WO2021255518A1

WO2021255518A1 - Compositions and methods for treating acute myeloid leukemia

Info

Publication number: WO2021255518A1
Application number: PCT/IB2021/000396
Authority: WO
Inventors: Fumihiko Ishikawa; Yoriko Saito; Mari Hashimoto; Michiel De Hoon; Yukihide MOMOZAWA
Original assignee: Riken
Priority date: 2020-06-15
Filing date: 2021-06-14
Publication date: 2021-12-23

Abstract

The present invention provides methods for identifying correlations between genetic abnormalities and effective combination treatments and treating a disease or disorder associated with inhibition of apoptosis, such as acute myeloid leukemia with compounds that block inhibition of apoptosis.

Description

COMPOSITIONS AND METHODS FOR TREATING ACUTE MYELOID

LEUKEMIA

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/039,250, filed on June 15, 2020.

BACKGROUND

Myelodysplastic syndromes (MDS) are conditions that can occur when the blood- forming cells in the bone marrow become abnormal. This leads to low numbers of one or more types of blood cells. In about 1 in 3 patients, MDS can progress to a rapidly growing cancer of the bone marrow cells called acute myeloid leukemia (AML).

AML is a cancer of the myeloid line of blood cells that is typically fatal within weeks or months if left untreated. AML affected approximately one million people globally in 2015. It is estimated that there will be 19,520 new cases in the United States alone in 2018. Though the disease most commonly occurs in older adults, AML is curable in only 10% of those affected over the age of 60 years old. AML is initially typically treated with chemotherapy aimed at inducing remission. People may then go on to receive additional chemotherapy, radiation therapy, or a stem cell transplant. The five-year survival rate is only 24%.

For decades the backbone of treatment for AML has remained the anthracyclines (DNA intercalating and topoisomerase II inhibitors) and the nucleoside analogue cytarabinel. More recently, clinical studies have led to the approval of novel specific targeted inhibitors of pathogenic mutant proteins (e.g. inhibitors of mutant FLT3, IDH1, IDH2) and of survival pathways (e.g. inhibitors of BCL2 inhibitor and hedgehog pathway). Though these novel therapeutics have broadened treatment options the outlook for poor risk and relapsed/refractory disease remain very limited. Clonal heterogeneity within any one patient, and between patients, coupled with a lack of deep understanding of resistance mechanisms are significant barriers to in improving clinical outcome. For adverse prognostic risk, refractory and relapsed patients, allogeneic stem cell transplantation (allo-SCT) remains the most effective curative treatment. However, this option is only suitable for a minority of patients able to tolerate the appreciable toxicity of allo-SCT. In contrast, most AML patients who are older, unfit and often have poor risk features are unsuitable for allo-SCT and die of their disease. Furthermore, there are a certain number of patients whose leukemia is too aggressive to undertake allo-SCT or undergo relapse even after allo-SCT. Currently, there is an unmet need for treatment of AML patients with aggressive poor prognosis disease.

SUMMARY

In certain aspects, provided herein are methods related to treating a subject with acute myeloid leukemia (AML). For example, the methods comprise determining the presence or absence of one or more genetic mutations in FLT3, IDH1, CBL, and/or NRAS in an AML cell of the subject and, if one or more of the genetic mutations are present, individually or conjointly administering an effective amount of a XIAP inhibitor, BCL2 inhibitor, MCL1 inhibitor, or aurora kinase B inhibitor to the subject.

In certain aspects, provided herein are methods of identifying a subject-specific combination drug regimen. For example, the methods may comprise collecting AML cells from the subject, measuring a level of annexin V expression in the AML cells following exposure to a combination of two inhibitors selected from the group of: XIAP inhibitor,

BCL2 inhibitor, MCL1 inhibitor, or aurora kinase B inhibitor. Such methods may further comprise determining whether the level of annexin V expression is increased in AML cells exposed to the two inhibitors compared to a level of annexin V expression in AML cells not exposed to the two inhibitors or AML cells exposed to only one inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows RNAseq results from functionally-defined AML-engrafting cells of high-risk AML patients.

Figure 2 shows RNAseq volcano plot results of AML-engrafting cells obtained from deceased patients (FLT3-WT n=54; FLT3-mutated, n=32) and of normal CD34+ hematopoietic stem/progenitor cells obtained from healthy donors (n=44).

Figure 3 shows results of 35-compound chemical screening in a 96-well format to identify compounds with in vitro efficacy against high-risk poor-outcome AML.

Figure 4 shows representative flow cytometry dot plot and graph demonstrating efficacy of AZD5582 in leukemia cells.

Figure 5 shows results of targeted DNA sequencing to factors correlating with responsiveness and resistance to AZD5582 in high-risk poor-outcome AML cells.

Figure 6 shows in vitro responsiveness of leukemia cells to five compounds in order of elimination efficacy of 563845, barasertib, and GSK923295. Figure 7 shows exemplary scatter plots showing correlation between response to two drugs among AZD5582, venetoclax, S63845, barasertib, and GSK923295.

Figure 8 shows correlation of drug sensitivity/resistance with somatic mutations and karyotypical abnormalities.

Figure 9 shows XIAP dependence is linked to TP53 transcriptional activity and BCL2 dependence is linked to activation of EVI1 in leukemia cells.

Figure 10 shows in vivo elimination of FLT3-WT and FLT3-ITD+ AML as predicted by in vitro drug response profile.

Figure 11 shows in vivo treatment of FLT3-WT and -ITD+ AML in PDX-models and recovery of murine hematopoiesis with successful elimination of human leukemia cells.

Figure 12 shows representative plot demonstrating TP53 motif activity predicts XIAP dependence and AZD5582 responsiveness.

Figure 13 shows a schematic for correlation of genetic abnormalities and responsiveness to AZD5582 and venetoclax.

Figure 14 in vivo elimination of Ph(-)ALL, T(-)ALL, and Ph-B(-)MPAL by AZD5582 and venetoclax.

Figure 15 in vivo elimination of Ph(+)ALL, Ph(+)MPAL, CML, and Atypical CML (Ph-) by AZD5582 and venetoclax.

Figure 16 shows a representative heat map demonstrating in vitro efficacy of inhibitor compounds against ALL, MPAL, and CML patient samples.

DETAILED DESCRIPTION

General

Disclosed herein are methods related to treating a subject with acute myeloid leukemia (AML). In particular, it has been discovered that certain types of AML characterized by certain mutations are responsive to treatment with certain inhibitors or combinations of inhibitors. For example, AML characterized by FLT3-ITD+, DNMT3A, NPM1, and/or PPM1D mutations shows a strong response when treated with a XIAP inhibitor individually or conjointly with a BCL2 inhibitor. Similarly, AML characterized by FLT3 WT, mutated CBL, NRAS, IDH2, TET2, DNMT3A, NPM1, and/or SETBP1 shows a strong response when treated with a XIAP inhibitor individually or conjointly with a MCL1 inhibitor. AML characterized by FLT3-ITD+, mutated KMT2A, t(3;3)(q21;q26.2), and/or monosomy 7, however, shows a strong response when treated conjointly with a XIAP inhibitor and an aurora kinase B inhibitor. In contrast, AML characterized by FLT3-ITD+, IDH1, NPM1, and/or DNMT3A shows a strong response when treated conjointly with a BCL2 inhibitor and a MCL1 inhibitor.

Provided herein are methods of identifying genetic mutations in certain genes, for example FLT3, IDH1, CBL, and NRAS, as well as identifying chromosomal defects in AML cells. Accordingly, the invention provides various methods for tailoring therapeutic regimens for treating AML according to the presence or absence of these genetic markers in a particular patient’s AML. Exemplary chemical inhibitors that may be used individually or in combination treatment include AZD5582, venetoclax, S63845, and barasertib.

Also disclosed herein are methods for identifying a subject-specific combination drug regimen. The methods are based, in part, on the discovery that quantification of apoptotic marker annexin V following drug exposure in patient cells can rapidly predict drug responsiveness. For example, annexin V-positive apoptotic cells increased significantly at 4 hr and 6 hr post-exposure to AZD5582. Various methods described herein shorten the time from clinical presentation to determination of drug sensitivity and/or allow identification of subject-specific optimized combination drug regimen in a clinically feasible timescale. These methods may include collecting AML cells from the subject, measuring a level of annexin V expression in the AML cells following exposure to a combination of two inhibitors, and determining whether the level of annexin V expression is increased in AML cells exposed to the two inhibitors compared to a level of annexin V expression in AML cells not exposed to the two inhibitors or AML cells exposed to only one inhibitor. The two inhibitors, for example, may be selected from the group of: XIAP inhibitor, BCL2 inhibitor, MCL1 inhibitor, or aurora kinase B inhibitor.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated in order to facilitate the understanding of the present invention.

The singular forms “a”, “an” and “the” encompass plural references unless the context clearly indicates otherwise.

As used herein, the term “biomarker” or “marker” refers to a biological molecule, such as, for example, a nucleic acid, peptide, protein, or hormone, whose presence or concentration can be detected and correlated with a known condition, such as a disease state. It can also be used to refer to a differentially expressed gene whose expression pattern can be utilized as part of a predictive, prognostic or diagnostic process in healthy conditions or a disease state, or which, alternatively, can be used in methods for identifying a useful treatment or prevention therapy.

As used herein, the term “blood” can include, for example, plasma, serum, whole blood, blood lysates, and the like.

As used herein, the term “comprise” or “comprising” is generally used in the sense of include, that is to say permitting the presence of one or more additional features or components.

As used herein, the term “expression levels” refers, for example, to a determined level of biomarker expression. The term “pattern of expression levels” refers to a determined level of biomarker expression compared either to a reference (e.g., a housekeeping gene or inversely regulated genes, or other reference biomarker) or to a computed average expression value (e.g. in DNA-chip analyses). A pattern is not limited to the comparison of two biomarkers but is more related to multiple comparisons of biomarkers to reference biomarkers or samples.

As used herein, an mRNA “isoform” is an alternative transcript for a specific mRNA or gene. This term includes pre-mRNA, immature mRNA, mature mRNA, cleaved or otherwise truncated, shortened, or aberrant mRNA, and modified mRNA (e.g., containing any residue modifications, capping variants, polyadenylation variants, etc.).

As used herein, the terms “modulated” or “modulation,” or “regulated” or “regulation” and “differentially regulated” can refer to both up regulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and down regulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting), unless otherwise specified or clear from the context of a specific usage.

As used herein, the term “or” means “and/or” unless stated otherwise.

As used herein, the term “including” as well as other forms, such as “include”, “includes” and “included” is not limiting.

As used herein, the term “sample” encompasses a sample obtained from a subject or patient. The sample can be of any biological tissue or fluid. Such samples include, but are not limited to, sputum, saliva, buccal sample, oral sample, blood, serum, mucus, plasma, urine, blood cells (e.g., white cells), circulating cells (e.g., stem cells or endothelial cells in the blood), tissue, core or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, stool, peritoneal fluid, pleural fluid, tear fluid, or cells therefrom. Samples can also include sections of tissues such as frozen or fixed sections taken for histological purposes or microdissected cells or extracellular parts thereof. A sample to be analyzed can be tissue material from a tissue biopsy obtained by aspiration or punch, excision or by any other surgical method leading to biopsy or resected cellular material. Such a sample can comprise cells obtained from a subject or patient.

The term “treatment” or “treating” means any treatment of a disease in a mammal, including: (a) inhibiting the disease, i.e., slowing or arresting the development of clinical symptoms; and/or (b) relieving the disease, i.e., causing the regression of clinical symptoms and/or (c) alleviating or abrogating a disease and/or its attendant symptoms.

As used herein, the term “subject” refers to an animal, preferably a mammal, and most preferably a human. In some embodiments, a subject is a pediatric patient, e.g., a patient under 18 years of age, while an adult patient is 18 or older.

As used herein, the term "assessing" includes any form of measurement and includes determining if an element is present or not. The terms "determining," "measuring," "evaluating," "assessing," "analyzing," and "assaying" can be used interchangeably and can include quantitative and/or qualitative determinations.

As used herein, the terms "modulated" or "modulation," or "regulated" or "regulation" and "differentially regulated" can refer to both up regulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and down regulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting), unless otherwise specified or clear from the context of a specific usage.

In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the subject, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially.

Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic compounds.

The term "genetic mutation" or “genetic variant,” as used herein, generally refers to an alteration, variant or polymorphism in a nucleic acid sample or genome of a subject. Such alteration, variant or polymorphism can be with respect to a reference genome, which may be a reference genome of the subject or other individual. Single nucleotide polymorphisms (SNPs) are a form of polymorphisms. In some examples, one or more polymorphisms comprise one or more single nucleotide variations (SNVs), insertions, deletions, repeats, small insertions, small deletions, small repeats, structural variant junctions, variable length tandem repeats, and/or flanking sequences. Copy number variants (CNVs), transversions and other rearrangements are also forms of genetic variation. A genomic alternation may be a base change, insertion, deletion, repeat, copy number variation, or transversion.

