Methods of treating aml by identifying and targeting a myeloid / lymphoid leukemia stem cell

Abstract

The present disclosure provides methods of treating acute myeloid leukemia (AML) and methods of determining responsiveness to AML treatment regimens, the methods comprising identifying the presence or absence of Myeloid / Lymphoid leukemia stem cells (M / L LSC) in a sample from a subject.

Description

Attorney Docket No.: UNCO-050 / 001WO (300978-2221) METHODS OF TREATING AML BY IDENTIFYING AND TARGETING A MYELOID / LYMPHOID LEUKEMIA STEM CELL RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application No. 63 / 386,505, filed on December 8, 2022, the contents of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0002] Acute myeloid leukemia (AML) is a blood cancer in which the bone marrow of a subject makes abnormal myeloblasts, red blood cells, or platelets. AML is one of the most common forms of acute leukemia in adults. The build-up of AML cells in bone marrow and blood can rapidly lead to infection, anemia, excessive bleeding and death. BCL-2 inhibitor venetoclax has recently emerged as an important component of therapy for acute myeloid leukemia (AML). In combination with a number of backbone chemotherapy treatments, venetoclax can induce responses in approximately 60-70% of older previously untreated AML patients, many of whom are unfit for conventional induction therapy. However, resistance to venetoclax-based therapy has been documented, as well as relapse following initial response. There is a need in the art for methods of predicting response to venetoclax treatment and methods of treating AML in patients who are predicted to be refractory to or to relapse after treatment with venetoclax. There is also a need in the art for methods of treating AML, particularly in elderly patients that are unfit for conventional induction therapy and for patients with relapsed AML. SUMMARY OF THE INVENTION

[0003] The present disclosure provides methods of treating acute myeloid leukemia (AML) in a subject, the methods comprising the identification of Myeloid / Lymphoid Leukemia Stem Cells (M / L LSCs) in a sample from the subject, wherein a cell is identified as an M / L LSC if the cell is at least CD34+ and CD7+.

[0004] The present disclosure provides methods of identifying whether a subject having AML will be responsive to treatment with a combination comprising of at least one BCL-2 inhibitor and at least one hypomethylating agent, the methods comprising the identification ofAttorney Docket No.: UNCO-050 / 001WO (300978-2221) Myeloid / Lymphoid LSCs o(M / L LSC) in a sample from the subject, wherein a cell is identified as an M / L LSC if the cell is at least CD34+ and CD7+.

[0005] The present disclosure provides methods of treating acute myeloid leukemia (AML) in a subject, the methods comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphoid leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent when at least one M / L LSC is identified; or administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent when no M / L LSCs are identified.

[0006] The present disclosure provides methods of identifying if a subject having AML will be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphoid leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) identifying that the subject will not be responsive to the treatment when the presence of at least one M / L LSC is identified; or identifying that the subject will be responsive to the treatment when no M / L LSCs are identified.

[0007] The present disclosure provides methods of treating AML in a subject, the methods comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the number and / or percentage of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) comparing the number and / or percentage of m M / L LSCs identified in step (b) to a predetermined cutoff value; and d) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent when the number and / or percentage of M / L LSCs is equal to or greater than the predetermined cutoff value; or administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent when the number and / or percentage of M / L LSCs is less than the predetermined cutoff value.

[0008] The present disclosure provides methods of identifying if a subject having AML will be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the methods comprising: a) measuring the expression of at least CD34Attorney Docket No.: UNCO-050 / 001WO (300978-2221) and CD7 in a plurality of cells in a sample from the subject; b) identifying the number and / or percentage of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) comparing the number and / or percentage of m M / L LSCs identified in step (b) to a predetermined cutoff value; and d) identifying that the subject will not be responsive to the treatment when the number and / or percentage of M / L LSCs is equal to or greater than the predetermined cutoff value; or identifying that the subject will be responsive to the treatment when the number and / or percentage of M / L LSCs is less than the predetermined cutoff value.

[0009] In some aspects, the at least one M / L LSC targeting agent comprises the administration of at least one of: i) an anti-CD7 antibody; ii) an anti-CD7 immunotherapy, preferably wherein the immunotherapy comprises CAR-T and / or NK Cells that are directed specifically at CD7; iii) an agent that blocks CD7 signaling, preferably wherein the agent that blocks CD7 signaling is an inhibitor of the PI3Kinase and associated pathways; iv) an agent that inhibits CD7 activation, preferably wherein the agent that inhibits CD7 activation inhibits activation of CD7 by K12 and / or any additional CD7 ligands; and v) an agent that targets an M / L LSC-specific pathway and / or surface marker.

