red). X-axis represents the distance from the start codon (left) and stop codon (right). FIG. 3C is a correlation plot showing the normalized enrichment scores of RNA-seq (x-axis) and Ribo-seq (y-axis) following GSEA (Hallmark pathways), with selected biological processes annotated and color-coded. Shape indicates significance (P<0.05). For a full list, refer to Table 2. FIG. 3D is a volcano plot showing the number of transcripts with upregulated or downregulated translation efficiency (TE) following Zotatifin treatment (P<0.05, 2-fold change). FIGS. 3E-3F are a series of box plots showing the 5’ UTR length (FIG. 3E) and 5’ UTR complexity (FIG. 3F) (more negative Gibbs free energy means more complex 5’ UTR) of transcripts, categorized by transcripts with significantly increased or decreased TE. FIG. 3G is a bar plot showing normalized enrichment scores of Hallmark pathways following GSEA based on changes in TE following Zotatifin treatment. FIGS. 3H-3I show Western blot analysis of target protein expression following 4 or 24 hours treatment of MOLM-13, MV4-11 and HL-60 cells with 100 nM Zotatifin. FIG. 3H is a series of representative images of 3-4 independent experiments. Each experimental condition was performed with technical duplicates. FIG. 3I is a series of graphs showing quantified protein expression across 3-4 independent experiments as in FIG. 3H, normalized to total protein and DMSO control. Data represent mean±SEM across n=3-4 independent experiments each with technical duplicates. * P<0.05, ** P<0.01 , *** PcO.001 , **** PcO.0001 , ns not significant, P-values between 0.05 and 0.1 are indicated. Welch’s t-test. For FIG. 3I, the order of data shown for each protein, from left to right, is as follows: DMSO, and zotatifin (100 nM).

[0041] FIGS. 4A-4D are a series of graphs showing ribosome profiling of MOLM-13 cells treated with Zotatifin. FIG. 4A is a pie chart showing the distribution of ribosome-protected fragments (RPFs) across the coding sequence (CDS), 5’ UTR, 3’ UTR, or intronic region of mRNAs assessed by ribosome profiling. A representative example of technical triplicates is shown for DMSO control. FIG. 4B is a heatmap showing correlation of gene counts between technical triplicates for paired RNA-seq (left) and Ribo-seq (right) of DMSO control and Zotatifin-treated MOLM-13 cells (4 hours). FIGS. 4C-4D are a series of bar plots showing normalized enrichment scores of Hallmark pathways following GSEA of Zotatifin-treated MOLM-13 cells analyzed by RNA-seq (FIG. 4C) or Ribo-seq (FIG. 4D).

[0042] FIGS. 5A-5B are a series of graphs showing Zotatifin and Venetoclax synergize to kill AML cells in vitro. Bar plots show viability of MOLM-13, MV4-11 , HL-60 and 0CI-AML3 cells treated for 24 hours with indicated doses of Zotatifin or Venetoclax, alone or in combination. Viability was measured as the percentage of DAPI negative cells by flow cytometry. Representative plots of at least 2 independent experiments are shown. Data points represent technical triplicates within each experiment. Significance is only shown for comparisons between the top dose of Zotatifin or Venetoclax with their combination at 50 nM each or 100 nM each. **** P<0.0001 ; one-way ANOVA.

[0043] FIGS. 6A-6D are a series of graphs and images showing Zotatifin and Venetoclax synergize to kill AML cell lines. FIG. 6A is a series of synergy plots generated using SynergyFinder 3.0 for MOLM-13, MV4-11 , HL-60 and OCI-AML3 cells treated for 24 hours with 10 nM, 50 nM, or 100 nM of Zotatifin or Venetoclax, alone or in combination, with DMSO as control. Representative plots of at least 2 independent experiments are shown. FIG. 6B is a series of representative flow cytometry plots showing % viable MV4-11 cells following 24-hour treatment of DMSO control, Zotatifin alone (50 nM), Venetoclax alone (50 nM), or their combination (50 nM each). FIGS. 6C-6D show Western blot analysis of target protein expression following 24 hours treatment of MOLM-13, MV4-11 and HL-60 cells with 100 nM Zotatifin, 100 nM Venetoclax, or their combination at 50 nM each. FIG. 6C is a series of representative images of 3 independent experiments. Each experimental condition was performed with technical duplicates. FIG. 6D is a series of graphs showing quantified protein expression across 3 independent experiments as in FIG. 6C, normalized to total protein and DMSO control. Data represent mean±SEM across n=3 independent experiments each with technical duplicates. * P<0.05, ** P<0.01 , *“ P<0.001 , **** P<0.0001 , one-way ANOVA. For each protein in FIG. 6D, the order of data shown, from left to right, is as follows: DMSO, 100 nM zotatifin, 100 nM venetoclax, and the combination of 50 nM zotatifin and 50 nM venetoclax.

[0044] FIGS. 7A-7F are a series of graphs showing Zotatifin and Venetoclax synergize to kill primary AML cells in vitro and in vivo. FIG. 7A is a series of graphs showing viability of healthy control (n=13) and AML bulk BM cells (n=11 ) treated for 24 hours with Zotatifin 100 nM, Venetoclax 100 nM, or their combination at 50 nM each (Z50 V50). Data normalized to DMSO control. Significance is shown for comparisons between combination and monotherapies only. The data for healthy CD34+ and AML CD34+CD33+ cells with Zotatifin monotherapy are also in FIG. 1 E and repeated for completeness. Some missing data points are due to cell types that did not reach a minimum of 300 cells to quantify viability (e.g., several AML samples were devoid of T, B and NK cells). For each cell type in FIG. 7A, the order of data shown, from left to right, is as follows: DMSO, 100 nM zotatifin, 100 nM venetoclax, and the combination of 50 nM zotatifin and 50 nM venetoclax. FIG. 7B is a schematic of experimental design. Immunodeficient NSG mice (n=8 per group) were engrafted intravenously with 1 million AML cells from the indicated PDX models and treated for 28 days with Zotatifin intraperitoneally every 4 days (Q4D), Venetoclax once daily (QD) by oral gavage, or their combination at the indicated doses. Tumor burden was measured as the % human CD45+ cells in mouse peripheral blood every 14 days. Created with Biorender. FIG. 7C is a scatter plot showing tumor burden across study days by flow cytometry in DFAM- 16835. Significance is only shown for comparisons between combination and other groups. For each study day in FIG. 7C, the order of data shown, from left to right, is as follows: vehicle, zotatifin (1 mg / kg Q4D), venetoclax (100 mg / kg QD), and the combination of zotatifin (1 mg / kg) and venetoclax (100 mg / kg). FIG. 7D is a Kaplan-Meier plot showing mouse survival curves across groups for DFAM-16835. For FIG. 7D, shown, from left to right at the survival of 0%, are zotatifin (1 mg / kg Q4D), venetoclax (100 mg / kg QD), vehicle, and the combination of zotatifin (1 mg / kg) and venetoclax (100 mg / kg). FIG. 7E is a scatter plot showing tumor burden across study days in MDAM-13466. Significance is only shown for comparisons between combination and other groups. For each study day in FIG. 7E, the order of data shown, from left to right, is as follows: vehicle, zotatifin (0.6 mg / kg Q4D), venetoclax (50 mg / kg QD), and the combination of zotatifin (0.6 mg / kg) and venetoclax (50 mg / kg). FIG. 7F is a Kaplan-Meier plot showing mouse survival curves across groups for MDAM-13466. * P<0.05, ** P<0.01 , *“ P<0.001 , *“* PcO.0001 ; two-way ANOVA in (A), (C) and (E); log-rank test in (D) and (F). For FIG. 7F, shown, from left to right at the survival of 0%, are vehicle, zotatifin (0.6 mg / kg Q4D), venetoclax (50 mg / kg QD), and the combination of zotatifin (0.6 mg / kg) and venetoclax (50 mg / kg).

