EP4626439A2 - Methods of treating acute myeloid leukemia - Google Patents

Methods of treating acute myeloid leukemia

Info

Publication number
EP4626439A2
EP4626439A2 EP23837488.8A EP23837488A EP4626439A2 EP 4626439 A2 EP4626439 A2 EP 4626439A2 EP 23837488 A EP23837488 A EP 23837488A EP 4626439 A2 EP4626439 A2 EP 4626439A2
Authority
EP
European Patent Office
Prior art keywords
subject
lsc
aml
identified
cd11b
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23837488.8A
Other languages
German (de)
French (fr)
Inventor
Craig T. Jordan
Clayton Smith
Shanshan Pei
Austin E. GILLEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Colorado Denver
Original Assignee
University of Colorado System
University of Colorado Colorado Springs
University of Colorado Denver
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Colorado System, University of Colorado Colorado Springs, University of Colorado Denver filed Critical University of Colorado System
Publication of EP4626439A2 publication Critical patent/EP4626439A2/en
Pending legal-status Critical Current

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    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Definitions

  • Acute myeloid leukemia 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).
  • venetoclax can induce responses in approximately 60-70% of older previously untreated AML patients, many of whom are unfit for conventional induction therapy.
  • the present disclosure provides a method of treating acute myeloid leukemia (AML) in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression Attorney Docket No.: UNCO-049/001WO (300978-2220) measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) administering to the subject a combination of at least one BCL- 2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent when at least one m-LSC is identified; or administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating
  • AML acute myeloid leukemia
  • the present disclosure provides 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, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) identifying that the subject will not be responsive to the treatment when the presence of at least one m-LSC is identified; or identifying that the subject will be responsive to the treatment when no m-LSCs are identified.
  • m-LSC monocytic leukemia stem cell
  • 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, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of m-LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) comparing the number and/or percentage of m-LSCs identified in step (b) to a predetermined cutoff value; d) identifying that the subject will not be responsive to the treatment when the number and/or percentage of m-LSCs is equal to or greater than the predetermined cutoff value; or identifying that the subject will be Attorney Docket No.:
  • step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the number and/or percentage of m-LSCs in the plurality of cells is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+.
  • 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.
  • 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.
  • the present disclosure provides methods of identifying if a subject having AML will be responsive to treatment with a CD70-targeting agent, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, and CD70+; c) identifying that the subject will be responsive to the treatment when at least one CD70+ m-LSC is identified.
  • m-LSC monocytic leukemia stem cell
  • step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, CD64 and CD70
  • step (b) can comprise identifying the presence of at least one Attorney Docket No.: UNCO-049/001WO (300978-2220) CD70+ m-LSC, wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD64+ and CD70+.
  • step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e.
  • the preceding method can further comprise identifying that the subject will not be responsive to the treatment when the number and/or percentage of CD70+ m-LSCs is less than the predetermined cutoff value.
  • step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e.
  • step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, CD64 and CD70
  • step (b) can comprise identifying the number and/or percentage of CD70+ m-LSCs, wherein a cell is identified as a CD70+ m- LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD64+ and CD70.
  • step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e.
  • a predetermined cutoff value can be determined by comparing the number and/or percentage of CD70+ m-LSCs in samples obtained from one or more subjects known to be responsive to treatment with a CD70- targeting agent and the number and/or percentage of CD70+ m-LSCs samples from one or more subjects known to be not responsive to treatment with a CD70-targeting agent.
  • the skilled artisan can used methods known in the art to make such comparisons and determine a suitable predetermined cutoff value that will allow for the discrimination between responders and non-responders.
  • the preceding methods can further comprise a step of providing a treatment recommendation to a clinician and/or subject.
  • step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the presence of at least one m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+.
  • step (b) can further comprise identifying the presence of at least one m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and GPR56-.
  • the present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of m-LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) comparing the number and/or percentage of m-LSCs identified in step (b) to a predetermined cutoff value; d) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent when the number and/or percentage of m-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
  • step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the number and/or percentage of m-LSCs in the plurality of cells is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+.
  • step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and CD64
  • step (b) can comprise identifying the number and/or percentage of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD64+.
  • step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e.
  • step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and GPR56
  • step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and GPR56-.
  • the present disclosure provides methods of treating a subject having AML, the method 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-LSC targeting agent.
  • the present disclosure provides methods of treating a subject having AML, wherein the subject has AML that exhibits the presence of at least one m-LSC, the method comprising administering to said subject at least one m-LSC targeting agent.
  • the present disclosure provides a methods of treating a subject having AML, wherein the subject has AML that exhibits a number and/or percentage of m-LSC cells that is greater than or equal to a predetermined cutoff value, the method comprising administering to said subject at least one m-LSC targeting agent.
  • the present disclosure provides methods of treating a subject having AML, wherein the subject has AML that exhibits the presence of at least one m-LSC, the method comprising administering to said subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent.
  • the present disclosure provides a methods of treating a subject having AML, wherein the subject has AML that exhibits a number and/or percentage of m-LSC cells that is greater than or equal to a predetermined cutoff value, the method comprising administering to said Attorney Docket No.: UNCO-049/001WO (300978-2220) subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent.
  • the present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) administering to the subject a treatment comprising at least one CD70-targeting agent when at least one CD70+ m-LSC is identified.
  • m-LSC monocytic leukemia stem cell
  • the treatment can further comprise at least one BCL-2 inhibitor and at least one hypomethylating agent.
  • step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36 and CD70+) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and CD70+.
  • step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, CD64 and CD70+
  • step (b) can comprise identifying the presence of at least one CD70+ m-LSC wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD64+ and CD70+.
  • step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e.
  • step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, GPR56- and CD70+.
  • step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, GPR56 and CD70
  • step (b) can comprise identifying the presence of at least one CD70+ m-LSC, wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, GPR56- and CD70+.
  • the present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of CD70+ m-LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b- Attorney Docket No.: UNCO-049/001WO (300978-2220) , CD14-, CD36- and CD70+; c) comparing the number and/or percentage of CD70+ m-LSCs identified in step (b) to a predetermined cutoff value; d) administering to the subject treatment comprising at least one CD70-targeting agent when the number and/or percentage of CD70+ m-LSCs is equal to or greater than the predetermined cut
  • the treatment can further comprise at least one BCL-2 inhibitor and at least one hypomethylating agent.
  • step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36 and CD70) and step (b) can further comprise identifying the number and/or percentage of CD70+ m-LSCs in the plurality of cells is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and CD70+.
  • step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, GPR56- and CD70+.
  • step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, GPR56 and CD70
  • step (b) can comprise identifying the presence of at least one CD70+ m-LSC, wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, GPR56- and CD70+.
  • the present disclosure provides methods of treating a subject having AML, the method comprising administering to the subject having AML a treatment comprising at least one anti-CD70 target agent, wherein the subject has at least one CD70+ m-LSCs.
  • m-LSCs monocytic leukemia stem cells
  • the user performing the method can measure the expression of CD34, CD4, CD11b, CD14 and CD36 (and optionally one or more of CD117, CD244 and CD64) in cells from a sample from the subject in order to determine whether m-LSCs are present in the sample, and optionally, the percentage and/or number of m-LSCs in the sample.
  • the CD34-, CD4+, CD11b-, CD14- and CD36- immunophenotype can be optionally further supplemented with one or more markers selected from CD117-, CD244-, CD64+ and GPR56.
  • the user performing the method can measure the expression of CD34, CD4, CD11b, CD14 and CD36 (and optionally one or more of CD117, CD244, CD64 and GPR56) in cells from a sample from the subject in order to determine whether m-LSCs are present in the sample, and optionally, the percentage and/or number of m-LSCs in the sample.
  • 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), or any combination thereof.
  • PCR high-throughput sequencing
  • next generation sequencing Northern Blot
  • RT-PCR reverse transcription PCR
  • qPCR real-time PCR
  • quantitative PCR quantitative PCR
  • qRT-PCR quantitative PCR
  • flow cytometry flow cytometry
  • mass spectrometry microarray analysis
  • digital droplet PCR Western Blot
  • CITE-SEQ Cellular Indexing of Transcriptomes and Epitopes by Sequencing
  • a transcriptomic feature that identifies an m-LSC can be an upregulation in the expression of at least one of the biomarkers put forth in Table 5. As would be appreciated by the skilled artisan, an upregulation corresponds to an expression level that is greater than a control expression value seen in other types of cells. [00106] In some aspects of the methods of the present disclosure, a transcriptomic feature that identifies an m-LSC can be at least one of the biomarkers, or any combination of the biomarkers, put forth in Table 1A.
  • Table 1B – GSEA gene signatures upregulated in m-LSCs Any of the transcriptomic features put forth in Tables 1A, 1B and 5 can be combined together in any combination.
  • an m-LSC cell may be identified by the upregulation of two of the biomarkers put forth in Table 1A and three of the biomarkers put forth in Table 5.
  • identifying an m-LSC using the transcriptomic features described herein can comprise measuring the expression of the one or more biomarkers that make up the transcriptomic feature.
  • This measurement of expression 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, RNA sequencing, flow cytometry, mass spectrometry, microarray analysis, digital droplet PCR, Western Blot, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-SEQ), or any combination thereof.
  • Identifying an m-LSC using the transcriptomic features described herein can also comprise performing a transcriptomic analysis using any of the standard methods known in the art for transcriptomic analysis, including, but not limited to the methods described herein, such as CITE-SEQ.
  • Identification of CD70+ m-LSCs [00114] 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 CD70+ monocytic leukemia stem cells (m-LSCs) in a plurality of cells in a sample from a subject.
  • this immunophenotype can optionally be further supplemented with one or more makers selected from CD117-, CD244-, CD64+, and GPR56-.
  • the user performing the method can measure the expression of CD34, CD4, CD11b, CD14, CD36 and CD70 (and optionally one or more of CD117, CD244, CD64 and GPR56) in cells from a sample from the subject in order to determine whether CD70+ m- LSCs are present in the sample, and optionally, the percentage and/or number of CD70+ m- LSCs in the sample.
  • 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), or any combination thereof.
  • CD70+ m-LSCs can be alternatively or further identified by one or more transcriptomic features described herein.
  • a transcriptomic feature that identifies a CD 70+ m-LSC can be an upregulation in the expression of at least one of the biomarkers put forth in Table 5. As would be appreciated by the skilled artisan, an upregulation corresponds to an expression level that is greater than a control expression value seen in other types of cells.
  • a transcriptomic feature that identifies a CD70+ m-LSC can be at least one of the biomarkers, or any combination of the biomarkers, put forth in Table 1A.
  • Any of the transcriptomic features put forth in Tables 1A, 1B and 5 can be combined together in any combination.
  • a CD70+ m-LSC cell may be identified by the upregulation of two of the biomarkers put forth in Table 1A and three of the biomarkers put forth in Table 5, in addition to the measurement of CD70 expression.
  • identifying a CD70+ m-LSC using the transcriptomic features described herein can comprise measuring the expression of the one or more biomarkers that make up the transcriptomic feature. This measurement of expression can be accomplished using any suitable method known in the art by the skilled artisan.
  • Identifying a CD70+ m-LSC using the transcriptomic features described herein can also comprise performing a transcriptomic analysis using any of the standard methods known in the art for transcriptomic analysis, including, but not limited to the methods described herein, such as CITE-SEQ.
  • hypomethylating agents are agents that inhibit DNA methylation.
  • a hypomethylating agent can be selected from azacitidine, cytarabine and decitabine.
  • a hypomethylating agent can be any hypomethylating agent known in the art.
  • 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 cytarabine, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m- LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) identifying that the subject will not be responsive to the treatment when the presence of at least one m-LSC is identified; or identifying that the subject will be responsive to the treatment when no m-LSCs are identified.
  • m- LSC monocytic leukemia stem cell
  • the present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) administering to the subject a combination of at least one BCL-2 inhibitor, cytarabine, and at least one m-LSC targeting agent when at least one m-LSC is identified; or administering to the subject a combination of at least one BCL-2 inhibitor and cytarabine when no m-LSCs are identified.
  • m-LSC monocytic leukemia stem cell
  • the present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of m-LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) comparing the number and/or percentage of m-LSCs identified in step (b) to a predetermined cutoff value; d) administering to the subject a combination of at least one BCL-2 inhibitor, cytarabine, and at least one m-LSC targeting agent when the number and/or percentage of m-LSCs is equal to or greater than the predetermined cutoff value; or administering to the subject a combination of at least one
  • 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. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00136] In some aspects, a BCL-2 inhibitor can be venetoclax: pharmaceutically acceptable salt, analog, derivative, salt or ester thereof.
  • venetoclax may be identified by any one of the following 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.
  • a BCL-2 inhibitor can be navitoclax: pharmaceutically acceptable salt, analog, derivative, salt or ester thereof.
  • 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- Attorney Docket No.: UNCO-049/001WO (300978-2220) sulfonyl)benzamide.
  • navitoclax may be identified as CAS No.923564-51-6.
  • a BCL-2 inhibitor can be BGB-11417.
  • a BCL-2 inhibitor can be ZN-d5.
  • CD70-targeting agents can be at least one of: i) an anti-CD70 antibody; ii) an anti-CD70 immunotherapy, preferably wherein the immunotherapy comprises CAR-T and/or NK Cells that are directed specifically at CD70; iii) an agent that blocks CD70 signaling, preferably wherein the agent that blocks CD70 signaling prevents binding of CD27 and CD70.
  • an m-LSC targeting agent can be an agent that modulates one-carbon metabolism.
  • an m-LSC targeting agent can be an agent that modulates at least one of purine synthesis and pyrimidine synthesis.
  • An agent that modulates one-carbon metabolism is selected from methotrexate, brequinar and cladribine.
  • an m-LSC targeting agent can be cladribine: Attorney Docket No.: UNCO-049/001WO (300978-2220) , or a pharmaceutically acceptable salt, analog, derivative, salt or ester thereof.
  • cladribine may be identified by any one of the following names: 5-(6-Amino- 2-chloro-purin-9-yl)-2-(hydroxymethyl)oxolan-3-ol, 2-Chloro-2 ⁇ -deoxyadenosine, 2-Chloro- 2-Chlorodeoxyadenosine.
  • cladribine may be identified as CAS No.4291-63-8.
  • an m-LSC targeting agent can be brequinar: pharmaceutically acceptable salt, analog, derivative, salt or ester thereof.
  • brequinar may be identified by any one of the following names: 6-fluoro-2- (2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinolinecarboxylic acid, Biphenquinate and BPQ. As would be appreciated by the skilled artisan, brequinar may be identified as CAS No. 96187-53-0. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00151]
  • an m-LSC targeting agent can be methotrexate: pharmaceutically acceptable salt, analog, derivative, salt or ester thereof.
  • methotrexate may be identified by any one of the following names: (2S)-2-[(4- ⁇ [(2,4-Diaminopteridin-6- yl)methyl](methyl)amino ⁇ benzoyl)amino]pentanedioic acid, MTX, 4-Amino-N10- methylpteroylglutamic acid and N-[p-[[2,4-Diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-(+)-glutamic acid.
  • methotrexate may be identified as CAS No.59-05-2.
  • an m-LSC targeting agent can be an agent that inhibits MCL1.
  • agents that inhibit MCL1 are VU661013 and S63845.
  • an m-LSC targeting agent can be an immunotherapy.
  • an m-LSC targeting therapy can be an antifolate.
  • antifolates include methotrexate, pralatrexate and pemetrexed.
  • 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.
  • an immunotherapy can be an immunotherapy that specifically targets at least one monocytic antigen, including, but not limited to CD64 and LILRB4.
  • 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 CD64 and/or LILRB4.
  • 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 of 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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.
  • an agent described in the present disclosure can or may also be used to prevent a relevant disease, condition or disorder, or used to identify suitable candidates for such purposes.
  • the term “preventing,” “prevent,” or “protecting against” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.
  • 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.
  • the agents described herein can be administered to a subject in at least one therapeutically effective amount.
  • 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.
  • the therapeutically effective amount can be 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 LD 50 (the dose lethal to 50 % of the Attorney Docket No.: UNCO-049/001WO (300978-2220) 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.
  • 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.
  • 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.
  • 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,
  • compositions 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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.
  • a metal ion e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion
  • organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
  • 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.
  • references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein, of the same salt.
  • 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.
  • 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.
  • 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) which the therapeutic agents are administered.
  • “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.
  • multiple administration of one therapeutic agent can occur in temporal proximity to a single administration of another therapeutic agent.
  • temporal proximity may change during a treatment cycle or within a dosing regimen.
  • a method of treating acute myeloid leukemia (AML) in a subject comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent when at least one m-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-LSCs are identified.
  • m-LSC monocytic leukemia stem cell
  • Embodiment 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, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) identifying that the subject will not be responsive to the treatment when the presence of at least one m-LSC is identified; or identifying that the subject will be responsive to the treatment when no m-LSCs are identified.
  • m-LSC monocytic leukemia stem cell
  • Embodiment 3a The method of embodiment 1 or embodiment 2, wherein step (a) further comprises measuring the expression of at least CD117, CD244 and CD64, wherein a cell is identified as an m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+.
  • step (a) further comprises measuring the expression of at least CD117, CD244 and CD64, wherein a cell is identified as an m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+.
  • step (a) further comprises measuring the expression of at least CD117, CD244, CD64 and GPR56, wherein a cell is identified as an m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, and GPR56-.
  • Embodiment 4 The method of any one of the preceding embodiments, wherein the at least one m-LSC targeting agent is an agent modulates one-carbon metabolism, is an agent that modulates purine synthesis, is an agent that modulates pyrimidine synthesis, or any combination thereof.
  • Embodiment 6 The method of any one of the preceding embodiments, wherein the at least one m-LSC targeting agent is selected from methotrexate, brequinar and cladribine.
  • Embodiment 6 The method of any one of the preceding embodiments, wherein the at least one hypomethylating agent is selected from azacitidine and decitabine.
  • Embodiment 7. The method of any one of the preceding embodiments, wherein the at least one BCL-2 inhibitor is selected from venetoclax and navitoclax.
  • 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), or any combination thereof.
  • 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), or any combination thereof.
  • RT-PCR reverse transcription PCR
  • qPCR real-time PCR
  • quantitative PCR quantitative PCR
  • qRT-PCR quantitative PCR
  • Embodiment 11 The method of embodiment 10, wherein the at least one AML treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.
  • Embodiment 12 The method of any one of the preceding embodiments, wherein identifying that a subject will be responsive to treatment with a combination of at least one Attorney Docket No.: UNCO-049/001WO (300978-2220) 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.
  • Embodiment 13 The method of any one of the preceding embodiments, 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.
  • Embodiment 14 Embodiment 14.
  • 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.
  • Embodiment 15 The method of embodiment 14, wherein the sample is a bone marrow biopsy.
  • Embodiment 16 The method of embodiment 14, wherein the sample is a bone marrow aspirate.
  • Embodiment 17. The method of embodiment 14, wherein the sample is a biopsy of a chloroma.
  • step (a) further comprises performing a transcriptomic analysis of the plurality of cells in the sample; and step (b) further comprises identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on transcriptomic analysis performed in step (a), wherein a cell is identified as an m-LSC based on at least one of the following: i) an upregulation in the expression of at least one of biomarker from Table 5; ii) the expression of at least one biomarker from Table 1A; iii) an upregulation in the expression of at least one GSEA gene signature from Table 1B.
  • m-LSC monocytic leukemia stem cell
  • Embodiment 21 A method of treating acute myeloid leukemia (AML) in a subject, the method comprising: Attorney Docket No.: UNCO-049/001WO (300978-2220) a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) administering to the subject a treatment comprising at least one CD70-
  • AML acute myeloid leukemia
  • Embodiment 22 A method of identifying if a subject having AML will be responsive to treatment with a CD70-targeting agent, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m- LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m- LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) identifying that the subject will be responsive to the treatment when the presence of at least one CD70+ m-LSC is identified.