[0010] In some aspects, the at least one hypomethylating agent is selected from azacitidine and decitabine.

[0011] In some aspects, the at least one BCL-2 inhibitor is selected from venetoclax and navitoclax.

[0012] In some aspects, step (a) comprises performing PCR, high-throughput sequencing, next generation sequencing, Northern Blot, reverse transcription PCR (RT-PCR), real-time PCR (qPCR), quantitative PCR, qRT-PCR, flow cytometry, mass spectrometry, microarray analysis, digital droplet PCR, Western Blot, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-SEQ), any single cell omics technologies, or any combination thereof.

[0013] In some aspects, the subject is: i) a subject having AML who has not received any treatment for AML; or ii) a subject having AML who has received previously received at least one AML treatment, preferably wherein the at least one AML treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0014] In some aspects, identifying that a subject will be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying that the subject will have a durable remission after receiving the treatment of a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.Attorney Docket No.: UNCO-050 / 001WO (300978-2221)

[0015] In some aspects, identifying that a subject will not be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying that the subject will be refractory to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent and / or that the subject will suffer a relapse after treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0016] In some aspects, the biological sample comprises blood, a bone marrow biopsy, a bone marrow aspirate, a biopsy of a chloroma, a tissue biopsy, cerebrospinal fluid or any combination thereof.

[0017] Any of the above aspects, or any of the aspects described herein, can be combined with any other aspect.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.” Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

[0019] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claim.Attorney Docket No.: UNCO-050 / 001WO (300978-2221) BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and further features will be more clearly appreciated from the following detailed description when taken in conjunction with the accompanying drawings.

[0021] FIG.1A shows the survival probability following treatment with a combination of venetoclax and azacitidine for groups of AML patients who exhibit one of: CD7- / CD34-, CD7+ / CD34-, CD7- / CD34+ and CD7+ / CD34+ cells.

[0022] FIG.1B shows the survival probability following treatment with a combination of venetoclax and azacitidine for groups of AML patients who did not receive a stem cell transplantation and who exhibit one of: CD7- / CD34-, CD7+ / CD34-, CD7- / CD34+ or CD7+ / CD34+ cells.

[0023] FIG.1C shows the survival probability following treatment with a combination of venetoclax and azacitidine for groups of AML patients censored by those who received a stem cell transplant and who exhibit one of: CD7- / CD34-, CD7+ / CD34-, CD7- / CD34+ and CD7+ / CD34+ cells. DETAILED DESCRIPTION OF THE INVENTION

[0024] Acute myeloid leukemia is a blood cancer that is one of the most commonly diagnosed types of leukemia in adults. It is estimated that there will be approximately 11,000 deaths from AML in the United States in 2020, along with 20,000 newly diagnosed cases. The average age of a person diagnosed with acute myeloid leukemia is about 68, with most cases occurring after the age of 45. However, acute myeloid leukemia has also been diagnosed in younger patients, including children. Prognosis for patients diagnosed with acute myeloid leukemia is generally poor, with a long-term survival of only 40-50% in younger patients and a median overall survival of less than one year for older patients. New therapies aimed at supplementing the standard remission induction regimen of infusional cytarabine with intermittent dosing of an anthracycline has provided some improvement to treatment outcomes, but the improvements have been modest. Thus, there exists a need for more specialized and personalized treatment methods, particularly in older patients who are unfit for induction therapy.

[0025] Recent research has demonstrated that acute myeloid leukemia exhibits a high level of biological heterogeneity, potentially explaining the difficulty in finding effective therapeutic strategies for the treatment of AML. Furthermore, it has been recently recognized that leukemiaAttorney Docket No.: UNCO-050 / 001WO (300978-2221) stem cells (LSCs), which are capable of giving rise to identical daughter cells as well as differentiated cells, perpetuate and maintain acute myeloid leukemia.

[0026] As an alternative to standard induction therapy in, the current FDA-approved standard of care for elderly patients or patients who are otherwise unfit for such an aggressive chemotherapy is treatment with a combination of the BCL-2 inhibitor venetoclax and a hypomethylating agent (HMA), such as azacitidine or decitabine. Specifically, treatment with a combination of venetoclax and azacitidine (hereafter referred to as “Ven / aza treatment” or “treatment with Ven / aza”) is estimated to induce a complete remission (CR) of AML in approximately 60-70% of treated patients.