[0045] FIGS. 8A-8B are a series of graphs showing gating strategy for primary human bone marrow and in vivo safety of Zotatifin and Venetoclax in NSG mice. FIG. 8A is a series of flow plots showing gating strategy for a representative healthy BM sample delineating total CD45+ leukocytes, CD3+ T cells, CD19+ B cells, CD33+ myeloid cells, CD34+ HSPCs, and CD56+ NK cells. FIG. 8B is a series of line plots showing in vivo safety study of Zotatifin and Venetoclax combination in NSG mice. Mice (n=4 per group) were treated with (1 ) Zotatifin 1 mg / kg every 4 days intravenously and Venetoclax 50 mg / kg daily by oral gavage, (2) Zotatifin 1 mg / kg and Venetoclax 100 mg / kg, or (3) Zotatifin 3 mg / kg and Venetoclax 100 mg / kg via the same routes of administration for 14 days or until mice were euthanized for ethical reasons. Body weight was measured daily and reported relative to that on day 1 of treatment.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] The invention provides methods for treatment of cancer, involving administering to the subject a combination of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof.

[0048] The present invention is based, in part, on the discovery that the combination of venetoclax and zotatifin efficiently eradicates AML cells, such as AML cells with the FLT3 mutation (the most common genetic alteration in AML), nucleophosmin 1 (NPM1 ) mutation, neuroblastoma RAS viral oncogene homolog (NRAS) mutation, or adenomatous polyposis coli (APC) mutation. Combining venetoclax with zotatifin, an elF4A inhibitor, results in synergistic AML cell death. At low doses (10 nM), either drug alone has a modest effect on the viability of FLT3 mutated AML cell lines MOLM-13 and MV4-11 . In contrast, combining the drugs at these therapeutically relevant doses results in highly effective killing of the same cell lines. These data indicate that co-administration of venetoclax and zotatifin could be used to treat subjects with cancer (e.g., FLT3-mutated AML patients, NPM1 -mutated AML patients, NRAS-mutated AML patients, APC-mutated AML patients, primary refractory AML patients, AML patients who have developed resistance to existing venetoclax-containing regimens, solid tumor, breast cancer, lung cancer, colorectal cancer, or pancreatic cancer). I. Venetoclax

[0049] Venetoclax (VENCLEXTA®) (ABT-199; GDC-0199; RG7601 ) is a BCL-2 inhibitor. The structure of venetoclax is shown below.

[0050] II. Zotatifin Zotatifin (eFT226) inhibits the helicase activity of elF4A and curtails growth of tumors (e.g., receptor tyrosine kinase-driven tumors). The structure of zotatifin is shown below. By unwinding complex structures in the 5’ UTR of transcripts, elF4A promotes the splicing, nuclear export, and translation of transcripts. III. Methods of Treatment

[0051] The disclosure provides a method of treating cancer by administering a combination of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof. In some embodiments, the combined effect of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof co-administration is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent delivered alone or in the absence of the other. The effect of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof can be partially additive, wholly additive, or greater than additive (e.g., synergistic). A synergistic effect is where the combination is more therapeutically effective than the sum of the effect observed when either agent is administered alone. In some embodiments, the synergistic effect is greater than additive results. Any method for determining whether two or more therapeutic agents exhibit synergy may be used for determining the synergistic effect of the combination, such as methods described herein. For example, SynergyFinder Plus (PMID: 35085776) and SynergyFinder 2.0 (PMID: 32246720) may be used to determine drug synergy based on viable cell counts. The summary synergy scores may be interpreted as the average excess response due to drug interactions (i.e. a synergy score of 15 corresponds to 15% of response beyond expectation). A synergy score at or around 0 indicates limited confidence on synergy or antagonism, i.e., a synergy score of between -10 to 10 indicates that the interaction between two drugs is likely to be additive. A synergy score greater than 10 indicates that the interaction between two drugs is likely to be synergistic.

[0052] In some embodiments, co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a greater increase in progression-free survival (PFS) compared to administration of either agent alone. PFS refers to the length of time during and after treatment during which the disease being treated (e.g., cancer, e.g., leukemia, e.g., AML) does not get worse. Progression-free survival may include the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease.

[0053] In some embodiments, co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a greater increase in duration of objective response (DOR) compared to administration of either agent alone. The DOR is the time from the first occurrence of a documented objective response to disease progression, or death from any cause within 30 days of the last dose of a treatment, whichever occurs first.

[0054] In some embodiments, the cancer is a hematological cancer (e.g., a leukemia, a lymphoma, or a myeloma). In some embodiments, the hematological cancer is an acute myeloid leukemia (AML). In some embodiments, the hematological cancer is a chronic lymphocytic leukemia (CLL). In some embodiments, the hematological cancer is a small lymphocytic lymphoma (SLL). In some embodiments, the hematological cancer is a multiple myeloma (MM).

[0055] AML is the most common type of acute leukemia in adults. In some embodiments, the AML is an FLT3-mutated AML. FLT-3 (FMS-like tyrosine kinase 3) mutations are a common genetic aberration in patients with AML. In some embodiments, the AML is a nucleophosmin 1 (NPM1 ) mutated AML. In some embodiments, the AML is a neuroblastoma RAS viral oncogene homolog (NRAS) mutated AML. In some embodiments, the AML is an adenomatous polyposis coli (APC) mutated AML. In some embodiments, the subject previously received chemotherapy and is primary refractory (e.g., a subject who has previously received chemotherapy for AML and has not achieved a complete remission). In some embodiments, the subject is resistant to venetoclax or a pharmaceutically acceptable salt thereof, such that venetoclax is ineffective or where venetoclax or a pharmaceutically acceptable salt thereof was previously effective and has become less effective over time. In some embodiments, the AML is a relapsed AML. In some embodiments, the AML is a refractory AML. In some embodiments, the patient has not previously received treatment for cancer (e.g., co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof is used as an up-front therapy).

[0056] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a breast cancer, a lung cancer, a colorectal cancer, or a pancreatic cancer.

