Biomarkers and methods for treatment with NAE inhibitors

Abstract

Disclosed herein are markers whose mutational status is associated with sensitivity to treatment with NAE inhibitors. Mutational status is determined by measurement of characteristics of markers corresponding to the marker genes. Compositions and methods are provided to assess markers of marker genes to predict response to NAE inhibition treatment.

Description

Attorney Docket No.223266-549182 / PAT27156PCT01 BIOMARKERS AND METHODS FOR TREATMENT WITH NAE INHIBITORS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the priority benefit under 35 U.S.C. §l.19(e) of U.S. Provisional Application No.63 / 599,169 filed on November 15, 2023, and U.S. Provisional Application No.63 / 508,842, filed on June 16, 2023, the contents of the aforementioned applications are herein incorporated by reference in their entireties. SEQUENCE LISTING

[0002] This application contains a Sequence Listing which is submitted herewith in electronically readable format. The Sequence Listing file was created on June 16, 2023. The entire contents of the Sequence Listing in the 223266-518989_Sequence-Listing.xml (23,004 bytes) file are incorporated herein by reference. BACKGROUND

[0003] Cells become cancerous when their genotype or phenotype alters in a way that there is uncontrolled growth that is not subject to the confines of the normal tissue environment. Genetic alterations may be seen when one or more genes is mutated, amplified, deleted, overexpressed or underexpressed. These alterations may result in changes to the protein corresponding to the gene. Additionally, chromosome portions can be lost or moved from one location to another. Some cancers have characteristic patterns by which genotypes or phenotypes are altered. Genetic alterations can facilitate tumor progression, tumor growth rate or whether a tumor will metastasize. Some genetic alterations can affect whether a tumor cell will respond to therapy.

[0004] Hematological cancers of the hematopoietic and lymphoid tissues have a variety of treatment options, such as chemotherapy, radiation therapy, immunotherapy and stem cell transplantation. Depending on the origin, location or severity of the cancer, the chemotherapy might comprise treatment with antimetabolites, antimitotics, alkylating agents, histone deacetylase inhibitors, hypomethylating agents, proteasome inhibitors, kinase inhibitors, immunomodulators, and / or other agents, such as new agents acting in recently studied pathways.

[0005] One set of recently studied pathways relates to E1 enzyme activity. Ubiquitin and other ubiquitin-like molecules (ubls) are activated by a specific enzyme (an E1 enzyme, an ATP-dependent activating enzyme) which catalyzes the formation of an acyl-adenylate intermediate with the C-terminal glycine of the ubl. The activated ubl is then transferred to a - 1 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 catalytic cysteine residue within the E1 enzyme through formation of a thioester bond intermediate. After additional enzymatic steps, the ubl is then conjugated to the target protein, through isopeptide bond formation with the amino group of a lysine side chain in the target protein. Inhibiting E1 enzymatic activity means reducing the ability of an E1 enzyme to activate ubiquitin like (ubl) conjugation to a substrate peptide or protein (e.g., ubiquitination, neddylation, sumoylation). The ubl named Neural precursor cell-Expressed Developmentally Downregulated 8 (NEDD8) is activated by the heterodimer NEDD8-activating enzyme (NAE, also known as APPBP1-UBA3, UBE1C (ubiquitin-activating enzyme E1C)) and is transferred to one of two E2 conjugating enzymes (ubiquitin carrier protein 12 (UBC12) and UBC17), ultimately resulting in ligation of NEDD8 to cullin proteins by the cullin-RING subtype of ubiquitin ligases. A function of neddylation is the activation of cullin-based ubiquitin ligases involved in the turnover of many cell cycle and cell signaling proteins, including p27 and I- ^B. See Pan et al., Oncogene 23:1985-97 (2004). Inhibition of NAE can disrupt cullin-RING ligase- mediated protein turnover and can lead to apoptotic death in cells, e.g., tumor cells or cells of a pathogenic organism, e.g. a parasite. See Soucy et al., (2010) Genes & Cancer 1:708-716.

[0006] Some patients being treated with chemotherapeutic agents respond to one agent better than another. Valuable time in a patient’s treatment program can be lost pursuing therapy with an agent which eventually is proven ineffective for that patient or leads to treatment resistance of the cancer. As cancer is a progressive, deadly disease, many patients cannot afford the time for trial-and-error choices of therapeutic regimens. Expedient and accurate treatment based on analytical results for patients likely to have favorable therapeutic outcomes lead to effective management of the cancer. SUMMARY

[0007] The present disclosure relates to methods and kits for treatment of cancer, e.g., a hematological cancer in patients, e.g., human patients, who are characterized by marker analysis for favorable outcome of treatment. Marker analysis can include measurement of the amount, presence or changes of markers provided herein. The markers are predictive of whether there will be an improved outcome (e.g., improvement in good response, long time-to- progression, negative minimal residual disease, long progression-free survival and / or long term survival) after treatment with a regimen comprising a NEDD8-activating enzyme (NAE) inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, compared with similar treatment lacking an NAE inhibitor. Other benefits may include reduced side effects, a shorter course of treatment, or other benefits related to patient quality of life that may not - 2 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 directly reflect benefits in disease outcome. A treatment regimen comprising pevonedistat treatment can further comprise treatment with one or more additional agent(s), such as a BCL2 inhibitor, such as venetoclax (ABT-199); and / or a hypomethylating agent, e.g., azacitidine or a pharmaceutically acceptable salt thereof. A treatment regimen that incorporates administration to a hematological cancer patient with a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199) and a hypomethylating agent to the patient, reduces one or more symptoms of the hematological cancer, or delays or terminates the progression of the hematological cancer. Testing a biological sample, e.g., a sample obtained from a patient, e.g., a sample comprising tumor cells or contents or products thereof, e.g., in vitro, to detect or measure the presence, amounts or changes of genetic or phenotypic markers, e.g., the mutational status of at least one marker gene, identifies particular patients whose outcomes are expected to benefit from including an NAE inhibitor in their treatment regimen. More specifically, among a group of patients who are expected to benefit from a particular treatment, e.g., treatment with venetoclax (ABT-199) and a hypomethylating agent such as azacitidine or a pharmaceutically acceptable salt thereof, the test results identify those patients who are expected to gain additional benefit from including an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, in the treatment. Conversely, the test results also identify those patients who are not expected to gain an additional benefit from including an NAE inhibitor in the treatment.

[0008] In one aspect, the invention provides compositions, such as kits, or methods useful in detecting or measuring characteristics, e.g., amounts, presence or changes, of markers in a biological sample, e.g., a sample obtained from a cancer patient, e.g., a human patient. Such compositions and methods can determine the mutational status of marker genes described herein.

[0009] In another aspect, the invention provides disease, e.g., cancer, management strategies. In the foregoing aspects, the characteristic, e.g., size, sequence, composition or amount of marker, e.g., nucleic acid or protein, in a biological sample comprising tumor cells, e.g., hematological tumor cells, or contents or products thereof is measured. In one embodiment, the cancer is a hematological cancer, such as leukemia, lymphoma or myeloma. In some embodiments, the hematological cancer is acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) or chronic myelomonocytic leukemia (CMML). In some embodiments, the MDS is high risk MDS. In some embodiments, the AML is low-blast acute AML.