  • m- LSC monocytic leukemia stem cell
  • Embodiment 23 The method of embodiment 21 or embodiment 22, wherein step (a) further comprises measuring the expression of at least CD117, CD244 and CD64, wherein a cell is identified as a CD70+ m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and CD70+.
  • Embodiment 24 Embodiment 24.
  • the at least one CD70-targeting agent is: i) an anti-CD70 antibody, preferably wherein the anti-CD70 antibody is cusatuzumab; ii) an anti-CD70 immunotherapy, preferably wherein the immunotherapy comprises CAR-T and/or NK Cells that are directed specifically at CD70; or iii) an agent that blocks CD70 signaling, preferably wherein the agent that blocks CD70 signaling prevents binding of CD27 and CD70.
  • the at least one hypomethylating agent is selected from azacitidine and decitabine.
  • step (a) comprises performing PCR, high-throughput sequencing, next generation sequencing, Attorney Docket No.: UNCO-049/001WO (300978-2220) 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, [00225] Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-SEQ), or any combination thereof.
  • Embodiment 28 The method of any one of the preceding embodiments, wherein the subject is a subject having AML who has not received any treatment for AML.
  • Embodiment 29 The method any one of the preceding embodiments, wherein the subject is a subject having AML who has received previously received at least one AML treatment.
  • Embodiment 30 The method of embodiment 29, wherein the at least one AML treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.
  • Embodiment 31 Embodiment 31.
  • 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.
  • Embodiment 32 The method of embodiment 31, wherein the sample is a bone marrow biopsy.
  • Embodiment 33 The method of embodiment 31, wherein the sample is a bone marrow aspirate.
  • Embodiment 34 The method of embodiment 31, wherein the sample is a biopsy of a chloroma.
  • Embodiment 35 Embodiment 35.
  • step (a) further comprises performing a transcriptomic analysis of the plurality of cells in the sample; and step (b) further comprises identifying the presence of at least one CD70+ monocytic leukemia stem cell (m-LSC) based on transcriptomic analysis performed in step (a), wherein a cell is identified as a CD70+ m-LSC based on at least one of the following: i) an upregulation in the expression of at least one of biomarker from Table 5; ii) the expression of at least one biomarker from Table 1A; iii) an upregulation in the expression of at least one GSEA gene signature from Table 1B.
  • m-LSC monocytic leukemia stem cell
  • Mono specimens displayed very few blast-like cells ( ⁇ 1%) and a predominant monocyte-like profile (CD45-bright, SSC-high relative to lymphocytes). Mono specimens down-regulated CD34 and CD117, and up-regulated monocytic markers to varying degrees, including CD64. Finally, the MMP specimens were characterized as having discrete blast-like and monocytic-like subpopulations (range, 26%-79% for primitive and 14%-35% for monocytic), which were evident in both the CD45/SSC gate and phenotypic analysis of stem/progenitor makers and monocytic antigens contained a mixture of monocytic and primitive cells, occupying the middle range of the developmental spectrum ().
  • Multi-MMP the second type of MMP AML was Attorney Docket No.: UNCO-049/001WO (300978-2220) designated “Multi-MMP,” where readily detectable LSC activity was evident in both prim and mono subpopulations (FIG.1B).
  • WES whole-exome sequencing
  • Multi-MMP AML arises due to underlying mutational variation, consistent with the understanding that phenotypically distinct subpopulations of LSCs can simultaneously exist in certain patients with AML .
  • Prim vs mono engrafted cells from two representative specimens (AML-07 and AML-13) consistently differed in their developmental spectrum.
  • the prim subpopulation was able to recapitulate the full developmental spectrum of disease, with both prim and mono cells evident in each transplanted mouse.
  • LSCs that initiate and drive disease in the monocytic Attorney Docket No.: UNCO-049/001WO (300978-2220) subpopulation demonstrated a more restricted developmental hierarchy that resides toward the mature end of the myeloid developmental spectrum and distinct resistance to VEN+AZA therapy.
  • This subclass of LSCs was designated as mono-LSCs (m-LSCs).
  • m-LSCs mono-LSCs
  • p-LSCs prim-LSCs
  • these findings demonstrate that heterogeneous LSC subpopulations with distinct developmental phenotypes co-reside in the same patient.
  • LSC heterogeneity gives rise to bulk tumor populations with differing therapeutic response in PDX models, demonstrating clinical significance.
  • Example 2 [00243] In the following non-limiting example, experiments were performed to predict clinical outcomes as a function of m-LSCs. Without wishing to be bound by theory, it was hypothesized that de novo AML patients would differ in pathogenesis and clinical outcome as a function of the presence of m-LSCs. As illustrated schematically in FIG.3A, it was predicted that upon receiving VEN+AZA therapy, Uni-MMP patients that initially presented with only p-LSCs should achieve more durable remission due to intrinsic reliance of p-LSCs on venetoclax target BCL-2. In contrast, Multi-MMP patients with a distinct m-LSC population were predicted to relapse relatively quickly with monocytic disease.
  • Pt-69 had chromosome 11 and 16 duplication events in the prim compartment at diagnosis that were not present at relapse, again consistent with the outgrowth of the genetically distinct monocytic Attorney Docket No.: UNCO-049/001WO (300978-2220) subclone.
  • data from Pt-65 also shows the outgrowth of genetically distinct monocytic clones at relapse marked by two KRAS mutations.
  • both of the KRAS mutations that were dominant at relapse were not readily detectable at 400 ⁇ sequencing depth in the diagnosis specimen and were seen only using higher resolution methods (droplet-digital PCR).
  • Example 3 In the following non-limiting example, m-LSC immunophenotypes were identified and characterized in primary AML specimens. CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) analysis was performed, allowing simultaneous measurement of protein-based surface antigens and RNA-based transcriptome analysis at a single cell level. CITE-seq analysis was performed on a cohort of 27 primary AML specimens containing immunophenotypically defined Prim, MMP, and Mono AMLs (listed in Table 3).
  • CITE-seq Cellular Indexing of Transcriptomes and Epitopes by Sequencing
  • the CITE-seq data was first analyzed using Clustifyr, an application that assigns phenotypes based on comparison to the transcriptome of normal human hematopoiesis (see Fu et al. F1000Res.2020;9:223).
  • This analysis classifies myeloid cells into HSPC, MPP, Early Promyelocyte-like, Late promyelocyte-like, Myelocyte-like, classical monocyte-like, and Nonclassical monocyte-like subclusters, and revealed the myeloid developmental spectrum within primary AML specimens.
  • the MMP group was also further divided into Uni- MMP and Multi-MMP subgroups based on each functional LSC activity, as measured by xenograft assay (FIG.4A).
  • the relative proportion of each myeloid subcluster within Prim, Uni-MMP, Multi-MMP, and Mono groups was highly concordant with expected identity.
  • the Prim group almost exclusively contained the highest proportion of the three LPSC cell types (quiescent, primed, and cycling), which reside at the apex of the myeloid developmental hierarchy.
  • the Mono group presented a developmental hierarchy with almost no LSPC cell types but was rather comprised of a dominant ProMono-like cell type, suggesting the latter could be enriched for m-LSCs.
  • KMT2A-r_LSC_signature A KMT2A-rearranged leukemia-specific LSC signature (KMT2A-r_LSC_signature; Table 4; see Somervailee et al. Cancer Cell 2006;10:257–68; see Somervaille et al.
  • Example 4 [00253] In the following non-limiting example, experiments were performed to functionally validate m-LSCs. A series of flow sorting and transplant studies to functionally validate the m-LSC immunophenotype of the CITE-seq analysis. Two mono specimens AML-16 and AML-20 were employed, from which various subpopulations of cells were isolated using expression of CD45, CD34, CD4, CD14, CD11b, and CD36 (FIGS.5A-B and FIGS.8A-8B). To avoid contamination from conventional CD34+ p-LSCs, the CD34- fraction was gated when validating the m-LSCs.
  • AML-20 some engraftment potential was detected in the CD14-, CD11b+, and CD36+ counterparts (FIG.5E), suggesting that in certain patients the developmental hierarchy of m-LSC-driven AMLs is shallower than others (e.g., AML-16).
  • FIG.5C, FIG.5F, FIG.8C and FIG.8F serial transplant experiments demonstrated that m-LSCs in this Multi-MMP AML were also exclusively enriched by the CD34-, CD14-, CD11b-, and CD36- immunophenotype.
  • CD4 was not included in this sort due to limited cell numbers. Together, these data revealed an m-LSC immunophenotype that was applicable to both Mono and Multi-MMP AMLs, entirely distinct from profiles previously described for the more conventional primitive LSCs (i.e. CD34+ and CD38-). [00255] To further explore the immunophenotype of m-LSCs, analytical flow cytometry was used to evaluate the expression of several additional cell surface antigens associated with stem cell activity. Both CD117 and CD244 have been shown to be frequently expressed specifically in CD34- AML specimens. However, there was no detectable expression of CD117 in any of the seven specimens that underwent functional evaluation for the presence of m-LSCs.
  • Methotrexate (MTX), brequinar (BRQ), and cladribine (CdA) were selected based on their activities in inhibiting one-carbon metabolism enzymes DHFR and TYMS, pyrimidine synthesis enzyme DHODH, and purine-based DNA/RNA synthesis, respectively.
  • Coldrexate (MTX), brequinar (BRQ), and cladribine (CdA) were selected based on their activities in inhibiting one-carbon metabolism enzymes DHFR and TYMS, pyrimidine synthesis enzyme DHODH, and purine-based DNA/RNA synthesis, respectively.
  • Coldy- forming unit assays were performed on m-LSCs and p-LSCs isolated from Mono, Multi-MMP, and Prim specimens, along with normal CD34+ hematopoietic stem and progenitor cells (HSPC) from two healthy donors.
  • HSPC normal CD34+ hematopoietic stem and progenitor cells
  • CdA showed remarkable specificity against m-LSCs while sparing p-LSCs as well as normal HSPC controls. This selectivity was not seen by the other chemotherapy agents cytarabine and daunorubicin (FIG. 6C), suggesting the unique sensitivity of m-LSCs to CdA. In addition, CdA also outperformed BRQ and MTX in potency against m-LSCs (FIG.6D).
  • CdA was selected for in vivo proof-of concept studies in combination Attorney Docket No.: UNCO-049/001WO (300978-2220) with VEN+AZA in two functionally validated MMP AMLs.
  • Fig.6E Multi-MMP AML-13 and AML-07 were transplanted into NSG-S mice and treated with VEN+AZA alone, CdA alone, or the triple-drug combination in vivo.
  • Analysis of AML cells in both bone marrow and spleen demonstrated that the addition of CdA clearly improved the clearance of tumor that was otherwise resistant to VEN + AZA in both PDX models (Fig. 6F-6H).
  • Examples 1-5 describe the identification and characterization of a previously unrecognized type of human acute myeloid leukemia (AML) stem cell defined as “m-LSC.” This particular subclass of LSC is distinguished from more primitive subtypes by virtue of a unique immunophenotype (CD34-, CD4+, CD14-, CD11b-, and CD36-), a relatively narrow developmental profile that is limited to the creation of monocytic progeny, and a gene expression profile that is roughly analogous to normal human promyelocytes.
  • AML acute myeloid leukemia
  • m-LSC is distinct from the CD34- LSC populations described previously, where expression of both CD177 and CD244 were prevalent.
  • the molecular biology of m-LSCs differs from more primitive LSCs in that BCL-2 dependency seems to be largely dispensable, making this type of LSC resistant to treatment with venetoclax and azacytidine.
  • m-LSCs demonstrate selective reliance on one-carbon metabolism and purine/pyrimidine metabolism, adding to the importance of cellular metabolism in the context of AML pathogenesis and therapeutic resistance.
  • m-LSC reliance on purine metabolism mediates increased sensitivity to agents such as cladribine, a well-known purine analogue.
  • FIGS.7A-7C shows the tree as an analogy to describe developmental hierarchy of AML, where the underground roots represent LSCs and the branches above the ground symbolize more differentiated blasts.
  • FIG.7A a single class of more primitive LSCs (p-LSCs) may be present in newly diagnosed AML patients, with varying degrees of monocytic differentiation potential.
  • p-LSCs primitive LSCs
  • FIG.7B some newly diagnosed Multi-MMP AML patients present with at least two distinct subtypes of LSC with primitive vs. monocytic characteristics (p-LSC and m-LSC).
  • p-LSC and m-LSC primitive vs. monocytic characteristics
  • these patients would clinically be expected to respond and then relapse, or to be refractory to venetoclax-based therapies.
  • FIGS.7A-7C show the highest frequency of refractory disease.
  • FAB-M5 monocytic phenotypes
  • FAB-M4 myelomonocytic phenotypes
  • FIGS.7A-7C The novel model described in FIGS.7A-7C can be used to design future therapies.
  • Detection of any m-LSC population at diagnosis in a patient who is being considered for a venetoclax-based therapy may warrant consideration of additional therapies designed to selectively target monocytic population, in the hopes that relapse, which carries a very poor prognosis in this setting, can be avoided.
  • additional therapies designed to selectively target monocytic population, in the hopes that relapse, which carries a very poor prognosis in this setting, can be avoided.
  • Such potential agents include immunotherapies directed towards monocytic antigens such as CD64 and LILRB4, or small molecules that selectively impair unique biology of phenotypically monocytic AML.
  • MCL1 inhibitor drugs appear to be more active in the context of monocytic AML. The results provided herein demonstrate that m-LSCs depend on MCL1 for energy metabolism.
  • Normal CD34+ HSPCs were enriched from thawed MPB samples using the CD34 MicroBead kit (Miltenyi Biotec). Cells were cultured in complete serum-free media (SFM) in 37°C, 5% CO2 incubator. SFM is composed of IMDM (GIBCO), 20% BIT 9500 (STEMCELL Technologies), 10ug/ml LDL (Low Density Lipoprotein, Millipore), 55uM 2-Mercaptoethanol (GIBCO) and 1% Pen/Strep (GIBCO). Complete SFM were made by supplementing the SFM with FLT-3, IL-3 and SCF cytokines (PeproTech), each at 10 ng/ml.
  • SFM complete serum-free media
  • Colony-forming assays Freshly sorted m-LSCs from primary AMLs or prepared CD34+ HSPCs isolated from normal mobilized peripheral samples were plated in human methylcellulose (R&D systems) at about 100K/ml and 2K/ml, respectively. Small molecule inhibitors were directly added into the methylcellulose at the desired final concentration at the plating. Colonies were counted 2- 3 weeks after the initial plating.
  • NSG-S mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV- IL3,CSF2,KITLG)1Eav/MloySzJ) mice (The Jackson Laboratory) were used for xenograft studies in this study. Male or female mice ranging in age from 6 to 8 weeks were started on experiment. Littermates of the same sex were randomly assigned to experimental groups. NSG-S mice were pre-conditioned 24 hours prior to transplant with 30mg/kg busulfan (Alfa Aesar) via intraperitoneal (IP) injection.
  • busulfan Alfa Aesar
  • the busulfan stock was made fresh at 25 mg/ml in 100% DMSO, then the stock was diluted 1:10 in pre-warmed saline (0.9% NaCl) down to 2.5 mg/ml right before use.
  • the diluted busulfan solution was kept in 37°C water bath before IP injection to prevent precipitation of busulfan due to low solubility.
  • each subpopulation was sorted according to their percentage of total viable cells (detailed in Table 6). When the cell dose was less than 0.5e6/mouse, mononuclear cells isolated from bone marrow and spleens of na ⁇ ve NSG-S mice were used as carrier cells.
  • mice were then washed, pelleted, and resuspended in saline buffer to allow tail vein injection into NSG-S mice at 0.1ml per mouse. Fifteen minutes prior to injection, in vivo anti-human CD3 antibody (BioCell) was added at a final concentration of 1ug/e6 cells to prevent potential graft versus host disease. During all experiments, the weight of mice was approximately 20-30 grams with no animals losing greater than 10% body weight. The mice were kept in ventilated cages and given in vivo treatments when needed in the vivarium at University of Colorado. The majority of the experiments lasted for 6 to 12 weeks. At the end of the experiments, mice were euthanized using carbon dioxide.
  • in vivo anti-human CD3 antibody BioCell
  • Bone marrow and spleen were harvested, subjected to red blood cell lysis, and the mononuclear cells were stained with Attorney Docket No.: UNCO-049/001WO (300978-2220) human CD45, mouse CD45 antibodies, and DAPI to determine percentage of engraftment within viable cells. All animal work were performed in accordance with Institutional Animal Care and Use Committee protocol number 00308. [00284] In vivo treatments [00285] About 2-4 weeks post initial transplant, tumor burden in the bone marrow was determined to be above 5% in sentinel mice. Mice were then treated with various in vivo regimens as follows.
  • Venetoclax was given at 100mg/kg via oral gavage, five days/week for two weeks; Azacitidine was given at 3mg/kg via intraperitoneal injection, three days/week for two weeks; Cladribine was given at 10mg/kg via intraperitoneal injection, three days/week for two weeks. All treatments were given in the same two-week time window when stated together. [00286] CITE-seq sample preparation and library construction [00287] Mononuclear cell suspensions were prepared from freshly thawed primary AML specimens cryopreserved in liquid nitrogen.
  • cells were processed with the 10x Genomics 3’ dual index v3.1 library kit with feature barcoding technology for cell surface proteins. Briefly 10,000 cells were targeted from stock suspension of 1000 cells/ ⁇ l. Cells were processed following the protocol to generate 3’ gene expression libraries as well as Feature Barcode cell surface libraries. Both libraries were dual indexed, and samples were quantified by Qubit (Life Technologies) and assessed for size and quality by Tapestation (Agilent). Libraries were normalized and pooled for sequencing on a Novaseq 6000 (Illumina) for paired-end 2x150 bp sequencing. Targeted read depth for gene expression libraries was 100,000 reads/cell or ⁇ 500 million paired-end reads/library.
  • Targeted read depth for cell surface libraries was 40,000 reads/cell or 200 million paired end reads/library.
  • CITE-seq data pre-processing [00289] Raw sequencing data for gene expression, antibody derived tag (ADT; surface protein), and hashing libraries were processed using STARsolo 2.7.8a with the 10X Genomics GRCh38/GENCODE v32 genome and transcriptome reference (version GRCh38_2020A) or a TotalSeq barcode reference, as appropriate. Hashed samples were demultiplexed using GMM-Demux.
  • Scanpy and Seurat 4.1.1 were then used to generate uniform manifold approximation and projections from the TotalVI embeddings and perform exploratory analysis, data visualization, etc.
  • the myeloid subpopulation in the CITE-seq data was reannotated using scArches 0.5.7 and a leukemia reference dataset (see Zeng et al. Nat Med 2022;28: 1212–23).
  • the reference model was trained for 400 epochs based on the 3,000 most highly variable genes determined by scanpys’ pp.highly_variable_ genes() function.
  • CD34+_LSCs and KMT2A-r_LSCs were identified through scoring each individual cell using the AddModuleScore() function of the Seurat software and custom-generated candidate CD34+_LSC and KMT2A-r_LSC gene expression signatures, as stated in the main text.
  • WES Analysis [00293] WES libraries were generated using the Agilent SureSelect XT exome prep kit with 200 ng of input as per protocol (Agilent).
  • the probe used was SureSelect XT Human All Exon Attorney Docket No.: UNCO-049/001WO (300978-2220) V7 (Agilent). Libraries were normalized by Qubit (Invitrogen) and Tapestation (Agilent), and 2 ⁇ 150 bp reads were sequenced on a Novaseq 6000 (Illumina) to obtain 400 ⁇ coverage. Fastqc v0.11.9 was used to assess overall sequencing quality, and reads were trimmed using cutadapt (cutadapt, RRID:SCR_011841) v2.9 to remove the Illumina universal adapters, bases of poor quality (phred ⁇ 30), and any reads in which the minimum read length was ⁇ 10 base pairs in length.