[0057] IV. Dosing and Administration

[0058] Venetoclax or a pharmaceutically acceptable salt thereof may be administered at the same time as zotatifin or a pharmaceutically acceptable salt thereof. The agents can also be administered sequentially. For example, venetoclax can be administered before zotatifin or after zotatifin. Sequential or substantially simultaneous administration of each of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof treatment can be performed by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, local routes, and direct absorption through mucous membrane tissues. Venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof can be administered by the same route or by different routes. For example, venetoclax or a pharmaceutically acceptable salt thereof may be administered by orally while zotatifin or a pharmaceutically acceptable salt thereof can be administered intravenously. In some embodiments, venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof are administered at different frequencies. In some embodiments, venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof are administered at the same or at similar frequencies.

[0059] In some embodiments, venetoclax or a pharmaceutically acceptable salt thereof can be administered orally as a tablet at a dose of between about 50 mg to about 1500 mg (e.g., between about 50 mg to about 100 mg, between about 100 mg to about 200 mg, between about 200 mg to about 300 mg, between about 300 mg to about 400 mg, between about 400 mg to about 500 mg, between about 500 mg to about 600 mg, between about 600 mg to about 700 mg, between about 700 mg to about 800 mg, between about 800 mg to about 900 mg, between about 900 mg to about 1000 mg, between about 1000 mg to about 1100 mg, between about 1100 mg to about 1200 mg, between about 1200 mg to about 1300 mg, between about 1300 mg to about 1400 mg, or between about 1400 mg to about 1500 mg). In some embodiments, venetoclax or a pharmaceutically acceptable salt thereof can be administered at a dose of about 400 mg. In some embodiments, venetoclax or a pharmaceutically acceptable salt thereof can be administered at a dose of about 800 mg. In some embodiments, venetoclax or a pharmaceutically acceptable salt thereof can be administered at a dose of about 1200 mg. In some embodiments, venetoclax or a pharmaceutically acceptable salt thereof can be administered once daily.

[0060] In some embodiments, zotatifin or a pharmaceutically acceptable salt thereof can be administered intravenously, intraperitoneally, or subcutaneously at a dose of between about 0.001 mg / kg to about 0.1 mg / kg (e.g., between about 0.001 mg / kg to about 0.005 mg / kg, between about 0.005 mg / kg to about 0.01 mg / kg, between about 0.01 mg / kg to about 0.05 mg / kg, or between about 0.05 mg / kg to about 0.1 mg / kg). In some embodiments, zotatifin or a pharmaceutically acceptable salt thereof can be administered once every week for 3 weeks (i.e. , administered on days 1 , 8, and 15 of a 21 day cycle). In some embodiments, zotatifin or a pharmaceutically acceptable salt thereof can be administered on days 1 and 8 of a 21 day cycle).

[0061] EXAMPLES

[0062] Example 1. Leukemia Cell Vulnerability That Can Be Co-Targeted With BCL-2 Inhibition

[0063] This Example describes that the translation initiation factor EIF4A1 , which unwinds complex mRNA structures in the 5’ UTR of oncogenic transcripts, is highly expressed in AML stem- and progenitor-like cells. Inhibiting elF4A with the small molecule Zotatifin reduces translation of transcripts related to the cell cycle and survival. This results in downregulation of AKT, STAT-5, and MCL-1 and underlies synergy of Zotatifin with Venetoclax. The drug combination promotes apoptosis across AML genotypes, while the effect on healthy blood cells is limited. Using in vivo relapsed and refractory AML patient-derived xenograft models, the combination significantly suppressed tumor burden and prolonged survival of xenografted mice. These results support elF4A-mediated protein translation as a therapeutic target in AML.

[0064] Results

[0065] EIF4A 1 is highly expressed and can be targeted in primitive AML cells

[0066] Leukemia cells exhibit increased protein synthesis compared to healthy hematopoietic stem cells (HSCs), resulting in dependence on protein translation elongation and termination pathways that can be therapeutically targeted (Stevens et al., Nat Commun. 2018;9:3694; Sellar et al., J Clin Invest. 2022). We hypothesized that protein translation initiation factors may be upregulated in leukemia cells. In single-cell gene expression data of healthy BM and AML cells, the elF4F complex member EIF4A1 was upregulated in primitive leukemia cells (FIG. 1 A, FIG. 2) (van Galen et al., Cell. 2019;176:1265-81 .e24), which is supported by previous work that evaluated elF4A expression in bulk primary AML cells (Nishida et al., Leukemia. 2021 ;35:2469-81 ).

[0067] Mining DepMap bulk RNA-seq data showed that EIF4A1 is also highly expressed across 43 AML cell lines, and integration with PRISM drug treatment data indicated a correlation between EIF4A1 expression and sensitivity to the elF4A inhibitor Zotatifin (FIGS. 1 B-1 C) (DepMap 24Q2 Public, plus.figshare.com / articles / dataset / DepMap_24Q2_Public / 25880521 / 1 ). The PRISM drug studies were performed with Zotatifin at 2.5 pM for five days (Corsello et al., Nature Cancer. 2020;1 :235-48). To determine the extent to which it is effective at lower doses and shorter exposures, we treated 5 AML cell lines with titrated doses of Zotatifin for 24 hours and 72 hours and assessed viability using Cell-TiterG Io assay. Zotatifin reduced viability in all five cell lines, with a mean IC50 of 113.1 nM (range 11 .0-399.0 nM) at 24 hours and 63.8 nM (range 12.8-138.6 nM) at 72 hours, suggesting applicability against AML across diverse genotypes and differentiation states (FIG. 1 D and Table 1 ).

[0068] Table 1. IC50 of Zotatifin at 24 hours and 72 hours across 5 AML cell lines.

[0069] LOH= loss of heterozygosity; M= male; F= female; Dx= diagnosis

[0070] Finally, to test the potential presence of a therapeutic window, we cultured healthy BM cells and AML cells in StemSpan SFEM-II medium that supports hematopoietic cell / leukemic cell growth and treated the cells with Zotatifin (100 nM). We compared the viability of CD34+ hematopoietic stem and progenitor cells (HSPCs) from healthy BM and CD34+CD33+ cells from AML BM. This aberrant immunophenotype, which was largely absent in healthy BM but comprised 12.3% to 67.1% of CD45+ cells in AML samples, was used as a potential proxy for AML stem and progenitor cells. After 24 hours of treatment, we found increased cell death in Zotatifin-treated AML cells compared to healthy BM cells by flow cytometry (P=0.010, FIG. 1 E). These results suggest that AML cell survival selectively depends on the activity of elF4A.