[0010] In various embodiments, the marker characteristic, e.g., size, sequence, composition or amount of DNA, the size, sequence, composition or amount of RNA and / or the size, sequence, composition or amount of protein corresponding to a marker gene, either wild - 3 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 type or with one or more genotypic or phenotypic alteration, e.g., a polymorphism or mutation, e.g., somatic mutation, described herein is measured. In some embodiments, the measurement indicates that a gene is underexpressed or overexpressed. In some embodiments, the measurement indicates that a gene is amplified, deleted or translocated. In some embodiments, the measurement indicates that the gene is misexpressed or the altered gene product alters a signaling or functional pathway contributing to the cancer state. Disease management strategy is undertaken when assay results reveal information about a marker gene or marker genes, e.g., whether a gene is altered, or not, the identity of the alteration, and / or whether the RNA or protein amount of an altered marker gene or marker genes indicates favorable outcome to therapy comprising NAE inhibition, e.g., pevonedistat therapy. By examining the marker characteristic, e.g., size, sequence, composition or amount of one or more of the identified markers, it is therefore possible to eliminate ineffective or inappropriate therapeutic agents or regimens. Thus, one can undertake a therapeutic regimen which is likely to benefit a particular patient or type of patient, e.g., whether a particular regimen should be started or avoided, continued, discontinued or altered. Such analyses can be made on a patient-by-patient basis, e.g., identifying and / or selecting for treatment a cancer patient who is expected to demonstrate a favorable outcome upon administration of a therapeutic regimen, e.g., a therapeutic regimen comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof.

[0011] A marker gene useful for analysis in methods or kits described herein is a cancer marker gene, e.g., a marker gene selected from the group consisting of isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2). A result with mutational status, such as wild type status or alterations in DNA or effects, e.g., on marker RNA and / or protein characteristics, e.g., amounts, size, sequence or composition, provides a selection, treatment or disease management of cancer patients. In some embodiments, a gene or a mutant or genetically altered form thereof is useful, has a DNA, an RNA and / or protein characteristic, e.g., size, sequence, composition or amount, e.g., in a biological sample comprising tumor cells, e.g., hematological tumor cells, or contents or products thereof, if it is different than a normal DNA, RNA and / or protein. Described herein are examples of modifications of these genes, referred to as “marker genes” whose analysis can provide such results.

[0012] In some embodiments therapeutic methods include the step of beginning, continuing, or commencing a therapy accordingly where the presence of an alteration in a marker gene or the characteristic, e.g., size, sequence, composition or amount of a patient’s marker or markers, e.g., in a first and / or subsequent biological sample, e.g., a sample obtained from the patient, e.g., comprising tumor cells, e.g., hematological tumor cells, or contents or products - 4 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 thereof, indicates that the patient is expected to have a favorable outcome with the therapy, e.g., the NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapeutic regimen. In addition, the methods include therapeutic methods which further include the step of stopping, discontinuing, altering or halting a therapy accordingly where the presence of an alteration in a marker gene or the characteristic, e.g., size, sequence, composition or amount of a patient’s marker, e.g., in a second sample obtained from the patient, indicates that the patient is expected to demonstrate an unfavorable outcome with the treatment, e.g., with the NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof regimen, e.g., as compared to a patient identified as being expected to have a favorable outcome receiving the same therapeutic regimen. In another aspect, methods are provided for analysis and treatment of a patient not yet being treated with a therapy, e.g., an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapy and identification and report of predicted outcome of the treatment based upon the presence of an alteration in a marker gene or characteristic, e.g., size, sequence, composition or amount of one or more of a patient’s marker described herein. Such methods can include not being treated with the therapy, being treated with the therapy, being treated with the therapy in combination with one or more additional therapies, or being treated with a more aggressive dosing and / or administration regimen, e.g., a more frequent dosing regimen or higher dose with the NAE inhibitor therapy, as compared to the dosing and / or administration regimen of a patient identified as having a favorable outcome to standard treatment with an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof, optionally in combination with one or more additional therapies. Thus, the provided methods of the invention can eliminate ineffective or inappropriate use of therapy, e.g., NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof therapy regimens.

[0013] Additional methods include methods to identify new therapeutic combinations. Such methods include methods to identify an agent, or more than one agent, as useful in combination with an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, for treating a cancer, e.g., a hematological cancer (e.g., myeloma, leukemias, lymphoma, etc.), based on its ability to affect the presence of a genotypic or phenotypic alteration in a marker gene or characteristic, e.g., size, sequence, composition or amount of a marker or markers of the invention. For example, an agent or agents which in combination with an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, decreases or increases the presence of an alteration in a marker gene or characteristic, e.g., size, sequence, composition or amount of a marker or markers provided herein in a manner that indicates favorable outcome of a - 5 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 patient having a cancer, e.g., a hematological cancer would be a candidate agent for the combination. Alternatively, an agent or agents which in combination with an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, is able to decrease the viability of a tumor cell comprising a marker indicative of a favorable outcome would be a candidate agent for the combination.

[0014] The present invention is also directed to methods of treating a cancer patient, e.g., a human patient, with a therapeutic regimen, e.g., with an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapy regimen (e.g., alone, or in combination with one or more additional agent(s) such as a chemotherapeutic agent, e.g., a proteasome inhibitor, an alkylating agent, a BCL2 inhibitor, a hypomethylating agent, an antibiotic or an antimetabolite), which includes the step of selecting for treatment a patient whose marker characteristic, e.g., size, sequence, composition or amount, indicates that the patient is expected to have a favorable outcome with the therapeutic regimen, and treating the patient with the therapy, e.g., NAE inhibition, by an inhibitor such as pevonedistat or a pharmaceutically acceptable salt thereof therapy, with or without one or more additional agent(s). In some embodiments, the method can include the step of administering therapy to a patient whose cancer is characterized by marker characteristic, e.g., size, sequence, composition or amount or amounts indicative of a favorable outcome. In some embodiments a favorable outcome in the methods includes the use of pevonedistat or a pharmaceutically acceptable salt thereof, in combination with one or more additional agent(s), for treating a patient characterized as having tumor cells, e.g., hematological tumor cells, wherein IDH1 gene and / or IDH2 gene has a genetic alteration. In some embodiments a favorable outcome in the methods include the use of pevonedistat or a pharmaceutically acceptable salt thereof, in combination with one or more additional agent(s), for treating a patient characterized as having tumor cells, e.g., hematological tumor cells, wherein has a genetic alteration. In some embodiments, a favorable outcome in the methods further includes the use of a BCL2 inhibitor, e.g. venetoclax (ABT-199) and / or a hypomethylating agent, e.g. azacitidine.

[0015] The entire contents of all publications, patent applications, patents and other references mentioned herein are incorporated by reference.