  • Trimmed fastq files were then aligned to the GRCh38 p.13 genome using BWA v0.7.17 (BWA, RRID:SCR_010910).
  • Stringent quality control of alignments and read duplicate removal were performed using the Picard suite of tools v2.21.1 (Picard, RRID:SCR_006525) and samtools v1.8 (samtools, RRID:SCR_002105).
  • Variants were called on alignments using DeepVariant v1.0.0, followed by BCFtools (BCFtools, RRID:SCR_005227) v1.11 to filter variants of low quality. SNPs were removed if the raw unfiltered read depth was ⁇ 20 reads and the mapping quality ⁇ 30.
  • Pathway enrichment analysis was performed on metabolites that were ⁇ 1.2-fold higher in population D compared with E and with a P value of less than 0.1. All analyses were performed using the MetaboAnalyst 5.0 software (MetaboAnalyst, RRID:SCR_015539). [00296] Statistical Analysis [00297] Statistical analyses were performed in GraphPad Prism 9.3.1 (GraphPad Prism, RRID:SCR_002798). Median ⁇ interquartile range was used to describe summary statistics. One-tailed or two-tailed Mann–Whitney tests were used to compare two groups when applicable.
  • FIG.10A sows the percentage of total blast cells in each AML sample that were CD70+ (left side of graph) as well as the percentage of m-LSCs in each AML Sample that were CD70+ (right side of graph).
  • the results shown in FIG.10A demonstrate that CD70 is expressed on m-LSCs, with some samples exhibiting more than 50% m-LSCs that are CD70 positive.
  • FIG.10A shows the results from the analysis shown in FIG.10A based on whether the AML samples were obtained from patients that were sensitive to treatment with a combination of venetoclax and azacitidine or resistant to treatment with a combination of venetoclax and azacitidine.
  • FIG.10B shows the percentage of total blast cells and m-LSCs that were CD70+ in AML samples derived from Ven+Aza resistant samples and Ven+Aza sensitive samples. As shown in FIG.10B, patients who were sensitive to Ven+Aza treatment had a lower percentage of m-LSCs that were CD70+ as compared to patients that were Ven+Aza resistant. That is, the m-LSC populations of Ven+Aza resistant patients exhibited a higher percentage of cells that were CD70+.
  • these results indicate that patients who exhibit higher levels of CD70+ m-LSCs may be more resistant to treatment with Ven+Aza, and therefore would be benefit from alternative treatments, including those that incorporate the use of a CD70-targeting agent. Moreover, these results indicate that a patient’s response to treatment with a combination of venetoclax and azacitidine can be predicted by determining the number of CD70+ m-LSCs (thus, CD34-, CD4+, CD11b-, CD14-, CD36-, and CD70+ cells) in biological sample obtained from the patient.

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Abstract

The present disclosure provides methods of treating acute myeloid leukemia (AML) and methods of determining responsive to AML treatment regimes, the methods comprising identifying the presence or absence of monocytic leukemia stem cells (m-LSCs), including CD70+ m-LSCs, in a sample from a subject.

Description

Attorney Docket No.: UNCO-049/001WO (300978-2220) METHODS OF TREATING ACUTE MYELOID LEUKEMIA RELATED APPLICATIONS [0001] This application claims priority to, and the benefit of, U.S. Provisional Application No. 63/385,699, filed on December 1, 2022, and U.S. Provisional Application No.63/490,270, filed on March 15, 2023. The contents of each of the aforementioned patent applications are incorporated herein by reference in their entireties. GOVERNMENT SUPPORT [0002] This invention was made with government support under grant number R35CA242376 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION [0003] 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 [0004] The present disclosure provides a method of treating acute myeloid leukemia (AML) in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression Attorney Docket No.: UNCO-049/001WO (300978-2220) measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) administering to the subject a combination of at least one BCL- 2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent when at least one m-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-LSCs are identified. [0005] The present disclosure provides 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, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) identifying that the subject will not be responsive to the treatment when the presence of at least one m-LSC is identified; or identifying that the subject will be responsive to the treatment when no m-LSCs are identified. [0006] In some aspects of the methods of the present disclosure, step (a) further comprises measuring the expression of at least CD117, CD244 and CD64, wherein a cell is identified as an m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+. In some aspects of the methods of the present disclosure, step (a) further comprises measuring the expression of at least CD117, CD244, CD64 and GPR56, wherein a cell is identified as an m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, and GPR56-. [0007] The present disclosure provides methods of treating acute myeloid leukemia (AML) in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; and c) administering to the subject a treatment comprising at least one CD70-targeting agent when at least one CD70+ m-LSC is identified, preferably wherein the treatment further comprises at least one BCL-2 inhibitor and at least one hypomethylating agent when no CD70+ m-LSCs are identified. [0008] The present disclosure provides methods of identifying if a subject having AML will be responsive to treatment with a CD70-targeting agent, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic Attorney Docket No.: UNCO-049/001WO (300978-2220) leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; and c) identifying that the subject will be responsive to the treatment when the presence of at least one CD70+ m-LSC is identified. [0009] In some aspects of the preceding methods, step (a) further comprises measuring the expression of at least CD117, CD244 and CD64, wherein a cell is identified as a CD70+ m- LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and CD70+. In some aspects of the preceding methods, step (a) further comprises measuring the expression of at least CD117, CD244, CD64, and GPR56- wherein a cell is identified as a CD70+ m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, GPR56-, and CD70+. [0010] In some aspects, the at least one CD70-targeting agent is: i) an anti-CD70 antibody, preferably wherein the anti-CD70 antibody is cusatuzumab; ii) an anti-CD70 immunotherapy, preferably wherein the immunotherapy comprises CAR-T and/or NK Cells that are directed specifically at CD70; or iii) an agent that blocks CD70 signaling, preferably wherein the agent that blocks CD70 signaling prevents binding of CD27 and CD70. [0011] In some aspects of the methods of the present disclosure, the at least one m-LSC targeting agent is an agent modulates one-carbon metabolism, is an agent that modulates purine synthesis, is an agent that modulates pyrimidine synthesis, or any combination thereof. [0012] In some aspects of the methods of the present disclosure, the at least one m-LSC targeting agent is selected from methotrexate, brequinar and cladribine. [0013] In some aspects of the methods of the present disclosure, the at least one hypomethylating agent is selected from azacitidine and decitabine. [0014] In some aspects of the methods of the present disclosure, the at least one BCL-2 inhibitor is selected from venetoclax and navitoclax. [0015] In some aspects of the methods of the present disclosure, 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, [0016] Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-SEQ), or any combination thereof. [0017] 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. Attorney Docket No.: UNCO-049/001WO (300978-2220) [0018] 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. [0019] In some aspects of the methods of the present disclosure, the at least one AML treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent. [0020] In some aspects of the methods of the present disclosure, 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. [0021] In some aspects of the methods of the present disclosure, 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. [0022] In some aspects of the methods of the present disclosure, 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. In some aspects, the sample is a bone marrow biopsy. In some aspects, the sample is a bone marrow aspirate. In some aspects, the sample is a biopsy of a chloroma. [0023] In some aspects of the methods of the present disclosure, step (a) further comprises performing a transcriptomic analysis of the plurality of cells in the sample; and step (b) further comprises identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on transcriptomic analysis performed in step (a), wherein a cell is identified as an m-LSC based on at least one of the following: i) an upregulation in the expression of at least one of biomarker from Table 5; ii) the expression of at least one biomarker from Table 1A; iii) an upregulation in the expression of at least one GSEA gene signature from Table 1B. [0024] In some aspects of the methods of the present disclosure, the transcriptomic analysis is performed using RNA sequencing. [0025] In some aspects of the methods of the present disclosure, is performed using CITE- SEQ. [0026] Any of the above aspects, or any of the aspects described herein, can be combined with any other aspect. Attorney Docket No.: UNCO-049/001WO (300978-2220) [0027] 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”. [0028] 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. BRIEF DESCRIPTION OF THE DRAWINGS [0029] The above and further features will be more clearly appreciated from the following detailed description when taken in conjunction with the accompanying drawings. [0030] FIGS.1A and 1B show the characterization of developmentally heterogeneous LSCs.. FIGS.1A and 1B show a summary of engraft% data for Uni-MMP AML-12, AML-08, and AML-14 and Multi-MMP AML-07, and AML-13. Bulk stands for unsorted bulk tumor; prim stands for primitive subpopulation; mono stands for monocytic subpopulation. Each dot represents a mouse. AML-12 (bulk, n=7; prim, n=6; mono, n=6). AML-08 (prim, n=7; mono, n=9). AML-14 (prim, n=9; mono, n=7). AML-07 (bulk, n=7; prim, n=5; mono, n=6). AML- 13 (prim, n=8; mono, n=8). Median+/- interquartile range. Two-tailed Mann-Whitney tests Attorney Docket No.: UNCO-049/001WO (300978-2220) were used for comparing two groups, Kruskal-Wallis tests were used when comparing more than two groups. ns, not significant. [0031] FIGS.2A-2C show the differing nature of disease arising from prim and mono subpopulations of Multi-MMP AMLs. FIG.2A is a diagram depicting workflow used to isolate primitive and monocytic subpopulations of AML-07 for injecting into PDX mice and subsequent determination of their relative sensitivity to the VEN+AZA regimen in vivo. FIG. 2B shows the design of the VEN/AZA in vivo regimen (VEN, 100mg/kg, oral gavage (OG), 5 days/week x 2 weeks; AZA, 3mg/kg, Intraperitoneal injection (IP), 3 days/week x 2 weeks). FIG.2C shows the impact of in vivo VEN+AZA treatments on leukemia engrafted from prim versus mono subpopulations of AML-07. Engraft% was determined by % of hCD45+/mCD45- cells within total viable bone marrow cells. Each dot represents a unique mouse. PDX-07-prim (Control, n=10; VEN/AZA, n=10), PDX-07-mono (Control, n=10; VEN/AZA, n=10). Box plots show median +/- interquartile. Two-tailed Mann-Whitney test is used. ns, not significant. [0032] FIGS.3A-3G show clinical outcomes as a function of m-LSCs. FIG.3A is a diagram describing the leukemogenesis process of Uni-MMP and Multi-MMP AML patients and their predicted clinical responses to VEN+AZA therapy. FIG.3B, FIG.3C, and FIG.3D are representative cases of Uni-MMP and Multi-MMP AML patients received VEN+AZA therapy. The left panels depict sorting strategies for obtaining diagnosis-prim (Dx-prim) and diagnosis-mono (Dx-mono) subpopulations from diagnosis bulk disease (Dx-bulk), as well as relapse-mono (Rl-mono) subpopulations from relapse bulk disease (Rl-bulk) when applicable. The right panel shows engraftment percentage (engraft%) in NSG-S mice determined by hCD45+/mCD45-% within total viable bone marrow cells. Each dot represents a unique mouse. Median +/- interquartile. Mann-Whitney tests. ns, not significant; *p<0.05, **p<0.01, ***p<0.001. FIG.3B shows Patient 20 (Pt-20), a case of Uni-MMP AML presenting prolonged remission after VEN+AZA therapy for more than 3.5 years. Dx-bulk (n=9), Dx-prim (n=7), Dx-mono (n=7). FIG.3C shows Patient 12 (Pt-12), a case of Multi- MMP AML presenting predominant monocytic relapse in 12 months after receiving VEN+AZA therapy. Dx-prim (n=10), Dx-mono (n=9), Rl-mono (n=8). FIG.3D shows Patient 69 (Pt-69), a case of Multi-MMP AML presenting quick relapse in 3 months post VEN+AZA therapy. In this particular case, prim and mono subpopulations were gated using a different sorting strategy based on primitive antigen CD34 and monocytic antigen CD11b. For the patient’s diagnosis sample, the CD34+/CD11b-, CD34+/CD11b+, and CD34- /CD11b-pp subpopulations were sorted as Dx-prim-A, Dx-prim-B, and Dx-mono Attorney Docket No.: UNCO-049/001WO (300978-2220) subpopulations, respectively. For the patient’s relapse sample, the CD34-/CD11b-pp subpopulation was sorted as the predominant Rl-mono subpopulation. Dx-bulk (n=9), Dx- prim-A (n=3), Dx-prim-B (n=7), Dx-mono (n=7), Rl-bulk (n=9), Rl-mono (n=9). FIG.3E shows phenotypic changes from diagnosis to relapse in a cohort of AML patients received VEN+AZA therapy (N=25, Table 2A and Table 2B). FIG.3F shows remission duration for AML patients with monocytic relapse (N=9) versus non-monocytic relapse (N=16). FIG.3G shows remission duration of VEN/AZA relapsed AML patients. Shown are bar graphs of remission duration in days for a cohort of 25 AML patients who received the VEN/AZA therapy and experienced relapse response. The cohort contains five patients who sustained a monocytic phenotype between diagnosis and relapse (mono to mono), four patients transited from a primitive phenotype at diagnosis to monocytic phenotype at relapse (prim to mono), 15 patients sustained a primitive phenotype between diagnosis and relapse (prim to prim), and one patient transited from a monocytic phenotype at diagnosis to primitive phenotype at relapse (mono to prim). Each dot represents a unique patient. Median duration time of both groups are shown in days. In FIGS.3B, C, D, and F box plots represent median +/- interquartile. In FIGS.3B-D Kruskal-Wallis test was used. In FIG.3F, one-tailed Mann- Whitney test is used. ns, not significant. [0033] FIGS.4A-4B show identification of the m-LSC immunophenotype. FIG.4A is stacking bar graphs showing relative proportion of each subcluster within the highlighted “myeloid” region of the UMAP. FIG.4B shows protein expression of surface antigens CD45, CD34, CD4, CD14, CD11b, and CD36. The m-LSC enriched region is highlighted by dotted lines. [0034] FIGS.5A-5C show functional validation of m-LSC immunophenotypes. FIGS.5A- 5C, show gating strategies for sorting various subpopulations of Mono AML-16, Mono AML-20, and Multi-MMP AML-07 for determining their m-LSC activities using xenograft studies. The sorting is detailed in FIGS.12A-12C. Briefly, for AML-16 and AML-20, FIG. 5A (Live/mono), FIG.5B (Live/mono/CD34-/CD4+/CD14-), FIG.5C (Live/mono/CD34- /CD4+/CD14+), FIG.5D (Live/mono/CD34-/CD4+/CD14-/CD11b-CD36-), and FIG.5E (Live/mono/CD34-/CD4+/CD14-/CD11b+CD36+) were sorted and engrafted. For AML-07, CD4 was not included in the sort due to limitation of cells. FIGS.5E-5F show results from transplanting subpopulations of AML-16 (A(n=8), B(n=6), C(n=7), D(n=7), E(n=6)), AML- 20 (A(n=9), B(n=8), C(n=7), D(n=12), E(n=10)), AML-07 (A(n=10), B(n=10), C(n=9), D(n=10), E(n=8)) are shown as PDX-16, PDX-20, and PDX-07, respectively. Engraft% was determined by % of hCD45+/mCD45- cells within total viable bone marrow cells. Each dot Attorney Docket No.: UNCO-049/001WO (300978-2220) represents a unique mouse. Box plots represent median +/- interquartile. Two-tailed Mann- Whitney tests. [0035] Fig. 6A-6H shows molecular properties and targeting of m-LSCs. FIG. 6A shows impact of TYMS inhibitor methotrexate (MTX), DHODH inhibitor brequinar (BRQ), and purine analogue cladribine (CdA) on colony-forming unit (CFU) potential of CD34+ HSPCs isolated from two normal mobilized peripheral blood samples (MPB-1, 2) versus mono-LSCs isolated from mono AML-20 and mono AML-16.FIG.6B shows representative images of CFU assays measuring the efficacy of CdA on normal CD34+ HSPCs and m-LSCs. FIG. 6C and FIG. 6D show the impact of chemotherapy agents AraC, DNR, DHODH inhibitor brequinar (BRQ), and TYMS inhibitor methotrexate (MTX) on the CFU potential of m-LSCs, p-LSCs and CD34+ HSPCs.FIG. 6E shows a diagram depicting workflow and design of the regimens used for in vivo treatment. IP, intraperitoneal; OG, oral gavage.. FIG.6F shows the impact of in vivo VEN + AZA, CdA, or triple-drug combo treatments on the bone marrow tumor burden of PDX. Engraft% was determined by % of hCD45+/mCD45í cells within total viable bone marrow mononuclear cells. Each dot represents a unique mouse. For PDX of AML-13, control (n = 11), VEN + AZA (n = 9), CdA (n = 10), VEN + AZA + CdA (n = 10). For PDX of AML- 07, control (n = 11), VEN + AZA (n = 9), CdA (n = 12), VEN + AZA + CdA (n = 9). Box plots represent median ± interquartile range. Kruskal–Wallis test was used. ns, not significant. FIG. 6G shows the impact of VEN+AZA, CdA or combo treatments on the marrow tumor burden of PDX. hCD45+ count was determined by direct quantification of hCD45+/mCD45- cells within a set volume of marrow harvest using flow cytometry. FIG. 6H shows the impact of VEN+AZA, CdA or combo treatments on the spleen tumor burden of PDX. Engraft% was determined by % of hCD45+/mCD45- cells within total viable spleen mononuclear cells. hCD45+ count was determined by direct quantification of hCD45+/mCD45- cells within a set volume of spleen harvest using flow cytometry. Each dot represents a unique mouse. Control (n=6), VEN+AZA (n=6), CdA (n=6), VEN+AZA+CdA (n=6). Box plots represent median +/- interquartile. Kruskal-Wallis test was used. ns, not significant. [0036] FIGS.7A-7C show a model depicting interplay between LSC potential, immunophenotype, disease evolution, and clinical response to the venetoclax plus azacitidine therapy in AML patients. FIG.7A shows a first group of AML patients with disease solely driven by p-LSCs captured at various maturation stages with predominant Prim, MMP, and predominant Mono immunophenotypes. FIG.7B shows a second group of AML patients with multi-LSC activities (contains both p-LSCs and m-LSCs) presenting Prim, MMP, or Mono immunophenotypes depending on the relative degrees of maturation and ratio of the two Attorney Docket No.: UNCO-049/001WO (300978-2220) diseases rooted from the two distinct LSC subtypes. FIG.7C shows a last group of AML patients with disease solely driven by m-LSCs usually presenting a predominant mono immunophenotype due to the inherent nature of m-LSCs that are already resting at a relatively more mature promyelocyte-like developmental stage. In all graphs, teal colored symbols represent p-LSCs and their progenies, pink colored symbols represent m-LSCs and their progenies, circular arrows represent self-renewal capacity. [0037] FIGS.8A-8F show sorting strategies for determining m-LSC immunophenotype. FIGS.8A-8C show the immunophenotyping of A, B, C, D, and E subpopulations sorted from Mono AML-16 (FIG.8A), Mono AML-20 (FIG.8B), and Multi-MMP AML-07 (FIG.8C), and used for injection into NSG-S mice to determine their m-LSC potential. FIGS.8D-8F show the tumor burden in primary and secondary transplants shown by human CD45 and mouse CD45 staining. [0038] FIGS.9A and 9B show that M5 but not M4 patients showed significantly higher refractory rate to VEN/AZA therapy. FIG.9A shows a circular pie chart showing numbers of patients identified in different FAB subclasses. FIG.9B shows bar graphs showing percentage of patients had refractory or non-refractory responses to VEN+AZA therapy according to the ELN criteria . [0039] FIGS.10A and 10B show the analysis of CD70+ m-LSCs in AML samples. FIG.10A is a graph showing the percentage of total blast cells and percentage of m-LSCs that were CD70+ in the AML samples. FIG.10B shows the percentage of total blast cells and percentage of m-LSCs that were CD70+ in AML samples that were either resistant to treatment with a combination of venetoclax and azacitidine or responsive to treatment with a combination of venetoclax and azacitidine. DETAILED DESCRIPTION OF THE INVENTION [0040] 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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. [0041] 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 leukemia stem cells (LSCs), which are capable of giving rise to identical daughter cells as well as differentiated cells, perpetuate and maintain acute myeloid leukemia. [0042] 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. [0043] 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. [0044] 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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. [0045] 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 and 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. [0046] The present disclosure is based on, inter alia, the discovery a particular subpopulation of leukemia stems cells, denoted as monocytic leukemia stem cells (m-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, cytarabine and decitabine). [0047] 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-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-LSCs. [0048] Additionally, the present disclosure is based on, inter alia, the discovery a particular subpopulation of leukemia stems cells, denoted as CD70+ monocytic leukemia stem cells (m- LSCs) that can be used to predict a subject’s response to treatment with a CD70-targeting agent. [0049] Accordingly, the present disclosure provides, inter alia, a method of determining if a subject having AML will be responsive to treatment with a CD70-target agent based on whether CD70+ m-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 CD70+ m-LSCs. [0050] Methods of Predicting Response to Treatment [0051] 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) identifying that the subject will not be responsive to the treatment when the presence of at least one m-LSC is identified; or identifying that the subject will be responsive to the treatment when no m-LSCs are identified. [0052] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the presence of at least one m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and CD64, and step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD64+. [0053] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the presence of at least one m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and GPR56-. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and GPR56, and step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and GPR56-. [0054] 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, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of m-LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) comparing the number and/or percentage of m-LSCs identified in step (b) to a predetermined cutoff value; d) identifying that the subject will not be responsive to the treatment when the number and/or percentage of m-LSCs is equal to or greater than the predetermined cutoff value; or identifying that the subject will be Attorney Docket No.: UNCO-049/001WO (300978-2220) responsive to the treatment when the number and/or percentage of m-LSCs is less than the predetermined cutoff value. [0055] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the number and/or percentage of m-LSCs in the plurality of cells is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+. Thus, in a non- limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and CD64, and step (b) can comprise identifying the number and/or percentage of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD64+. [0056] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the presence of at least one m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and GPR56-. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and GPR56, and step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and GPR56-. [0057] In some aspects of the preceding methods, a predetermined cutoff value can be determined by comparing the number and/or percentage of m-LSCs in samples obtained from one or more subjects known to be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent and the number and/or percentage of m-LSCs samples from one or more subjects known to be not responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent. The skilled artisan can used methods known in the art to make such comparisons and determine a suitable predetermined cutoff value that will allow for the discrimination between responders and non-responders. [0058] 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, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m- LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ Attorney Docket No.: UNCO-049/001WO (300978-2220) m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) identifying that the subject will not be responsive to the treatment when the presence of at least one m-LSC is identified; or identifying that the subject will be responsive to the treatment when no m-LSCs are identified. [0059] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36, and CD70) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD70+, CD117-, CD244- and CD64+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and CD64, and step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD70+, and CD64+. [0060] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36, and CD70) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD70+, CD117-, CD244-, CD64+ and GPR56-. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, CD70 and GPR56, and step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD70+ and GPR56-. [0061] 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, CD4, CD11b, CD14, CD36, and CD70 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of CD70+ m-LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as a CD70+ m- LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) comparing the number and/or percentage of CD70+ m-LSCs identified in step (b) to a predetermined cutoff value; d) identifying that the subject will not be responsive to the treatment when the number and/or percentage of CD70+ m-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 CD70+ m-LSCs is less than the predetermined cutoff value. Attorney Docket No.: UNCO-049/001WO (300978-2220) [0062] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36, and CD70) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD70+, CD117-, CD244- and CD64+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and CD64, and step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD70+, and CD64+. [0063] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36, and CD70) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD70+, CD117-, CD244-, CD64+ and GPR56-. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, CD70 and GPR56, and step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD70+ and GPR56-. [0064] In some aspects of the preceding methods, a predetermined cutoff value can be determined by comparing the number and/or percentage of CD70+ m-LSCs in samples obtained from one or more subjects known to be responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent and the number and/or percentage of CD70+ m-LSCs samples from one or more subjects known to be not responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent. The skilled artisan can used methods known in the art to make such comparisons and determine a suitable predetermined cutoff value that will allow for the discrimination between responders and non-responders. [0065] 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-LSC targeting agent. Attorney Docket No.: UNCO-049/001WO (300978-2220) In some aspects, the alternative treatment can not include at least one BCL-2 and/or not include at least one hypomethylating 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. [0066] 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. [0067] 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. [0068] Methods of Predicting Response to anti-CD70 Treatment [0069] The present disclosure provides methods of identifying if a subject having AML will be responsive to treatment with a CD70-targeting agent, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, and CD70+; c) identifying that the subject will be responsive to the treatment when at least one CD70+ m-LSC is identified. In some aspects, the preceding method can further comprise identifying that the subject will not be responsive to the treatment when no CD70+ m-LSCs are identified. [0070] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36 and CD70) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and CD70+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, CD64 and CD70, and step (b) can comprise identifying the presence of at least one Attorney Docket No.: UNCO-049/001WO (300978-2220) CD70+ m-LSC, wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD64+ and CD70+. [0071] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36 and CD70) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, GPR56- and CD70+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, GPR56 and CD70, and step (b) can comprise identifying the presence of at least one CD70+ m-LSC, wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, GPR56- and CD70+. [0072] The present disclosure provides methods of identifying if a subject having AML will be responsive to treatment with a CD70-targeting agent: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of CD70+ m-LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as a CD70+ m- LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) comparing the number and/or percentage of CD70+ m-LSCs identified in step (b) to a predetermined cutoff value; d) identifying that the subject will be responsive to the treatment when the number and/or percentage of CD70+ m-LSCs is greater than or equal to the predetermined cutoff value. In some aspects, the preceding method can further comprise identifying that the subject will not be responsive to the treatment when the number and/or percentage of CD70+ m-LSCs is less than the predetermined cutoff value. [0073] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36 and CD70) and step (b) can further comprise identifying the number and/or percentage of CD70+ m-LSCs in the plurality of cells is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and CD70. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, CD64 and CD70, and step (b) can comprise identifying the number and/or percentage of CD70+ m-LSCs, wherein a cell is identified as a CD70+ m- LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD64+ and CD70. [0074] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e. in addition to CD34, Attorney Docket No.: UNCO-049/001WO (300978-2220) CD4, CD11b, CD14, CD36 and CD70) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, GPR56- and CD70+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, GPR56 and CD70, and step (b) can comprise identifying the presence of at least one CD70+ m-LSC, wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, GPR56- and CD70+. [0075] In some aspects of the preceding methods, a predetermined cutoff value can be determined by comparing the number and/or percentage of CD70+ m-LSCs in samples obtained from one or more subjects known to be responsive to treatment with a CD70- targeting agent and the number and/or percentage of CD70+ m-LSCs samples from one or more subjects known to be not responsive to treatment with a CD70-targeting agent. The skilled artisan can used methods known in the art to make such comparisons and determine a suitable predetermined cutoff value that will allow for the discrimination between responders and non-responders. [0076] 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 be responsive to treatment with a CD-70 targeting agent, the method can further comprise providing a treatment recommendation that comprises administering a CD70-targeting agent. In some aspects, the treatment recommendation can comprise administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least CD-70 targeting agent. [0077] In some aspects of the preceding methods, identifying that a subject will not be responsive to treatment with a CD70-targeting agent comprises identifying that the subject will be refractory to treatment with the at least one CD70-targeting agent and/or that the subject will suffer a relapse after treatment with the at least one CD70-targeting agent. [0078] In some aspects of the preceding methods, identifying that a subject will be responsive to treatment with a CD70-targeting agent comprises identifying that the subject will have a durable remission after receiving the treatment of at CD70-targeting agent. [0079] Methods of treating AML [0080] The present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), Attorney Docket No.: UNCO-049/001WO (300978-2220) wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent when at least one m-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-LSCs are identified. [0081] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the presence of at least one m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and CD64, and step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD64+. [0082] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the presence of at least one m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and GPR56-. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and GPR56, and step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and GPR56-. [0083] The present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of m-LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) comparing the number and/or percentage of m-LSCs identified in step (b) to a predetermined cutoff value; d) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent when the number and/or percentage of m-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-LSCs is less than the predetermined cutoff value. Attorney Docket No.: UNCO-049/001WO (300978-2220) [0084] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the number and/or percentage of m-LSCs in the plurality of cells is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+. Thus, in a non- limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and CD64, and step (b) can comprise identifying the number and/or percentage of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD64+. [0085] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e. in addition to CD34, CD4, CD11b, CD14 and CD36) and step (b) can further comprise identifying the presence of at least one m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and GPR56-. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36 and GPR56, and step (b) can comprise identifying the presence of at least m-LSC wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and GPR56-. [0086] The present disclosure provides methods of treating a subject having AML, the method 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-LSC targeting agent. [0087] The present disclosure provides methods of treating a subject having AML, wherein the subject has AML that exhibits the presence of at least one m-LSC, the method comprising administering to said subject at least one m-LSC targeting agent. [0088] The present disclosure provides a methods of treating a subject having AML, wherein the subject has AML that exhibits a number and/or percentage of m-LSC cells that is greater than or equal to a predetermined cutoff value, the method comprising administering to said subject at least one m-LSC targeting agent. [0089] The present disclosure provides methods of treating a subject having AML, wherein the subject has AML that exhibits the presence of at least one m-LSC, the method comprising administering to said subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent. [0090] The present disclosure provides a methods of treating a subject having AML, wherein the subject has AML that exhibits a number and/or percentage of m-LSC cells that is greater than or equal to a predetermined cutoff value, the method comprising administering to said Attorney Docket No.: UNCO-049/001WO (300978-2220) subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent. [0091] The present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) administering to the subject a treatment comprising at least one CD70-targeting agent when at least one CD70+ m-LSC is identified. In some aspects, the treatment can further comprise at least one BCL-2 inhibitor and at least one hypomethylating agent. [0092] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36 and CD70+) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and CD70+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, CD64 and CD70+, and step (b) can comprise identifying the presence of at least one CD70+ m-LSC wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD64+ and CD70+. [0093] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36 and CD70) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, GPR56- and CD70+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, GPR56 and CD70, and step (b) can comprise identifying the presence of at least one CD70+ m-LSC, wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, GPR56- and CD70+. [0094] The present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of CD70+ m-LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b- Attorney Docket No.: UNCO-049/001WO (300978-2220) , CD14-, CD36- and CD70+; c) comparing the number and/or percentage of CD70+ m-LSCs identified in step (b) to a predetermined cutoff value; d) administering to the subject treatment comprising at least one CD70-targeting agent when the number and/or percentage of CD70+ m-LSCs is equal to or greater than the predetermined cutoff value. In some aspects, the treatment can further comprise at least one BCL-2 inhibitor and at least one hypomethylating agent. [0095] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244 and CD64 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36 and CD70) and step (b) can further comprise identifying the number and/or percentage of CD70+ m-LSCs in the plurality of cells is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and CD70+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, CD64 and CD70, and step (b) can comprise identifying the number and/or percentage of CD70+ m-LSCs, wherein a cell is identified as a CD70+ m- LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD64+ and CD70+. [0096] In some aspects of the preceding method, step (a) can further comprise measuring the expression of at least one of CD117, CD244, CD64, and GPR56 (i.e. in addition to CD34, CD4, CD11b, CD14, CD36 and CD70) and step (b) can further comprise identifying the presence of at least one CD70+ m-LSC is identified based on the immunophenotype of CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, GPR56- and CD70+. Thus, in a non-limiting example, step (a) can comprise measuring the expression of CD34, CD4, CD11b, CD14, CD36, GPR56 and CD70, and step (b) can comprise identifying the presence of at least one CD70+ m-LSC, wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, GPR56- and CD70+. [0097] The present disclosure provides methods of treating a subject having AML, the method comprising administering to the subject having AML a treatment comprising at least one anti-CD70 target agent, wherein the subject has at least one CD70+ m-LSCs. In some aspects, the treatment further comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent. [0098] The present disclosure provides methods of treating a subject having AML, the method comprising administering to the subject having AML a treatment comprising at least one anti-CD70 target agent, wherein the subject has AML that exhibits a number of percentage of CD70+ m-LSCs that is greater than or equal to a predetermined cutoff value. In Attorney Docket No.: UNCO-049/001WO (300978-2220) some aspects, the treatment further comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent. [0099] Identification of m-LSCs [00100] 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 monocytic leukemia stem cells (m-LSCs) in a plurality of cells in a sample from a subject. [00101] It is understood that a step of identifying at least one m-LSC in a plurality of cells can result in the identification of no m-LSCs (i.e. the user can determine that there are actually no m-LSCs present in the plurality of cells). [00102] In the methods of the present disclosure, m-LSCs can be identified based on the novel immunophenotype of: CD34-, CD4+, CD11b-, CD14- and CD36-. In some aspects, this immunophenotype can optionally be further supplemented with one or more makers selected from CD117-, CD244- and CD64+. Accordingly, in methods in which m-LSCs are identified via the immunophenotype, the user performing the method can measure the expression of CD34, CD4, CD11b, CD14 and CD36 (and optionally one or more of CD117, CD244 and CD64) in cells from a sample from the subject in order to determine whether m-LSCs are present in the sample, and optionally, the percentage and/or number of m-LSCs in the sample. In some aspects, the CD34-, CD4+, CD11b-, CD14- and CD36- immunophenotype can be optionally further supplemented with one or more markers selected from CD117-, CD244-, CD64+ and GPR56. Accordingly, in methods in which m-LSCs are identified via the immunophenotype, the user performing the method can measure the expression of CD34, CD4, CD11b, CD14 and CD36 (and optionally one or more of CD117, CD244, CD64 and GPR56) in cells from a sample from the subject in order to determine whether m-LSCs are present in the sample, and optionally, the percentage and/or number of m-LSCs in the sample. [00103] 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), or any combination thereof. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00104] In addition to the immunophenotype described above, m-LSCs can be alternatively or further identified by one or more transcriptomic features described herein. [00105] In some aspects of the methods of the present disclosure, a transcriptomic feature that identifies an m-LSC can be an upregulation in the expression of at least one of the biomarkers put forth in Table 5. As would be appreciated by the skilled artisan, an upregulation corresponds to an expression level that is greater than a control expression value seen in other types of cells. [00106] In some aspects of the methods of the present disclosure, a transcriptomic feature that identifies an m-LSC can be at least one of the biomarkers, or any combination of the biomarkers, put forth in Table 1A. [00107] Table 1A – Biomarkers downregulated in m-LSCs Attorney Docket No.: UNCO-049/001WO (300978-2220) [00108] In some aspects of the methods of the present disclosure, a transcriptomic feature that identifies an m-LSC can be an upregulation in at least one of the GSEA gene signatures put forth in Table 1B. The skilled artisan would readily appreciate the biomarkers of each of the GSEA gene signatures put forth in Table 1B. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00109] Table 1B – GSEA gene signatures upregulated in m-LSCs [00110] Any of the transcriptomic features put forth in Tables 1A, 1B and 5 can be combined together in any combination. In a non-limiting example, an m-LSC cell may be identified by the upregulation of two of the biomarkers put forth in Table 1A and three of the biomarkers put forth in Table 5. [00111] As would be appreciated by the skilled artisan, identifying an m-LSC using the transcriptomic features described herein can comprise measuring the expression of the one or more biomarkers that make up the transcriptomic feature. This measurement of expression 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, RNA sequencing, flow cytometry, mass spectrometry, microarray analysis, digital droplet PCR, Western Blot, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-SEQ), or any combination thereof. [00112] Identifying an m-LSC using the transcriptomic features described herein can also comprise performing a transcriptomic analysis using any of the standard methods known in the art for transcriptomic analysis, including, but not limited to the methods described herein, such as CITE-SEQ. [00113] Identification of CD70+ m-LSCs [00114] 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 CD70+ monocytic leukemia stem cells (m-LSCs) in a plurality of cells in a sample from a subject. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00115] It is understood that a step of identifying at least one CD70+ m-LSC in a plurality of cells can result in the identification of no CD70+ m-LSCs (i.e. the user can determine that there are actually no CD70+ m-LSCs present in the plurality of cells). [00116] In the methods of the present disclosure, m-LSCs can be identified based on the novel immunophenotype of: CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+. In some aspects, this immunophenotype can optionally be further supplemented with one or more makers selected from CD117-, CD244- and CD64+. Accordingly, in methods in which CD70+ m- LSCs are identified via the immunophenotype, the user performing the method can measure the expression of CD34, CD4, CD11b, CD14, CD36 and CD70 (and optionally one or more of CD117, CD244 and CD64) in cells from a sample from the subject in order to determine whether CD70+ m-LSCs are present in the sample, and optionally, the percentage and/or number of CD70+ m-LSCs in the sample. In some aspects, this immunophenotype can optionally be further supplemented with one or more makers selected from CD117-, CD244-, CD64+, and GPR56-. Accordingly, in methods in which CD70+ m-LSCs are identified via the immunophenotype, the user performing the method can measure the expression of CD34, CD4, CD11b, CD14, CD36 and CD70 (and optionally one or more of CD117, CD244, CD64 and GPR56) in cells from a sample from the subject in order to determine whether CD70+ m- LSCs are present in the sample, and optionally, the percentage and/or number of CD70+ m- LSCs in the sample. [00117] 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), or any combination thereof. [00118] In addition to the immunophenotype described above, CD70+ m-LSCs can be alternatively or further identified by one or more transcriptomic features described herein. [00119] In some aspects of the methods of the present disclosure, a transcriptomic feature that identifies a CD 70+ m-LSC can be an upregulation in the expression of at least one of the biomarkers put forth in Table 5. As would be appreciated by the skilled artisan, an upregulation corresponds to an expression level that is greater than a control expression value seen in other types of cells. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00120] In some aspects of the methods of the present disclosure, a transcriptomic feature that identifies a CD70+ m-LSC can be at least one of the biomarkers, or any combination of the biomarkers, put forth in Table 1A. [00121] Any of the transcriptomic features put forth in Tables 1A, 1B and 5 can be combined together in any combination. In a non-limiting example, a CD70+ m-LSC cell may be identified by the upregulation of two of the biomarkers put forth in Table 1A and three of the biomarkers put forth in Table 5, in addition to the measurement of CD70 expression. [00122] As would be appreciated by the skilled artisan, identifying a CD70+ m-LSC using the transcriptomic features described herein can comprise measuring the expression of the one or more biomarkers that make up the transcriptomic feature. This measurement of expression 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, RNA sequencing, flow cytometry, mass spectrometry, microarray analysis, digital droplet PCR, Western Blot, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-SEQ), or any combination thereof. [00123] Identifying a CD70+ m-LSC using the transcriptomic features described herein can also comprise performing a transcriptomic analysis using any of the standard methods known in the art for transcriptomic analysis, including, but not limited to the methods described herein, such as CITE-SEQ. [00124] Hypomethylating agents [00125] 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, cytarabine and decitabine. In some aspects of the methods presented herein, a hypomethylating agent can be any hypomethylating agent known in the art.