[0071] Zotatifin downregulates AKT, metabolic, and cell cycle pathways, and upregulates inflammatory and apoptotic pathways

[0072] Next, we sought to determine how Zotatifin treatment affects gene expression and protein translation. To address this, we performed RNA-seq to measure gene expression and Ribo-seq to measure ribosome-protected fragments (RPFs, FIG. 3A) (Ingolia, Cell. 2016;165:22-33). We treated MOLM-13 cells with DMSO (control) or Zotatifin (100 nM) for 4 hours, lysed the cells, isolated total RNA and ribosome-protected mRNA, and performed sequencing. Following alignment, we first assessed the position of RPFs along transcripts. We observed increased ribosome occupancy in the 5’ UTR and around the start codon, but not around the stop codon (FIG. 3B, FIG. 4A). This is consistent with Zotatifin- induced immobilization of the elF4F complex at the beginning of transcripts (Li et al., Nat Commun. 2023;14:553). We evaluated the RNA-seq data to identify genes with altered expression and Ribo-seq to identify genes with altered ribosome occupancy in Zotatifin compared to DMSO conditions, using DE- Seq2 to analyze both datasets separately (Love et al., Genome Biol. 2014;15:550). To determine pathways and biological processes that are affected by Zotatifin, we evaluated Hallmark gene sets using gene set enrichment analysis (GSEA). Gene sets that were downregulated by Zotatifin transcriptionally (based on RNA-seq) and translational ly (Ribo-seq data) were related to metabolism, cell cycle and proliferation, and signaling pathways (FIG. 3C, FIG. 4B-4D, Table 2). Downregulated metabolic processes include fatty acid metabolism, oxidative phosphorylation, and genes encoding components of the peroxisome. Downregulated cell cycle and proliferation processes include MYC and E2F targets; while downregulated signaling pathways include PI3K / AKT / MTOR and MTORC1 signaling. Gene sets that were upregulated by Zotatifin in both RNA-seq and Ribo-seq were enriched with stress and immune responses including apoptosis, inflammation, and interferons. In addition, upregulated signaling pathways include TNFa, TGFp, and TP53 pathways. Collectively, we found that Zotatifin decreases processes and pathways relating to metabolism, cell survival, and proliferation, and increases stress / immune responses.

[0073] Table 2. Gene set enrichment analysis of Zotatifin-treated MOLM-13 cells.

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[0109] Considering that Zotatifin’s target elF4A promotes protein translation (Bhat et al., Nat Rev Drug Discov. 2015;14:261-78), we determined the translation efficiency of every transcript by calculating the ratio of RPFs to total mRNA counts within each gene’s coding sequence using DeltaTE (Chothani et al., Curr Protoc Mol Biol. 2019;129:e108). Zotatifin decreased the translation efficiency of 1 ,394 genes and increased the translation efficiency of 1 ,198 genes (FIG. 3D). Transcripts with decreased translation efficiency harbored significantly longer and more complex 5’ UTR structures as indicated by a more negative Gibbs free energy (FIG. 3E-3F). To determine if Zotatifin-induced translation efficiency alterations affect specific gene sets, we performed GSEA. This revealed that Zotatifin decreased the translation efficiency of transcripts in the PI3K / AKT / MTOR signaling and cell cycle pathways (MYC, E2F targets, G2M checkpoint, FIG. 3G), supporting that elF4A inhibition by Zotatifin causes translational downregulation of oncogenic pathways.

[0110] We next performed western blotting to measure proteins that promote AML cell survival and growth. We treated MOLM-13, MV4-11 , and HL-60 cells with 100 nM Zotatifin for 4 hours and 24 hours, and assessed key regulators of oncogenic signaling pathways and mitochondrial apoptosis. Representative images are shown in FIG. 3H and total protein-normalized levels from at least three separate experiments are shown in FIG. 3I. Total AKT was downregulated in all three cell lines at the 24- hour time point. STAT-5, representing JAK / STAT signaling, was also downregulated albeit to a lesser extent. We assessed the anti-apoptotic BCL-2 family proteins BCL-2 and MCL-1 . MCL-1 was downregulated in all three cell lines at the 4-hour time point, which aligns with its short half-life (Domina et al., Oncogene. 2004;23:5301-15). Both MCL-1 and elF4A initially decreased but rebounded or increased after 24 hours, highlighting a dynamic response to Zotatifin. There were no major changes to BCL-2 expression. Consistent with apoptosis, Zotatifin increased expression of BAK in MOLM-13 and HL-60 cells after 24 hours (FIG. 3I). Overall, Zotatifin decreases oncogenic drivers AKT, STAT-5 and elF4A, decreases anti-apoptotic MCL-1 , and increases pro-apoptotic BAK.

[0111] Zotatifin synergizes with Venetoclax in AML cell lines

[0112] Because Zotatifin decreased AKT and MCL1 expression, which have been implicated in resistance to the BCL-2 inhibitor Venetoclax (Choudhary et al., Cell Death Dis. 2015;6:e1593; Lin et al., Sci Rep. 2016;6:27696; Luedtke et al., Signal Transduct Target Ther. 2017;2:17012; Zhang et al., Signal Transduct Target Ther. 2022;7:51 ; Sullivan et al., Sci Transl Med. 2022;14:eabo6891 ), we sought to determine if combining Zotatifin with Venetoclax could be synergistic. To this end, we treated MOLM-13, MV4-11 , HL-60 and OCI-AML3 cell lines for 24 hours with titrated doses of Zotatifin and Venetoclax. These cell lines represent a range of AML differentiation states and genotypes, including FLT3, TP53, and NRAS mutations (Table 1 ). While either drug alone elicited a dose-dependent decrease in AML cell viability, the combination elicited greater efficacy (FIGS. 5A-5B). Applying synergy calculations using SynergyFinder, which employs the zero interaction potency (ZIP) model to quantify synergy by comparing observed combination responses to expected responses, demonstrated significant synergy between Zotatifin and Venetoclax across all four AML cell lines (ZIP synergy scores ranging from 29.7 to 57.2, FIG. 6A). In a representative example, 50 nM of either drug alone resulted in a viability of 68.6-84.4%, but both drugs together decreased viability to 0.97% in MV4-11 cells (FIG. 6B). For HL-60 cells, which are relatively primitive and sensitive to Venetoclax, we observed little further killing when combining Zotatifin with Venetoclax at 100 nM; however, strong synergy was still observed when using both drugs at 50 nM.

[0113] To dissect the mechanistic basis of this synergy, we treated MOLM-13, MV4-11 , and HL-60 cells for 24 hours with Zotatifin or Venetoclax at 100 nM, or their combination at 50 nM each as determined to be synergistic. Zotatifin alone decreased AKT and STAT-5, as did Venetoclax alone, consistent with previous findings (Ma et al., Clin Cancer Res. 2019;25:6815-26; Yamatani et al., Transl Oncol. 2022;18:101354). Both proteins were further decreased by the combination (FIGS. 6C-6D). Increased MCL-1 in MV4-11 cells treated with Zotatifin alone was similar to the 24-hour time point shown in FIGS. 3G-3H, and a decrease was appreciable with the combination, but these changes did not reach statistical significance based on total protein-normalized values (FIGS. 6C-6D). BAK was increased by the combination compared to monotherapies and DMSO control, consistent with apoptosis (FIGS. 6B-6C). Overall, the combination reduced oncogenic AKT and STAT-5 and enhanced pro-apoptotic BAK expression.

[0114] Zotatifin synergizes with Venetoclax against primary AML cells in vitro and in vivo

[0115] To test the clinical translatability of these findings, we wanted to determine if AML cells are selectively sensitive to Zotatifin plus Venetoclax compared to healthy cells. We cultured primary BM cells from healthy individuals (n=13) and AML patients (n=11 , blasts 67.3±4.7% mean±sem) and treated them for 24 hours with either drug alone or their combination (Tables 3 and 4).