[0016] Other features and advantages of the invention will be apparent from the following detailed description, drawings and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1. A bar graph is depicted representing the statistical significance of response rate (CR / CRi) of PEV+VEN+AZA versus VEN+AZA treatment in AML patients with - 6 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 IDH1 mutated, IDH1 / IDH2 mutated, IDH2 mutated, and wild-type IDH1 / 2 genes , in clinical trial NCT04266795.

[0018] Figure 2A. Kaplan-Meier curves for patients with EFS data, showing the statistical significance between PEV+VEN+AZA versus VEN+AZA in patients with mutated IDH1 or IDH2 in accordance with clinical trial NCT04266795.

[0019] Figure 2B Kaplan-Meier curves for patients with EFS data, showing no statistical significance between PEV+VEN+AZA versus VEN+AZA in patients with no mutated IDH1 nor IDH2 in accordance with clinical trial NCT04266795.

[0020] Figure 3 represents a bar chart illustrating 11 genes, from subjects who were response evaluable and had at least one mutation during the phase II study, that were considered for further analysis due to prevalence or interest. DETAILED DESCRIPTION

[0021] One of the continued problems with therapy in cancer patients is individual differences in response to therapies. While advances in development of successful cancer therapies progress, only a subset of patients respond to any particular therapy. With the narrow therapeutic index and the toxic potential of many available cancer therapies, such differential responses potentially contribute to patients undergoing unnecessary, ineffective and even potentially harmful therapy regimens. If a designed therapy could be targeted to treat individual patients, such situations could be reduced or even eliminated. Furthermore, targeted designed therapy may provide more focused, successful patient therapy overall. Accordingly, there is a need to identify, select and treat particular cancer patients, e.g., hematological cancer patients, who are expected to have a favorable outcome when administered particular cancer therapies as well as particular cancer patients who may have a favorable outcome using more aggressive and / or alternative cancer therapies, e.g., alternative to previous cancer therapies administered to the patient. It would also be beneficial to provide for the diagnosis, staging, prognosis, and monitoring of cancer patients, including, e.g., hematological cancer patients (e.g., patients suffering from leukemia, lymphoma, or myeloma) who would benefit from particular cancer treatment therapies as well as those who would benefit from a more aggressive and / or alternative cancer treatment therapy, e.g., alternative to a cancer therapy or therapies the patient has received, thus resulting in appropriate preventative and / or therapeutic measures.

[0022] The present invention is based, in part, on the mutational status of a marker gene in samples from cancer patients, e.g. hematological cancer patients, who had a favorable outcome of treatment with a regimen comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, in combination with one or more additional agent(s). In - 7 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 some embodiments, the regimen comprising an NAE inhibitor further comprises a hypomethylating agent, e.g., azacitidine in combination with pevonedistat or a pharmaceutically acceptable salt thereof. In some embodiments, the regimen comprising an NAE inhibitor further comprises a BCL2 inhibitor, e.g., venetoclax (ABT-199), and / or a hypomethylating agent, e.g., azacitidine, in combination with pevonedistat or a pharmaceutically acceptable salt thereof. In some embodiments, the marker gene, e.g., the hematological cancer marker gene, encodes a metabolic enzyme, e.g., is a gene whose encoded protein is an enzyme involved in a cellular metabolic pathway. Examples of cancer marker genes include IDH1 and IDH2. Other examples of marker genes are described in WO 2019 / 109016 (e.g., TET2, RUNX1, NRAS, KRAS, DNMT3A, TP53, IDH2, EZH2, IDH1, NPM1, and PHF6), which is incorporated by reference.

[0023] Such marker genes can be found with genetic alterations, e.g., mutations or truncations, in hematological cancers, such as AML, MDS, or CMML. In some embodiments, at least one marker gene, e.g., a hematological cancer marker gene, selected from the group consisting of IDH1 and IDH2 has a genetic alteration. Marker genes can be wild type or exhibit genotypic or phenotypic alteration, e.g., a copy number change, a polymorphism or mutation, e.g., somatic mutation, whose presence can affect expression or activity of the encoded gene product. In some embodiments, there can be more than one alteration in a marker gene or more than one marker gene with an alteration in a patient’s biological sample, such as a tumor cell, tumor or product, e.g., secretion, from a tumor cell. In additional embodiments, there can be marker gene alterations in cells which have alterations in additional genes, including alterations that can lead to tumorigenesis, but the additional altered genes may not be marker genes as considered herein. In some embodiments, the alteration is an inactivating mutation. In some embodiments, the alteration is an activating mutation. In other embodiments, the alteration affects the expression of the marker gene. In other embodiments, a genotypic alteration can result in an altered interaction of the encoded gene product with a cellular binding partner.

[0024] In some embodiments, alterations in a marker gene indicates a favorable outcome to treatment with a regimen comprising pevonedistat or a pharmaceutically acceptable salt thereof. In one marker gene alteration embodiment, IDH1 is altered in a sample from a patient, e.g., a human patient. For example, a patient with an alteration in IDH1 may respond more favorably to a therapeutic regimen comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof, in combination with a BCL2 inhibitor, such as venetoclax (ABT-199), and / or a hypomethylating agent, e.g. azacitidine.

[0025] In some embodiments, alterations in a marker gene indicates a favorable outcome to treatment with a regimen comprising pevonedistat or a pharmaceutically acceptable - 8 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 salt thereof. In one marker gene alteration embodiment, IDH2 is altered in a sample from a patient, e.g., a human patient. For example, a patient with an alteration in IDH2 may respond more favorably to a therapeutic regimen comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof, in combination with a BCL2 inhibitor, such as venetoclax (ABT-199), and / or a hypomethylating agent, e.g. azacitidine.

[0026] In some embodiments, alterations in a combination of marker genes indicates a favorable outcome to treatment with a regimen comprising pevonedistat or a pharmaceutically acceptable salt thereof. In one marker gene combination embodiment, both IDH1 and IDH2 are altered in a sample from a patient, e.g., a human patient. For example, a patient with an alteration in both IDH1 and IDH2 may respond more favorably to a therapeutic regimen comprising pevonedistat or a pharmaceutically acceptable salt, such as pevonedistat or a pharmaceutically acceptable salt thereof, in combination with a BCL2 inhibitor, such as venetoclax (ABT-199), and / or a hypomethylating agent, e.g. azacitidine.

[0027] In other embodiments, a favorable outcome to treatment with a regimen comprising pevonedistat or a pharmaceutically acceptable salt thereof in combination with one or more additional agent(s) is indicated from an altered marker gene combination where IDH1 is altered and IDH2 is wild type.

[0028] In other embodiments, a favorable outcome to treatment with a regimen comprising pevonedistat or a pharmaceutically acceptable salt thereof in combination with one or more additional agent(s) is indicated from an altered marker gene combination where IDH2 is altered and IDH1 is wild type.