The term "polynucleotide," as used herein, generally refers to a molecule comprising one or more nucleic acid subunits. A polynucleotide can include one or more subunits selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. A nucleotide can include A, C, G, T or U, or variants thereof. A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (i.e., A or G, or variant thereof) or a pyrimidine (i.e., C, T or U, or variant thereof). A subunit can enable individual nucleic acid bases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or uracil-counterparts thereof) to be resolved. In some examples, a polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or derivatives thereof. A polynucleotide can be single-stranded or double stranded.

The term "genome" generally refers to an entirety of an organism's hereditary information. A genome can be encoded either in DNA or in RNA. A genome can comprise coding regions that code for proteins as well as non-coding regions. A genome can include the sequence of all chromosomes together in an organism. For example, the human genome has a total of 46 chromosomes. The sequence of all of these together constitutes a human genome.

Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal cells that build up in the bone marrow and blood and interfere with normal blood cells. As an acute leukemia, AML progresses rapidly and is typically fatal within weeks or months if left untreated. Risk factors of AML include exposure to benzene, previous chemotherapy or radiation therapy, myelodysplastic syndrome, and smoking. Diagnosis of the disease is generally based on bone marrow aspiration and specific blood tests. AML has several subtypes. AML with recurrent genetic abnormalities includes AML with translocation between chromosomes 8 and 21. Other subtypes include AML with translocation or inversion in chromosome 16, AML with translocation between chromosome 9 and 11, APL (M3) with translocation between chromosomes 15 and 17, AML with translocation between chromosomes 6 and 9, AML with translocation or inversion in chromosome 3), AML with translocation or inversion in chromosome 3), AML (megakaryoblastic) with a translocation between chromosomes 1 and 22, AML with myelodysplasia-related changes, AML related to previous chemotherapy or radiation (alkylating agent-related AML, topoisomerase II inhibitor-related AML), AML not otherwise categorized (AML minimally differentiated (MO)), AML with minimal maturation (Ml), AML with maturation (M2), acute myelomonocytic leukemia (M4), acute monocytic leukemia (M5), acute erythroid leukemia (M6), acute megakaryoblastic leukemia (M7), acute basophilic leukemia, acute panmyelosis with fibrosis, myeloid sarcoma (also known as granulocytic sarcoma, chloroma or extramedullary myeloblastoma), and undifferentiated and biphenotypic acute leukemias (also known as mixed phenotype acute leukemias).

There are two commonly used classification systems for AML, the French-American- British (FAB) system, and the World Health Organization (WHO) system.

French-American-British (FAB) classification of AML

The FAB system divides AML into subtypes, MO through M7, based on the type of cell from which the leukemia develops and how mature the cells are. This is based largely on how the leukemia cells look under the microscope after routine staining. Subtypes MO through M5 all start in immature forms of white blood cells. M6 AML starts in very immature forms of red blood cells, while M7 AML starts in immature forms of cells that make platelets. MO is undifferentiated acute myeloblastic leukemia, Ml is acute myeloblastic leukemia with maturation, M2 is acute myeloblastic leukemia with maturation, M3 is acute promyelocytic leukemia (APL), M4 is acute myelomonocytic leukemia, M4 eos is acute myelomonocytic leukemia with eosinophilia, M5 is acute monocytic leukemia, M6 is acute erythroid leukemia, and M7 is acute megakaryoblastic leukemia. Though the FAB classification system is useful and still commonly used to group AML into subtypes, it does not take into account many of the factors that are known to affect prognosis. The FAB classification requires a blast percentage of at least 30% in bone marrow (BM) or peripheral blood (PB) for the diagnosis of AML. AML must be carefully differentiated from "preleukemic" conditions such as myelodysplastic or myeloproliferative syndromes, which are treated differently. World Health Organization (WHO) classification of AML

The World Health Organization has developed a newer system that includes some of the factors not included in the FAB system, in an effort to better classify AML by providing more descriptive subcategories of interest to clinicians. The WHO system divides AML into several groups: AML with certain genetic abnormalities, AML with myelodysplasia-related changes, AML related to previous chemotherapy or radiation, AML not otherwise specified (includes cases of AML that do not fall into one of the aforementioned groups, and is similar to the FAB classification), myeloid sarcoma (also known as granulocytic sarcoma or chloroma), myeloid proliferations related to Down syndrome, undifferentiated and bi- phenotypic acute leukemias (leukemias that have both lymphocytic and myeloid feature; sometimes called ALL with myeloid markers, AML with lymphoid markers, or mixed phenotype acute leukemias).

The WHO criteria establishes the diagnosis of AML by demonstrating involvement of more than 20% of the blood and/or bone marrow by leukemic myeloblasts, except in the three best prognosis forms of acute myeloid leukemia with recurrent genetic abnormalities: translocation between chromosomes 8 and 21 (seen most often in patients with M2), inversion of chromosome 16 (seen most often in patients with M4 eos) or a translocation between chromosome 16 and itself, and translocation between chromosome 15 and 17 (seen most often in patients with M3), in which the presence of the genetic abnormality is diagnostic.

Pharmaceutical Compositions & Administration

In certain embodiments, provided herein are pharmaceutical compositions and methods of using pharmaceutical compositions. In some embodiments, the pharmaceutical compositions provided herein comprise an XIAP inhibitor. In some embodiments, the pharmaceutical compositions provided herein comprise a BCL-2 inhibitor. In some embodiments, the pharmaceutical compositions provided herein comprise an MCL1 inhibitor. In some embodiments, the pharmaceutical compositions provided herein comprise an aurora kinase B inhibitor.

In certain embodiments, the compositions and methods provided herein may be utilized to treat a subject in need thereof. In certain embodiments, the subject is a mammal such as a human, or a non-human mammal. In some embodiments, the subject has cancer. When administered to a subject, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a therapeutic compound and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In certain embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an eye drop.

In certain embodiments, the pharmaceutical compositions provided herein comprise a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self- microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a therapeutic compound. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, 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.

In certain embodiments, the pharmaceutical compositions provided herein can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water- in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active compounds with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment. Alternatively or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracap sular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

In certain embodiments, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site. Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments, the active compound may be administered two or three times daily. In some embodiments, the active compound will be administered once daily.

In certain embodiments, compounds may be used alone or conjointly administered with another type of therapeutic agent (e.g., an immuno-oncology agent or a chemotherapeutic agent disclosed herein). As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.

In certain embodiments, conjoint administration of therapeutic compounds with one or more additional therapeutic agent(s) (e.g., one or more additional chemotherapeutic agent(s)) provides improved efficacy relative to each individual administration of the compound (e.g., copper ionophore) or the one or more additional therapeutic agent(s). In certain such embodiments, the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of individual administration of the therapeutic compound and the one or more additional therapeutic agent(s).

Pharmaceutically acceptable salts of compounds in the methods provided herein. In certain embodiments, contemplated salts include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, lH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts include, but are not limited to, Na, Ca, K, Mg, Zn, copper, cobalt, cadmium, manganese, or other metal salts.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In some embodiments, the therapeutic compound used in the methods herein is a XIAP inhibitor. Inhibitor of Apoptosis Proteins (IAP) antagonists are compounds that can modulate the activity of certain proteins involved in apoptotic pathways, or signaling pathways associated with inflammation and/or autoimmune diseases and/or cell division and/or angiogenesis. The members of the IAP family are functionally and structurally related proteins, which inhibit apoptosis. IAPs share a Baculovirus IAP Repeat (BIR) domain, each having one to three copies. Eight members of the IAP family have currently been identified, in both baculovirus and humans. Human members of the IAP family include but are not limited to: XIAP, cIAPl (also, BIRC2), cIAP2 (also, BIRC3), NAIP, survivin, ML-IAP, apollon, and ILP2. In certain instances, XIAP inhibits apoptosis by binding to and inhibiting the activity of caspase-9, caspase-3 or caspase-7. Exemplary XIAP inhibitors are listed in

Table 1.

Table 1. Exemplary XIAP Inhibitors

In some embodiments, the XIAP inhibitor used in the methods herein is a SMAC mimetic. In some embodiments, the SMAC mimetic is AZD5582. One protein implicated in binding with IAPs is SMAC. SMAC is a mitochondrial protein that negatively regulates apoptosis or programmed cell death. When a cell is primed for apoptosis by the final execution step of caspase activation, SMAC binds to IAP, which prevents IAP from binding to, and deactivating caspases. SMAC promotes apoptosis by activating caspases. SMAC mimetics inhibit IAP proteins.

Non-limiting examples of a SMAC mimetic, or a salt or solvate thereof, comprise:

AZD5582 (also known as (2S,2'S)-N,N'-((lS,rS,2R,2'R)-(hexa-2,4-diyne-l,6- diylbi s(oxy))bi s(2,3 -dihydro- 1 H-indene-2, 1 -diyl))bi s( 1 -((S)-2-cyclohexyl -2-((S)-2- (methylamino)propanamido)acetyl)pynOlidine-2-carboxamide)):

birinapant (also known as TL-32711, or (2S)-N-[(2S)-l-[(2R,4S)-2-[[6-fluoro-2-[6- fluoro-3-[[(2R,4S)-4-hydroxy-l-[(2S)-2-[[(2S)-2-

(methylamino)propanoyl]amino]butanoyl]pyrrolidin-2-yl]methyl]-lH-indol-2-yl]-lH-indol- 3-yl]methyl]-4-hydroxypyrrolidin-l-yl]-l-oxobutan-2-yl]-2-(methylamino)propanamide, TetraLogic Pharmaceuticals):

LCL161 ((S)-N-((S)-l-cyclohexyl-2-((S)-2-(4-(4-fluorobenzoyl)thiazol-2- yl)pyrrolidin-l- yl)-2-oxoethyl)-2-(methylamino) propanamide; Novartis):

GDC-0152 ((S)4 -((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-N- (4- phenyl-l,2,3-thiadiazol-5-yl)pyrrolidine-2-carboxamide, Genentech):

GDC-0917 ((S)- 1 -((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)- N-(2- (oxazol-2-yl)-4-phenylthiazol-5-yl)pyrrolidine-2-carboxamide, Genentech):

HGS1029 (Human Genome Sciences), and AT -406 ((5S,8S,10aR)-N- (diphenylmethyl)decahydro-5-[[(2S)-2-(methylamino)-l- oxopropyl]amino]-3-(3-methyl-l- oxobutyl)-6-oxo-pyrrolo[l,2-a][l,5]diazocine-8- carboxamide, Ascenta):

BV-6 (also known as BV6, or ((S,S,2S,2'S)-N,N'-((2S,2'S)-(hexane-l,6- diylbis(azanediyl)) bis(3-oxo-l,l-diphenylpropane-3,2-diyl))bis(l-((S)-2-cyclohexyl-2-((S)- 2-(methylamino)propanamido)acetyl)pyrrolidine-2-carboxamide)), Genentech):

or a salt or solvate thereof.

In some embodiments, the therapeutic compound used in the methods herein is a BCL2 inhibitor. A BCL2 inhibitor is a potent and highly selective BH3 mimetic antagonist of BCL2 that blocks the anti-apoptotic activity of BCL-2. The BCL2 family proteins share several conserved "BH" domains termed BH1, BH2, BH3 and BH4, as in the inhibitors (BCL-2, BCL-XL, BCL-2 MCL-1, BFL/A1, BCL-B), instead the activators (BIM, BID, PUMA) and the sensitizers (BAD, BMF, NOXA) possess only the BH3 domain, and hence are often referred to as "BH3-only" proteins. BH3 mimetics can occupy the inhibitors (anti- apoptotic proteins), preventing them from binding the activators and can induce apoptosis through the intrinsic apoptosis pathway. BH3 mimetic antagonists are as described in C. Billard, Mol. Cancer Then, 2013, 9, 1691 -700, incorporated by reference. Exemplary BCL2 inhibitors are listed in Table 2.

Table 2. Exemplary BCL2 Inhibitors

Examples of BCL2 inhibitor structures are the following:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the BCL2 inhibitor used in the methods herein is venetoclax. Venetoclax (also called GDC-0199, ABT-199, and RG7601) is a BCL-2 inhibitor. Venetoclax is chemically known as 4-[4-[[2-(4-chlorophenyl)-4,4- dimethylcyclohexen-1 -yl]methyl]piperazin-l -yl]-N-[3- nitro-4-(oxan-4- ylmethylamino)phenyl]sulfonyl-2-(l H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide and has the structure shown below:

Venetoclax is marketed in the United States as VENCLEXTA™, which is indicated for the treatment of chronic lymphocytic leukemia. U.S. Patent No. 8,546,399, which is hereby incorporated by reference, discloses venetoclax and its preparation. U.S. Patent No. 8,722,657, which is hereby incorporated by reference, discloses anhydrous and hydrated crystalline forms A to N of venetoclax with characteristic powder X-ray diffraction data. PCT Publication Nos. WO 2012/121758 and WO 2012/58392 disclose non-crystalline solid dispersions of venetoclax.