[0116] Table 3. Patient demographics and genetic information.

[0117] Table 4. Patient demographics and genetic information.

[0118] * Different time points from the same patient t Source: Rapid Heme Panel (PMID: 27339098) HiDAC: High-Dose Cytarabine

[0119] HSCT: Hematopoietic Stem Cell Transplant MEC: Mitoxantrone, Etoposide, and Cytarabine

[0120] We quantified cell viability by flow cytometry on total CD45+ leukocytes. Within this limited time frame, the combination treatment decreased viability in healthy BM to 87.5% compared to 71 .2% in AML (FIG. 7A). To assess if the combination had a different effect on diverse cell types, we further gated on CD34+ HSPCs from healthy BM and CD34+CD33+ putative AML stem and progenitor cells from AML BM, as well as major cell populations including CD33+ myeloid, CD19+ B, CD3+ T, and CD56+ NK cells (FIG. 8A). In AML samples, the combination decreased the viability of CD33+ myeloid cells to 65.6% and CD34+CD33+ cells to 49.9%, compared to 95.3% in healthy BM CD33+ myeloid cells and 80.9% in healthy BM CD34+ HSPCs (FIG. 7A), highlighting a potential therapeutic window. In both healthy BM samples and AML samples, the effect of Zotatifin and Venetoclax on CD19+ B cells, CD3+ T cells, and CD56+ NK cells was limited, as the average viability measures all remained above 76%, further supporting limited toxicity in normal blood cells (FIG. 7A). The gold standard in preclinical drug efficacy studies is to test PDX models. We first determined the tolerability profile of Zotatifin and Venetoclax in immunodeficient Nod.Cg-PrkdcscidlL2rgtm1 Wjl / SzJ (NSG) mice (n=4 per group). We treated these mice with drug doses that have been used in other studies (Zhang et al., Signal Transduct Target Ther. 2022;7:51 ; Gerson-Gurwitz et al., Front Oncol. 2021 ;11 :766298) and demonstrated that a combination of Zotatifin at 1 mg / kg every four days and Venetoclax at 100 mg / kg daily for 14 days was tolerable in these animals, without significant impact on body weight (FIG. 8B).

[0121] Next, we evaluated the in vivo efficacy of Zotatifin and Venetoclax using one relapsed (DFAM- 16835) and one refractory (MDAM-13466) AML PDX model, reflecting clinical scenarios with a critical need for new therapies (FIG. 7B and Tables 3 and 4). In the first model (DFAM-16835), we engrafted mice with AML cells with complex cytogenetics from a 61 -year-old patient at relapse following chemotherapy and allogeneic stem cell transplant. When peripheral blood measurement of human CD45+ cells reached a mean of 3.8±2.5%, we initiated treatment with monotherapies or the combination for 28 days (n=8 mice per group), in line with 28-day treatment cycles with Venetoclax-based regimens in clinical practice (Jonas and Pollyea, Leukemia. 2019;33:2795-804). We monitored tumor burden at 14- day intervals by measuring the % human CD45+ cells in the peripheral blood (FIG. 7B). At the end of treatment (day 28), tumor burden was significantly decreased by the combination (mean 0.25%, range 0.10-0.50%) as compared to vehicle control (mean 38.4%, range 23.3-57.5%), Zotatifin monotherapy (mean 13.0%, range 8.5-24.1%), and Venetoclax monotherapy groups (mean 9.7%, range 4.7-13.7%, all PcO.001 , FIG. 7C). Strikingly, the median survival was 39 days for the vehicle control group, compared to 70 days for the Zotatifin + Venetoclax group (P<0.0001 ) (FIG. 7D). Neither monotherapy was sufficient to confer a significant survival benefit in this aggressive PDX model, supporting the synergistic activity of elF4A and BCL-2 inhibition.

[0122] In the second model (MDAM-13466), we engrafted mice with AML cells harboring NRAS and APC mutations from a 54-year-old patient with refractory disease following Decitabine treatment (FIG. 7B and Tables 3 and 4). When peripheral blood measurement of human CD45+ cells reached a mean of 0.4±0.2% (MDAM-13466), we initiated treatment. To assess the robustness of the response and maximize clinical translatability, we lowered the dose of Zotatifin to 0.6 mg / kg and Venetoclax to 50 mg / kg, maintaining the same dose frequency and route of administration. Across all time points of tumor burden measurement, both on-treatment and post-treatment, the combination significantly decreased the percentage of human CD45+ cells relative to all other treatment groups (FIG. 7E). For example, on study day 43, tumor burden remained significantly lower following the combination (mean 16.5%, range 7.4- 28.8%) as compared to vehicle control (mean 87.3%, range 80.0-92.4%, P<0.0001 ), Zotatifin monotherapy (mean 79.4%, range 70.4-83.7%, P<0.0001 ), and Venetoclax monotherapy groups (mean 35.0%, range 15.7-47.9%, P<0.01 , FIG. 7E). Accordingly, the combination significantly extended survival relative to all other treatment groups (FIG. 7F). Median survival was 51 days for the vehicle control group, compared to 79 days for the Zotatifin + Venetoclax group (P<0.0001 ) (FIG. 7F). Despite the lower dose, Zotatifin or Venetoclax monotherapy was sufficient to confer a significant survival benefit in this model. Together, these in vivo data support the translational potential of combining Zotatifin and Venetoclax in clinically challenging cases of AML. Summary

[0123] In this study, we found elF4A to be enriched in AML, particularly in primitive cell states relative to their healthy counterparts, underscoring its potential as a therapeutic target. Inhibition of elF4A by Zotatifin decreases AML cell viability across genetically diverse AML cell lines and primary cells, suggesting that Zotatifin may be broadly applicable in AML. Using ribosome profiling, we found that Zotatifin decreases metabolic and cell cycle / proliferation pathways, including PI3K / AKT / MT0R signaling, and increases stress signaling and inflammatory pathways, including TP53, TNFa, and interferons. At the protein level, Zotatifin decreased AKT, STAT-5, and MCL-1 , which have been associated with Venetoclax resistance, and was found to synergize with Venetoclax to kill AML cells both in vitro and in vivo. This lays the groundwork to develop Zotatifin and its combination with Venetoclax for the treatment of AML.

[0124] We found that Zotatifin downregulates metabolic pathways including fatty acid metabolism, oxidative phosphorylation, and peroxisome genes, and AKT levels were further decreased by the combination with Venetoclax. This could limit escape mechanisms in AML cells. Another level of synergy may come from targeting different cell states. While Venetoclax primarily targets primitive cells (Pei et al., Cancer Discov. 2020;10:536-51 ), the results described herein show that Zotatifin downregulates AKT and downstream mTOR signaling. Thus, Zotatifin may target and eliminate residual AML cells that are not efficiently cleared by Venetoclax alone. Overall, the pathways modulated by Zotatifin likely disrupt AML cell survival.