[0029] The mutational status result, e.g., identification, detection and / or measurement of the wild type gene or a mutation in a marker gene can be used to determine whether a favorable outcome can be expected by treatment of a hematological cancer, e.g., with a regimen comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, or whether an alternative therapy to and / or a more aggressive therapy with, e.g., an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof may enhance expected survival time. For example, the compositions and methods provided herein can be used to determine whether a patient is expected to have a favorable outcome to treatment with an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapeutic agent or to an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof dosing or administration regimen. In general, mutation in marker genes described herein is associated with sensitivity to or favorable outcome of treatment with a regimen comprising an NAE inhibitor. Examples of marker genes which can function as a cancer marker gene, e.g. a - 9 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 hematological cancer gene, and whose mutational status is associated with sensitivity to NAE inhibition include IDH1 and IDH2.

[0030] Described herein are, without limitation: 1) methods and compositions for determining whether a treatment regimen comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof will or will not be effective to achieve a favorable outcome and / or manage the cancer; 2) methods and compositions for monitoring the effectiveness of an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapy (alone or in a combination of agents) and dosing and administrations used for the treatment of cancer e.g., a hematological cancer; 3) methods and compositions for treatment of cancer e.g., a hematological cancer comprising administering an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof; 4) methods and compositions for identifying specific combinations of therapeutic agents as well as dosing and administration regimens that are effective for the treatment of cancer, e.g., a hematological cancer in specific patients; and 5) methods and compositions for treatment of cancer, e.g., hematological management strategies.

[0031] As used herein, the term “NAE inhibitor” refers to an inhibitor of the NEDD8-activating enzyme (NAE) heterodimer. Examples of NAE inhibitors include pevonedistat (formerly known as MLN4924 and TAK-924) or a pharmaceutically acceptable salt thereof. Langston S. et al. U.S. Patent no.8,207,177, whose PCT application was published as WO 2007 / 092213, WO 2006 / 084281 and WO 2008 / 019124 (the entire contents of which are hereby incorporated by reference), disclose compounds which are effective inhibitors of NAE. In some embodiments, NAE inhibitors do not inhibit, or are very poor at inhibiting, other (non- NAE) E1 enzymes. The compounds are useful for inhibiting NAE activity in vitro and in vivo and are useful for the treatment of disorders of cell proliferation, e.g., cancer, and other disorders associated with NAE activity, such as pathogenic infections and neurodegenerative disorders. One class of compounds described in Langston et al. are 4–substituted ((1S, 2S, 4R)-2-hydroxy- 4-{7H-pyrrolo[2,3-d]pyrimidin-7-yl}cyclopentyl)methyl sulfamates.

[0032] Pevonedistat (((1S,2S,4R)-4-{4-(1S)-2,3-dihydro-1H-inden-1-ylamino-7H- pyrrolo2,3-dpyrimidin-7-yl}-2-hydroxycyclopentyl)methyl sulfamate) is an NAE-specific E1 inhibitor which disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumor cells by perturbation of cellular protein homeostasis (Soucy et al. (2009) Nature 458:732-736). The evaluation of pevonedistat (MLN4924) in cellular and tumor xenograft studies has revealed two distinct mechanisms of action. The first is the induction of DNA re- replication, DNA damage and cell death through MLN4924-mediated dysregulation of the - 10 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 CRL1SKP2and CRL4DDB1substrate Cdt-1 (Milhollen et al. (2011) Cancer Res.71:3042-3051). It has been shown that p53 status does not impact the induction of DNA re-replication but may make cells more prone to undergo apoptosis or senescence depending on the appropriate genetic background (Milhollen et al. (2011) supra, Lin et al. (2010) Nature 464:374-379 and Lin et al. (2010) Cancer Res.70:10310-20). The second mechanism is the inhibition of NF-κB pathway activity in NF-κB dependent Diffuse Large B-Cell Lymphomas primarily through dysregulation of CRL1βTRCPmediated turnover of phosphorylated IκBα (Milhollen et al. (2010) Blood 116:1515-1523). In addition, pre-clinical models of Acute Myelogenous Leukemia (AML) are sensitive to pevonedistat inhibition in both cell lines and primary patient blasts through mechanisms related to Cdt-1 dysregulation, NF-κB inhibition and induction of reactive oxygen species (Swords et al. (2010) Blood 115:3796-3800).

[0033] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In one embodiment, the pharmaceutically acceptable salt is a hydrochloride salt form. - 11 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0034] As used herein, the term “hypomethylating agent” refers to compounds which inhibit DNA methyltransferase. Examples of hypomethylating agents include azacitidine (also known as azacytidine) and decitabine.

[0035] As used herein, the term “BCL2 inhibitor” refers to compounds which inhibit BCL2. Examples of BCL2 inhibitors include venetoclax (ABT-199) and navitoclax.

[0036] As used herein, “combination” therapy refers to simultaneous, separate or sequential administration of two or more therapies (e.g., two or more different medications). In one aspect, "combination" refers to simultaneous administration of two or more different medications, where the two or more different medications are present in a single dosage form. In another aspect, “combination” refers to separate administration of two or more different medications, where the two or more different medications are found in different dosage forms (e.g., one medication in an oral tablet form and another in, e.g., a different oral tablet form, an oral capsule form or an injectable form). In another aspect, “combination” refers to sequential administration of two or more different medications, where the two or more different medications are given at different points in time.

[0037] As used herein, a “marker gene” refers to a gene which can have an alteration such that its marker nucleic acid (e.g., DNA or RNA) and / or its marker protein has a characteristic, e.g., size, sequence, composition or amount(s) which, alone or in combination with one or more marker genes, indicate outcome or prognosis upon treatment. Marker genes described herein as linked to outcome after NAE inhibitor treatment are examples of genes within chromosome locus markers and are provided in Table 1 and further described in subsequent paragraphs. A marker gene listed in Table 1 can have isoforms which are either ubiquitous or have restricted expression. The additional wild type isoforms and mature proteins are readily retrievable and understandable to one of skill in the art by reviewing the information provided under the Entrez Gene database (maintained by the National Center for Biotechnology Information, Bethesda, MD) identified by the Entrez Gene ID number listed in Table 1.