Attorney Docket No.: UNCO-049/001WO (300978-2220) [00126] In some aspects, a hypomethylating agent can be azacitidine: 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. [00127] 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-Azadeoxycytidine, 2-Desoxy-5-azacytidine and 2ƍ-Deoxy-5- Attorney Docket No.: UNCO-049/001WO (300978-2220) azacytidine. As would be appreciated by the skilled artisan, decitabine may be identified as CAS No.2353-33-5. [00128] Substitution of hypomethylating agent [00129] 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. [00130] Accordingly, in a non-limiting example, 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 cytarabine, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m- LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) identifying that the subject will not be responsive to the treatment when the presence of at least one m-LSC is identified; or identifying that the subject will be responsive to the treatment when no m-LSCs are identified. [00131] In another non-limiting example, 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 cytarabine, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of m-LSCs in the plurality of Attorney Docket No.: UNCO-049/001WO (300978-2220) cells based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) comparing the number and/or percentage of m-LSCs identified in step (b) to a predetermined cutoff value; d) identifying that the subject will not be responsive to the treatment when the number and/or percentage of m-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-LSCs is less than the predetermined cutoff value. [00132] In another non-limiting example, the present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) administering to the subject a combination of at least one BCL-2 inhibitor, cytarabine, and at least one m-LSC targeting agent when at least one m-LSC is identified; or administering to the subject a combination of at least one BCL-2 inhibitor and cytarabine when no m-LSCs are identified. [00133] In another non-limiting example, the present disclosure provides methods of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the number and/or percentage of m-LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) comparing the number and/or percentage of m-LSCs identified in step (b) to a predetermined cutoff value; d) administering to the subject a combination of at least one BCL-2 inhibitor, cytarabine, and at least one m-LSC targeting agent when the number and/or percentage of m-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 cytarabine when the number and/or percentage of m-LSCs is less than the predetermined cutoff value. [00134] BCL-2 inhibitors [00135] 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. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00136] 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 following 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. [00137] In some aspects, a BCL-2 inhibitor can be navitoclax: 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- Attorney Docket No.: UNCO-049/001WO (300978-2220) sulfonyl)benzamide. As would be appreciated by the skilled artisan, navitoclax may be identified as CAS No.923564-51-6. [00138] In some aspects, a BCL-2 inhibitor can be BGB-11417. [00139] In some aspects, a BCL-2 inhibitor can be ZN-d5. [00140] CD70-targeting agents [00141] In some aspects, CD70-targeting agent can be at least one of: i) an anti-CD70 antibody; ii) an anti-CD70 immunotherapy, preferably wherein the immunotherapy comprises CAR-T and/or NK Cells that are directed specifically at CD70; iii) an agent that blocks CD70 signaling, preferably wherein the agent that blocks CD70 signaling prevents binding of CD27 and CD70. [00142] In some aspects, a CD70-targeting agent can be cusatuzumab. [00143] 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. [00144] In some aspects, an immunotherapy can be an immunotherapy that specifically targets CD70. 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 CD70. [00145] m-LSC targeting agents [00146] In some aspects of the methods presented herein, an m-LSC targeting agent can be an agent that modulates one-carbon metabolism. [00147] In some aspects of the methods presented herein, an m-LSC targeting agent can be an agent that modulates at least one of purine synthesis and pyrimidine synthesis. [00148] An agent that modulates one-carbon metabolism is selected from methotrexate, brequinar and cladribine. [00149] In some aspects, an m-LSC targeting agent can be cladribine: Attorney Docket No.: UNCO-049/001WO (300978-2220) , or a pharmaceutically acceptable salt, analog, derivative, salt or ester thereof. As would be appreciated by the skilled artisan, it is understood that cladribine may be identified by any one of the following names: 5-(6-Amino- 2-chloro-purin-9-yl)-2-(hydroxymethyl)oxolan-3-ol, 2-Chloro-2ƍ-deoxyadenosine, 2-Chloro- 2-Chlorodeoxyadenosine. As would be appreciated by the skilled artisan, cladribine may be identified as CAS No.4291-63-8. [00150] In some aspects, an m-LSC targeting agent can be brequinar: pharmaceutically acceptable salt, analog, derivative, salt or ester thereof. As would be appreciated by the skilled artisan, it is understood that brequinar may be identified by any one of the following names: 6-fluoro-2- (2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinolinecarboxylic acid, Biphenquinate and BPQ. As would be appreciated by the skilled artisan, brequinar may be identified as CAS No. 96187-53-0. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00151] In some aspects, an m-LSC targeting agent can be methotrexate: pharmaceutically acceptable salt, analog, derivative, salt or ester thereof. As would be appreciated by the skilled artisan, it is understood that methotrexate may be identified by any one of the following names: (2S)-2-[(4-{[(2,4-Diaminopteridin-6- yl)methyl](methyl)amino}benzoyl)amino]pentanedioic acid, MTX, 4-Amino-N10- methylpteroylglutamic acid and N-[p-[[2,4-Diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-(+)-glutamic acid. As would be appreciated by the skilled artisan, methotrexate may be identified as CAS No.59-05-2. [00152] In some aspects, an m-LSC targeting agent can be an agent that inhibits MCL1. Non- limiting examples of agents that inhibit MCL1 are VU661013 and S63845. [00153] In some aspects, an m-LSC targeting agent can be an immunotherapy. [00154] In some aspects, an m-LSC targeting therapy can be an antifolate. Non-limiting examples of antifolates include methotrexate, pralatrexate and pemetrexed. [00155] 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. [00156] In some aspects, an immunotherapy can be an immunotherapy that specifically targets at least one monocytic antigen, including, but not limited to CD64 and LILRB4. 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 CD64 and/or LILRB4. [00157] 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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. 108 M or less, e.g. from 108 M to 1013 M, e.g., from 109 M to 1013 M). In certain embodiments, an anti-target antibody binds to an epitope of a target that is conserved among different species. [00158] 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. [00159] 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. For 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. [00160] 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. [00161] 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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. [00162] 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. [00163] 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 as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. CAR-T cells and related compositions can be administered to a subject intravenously. [00164] 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. [00165] Subjects [00166] 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. [00167] 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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. [00168] In some aspects of the methods of the present disclosure, the subject is a human. [00169] 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. [00170] 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. [00171] In some aspects of the methods of the present disclosure, a subject is a subject who has at least one m-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-LSCs 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-LSCs that are greater than a predetermined cutoff number. [00172] In some aspects of the methods of the present disclosure, a subject is a subject who has at least one CD70+ m-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 CD70+ m-LSCs 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 CD70+ m-LSCs that are greater than a predetermined cutoff number. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00173] Samples [00174] 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. [00175] In some aspects, a sample can be a bone marrow biopsy. [00176] In some aspects, a sample can be a bone marrow aspirate. [00177] In some aspects, a sample can be a biopsy of a chloroma. [00178] General Definitions [00179] 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. [00180] 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. [00181] 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 of 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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. [00182] 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. [00183] 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. [00184] 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. [00185] 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. [00186] It is to be understood that, for any agent, the therapeutically effective amount can be 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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. [00187] 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. [00188] 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., glycine, alanine, phenylalanine, arginine, etc. [00189] 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. [00190] 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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. [00191] 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. [00192] 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. [00193] 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. [00194] 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 another 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 Attorney Docket No.: UNCO-049/001WO (300978-2220) 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. [00195] Exemplary Embodiments [00196] Embodiment 1. A method of treating acute myeloid leukemia (AML) in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent when at least one m-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-LSCs are identified. [00197] Embodiment 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, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) identifying that the subject will not be responsive to the treatment when the presence of at least one m-LSC is identified; or identifying that the subject will be responsive to the treatment when no m-LSCs are identified. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00198] Embodiment 3a. The method of embodiment 1 or embodiment 2, wherein step (a) further comprises measuring the expression of at least CD117, CD244 and CD64, wherein a cell is identified as an m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244- and CD64+. [00199] Embodiment 3b. The method of embodiment 1 or embodiment 2, wherein step (a) further comprises measuring the expression of at least CD117, CD244, CD64 and GPR56, wherein a cell is identified as an m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, and GPR56-. [00200] Embodiment 4. The method of any one of the preceding embodiments, wherein the at least one m-LSC targeting agent is an agent modulates one-carbon metabolism, is an agent that modulates purine synthesis, is an agent that modulates pyrimidine synthesis, or any combination thereof. [00201] Embodiment 5. The method of any one of the preceding embodiments, wherein the at least one m-LSC targeting agent is selected from methotrexate, brequinar and cladribine. [00202] Embodiment 6. The method of any one of the preceding embodiments, wherein the at least one hypomethylating agent is selected from azacitidine and decitabine. [00203] Embodiment 7. The method of any one of the preceding embodiments, wherein the at least one BCL-2 inhibitor is selected from venetoclax and navitoclax. [00204] Embodiment 8. The method of any one of the preceding embodiments, 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), or any combination thereof. [00205] Embodiment 9. The method of any one of the preceding embodiments, wherein the subject is a subject having AML who has not received any treatment for AML. [00206] Embodiment 10. The method any one of the preceding embodiments, wherein the subject is a subject having AML who has received previously received at least one AML treatment. [00207] Embodiment 11. The method of embodiment 10, wherein the at least one AML treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent. [00208] Embodiment 12. The method of any one of the preceding embodiments, wherein identifying that a subject will be responsive to treatment with a combination of at least one Attorney Docket No.: UNCO-049/001WO (300978-2220) 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. [00209] Embodiment 13. The method of any one of the preceding embodiments, 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. [00210] Embodiment 14. The method of any one of the preceding embodiments, 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. [00211] Embodiment 15. The method of embodiment 14, wherein the sample is a bone marrow biopsy. [00212] Embodiment 16. The method of embodiment 14, wherein the sample is a bone marrow aspirate. [00213] Embodiment 17. The method of embodiment 14, wherein the sample is a biopsy of a chloroma. [00214] Embodiment 18. The method of any one of the preceding embodiments, wherein [00215] step (a) further comprises performing a transcriptomic analysis of the plurality of cells in the sample; and step (b) further comprises identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on transcriptomic analysis performed in step (a), wherein a cell is identified as an m-LSC based on at least one of the following: i) an upregulation in the expression of at least one of biomarker from Table 5; ii) the expression of at least one biomarker from Table 1A; iii) an upregulation in the expression of at least one GSEA gene signature from Table 1B. [00216] Embodiment 19. The method of embodiment 18, wherein the transcriptomic analysis is performed using RNA sequencing. [00217] Embodiment 20. The method of embodiment 19, wherein the RNA sequencing is performed using CITE-SEQ. [00218] Embodiment 21. A method of treating acute myeloid leukemia (AML) in a subject, the method comprising: Attorney Docket No.: UNCO-049/001WO (300978-2220) a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) administering to the subject a treatment comprising at least one CD70-targeting agent when at least one CD70+ m-LSC is identified, preferably wherein the treatment further comprises at least one BCL-2 inhibitor and at least one hypomethylating agent. [00219] Embodiment 22. A method of identifying if a subject having AML will be responsive to treatment with a CD70-targeting agent, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m- LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m- LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) identifying that the subject will be responsive to the treatment when the presence of at least one CD70+ m-LSC is identified. [00220] Embodiment 23. The method of embodiment 21 or embodiment 22, wherein step (a) further comprises measuring the expression of at least CD117, CD244 and CD64, wherein a cell is identified as a CD70+ m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+ and CD70+. [00221] Embodiment 24. The method of any one of the preceding embodiments, wherein the at least one CD70-targeting agent is: i) an anti-CD70 antibody, preferably wherein the anti-CD70 antibody is cusatuzumab; ii) an anti-CD70 immunotherapy, preferably wherein the immunotherapy comprises CAR-T and/or NK Cells that are directed specifically at CD70; or iii) an agent that blocks CD70 signaling, preferably wherein the agent that blocks CD70 signaling prevents binding of CD27 and CD70. [00222] Embodiment 25. The method of any one of the preceding embodiments, wherein the at least one hypomethylating agent is selected from azacitidine and decitabine. [00223] Embodiment 26. The method of any one of the preceding embodiments, wherein the at least one BCL-2 inhibitor is selected from venetoclax and navitoclax. [00224] Embodiment 27. The method of any one of the preceding embodiments, wherein step (a) comprises performing PCR, high-throughput sequencing, next generation sequencing, Attorney Docket No.: UNCO-049/001WO (300978-2220) 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, [00225] Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-SEQ), or any combination thereof. [00226] Embodiment 28. The method of any one of the preceding embodiments, wherein the subject is a subject having AML who has not received any treatment for AML. [00227] Embodiment 29. The method any one of the preceding embodiments, wherein the subject is a subject having AML who has received previously received at least one AML treatment. [00228] Embodiment 30. The method of embodiment 29, wherein the at least one AML treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent. [00229] Embodiment 31. The method of any one of the preceding embodiments, 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. [00230] Embodiment 32. The method of embodiment 31, wherein the sample is a bone marrow biopsy. [00231] Embodiment 33. The method of embodiment 31, wherein the sample is a bone marrow aspirate. [00232] Embodiment 34. The method of embodiment 31, wherein the sample is a biopsy of a chloroma. [00233] Embodiment 35. The method of any one of the preceding embodiments, wherein step (a) further comprises performing a transcriptomic analysis of the plurality of cells in the sample; and step (b) further comprises identifying the presence of at least one CD70+ monocytic leukemia stem cell (m-LSC) based on transcriptomic analysis performed in step (a), wherein a cell is identified as a CD70+ m-LSC based on at least one of the following: i) an upregulation in the expression of at least one of biomarker from Table 5; ii) the expression of at least one biomarker from Table 1A; iii) an upregulation in the expression of at least one GSEA gene signature from Table 1B. [00234] Embodiment 36. The method of embodiment 35, wherein the transcriptomic analysis is performed using RNA sequencing. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00235] Embodiment 37. The method of embodiment 36, wherein the RNA sequencing is performed using CITE-SEQ. [00236] Experimental Examples [00237] Example 1 [00238] In the following non-limiting example, experiments were performed to characterize developmentally heterogenous LSCs. A cohort of 20 primary human patient specimens selected to represent the spectrum of phenotypes commonly encountered in AML were analyzed by standard flow cytometric blood cell gating (see Table 1C for patient and specimen information). The analysis revealed three broad classes of specimens were defined: 1) predominantly primitive (Prim), 2) mixed monocytic-primitive (MMP), and 3) predominantly monocytic (Mono). As indicated by the nomenclature, the Prim specimens displayed almost no differentiated monocytic features (<1%) and a predominant blast-like profile (CD45-medium and low internal side scatter (SSC) relative to lymphocytes from the same patient). Prim specimens expressed high levels (>90% in most) of the canonical stem/progenitor associated markers (CD34 and CD117), and low expression of monocytic antigens (CD11b, CD64, CD14, CD36, and LILRB4). In contrast, on the most differentiated end of the spectrum, Mono specimens displayed very few blast-like cells (<1%) and a predominant monocyte-like profile (CD45-bright, SSC-high relative to lymphocytes). Mono specimens down-regulated CD34 and CD117, and up-regulated monocytic markers to varying degrees, including CD64. Finally, the MMP specimens were characterized as having discrete blast-like and monocytic-like subpopulations (range, 26%-79% for primitive and 14%-35% for monocytic), which were evident in both the CD45/SSC gate and phenotypic analysis of stem/progenitor makers and monocytic antigens contained a mixture of monocytic and primitive cells, occupying the middle range of the developmental spectrum (). Without wishing to be bound by theory, it was hypothesized that heterogeneity within the LSC compartment contributed to the developmental heterogeneity observed in the analysis described above. To begin to characterize potential LSC heterogeneity, flow cytometric cell sorting was performed to isolate primitive (prim) and monocytic (mono) subpopulations from several MMP AMLs. Each subpopulation was independently transplanted into immunodeficient NSG-S mice and evaluated for engraftment of leukemic disease by measuring the percentage of human CD45+ cells in the bone marrow of each experimental animal. These studies revealed two subgroups of MMP AML patients. One subgroup was designated “Uni-MMP,” where LSC activity was exclusively detected in the prim but not in the mono subpopulation (FIG.1A). In contrast, the second type of MMP AML was Attorney Docket No.: UNCO-049/001WO (300978-2220) designated “Multi-MMP,” where readily detectable LSC activity was evident in both prim and mono subpopulations (FIG.1B). [00239] To assess the underlying mutational profile of the Uni- and Multi-MMP types of AML, whole-exome sequencing (WES) was performed on flow-sorted prim and mono subpopulations. The WES analysis indicated that the prim and mono subpopulations from three independent Uni-MMP specimens had a comparable mutational profile in the 49 commonly mutated genes of AML, which include ASXL1, CEBPA, GATA2, JAK3, MYD88, PTPN11, SRSF2, BCOR, CSF3R, GNAS, KDM6A, NOTCH1, RAD21, STAG2, BCOR1, DNMT3A, HRAS, KIT, NPM1, RUNX1, TET2, BRAF, ETV6, IDH1, KMT2A, NRAS, SETBP1, TP53, CALR, EZH2, IDH2, KRAS, PHF6, SF3B1, U2AF1, CBL, FLT3, JAK1, MEK1, PML, SMC1A, WT1, CBLB, GATA1, JAK2, MPL, PTEN, SMC3, ZRSR2. A few differences in variant allele frequency were noted (FLT3 and SETBP1 mutations), but overall, the analyses were consistent with a common genetic origin. WES of three Multi- MMP specimens showed two specimen with similar profiles between prim and mono subpopulations; however, the third specimen demonstrated a clear difference, with differing NRAS and SMC1A mutations in the two fractions. One specimen also showed multiple mutations in common including IDH2 and SRSF2 between the prim and mono subpopulations, suggesting that divergence occurred at a relatively late stage of pathogenesis from a common ancestor clone. Without wishing to be bound by theory, these data indicate that in at least some instances Multi-MMP AML arises due to underlying mutational variation, consistent with the understanding that phenotypically distinct subpopulations of LSCs can simultaneously exist in certain patients with AML . [00240] To further characterize the leukemogenic properties of LSCs in Multi-MMP patients, the nature of disease arising in transplanted NSG-S mice was evaluated. Prim vs mono engrafted cells from two representative specimens (AML-07 and AML-13) consistently differed in their developmental spectrum. For both AML specimens, the prim subpopulation was able to recapitulate the full developmental spectrum of disease, with both prim and mono cells evident in each transplanted mouse. In contrast, the mono subpopulation only gave rise to monocytic disease, with no evidence of more primitive cells. [00241] Notably, the observed distinct differences in developmental spectrum of engrafted disease translated into clear differences in therapeutic sensitivity. When prim vs mono engrafted groups were treated with a regimen of venetoclax plus azacitidine (VEN+AZA) in vivo, the mono derived disease was significantly more resistant to VEN+AZA than were the prim cells (FIGS. 2A-C). Thus, the LSCs that initiate and drive disease in the monocytic Attorney Docket No.: UNCO-049/001WO (300978-2220) subpopulation demonstrated a more restricted developmental hierarchy that resides toward the mature end of the myeloid developmental spectrum and distinct resistance to VEN+AZA therapy. This subclass of LSCs was designated as mono-LSCs (m-LSCs). in contrast to the more conventional prim-LSCs (p-LSCs). Without wishing to be bound by theory, these findings demonstrate that heterogeneous LSC subpopulations with distinct developmental phenotypes co-reside in the same patient. Moreover, LSC heterogeneity gives rise to bulk tumor populations with differing therapeutic response in PDX models, demonstrating clinical significance. [00242] Example 2 [00243] In the following non-limiting example, experiments were performed to predict clinical outcomes as a function of m-LSCs. Without wishing to be bound by theory, it was hypothesized that de novo AML patients would differ in pathogenesis and clinical outcome as a function of the presence of m-LSCs. As illustrated schematically in FIG.3A, it was predicted that upon receiving VEN+AZA therapy, Uni-MMP patients that initially presented with only p-LSCs should achieve more durable remission due to intrinsic reliance of p-LSCs on venetoclax target BCL-2. In contrast, Multi-MMP patients with a distinct m-LSC population were predicted to relapse relatively quickly with monocytic disease. To test this concept, three de novo AML patients who had been treated with the VEN+AZA therapy were evaluated. As illustrated in FIGS.3B-D, at diagnosis, patient Pt-20 had no detectable m-LSC activity, whereas patients Pt-12 and Pt-69 had readily detectable m-LSC activities as revealed by xenograft assays. The presence of functionally defined m-LSCs directly correlated with clinical outcomes, where Uni-MMP patient Pt-20 experienced prolonged remission for more than 3.5 years, and Multi-MMP patients Pt-12 and Pt-69 had relatively rapid relapse of disease with a monocytic phenotype within 12 and 3 months, respectively. [00244] To assess mutational changes that may occur during pathogenesis, WES analysis was performed on the sorted prim and mono subpopulations of Pt-20, 12, and 69. As expected for a Uni-MMP AML, Pt-20, a Uni-MMP AML patient, showed a very similar mutation profile between prim- and mono-sorted cell populations. In contrast, both patients 12 and 69 demonstrated differing mutations. More specifically, Pt-12 had an NRAS mutation specific to the mono subpopulation at diagnosis that remained at relapse. Conversely, Pt-12 had an SMCA1 mutation in the prim subpopulation at diagnosis that was absent at relapse. The data clearly suggest the outgrowth of the genetically defined monocytic subclone. Similarly, Pt-69 had chromosome 11 and 16 duplication events in the prim compartment at diagnosis that were not present at relapse, again consistent with the outgrowth of the genetically distinct monocytic Attorney Docket No.: UNCO-049/001WO (300978-2220) subclone. Lastly, for comparative purposes, data from Pt-65, also shows the outgrowth of genetically distinct monocytic clones at relapse marked by two KRAS mutations. Notably, both of the KRAS mutations that were dominant at relapse were not readily detectable at 400× sequencing depth in the diagnosis specimen and were seen only using higher resolution methods (droplet-digital PCR). Without wishing to be bound by theory, these data suggest even minor monocytic subclones can be selected by the strong selective pressure of VEN + AZA in vivo. [00245] Additionally, a cohort of 25 AML patients who relapsed from the VEN+AZA therapy were subsequently analyzed (Table 2A and Table 2B). Nine patients, including five phenotypically monocytic and four who were primitive at diagnosis, relapsed with monocytic features, representing 36% of overall cases (FIG.3E). [00246] Notably, patients who relapsed with monocytic features also had a significantly shorter remission duration than patients who relapsed with a primitive immunophenotype (FIG.3F and FIG.3G), suggesting that patients with pre-existing m-LSCs may represent an especially poor prognosis subgroup when receiving VEN+AZA therapy. Without wishing to be bound by theory, these findings indicate that the presence of m-LSCs in newly diagnosed AML patients represents a distinct disease entity that has not been identified previously and that these m-LSCs can be used to predict pathogenesis of disease in response to VEN+AZA therapy. [00247] Example 3 [00248] In the following non-limiting example, m-LSC immunophenotypes were identified and characterized in primary AML specimens. CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) analysis was performed, allowing simultaneous measurement of protein-based surface antigens and RNA-based transcriptome analysis at a single cell level. CITE-seq analysis was performed on a cohort of 27 primary AML specimens containing immunophenotypically defined Prim, MMP, and Mono AMLs (listed in Table 3). The CITE-seq data was first analyzed using Clustifyr, an application that assigns phenotypes based on comparison to the transcriptome of normal human hematopoiesis (see Fu et al. F1000Res.2020;9:223). This analysis classifies myeloid cells into HSPC, MPP, Early Promyelocyte-like, Late promyelocyte-like, Myelocyte-like, classical monocyte-like, and Nonclassical monocyte-like subclusters, and revealed the myeloid developmental spectrum within primary AML specimens. The lineage assignments were strongly supported by the expression patterns of classic lineage-specific markers, for example, of CD7, CD56, CD19, CD33, CD34, CD11b, and CD123 at both protein and RNA levels, as well as lineage- Attorney Docket No.: UNCO-049/001WO (300978-2220) specific transcriptional factors such as GATA2, CEBPA, SP11/PU.1, AZU1, TCF7 and PAX5. [00249] Next, scArches analysis on the myeloid subpopulation to identify cell types that are indicative of the myeloid developmental hierarchy (see Zeng et al. Nat Med 2022;28: 1212– 23 and Lotfollahi et al. Nat Biotechnol 2022;40:121–30). This analysis revealed a diverse mixture of cell types, including leukemia stem and progenitor cell (LSPC)-quiescent, LSPCprimed, LSPC-cycling, granulocyte-macrophage progenitor (GMP)–like, promonocyte (ProMono)-like, Mono-like, and conventional dendritic cell (cDC)–like leukemic cells), demonstrating a clear developmental hierarchy from top left to bottom right in the myeloid subpopulation of our primary AML cohort [00250] Next, the overall CITE-seq data were segregated into Prim, MMP, and Mono groups according to their immunophenotype. The MMP group was also further divided into Uni- MMP and Multi-MMP subgroups based on each functional LSC activity, as measured by xenograft assay (FIG.4A). The relative proportion of each myeloid subcluster within Prim, Uni-MMP, Multi-MMP, and Mono groups was highly concordant with expected identity. The Prim group almost exclusively contained the highest proportion of the three LPSC cell types (quiescent, primed, and cycling), which reside at the apex of the myeloid developmental hierarchy. In contrast the Mono group presented a developmental hierarchy with almost no LSPC cell types but was rather comprised of a dominant ProMono-like cell type, suggesting the latter could be enriched for m-LSCs. Consistent with this hypothesis, both LSPC and ProMono-like cell types were present in the Multi-MMP, whereas only the former is present in the Uni-MMP group (FIG.4A). [00251] To further test which cell type might be enriched for LSC activities, several known LSC signatures were screened using the CITE-seq dataset. A KMT2A-rearranged leukemia-specific LSC signature (KMT2A-r_LSC_signature; Table 4; see Somervailee et al. Cancer Cell 2006;10:257–68; see Somervaille et al. Cell Stem Cell 2009;4:129–40; see Hess et al., Blood 2006;108:297–304) matched well with the ProMono-like cell type uniquely present in the Mono and Multi-MMP groups. Previous studies have shown that KMT2A-rearranged leukemia is distinct from other AMLs, is frequently associated with a monocytic immunophenotype, and has LSC characteristics that are distinct from conventional CD34+ primitive LSCs. Consistently, a CD34+ primitive LSC signature (CD34+_LSC_signature, Table 4; see Ng et al., Nature 2016;540:433–7; see Eppert et al. Nat Med 2011;17:1086–93) matched well with LSPCprimed but not ProMono-like clusters in the CITE-seq dataset, further supporting the concept that the ProMono-like cell type could be enriched for m-LSC activities. GSEA was Attorney Docket No.: UNCO-049/001WO (300978-2220) also preformed and significantly up-regulated genes in m-LSCs were identified (Table 5). Finally, expression analysis of the surface antigens revealed that the ProMono-like cells are largely CD4+, CD14í, CD11bí, and CD36í relative to other myeloid leukemia cells, providing a candidate immunophenotype for m-LSCs (FIG.4B). [00252] Example 4 [00253] In the following non-limiting example, experiments were performed to functionally validate m-LSCs. A series of flow sorting and transplant studies to functionally validate the m-LSC immunophenotype of the CITE-seq analysis. Two mono specimens AML-16 and AML-20 were employed, from which various subpopulations of cells were isolated using expression of CD45, CD34, CD4, CD14, CD11b, and CD36 (FIGS.5A-B and FIGS.8A-8B). To avoid contamination from conventional CD34+ p-LSCs, the CD34- fraction was gated when validating the m-LSCs. As shown in FIGS.5D-E and FIGS.8D-8E, functional analysis of each subpopulation demonstrated that the majority of m-LSCs were enriched in the CD34-, CD4+, CD14-, CD11b-, and CD36- immunophenotype. Further, secondary transplantation of animals originally engrafted with m-LSCs (designated as population D) also demonstrated robust engraftment, an indication of strong self-renewal potential. [00254] Notably, in AML-20, some engraftment potential was detected in the CD14-, CD11b+, and CD36+ counterparts (FIG.5E), suggesting that in certain patients the developmental hierarchy of m-LSC-driven AMLs is shallower than others (e.g., AML-16). A similar approach to validate m-LSCs in Multi-MMP patients was also applied: AML-07 was functionally defined to be a Multi-MMP specimen. As shown in FIG.5C, FIG.5F, FIG.8C and FIG.8F, serial transplant experiments demonstrated that m-LSCs in this Multi-MMP AML were also exclusively enriched by the CD34-, CD14-, CD11b-, and CD36- immunophenotype. Of note, CD4 was not included in this sort due to limited cell numbers. Together, these data revealed an m-LSC immunophenotype that was applicable to both Mono and Multi-MMP AMLs, entirely distinct from profiles previously described for the more conventional primitive LSCs (i.e. CD34+ and CD38-). [00255] To further explore the immunophenotype of m-LSCs, analytical flow cytometry was used to evaluate the expression of several additional cell surface antigens associated with stem cell activity. Both CD117 and CD244 have been shown to be frequently expressed specifically in CD34- AML specimens. However, there was no detectable expression of CD117 in any of the seven specimens that underwent functional evaluation for the presence of m-LSCs. Similarly, CD244 was only detected at low levels in two of seven specimens. Expression of the monocytic marker, CD64, was also investigated, as this antigen was shown Attorney Docket No.: UNCO-049/001WO (300978-2220) to be prevalent in monocytic relapse. In contrast, CD64 was strongly expressed in six of seven specimens. Without wishing to be bound by theory, these results indicated that m- LSCs can be further identified using the immunophenotypic markers of CD117-, CD244-, and CD64+, in addition to those discussed above. [00256] Finally, expression of another LSC marker, GPR56, was also evaluated. mRNA expression of GPR56 was almost absent in monocytic AML at both bulk and single-cell resolution. Moreover, flow analysis of GPR56 also showed that its protein expression was high in primitive AML cells but is entirely absent in the m-LSC-enriched population. Without wishing to be bound by theory, these results indicated that m-LSCs can be further identified by the immunophenotype of CD117-, CD244-, GPR56- and CD64+. [00257] Example 5 [00258] In the following non-limiting example, experiments were performed to target purine metabolism in m-LSCs. m-LSCs drive refractory/relapse response to venetoclax based regimens, and to address this, therapies that selectively target m-LSCs were developed. Using the CITE-seq data described above, several gene expression signatures were evident in the m-LSC compartment including pyrimidine metabolism and purine metabolism. These results were corroborated by expression patterns of key enzymes known to modulate one- carbon metabolism (e.g., TYMS and DHFR) that feed into purine (e.g., HPRT1) and pyrimidine synthesis (e.g., DHODH) pathways. Consistent with gene expression analyses, metabolic analysis also revealed purine metabolism was enriched in flow-sorted m-LSCs relative to non m-LSCs. [00259] Functional studies were then performed to evaluate agents known to impact these pathways. [00260] Methotrexate (MTX), brequinar (BRQ), and cladribine (CdA) were selected based on their activities in inhibiting one-carbon metabolism enzymes DHFR and TYMS, pyrimidine synthesis enzyme DHODH, and purine-based DNA/RNA synthesis, respectively.Colony- forming unit assays were performed on m-LSCs and p-LSCs isolated from Mono, Multi-MMP, and Prim specimens, along with normal CD34+ hematopoietic stem and progenitor cells (HSPC) from two healthy donors. As shown in Fig.6A and FIG.6B, CdA showed remarkable specificity against m-LSCs while sparing p-LSCs as well as normal HSPC controls. This selectivity was not seen by the other chemotherapy agents cytarabine and daunorubicin (FIG. 6C), suggesting the unique sensitivity of m-LSCs to CdA. In addition, CdA also outperformed BRQ and MTX in potency against m-LSCs (FIG.6D). Thus, given its superior selectivity and extensive clinical use, CdA was selected for in vivo proof-of concept studies in combination Attorney Docket No.: UNCO-049/001WO (300978-2220) with VEN+AZA in two functionally validated MMP AMLs. As shown in Fig.6E, Multi-MMP AML-13 and AML-07 were transplanted into NSG-S mice and treated with VEN+AZA alone, CdA alone, or the triple-drug combination in vivo. Analysis of AML cells in both bone marrow and spleen demonstrated that the addition of CdA clearly improved the clearance of tumor that was otherwise resistant to VEN + AZA in both PDX models (Fig. 6F-6H). Notably, flow analysis of residual engrafted human cells after treatment revealed that the VEN+AZA regimen selectively cleared the primitive subpopulation, whereas the CdA regimen targeted the monocytic counterpart, and the triple-drug regimen eradicated both. [00261] Summary of Examples 1-5 [00262] Examples 1-5 describe the identification and characterization of a previously unrecognized type of human acute myeloid leukemia (AML) stem cell defined as “m-LSC.” This particular subclass of LSC is distinguished from more primitive subtypes by virtue of a unique immunophenotype (CD34-, CD4+, CD14-, CD11b-, and CD36-), a relatively narrow developmental profile that is limited to the creation of monocytic