[0125] The studies described herein using primary cells are supportive of a therapeutic window. In a time frame of 24 hours, AML cells with myeloid and progenitor cell phenotypes were selectively killed while healthy cells were spared. Further supporting the translational potential of these findings, combining Venetoclax and Zotatifin to treat two different clinically challenging PDX models prolonged survival reminiscent of long-term clinical benefit. This survival benefit in combination-treated mice relative to vehicle control and monotherapy groups provides a rationale for further exploration of tumor cell sensitivity at disease recurrence and the survival benefit of continuous treatment. Overall, these results represent a significant step forward by contributing mechanistic insight and pre-clinical data that strongly support targeting elF4A-mediated translation initiation in AML.

[0126] In summary, the results described herein show identification of elF4A as a promising therapeutic target enriched in AML stem- and progenitor-like cell states, and that its inhibition by Zotatifin is applicable to AML across diverse genotypes and differentiation states. By ribosome profiling, Zotatifin decreases the translation efficiency of transcripts relating to PI3K / AKT / MTOR signaling and cell cycle pathways.

[0127] Further, Zotatifin downregulates metabolic pathways and increases apoptosis and inflammation. By immunoblotting, Zotatifin was found to decrease expression of MCL-1 at 4 hours and of AKT and STAT-5 at 24 hours, concomitant with BAK upregulation. The combination of Zotatifin and Venetoclax synergistically kills AML cells whilst sparing healthy human BM cells, indicating a therapeutic window for selective eradication of tumor cells. Finally, the combination achieved therapeutic efficacy in two clinically relevant AML patient-derived xenograft (PDX) models in vivo by curtailing tumor growth and prolonging survival. Taken together, these findings demonstrate a new therapeutic approach for treating AML patients. Methods

[0128] Inhibitors

[0129] Zotatifin (HY-112163) and Venetoclax (HY-15531 ) were purchased from MedChemExpress.

[0130] Cell culture

[0131] AML cell lines MOLM-13, MV4-11 , HL-60, OCI-AML3, and U937 were purchased from DSMZ or ATCC and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). Cell line authentication was confirmed by short tandem repeat (STR) DNA fingerprinting every 6 months. Mycoplasma testing was routinely conducted once every 3 months and cultures were confirmed to be negative by PCR. Primary BM samples were cultured in STEMSPAN™ Serum-Free Expansion Medium II (SFEM II) (StemCell Technologies #09605) containing a cytokine cocktail and UM729 in accordance with the manufacturer’s recommendations.

[0132] Primary samples

[0133] Healthy BM samples were obtained from the sternum of individuals undergoing cardiac surgery at the Brigham and Women’s Hospital following informed consent in accordance with the institutional review board (IRB) protocol 2020P004103 at Mass General Brigham. In addition, human BM mononuclear cells (MNCs) were purchased from StemCell Technologies (#70001 .2), while human BM CD34+ purified hematopoietic stem and progenitor cells (HSPCs) were purchased from Lonza Bioscience (#2M-101 B) or StemCell Technologies (#70002.3). AML BM samples were obtained from the Pasquarello Tissue Bank at the Dana-Farber Cancer Institute, following informed consent in accordance with the IRB protocol 01 -206 and secondary use protocol 20-123.

[0134] Gene expression analysis

[0135] Single-cell RNA-sequencing data from 16 AML and 5 healthy BM samples was analyzed for genes related to eukaryotic translation initiation (van Galen et al., Cell. 2019;176:1265-81 .e24). In malignant cells and their healthy counterparts, the expression of these genes was normalized using the Seurat function NormalizeData. For sina / violin plots of EIF4A1 , the adjusted P-value was calculated using the Wilcoxon test with Bonferroni correction as implemented by the Seurat function FindMarkers (Hao et al., Cell. 2021 ;184:3573-87. e29). To generate a heatmap of translation initiation factors, candidate genes were selected from the literature (Aitken and Lorsch, Nat Struct Mol Biol. 2012;19:568-76). Data was scaled using the Seurat function ScaleData and for each cell type, the average of scaled expression values was visualized using the ComplexHeatmap package (Gu, Imeta. 2022;1 :e43). These analyses were performed in R for statistical computing version 4.3.1 .

[0136] Cell viability assays

[0137] For determination of Zotatifin half-maximal inhibitory concentration (IC50) values, AML cell lines MV4-11 , HL-60, U937, OCI-AML3 and MOLM-13 were seeded at 0.5x106 cells per ml (200 pl) in triplicate wells of 96-well plates and treated for 24 hours or 72 hours with serial 10-fold titrated doses of Zotatifin starting with a top dose of 10 pM. Cell viability was assessed by CellTiter-Glo assay (Promega G7570) according to the manufacturer’s recommendations and luminescence was measured using the Synergy H1 microplate reader (Agilent BioTek). IC50 curves were plotted based on the non-linear fit of [Inhibitor] vs. response (three parameters) model using GraphPad Prism version 8.0. For drug synergy experiments, AML cell lines were seeded as described above and treated for 24 hours with titrated doses of Zotatifin or Venetoclax alone (10 nM, 50 nM, and 100 nM), or their combination in various combinations. Cell viability was assessed on a Cytek Aurora flow cytometer (Cytek Biosciences) after staining with DAPI or propidium iodide at 1 :2000 dilution for 15 mins. Flow cytometry data was analyzed using FlowJo version 10.8.1 . Synergy scores and plots were generated using SynergyFinder 3.0 by uploading data to the web application (synergyfinder.fimm.fi / ) (lanevski et al., Nucleic Acids Res.

[0138] 2020;48:W488-93). We used the viability readout under default settings including outlier detection and LL4 for curve fitting. For primary healthy and AML BM samples or purified CD34+ HSPCs, samples were treated for 24 hours with Zotatifin or Venetoclax alone at 100 nM, or their combination at 50 nM each, and viability was assessed by DAPI staining and flow cytometry using an antibody panel comprising CD3- FITC (BD #555332), CD14-PE-Cy7 (Beckman Coulter A22331 ), CD19-BV785 (BioLegend #302240), CD33-PE (BD #347787), CD34-APC-eFluor780 (Invitrogen #47-0349-42), CD45-PerCP (BD #340665), and CD56-APC (BD #341026).

[0139] Western blot

[0140] AML cell lines were treated with 100 nM Zotatifin for 4 hours or 24 hours in parallel with 0.01 % DMSO as the negative control and cell lysates were freshly collected in RIPA lysis buffer (EMD Millipore 20-188) containing protease inhibitor cocktail (ThermoFisher 78430) following the manufacturer’s recommendations. Protein concentration was measured using Bradford assay (Biorad 5000006) and western blotting was performed using Bio-Techne ProteinSimple JessTM system (SM-W004) following manufacturer’s recommendations, in technical duplicates across at least 3 independent experiments.