[0038] Wild-Type IDH1 gene encoded protein from human cells has the following primary amino acid sequence: MSKKISGGSV VEMQGDEMTR IIWELIKEKL IFPYVELDLH SYDLGIENRD ATNDQVTKDA AEAIKKHNVG VKCATITPDE KRVEEFKLKQ MWKSPNGTIR NILGGTVFRE AIICKNIPRL VSGWVKPIII GRHAYGDQYR ATDFVVPGPG KVEITYTPSD GTQKVTYLVH NFEEGGGVAM GMYNQDKSIE DFAHSSFQMA LSKGWPLYLS TKNTILKKYD GRFKDIFQEI YDKQYKSQFE AQKIWYEHRL IDDMVAQAMK SEGGFIWACK NYDGDVQSDS VAQGYGSLGM MTSVLVCPDG KTVEAEAAHG TVTRHYRMYQ KGQETSTNPI ASIFAWTRGL AHRAKLDNNK ELAFFANALE EVSIETIEAG FMTKDLAACI KGLPNVQRSD YLNTFEFMDK LGENLKIKLA QAKL - SEQ ID NO: 1. - 12 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0039] Wild-Type IDH2 gene encoded protein from human cells has the following primary amino acid sequence: MAGYLRVVRS LCRASGSRPA WAPAALTAPT SQEQPRRHYA DKRIKVAKPV VEMDGDEMTR IIWQFIKEKL ILPHVDIQLK YFDLGLPNRD QTDDQVTIDS ALATQKYSVA VKCATITPDE ARVEEFKLKK MWKSPNGTIR NILGGTVFRE PIICKNIPRL VPGWTKPITI GRHAHGDQYK ATDFVADRAG TFKMVFTPKD GSGVKEWEVY NFPAGGVGMG MYNTDESISG FAHSCFQYAI QKKWPLYMST KNTILKAYDG RFKDIFQEIF DKHYKTDFDK NKIWYEHRLI DDMVAQVLKS SGGFVWACKN YDGDVQSDIL AQGFGSLGLM TSVLVCPDGK TIEAEAAHGT VTRHYREHQK GRPTSTNPIA SIFAWTRGLE HRGKLDGNQD LIRFAQMLEK VCVETVESGA MTKDLAGCIH GLSNVKLNEH FLNTTDFLDT IKSNLDRALG RQ -SEQ ID NO: 2

[0040] Exemplary Wild-Type IDH1 genomic sequence (SEQ ID NO: 3) gggctgagga ggcggggcct gggaggggac aaagccggga agaggaaaag ctcggaccta 60 ccctgtggtc ccgggtttct gcagagtcta cttcagaagc ggaggcactg ggagtccggt 120 ttgggattgc caggctgtgg ttgtgagtct gagcttgtga gcggctgtgg cgccccaact 180 cttcgccagc atatcatccc ggcaggcgat aaactacatt cagttgagtc tgcaagactg 240 ggaggaactg gggtgataag aaatctattc actgtcaagg tttattgaag tcaaaatgtc 300 caaaaaaatc agtggcggtt ctgtggtaga gatgcaagga gatgaaatga cacgaatcat 360 ttgggaattg attaaagaga aactcatttt tccctacgtg gaattggatc tacatagcta 420 tgatttaggc atagagaatc gtgatgccac caacgaccaa gtcaccaagg atgctgcaga 480 agctataaag aagcataatg ttggcgtcaa atgtgccact atcactcctg atgagaagag 540 ggttgaggag ttcaagttga aacaaatgtg gaaatcacca aatggcacca tacgaaatat 600 tctgggtggc acggtcttca gagaagccat tatctgcaaa aatatccccc ggcttgtgag 660 tggatgggta aaacctatca tcataggtcg tcatgcttat ggggatcaat acagagcaac 720 tgattttgtt gttcctgggc ctggaaaagt agagataacc tacacaccaa gtgacggaac 780 ccaaaaggtg acatacctgg tacataactt tgaagaaggt ggtggtgttg ccatggggat 840 gtataatcaa gataagtcaa ttgaagattt tgcacacagt tccttccaaa tggctctgtc 900 taagggttgg cctttgtatc tgagcaccaa aaacactatt ctgaagaaat atgatgggcg 960 ttttaaagac atctttcagg agatatatga caagcagtac aagtcccagt ttgaagctca 1020 aaagatctgg tatgagcata ggctcatcga cgacatggtg gcccaagcta tgaaatcaga 1080 gggaggcttc atctgggcct gtaaaaacta tgatggtgac gtgcagtcgg actctgtggc 1140 ccaagggtat ggctctctcg gcatgatgac cagcgtgctg gtttgtccag atggcaagac 1200 agtagaagca gaggctgccc acgggactgt aacccgtcac taccgcatgt accagaaagg 1260 acaggagacg tccaccaatc ccattgcttc catttttgcc tggaccagag ggttagccca 1320 cagagcaaag cttgataaca ataaagagct tgccttcttt gcaaatgctt tggaagaagt 1380 ctctattgag acaattgagg ctggcttcat gaccaaggac ttggctgctt gcattaaagg 1440 tttacccaat gtgcaacgtt ctgactactt gaatacattt gagttcatgg ataaacttgg 1500 agaaaacttg aagatcaaac tagctcaggc caaactttaa gttcatacct gagctaagaa 1560 ggataattgt cttttggtaa ctaggtctac aggtttacat ttttctgtgt tacactcaag 1620 gataaaggca aaatcaattt tgtaatttgt ttagaagcca gagtttatct tttctataag 1680 tttacagcct ttttcttata tatacagtta ttgccacctt tgtgaacatg gcaagggact 1740 tttttacaat ttttatttta ttttctagta ccagcctagg aattcggtta gtactcattt 1800 gtattcactg tcactttttc tcatgttcta attataaatg accaaaatca agattgctca 1860 aaagggtaaa tgatagccac agtattgctc cctaaaatat gcataaagta gaaattcact 1920 - 13 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 gccttcccct cctgtccatg accttgggca cagggaagtt ctggtgtcat agatatcccg 1980 ttttgtgagg tagagctgtg cattaaactt gcacatgact ggaacgaagt atgagtgcaa 2040 ctcaaatgtg ttgaagatac tgcagtcatt tttgtaaaga ccttgctgaa tgtttccaat 2100 agactaaata ctgtttaggc cgcaggagag tttggaatcc ggaataaata ctacctggag 2160 gtttgtcctc tccatttttc tctttctcct cctggcctgg cctgaatatt atactactct 2220 aaatagcata tttcatccaa gtgcaataat gtaagctgaa tcttttttgg acttctgctg 2280 gcctgtttta tttcttttat ataaatgtga tttctcagaa attgatatta aacactatct 2340 tatcttctcc tgaactgttg attttaatta aaattaagtg ctaattacca ttaaaaaaaa 2400 aa 2402