In some embodiments, the therapeutic compound used in the methods herein is a MCL1 inhibitor. Bone marrow cell leukemia 1 (MCL1) is an important anti-apoptotic member of the BCL-2 protein family and a master regulator of cell survival. Amplification of the MCL1 gene and/or overexpression of the MCL1 protein has been observed in several cancer types and is mainly associated with tumor development. MCL1 binds to pro-apoptotic proteins such as Bim, Noxa, Bak and Bax and promotes cell survival by neutralizing their extinction-inducing activity. As a result, inhibition of MCL1 releases these apoptotic proteins, which often lead to induction of apoptosis in MCL1 -dependent tumor cells for survival. Exemplary MCL1 inhibitors are listed in Table 3.

Table 3: Exemplary MCL1 Inhibitors

In one example, the MCL-1 inhibitor is a small molecule, such as a small molecule BH3 mimetic capable of binding to the hydrophobic pocket of MCL-1 protein and stabilizing MCL-1 levels. A number of small molecules which preferentially bind MCL-1 are known in the art and contemplated for use herein. In one example, the small molecule inhibitor of MCL-1 protein suitable for use in the method of the disclosure is an indol-2-carboxylic acid having the formula:

(A 1210477) or an analog thereof selected from the group consisting of:

(A1208746).

The small molecule inhibitors of MCL-1 designated A1210477, A1 155905, A1248767 and A1208746 are described in Leverson et al., (2015) Cell Death Dis., 6:el590, the contents of which are incorporated herein in its entirety. Pharmaceutically acceptable derivatives, polymorphs or salts of A1210477, Al 155905, A1248767 and A1208746 which bind MCL-1 and inhibit activity thereof are also contemplated for use in the method of the disclosure.

In another example, the small molecule inhibitor of MCL- 1 which is suitable for use in the method of the disclosure is:

Compound UMI-77 is described in Abulwerdi et al, (2014) Mol. Cancer Ther., 13(3):565-575, the entire contents of which is incorporated herein in its entirety. Pharmaceutically acceptable derivatives, polymorphs or salts of the UMI-77 which bind MCL-1 and inhibit activity thereof are also contemplated for use in the method of the disclosure.

In another example, the small molecule inhibitor of MCL-1 suitable for use in the method of the disclosure is a molecule as described in Belmar et al. (2015) Pharmacology & Therapeutics, 145:76-84, the contents of which is incorporated herein in its entirety. For example, the small molecule inhibitor of MCL-1 may be a SI molecule having a 1-oxo-lH- phenalene-2,3-dicarbonitrile backbone. An exemplary SI molecule for use in the method of the disclosure is SI:

Also contemplated for use in the method of the disclosure are pharmaceutically acceptable derivatives, polymorphs or salts of the SI compound described herein which bind MCL-1 and inhibit activity thereof.

In another example, the small molecule inhibitor of MCL-1 suitable for use in the method of the disclosure is a SI derivative, as described in Belmar et al. (2015), which is selected from the group consisting of:

Also contemplated for use in the method of the disclosure are pharmaceutically acceptable derivatives, polymorphs or salts of the SI derivative described herein which bind MCL-1 and inhibit activity thereof. In another example, the small molecule inhibitor of MCL-1 suitable for use in the method of the disclosure is an A* STAR compound, as described in Belmar et al. (2015), which is selected from the group consisting of:

Also contemplated for use in the method of the disclosure are pharmaceutically acceptable derivatives, polymorphs or salts of the A* STAR compounds described herein which bind MCL- 1 and inhibit activity thereof.

In another example, the small molecule inhibitor of MCL- 1 suitable for use in the method of the disclosure is:

wherein R is H or Me. In one example, R is H. In another example, R is Me. As described in Belmar et al. (2015), compounds in accordance with this example bind to MCL-1.

In another example, the small molecule inhibitor of MCL-1 suitable for use in the method of the disclosure is:

or a pharmaceutically acceptable derivative, polymorph or salt thereof.

or a pharmaceutically acceptable derivative, polymorph or salt thereof.

In another example, the small molecule inhibitor of MCL-1 suitable for use in the method of the disclosure is Marinopyrrole A (named Maritoclax):

Also contemplated for use in the method of the disclosure are pharmaceutically acceptable derivatives, polymorphs or salts of Marinopyrrole A which bind MCL-1 and inhibit activity thereof.

As discussed in Belmar et al. (2015), Marinopyrrole A is a natural product isolated from an obligate marine Streptomyces which has been shown to selectively bind MCL-1 over other BCL- 2 family proteins, without leading to degradation of MCL-1 protein or downregulation in expression of MCL- 1. In another example, the small molecule inhibitor of MCL-1 suitable for use in the method of the disclosure is:

or a pharmaceutically acceptable derivative, polymorph or salt thereof.

In another example, the small molecule inhibitor of MCL-1 suitable for use in the method of the disclosure is selected from:

or pharmaceutically acceptable derivatives, polymorphs or salts thereof.

or pharmaceutically acceptable derivatives, polymorphs or salts thereof.

and

or pharmaceutically acceptable derivatives, polymorphs or salts thereof.

In yet another example, the small molecule inhibitor of MCL-1 suitable for use in the method of the disclosure is a MCL-1 inhibitor molecule as described in Brumatti and Ekert (2013) Cell Death and Differentiation, 20: 1440-1441, including, for example, TW-37:

including pharmaceutically acceptable derivatives, polymorphs or salts thereof.

In another example, the small molecule inhibitor of MCL-1 suitable for use in the method of the disclosure is a MCL-1 inhibitor molecule as described in Kotschy et al, (2016) Nature, 538:477-482 including, for example, compound S63845:

Also contemplated for use in the method of the disclosure is a pharmaceutically acceptable derivative, polymorph, salt or prodrug of S63845.

Other small molecules which selectively inhibit MCL-1 activity and which are contemplated for use in the method of the present disclosure are described in WO2013/149124, WO2013052943 and WO2015/031608, the entire contents of which are incorporated herein in their entirety.

In some embodiments, the MCL1 inhibitor used in the methods herein is S63845.

In some embodiments, the therapeutic compound used in the methods herein is an aurora Kinase B inhibitor. Exemplary aurora kinase B inhibitors are listed in Table 4.

Table 4. Exemplary Aurora Kinase B Inhibitors

Aurora Kinases (Aurora- A, Aurora-B and Aurora-C) encode cell cycle regulated serine-threonine protein kinases (summarized in Adams et al., 2001, Trends in Cell Biology.11(2): 49-54). These show a peak of expression and kinase activity through G2 and mitosis and a role for human Aurora kinases in cancer has long been implicated.

The Aurora Kinase inhibitor known as AZD1152 (2-(ethyl(3-((4-((5-(2-((3- fluorophenyl)amino)-2-oxoethyl)-lH-pyrazol-3-yl)amino)quinazolin-7- yl)oxy)propyl)amino)ethyl dihydrogen phosphate), pictured below, was first disclosed in International Patent Application W02004/058781.

AZD1152 In some embodiments, the aurora kinase B inhibitor used in the methods herein is AZD1 152 (barasertib).

Methods of Identifying Genetic Abnormalities

In certain embodiments, provided herein are methods of determining the presence or absence of genetic abnormalities in a subject with AML. In some embodiments, the methods comprise acquiring genetic material from the cells of the subject with AML. In some embodiments, the methods comprise sequencing the genetic material by methods known to those skilled in the art. Any suitable sequencing techniques may be employed to determine the sequence of target DNA.

In some embodiments, the methods used to determine whether the subject has a mutation in a gene comprises the use of one or more of the following methods: quantitative PCR (q-PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time PCR (RT-PCR), polyacrylamide gel electrophoresis (PAGE), multiplex ligation dependent probe amplification (MLPA), sanger sequencing, targeted DNA sequencing, whole genome DNA sequencing, exome sequencing, pyrosequencing, comparative genomic hybridization array (CGH), restriction fragment length polymorphism (RFLP), short tandem repeat analysis (STR), dynamic allele-specific hybridization (DASH), genotyping, variable number tandem repeats (VNTRs), molecular beacons, amplification refractory mutation system PCR (ARMS- PCR), Invader.TM. assay (flap endonuclease (FEN)), primer extension, mass spectrometry, TaqMan.TM., denaturing high performance liquid chromatography (DHPLC), and high resolution melting analysis.

In some embodiments, the use of high-throughput, so-called "second generation", "third generation" and "next generation" techniques are preferred. In second generation techniques, large numbers of DNA molecules are sequenced in parallel. Typically, tens of thousands of molecules are anchored to a given location at high density and sequences are determined in a process dependent upon DNA synthesis. Reactions generally consist of successive reagent delivery and washing steps, e.g. to allow the incorporation of reversible labelled terminator bases, and scanning steps to determine the order of base incorporation. Array-based systems of this type are available commercially e.g. from Illumina, Inc. (San Diego, Calif.; http://www.illumina.com/). Third generation techniques are typically defined by the absence of a requirement to halt the sequencing process between detection steps and can therefore be viewed as real-time systems. For example, the base-specific release of hydrogen ions, which occurs during the incorporation process, can be detected in the context of microwell systems (e.g. see the Ion Torrent system available from Life Technologies; http://www.lifetechnologies.com/). Similarly, in pyrosequencing the base-specific release of pyrophosphate (PPi) is detected and analysed. In nanopore technologies, DNA molecules are passed through or positioned next to nanopores, and the identities of individual bases are determined following movement of the DNA molecule relative to the nanopore. Systems of this type are available commercially e.g. from Oxford Nanopore

(https://www.nanoporetech.com/). In an alternative method, a DNA polymerase enzyme is confined in a "zero-mode waveguide" and the identity of incorporated bases are determined with florescence detection of gamma-labeled phosphonucleotides (see e.g. Pacific Biosciences; http://www.pacificbiosciences.com/).

In some embodiments, the method comprises identifying genetic mutations in certain genes. Exemplary genes assessed for genetic mutations are listed in Table 5.

Table 5: Exemplary Genes Assessed for Genetic Mutations

In some embodiments, the methods comprise identifying a genetic mutation in FLT3. Common gene mutations of FLT3 are the internal tandem duplication (ITD) mutation and the tyrosine kinase domain (FLT3-TKD) mutation. In some embodiments, the methods comprise identifying an FLT3-ITD+ mutation. In some embodiments, the methods comprise identifying an FLT3-TKD+ mutation.

In some embodiments, the methods comprise identifying a genetic mutation in IDH1. A common gene mutation of IDH1 is R132. In some embodiments, the methods comprise identifying an IDHl R132 mutation. In some embodiments, the methods comprise identifying a genetic mutation in CBL.

In some embodiments, the methods comprise identifying a genetic mutation is NRAS.

In some embodiments, the methods comprise identifying chromosome abnormalities.

Chromosomal abnormalites herein include monosomy of one or more chromosomes (X chromosome monosomy, also known as Turner's syndrome), trisomy of one or more chromosomes (13, 18, 21, and X), tetrasomy and pentasomy of one or more chromosomes (which in humans is most commonly observed in the sex chromosomes, e.g., XXXX, XXYY, XXXY, XYYY, XXXXX, XXXXY, XXXYY, XYYYY and XXYYY), monoploidy, triploidy (three of every chromosome, e.g. 69 chromosomes in humans), tetraploidy (four of every chromosome, e.g. 92 chromosomes in humans), pentaploidy and multiploidy.The most common chromosomal abnormalities in MDS/AML are Del(5q), trisomy 8, del(20q), del(7q), monosomy 7, and complex karyotypes (Table 6). Loss and/or gain of genomic materials in these chromosomes are associated with the initiation and progression of MDS/AML.

Table 6: Chromosomal aberrations in MDS/AML

Non-limiting examples of techniques to identify chromosomal abnormalities include metaphase cytogenetic (MC) analysis, FISH, Array-CGH, SNP-Array, SKY, and NGS (Table 7)·

Table 7: Techniques for detecting chromosomal aberrations in MDS/AML

In some embodiments, the methods comprise measuring expression levels of genes with identified mutations. Expression levels of genes can be measured by measuring nucleic acid amounts (e.g., mRNA amounts and/or genomic DNA). The determination of nucleic acid amounts can be performed by a variety of techniques known to the skilled practitioner. For example, expression levels of nucleic acids, alternative splicing variants, chromosome rearrangement and gene copy numbers can be determined by microarray analysis (see, e.g., U.S. Pat. Nos. 6,913,879, 7,364,848, 7,378,245, 6,893,837 and 6,004,755) and quantitative PCR. In some embodiments, expression levels of genes can be measured by RNA sequencing. Copy number changes may be detected, for example, with the Illumina Infmium II whole genome genotyping assay or Agilent Human Genome CGH Microarray (Steemers et ah, 2006). Examples of methods to measure mRNA amounts include reverse transcriptase- polymerase chain reaction (RT-PCR), including real time PCR, microarray analysis, nanostring, Northern blot analysis, differential hybridization, and ribonuclease protection assay. Such methods are well-known in the art and are described in, for example, Sambrook et ah, Molecular Cloning: A Laboratory Manual, current edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Ausubel et ah, Current Protocols in Molecular Biology, John Wiley & sons, New York, N.Y.