[0141] STAT-5 (Cat. 94205), AKT (Cat. 9272S), elF4A (Cat. 2013S), MCL-1 (Cat. 5453T), BAK (Cat. 12105S) and GAPDH (Cat. 21 18S) antibodies were purchased from Cell Signaling Technologies. BCL-2 (12789-1 -AP) antibody was purchased from Proteintech. Quantification of protein expression was expressed relative to DMSO control, after normalization to total protein load. AML cell lines were also treated with 100 nM Zotatifin alone, 100 nM Venetoclax alone, or their combination at 50 nM each for 24 hours before cell lysis and western blotting as described above.

[0142] In vivo study

[0143] In vivo experiments were performed at Dana-Farber Cancer Institute at the Experimental Therapeutics core, in accordance with Dana-Farber Institutional Animal Care and Use Committee (IACUC) protocol 04-1 1 1 . The tolerability profile of Zotatifin and Venetoclax combination was determined in immunodeficient Nod.Cg-PrkdcscidlL2rgtm1 Wjl / SzJ (NSG) mice (n=4 per group) in which animals were treated with (1 ) Zotatifin 1 mg / kg every 4 days intravenously and Venetoclax 50 mg / kg daily by oral gavage, (2) Zotatifin 1 mg / kg and Venetoclax 100 mg / kg, or (3) Zotatifin 3 mg / kg and Venetoclax 100 mg / kg for 14 days or until mice were euthanized for ethical reasons. Zotatifin was formulated as a solution in 5% dextrose, while Venetoclax was formulated with 10% ethanol, 60% Phosal 50PG and 30% PEG400. Body weight of mice was monitored daily. The in vivo efficacy study was performed in two independent PDX models. The first PDX (DFAM-16835) is a relapsed AML model following high-dose chemotherapy and allogeneic stem cell transplant with complex cytogenetics, FLT3-ITD, FLT3-TKD, and NPM1 mutations. The second (MDAM-13466) is a refractory AML model following Decitabine treatment with mutations in NRAS and APC (refer to Tables 3 and 4 for more information on both models). Both models are derived from the Public Repository of Xenografts (PRoXe) (Townsend et al., Cancer Cell. 2016;29:574-86). Mice were engrafted with 1 million cells intravenously before reaching 8 weeks of age. For DFAM-16835, treatment groups were vehicle control, Zotatifin 1 mg / kg every 4 days intraperitoneally, Venetoclax 100 mg / kg daily by oral gavage, and their combination at the same doses and routes of administration (n=8 mice per group). For MDAM-13466, treatment groups were vehicle control, Zotatifin 0.6 mg / kg every 4 days intraperitoneally, Venetoclax 50 mg / kg daily by oral gavage, and their combination at the same doses and routes of administration (n=8 mice per group). The % human CD45+ cells in mouse peripheral blood was monitored by flow cytometry every 14 days until the mice reached endpoint. Survival was also monitored until the mice reached endpoint.

[0144] Ribosome profiling

[0145] MOLM-13 cells were treated for 4 hours with 100 nM Zotatifin or 0.01% DMSO as a control. To prepare samples for ribosome sequencing, 5-10 million cells were pelleted at 500 g for 5 minutes, the supernatant was discarded, and pellets were gently mixed with room temperature culture medium containing cycloheximide (final concentration 0.1 mg / ml). Cells were pelleted again, supernatant discarded, and pellets were gently mixed with pre-cooled PBS containing 0.1 mg / ml cycloheximide. Cells were pelleted again, supernatant discarded, and pellets were snap-frozen in liquid nitrogen for ribosome sequencing. To prepare samples for RNA sequencing, 5-10 million cells were pelleted and washed with pre-cooled PBS. Cells were pelleted again and TRIzol was added at 1 ml per million cells and pipetted repeatedly until no cell clumps remained. Samples were then snap-frozen in liquid nitrogen and sent to CD Genomics for ribosome sequencing and RNA-sequencing (cd-genomics.com / ribosome-profiling.html).

[0146] Identification of differential translation efficiency genes

[0147] The quantification of gene expression was performed with the assistance of CD Genomics services who provided Ribo-seq and RNA-seq read counts. These gene counts were normalized using the DESeq2 statistical model through size factor estimation, followed by model fitting to account for the condition, sequencing type, and their interaction. Translation efficiency for all genes was then assessed using the deltaTE (ATE) integrative analysis approach in R, where ATE was calculated as the ratio of RPFs to total mRNA counts (ATE=RPF / mRNA) (Chothani et al., Curr Protoc Mol Biol. 2019;129:e108). As part of the DESeq2 package, the empirical Bayes technique was used to calculate log fold changes, and a Benjamini-Hochberg (BH) procedure was performed to control for the false discovery rate (FDR). An adjusted P-value threshold of 0.05 was used to identify significant changes in translation efficiency with a minimum 2-fold change. Differential gene expression and pathway enrichment analysis

[0148] Differential gene expression analysis of RNA-seq and Ribo-seq data was performed using DESeq2 (v.1 .40.2) in R. Using NCBI identifications from the MANE select dataset

[0149] (GRCh38.v1 ,3.ensembl), the genes were filtered for canonical transcripts to resolve any duplicates. The log2FoldChanges for ATE, RNA-seq, and Ribo-seq were then sorted in descending order to use as input for pathway enrichment analysis. This analysis was conducted using fast gene set enrichment analysis (FGSEA v.1 .28.0) in R with a threshold of Benjamini-Hochberg (BH)-adjusted P-value < 0.05 and 10,000 gene permutations (Korotkevich et al., bioRxiv. 2016. dx.doi.org / 10.1 101 / 060012). We considered the Hallmark pathways (MsigDB v.7.5.1 ). The results of FGSEA were derived as normalized enrichment scores and their respective P-values. To assess the relationship between mRNA abundance and protein synthesis in significantly enriched pathways (P < 0.05), we plotted the normalized enrichment scores for RNA-seq against Ribo-seq data.

[0150] Statistical analysis

[0151] Statistical analysis was performed using Graph Pad Prism 8.0. Normality of data was assessed by Shapiro-Wilk test. Comparisons between two groups were performed using paired or unpaired t-tests according to experimental design. For comparisons between three or more groups, one-way or two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test was used. To assess synergy and calculate zero interaction potency (ZIP) scores, we used SynergyFinder 3.0. ZIP scores exceeding 10 are strongly indicative of an interaction between two drugs being synergistic (lanevski et al., Nucleic Acids Res. 2020;48:W488-93). The Kaplan-Meier method was used to generate survival curves, and logrank test for comparison between groups. Results are considered statistically significant when the P-value is <0.05.

[0152] OTHER EMBODIMENTS

[0153] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[0154] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0155] Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments:

[0156] E1 . A method for treating a subject having a cancer, the method comprising administering to the subject: a therapeutically effective amount of venetoclax, or a pharmaceutically acceptable salt thereof; and a therapeutically effective amount of zotatifin, or a pharmaceutically acceptable salt thereof.

[0157] E2. The method of E1 , wherein the cancer is leukemia. E3. The method of E2, wherein the leukemia is acute myeloid leukemia (AML).

[0158] E4. The method of E3, wherein the AML is an FMS-like tyrosine kinase 3 (FLT3) mutated AML.