[0027] However, this means that approximately 30-40% of patients do not end up responding to Ven / aza treatment and therefore do not achieve complete remission. There is a need in the art for methods of identifying this 30-40% of patients who are unlikely to respond to treatment with Ven / aza. The ability to identify these patients, prior to treatment, would allow clinicians to avoid the toxicity, expense and negative quality of life associated with an ineffective therapy. Moreover, these patients could be directed to other therapies, increasing their odds of survival. Finally, having a reliable method of identifying these patients will allow for the design of clinical trials aimed at testing personalized therapies for this specific AML patient population.

[0028] Numerous studies have described the properties of malignant stem cells that drive the pathogenesis of myeloid leukemias. Analyses of primary human tissue specimens, as well as many different mouse models have consistently shown that leukemia stem cells (LSCs) are biologically distinct from bulk tumor populations, and frequently demonstrate drug sensitivity / resistance profiles that differ from the majority of leukemic cell types. Analogous to normal hematopoietic stem cells, conventional LSCs are also thought to be mostly quiescent, and capable of giving rise to progeny that comprise the overall tumor population. As such, LSCs represent an important target in the development of novel therapies. Previous attempts to target the LSC population have been made, including focusing on specific cell surface antigens, metabolic interventions, epigenetic strategies, mutation-targeted approaches, and immune-based therapies. While multiple strategies derive from robust experimental evidence, as yet improvement of clinical outcomes due to direct eradication of LSCs has remained limited.

[0029] A major challenge in targeting LSCs is the inherent heterogeneity of malignant stem cells. Most notably, LSC populations derived from human AML patients demonstrate significant intra- and inter-patient heterogeneity in developmental stages andAttorney Docket No.: UNCO-050 / 001WO (300978-2221) immunophenotypes, which is thought to be in at least part driven by underlying genetic diversity. Importantly, recent studies have demonstrated that the existence of heterogeneous underlying LSC populations can mediate differing therapeutic outcomes of conventional chemotherapy and venetoclax-based therapies.

[0030] The present disclosure is based on, inter alia, the discovery of a particular subpopulation of leukemia stems cells, denoted as Myeloid / Lymphoid LSCs (M / L LSCs) that can be used to predict a subject’s response to treatment with a combination of a BCL-2 inhibitor (e.g. venetoclax) and a hypomethylating agent (e.g. azacitidine and decitabine).

[0031] Accordingly, the present disclosure provides, inter alia, a method of determining if a subject having AML will be responsive to treatment with a combination of at least one BCL- 2 inhibitor and at least one hypomethylating agent based on whether M / L LSCs are identified in samples from a subject, as well as methods of treating AML in a subject comprising administering a particular treatment to the subject based on whether a sample from a subject contains M / L LSCs.

[0032] Methods of Predicting Response to Treatment

[0033] The present disclosure provides methods of identifying if a subject having AML will be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the methods comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphoid leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; and c) identifying that the subject will not be responsive to the treatment when the presence of at least one M / L LSC is identified; or identifying that the subject will be responsive to the treatment when no M / L LSCs are identified.

[0034] The present disclosure provides methods of identifying if a subject having AML will be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the methods comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the number and / or percentage of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) comparing the number and / or percentage of m M / L LSCs identified in step (b) to a predetermined cutoff value ; and d) identifying that the subject will not be responsive to the treatment when the number and / or percentage of M / L LSCs is equal to or greater than theAttorney Docket No.: UNCO-050 / 001WO (300978-2221) predetermined cutoff value; or identifying that the subject will be responsive to the treatment when the number and / or percentage of M / L LSCs is less than the predetermined cutoff value.

[0035] The preceding methods can further comprise a step of providing a treatment recommendation to a clinician and / or subject. Accordingly, if a subject is identified as a subject who will not be responsive to a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method can further comprise providing a treatment recommendation that comprises administering an alternative treatment. In some aspects, the alternative treatment can comprise administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent. If a subject is identified as a subject that will be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, then the method can further comprise providing a treatment recommendation comprising administering said combination to the subject.

[0036] In some aspects of the preceding methods, identifying that a subject will be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying that the subject will have a durable remission after receiving the treatment of a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0037] In some aspects of the preceding methods, identifying that a subject will not be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying that the subject will be refractory to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent and / or that the subject will suffer a relapse after treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0038] Methods of treating AML

[0039] The present disclosure provides methods of treating acute myeloid leukemia (AML) in a subject, the methods comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphoid leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent when at least one M / L LSC is identified; or administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent when no M / L LSCs are identified.Attorney Docket No.: UNCO-050 / 001WO (300978-2221)

[0040] The present disclosure provides methods of treating acute myeloid leukemia (AML) in a subject, the methods comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the number and / or percentage of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) comparing the number and / or percentage of m M / L LSCs identified in step (b) to a predetermined cutoff value; and d) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent when the number and / or percentage of M / L LSCs is equal to or greater than the predetermined cutoff value; or administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent when the number and / or percentage of M / L LSCs is less than the predetermined cutoff value.