Methods of Treatment

Embodiments of the methods relate to administration of a compound or composition to treat any disease or disorder characterized by genetic mutations in genes that regulate apoptosis.

In some embodiments, treating a disease or disorder characterized by genetic mutations in genes that regulate apoptosis, such as MDS/AML/a type of cancer, can involve disease prevention, reducing the risk of the disease, ameliorating or relieving symptoms of the disease, eliciting a bodily response against the disease, inhibiting the development or progression of the disease, inhibiting or preventing the onset of symptoms of the disease, reducing the severity of the disease, causing a regression of the disease or a disease symptom, causing remission of the disease, preventing relapse of the disease, and the like. In some embodiments, treating includes prophylactic treatment. In some embodiments, treating does not include prophylactic treatment.

In some embodiments, the MDS can be selected from Fanconi Anemia, refractory anemia, refractory neutropenia, refractory thrombocytopenia, refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), refractory anemia with excess blasts I and II (RAEB), myelodysplastic syndrome, unclassified (MDS-U), MDS associated with isolated del(5q)-syndrome, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), refractory cytopenia of childhood, or a combination thereof. In some embodiments, the MDS is primary MDS. In some embodiments, the MDS is secondary MDS.

In some embodiments, the AML can be selected from AML with recurrent genetic abnormalities (such as, for example, AML with translocation between chromosomes 8 and 21, AML with translocation or inversion in chromosome 16, AML with translocation between chromosomes 9 and 11, APL (M3) with translocation between chromosomes 15 and 17, AML with translocation between chromosomes 6 and 9, AML with translocation or inversion in chromosome 3, and the like), AML (megakaryoblastic) with a translocation between chromosomes 1 and 22, AML with myelodysplasia-related changes, AML related to previous chemotherapy or radiation (such as, for example, alkylating agent-related AML, topoisomerase II inhibitor-related AML, and the like), AML not otherwise categorized (does not fall into above categories - similar to FAB classification; such as, for example, AML minimally differentiated (MO), AML with minimal maturation (Ml), AML with maturation (M2), acute myelomonocytic leukemia (M4), acute monocytic leukemia (M5), acute erythroid leukemia (M6), acute megakaryoblastic leukemia (M7), acute basophilic leukemia, acute panmyelosis with fibrosis, and the like), myeloid sarcoma (also known as granulocytic sarcoma, chloroma or extramedullary myeloblastoma), undifferentiated and biphenotypic acute leukemias (also known as mixed phenotype acute leukemias), and the like.

AML is an aggressive type of cancer in which the bone marrow makes abnormal myeloblasts, red blood cells, or platelets. This type of cancer is fatal if left untreated. AML is the most common type of acute leukemia in adults. AML is also called acute myelogenous leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, and acute non- lymphocytic leukemia. There are different subtypes of AML. Most AML subtypes are based on how developed the cancer cells are at the time of diagnosis and different they are from normal cells. Certain factors affect prognosis and treatment options for AML. The prognosis and treatment options depend on the age of the patient, the subtype of AML, whether the patient received chemotherapy in the past to treat a different cancer, whether there is a history of a blood disorder such as myelodysplastic syndrome, whether the cancer has spread to the central nervous system, and whether the cancer has been treated before or recurred.

In some embodiments, the type of cancer can be selected from breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, stomach cancer, testicular cancer, thyroid cancer, urothelial cancer, and the like.

In some embodiments, the administration may decrease the incidence of one or more symptoms associated with MDS/AML/a type of cancer. In some embodiments, the administration may decrease marrow failure, immune dysfunction, transformation to overt leukemia, or combinations thereof in said individual, as compared to an individual not receiving said composition.

In some embodiments, the method may decrease a marker of viability of MDS cells or cancer cells. In one aspect, the method may decrease a marker of viability of MDS, AML, and/or cancer cells. The marker may be selected from survival over time, proliferation, growth, migration, formation of colonies, chromatic assembly, DNA binding, RNA metabolism, cell migration, cell adhesion, inflammation, or a combination thereof.

Combination Treatment & Determining Drug Responsiveness

The present invention provides methods of treating AML by identifying specific genetic mutations and chromosomal abnormalities in aggressive, poor prognosis AML subjects and determining which drug combinations a subject will likely respond to during treatment. In particular, the present invention is directed to determining responsiveness to treatment with a XIAP inhibitor, a BCL2 inhibitor, a MCL1 inhibitor, an aurora kinase B inhibitor, or a combination thereof. The subject may be administered an inhibitor individually or conjointly with another inhibitor. The present invention provides a link between genetic information and drug combination sensitivity.

In some embodiments, the methods comprise determining the presence of a FLT3- ITD+ mutation and the absence of an IDH1 mutation, and then administering a XIAP inhibitor individually or conjointly with a BCL2 inhibitor to the subject.

In some embodiments, the chromosomal abnormalities assessed for in AML subjects include complex karyotype and monosomy 7. In some such embodiments, the methods further comprise determining the presence of a FLT3-ITD+ mutation, absence of an IDH1 mutation, and presence of complex karyotype and/or monosomy 7, and then administering a XIAP inhibitor individually or conjointly with a BCL2 inhibitor to the subject.

In some embodiments, the genes assessed for genetic mutations in AML subjects include DNMT3A, NPM1, and/or TET2. In some such embodiments, the methods further comprise determining the presence of a FLT3-ITD+ mutation, absence of an IDHl mutation, presence of a mutation in DNMT3A, NPM1, and/or TET2, and then administering a XIAP inhibitor individually or conjointly with a BCL2 inhibitor to the subject. In other such embodiments, the methods further comprise determining the presence of a FLT3-ITD+ mutation, absence of an IDHl mutation, presence of complex karyotype and/or monosomy 7, presence of a mutation in DNMT3A, NPM1, and/or TET2 in the subject, and then administering a XIAP inhibitor or conjointly administering a XIAP inhibitor and a BCL2 inhibitor to the subject.

In some embodiments, the genes assessed for genetic mutations in AML subjects include PPM1D, MGA, and/or WT1. In some such embodiments, the methods further comprise determining the presence of a FLT3-ITD+ mutation, absence of an IDHl mutation, presence of a mutation in PPM1D, MGA, and/or WT1, and then administering a XIAP inhibitor individually or conjointly with a BCL2 inhibitor to the subject. In other such embodiments, the methods further comprise determining the presence of a FLT3-ITD+ mutation, absence of an IDHl mutation, presence of complex karyotype and/or monosomy 7, presence of a mutation in PPM1D, MGA, and/or WT1 in the subject, and then administering a XIAP inhibitor individually or conjointly with a BCL2 inhibitor. In yet other such embodiments, the methods further comprise determining the presence of a FLT3-ITD+ mutation, absence of an IDHl mutation, presence of complex karyotype and/or monosomy 7, presence of a mutation in DNMT3A, NPM1, and/or TET2, presence of a mutation in PPM ID, MGA, and/or WT1 in the subject, and then administering a XIAP inhibitor individually or conjointly with a BCL2 inhibitor to the subject.

In some embodiments, the genes assessed for genetic mutations in AML subjects include PTPN11, CEBPA, CUX1, NF1, ZASR2, TP53, and/or ETV6. In some such embodiments, the methods further comprise determining the presence of a FLT3-ITD+ mutation, absence of an IDHl mutation, presence of a mutation in PTPN11, CEBPA, CUX1, NF1, ZASR2, TP53, and/or ETV6, and then administering a XIAP inhibitor individually or conjointly with an MCL1 inhibitor to the subject. In other such embodiments, the methods further comprise determining the presence of FLT3-ITD+ mutation, absence of an IDHl mutation, presence of complex karyotype and/or monosomy 7, presence of a mutation in PTPN11, CEBPA, CUX1, NF1, ZASR2, TP53, and/or ETV6 in the subject, and then administering a XIAP inhibitor individually or conjointly with an MCL1 inhibitor to the subject. In yet other such embodiments, the methods further comprise determining the presence of FLT3-ITD+ mutation, absence of an IDHl mutation, presence of complex karyotype and/or monosomy 7, presence of a mutation in DNMT3A, NPM1, and/or TET2, presence of a mutation in PTPN11, CEBPA, CUX1, NF1, ZASR2, TP53, and/or ETV6 in the subject, and then administering a XIAP inhibitor individually or conjointly with an MCL1 inhibitor to the subject.

In some embodiments, the methods comprise determining the presence of a FLT3- ITD+ mutation and the presence of an IDHl mutation, and then conjointly administering a BCL2 inhibitor and a MCL1 inhibitor to the subject. In some embodiments, the genes assessed for genetic mutations in AML subjects are DNMT3A and/or NPM1. In some such embodiments, the methods comprise determining the presence of a FLT3-ITD+ mutation, presence of an IDHl mutation, presence of a mutation in DNMT3 A and/or NPM1, and then conjointly administering a BCL2 inhibitor with a MCL1 inhibitor to the subject.

In some embodiments, the methods comprise determining the presence of a FLT3- TKD+ mutation and then administering a XIAP inhibitor individually or conjointly with a BCL2 inhibitor or an MCL1 inhibitor to the subject.

In some embodiments, the chromosomal abnormalities assessed for in AML subjects include complex karyotype, monosomy 7, and/or monosomy 17. In some embodiments, the methods further comprise determining the presence of a FLT3-TKD+ mutation, presence of complex karyotype, monosomy 7, and/or monosomy 17, and then administering a XIAP inhibitor individually or conjointly with a BCL2 inhibitor or an MCL1 inhibitor to the subject.

In some embodiments, the genes assessed for genetic mutations in AML subjects include TET2, WT1, SMC3A, MLL-ENL, and/or NF1. In some embodiments, the methods further comprise determining the presence of a FLT3-TKD+ mutation, presence of a mutation in TET2, WT1, SMC3A, MLL-ENL, and/or NF1, and then administering a XIAP inhibitor individually or conjointly with a BCL2 inhibitor or an MCL1 inhibitor to the subject. In some embodiments, the methods further comprise determining the presence of a FLT3-TKD+ mutation, presence of complex karyotype, monosomy 7, and/or monosomy 17, presence of a mutation in TET2, WT1, SMC3A, MLL-ENL, and/or NF1, and then administering a XIAP inhibitor individually or conjointly with a BCL2 inhibitor or an MCL1 inhibitor to the subject. In some embodiments, the methods comprise determining the absence of a mutation in FLT3, presence of a mutation in PTPN11, MGA, and/or PHF, and then administering a BCL2 inhibitor to the subject.

In some embodiments, the chromosomal abnormalities assessed in AML subjects include complex karyotype, chromosome 17 abnormality, trisomy 21, and/or monosomy 7, In some such embodiments, the methods further comprise determining the absence of a mutation in FLT3, presence of a mutation in PTPN11, MGA, and/or PHF, presence of complex karyotype, chromosome 17 abnormality, trisomy 21, and/or monosomy 7, and then administering a BCL2 inhibitor to the subject.

In some embodiments, the genes assessed for genetic mutations in AML subjects include CEBPA, PPM1D, and/or GATA2. In some such embodiments, the methods comprise determining the absence of a mutation in FLT3, presence of a mutation in CEBPA, PPM1D, and/or GAT A, and then conjointly administering a BCL2 inhibitor and a XIAP inhibitor. In yet other such embodiments, the methods further comprise determining the absence of a mutation in FLT3, presence of a mutation in CEBPA, PPM1D, and/or GATA, presence of complex karyotype, chromosome 17 abnormality, trisomy 21, and/or monosomy 7, and then administering a BCL2 inhibitor to the subject.

In some embodiments, the genes assessed for genetic mutations in AML subjects include CBL. In some such embodiments, the methods comprise determining the absence of a mutation in FLT3, presence of a mutation in CBL, and then conjointly administering a XIAP inhibitor with an MCL1 inhibitor or a BCL2 inhibitor to the subject. In other such embodiments, the methods further comprise determining the absence of a mutation in FLT3, presence of a mutation in CBL, presence of complex karyotype, chromosome 17 abnormality, trisomy 21, and/or monosomy 7, and then conjointly administering a XIAP inhibitor and a MCL1 inhibitor or a BCL2 inhibitor to the subject.

In some embodiments, the genes assessed for genetic mutations in AML subjects include IDH2, TET2, DNMT3A, NPM1, and/or SETBP1. In some embodiments, the methods comprise determining the absence of a mutation in FLT3, presence of a mutation in CBL, presence of a mutation in IDH2, TET2, DNMT3A, NPM1, and/or SETBP1, and then conjointly administering a XIAP inhibitor and an MCL1 inhibitor or a BCL2 inhibitor to the subject.

In some embodiments, the genes assessed for genetic mutations in AML subjects include NRAS. In some such embodiments, the methods comprise determining the absence of a mutation in FLT3, presence of a mutation in NRAS, and then conjointly administering a XIAP inhibitor and an aurora kinase B inhibitor to the subject. In other such embodiments, the methods further comprise determining the absence of a mutation in FLT3, presence of a mutation in NRAS, presence of complex karyotype, chromosome 17 abnormality, trisomy 21, and/or monosomy 7, and then conjointly administering a XIAP inhibitor and an aurora kinase B inhibitor to the subject.