[0159] E5. The method of any one of E1 -E4, wherein the subject previously received chemotherapy and is primary refractory.

[0160] E6. The method of any one of E1 -E5, wherein the patient is resistant to venetoclax.

[0161] E7. The method of any one of E1 -E6, wherein co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a synergistic effect.

[0162] E8. The method of any one of E1 -E7, wherein co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a greater increase in progression-free survival (PFS) compared to administration of either agent alone.

[0163] E9. The method of any one of E1 -E8, wherein co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a greater increase in duration of objective response (DOR) compared to administration of either agent alone.

[0164] E10. The method of any one of E1 -E9, wherein venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof are administered simultaneously.

[0165] E11 . The method of any one of E1 -E9, wherein venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof are administered sequentially.

[0166] E12. The method of any one of E1 -E11 , wherein venetoclax or a pharmaceutically acceptable salt thereof is administered orally as a tablet.

[0167] E13. The method of any one of E1 -E12, wherein zotatifin or a pharmaceutically acceptable salt thereof is administered intravenously, intraperitoneally, or subcutaneously.

[0168] E14. The method of any one of E1 -E13, wherein the subject is a human subject.

[0169] E15. Use of a therapeutically effective amount of venetoclax or a pharmaceutically acceptable salt thereof in treating a patient who has been diagnosed with AML in combination with zotatifin or a pharmaceutically acceptable salt thereof.

[0170] E16. A method for treating a human subject having relapsed or refractory acute myeloid leukemia, comprising administering to the subject venetoclax in combination with zotatifin, wherein a dose of between about 50 mg to about 1500 mg of venetoclax is orally administered once daily to the subject.

[0171] E17. The method of E16, wherein a dose of between about 0.001 mg / kg to about 0.1 mg / kg of zotatifin is intravenously administered once every week for 3 weeks to the subject.

[0172] E18. The method of E17, wherein the AML of the subject comprises an FLT3 mutation. Other embodiments of the technology described herein can be defined according to any of the following numbered embodiments:

[0173] E1 . A method for treating a subject having a cancer, the method comprising administering to the subject: a therapeutically effective amount of venetoclax, or a pharmaceutically acceptable salt thereof; and a therapeutically effective amount of zotatifin, or a pharmaceutically acceptable salt thereof.

[0174] E2. The method of E1 , wherein the cancer is leukemia.

[0175] E3. The method of E2, wherein the leukemia is acute myeloid leukemia (AML).

[0176] E4. The method of E3, wherein the AML has mutations in FMS-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1 ), neuroblastoma RAS viral oncogene homolog (NRAS), or adenomatous polyposis coli (APC).

[0177] E5. The method of any one of E1 -E4, wherein the subject previously received chemotherapy and is primary refractory.

[0178] E6. The method of any one of E1 -E5, wherein the patient is resistant to venetoclax.

[0179] E7. The method of any one of E1 -E6, wherein co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a synergistic effect.

[0180] E8. The method of any one of E1 -E7, wherein co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a greater increase in progression-free survival (PFS) compared to administration of either agent alone.

[0181] E9. The method of any one of E1 -E8, wherein co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a greater increase in duration of objective response (DOR) compared to administration of either agent alone.

[0182] E10. The method of any one of E1 -E9, wherein venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof are administered simultaneously.

[0183] E11 . The method of any one of E1 -E9, wherein venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof are administered sequentially.

[0184] E12. The method of any one of E1 -E11 , wherein venetoclax or a pharmaceutically acceptable salt thereof is administered orally as a tablet.

[0185] E13. The method of any one of E1 -E12, wherein zotatifin or a pharmaceutically acceptable salt thereof is administered intravenously, intraperitoneally, or subcutaneously.

[0186] E14. The method of any one of E1 -E13, wherein the subject is a human subject.

[0187] E15. Use of a therapeutically effective amount of venetoclax or a pharmaceutically acceptable salt thereof in treating a patient who has been diagnosed with AML in combination with zotatifin or a pharmaceutically acceptable salt thereof.

[0188] E16. A method for treating a human subject having relapsed or refractory acute myeloid leukemia, comprising administering to the subject venetoclax in combination with zotatifin, wherein a dose of between about 50 mg to about 1500 mg of venetoclax is orally administered once daily to the subject.

[0189] E17. The method of E16, wherein a dose of between about 0.001 mg / kg to about 0.1 mg / kg of zotatifin is intravenously administered once every week for 3 weeks to the subject.

[0190] E18. The method of E17, wherein the AML of the subject comprises an FLT3 mutation, an NPM1 mutation, an NRAS mutation, or an APC mutation.

[0191] Other embodiments are within the following claims.

[0192] What is claimed is:

Claims

CLAIMS1 . A method for treating a subject having a cancer, the method comprising administering to the subject: a therapeutically effective amount of venetoclax, or a pharmaceutically acceptable salt thereof; and a therapeutically effective amount of zotatifin, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1 , wherein the cancer is leukemia.

3. The method of claim 2, wherein the leukemia is acute myeloid leukemia (AML).

4. The method of claim 3, wherein the AML has mutations in FMS-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1 ), neuroblastoma RAS viral oncogene homolog (NRAS), or adenomatous polyposis coli (APC).

5. The method of claim 1 , wherein the subject previously received chemotherapy and is primary refractory.

6. The method of claim 1 , wherein the patient is resistant to venetoclax.

7. The method of claim 1 , wherein co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a synergistic effect.

8. The method of claim 1 , wherein co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a greater increase in progression-free survival (PFS) compared to administration of either agent alone.

9. The method of claim 1 , wherein co-administration of venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof leads to a greater increase in duration of objective response (DOR) compared to administration of either agent alone.

10. The method of claim 1 , wherein venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof are administered simultaneously.11 . The method of claim 1 , wherein venetoclax or a pharmaceutically acceptable salt thereof and zotatifin or a pharmaceutically acceptable salt thereof are administered sequentially.

12. The method of claim 1 , wherein venetoclax or a pharmaceutically acceptable salt thereof is administered orally as a tablet.

13. The method of claim 1 , wherein zotatifin or a pharmaceutically acceptable salt thereof is administered intravenously, intraperitoneally, or subcutaneously.

14. The method of claim 1 , wherein the subject is a human subject.

15. Use of a therapeutically effective amount of venetoclax or a pharmaceutically acceptable salt thereof in treating a patient who has been diagnosed with AML in combination with zotatifin or a pharmaceutically acceptable salt thereof.

16. A method for treating a human subject having relapsed or refractory acute myeloid leukemia, comprising administering to the subject venetoclax in combination with zotatifin, wherein a dose of between about 50 mg to about 1500 mg of venetoclax is orally administered once daily to the subject.

17. The method of claim 16, wherein a dose of between about 0.001 mg / kg to about 0.1 mg / kg of zotatifin is intravenously administered once every week for 3 weeks to the subject.

18. The method of claim 17, wherein the AML of the subject comprises an FLT3 mutation, an NPM1 mutation, an NRAS mutation, or an APC mutation.

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