[0041] The present disclosure provides methods of treating a subject having AML, the methods comprising administering to the subject having AML a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent.

[0042] Identification of M / L LSCs

[0043] From the descriptions of the methods presented herein, it is appreciated that the methods of the present disclosure incorporate a step of identifying at least one, the number of, and / or the percentage of M / L LSCs in a plurality of cells in a sample from a subject.

[0044] It is understood that a step of identifying at least one of M / L LSC in a plurality of cells can result in the identification of no of M / L LSCs (i.e. the user can determine that there are no M / L LSCs present in the plurality of cells).

[0045] In the methods of the present disclosure, m-LSCs can be identified based on the novel immunophenotype of: CD34+ and CD7+.

[0046] The expression of the biomarkers described above, or any other biomarker describe herein, can be accomplished using any suitable method known in the art by the skilled artisan. Such methods include, but are not limited to, PCR, high-throughput sequencing, next generation sequencing, Northern Blot, reverse transcription PCR (RT-PCR), real-time PCR (qPCR), quantitative PCR, qRT-PCR, flow cytometry, mass spectrometry, microarray analysis, digital droplet PCR, Western Blot, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-SEQ), any single cell omics technologies, or any combination thereof.Attorney Docket No.: UNCO-050 / 001WO (300978-2221)

[0047] Hypomethylating agents

[0048] As would be appreciated by the skilled artisan, hypomethylating agents are agents that inhibit DNA methylation. In some aspects of the methods presented herein, a hypomethylating agent can be selected from azacitidine and decitabine. In some aspects of the methods presented herein, a hypomethylating agent can be any hypomethylating agent known in the art.

[0049] In some aspects, a hypomethylating agent can be azacitidine:, or a pharmaceutically acceptable salt, analog, derivative, salt or ester thereof. As would be appreciated by the skilled artisan, it is understood that azacitidine may be identified by any one of the following names: 5-Azacytidine, Azacytidine, Ladakamycin, 4-Amino-1-ȕ-D-ribofuranosyl-s-triazin-2(1H)-one, U-18496, CC-486 and 4- Amino-1-ȕ-D-ribofuranosyl-1,3,5-triazin-2(1H)-one. As would be appreciated by the skilled artisan, azacitidine may be identified as CAS No.320-67-2.

[0050] In some aspects, a hypomethylating agent can be decitabine:, or a pharmaceutically acceptable salt, analog, derivative, salt or ester thereof. As would be appreciated by the skilled artisan, it is understood that decitabine may be identified by any one of the following names: 5-aza-2'-deoxycytidine, 4-Amino-1-(2- deoxy-ȕ-D-erythro-pentofuranosyl)-1,3,5-triazin-2(1H)-one, 5-Aza-2ƍ-deoxycytidine, 5-Attorney Docket No.: UNCO-050 / 001WO (300978-2221) Azadeoxycytidine, 2-Desoxy-5-azacytidine and 2ƍ-Deoxy-5-azacytidine. As would be appreciated by the skilled artisan, decitabine may be identified as CAS No.2353-33-5.

[0051] Substitution of hypomethylating agent

[0052] In any of the methods described herein, a hypomethylating agent can be replaced by cytarabine:, or a pharmaceutically acceptable salt, analog, derivative, salt or ester thereof. As would be appreciated by the skilled artisan, it is understood that cytarabine may be identified by any one of the following names: 4-amino-1-[(2R,3S,4S,5R)- 3,4-dihydroxy-5- (hydroxymethyl)oxolan-2-yl] pyrimidin-2-one, Aracytidine and cytosine arabinoside . As would be appreciated by the skilled artisan, cytarabine may be identified as CAS No.147-94-4.

[0053] BCL-2 inhibitors

[0054] In some aspects of the methods presented herein, a BCL-2 inhibitor can be selected from venetoclax and navitoclax. In some aspects of the methods presented herein, a BCL-2 inhibitor can be any BCL-2 inhibitor known in the art.

[0055] In some aspects, a BCL-2 inhibitor can be venetoclax:pharmaceutically acceptable salt, analog, derivative, salt or ester thereof. As would be appreciated by the skilled artisan, it is understood that venetoclax may be identified by any one of the followingAttorney Docket No.: UNCO-050 / 001WO (300978-2221) names: GDC-0199, ABT-199, RG-7601, 4-(4-{[2-(4-Chlorophenyl)-4,4-dimethyl-1- cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4- ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide, Venclexta and Venclyxto. As would be appreciated by the skilled artisan, venetoclax may be identified as CAS No.1257044-40-8.