In some embodiments, the genes assessed for genetic mutations in AML subjects include DNMT3 A and/or BCOR. In some such embodiments, the methods further comprise determining the absence of a mutation in FLT3, presence of a mutation in NRAS, presence of complex karyotype, chromosome 17 abnormality, trisomy 21, and/or monosomy 7, presence of a mutation in DNMT3A and/or BCOR, and then conjointly administering a XIAP inhibitor and an aurora kinase B inhibitor to the subject.

In some embodiments, the methods described herein comprise quantification of annexin V in AML cells following drug exposure to determine subject-specific drug responsiveness. Annexins represent a highly conserved family of proteins that selectively bind to negatively charged, phosphatidylserine containing phospholipid membranes in the presence of calcium ion. Dying cells undergoing apoptosis expose these negatively charged lipids on the outer leaflet of the plasma membrane. Therefore, annexins selectively bind to apoptotic cells. This diagnostic application of annexins was first demonstrated using fluorescently labeled annexin V. In some embodiments, flow cytometric detection of phosphatidylserine expression on apoptotic cells with fluorescein labeled Annexin V may be used to determine drug sensitivity.

In some embodiments, the methods comprise collecting AML cells from the subject, measuring a level of annexin V expression in AML cells following exposure to conjointly administering an XIAP inhibitor and BCL2 inhibitor, and determining whether the level of annexin V expression is increased in AML cells exposed to the two inhibitors compared to a level of annexin V expression in AML cells not exposed to the two inhibitors or AML cells exposed to only the XIAP inhibitor or the BCL2 inhibitor.

In some embodiments, the methods described herein comprise collecting AML cells from the subject, measuring a level of annexin V expression in AML cells following exposure to conjointly administering an XIAP inhibitor and MCL1 inhibitor, and determining whether the level of annexin V expression is increased in AML cells exposed to the two inhibitors compared to a level of annexin V expression in AML cells not exposed to the two inhibitors or AML cells exposed to only the XIAP inhibitor or the MCL1 inhibitor. In some embodiments, the methods described herein comprise collecting AML cells from the subject, measuring a level of annexin V expression in AML cells following exposure to conjointly administering an XIAP inhibitor and aurora kinase B inhibitor, and determining whether the level of annexin V expression is increased in AML cells exposed to the two inhibitors compared to a level of annexin V expression in AML cells not exposed to the two inhibitors or AML cells exposed to only the XIAP inhibitor or the aurora kinase B inhibitor.

EXAMPLES

The following study focused on an AML patient population with the most aggressive clinical course and poor clinical outcome and aimed to identify therapeutic targets actionable in a broad population of AML patients with aggressive poor prognosis disease. To identify therapeutic targets, RNA sequencing was performed from functionally-defined AML engrafting cells from high risk AML patients followed by in vitro testing of directed libraries of small molecules. A matrix of five small molecule inhibitors was then taken forward for extensive in vivo testing in AML NSG PDX models. In summary, the data show that combination treatment strategy based on AZD5582, a dimeric second mitochondrial activator of caspases (SMAC) mimetic which bivalently blocks inhibitor of apoptosis proteins (IAPs), was highly effective in vivo against a range of poor risk leukemia samples20. Inhibition of IAPs activated apoptotic mechanisms and, in combination with additional small molecules, that were patient-specific and therapeutically efficacious, despite diverse mutations and chromosomal abnormalities. Correlations were identified between sensitivity to XIAP or BCL2 inhibition with mutations (FLT3, IDH1, CBL, NRAS, TET2), chromosomal aberrations (rearrangements involving MLL1 or EVI1/MECOM) and transcription regulatory function of TP53 in the majority of patients. These findings will facilitate curative precision medicine for the most clinically-aggressive AML in the future.

Example 1: Identification of therapeutic targets in clinically aggressive adverse risk AML

Bone marrow (BM) or peripheral blood (PB) samples from 136 patients with clinically aggressive leukemias including FLT3-WT (n=89), FLT3-mutated AML (n=38; ITD+ n=26, TKD n=12), MDS (n=3), and Ph+ mixed phenotype acute leukemia (MPAL) and Ph+ leukemia (n=6) (Patient characteristics summarized in Table 8). Risk stratification by European LeukemiaNet (ELN) 2010 criteria showed an excess proportion of adverse risk AML patients in the study population (n=126) compared with previously reported populations of AML patients enrolled in multicenter clinical trials (Figure 1, Panel A). Among AML patients, 66.7% (84 out of 126) came to the hospital at the time of relapse or were refractory to treatment. Consistent with the risk stratification, 92.1% (116 out of 126) did not achieve long-term remission, and 84.9% (107 out of 126) had died of the disease (Figure 1, Panel B).

Table 8: Patient Characteristics

The aim of the study was to discover potential drug targets in highly aggressive AML refractory to currently available treatment options. Therefore, leukemia-engrafting cells from AML samples obtained from the deceased patients (Figure 1, Panel C). In 86 of 119 patients, the cellular subpopulation with leukemia engraftment capacity was determined and RNAseq global transcriptome analysis was performed on samples derived from the AML engrafting cell populations (FLT3- WT patients n=54, FLT3-mutated patients n=32). Leukemia- engrafting cells from FLT3-WT AML were compared with cord blood CD34+ hematopoietic stem/progenitor cells (HSPCs) obtained from healthy donors (HSPCs; n=44), and 3616 differentially expressed genes (DEGs) were identified (|Log2FC|>l, padj<0.05; principal component analysis (PCA) plot shown in Figure 1, Panel D, Volcano plot shown in Figure 2). Of those, 2904 genes were upregulated (Log2FC>l, padj<0.05, Figure 1, Panel D) and 712 genes were downregulated (Log2FC<-l, padj<0.05) in FLT3-WT AML leukemia-engrafting cells. Analogous analysis comparing FLT3-mutated AML-engrafting cells with cord blood HSPCs yielded 4346 DEGs (|Log2FC|>l, padj<0.05; principal component analysis (PCA) plot shown in Figure 1, Panel D, Figure 2, Panel B). Of those, 3216 genes were upregulated (Log2FC>l, padj<0.05) and 1130 genes were downregulated (Log2FC<-l, padj<0.05) in FLT3- mutated AML-engrafting cells.

To identify functions and pathways with potential pathophysiological relevance, the study focused on 1284 DEGs up-regulated both in FLT3 WT and mutated AML samples (Log2F01.5, padj<0.05, base mean>20 RPKM) (Figure 1, Panel E). Gene Ontology (GO) pathway analysis using GOrilla software revealed 325 GO terms that were significantly enriched in FLT3-WT and -mutated AML cells (p<0.001, adjusted by Benjamini method). The REViGO software was used to summarize the GO results, revealing the cluster of GO terms related to gene regulation, such as regulation of cell proliferation and apoptotic process (Figure 1, Panels F, G). Protein-protein interaction analysis using STRING was performed on products of the common 1284 DEGs, resulting in subnetworks of inter- related proteins (Figure 1, Panel H left). Strong interactions were observed in the subnetwork of proteins associated with mitotic cell cycle (i.e. CDK1, CDC20, CCNBl, CCNB2, KIF20A, NCAPG), as well as BIRC5, a member of the inhibitor of apoptosis (LAP) gene family (Figure 1, Panel H right, Panel I). These analyses highlighted potential dependencies of leukemia-engrafting cells from highly aggressive, poor prognosis AML upon proteins and pathways involved in cell defense, cell cycle, and inhibition of apoptosis. To examine how these vulnerabilities may be exploited, chemical screening of small molecule inhibitors targeting these pathways in primary cells derived from clinically aggressive relapsed/refractory leukemia patients was performed.

Example 2: Treatment-resistant FLT3-WT and FLT3-ITD+ AML demonstrate dependencies to distinct survival- and proliferation-related pathways

To assess whether disrupting differentially activated pathways leads to elimination of treatment- resistant leukemia cells, 35 available small molecule inhibitors targeting products of upregulated DEGs and enriched pathways were evaluated (data not shown) against cells derived from relapsed/refractory FLT3-WT (n=8) and -ITD+ (n=2) AML patients (data not shown, Patients 1-10). These samples were from clinically highly aggressive cases where patients had either failed induction chemotherapy, relapsed multiple times or relapsed following hematopoietic stem cell transplantation (HSCT) and all patients have since deceased. To quantitatively assess AML cell elimination, an in vitro culture assay was developed in which the cells were exposed to compounds at multiple concentrations for 72 hours (representative data shown in Figure 3, Panel A) and the absolute number of viable AML cells quantitated by flow cytometry. Efficacy of AML elimination for each compound against each patient sample, as compared with vehicle control (DMSO), is shown as a heat map in Figure 3, Panel B. Of the 35 compounds, exposure to eight (AZD5582, YM155, venetoclax, S63845, dinaciclib, GSK923295, SB743921, barasertib) resulted in greater than 70% killing of AML cells at 300nM in at least six of ten patients tested. Among them, AZD5582, a bivalent SMAC-mimetic antagonist of IAP proteins, particularly BIRC4/XIAP, exhibited the greatest efficacy at the lowest concentrations, with greater than 70% killing achieved in nine of ten AML patient samples at 300nM and seven of ten even at lOnM.

Next, in vitro chemical screening against an additional 49 samples from AML patient samples with similar characteristics (total FLT3-WT n=43 and -ITD+ AML n=16) was performed using five compounds (AZD5582 at 10nM/30nM/100nM; venetoclax, S63845, barasertib and GSK923295 each at 100nM/300nM) (Figure 3, Panel C; data no shown,

Patient 1-59). Each of these compounds inhibit distinct targets and pathways: XIAP, BCL2, MCL1, aurora kinase B and KIF10, respectively. In vitro efficacy was defined as elimination of greater than 70% of AML cells at 30nM for AZD5582, 300nM for venetoclax and S63845 and lOOnM for barasertib and GSK923295. Dinaciclib, YM155 and SB743921 were not evaluated further because of their relative high toxicity against human CB HSPCs (residual human HSPCs at 30nM drug exposure, dinaciclib: 4.8+/- 3.0%, YM155: 11.8+/- 5.0%, SB743921 : 8.9 +/- 2.7%) at the dose where 70% or greater leukemia cell elimination was achieved. Among FLT3-WT cases, AZD5582 demonstrated greatest efficacy, eliminating greater than 70% of AML cells at 30nM in 31 of 43 cases (72.1%), while venetoclax,

S63845, barasertib and GSK923295 achieved greater than 70% AML cell elimination in 20,

9, 11 and 4 of 43 cases (46.5%, 20.9%, 25.6%, 9.3%), respectively. Surprisingly, although AZD5582 does not directly target normal or mutated FLT3, greater than 70% leukemia elimination was achieved at 30nM in 14 of 16 clinically aggressive FLT3-ITD+ cases (87.5%). In some cases, venetoclax at 300nM (10 of 16 cases), S63845 at 300nM (11 of 16 cases), barasertib at lOOnM (9 of 16 cases) and GSK923295 at lOOnM (7 of 16 cases) were also found effective. Importantly, cases with adverse risk chromosomal abnormalities showed responsiveness to AZD5582: 16 of 24 FLT3-WT and 7 of 7 ITD+ with complex karyotype,

17 of 23 FLT3-WT and 3 of 3 ITD+ with abnormalities involving chromosomes 5, 7 and/or 17, 3 of 3 FLT3-WT and 1 of 1 ITD+ with t(3;3)(q21;q26.2) or inv(3)(q21q26) and 3 of 3 FLT3-WT and 1 of 1 FLT3-TKD mutant with t(v;l lq23.3) KMT2A/MLL translocation (Figure 3, Panel C). AZD5582 was effective against the highest frequency of cases with adverse risk chromosomal abnormalities among the five compounds examined (Figure 3, Panel D). In primary AML cells, exposure to AZD5582 lead to apoptotic cell death through downregulation of XIAP, as evidenced by loss of BIRC4/XIAP protein expression and increased protein levels of activated caspase-3 (Figure 3, Panel E). In patient samples containing both AML cells and native T cells, native T cells were substantially more resistant to AZD5582 while co-existing AML cells were effectively eliminated (viable CD33+ human AML cells 9.75 +/- 1.6%, viable CD3+ human T cells 78.6 +/- 5.6%; n=20 cases, p<0.0001 by paired two-tailed t-test) (Figure 3, Panel F; representative flow cytometry plots in Figure

4, Panel A). Interestingly, AZD5582 showed greater efficacy compared with birinapant (a second-generation SMAC-mimeti c/bivalent antagonist of IAP proteins) and AT406 (a monovalent IAP inhibitor) (Figure 4, Panel B). Against FLT3-ITD+ cases, AZD5582 was more effective than FLT3 inhibitors quizartinib, crenolanib, gilteritinib and midostaurin (Figure 3, Panel G). Surprisingly, even though Bcr-Abl kinase is not directly targeted, AZD5582 at 30nM demonstrated greater than 70% leukemia elimination of leukemia cells in five of six poor prognosis imatinib-resistant Ph+ ALL and MPAL cases (Figure 3, Panel H; additional data not shown, Patient 60-65). Venetoclax (4 of 6 cases) and S63845 (2 of 6 cases) were also found effective while most leukemias were relatively resistant to barasertib or GSK923295. Through in vitro chemical screening, an efficacy matrix of five compounds targeting distinct targets and pathways in highly aggressive poor risk AML was established. AZD5582, an inhibitor of XIAP, demonstrated the greatest capacity for leukemia cell elimination for FLT3-WT and -mutated as well as Ph+ leukemia cells.