[0041] Exemplary Wild-Type IDH1 ORF sequence (SEQ ID NO: 4) ATG TCC AAA AAA ATC AGT GGC GGT TCT GTG GTA GAG ATG CAA GGA GAT GAA ATG ACA CGA 60 ATC ATT TGG GAA TTG ATT AAA GAG AAA CTC ATT TTT CCC TAC GTG GAA TTG GAT CTA CAT 120 AGC TAT GAT TTA GGC ATA GAG AAT CGT GAT GCC ACC AAC GAC CAA GTC ACC AAG GAT GCT 180 GCA GAA GCT ATA AAG AAG CAT AAT GTT GGC GTC AAA TGT GCC ACT ATC ACT CCT GAT GAG 240AAG AGG GTT GAG GAG TTC AAG TTG AAA CAA ATG TGG AAA TCA CCA AAT GGC ACC ATA CGA 300AAT ATT CTG GGT GGC ACG GTC TTC AGA GAA GCC ATT ATC TGC AAA AAT ATC CCC CGG CTT 360 GTG AGT GGA TGG GTA AAA CCT ATC ATC ATA GGT CGT CAT GCT TAT GGG GAT CAA TAC AGA 420 GCA ACT GAT TTT GTT GTT CCT GGG CCT GGA AAA GTA GAG ATA ACC TAC ACA CCA AGT GAC 480 GGA ACC CAA AAG GTG ACA TAC CTG GTA CAT AAC TTT GAA GAA GGT GGT GGT GTT GCC ATG 540GGG ATG TAT AAT CAA GAT AAG TCA ATT GAA GAT TTT GCA CAC AGT TCC TTC CAA ATG GCT 600CTG TCT AAG GGT TGG CCT TTG TAT CTG AGC ACC AAA AAC ACT ATT CTG AAG AAA TAT GAT 660 GGG CGT TTT AAA GAC ATC TTT CAG GAG ATA TAT GAC AAG CAG TAC AAG TCC CAG TTT GAA 720 GCT CAA AAG ATC TGG TAT GAG CAT AGG CTC ATC GAC GAC ATG GTG GCC CAA GCT ATG AAA 780 TCA GAG GGA GGC TTC ATC TGG GCC TGT AAA AAC TAT GAT GGT GAC GTG CAG TCG GAC TCT 840GTG GCC CAA GGG TAT GGC TCT CTC GGC ATG ATG ACC AGC GTG CTG GTT TGT CCA GAT GGC 900AAG ACA GTA GAA GCA GAG GCT GCC CAC GGG ACT GTA ACC CGT CAC TAC CGC ATG TAC CAG 960 AAA GGA CAG GAG ACG TCC ACC AAT CCC ATT GCT TCC ATT TTT GCC TGG ACC AGA GGG TTA 1020 GCC CAC AGA GCA AAG CTT GAT AAC AAT AAA GAG CTT GCC TTC TTT GCA AAT GCT TTG GAA 1080 GAA GTC TCT ATT GAG ACA ATT GAG GCT GGC TTC ATG ACC AAG GAC TTG GCT GCT TGC ATT 1140AAA GGT TTA CCC AAT GTG CAA CGT TCT GAC TAC TTG AAT ACA TTT GAG TTC ATG GAT AAA 1200CTT GGA GAA AAC TTG AAG ATC AAA CTA GCT CAG GCC AAA CTT 1242

[0042] Exemplary Wild-Type IDH2 genomic sequence (SEQ ID NO: 5) tccccggcaa ggcccaatgg ggcggcaggc ccggcagccc cgccccggtg gtgcccgcgc 60 ggccagcgcc cgccaggccc agcgttagcc cgcggccagg cagccgggag gagcggcgcg 120 cgctcggacc tctcccgccc tgctcgttcg ctctccagct tgggatggcc ggctacctgc 180 gggtcgtgcg ctcgctctgc agagcctcag gctcgcggcc ggcctgggcg ccggcggccc 240 tgacagcccc cacctcgcaa gagcagccgc ggcgccacta tgccgacaaa aggatcaagg 300 tggcgaagcc cgtggtggag atggatggtg atgagatgac ccgtattatc tggcagttca 360 tcaaggagaa gctcatcctg ccccacgtgg acatccagct aaagtatttt gacctcgggc 420 tcccaaaccg tgaccagact gatgaccagg tcaccattga ctctgcactg gccacccaga 480 agtacagtgt ggctgtcaag tgtgccacca tcacccctga tgaggcccgt gtggaagagt 540 tcaagctgaa gaagatgtgg aaaagtccca atggaactat ccggaacatc ctggggggga 600 ctgtcttccg ggagcccatc atctgcaaaa acatcccacg cctagtccct ggctggacca 660 agcccatcac cattggcagg cacgcccatg gcgaccagta caaggccaca gactttgtgg 720 cagaccgggc cggcactttc aaaatggtct tcaccccaaa agatggcagt ggtgtcaagg 780 agtgggaagt gtacaacttc cccgcaggcg gcgtgggcat gggcatgtac aacaccgacg 840 agtccatctc aggttttgcg cacagctgct tccagtatgc catccagaag aaatggccgc 900 tgtacatgag caccaagaac accatactga aagcctacga tgggcgtttc aaggacatct 960 tccaggagat ctttgacaag cactataaga ccgacttcga caagaataag atctggtatg 1020 agcaccggct cattgatgac atggtggctc aggtcctcaa gtcttcgggt ggctttgtgt 1080 gggcctgcaa gaactatgac ggagatgtgc agtcagacat cctggcccag ggctttggct 1140 cccttggcct gatgacgtcc gtcctggtct gccctgatgg gaagacgatt gaggctgagg 1200 ccgctcatgg gaccgtcacc cgccactatc gggagcacca gaagggccgg cccaccagca 1260 ccaaccccat cgccagcatc tttgcctgga cacgtggcct ggagcaccgg gggaagctgg 1320 atgggaacca agacctcatc aggtttgccc agatgctgga gaaggtgtgc gtggagacgg 1380 tggagagtgg agccatgacc aaggacctgg cgggctgcat tcacggcctc agcaatgtga 1440 agctgaacga gcacttcctg aacaccacgg acttcctcga caccatcaag agcaacctgg 1500 acagagccct gggcaggcag tagggggagg cgccacccat ggctgcagtg gaggggccag 1560 - 14 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 ggctgagccg gcgggtcctc ctgagcgcgg cagagggtga gcctcacagc ccctctctgg 1620 aggcctttct aggggatgtt tttttataag ccagatgttt ttaaaagcat atgtgtgttt 1680 cccctcatgg tgacgtgagg caggagcagt gcgttttacc tcagccagtc agtatgtttt 1740 gcatactgta atttatattg cccttggaac acatggtgcc atatttagct actaaaaagc 1800 tcttcacaaa aaaaaaaa 1818