[0056] In some aspects, a BCL-2 inhibitor can be navitoclax:, or a pharmaceutically acceptable salt, analog, derivative, salt or ester thereof. As would be appreciated by the skilled artisan, it is understood that navitoclax may be identified by any one of the following names: ABT263, ABT-263 and 4-(4-{[2-(4-Chlorophenyl)-5,5- dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1- (phenylsulfanyl)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1- sulfonyl)benzamide. As would be appreciated by the skilled artisan, navitoclax may be identified as CAS No.923564-51-6.

[0057] In some aspects, a BCL-2 inhibitor can be BGB-11417.

[0058] In some aspects, a BCL-2 inhibitor can be ZN-d5.

[0059] M / L LSC targeting agents

[0060] In some aspects, an M / L LSC targeting agent can be at least one of: i) an anti-CD7 antibody; ii) an anti-CD7 immunotherapy, preferably wherein the immunotherapy comprises CAR-T and / or NK Cells that are directed specifically at CD7; iii) an agent that blocks CD7 signaling, preferably wherein the agent that blocks CD7 signaling is an inhibitor of the PI3Kinase and associated pathways; iv) an agent that inhibits CD7 activation, preferably wherein the agent that inhibits CD7 activation inhibits activation of CD7 by K12 and / or anyAttorney Docket No.: UNCO-050 / 001WO (300978-2221) additional CD7 ligands; and v) an agent that targets an M / L LSC-specific pathway and / or surface marker.

[0061] In some aspects of the methods of the present disclosure, immunotherapy can comprise administering a therapeutically effective amount of at least one antibody, at least one checkpoint inhibitor, at least one chimeric antigen receptor-modified T-Cell (CAR-T cell), or any combination thereof. Immunotherapy can comprise adoptive cell transfer therapy.

[0062] In some aspects, an immunotherapy can be an immunotherapy that specifically targets at least one M / L LSC antigen, including, but not limited to CD7. Accordingly, a non-limiting example of an immunotherapy that specifically targets at least one monocytic antigen can be a CAR-T cell that comprises a chimeric antigen receptor comprising an antigen binding domain that binds to CD7.

[0063] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An antibody that binds to a target refers to an antibody that is capable of binding the target with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting the target. In one embodiment, the extent of binding of an anti-target antibody to an unrelated, non-target protein is less than about 10% of the binding of the antibody to target as measured, e.g., by a radioimmunoassay (RIA) or biacore assay. In certain embodiments, an antibody that binds to a target has a dissociation constant (Kd) of < 1 ^Ȃ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 108M or less, e.g. from 108M to 1013M, e.g., from 109M to 1013M). In certain embodiments, an anti-target antibody binds to an epitope of a target that is conserved among different species.

[0064] A "blocking antibody" or an "antagonist antibody" is one that partially or fully blocks, inhibits, interferes, or neutralizes a normal biological activity of the antigen it binds. For example, an antagonist antibody may block signaling through an immune cell receptor (e.g., a T cell receptor) so as to restore a functional response by T cells (e.g., proliferation, cytokine production, target cell killing) from a dysfunctional state to antigen stimulation.

[0065] An "agonist antibody" or "activating antibody" is one that mimics, promotes, stimulates, or enhances a normal biological activity of the antigen it binds. Agonist antibodies can also enhance or initiate signaling by the antigen to which it binds. In some embodiments, agonist antibodies cause or activate signaling without the presence of the natural ligand. ForAttorney Docket No.: UNCO-050 / 001WO (300978-2221) example, an agonist antibody may increase memory T cell proliferation, increase cytokine production by memory T cells, inhibit regulatory T cell function, and / or inhibit regulatory T cell suppression of effector T cell function, such as effector T cell proliferation and / or cytokine production.

[0066] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

[0067] CAR-T cells are T cells that are genetically modified to stably express at least one chimeric antigen receptor (CAR). A CAR can comprise an extracellular domain, transmembrane domain and a cytoplasmic domain. A CAR can comprise an antigen binding domain. An antigen binding domain can be located in an extracellular domain. In some aspects of the methods of the present disclosure, the antigen binding domain binds to at least one AML cell surface protein. In some aspects of the methods of the present disclosure, the antigen binding domain binds to CD64 and / or LILRB4. A CAR can also comprise an extracellular spacer (hinge) domain. An extracellular spacer can be located in an extracellular domain. A CAR can comprise a signaling domain. A signaling domain can be a T-cell activation domain. A signaling domain can be located in a cytoplasmic domain. A CAR can comprise at least one costimulatory domain. A CAR can comprise at least two costimulatory domains. A CAR can comprise at least three costimulatory domains. A costimulatory domain can be located in a cytoplasmic domain.