Example 3: AZD5582 responsiveness is modulated by AML-associated somatic mutations and aberrant chromosomes

The correlation between mutational landscape and in vitro leukemia elimination in a total of 107 AML cases (FLT3-WT n=79, FLT3-ITD+ n=17, FLT3-TKD n=l 1) was examined through targeted DNA sequencing for 41 AML-associated somatic mutations (data not shown). AZD5582 showed efficacy across samples with differing somatic mutations (Figure 5, Panel A). In vitro sensitivity of each case to AZD5582 in the order of responsiveness is shown in (Figure 5, Panel B upper panel, n=107), with presence of somatic mutations indicated below. The same data with cases rearranged in the order of sensitivity to venetoclax is shown (Figure

5, lower Panel B, n=103; Figure 6 shows data rearranged by S63845, barasertib and GSK923295 sensitivity). Responsiveness to each compound was: 73 of 107 for AZD5582 (68.2%), 61 of 103 for venetoclax (59.2%), 23 of 66 for S63845 (34.8%), 21 of 66 for barasertib (31.8%) and 11 of 66 for GSK923295 (16.7%). Of note, 21 barasertib responsive cases included 9 of 11 GSK923295 responsive cases, indicating that barasertib responsiveness correlates highly with GSK923295 responsiveness (Figure 7). Effects of mutations on sensitivity/resistance to AZD5582 and venetoclax is summarized (Figure 8, additional data not shown). Consistent with previous reports, mutations in IDH1 and IDH2 correlated with venetoclax responsiveness. Other statistically significant correlations included mutated TP53 and mutated IDH1 with AZD5582 resistance and mutated TP53 and mutated CBL with venetoclax resistance. NPM1 and TET2 mutations correlated with venetoclax sensitivity; mutated TET2 with barasertib sensitivity; and FLT3- ITD+ with S63845, barasertib and GSK923295 sensitivity. While mutated TP53 showed a correlation with resistance to both AZD5582 and venetoclax, it does not determine responsiveness to these drugs completely, as 13 of 73 AZD5582 sensitive and 11 of 61 venetoclax sensitive cases were found to carry TP53 mutations and responsiveness to these compounds among TP53 mutated cases were highly variable.

Next, the effects of chromosomal abnormalities on responsiveness to the five compounds (Figure 5, Panel C). Again, AZD5582 was found to be effective against a majority of cases with chromosome 5, 7 or 17 abnormalities (-5, del(5q), -7, -17, abnormal 17p) and/or complex karyotype. In addition, all six MLL-rearranged cases (Patient 18, 32, 33, 36, 68, 91) showed AZD5582 responsiveness. Moreover, the presence of t(3;3), inv(3) and monosomy 7 were associated with venetoclax resistance and monosomy 7 with S63845 resistance (Figure 5, Panel C, Figure 8, additional data not shown). Interestingly, all eight cases with abnormalities involving chromosome 3 (t(3;3) patient 13, 51; inv(3) patient 26, 29; t(l;3)(p36;q21) patient 16, 93; t(3;21)(q26;q21) patient 14; t(3;17)(q26.2;ql 1.2) patient 73) were sensitive to AZD5582 but showed resistance to venetoclax and S63845. Impact of mutations and chromosome aberrations on sensitivity to five inhibitors are summarized in Figure 5, Panel D. While these findings demonstrate that XIAP dependence is an important mechanism for survival of highly aggressive AML, no single somatic mutation or chromosomal abnormality completely defines responsiveness of human AML cells to these molecular targeting drugs. Combinations of multiple gene mutations and karyotype abnormalities correlated with responsiveness to AZD5582 and venetoclax were examined. 103 cases were separated into four groups based on AZD5582 and venetoclax responsiveness (Figure 5, Panel E).

Overall, mutations in FLT3, NRAS and CBL were associated with XIAP dependence and responsiveness to AZD5582. 22 of 27 FLT3-mutated AML was AZD5582-responsive and five AZD5582 resistant cases carried mutated IDH1, mutated IDH2 or both. On the other hand, all 20 IDHl WT FLT3-mutated cases were AZD5582 sensitive, suggesting that mutant IDH1/2 diminishes XIAP dependence in FLT3-mutated leukemia cells. In addition, 11 of 14 NRAS- mutated cases were AZD5582 sensitive. Three NRAS-mutated AZD5582-resistant cases were associated with complex karyotype with TP53, IDH1 or RUNX1 mutation. Similarly, eight of nine CBL-mutated cases were AZD5582 sensitive and the resistant case carried both TP53 and RUNX1 mutations. These findings suggest mutated FLT3, NRAS and CBL confers XIAP dependence while TP53, IDH1 and RUNX1 mutations and complex karyotype diminishes it. Finally, in 9 cases with resistance to both AZD5582 and venetoclax, the most frequent genetic abnormalities were complex karyotype (n=7) and mutated TP53 (n=7), with six cases having both. Six cases with both complex karyotype and mutated-TP53 were resistant to all five compounds showing that these two genetic abnormalities contributed to diminished dependence to multiple survival pathways in highly aggressive AML. In addition, aberrant chromosome 3 showed a strong link with venetoclax resistance.

Example 4: Dependence of leukemic cells to XIAP for survival positively correlate with TP53 transcriptional activity while EVI1 activation leads to resistance to BCL2 inhibition

These findings showed that aberrant TP53 contributes to diminished dependence to multiple survival pathways in highly aggressive AML. In addition, chromosome 3 abnormality t(3;3)/inv(3) showed a strong link with venetoclax resistance. Therefore, the interactions between these genetic abnormalities and XIAP and BCL2 dependence in high-risk poor- outcome AML was examined next.

First, to assess how each mutation disrupts the biological function of TP53, motif activity response analysis was used. Motif activity 31 quantifies the regulatory function of a transcription factor (TF) based on the RNA-seq expression level of genes with predicted DNA binding site motifs for the TF in their promoter region. Interestingly, a correlation was found between motif activity of TP53 and responsiveness to AZD5582. Focusing on TP53-mutated cases (pink, lavender and purple circles, n=26), TP53 motif activity and AZD5582 responsiveness showed a positive correlation, where AZD5582 sensitivity was associated with higher TP53 motif function (Figure. 9, Panel A, Pearson’s correlation coefficients.5063, p=0.0071). Furthermore, TP53 mutants with high impact mutations, including frameshift and deletion, reduced TP53 motif activity (Figure 9, upper Panel B). In addition, several TP53 missense mutations appeared to be associated with diminished AZD5582 responsiveness (Figure 9, lower Panel B). Next, potential correlation among location of TP53 mutations, impact of mutations on TP53 motif activity, and impact of mutations on responsiveness to AZD5582 were examined. Mutations close to the DNA-binding surface of TP53 such as Glu286Val and mutations near Zn- finger domain such as Hisl79Tyr resulted in diminished TP53 motif activity and diminished AZD5582 responsiveness while mutations away from the DNA-binding surface such as Tyr220Cys preserved TP53 motif activity and AZD5582 responsiveness was maintained (Figure 9, Panel C). These findings suggest that identification of the specific TP53 mutations present in individual patients may inform treatment choices based on AZD5582 responsiveness. Next, to link TP53 TF function with XIAP dependence, expression of 500 TP53 target genes (genes containing TP53 binding motif in the promoter regions) in AML cells was analyzed. In AZD5582-resistant AML cells with low TP53 motif activity, genes such as BCL2 Binding Component 3 (BBC3) and Lamin A/C (LMNA) were down-regulated, suggesting that reduced TP53 TF function is associated with decreased expression of these genes led to diminished pro-apoptosis (Figure 9, Panel D). These findings were consistent with a previous report showing that in Molml3 cells, TP53 binding to BBC3 was maintained in mutants such as R282W and Y220C, which were found to have preserved TP53 motif activity (Figure 9, Panels B, E). These findings suggest BBC3 as one of the determinants of XIAP dependence and sensitivity to XIAP inhibition in poor prognosis AML.

Next, the mechanism of venetoclax resistance in AML cells with t(3;3)(q21;q26) and inv(3q21) was explored. Chromosomal abnormalities involving 3q21, such as t(3;3)(q21;q26) and inv(3q21), result in activation of EVI1/MECOM transcription by the GATA2 enhancer33- 35. Consistent with this, high levels of EVIl/MECOM expression in AML cells with t(3;3)(q21;q26) and inv(3q21) (green/orange circles, Fig. 9, Panel F), and lower levels of EVIl/MECOM expression in venetoclax-sensitive AML cells were found (left upper quadrant, Fig. 9, Panel F At the same time, GATA motif activity was low in t(3 ; 3 ) and inv(3) AML cells (green/orange circles, Fig. 9, Panel G). Therefore, differentially-expressed genes between the following two groups were examined: 1) venetoclax-resistant, EVIl/MECOM expression high and GATA motif activity low t(3;3)/inv(3) AML (n=2 each, green/orange circles in Fig. 9, Panel G) and 2) venetoclax-sensitive, EVIl/MECOM expression low and GATA motif activity high AML (n=23, blue circles in Fig. 9, Panel G) and identified 2268 genes up-regulated in t(3;3)/inv(3) AML cells. Interestingly, the GATA motif is recognized by both GATA2 and EVIl/MECOM and these two TFs have target genes in common. However, promoters containing ETS-like motif is bound and regulated specifically by EVIl/MECOM, not by GATA236. Since t(3;3)/inv(3) AML cells showed elevated EVIl/MECOM expression and low GATA motif activity, it was hypothesized that genes that bind EVIl/MECOM via promoters containing the ETS-like motif, but not the GATA motif, may specifically play a role in venetoclax resistance in t(3;3)/inv(3) AML. Therefore, 2268 up-regulated genes in t(3;3)/inv(3) had EVIl/MECOM-binding ETS-like motifs in their promotors were examined by using previously published ChIPseq data identifying 2350 genes with ETS-like motifs in their promoters known to bind EVI1/MECOM36, and it was found that 135 such EVI1/MECOM- regulated genes (Fig. 9, Panel H). Among those 135 genes, an enrichment of genes related to regulation of apoptotic processes was found, including NR4A2, SOCS3 and EGR1 (Fig. 9, Panels I, J). Since t(3;3)/inv(3) AML were venetoclax-resistant but AZD5582-sensitive, these apoptosis regulators may control distinct responses associated with XIAP dependence and BCL2 dependence through overexpression of EVIl/MECOM.

To determine which other drugs can be used in patient-specific combination treatment regimen, evaluation of in vitro AML elimination is required. For such in vitro evaluation to be feasible for clinical use, determination of the efficacy must be performed in as short a time as possible at clinical presentation. Therefore, short-term time-course of drug-induced apoptosis by flow cytometry for annexin V expression following exposure to AZD5582 in both sensitive (n=9) and resistant (n=6) cases was examined (Fig. 9, Panel K). In AZD5582-sensitive cases, the frequency of 7AAD-annexin V+ apoptotic cells increased significantly at 4 hours and 6 hours post-exposure (p<0.05 and p<0.005 by paired two-tailed t-test compared with 2 hours post-exposure), reaching above 8% at 6 hours. Consistent with this finding, the frequency of 7AAD+annexin V+ dead cells increased starting at 6 hours post-exposure (p<0.05 by paired two-tailed t-test compared with 2 hours post-exposure), exceeding 20% at 24 hours post exposure (p<0.0005 by paired two-tailed t-test compared with 2 hours post-exposure). On the other hand, in cases that were relatively resistant to AZD5582, at 24 hours post-exposure, frequencies of apoptotic cells at 6 hours and dead cell fraction at 24 hours remained less than 5% and 10%, respectively. This indicates that flow cytometric annexin V quantification following 6-hour in vitro exposure can predict AZD5582 responsiveness. This substantially shortens the time from clinical presentation to determination of drug sensitivity, allowing identification of patient-specific optimized combination drug regimen in a clinically feasible timescale.