progeny, and a gene expression profile that is roughly analogous to normal human promyelocytes. Notably, the m-LSC is distinct from the CD34- LSC populations described previously, where expression of both CD177 and CD244 were prevalent. The molecular biology of m-LSCs differs from more primitive LSCs in that BCL-2 dependency seems to be largely dispensable, making this type of LSC resistant to treatment with venetoclax and azacytidine. Further, m-LSCs demonstrate selective reliance on one-carbon metabolism and purine/pyrimidine metabolism, adding to the importance of cellular metabolism in the context of AML pathogenesis and therapeutic resistance. [00263] Notably, m-LSC reliance on purine metabolism mediates increased sensitivity to agents such as cladribine, a well-known purine analogue. As demonstrated herein, addition of cladribine to the VEN+AZA regimen increases eradication of primary AML containing m- LSC activity in both in vitro and in vivo preclinical models. These findings provide important mechanistic insights that may explain results from several recent clinical trials reporting superior CR/CRi and MRD negativity rates when venetoclax is combined with chemotherapy regimens, especially ones that contain cladribine or its close analogue fludarabine, namely FLAG-Ida, CLIA or Clad-LDAC/AZA. Notably, the addition of cladribine in these three trials was mainly based on empirical clinical experience and the long history of using cladribine-containing chemotherapy regimens for relapse/refractory stages of AML. As demonstrated herein, the discovery of m-LSCs, the unique m-LSC resistance to Attorney Docket No.: UNCO-049/001WO (300978-2220) venetoclax, and the m-LSC sensitivity to cladribine, may explain the efficacy of cladribine/venetoclax-based regimens in eradicating the overall LSC population. [00264] The results provided herein demonstrate a stratification of primary AML patients based on the nature of underlying LSC subpopulations. FIGS.7A-7C shows the tree as an analogy to describe developmental hierarchy of AML, where the underground roots represent LSCs and the branches above the ground symbolize more differentiated blasts. In FIG.7A, a single class of more primitive LSCs (p-LSCs) may be present in newly diagnosed AML patients, with varying degrees of monocytic differentiation potential. In this scenario, no m- LSCs are present, and venetoclax-based therapy would be predicted to confer relatively high CR rates and longer remissions. In contrast, as shown in FIG.7B, some newly diagnosed Multi-MMP AML patients present with at least two distinct subtypes of LSC with primitive vs. monocytic characteristics (p-LSC and m-LSC). Depending on the size of the m-LSC population, these patients would clinically be expected to respond and then relapse, or to be refractory to venetoclax-based therapies. Finally, in more extreme cases, only m-LSCs are present (FIG.7C), which would likely result in disease that is refractory to venetoclax. As shown in FIGS.9A-9B, patients that present with the most differentiated monocytic phenotypes (i.e., FAB-M5) but not those with myelomonocytic phenotypes (i.e., FAB-M4) show the highest frequency of refractory disease. Moreover, patients relapsed with a monocytic disease have significantly shorter duration of remission comparing to ones that had primitive relapse. [00265] The novel model described in FIGS.7A-7C can be used to design future therapies. Detection of any m-LSC population at diagnosis in a patient who is being considered for a venetoclax-based therapy, may warrant consideration of additional therapies designed to selectively target monocytic population, in the hopes that relapse, which carries a very poor prognosis in this setting, can be avoided. [00266] Aside from cladribine (and methotrexate), such potential agents include immunotherapies directed towards monocytic antigens such as CD64 and LILRB4, or small molecules that selectively impair unique biology of phenotypically monocytic AML. For example, MCL1 inhibitor drugs appear to be more active in the context of monocytic AML. The results provided herein demonstrate that m-LSCs depend on MCL1 for energy metabolism. Alternatively, for patients with pure p-LSC disease at diagnosis, no additional therapy beyond venetoclax and azacitidine may be appropriate to allow for durable remissions, and for those with pure m-LSCs, one could consider replacement of venetoclax Attorney Docket No.: UNCO-049/001WO (300978-2220) for another therapeutic strategy more likely to be effective. Future clinical trials designed in this way may serve to optimize outcomes and prevent over-treatment. [00267] m-LSC driven disease propagation and/or relapse is only observed in approximately 30% of AML patients. However, the emergence of drug resistant disease eventually occurs in the majority of newly diagnosed patients treated with the VEN+AZA regimen. This finding strongly implies that other subclasses of LSCs are present (or can evolve) in patients treated with venetoclax-based regimens. Thus, additional phenotypes associated with venetoclax-resistant LSCs may exist. [00268] Without wishing to be bound by theory, the results presented herein demonstrate the connection between disease-initiating mutations, LSC heterogeneity, developmental state of AML, and varying therapeutic response in the clinic. A novel type of venetoclax-resistant m- LSC can be found in both RAS-mutant mono (AML-20), KMT2A-rearranged mono (AML- 16), and Multi-MMP (AML-07) contexts. Thus, taken together, these lines of investigation suggest that complex oncogenic mutations likely converge on limited LSC subtypes that can shape the developmental hierarchy of bulk disease and ultimately affect therapeutic response in the clinic. This model implies that understanding LSC heterogeneity is at the core of improving therapeutic interventions for AML. Besides AML, malignant stem cell heterogeneity may also have ramifications in myeloid pathogenesis. Thus, successful development of the next generation of precision medicine towards AML requires careful dissection of human LSC heterogeneity and discovery of targeted therapies tailored to each LSC subtype. [00269] Materials and Methods of Examples 1-5 [00270] Primary AML and normal mobilized peripheral blood samples [00271] Primary human AML specimens were obtained from leukapheresis product, peripheral blood, or bone marrow of AML patients who gave informed consent for sample procurement on the University of Colorado tissue procurement protocols (Colorado Multiple Institutional Review Board Protocol #12-0173 & #06-0720). Normal mobilized peripheral blood (MPB) specimens were obtained from volunteer donors at the University of Colorado. Age, sex, cytogenetics, and mutation information of primary AML specimens used in the current study are detailed in Table 1C. [00272] Patients, Treatment and Responses [00273] Twenty-five newly diagnosed AML patients who received venetoclax + azacitidine therapy and had experienced relapse response were included in this study. The patients were diagnosed, treated, and monitored for relapse response over a period from January 2015 to Attorney Docket No.: UNCO-049/001WO (300978-2220) February 2020. The University of Colorado Institutional Review Board approved a request to retrospectively analyze these patients (#19-0115). Diagnosis and relapse phenotypes were determined through morphological review by hematologists and examination of clinical flow notes when available. Remission duration was calculated as the numbers of days in between the date of best response and the date of relapse. Cytogenetic, mutational information of diagnosis and relapse stages as well as remission duration time are detailed in Table 2. [00274] Processing and culturing of primary AML and normal CD34+ HSPCs [00275] Primary human AML specimens and normal MPB samples were resuspended at about 100-200 e6 cells per ml in freezing media composed of 50% FBS (GE Healthcare), 10% DMSO (Sigma) and 40% IMDM media (GIBCO) and then cryo-preserved in liquid nitrogen. Cells were thawed in 37°C water bath, washed twice in thawing media composed of IMDM (GIBCO), 2.5% FBS (GE Healthcare) and 10 ug/ml DNase (Sigma). Normal CD34+ HSPCs were enriched from thawed MPB samples using the CD34 MicroBead kit (Miltenyi Biotec). Cells were cultured in complete serum-free media (SFM) in 37°C, 5% CO2 incubator. SFM is composed of IMDM (GIBCO), 20% BIT 9500 (STEMCELL Technologies), 10ug/ml LDL (Low Density Lipoprotein, Millipore), 55uM 2-Mercaptoethanol (GIBCO) and 1% Pen/Strep (GIBCO). Complete SFM were made by supplementing the SFM with FLT-3, IL-3 and SCF cytokines (PeproTech), each at 10 ng/ml. [00276] Colony-forming assays [00277] Freshly sorted m-LSCs from primary AMLs or prepared CD34+ HSPCs isolated from normal mobilized peripheral samples were plated in human methylcellulose (R&D systems) at about 100K/ml and 2K/ml, respectively. Small molecule inhibitors were directly added into the methylcellulose at the desired final concentration at the plating. Colonies were counted 2- 3 weeks after the initial plating. [00278] Immunophenotyping of primary AML [00279] About 0.5-1e6 freshly thawed primary AML cells were stained with immunophenotyping panel containing antibodies against human CD45, CD34, CD117, CD11b, CD64, CD14, CD244, LILRRB4, CD36 or CD68 at 4°C for 15 mins, washed with ice cold FACS buffer, and resuspended in FACS buffer and analyzed on BD FACS Celesta flowcytometry (BD). FCS files were analyzed on Flowjo 10.5.3 (Flowjo).. [00280] Flow sorting of prim and mono subpopulations for xenograft studies [00281] Freshly thawed primary AML cells were stained with viability dyes and antibodies against human CD45 or CD34 and CD11b. For all specimens except for Pt-69, the prim Attorney Docket No.: UNCO-049/001WO (300978-2220) subpopulation was sorted as CD45-medium, SSC-medium, while the mono subpopulation was sorted as CD45-bright, SSC-medium/high. For Pt-69, prim and mono subpopulations were not readily separable using the CD45/SSC gating strategy. An alternative CD34/CD11b gating strategy was used. From the CD34/CD11b gate, two prim subpopulations were revealed from the diagnosis (Dx) specimen, one as Dx-prim-A displaying a CD34+/CD11b- phenotype, one as Dx-prim-B bearing a CD34+/CD11b+ phenotype. One mono subpopulation was also revealed from the diagnosis (Dx) specimen, named as Dx-mono exhibiting a CD34-/CD11b-partial positive (PP) phenotype. In contrast, at relapse (Rl), a single mono subpopulation showing a CD34-/CD11b-PP phenotype was revealed as Rl- mono. All subpopulations were sorted and used for xenograft studies. [00282] Xenograft studies [00283] NSG-S (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV- IL3,CSF2,KITLG)1Eav/MloySzJ) mice (The Jackson Laboratory) were used for xenograft studies in this study. Male or female mice ranging in age from 6 to 8 weeks were started on experiment. Littermates of the same sex were randomly assigned to experimental groups. NSG-S mice were pre-conditioned 24 hours prior to transplant with 30mg/kg busulfan (Alfa Aesar) via intraperitoneal (IP) injection. The busulfan stock was made fresh at 25 mg/ml in 100% DMSO, then the stock was diluted 1:10 in pre-warmed saline (0.9% NaCl) down to 2.5 mg/ml right before use. The diluted busulfan solution was kept in 37°C water bath before IP injection to prevent precipitation of busulfan due to low solubility. For comparing the engraftment potential of different subpopulations of a primary AML, each subpopulation was sorted according to their percentage of total viable cells (detailed in Table 6). When the cell dose was less than 0.5e6/mouse, mononuclear cells isolated from bone marrow and spleens of naïve NSG-S mice were used as carrier cells. The sorted cells with or without addition of carrier cells were then washed, pelleted, and resuspended in saline buffer to allow tail vein injection into NSG-S mice at 0.1ml per mouse. Fifteen minutes prior to injection, in vivo anti-human CD3 antibody (BioCell) was added at a final concentration of 1ug/e6 cells to prevent potential graft versus host disease. During all experiments, the weight of mice was approximately 20-30 grams with no animals losing greater than 10% body weight. The mice were kept in ventilated cages and given in vivo treatments when needed in the vivarium at University of Colorado. The majority of the experiments lasted for 6 to 12 weeks. At the end of the experiments, mice were euthanized using carbon dioxide. Bone marrow and spleen were harvested, subjected to red blood cell lysis, and the mononuclear cells were stained with Attorney Docket No.: UNCO-049/001WO (300978-2220) human CD45, mouse CD45 antibodies, and DAPI to determine percentage of engraftment within viable cells. All animal work were performed in accordance with Institutional Animal Care and Use Committee protocol number 00308. [00284] In vivo treatments [00285] About 2-4 weeks post initial transplant, tumor burden in the bone marrow was determined to be above 5% in sentinel mice. Mice were then treated with various in vivo regimens as follows. Venetoclax was given at 100mg/kg via oral gavage, five days/week for two weeks; Azacitidine was given at 3mg/kg via intraperitoneal injection, three days/week for two weeks; Cladribine was given at 10mg/kg via intraperitoneal injection, three days/week for two weeks. All treatments were given in the same two-week time window when stated together. [00286] CITE-seq sample preparation and library construction [00287] Mononuclear cell suspensions were prepared from freshly thawed primary AML specimens cryopreserved in liquid nitrogen. For each specimen, about 1-2e6 mononuclear cell suspension was stained with a panel of Total-seq B antibodies (1ug/each, Biolegend) and fluorochrome conjugated flow antibodies for CD45 (BD Biosciences), CD235a (BD Biosciences), and DAPI. Cells were stained in staining buffer (PBS + 2% FBS) for 20 minutes at 4°C. Viable and red blood cell excluded cells were obtained through sorting DAPI-/CD235a- cells on BD Biosciences ARIA II cell sorter. After sorting, cells were washed twice using staining buffer and resuspended to 1000 cells/μl in PBS with 2% FBS and used immediately for capture. Following cell collection and counting, cells were processed with the 10x Genomics 3’ dual index v3.1 library kit with feature barcoding technology for cell surface proteins. Briefly 10,000 cells were targeted from stock suspension of 1000 cells/μl. Cells were processed following the protocol to generate 3’ gene expression libraries as well as Feature Barcode cell surface libraries. Both libraries were dual indexed, and samples were quantified by Qubit (Life Technologies) and assessed for size and quality by Tapestation (Agilent). Libraries were normalized and pooled for sequencing on a Novaseq 6000 (Illumina) for paired-end 2x150 bp sequencing. Targeted read depth for gene expression libraries was 100,000 reads/cell or ~ 500 million paired-end reads/library. Targeted read depth for cell surface libraries was 40,000 reads/cell or 200 million paired end reads/library. Attorney Docket No.: UNCO-049/001WO (300978-2220) [00288] CITE-seq data pre-processing [00289] Raw sequencing data for gene expression, antibody derived tag (ADT; surface protein), and hashing libraries were processed using STARsolo 2.7.8a with the 10X Genomics GRCh38/GENCODE v32 genome and transcriptome reference (version GRCh38_2020A) or a TotalSeq barcode reference, as appropriate. Hashed samples were demultiplexed using GMM-Demux. Next, cell-containing droplets were identified using dropkick 1.2.6 using manual thresholds when automatic thresholding failed, ambient RNA was removed using DecontX 1.12 (www.github.com/campbio/celda) and cells estimated to contain >50% ambient RNA were removed, and doublets were identified using DoubletFinder 2.0.3 and removed. Remaining cells were then filtered to retain only those with > 200 genes, 500-80,000 UMIs, < 10-20% of UMIs from genes encoded by the mitochondrial genome (sample dependent based on UMI distributions), < 5% of UMIs derived from HBB, < 20,000 UMIs from antibody-derived tags (ADTs), and >100-2,750 UMIs from antibody derived tags (sample dependent based on UMI distribution). Filtered cells were modeled in latent space using TotalVI 0.18.0 to create a joint embedding derived from both RNA and ADT expression data, corrected for batch effects, mitochondrial proportion, and cell cycle. Scanpy 1.8.2 ) was used to cluster the data in latent space using the leiden algorithm and marker genes were identified in latent space using TotalVI. [00290] CITE-seq Data Analysis [00291] Clusters were annotated using clustifyr 1.9.1 and the leukemic/normal bone marrow reference dataset (see Triana et al. Nat Immunol 2021;22:1577–89). Scanpy and Seurat 4.1.1 (ref.58; RRID:SCR_007322) were then used to generate uniform manifold approximation and projections from the TotalVI embeddings and perform exploratory analysis, data visualization, etc. The myeloid subpopulation in the CITE-seq data was reannotated using scArches 0.5.7 and a leukemia reference dataset (see Zeng et al. Nat Med 2022;28: 1212–23). The reference model was trained for 400 epochs based on the 3,000 most highly variable genes determined by scanpys’ pp.highly_variable_ genes() function. Then the model was updated with transfer learning of the scArches algorithm to annotate the query myeloid subpopulation. CD34+_LSCs and KMT2A-r_LSCs were identified through scoring each individual cell using the AddModuleScore() function of the Seurat software and custom-generated candidate CD34+_LSC and KMT2A-r_LSC gene expression signatures, as stated in the main text. [00292] WES Analysis [00293] WES libraries were generated using the Agilent SureSelect XT exome prep kit with 200 ng of input as per protocol (Agilent). The probe used was SureSelect XT Human All Exon Attorney Docket No.: UNCO-049/001WO (300978-2220) V7 (Agilent). Libraries were normalized by Qubit (Invitrogen) and Tapestation (Agilent), and 2 × 150 bp reads were sequenced on a Novaseq 6000 (Illumina) to obtain 400× coverage. Fastqc v0.11.9 was used to assess overall sequencing quality, and reads were trimmed using cutadapt (cutadapt, RRID:SCR_011841) v2.9 to remove the Illumina universal adapters, bases of poor quality (phred <30), and any reads in which the minimum read length was <10 base pairs in length. Trimmed fastq files were then aligned to the GRCh38 p.13 genome using BWA v0.7.17 (BWA, RRID:SCR_010910). Stringent quality control of alignments and read duplicate removal were performed using the Picard suite of tools v2.21.1 (Picard, RRID:SCR_006525) and samtools v1.8 (samtools, RRID:SCR_002105). Variants were called on alignments using DeepVariant v1.0.0, followed by BCFtools (BCFtools, RRID:SCR_005227) v1.11 to filter variants of low quality. SNPs were removed if the raw unfiltered read depth was <20 reads and the mapping quality <30. All remaining variants were then annotated using Annovar v2020- 06-07 (Annovar, RRID:SCR_012821). Of all the databases that were used for annotations, the most notable ones used for data interpretation were the COSMIC database v92 (COSMIC, RRID:SCR_002260), Clinvar v2020031 (Clinvar, RRID:SCR_006169), SIFT (SIFT, RRID: SCR_012813), PolyPhen (PolyPhen, RRID:SCR_013189), and nci60. For comparing mutational profile between prim and mono cells, we focused on nonsynonymous exonic mutations that occurred in the 49 commonly mutated genes of AML. [00294] Metabolomic Analysis [00295] For each Mono-AML specimen, five replicates of 500,000 cells each were freshly sorted from the m-LSC–enriched D subpopulation (CD34í, CD4+, CD14í, CD11bí, CD36í m-LSC) and the non–m- LSC E subpopulation (CD34í, CD4+, CD14í, CD11b+, CD36+ non– m-LSC). Sorted cells were washed in ice-cold PBS, and the cell pellets were snap-frozen for ultra-high performance liquid chromatographymass spectrometry analysis as