[0068] In some aspects of the methods of the present disclosure CAR-T cells can be autologous with respect to a subject. In some aspects, CAR-T cells can be allogeneic with respect to a subject.

[0069] In some aspects of the methods of the present disclosure, CAR-T cells may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions can comprise a plurality of CAR-T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such asAttorney Docket No.: UNCO-050 / 001WO (300978-2221) EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. CAR-T cells and related compositions can be administered to a subject intravenously.

[0070] A CAR-T cell can comprise a chimeric antigen receptor. A chimeric antigen receptor can comprise an antigen binding domain. An antigen binding domain can bind to CD64 and / or LILRB4.

[0071] Subjects

[0072] As used herein, the term “subject” includes human and non-human animals, as well as cell lines, cell cultures, tissues, and organs. In some aspects, the subject is a mammal. The mammal can be e.g., a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig. The subject can also be a bird or fowl. In some aspects, the subject is a human.

[0073] As used herein, the term “subject in need thereof” refers, both of which refer to a subject having a disease or having an increased risk of developing the disease. A “subject” includes a mammal. The mammal can be e.g., a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig. The subject can also be a bird or fowl. In some aspects, the mammal is a human. A subject in need thereof can be one who has been previously diagnosed or identified as having a disease or disorder disclosed herein. A subject in need thereof can also be one who is suffering from a disease or disorder disclosed herein. Alternatively, a subject in need thereof can be one who has an increased risk of developing such disease or disorder relative to the population at large (i.e., a subject who is predisposed to developing such disorder relative to the population at large). A subject in need thereof can have a refractory or resistant a disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that does not respond or has not yet responded to treatment). The subject may be resistant at start of treatment or may become resistant during treatment. In some aspects, the subject in need thereof received and failed all known effective therapies for a disease or disorder disclosed herein. In some aspects, the subject in need thereof received at least one prior therapy.

[0074] In some aspects of the methods of the present disclosure, the subject is a human.

[0075] In some aspects of the methods of the present disclosure, the subject is a subject having AML who has not received any treatment for AML.

[0076] In some aspects of the methods of the present disclosure, the subject is a subject having AML who has received previously received at least one AML treatment. In some aspects, the at least one treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.Attorney Docket No.: UNCO-050 / 001WO (300978-2221)

[0077] In some aspects of the methods of the present disclosure, a subject is a subject who has at least one M / L LSC present in their population of AML cells. In some aspects of the methods of the present disclosure, a subject is a subject whose AML cell population contains a percentage of M / L LSC that are greater than a predetermined cutoff percentage. In some as aspects of the methods of the present disclosure, a subject is a subject whose AML cell population contains a number of M / L LSC that are greater than a predetermined cutoff number.

[0078] Samples

[0079] In some aspects of the methods of the present disclosure, a sample can comprise blood, a bone marrow biopsy, a bone marrow aspirate, a biopsy of a chloroma, a tissue biopsy, cerebrospinal fluid or any combination thereof.

[0080] In some aspects, a sample can be a bone marrow biopsy.

[0081] In some aspects, a sample can be a bone marrow aspirate.

[0082] In some aspects, a sample can be a biopsy of a chloroma.

[0083] General Definitions

[0084] As used herein, the expressions “one or more of A, B, or C,” “one or more A, B, or C,” “one or more of A, B, and C,” “one or more A, B, and C,” “selected from the group consisting of A, B, and C”, “selected from A, B, and C”, and the like are used interchangeably and all refer to a selection from a group consisting of A, B, and / or C, i.e., one or more As, one or more Bs, one or more Cs, or any combination thereof, unless indicated otherwise.

[0085] It is to be understood that, unless otherwise stated, any description of a method of treatment includes use of the agents to provide such treatment as is described herein. It is to be further understood, unless otherwise stated, any description of a method of treatment includes use of the agents to prepare a medicament to treat such condition. The treatment includes treatment of human or non-human animals including rodents and other disease models used herein.

[0086] As used herein, the term “treating” or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of an agent described in the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model. It is to be appreciated that references to “treating” or “treatment” include the alleviation of established symptoms ofAttorney Docket No.: UNCO-050 / 001WO (300978-2221) a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) preventing the appearance of clinical symptoms of the state or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

[0087] It is to be understood that an agent described in the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, can or may also be used to prevent a relevant disease, condition or disorder, or used to identify suitable candidates for such purposes.