Example 5: Effective in vivo treatment choices as informed by in vitro treatment efficacy and genetic information

Through in vitro screening, an efficacy matrix of AZD5582, venetoclax, S63845, barasertib and GSK923295 against leukemia cells derived from high-risk poor-outcome AML patients was established. This suggests that optimized drug combination against highly aggressive AML can be informed by patterns of in vitro responsiveness as well as genetic events including somatic mutations, chromosome abnormalities and altered gene regulatory networks inherent to each patient. To explore this possibility, PDX models of 22 high-risk poor-outcome AML cases with various somatic mutations and karyotypic abnormalities were created for in vivo therapeutic testing. All 22 patients experienced primary refractoriness or relapse and 21 had succumbed to the disease (one patient was lost to follow-up following disease relapse). Clinical information was summarized (data not shown). Among 67 cases in which all five compounds were tested, 61 (91.0%) showed responsiveness to at least one compound, most frequently to AZD5582 (n=49), then to venetoclax (n=34); 39 cases showed responsiveness to at least two agents, most commonly to AZD5582 and venetoclax (n=22), followed by other AZD5582-based combinations (n=l l) and venetoclax-based combinations (n=7). Therefore, to determine the optimal in vivo treatment in the PDX models, in vitro sensitivity/resistance to AZD5582 and venetoclax was first focused on (Figure 10, Panel A leftmost panel), and then how the cases responded to S63845 and barasertib was assessed (Figure 10, Panel A middle and right panels). Human AML chimerism in PB over treatment time-course and in BM and spleen at the end of treatment course was assessed (Figure 10, Panels B-D, Figure 11). For cases that showed in vitro responsiveness to both AZD5582 and venetoclax, in vivo effect of each compound as single agent and in combination was assessed (Figure 10, Panel B). For case 9 (FLT3-ITD+, mutated DNMT3A, NPM1 and PPM1D, complex karyotype), complete elimination of AML cells in the BM, spleen and PB with AZD5582 alone was found. In case 1 (FLT3 WT, mutated CBL, NRAS, IDH2, TET2, DNMT3A, NPM1 and SETBP1, complex karyotype), single agent AZD5582 and venetoclax resulted in elimination of AML cells in some recipients but not in all. However, combination treatment with AZD5582 and venetoclax resulted in highly efficacious in vivo elimination. This case also showed high responsiveness to MCL inhibition. Combination treatment with AZD5582 and S63845 resulted in complete elimination of AML cells in the BM, spleen and PB. Similarly, patient 53 (FLT3-ITD+ and mutated WT1, monosomy 7, complex karyotype), patient 33 (FLT3-TKD+, mutated TET2, WT1 and SMC3A, MLL-ENL), patient 22 (FLT3 WT, mutated TP53, GATA2 and monoallelic CEBPA, chromosome 17 abnormality, trisomy 21, monosomy 7, complex karyotype) and patient 2 (mutated monoallelic CEBPA, PPM1D, trisomy 21) were highly responsive in vivo to combined XIAP and BCL2 inhibition by AZD5582 and venetoclax. Combination treatment with AZD5582 and S63845 in PDX-models of patient 47 (FLT3-ITD+, mutant DNMT3A, NPM1, TET2, PTPN11, monoallelic CEBPA, CUX1, NF1, ZASR2) and patient 34 (FLT3-TKD, mutated NF1, monosomy 7, monosomy 17, complex karyotype) led to nearly complete elimination of leukemia cells in PB, BM and spleen. Next, AZD5582-sensitive, venetocl ax-resistant cases was focused on (Figure 10, Panel B). Patient 48 (FLT3-ITD+, mutated TET2, MGA, complex karyotype) was highly sensitive to AZD5582 alone in vivo. In other cases, responsiveness to S63845 and barasertib was examined to determine a combination partner to AZD5582. For instance, patient 51 (FLT3-ITD+, mutated KMT2A, t(3;3)(q21;q26.2) and monosomy 7) and patient 4 (FLT3-WT, mutated NRAS, DNMT3A, BCOR, complex karyotype) were responsive to barasertib in vitro and combination treatment with AZD5582 and barasertib in vivo resulted in clearance of AML cells from the PB, BM and spleen in PDX models. On the other hand, combination treatment with AZD5582 and S63845 in PDX-models of patient 52 (FLT3-ITD+, mutant TP53, DNMT3A, NPM1, monoallelic CEBPA and ETV6) resulted in elimination of AML cells in vivo.

Finally, among AML cases resistant to AZD5582 (XIAP inhibition), cases that were sensitive to venetoclax (BCL2 inhibition) and S63845 (MCL1 inhibition) were found (Figure 10, Panel D). In patient 7 (mutated PTPN11, MGA, PHF), venetoclax alone was highly effective in vivo. Moreover, inhibition of two anti-apoptotic molecules was found quite effective in vivo. For instance, in patient 58 (FLT3- ITD+, mutated IDH1, NPM1, DNMT3A) which was sensitive to venetoclax and S63845 but relatively resistant to AZD5582 in vitro , in vivo venetoclax/S63845 combination was very effective. While FLT3-ITD+ cases were highly responsive to AZD5582 overall, in this particular case, mutated IDH1 modified XIAP dependence of FLT-ITD+ AML. Importantly, in four patients (patients 1, 9, 22, 53), recipients underwent a three-week course of AZD5582/venetoclax in vivo followed by a four-week observation period without treatment. In these recipients, no evidence of AML relapse in the BM, spleen and PB was found (Figure 10, Panel E). In each case, it was confirmed that both leukemia clearance and recovery of murine trilineage hematopoietic cell recovery (erythrocytes, megakaryocytes and granulocytes) following AZD5582-based combination treatment in situ by immunohistochemistry (Figure 10, Panels F, G). In addition, murine Macl+Grl- monocytes and Macl+Grl+ granulocytes by flow cytometry were identified (Figure 11, Panel B). Efficacy of in vivo FLT3-WT (n=l l) and FLT3-mutated (n=10) AML elimination using AZD5582-based combination treatments are summarized in Figure 10, Panel H.

Conclusions

Over the past decade, diversity in somatic mutations in AML patients have become clear through DNA sequencing. This genetic complexity and heterogeneity complicate targeted drug treatment strategies for AML. Drugs that target specific aberrant proteins with more favorable toxicity profiles are now viable treatment options for patients with gain-of-function mutations such as FLT3-ITD, IDHl and IDH2, especially for the elderly with comorbidities or those that have failed multiple rounds of conventional therapies. However, long-term outcome for relap sed/refractory patients continues to be poor42. Based on this background, the aim of this study was to develop effective therapeutic strategies for high risk AML, focusing on a group of patients who not only presented with adverse genetic/karyotypic prognostic factors but also went on to demonstrate poor clinical outcomes, with the majority being refractory to induction chemotherapy or experiencing relapse following chemotherapy or HSCT and ultimately succumbing to the disease.

This group of high-risk poor-outcome patients was associated with multiple somatic mutations and complex karyotypic abnormalities. To identify a strategy to eradicate AML cells in such patients, the approach was to target critical pathways and regulatory networks rather than individual abnormal proteins produced through gene mutations or chromosomal abnormalities. Despite substantial heterogeneity in chromosomal events and somatic mutations among the study population, it was found that inhibition of five interrelating pathways led to effective in vitro elimination of AML cells. As would be expected, response to inhibition of these pathways varied among cases and patient-specific profile of vulnerabilities were identified through in vitro assessment. Surprisingly, maximal vulnerability for these genetically and biologically diverse patient-derived AML cells converged to two anti-apoptotic proteins: XIAP and BCL2. Among AML cells resistant to conventional chemotherapy and/or HSCT, prevalent dependence to these proteins for survival was found, as evidenced by effective targeting of AML cells in over 90% of patients examined.

These in vitro findings were confirmed in vivo using PDX models. In vivo therapeutic modeling demonstrated that XIAP inhibition was highly effective against leukemia cells resistant to conventional chemotherapy and HSCT. In addition, 72-hour in vitro exposure assay established patient-specific profile of responsiveness to multiple drugs that informed the selection of optimal treatment strategy. In vivo combined treatment based on this patient- specific responsiveness profile resulted in complete elimination of AML cells even in the NSG PDX environment lacking human anti-tumor immune cells. Importantly, in vitro sensitivity assessment could be performed in as little as 6 hours by assessing apoptotic cell death of AML cells by flow cytometry. This allows determination of patient-specific optimized combination drug regimen in a clinically feasible timescale using methodology already widely available in clinical laboratories.

In addition, determinants of drug responsiveness in high risk AML with complex genetic characteristics was explored, with the presence of aberrations at various levels including chromosome, gene, transcriptome and protein (Table 9). In vitro responsiveness to inhibition of XIAP and BCL2 and genetic abnormalities present in 103 patients are summarized in Table 9. Dependence to XIAP correlated with the presence of: MLL-rearrangement, WT IDH1 with mutated FLT3, inv(3) and t(3 ; 3 ) in FLT3 WT, mutated CBL in FLT3 WT, mutated NRAS in FLT3/CBL WT, and preserved TP53 motif activity in TP53 mutated cases. Dependence to BCL2 correlated with the presence of: concurrent mutations in FLT3 and IDHl, and mutated TET2 in FLT3 WT. Among 94 cases sensitive to either AZD5582, venetoclax or both, correlation of genetic abnormalities and responsiveness was identified in 70 cases. Among 9 cases resistant to both agents, 6 cases were associated with both TP53 (homozygous or heterozygous) mutations and complex karyotype, and one case each with homozygous TP53/RUNX1 mutation, complex karyotype/del(7) and trisomy 8. XIAP > BCL2, XIAP dependence more frequent; BCL2 > XIAP, BCL2 dependence more frequent.

It was found that mutated FLT3, either ITD or TKD mutation, conferred high responsiveness to both AZD5582 and venetoclax. Interestingly, concurrent IDHl mutation diminished dependence on XIAP while increasing vulnerability to BCL2 inhibition in FLT3- mutated AML. In contrast, AML with mutated CBL were resistant to venetoclax and sensitive to AZD5582. XIAP inhibition was also highly effective in patients with chromosomal aberrations. For instance, clinically-aggressive MLL-rearranged AML with MLL-AF9, MLL- ENL and MLL-AF6 translocations and concurrent mutations in genes such as FLT3, IDH2 and GATA2 demonstrated potent vulnerability to XIAP inhibition in every case, extending previously reported finding in genetically-engineered mouse model of MLL-AF9 leukemia. In addition, a majority of cases with monosomy 7 showed resistance to venetoclax, suggesting that loss of one chromosome 7 reduces BCL2 dependence in these cases (data not shown).

Multiple and complex genetic events underlie treatment resistance and poor clinical outcome of the patient population. Nevertheless, a set of criteria was found that could be applied to help predict the optimal treatment for over 80% of cases: 1) status of somatic mutations in six recurrently mutated genes (FLT3, IDHl, IDH2, CBL, NRAS and TET2), 2) abnormalities involving two chromosomes (translocation and inversion involving 3q21 and translocation involving chromosome 1 lq23) and 3) TP53 motif activity in TP53 mutated cases. TP53 motif activity positively correlated with XIAP dependence not only in all TP53 mutated cases but also in TP53 mutated cases not included in criteria 1) and 2) above (Figure 12). Determination of TP53 motif activity, a measure of transcriptional regulation by TP53, requires RNA-sequencing. However, by identifying a subset of TP 53 -regulated genes with expression profile that correlate strongly with TP53 motif activity, it might be possible to obtain a surrogate measure that only requires targeted PCR for a limited number of genes, simplifying implementation. In the remaining 18 of 103 cases, highly heterogeneous combinations of somatic mutations and chromosomal abnormalities were present. Identification of genetic mechanisms for their dependence on XIAP and/or BCL2 for survival will await future study. Correlation of genetic abnormalities and dependence to XIAP or BCL2 anti-apoptotic pathways are summarized in Figure 13.

Overall, among 103 high-risk poor-outcome patients, leukemia cells from 94 (91.3%) showed high responsiveness to AZD5582 and/or venetoclax. Integrative analysis of somatic mutations, chromosomal abnormalities, gene expression and regulatory networks in diversified international patient population will await future study. Through such efforts, patient-specific, precision medicine-approach for adverse-risk poor-outcome leukemia will become possible. Understanding the biology of stem cells in leukemia has paved a path for other malignancies. Similarly, identifying vulnerabilities in highly aggressive leukemia will lead to development of effective treatment strategies in other malignancies.

Table 9: Correlation of genetic abnormalities and dependence on XIAP and BCL2

Normal chr. 3, FLT3/CBL/N RAS/TET2 WT, TP53 mutated

Example 6: Methods of Study

Human samples

All patient samples were obtained from Toranomon Hospital under written informed consent. The study was performed with authorization from the Institutional Review Board for Human Research at RIKEN and Toranomon Hospital, in accordance with the ethical standards of responsible committees on human experimentation at each institution. CB cells were obtained from Chubu Cord Blood Bank.

Mice and xenogeneic transplantation

Immune-compromised NOD/SCID/I12rgKO (NSG) mice were bred and maintained under defined flora at the animal facility of RIKEN and at The Jackson Laboratory. All experiments were performed with authorization from and according to guidelines established by the Institutional Animal Committees at RIKEN and The Jackson Laboratory. Both female and male newborn NSG mice received 1.5Gy total body irradiation followed by intravenous injection of sorted human cells within 72 hours of birth. The extent of engraftment of human cells in the NSG recipients was assessed by retro-orbital phlebotomy and flow cytometry. Treatment studies were conducted when sufficient engraftment was observed, at approximately 6 weeks of age.