described previously (32). Results were normalized by cell number. Pathway enrichment analysis was performed on metabolites that were ^1.2-fold higher in population D compared with E and with a P value of less than 0.1. All analyses were performed using the MetaboAnalyst 5.0 software (MetaboAnalyst, RRID:SCR_015539). [00296] Statistical Analysis [00297] Statistical analyses were performed in GraphPad Prism 9.3.1 (GraphPad Prism, RRID:SCR_002798). Median ± interquartile range was used to describe summary statistics. One-tailed or two-tailed Mann–Whitney tests were used to compare two groups when applicable. Kruskal–Wallis tests with correction for multiple comparisons using the original Attorney Docket No.: UNCO-049/001WO (300978-2220) FDR method of Benjamini and Hochberg were used to compare three or more groups. The exact statistical analysis methods are provided in the figure legends. [00298] Example 6 [00299] In the following non-limiting example, CD70 expression in m-LSCs was analyzed by flow cytometry and correlated to resistance to treatment with a combination of venetoclax and azacitidine. [00300] Briefly, cells from fourteen AML samples were analyzed by flow cytometry to identify m-LSCs based on the immunophenotype CD34-, CD4+, CD11b-, CD14- and CD36- as well as for the expression of CD70. FIG.10A sows the percentage of total blast cells in each AML sample that were CD70+ (left side of graph) as well as the percentage of m-LSCs in each AML Sample that were CD70+ (right side of graph). The results shown in FIG.10A demonstrate that CD70 is expressed on m-LSCs, with some samples exhibiting more than 50% m-LSCs that are CD70 positive. The results from the analysis shown in FIG.10A were then further analyzed based on whether the AML samples were obtained from patients that were sensitive to treatment with a combination of venetoclax and azacitidine or resistant to treatment with a combination of venetoclax and azacitidine. FIG.10B shows the percentage of total blast cells and m-LSCs that were CD70+ in AML samples derived from Ven+Aza resistant samples and Ven+Aza sensitive samples. As shown in FIG.10B, patients who were sensitive to Ven+Aza treatment had a lower percentage of m-LSCs that were CD70+ as compared to patients that were Ven+Aza resistant. That is, the m-LSC populations of Ven+Aza resistant patients exhibited a higher percentage of cells that were CD70+. Without wishing to be bound by theory, these results indicate that patients who exhibit higher levels of CD70+ m-LSCs may be more resistant to treatment with Ven+Aza, and therefore would be benefit from alternative treatments, including those that incorporate the use of a CD70-targeting agent. Moreover, these results indicate that a patient’s response to treatment with a combination of venetoclax and azacitidine can be predicted by determining the number of CD70+ m-LSCs (thus, CD34-, CD4+, CD11b-, CD14-, CD36-, and CD70+ cells) in biological sample obtained from the patient. s 4 4 0 a 5 8 d 7 s x e S A / N M M M M F M F F eg A / 6 5 7 4 9 6 3 3 0 2 9 9 A N 7 7 8 5 4 4 , r si 3 , 3 , o t i r o) ) 3 e d ) 3 s o o v + 7 ) + 7 a z b i ) 2 i n o ( 4 r ) p a z + 7 , L L o o o + 7 ga N e T ( e A , h n i 2 ( ( M v 8 e A, e H A o v N o v o ( e i e s p Ms a B p a C E 2Ks p S s p N a r o N N v e e e s p D D l e l MMa l D l o i o D D Da l R e R MD ( e H R M e R r P n e R DI 1 0 2 L - 0 3 L - 0 4 5 6 7 8 9 0 L - 0 L - 0 L - 0 L - 0 L - 0 L - 0 1 L -L -L M M M M M M M M M MM A A A A A A A A A A A q 1 m 2 o q s o q 1 ( i n . ] 3 8 [ p ( ) o n o n q ( ) 6 1 ) r t ] [ q ; ,l d, d 1 s , ) 1 , ) 4 q 1 ( ) - ) 3 2 3 2 + ,l + , 9(t ] ) 6 q 1 ; 7 ] o 2 n 3 ( , 0 1 Y 1 2 p ( X 1 ; ] v 8 [ ) , 9 9 ] 1 0 4 2 1 ( X2 p 7 1 6 ( 4 / 3 2 ( ; 9 s , 8 ] p( q 1 d d ( t 4 + , 2[ 7 2( ) 2 2 [ ] . q( 2 x 6 1 n 6 ( t [ [ d d X , ) 1 4 ) 2 i y X6 1 4 ; Y X a , 6 4 1 ; [ 1 1 ( 2 p q ; a , , ) 5/ + r e 1 ) 1 l s [ ( 1 3 X4 2 ] 4 , ) d 1 1 ; X, m Xo X X s , , i 6 ( v Y- X , X , X 9 ( 2 d d ) p ( X , q ( [ ) 7 2 + , 6 ( X , r 4 n i , 6 6 X 4 4 X , t, + ,l a , 7 ( l ) 1 5 ) 8 q ( 0 2 t, 6 4 6 t , 4 5 4 6 4 X s Y X e 4 ( , X 6 , d, ) d ) d 3 + , X 4 6 4 3 3 a ( , d 5 1 X , 7 d a + , 6 4 F A / N F M M M M F F F M F F M A / A / 7 7 1 2 4 7 0 5 3 6 4 7 5 6 0 6 0 3 A / 9 N N 5 7 N 6 o o y ) o o o 3 + ) o o t v v r o a v v v 7 T v o v o v o v L d A / A o o t z a o o o ( C o o o o e s L N/ NN e N c e ar / f n e N e N N e s p S d N e N N Ms e N e Me r M e e e R ( a e n e D D C g o A D D v D D D l D D R a r p 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 1 0 2 1 2 2 2 3 2 4-L -L -L -L -L -L -L -L -L -L -L -L - 2 L -LMMMM M MM M M M M M M M A A A A A A A A A A A A A A B M 8 1 9 1 9 1 7 1 7 6 1 8 1 8 1 7 1 8 1 9 1 7 1 8 1 7 1 7 1 6 7 1 8 7 1 5 7 6 7 6 5 p ( a t , B 0 e 2 / 0 2 0 0 1 0 0 0 0 2 0 2 0 2 0 0 2 0 2 0 1 2 0 0 0 2 1 0 0 2 1 0 1 1 1 1 1 l 0 0 0 0 0 e X X n i 8 l 2 / 2 2 / 6 / / 2 1 5 / 1 1 / 1 / 2 / 3 / 2 / / / / 4 2 / 2 / / / / 2 / 2 / / 8 2 / / 1 2 2 9 / 2 4 / 1 / 2 / 5 / 0 2 2 2 / 2 0 / 2 2 / 2 1 / 3 7 / 1 / 1 / 7 1 / 2 / / 2 9 / 2 / 2 / 2 / 5 / 1 / 2 / R , 2 7 / 1 9 / 1 2 / 1 6 / 4 G s s s s b s s b s s d d d s s s d d s s n e q ; 1 y k r si e r v e r e r e a r e r e a d e r e r e e e e d e r e r e d e r e e e d e d e r e r e g 7 ; 6 B ] R d v Ad v Ad v r Ad o v Av a d v r Ad o mv Av a r e d v d mmmmv d v d mv mmmmv v o t 2 q( (t ) 1 [ t A Ar e t r e t r e t r e t A Ar e d Ar e r e r e r e d Ad A y ) 6X N F F n n n n n t n t n t n t n t n C 1 L I I I I I I I I I I e 1 ( r X, E n ; e il 6 ( e t d 6 + 4/ s , a X,l X s ] 8 [ , , 7 ) N B P 6 4 4/ 3 ] 2 q M/ S N N Y Y N Y N N N N Y N N N N Y N Y Y N N N N Y Y D e e s p c y i t t y M p a l o c e n o Re n h o x p M e S F F M F F F M F M F F M F F M F F M M F M M M M M si s e c i o p n y t t y g 2 5 6 7 7 1 8 4 5 8 0 3 6 0 6 8 3 o c e g n o n A 2 6 3 7 7 8 6 6 8 6 7 7 6 7 7 7 7 1 8 9 7 0 8 0 7 5 7 4 8 5 7 3 7 a i e o Dh p M D I tn 9 ei 6 5 6 3 9 9 4 6 4 1 0 8 1 6 5 2 1 3 2 1 9 2 0 7 5 5 4 7 4 9 2 3 1 2 5 4 1 7 0 8 4 4 0 5 0 B D 2 I t 9 6 t -t -t -t -t -t -t -t -t -t -t -t -t -t - - - - - - - - 3- 4- 3- 3 - e n e -t a P P P P P P P P P P P P P t P t P t P t P t P t P t P t P t P t P t P t P l i t P P b a a T P 1[) 3 Ht q l q 2 S n ( ; - ) d s 1 , 1 -, 7 4[) -, ) 5 1 q; I 1 F e 1 , 1 1- m ( i 9 4 p( , ) 1-, 2 1 6 2 + , Y 2 y e p( B n + , ) ; a l / ] ) r s , 4 7 3 ) [ 1 3 3 q 1 ; ? q ] 1 ] ] X, ] 5 1 ] r a 8 8 ] ; 3 q 3 - ( 2 ) 2 [ 2 q 0 0 ( ] 2 2 7 [ Y 1 4 ; [ e r 4 / ] + , 2 [ (t 1 1 1 97 1 1 l 2 ) 3 q() q ( ; ? / X ) 1 [ X, 3 ( 2 [ Y [ X X 4/ X ] 2X, 6 ( - t) ,l 6 3 s A [ d + , ( r 5 , 5 2 8 T + s , , ( ) 6 (t ) 6 v n 7 , 6 , 6 1 [ 4 / 9 e 4 d , l e 1 ( ?( 4 i, 4 4 8 + ] 8 / Xd, d d r e X , 2 q 4 / M Y] ) 2 a , d X , Y ;4 ] K X , 7 X , . 1 8 7 [ 4 ) 0 3 4 1 1 5 1 + , 1 5 2 4 X , 7 4 ci t c i t c i t c i t c i t c i c i c i y c y c y c y t t t c y y y y o o o o c c c c n o o o n o n o n o n o n o o n o o n o M M M M M M M M ci t c i y t c i t c i t e c y v e v e v e o c y y i o c c t i it i it v i it i no n o o o n o n o m i r m i m i m i P r r r M M M M P P P 5 6 3 9 6 1 8 6 2 - t 9- 4- 4- 0- 1- 5- 1- P t P t P t P t P t P t P t P m 66 K e N T n D , , F R , , 1 s f 7 RO C L 3 E G 4 d , HT 8 1 o 2 S X 6 n a 5 1 3 . 2 q I ; 3 . ] q 0 2 1 2 2 2 3 2 4 2 1- 2- 3- x l D R x D x D x l D R x D 61 ] ] ] ] ] e s - L -L -L -L -L C C C 3 [ 9 MM _ 2 _ 2 _ 6 _ 0 _ 9 _ 9 _ 9 pX 1 9 ( [ 1 7 [ 1 0 [ 2 0 [ 2 [ E M T MMMMM 1- 1- 1- 2- 6- 6- 8-) X, I A A A A A M M M t t P t t t t P t 6 Y Y Y X Y Y C B B B P P P P P 3 ; 1 4 ( t / , ] , 9 6 X , 4 6 X , 4 6 X , 4 6 X , 4 6 4 X X , 6 sl 4 l e c 7 7 7 7 f 9 1 9 1 1 9 1 1 9 7 7 7 7 7 7 7 7 1 1 9 1 1 9 1 1 9 1 1 9 9 9 1 1 1 1 1 1 9 9 7 7 7 1 1 1 1 9 9 1 1 1 1 9 1 1 1 ev e e t v e e o e # i v i i ti i t v i i t v i i t v i t mr m m i i i i m m m P r i P r i P r i P r i P r P e p y P P t e v e c b MP P P P P u m MMMMMMo o o o i m i m i m i M- M M M M- n n n n it v e v e v e v i t s r i it m i it i it i it i y c p r p r p r p it l -i -i -i M-i it M-i o o o o i o CS u n n n n l u n m m m m r m i m i m i m i n M U U U U U P P P P o L M P r r r r M D I 1 2 3 4 7 8 9 0 2 3 4 6 7 7 8 4 0 5 0 q e 0- 0- 0- 0- 0- 0- 0- 1- 1- 1- 1- 1- 1 8 - 1 9 - 1- 0-t 4- P t 3- P t 4- P t 3- P t 3- P t 3 P e s l E L L L L L L L L L L L L L L L T MMMMMMMMMMMMMMM b A A A A A A A A A A A A A A A a I T C Table 4
AL M 6 C A2 E 1 F 1 L S 1 T 1 N 3 5 L 2 2 P E 1 C R B S N N I 1 U C K R T T T T O N I D F A A C L C P O P K T R I 1 P H4 M Y I P P Z P P O M M M C G C P N C C T C S B P P P T R E R A S E S 1 L 2 2 2 9 1 L P A X M C P 1 K L T Z L D A C 2 6 S 1 N 1 5 A 7 P D P R T 1 E 1 O B G M D L P R T A T F R N NP P R X 1 G D A B F N R S G S T E P N C Y S V R P B A R V E M A B A R F S C N G W T B T M F M I N H M D 4 5 0 1 B 7 T S 1 7 A 3 1 T A P C 6 0 B 1 1 P P 1 0 N L M 6 6 1 8 N A 1 1 Z N B B M C B P B M 1 N I C G L A I K A4 N E A A X G R 1 5 I P M A B Y A G E C M A F F M M A W F Z R S E H G P M S M F L P O C N D A T E U L R M R M H T T A3 3 1 J 0 1 1 N 0 D A I A D O 1 1 V 1 3 7 3 D 1 B A 8 1 1 S S 8 S 4 R C F A D F N 8 6 B F F B 1 R B F D H A F P N 7 P S P K P F AT D F I E 2 S F H T E L D C U U M D S R L U M D S C U R E T C U M U D A P E T H M M E D S N N P N P H C D N N C C G H N N P 0 A 1 R S 2 A 0 N 2 N 5 2 1 T O P AI 1 1 8 6 A A A A 2 6 D L M A 2 L 9 Z Q 3 F A B B R B G C G K S R 0 0 I M A L F N G 0 C 0 1 0 1 0 1 N T M D R R C M M T C L P A 2 X G N N M M C T Q C A A M H C F S S S M M P M R H A H L U U A A C F C G K A R S O P N N C S A P P U A H G G C H P N K A E 2 S C P A 3 4 B P R 1 N 1 T F 1 1 6 1 S 0 1 1 A D 2 1 5 L 5 A 3 5 4 1 B 3 C A C S R S R A H US B I P F D F H M Z B 3 K M B K Y L B M B V N R H K D S R S D R C R H P U D S M O U S F S F P S U B S U E G R X K C S M C P M T T I 1 9 3 . 2 2 2 2 -2 2 1 1 1 B C 2 7 0 3 I H E D S 2 9 E S G T L 2 A P 1 T 1 C 1 1 L C A P L 1 B F 1 2 L D 1 2 R C A 5 5 A S T U D M M LAA X E P F I G R 1 P N 1 C K U D A P P A C L P N R 3 L N R C D A S P AK A P A H B N K S A P P R N G N P D N D R M C Y C O P A M A N H 1S F 2 1 R E 1 A 8 C A B P 1 5 C M L M M R E C 8 1 6 5 1 1 C 6 R 3 H 1 B R S B 3 4 2 P 5 P P A A 0 2 A R B A C AS 3 C 1 A A 2 O M 3 F O 2 T G C X T C Y H L O R M R 1 C L H Q N O U P E A L C F R L R N S M P S C L S N A A M W A Y N F S P T P P R C N K S A 7 1 I 9 M B 2 2 1 K 2 1 f r C P 2 D 0 1 2 L 2 2 4 2 C 3 C 4 2 2 5 A 3 1 O P P M M D 2 D P 5 f r M S 1 1 A F M D S S E F P P C F M E S P D o L T D V C X C E C R M R S S A o 1 5 P P O C U M C S T D N S I T N N M D T G S M C N C T E D A C S N O T P P S T P O R L O 0 R R K R P R N E S L T E U G P L C X P C M P N E A C A U N O A O C A C C H P 1 0 D 4 2 2 3 3 2 2 6 5 K P 9 Q 0 2 A 2 1 A 1 P 2 M F T R A B 3 2 1 4 P L P B 3 6 A1 A C S A K3 3 2 P 6 B S G 1 L 2 S 2 1 T F M U T C F A S O D P B R U B U D C K P 2 O H P D O H T C T B A E F X I D T N A I E M N T C L S A X W A A T C C U T N B E D M E Y M R B E U 1 8 P 5 2 L 1 B 1 F X 1 3 V D 7 P 1 1 1 P 1 P 1 2 7 2 1 T N C 2 M M G 2 B 0 1 R 3 1 B 1 0 L 1 9 2 P A A M E X 6 X X B A L O R L N M 9 F 0 R E T S E D M C H R P T R N O B R R E G P C P T R S 5 P C C C Q C P F S D T 0 1 T P A S C S Q I R A P T U A S C U M H S M S 1 A 2 A C S 1 0 1 3 2 8 1 7 1 P 5 1 4 B 1 1 B 1 1 B 3 1 X P E B 2 L 6 C 1 1 C 8 1 1 2 1 D 1 L B 7 C C P I H1 A K C F F S H C N 2 4 K B 3 F L P T A C N HP L A F M X A B Y R R N T F F M R L S P I S A O H T D C R A C NS V U T R O G P R T S L T H H D C G L C A D C C R G M S P H T 2 1 7 7 1 0 6 6 7 6 T P A S F A G P 4 1 1 7 A A 2 C 2 r a 6 L B 1 E 1 A M K P 6 3 7 B 5 X E R A U M P A B P 2 I M E 0 4 S O A M Y B Y P F S 2 T S P N F R D X N N P G D S N P C O E 0 T M S C - 1 E C C E N O T 1 S M I M C H C Z W C R R N P R L A N O D A N P S F G R R WF O 3 C U A O U S Y R Y Z B P A B C U U S P V S T C D M A T P R X E L S H T S N M G P D N E D P D N N 2 1 C 8 P 2 L 2 Y 1 S 2 1 8 5 P 1 1 P M P N B 2 5 1 2 2 1 Y 3 S L 7 2 T 1 P 1 1 E H S C C L F C 3 S X P A Z A X T N R N T T X M I XP F 1 2 S M T N S 1 F N O P F R A A R S D F U P N A P M N R N E R Z H M D R T D P A P H S G D E D H H T W R S E 4 4 E 1 2 A 1 A B 3 4 1 U L 2 1 D 2 E 4 3 1 1 1 S D 3 7 D 3 L M 3 A 1 H 0 F M P O X S P H R 2 N G 2 I A N F S G P M 1 C R A X H B T P E P O F D 9 L P S F P R A N E R E L B E F P M R N A L S A T DF E O C R L C L E A S H N T T B C O P M C A E R A T S L Y G H C R C B P C 0 8 7 3 3 7 3 1 1 5 B 1 B 0 1 F B 1 1 3 1 4 4 3 2 f P 2 A J A 1 P 3 A U 1 C 2 1 C 6 0 L D P E B 5 r o L R M P S C E 5 2 2 E H P R X F L 6 P C D B D L B P I O L P N 5 F T A 1 2 C T L C C L B W O S C B C U A C E N T P O E Z R B M L B R R L C N S U Y N D N S X T P A Z X 4 4 H A 1 H B P 2 T 6 1 G 2 I 3 3 P 6 2 3 R 1 R 2 S A N Y R A 1 L M 1 P B X1 C X G B B G R K D 1 C A 7 T O A1 A A F C K M C I Q D F R M E X T N A D R N G 3 M I X R G D N 0 0 S T M C M U M T N D C K P U P C P C F T S M F P U H B D A P A F 1 S C A O P G D L I S N 2 4 7 6 0 1 D L f r A F D H O o 6 U P 1 C D C N H C 5 H B L R 1 P T K P ML A T M A P I R X D 0 2 X F A V 6 1 T C N D 2 P T I H H A 1 M 2 1 I B 2 N A 3 C R P G 1 F F R 3 H P 3 N A 1 R 2 AR M E PN S R P H P 1 5 L 2 1 0 P F O 1 2 R L I N P M E U N 1 2 L S C P M A S L N D R R M B G P A 2 CH P 3 L S Z R C F N E N S R O T 3 T A 8 5 2 2 M PN D B N C L S R E P H 1 B 6 4 6 UX F L 1 P S N O K P R C R N H B 3 AH A 1 7 1 5 F A T 2 S B A P S I A H H H R C t l s fo n oit c a r F no i t a l k u k l p l m o m o n m on o k n m on m on k l A- B- o l m o n k o n m o u i r o i r o n o u m i m i o l u n o o u i r o i r o i r o u i r o i r n o b- p- m p- m m b - r p b p m p m p m b p m p m x p r p m b- m b D x - D x x - - x D x l u D D R D -x - D x -x l D D R - l R S ne 2 m 1 2 1 2 1 8 0 8 0 4 1 4 1 7 0 7 0 7 0 3 1 3 1 i - 0 0 0 c L -L -L -L -L -L -L -L -L -L -L -L 2 2 2 21 21 21 96 96 96 96 96 96 e - - - - - - - - - - - M M M M M t t t t t t t t t t t -t p M M M M M M M P P P P P P P P P P P P S A A A A A A A A A A A A 6 e l b a T 1 2 5 2 . 1 0 . 0 %6 % . 8 . 0 5 . 9 9 4 1 1 - ,- , 4 + 1 4 b + 1 1 b - ,- , 1 1 4 + 1 4 b + 1 1 b D 1 C D ,- C , D D 1 D D 1 1 - + b + b- C 4 / C D / C D C C / C D / C D 4 1 4 1 1 1 1 1 3 + / D 4 + - /- -4 + / D 4 4 1 4 4 + - C/- -4 D D D D 1 3 D 4 4 1 4 1 3 C/- C/- C/ C C / h C D / g - C/ D - C / D D C C C D / i 4 3 4 3 + / / + h g - C/ D - C / D D C C / 4 4 - h 3 4 /- i 4 3 4 3 + / + g i D 3 1 4 1 C D C D D h- D D 4 D 4 h- D D 4 4 h- / h / h C/- C/C C S / S h C / C/ D C C C / C D / C D C g i g i 4 -4 , g h - / - S h h /- C/ - S h- h - 3 D 3 t i g i 4 h h g - h i - 3 4 S D 3 , g i g i 4 3 4 r C C C D t h h- h- D 3 S , t h C S C S C / D h C / b S - S S / h C / g 5 , h i r C C C D b S S S / h C / g h i r S , t S h , t g i h h h g i h 4 t S h , t g i h h- g i - h 5 , 4 t S h , t g i h h g i b- g h 5 4 i r gi - r C - S C D g C i / r g - b ir C - D gi gi - C - D b- b- S S S I -5 b - S C S S C r , S /- b r I - b - S C S S S C/ 5 -I 4 5 D 4 , t , t D h g h P 4 5 4 t h , t P 5 4 5 4 , t , t P C C ir giA D D D C g /- C I / i h - r g b i A D D h r D C g h / C/ i r gi A /- r D I /- b P I - r P 5 b- 4 5 P I -5 b - - I -I b-5 b- A A D 4 D A P A 4 5 D D D 4 P P 4 5 C/ D A A D 4 D D C/- C/- - I C D / D - C/ D -I C I / - P I A P P I P I D A A P A P D D A D A D D A B C D E A B C D E A B C D E 6 1 6 1 6 6 6 0 0 0 0 0 7 7 7 7 7 - - 1- 1- 1- 2- 2- 2- 2- 2- 0- 0 0 0 0 L L L L L L L L L L L -L -L -L -LM M M M M M M M M M M M M M M A A A A A A A A A A A A A A A

Claims

Attorney Docket No.: UNCO-049/001WO (300978-2220) 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, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one m-LSC targeting agent when at least one m-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-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, CD4, CD11b, CD14 and CD36 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one monocytic leukemia stem cell (m-LSC) based on the expression measured in step (a), wherein a cell is identified as an m-LSC if it is at least CD34-, CD4+, CD11b-, CD14- and CD36-; c) identifying that the subject will not be responsive to the treatment when the presence of at least one m-LSC is identified; or identifying that the subject will be responsive to the treatment when no m-LSCs are identified. 3. The method of claim 1 or claim 2, wherein step (a) further comprises measuring the expression of at least CD117, CD244, CD64 and GPR56-, wherein a cell is identified as an m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, and GPR56-. Attorney Docket No.: UNCO-049/001WO (300978-2220) 4. A method of treating acute myeloid leukemia (AML) in a subject, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m- LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) administering to the subject a treatment comprising at least one CD70-targeting agent when at least one CD70+ m-LSC is identified, preferably wherein the treatment further comprises at least one BCL-2 inhibitor and at least one hypomethylating agent. 5. A method of identifying if a subject having AML will be responsive to treatment with a CD70-targeting agent, the method comprising: a) measuring the expression of at least CD34, CD4, CD11b, CD14, CD36 and CD70 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one CD70+ monocytic leukemia stem cell (m- LSC) based on the expression measured in step (a), wherein a cell is identified as a CD70+ m-LSC if it is at least CD34-, CD4+, CD11b-, CD14-, CD36- and CD70+; c) identifying that the subject will be responsive to the treatment when the presence of at least one CD70+ m-LSC is identified. 6. The method of claim 4 or claim 5, wherein step (a) further comprises measuring the expression of at least CD117, CD244, CD64 and GPR56, wherein a cell is identified as a CD70+ m-LSC is identified if it is at least CD34-, CD4+, CD11b-, CD14-, CD36-, CD117-, CD244-, CD64+, GPR56-, and CD70+. 7. The method of any one of the preceding claims, wherein the at least one CD70- targeting agent is: i) an anti-CD70 antibody, preferably wherein the anti-CD70 antibody is cusatuzumab; ii) an anti-CD70 immunotherapy, preferably wherein the immunotherapy comprises CAR-T and/or NK Cells that are directed specifically at CD70; or iii) an agent that blocks CD70 signaling, preferably wherein the agent that blocks CD70 signaling prevents binding of CD27 and CD70. Attorney Docket No.: UNCO-049/001WO (300978-2220) 8. The method of any one of the preceding claims, wherein the at least one m-LSC targeting agent is an agent modulates one-carbon metabolism, is an agent that modulates purine synthesis, is an agent that modulates pyrimidine synthesis, or any combination thereof. 9. The method of any one of the preceding claims, wherein the at least one m-LSC targeting agent is selected from methotrexate, brequinar and cladribine. 10. The method of any one of the preceding claims, wherein the at least one hypomethylating agent is selected from azacitidine and decitabine. 11. The method of any one of the preceding claims, wherein the at least one BCL-2 inhibitor is selected from venetoclax and navitoclax. 12. 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), or any combination thereof. 13. The method of any one of the preceding claims, wherein the subject is a subject having AML who has not received any treatment for AML. 14. The method any one of the preceding claims, wherein the subject is a subject having AML who has received previously received at least one AML treatment. 15. The method of claim 10, wherein the at least one AML treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent. 16. 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. Attorney Docket No.: UNCO-049/001WO (300978-2220) 17. 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. 18. 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. 19. The method of claim 18, wherein the sample is a bone marrow biopsy. 20. The method of claim 18, wherein the sample is a bone marrow aspirate. 21. The method of claim 18, wherein the sample is a biopsy of a chloroma.
EP23837488.8A 2022-12-01 2023-12-01 Methods of treating acute myeloid leukemia Pending EP4626439A2 (en)

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