[0043] Exemplary Wild-Type IDH2 ORF sequence (SEQ ID NO: 6) ATG GCC GGC TAC CTG CGG GTC GTG CGC TCG CTC TGC AGA GCC TCA GGC TCG CGG CCG GCC 60 TGG GCG CCG GCG GCC CTG ACA GCC CCC ACC TCG CAA GAG CAG CCG CGG CGC CAC TAT GCC 120 GAC AAA AGG ATC AAG GTG GCG AAG CCC GTG GTG GAG ATG GAT GGT GAT GAG ATG ACC CGT 180ATT ATC TGG CAG TTC ATC AAG GAG AAG CTC ATC CTG CCC CAC GTG GAC ATC CAG CTA AAG 240TAT TTT GAC CTC GGG CTC CCA AAC CGT GAC CAG ACT GAT GAC CAG GTC ACC ATT GAC TCT 300 GCA CTG GCC ACC CAG AAG TAC AGT GTG GCT GTC AAG TGT GCC ACC ATC ACC CCT GAT GAG 360 GCC CGT GTG GAA GAG TTC AAG CTG AAG AAG ATG TGG AAA AGT CCC AAT GGA ACT ATC CGG 420 AAC ATC CTG GGG GGG ACT GTC TTC CGG GAG CCC ATC ATC TGC AAA AAC ATC CCA CGC CTA 480 GTC CCT GGC TGG ACC AAG CCC ATC ACC ATT GGC AGG CAC GCC CAT GGC GAC CAG TAC AAG 540 GCC ACA GAC TTT GTG GCA GAC CGG GCC GGC ACT TTC AAA ATG GTC TTC ACC CCA AAA GAT 600 GGC AGT GGT GTC AAG GAG TGG GAA GTG TAC AAC TTC CCC GCA GGC GGC GTG GGC ATG GGC 660 ATG TAC AAC ACC GAC GAG TCC ATC TCA GGT TTT GCG CAC AGC TGC TTC CAG TAT GCC ATC 720 CAG AAG AAA TGG CCG CTG TAC ATG AGC ACC AAG AAC ACC ATA CTG AAA GCC TAC GAT GGG 780CGT TTC AAG GAC ATC TTC CAG GAG ATC TTT GAC AAG CAC TAT AAG ACC GAC TTC GAC AAG 840AAT AAG ATC TGG TAT GAG CAC CGG CTC ATT GAT GAC ATG GTG GCT CAG GTC CTC AAG TCT 900 TCG GGT GGC TTT GTG TGG GCC TGC AAG AAC TAT GAC GGA GAT GTG CAG TCA GAC ATC CTG 960 GCC CAG GGC TTT GGC TCC CTT GGC CTG ATG ACG TCC GTC CTG GTC TGC CCT GAT GGG AAG 1020 ACG ATT GAG GCT GAG GCC GCT CAT GGG ACC GTC ACC CGC CAC TAT CGG GAG CAC CAG AAG 1080 GGC CGG CCC ACC AGC ACC AAC CCC ATC GCC AGC ATC TTT GCC TGG ACA CGT GGC CTG GAG 1140 CAC CGG GGG AAG CTG GAT GGG AAC CAA GAC CTC ATC AGG TTT GCC CAG ATG CTG GAG AAG 1200 GTG TGC GTG GAG ACG GTG GAG AGT GGA GCC ATG ACC AAG GAC CTG GCG GGC TGC ATT CAC 1260 GGC CTC AGC AAT GTG AAG CTG AAC GAG CAC TTC CTG AAC ACC ACG GAC TTC CTC GAC ACC 1320 ATC AAG AGC AAC CTG GAC AGA GCC CTG GGC AGG CAG TAG 1359 Table 1 Marker Gene Description M pe G ID ID. ,

[0044] In some embodiments, the mutation in IDH1 is a mutation of arginine 132 to cysteine, glycine, histidine, or serine (R132C, R132G, R132H, or R132S). In these embodiments, the mutation is a substitution in a codon of the IDH1 gene and the mutant codon is nucleotides 689-691 of SEQ ID NO:3 or 394-396 of SEQ ID NO:4. In these embodiments, the mutant nucleotide is base 689 and / or 690 of SEQ ID NO:3 or 394 and / or 395 of SEQ ID NO:4.

[0045] In one embodiment, the mutation in IDH2 is a mutation of leucine 86 to proline (L86P). In this embodiment, the mutation is a substitution in a codon of the IDH2 gene and the mutant codon is nucleotides 420-422 of SEQ ID NO:5 or 256-258 of SEQ ID NO:6. In this embodiment, the mutant nucleotide is base 421 of SEQ ID NO:5 or 257 of SEQ ID NO:6.

[0046] In one embodiment, the mutation in IDH2 is a mutation of glutamine 316 to leucine (Q316L). In this embodiment, the mutation is a substitution in a codon of the IDH2 gene and the mutant codon is nucleotides 1110-1112 of SEQ ID NO:5 or 946-948 of SEQ ID NO:6. - 15 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 In this embodiment, the mutant nucleotide is base 1111 of SEQ ID NO:5 or 947 of SEQ ID NO:6.

[0047] In some embodiments, the mutation in IDH2 is a mutation of arginine 140 to glycine, or glutamine (R140G or R140Q). In these embodiments, the mutation is a substitution in a codon of the IDH2 gene and the mutant codon is nucleotides 582-584 of SEQ ID NO:5 or 418-420 of SEQ ID NO:6. In these embodiments, the mutant nucleotide is base 582, or 583 of SEQ ID NO:5 or 418, or 419 of SEQ ID NO:6.

[0048] In some embodiments, the mutation in IDH2 is a mutation of arginine 172 to lysine, (R172K). In these embodiments, the mutation is a substitution in a codon of the IDH2 gene and the mutant codon is nucleotides 678-680 of SEQ ID NO:5 or 514-516 of SEQ ID NO:6. In these embodiments, the mutant nucleotide is base 679 of SEQ ID NO:5 or 515 of SEQ ID NO:6.

[0049] In one embodiment, the mutation in IDH2 is a deletion of leucine 143, glycine 144, glycine 145 and threonine 146. In this embodiment, the mutation is an in-frame deletion of codons of the IDH2 gene, deletion of nucleotides 591-602 of SEQ ID NO:5 or 427-438 of SEQ ID NO:6. In this embodiment, the amino acid sequence comprises a new NIVF motif (SEQ ID NO:17, residues 141, 142, 147, and 148 of SEQ ID NO:2) resulting from the deletion of amino acids 143-146 of SEQ ID NO:2.

[0050] Table 2. Exemplary codon mutations leading to IDH1 and IDH2 mutations used in embodiments of the disclosure. I A A n R R R Rdeletion *the capitalized bold nucleotides are the mutant codon - 16 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0051] In some embodiments, a gene is defined as “genetically altered” or “mutated” “mutant” or as having a “genetic alteration” or a “mutation” if a change from wild type, e.g., a cancer-driving alteration, e.g., not normal allelic variation, is detected by sequencing a nucleic acid marker corresponding to a marker gene. In one embodiment, the sequencing method is Next Generation Sequencing (NGS). A “truncating mutation” as used herein refers to a sequence change selected from the group consisting of a frameshift insertion, a frameshift deletion, an in- frame deletion, a nonsense mutation, and a splice site mutation in a codon such that the marker nucleic acid encodes, e.g., its expression results in, a shortened, sometimes nonfunctional, altered version of a protein corresponding to the marker gene. A “single amino acid change” as used herein in the context of cancer results from a substituted nucleotide in a codon that encodes, e.g., its expression results in, a non-reference, cancer driving amino acid in an altered version of a protein corresponding to the marker gene.

[0052] As used herein, “IDH1” or “isocitrate dehydrogenase (NADP(+)) 1, cytosolic” refers to Gene ID 3417, the gene corresponding to at least three expressed mRNA variants, one of which is the mRNA described in GenBank Accession No. NM_005896, encoding GenPept Accession No. NP_005887. The open reading frame of the IDH1 gene is nucleotides 296 to 1540 of SEQ ID NO: 3. Other names for IDH1 include epididymis luminal protein-216 and oxalosuccinate decarboxylase. IDH1 catalyzes the oxidative decarboxylation of isocitrate to 2- oxoglutarate in the cytoplasm and peroxisome and can play a role in NADPH production. The IDH1 gene can be found on chromosome 2 (2q34).