Flow cytometry

The following monoclonal antibodies (mAbs) were used for flow cytometry: Mouse anti human CD19 (Catalog No. 555412, 341093), CD3 (Catalog No. 563800, 562426), CD33 (Catalog No. 562854, 555450), CD56 (Catalog No. 563169), CD4 (Catalog No. 563875), CD 8 (Catalog No. 348793), CD34 (Catalog No. 348791, 555822), CD38 (Catalog No. 340439), CD45RA (Catalog No. 555488), and CD45 (Catalog No. 563204, 555482, 555485, 563879); Rat anti-mouse Terl 19 (Catalog No. 557915), Macl (Catalog No. 561039), Gr-1 (Catalog No. 560453), and CD45 (Catalog No. 563410, 557659); 7-AAD (Catalog No. 559925) (BD Biosciences); Annexin V (Catalog No. 556422) (BD Biosciences) (Catalog No. 640941) (BioLegend); Trucount Tubes (Catalog No. 340334) (BD Biosciences). Analyses were performed with FACSArialll and FACSCantoII (BD). For xenogeneic transplantation, cells were labeled with BV510-conjugated anti-CD45 mAb, BV786- conjugated anti-CD3 mAb, PE-Cy7-conjugated anti-CD19 mAb, BV421 -conjugated anti-CD33 mAb, BV711 -conjugated anti-CD56 mAb, BV650-conjugated anti- CD4 mAb, APC-Cy7-conjugated anti-CD8 mAb, PE-conjugated anti-CD34 mAb, APC- conjugated anti-CD38 mAb, and FITC- conjugated anti-CD45RA mAb and isolated by sorting using FACSArialll (BD).

RNAseq and bioinformatic analyses

RNA extraction and RNAseq analysis were performed as described previously46. Briefly, RNA was extracted using TRIzol reagent (Catalog No. 15596018; Invitrogen) from original patient samples or from human CD45+ cells isolated from bone marrow cells of recipient mice. NEB Next Ultra RNA Fibrary Prep Kit for Illumina (Catalog No. E7530; New England Biolabs) was used for RNA library preparation. Final library size distribution was validated using Bioanalyzer (Agilent) and quantified using quantitative PCR. The DNA libraries were hybridized to a flow cell, amplified on the Illumina cBot, and subsequently run on the Hiseq 2500 (Illumina, using 50-base single-end read mode). The sequence reads were mapped to the human genome (NCBI version 19) using TopHat2 version 2.0.8 and botwie2 version 2.1.0 with default parameters, and gene annotation was provided by NCBI RefSeq. The transcript abundances were estimated using Cufflinks (version 2.1.1). DEseq247 was used for differential gene expression analysis. A required Benjamini-Hochberg adjusted p- value less than 0.05 and absolute log2 fold-change greater than 1.0 to identify differentially expressed genes. The programming framework R version 3.5.3 was used to call DEGs. Gene Ontology term enrichment analysis was performed using GOrilla25,26 and REViG027. To analyze DEGs regulated by EVIl/MECOM, EVIl -bound sites were obtained from previously published ChIPseq36. Hgl8 mapping data was converted into GCRh37 mapping using NCBI Genome Remapping Service (https://www.ncbi.nlm.nih.gov/genome/tools/remap/).

Target-genome sequencing

Target-genome sequencing was performed on DNA extracted from original patient samples or human CD45+ cells isolated from PDX-model BM. DNA extraction was conducted using DNeasy Blood and Tissue kit (Qiagen). Using the DNA, all coding regions and 2bp flanking intronic sequences of the 41 established genes related with AML were analyzed. A total length of the target region was 127,151 bp. A two-step PCR method was used to construct DNA libraries according to previously-published methods. After purification and quantification, pooled libraries were sequenced by 2x150-bp paired-end reads on HiSeq 2500 (Illumina). ClinVar50 was also used to discriminate pathogenic mutations from likely-benign mutations.

Compounds

The compounds used in this study are listed (table not shown).

In vitro chemical screening

For large scale chemical screening using 35 compounds, 8-10x104 patient-derived leukemia cells per well were seeded in Stemline II Hematopoietic Stem Cell Expansion Medium (SIGMA) supplemented with stem cell factor (50ng/ml), FLT3 ligand (50ng/ml), thrombopoietin (50ng/ml), IL-2 (5ng/ml), IL-3 (20ng/ml), IL-7 (20ng/ml) and IL-15 (lOng/ml) using 96-well plates. For in vitro treatment experiments using 5 compounds, 1x105 cells were plated in the same medium. Cells were exposed to small molecules at indicated concentrations for 72 hours at 37°C in humidified atmosphere containing 5% C02. Cells were harvested and stained with BV421- labeled anti-hCD45, and 7AAD, collected in BD TruCountTM Tubes (Catalog No. 340334, BD) and analyzed using FACSCanto II (BD).

In vivo treatment

In vivo treatment experiments were performed with AML-engrafted NSG recipients using compounds listed (table not shown). The recipients were treated with AZD5582 (SIGMA) (30 mg/kg) intraperitoneally once a day, venetoclax (ABT-199, Active Biochem) (70 mg/kg) orally once a day, S-63845 (Activebiochem), barasertib (Selleck), GSK293295 (Selleck), or quizartinib (Selleck). For combination treatment, doses of AZD5582 and venetoclax were halved. The mice were euthanized when they became moribund or after 4-6 weeks of treatment. Human AML chimerism in BM, spleen, and PB was determined using flow cytometry. All treated recipients and their pre- and post- treatment engraftment data are tabulated (table not shown).

Imm unohistochem istry

Tissue sections (3 pm) were cut from 4% paraformaldehyde (PFA)-fixed paraffin- embedded recipient organs. Sections were deparaffinized using xylene and ethanol and antigen retrieval was performed (Retrievagen A (pH 6.0), BD PharmingenTM). Non-specific background was reduced by incubating the slides in methanol + H202 (Wako). After blocking with horse serum, slides were incubated with mouse anti-human CD45 antibody (DAKO, M0701) (1:150) then HRP-conjugated horse anti- rabbit/mouse IgG antibody (ImmPRESSTM, Cat. MP-7500). Slides were then stained with 3,3’- Diaminobenzine (DAB) or Hematoxylin/Eosin, dehydrated, mounted using Vectamount (Vector Laboratories) and analyzed with Axi overt 200 microscope (Zeiss). Photos were taken using AxioCam MRc 5 (Zeiss) using the AxioVision rel. 4.6 software.

Statistical analysis

In comparison of gene expression levels between two groups, difference was tested by Student’s t-test. Impact of DNA mutations and chromosomal aberration were evaluated by Fisher’s exact test. For in vivo treatment experiments, difference in the percentage of hCD45+ cells in the BM and spleen between the treatment groups was analyzed using a two-tailed t-test. P-value less than 0.05 was considered significant. Data are given as means ± SEM or a median ± SEM as described in figure legends respectively. All analyses were performed by GraphPad Prism version 8.3.1 (GraphPad Software).

Data availability

All RNA sequencing datasets produced in this study are deposited in the National Bioscience Database. Accession number will be available by the time of publication. Source data for targeted DNA-seq analysis are provided with this paper. Any other relevant data are available from the corresponding author upon reasonable request.

Example 7: In vitro efficacy of inhibitor compounds against ALL, MPAL, and CML patient samples

In vitro efficacy of inhibitor compounds was tested against ALL, MPAL, and CML patient samples. T-ALL were more sensitive to venetoclax rather than AZD5582, while the majority of CML cells were killed by AZD5582 efficiently rather than venetoclax (Figure 16).

Example 8: In vivo efficacy of dexamethasone, AZD5582, and venetoclax in Ph(-) and Ph(+) leukemia In vivo effect of dexamethasone, AZD5582, and venetoclax in combination was assessed for cases in Example 7 that showed in vitro responsiveness to both AZD5582 and venetoclax, (Figures 14 and 15). Combination treatment with AZD5582 and venetoclax resulted in highly efficacious in vivo elimination. Reproducible in vivo efficacy was observed for Ph(-) B-ALL and T-ALL. For Ph(+) B-AFF, better efficacy was observed using AZD5582 and venetoclax in ABF inhibitors and steroid.

Incorporation by Reference

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

What is claimed:

1. A method of treating a subject with acute myeloid leukemia (AML), comprising:

(a) determining whether an AML cell of the subject is characterized by the presence or absence of one or more genetic mutations in:

(l) FLT3;

(n) IDH1;

(iii) CBL; and/or

(iv) NRAS; and

(b) if one or more of the genetic mutations are present, individually or conjointly administering an effective amount of a XIAP inhibitor, BCL2 inhibitor, MCL1 inhibitor, or aurora kinase B inhibitor to the subject.

2. The method of claim 1, wherein a genetic mutation is identified in FLT3.

3. The method of claim 2, wherein the FLT3 genetic mutation is a FLT3-ITD+ mutation.

4. The method of claim 3, wherein no mutation is identified in IDH1.

5. The method of claim 4, wherein the AML cells further comprise complex karyotype and/or monosomy 7.

6. The method of claim 4 or 5, wherein a genetic mutation is identified in DNMT3A,

NPM1, and/or TET2.

7. The method of any one of claims 4-6, wherein a genetic mutation is identified in PPM1D, MGA, and/or WT1.

8. The method of any one of claims 4-7, wherein the method comprises administering the XIAP inhibitor individually or conjointly with the BCL2 inhibitor.

9. The method of any one of claims 4-6 or, wherein a genetic mutation is identified in PTPN11, CEBPA, CUX1, NF1, ZASR2, TP53, and/or ETV6.

10. The method of any one of claims 4-6 or 9, wherein the method comprises administering the XIAP inhibitor individually or conjointly with the MCL1 inhibitor.

11. The method of claim 3, wherein a mutation is identified in IDH1.

12. The method of claim 11, wherein a genetic mutation is identified in DNMT3A and/or NPM1.

13. The method of claim 11 or 12, wherein the method comprises conjointly administering the BCL2 inhibitor and the MCL1 inhibitor.

14. The method of claim 2, wherein the FLT3 genetic mutation is a FLT3-TKD+ mutation.

15. The method of claim 14, wherein the AML cells further comprise complex karyotype, monosomy 7, and/or monosomy 17.

16. The method of claim 14 or 15, wherein a genetic mutation is identified in TET2, WT1, SMC3A, MLL-ENL, and/or NF1.

17. The method of any one of claims 14-16, wherein the method comprises administering the XIAP inhibitor individually or conjointly with the BCL2 inhibitor or the MCL1 inhibitor.

18. The method of claim 1, wherein no mutation is identified in FLT3.

19. The method of claim 18, wherein the AML cells further comprise complex karyotype, chromosome 17 abnormality, trisomy 21, and/or monosomy 7.

20. The method of claim 18 or 19, wherein a genetic mutation is identified in PTPN11,

MGA, and/or PHF.

21. The method of claim 20, wherein the method comprises administering the BCL2 inhibitor.

22. The method of claim 18 or 19, wherein a genetic mutation is identified in CEBPA, PPM1D, and/or GATA2.

23. The method of claim 22, wherein the method comprises administering BCL2 inhibitor conjointly with the XIAP inhibitor.

24. The method of claim 18 or 19, wherein a genetic mutation is identified in CBL.

25. The method of claim 24, wherein a genetic mutation is identified in IDH2, TET2,

DNMT3A, NPM1, and/or SETBP1.

26. The method of claim 24 or25, wherein the method comprises administering the XIAP inhibitor conjointly with the MCL1 inhibitor or the BCL2 inhibitor.

27. The method of claim 18 or 19, wherein a genetic mutation is identified in NRAS.

28. The method of claim 27, wherein the AML cells further comprise complex karyotype.

29. The method of claim 27 or 28, wherein a genetic mutation is identified in DNMT3A and/or BCOR.

30. The method of any one of claims 27-29, wherein the method comprises administering the XIAP inhibitor conjointly with the aurora kinase B inhibitor.

31. The method of any one of claims 1 -30, wherein the XIAP inhibitor is AZD5582.

32. The method of any one of claims 1-30, wherein the BCL2 inhibitor is venetoclax.

33. The method of any one of claims 1-30, wherein the MCL1 inhibitor is S63845.

34. The method of any one of claims 1-30, wherein the aurora kinase B inhibitor is barasertib.

35. A method of identifying a subject-specific combination drug regimen, comprising:

(a) collecting AML cells from the subject;

(b) measuring a level of annexin V expression in the AML cells following exposure to a combination of two inhibitors, selected from the group of:

(l) XIAP inhibitor;

(ii) BCL2 inhibitor; (iii) MCL1 inhibitor; or

(iv) aurora kinase B inhibitor; and

(c) determining whether the level of annexin V expression is increased in AML cells exposed to the two inhibitors compared to a level of annexin V expression in AML cells not exposed to the two inhibitors or AML cells exposed to only one of the two inhibitors.

36. The method of claim 35, wherein the XIAP inhibitor is AZD5582.

37. The method of claim 35, wherein the BCL2 inhibitor is venetoclax.

38. The method of claim 35, wherein the MCL1 inhibitor is S63845.

39. The method of claim 35, wherein the aurora kinase B inhibitor is barasertib.