[0088] As used herein, the term “preventing,” “prevent,” or “protecting against” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.

[0089] As used herein, the term “pharmaceutically acceptable” refers to those compounds, anions, cations, materials, compositions, carriers, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0090] It is understood that the agents described herein can be administered to a subject in at least one therapeutically effective amount. As used herein, the term “therapeutically effective amount”, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject’s body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician. A clinician can also determine the therapeutically effective amounts of the agents described herein using established dosing and administration protocols for the agents described herein.

[0091] It is to be understood that, for any agent, the therapeutically effective amount can beAttorney Docket No.: UNCO-050 / 001WO (300978-2221) estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic / prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50(the dose therapeutically effective in 50 % of the population) and LD50(the dose lethal to 50 % of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0092] Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance / response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

[0093] As used herein, the term “pharmaceutically acceptable salts” refer to derivatives of the agents described herein wherein the agent is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent agent formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurring amine acids, e.g.,Attorney Docket No.: UNCO-050 / 001WO (300978-2221) glycine, alanine, phenylalanine, arginine, etc.

[0094] In some aspects, the pharmaceutically acceptable salt is a sodium salt, a potassium salt, a calcium salt, a magnesium salt, a diethylamine salt, a choline salt, a meglumine salt, a benzathine salt, a tromethamine salt, an ammonia salt, an arginine salt, or a lysine salt.

[0095] Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like. The present disclosure also encompasses salts formed when an acidic proton present in the parent agent either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. In the salt form, it is understood that the ratio of the agent to the cation or anion of the salt can be 1:1, or any ratio other than 1:1, e.g., 3:1, 2:1, 1:2, or 1:3.

[0096] It is to be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein, of the same salt.

[0097] The term “refractory” as used herein, is used in its broadest sense to refer to instances in which the disease present in a subject does not respond to a particular therapy, i.e. the therapy provides no or decreased clinical benefit to that particular subject.

[0098] As used herein, the term “combination therapy” or “co-therapy” includes the administration of an agent disclosed herein, or a pharmaceutically acceptable salt, polymorph or solvate thereof, and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.

[0099] As used herein, the term “temporal proximity” refers to that administration of one therapeutic agent occurs within a time period before or after the administration of another therapeutic agent, such that the therapeutic effect of the one therapeutic agent overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, the therapeutic effect of the one therapeutic agent completely overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, “temporal proximity” means that administration of one therapeutic agent occurs within a time period before or after the administration of anotherAttorney Docket No.: UNCO-050 / 001WO (300978-2221) therapeutic agent, such that there is a synergistic effect between the one therapeutic agent and the other therapeutic agent. “Temporal proximity” may vary according to various factors, including but not limited to, the age, gender, weight, genetic background, medical condition, disease history, and treatment history of the subject to which the therapeutic agents are to be administered; the disease or condition to be treated or ameliorated; the therapeutic outcome to be achieved; the dosage, dosing frequency, and dosing duration of the therapeutic agents; the pharmacokinetics and pharmacodynamics of the therapeutic agents; and the route(s) through which the therapeutic agents are administered. In some embodiments, “temporal proximity” means within 15 minutes, within 30 minutes, within an hour, within two hours, within four hours, within six hours, within eight hours, within 12 hours, within 18 hours, within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within a week, within 2 weeks, within 3 weeks, within 4 weeks, with 6 weeks, or within 8 weeks. In some embodiments, multiple administration of one therapeutic agent can occur in temporal proximity to a single administration of another therapeutic agent. In some embodiments, temporal proximity may change during a treatment cycle or within a dosing regimen.

[0100] Experimental Examples

[0101] Example 1 – CD7 and CD34 co-expression segregate subpopulations with different overall survival outcomes

[0102] The following is a non-limiting example that demonstrates that subjects having AML that co-expresses CD7 and CD34 exhibit poorer response to treatment with a combination of venetoclax and azacitidine.

[0103] A series of samples from subject having AML and who have been treated with a combination of venetoclax and azacitidine was analyzed by flow cytometry to determine the expression of CD7 and CD34 on AML cells. The survival probability of the patients was then plotted as a function of time (in days) post-treatment.

[0104] FIG.1A shows the analysis of all of the AML samples, regardless of whether the subjects also received a stem cell transplant (SCT). As shown in FIG.1A, subjects who had AML that exhibited cells that co-expressed CD7+ and CD34+ (i.e. M / L LSCs) exhibited poorer outcomes following venetoclax / azacitidine treatment as compared to subjects who exhibited cells that were CD7- / CD34- or CD7- / CD34+. Without wishing to be bound by theory, these results demonstrate that the presence of CD7+ / CD34+ cells in biological samples from subjects having AML can be used to determine that they will have a poor response to treatment with a combination of venetoclax and azacitidine.Attorney Docket No.: UNCO-050 / 001WO (300978-2221)

[0105] The data was further analyzed to either exclude subjects who had received an SCT (FIG.1B) or to censor the data by the subjects who had received an SCT (FIG.1C). Similar to the results presented in FIG.1A, FIGs.1B and 1C show that subjects who had AML that exhibited cells that co-expressed CD7+ and CD34+ (i.e. M / L LSCs) exhibited poorer outcomes following venetoclax / azacitidine treatment as compared to subjects who exhibited cells that were CD7- / CD34- or CD7- / CD34+.

[0106] Without wishing to be bound by theory, the results presented in this example demonstrate that the methods of the present disclosure that are based on the identification of CD7+ / CD34+ M / L LSCs can be used to predict the response that a subject having AML will have to treatment with a combination of venetoclax and azacitidine.

Claims

Attorney Docket No.: UNCO-050 / 001WO (300978-2221) What is claimed is:

1. A method of treating acute myeloid leukemia (AML) in a subject, the method comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphoid leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent when at least one M / L LSC is identified; or administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent when no M / L LSCs are identified.

2. A method of identifying if a subject having AML will be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphoid leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) identifying that the subject will not be responsive to the treatment when the presence of at least one M / L LSC is identified; or identifying that the subject will be responsive to the treatment when no M / L LSCs are identified.

3. A method of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the number and / or percentage of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+;Attorney Docket No.: UNCO-050 / 001WO (300978-2221) c) comparing the number and / or percentage of m M / L LSCs identified in step (b) to a predetermined cutoff value; and d) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent when the number and / or percentage of M / L LSCs is equal to or greater than the predetermined cutoff value; or administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent when the number and / or percentage of M / L LSCs is less than the predetermined cutoff value.

4. A method of identifying if a subject having AML will be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method comprising: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the number and / or percentage of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) comparing the number and / or percentage of m M / L LSCs identified in step (b) to a predetermined cutoff value ; and d) identifying that the subject will not be responsive to the treatment when the number and / or percentage of M / L LSCs is equal to or greater than the predetermined cutoff value; or identifying that the subject will be responsive to the treatment when the number and / or percentage of M / L LSCs is less than the predetermined cutoff value.

5. The method of any one of the preceding claims, wherein the at least one M / L LSC targeting agent comprises the administration of at least one of: i) an anti-CD7 antibody; ii) an anti-CD7 immunotherapy, preferably wherein the immunotherapy comprises CAR-T and / or NK Cells that are directed specifically at CD7; iii) an agent that blocks CD7 signaling, preferably wherein the agent that blocks CD7 signaling is an inhibitor of the PI3Kinase and associated pathways; iv) an agent that inhibits CD7 activation, preferably wherein the agent that inhibits CD7 activation inhibits activation of CD7 by K12 and / or any additional CD7 ligands; and v) an agent that targets an M / L LSC-specific pathway and / or surface marker.Attorney Docket No.: UNCO-050 / 001WO (300978-2221) 6. The method of any one of the preceding claims, wherein the at least one hypomethylating agent is selected from azacitidine and decitabine.

7. The method of any one of the preceding claims, wherein the at least one BCL-2 inhibitor is selected from venetoclax and navitoclax.

8. The method of any one of the preceding claims, wherein step (a) comprises performing PCR, high-throughput sequencing, next generation sequencing, Northern Blot, reverse transcription PCR (RT-PCR), real-time PCR (qPCR), quantitative PCR, qRT-PCR, flow cytometry, mass spectrometry, microarray analysis, digital droplet PCR, Western Blot, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-SEQ), any single cell omics technologies, or any combination thereof.

9. The method of any one of the preceding claims, wherein the subject is: i) a subject having AML who has not received any treatment for AML; or ii) a subject having AML who has received previously received at least one AML treatment, preferably wherein the at least one AML treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

10. The method of any one of the preceding claims, wherein identifying that a subject will be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying that the subject will have a durable remission after receiving the treatment of a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

11. The method of any one of the preceding claims, wherein identifying that a subject will not be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying that the subject will be refractory to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent and / or that the subject will suffer a relapse after treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.Attorney Docket No.: UNCO-050 / 001WO (300978-2221) 12. The method of any one of the preceding claims, wherein the biological sample comprises blood, a bone marrow biopsy, a bone marrow aspirate, a biopsy of a chloroma, a tissue biopsy, cerebrospinal fluid or any combination thereof.

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