[0053] As used herein, “IDH2” or “isocitrate dehydrogenase (NADP(+)) 2” refers to Gene ID 3418, the gene corresponding to at least three expressed mRNA variants, one of which is the mRNA described in GenBank Accession No. NM_002168, encoding GenPept Accession No. NP_002159. The open reading frame of the IDH2 gene is nucleotides 165 to 1523 of SEQ ID NO: 5. Other names for IDH2 include isocitrate dehydrogenase-mitochondrial (ICD-M). IDH2 catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate and can play a role in metabolism. The IDH2 gene can be found on chromosome 15 (15q26).

[0054] Mutations in IDH1 and IDH2 can permit tumors to utilize alternative energy pathways. For example, the structures of IDH1 and IDH2 have arginines (R100 and R132 for IDH1 and R140 and R172 for IDH2) in their active sites. Mutations of, or near, these arginines can confer the ability of these enzymes to catalyze the reverse reaction and generate R(-)-2- hydroxyglutarate in tumors. In addition to the mutations described above and in Table 2, non- limiting examples of mutations found in AML tumor samples include IDH1 R132L and IDH2 R140L and R140W. - 17 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0055] There has been interest in public cataloging alterations associated with cancers. Examples of public databases which include information about alterations associated with cancers are the Database of Genotypes and Phenotypes (dbGaP) maintained by the National Center for Biotechnology Information (Bethesda, MD) and Catalogue of Somatic Mutations in Cancer (COSMIC) database maintained by the Wellcome Trust Sanger Institute (Cambridge, UK). The website maintained by the Exome Aggregation Consortium (ExAC) accessible at the website maintained the Broad Institute, Cambridge, MA, harmonizes exome sequencing data and provides alternative amino acid frequencies in healthy individuals whose exomes were sequenced as part of various disease-specific and population genetic studies.

[0056] The markers and marker genes described herein were identified based on genetic profiles of samples from patients in clinical trials, such as a trial identified on the clinical trials website maintained by the U.S. National Library of Medicine, NCT04266795. The patients in this trial were suffering from acute myelogenous leukemia (AML). About 30% of patients suffering from myelodysplastic syndrome (MDS) or chronic myelomonocytic leukemia (CMML) progress to AML, for which there is no cure.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the meanings which are commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, nomenclature utilized in connection with, and techniques of cell and tissue culture, molecular biology and protein and oligo- or polynucleotide chemistry and hybridization described herein are those known in the art. GenBank or GenPept accession numbers and useful nucleic acid and peptide sequences can be found at the website maintained by the National Center for Biotechnology Information, Bethesda, MD. Ensemble Annotation Transcripts are from the GRCH37 release 94 available at the website maintained by the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI). The content of all database accession records (e.g., Entrez Gene, GenBank, RefSeq, Ensembl, COSMIC) cited throughout this application (including the Tables) are hereby incorporated by reference. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, protein purification, tissue culture and transformation and transfection (e.g., electroporation, lipofection, etc.). Enzymatic reactions are performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures generally are performed according to methods known in the art, e.g., as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. (2000) Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or Harlow, E. and Lane, D. - 18 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of patients. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In the case of conflict, the present specification, including definitions, will control.

[0058] The articles "a," "an" and “at least one” are used herein to refer to one or to more than one of the grammatical object of the article. By way of example, "an element" means one or more than one element, at least one element. In the case of conflict, the present specification, including definitions, will control.

[0059] As used herein, the term "about" denotes that the thereafter following value is no exact value but is the center point of a range that is + / -5% of the value of the value. If the value is a relative value given in percentages the term "about" also denotes that the thereafter following value is no exact value but is the center point of a range that is + / -5% of the value, whereby the upper limit of the range cannot exceed a value of 100%.

[0060] As used herein, a “favorable” outcome or prognosis refers to complete remission (CR), incomplete remission (CRi), long term survival, long progression-free survival (PFS), long time-to-progression (TTP), a negative minimal residual disease (MRD), e.g., at a 10- 5 threshold, and / or good response. Conversely, an “unfavorable” prognosis refers to short term survival, short time-to-progression (TTP), short progression-free survival, a positive minimal residual disease and / or poor response.

[0061] A “marker” as used herein, includes a material corresponding to a marker gene whose mutational status has been identified in a biological sample, e.g., tumor cells or contents or products thereof of a patient and furthermore that status is characteristic of a patient whose outcome is favorable or unfavorable with treatment e.g., treatment comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof. Examples of a marker include a material, e.g., marker nucleic acid or marker protein, e.g., a chromosome locus, DNA for a gene, RNA for a gene or protein for or corresponding to a gene. Outcome can be determined using each single marker individually as a marker; or alternatively can include one or more, or all of the characteristics collectively when reference is made to “markers” or “marker sets.” Marker sets can be combinations of chromosome locus, DNA, RNA or protein from more than one marker gene, combinations of chromosome locus, DNA, RNA or protein from a single gene, - 19 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 or any combination of the foregoing. A marker DNA, marker RNA or marker protein can correspond to base pairs on a chromosome locus marker. For example, a marker DNA can include genomic DNA from a chromosome locus marker, marker RNA can include a polynucleotide transcribed from a locus marker, and a marker protein can include a polypeptide resulting from expression at a chromosome locus marker in a biological sample, e.g., comprising tumor cells or contents or products thereof.

[0062] A “marker nucleic acid” is a nucleic acid (e.g., genomic DNA, RNA, cDNA) encoded by or corresponding to a marker gene of the invention. Such marker nucleic acids include DNA, e.g., sense and anti-sense strands of genomic DNA (e.g., including any introns occurring therein), comprising the entire or a partial sequence, e.g., one or more of the exons of the genomic DNA, up to and including the open reading frame of any of the marker genes or the complement of such a sequence. The marker nucleic acids also include RNA comprising the entire or a partial sequence of any marker or the complement of such a sequence, wherein all thymidine residues are replaced with uridine residues, mRNA generated by transcription of genomic DNA (i.e. prior to splicing), and mRNA generated by splicing of RNA transcribed from genomic DNA. A “marker nucleic acid” may also include a cDNA made by reverse transcription of an RNA generated by transcription of genomic DNA (including spliced RNA).

[0063] A marker nucleic acid also includes sequences which differ from the wild type nucleotide sequence, e.g., as listed in Table 1, due to degeneracy of the genetic code, and encode wild type protein or protein whose sequence alteration does not affect the healthy state of the subject, e.g., is not a cancer-associated change. It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. As used herein, the phrase "allelic variant" refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence. Such naturally occurring allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals, e.g., in cells, e.g., germline cells, of individuals without cancer. Such changes are compiled in the ExAc database and can be readily identified by using hybridization probes to identify the same genetic locus in a variety of individuals. Detection of any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of naturally occurring allelic variation and that do not alter the functional activity of a wild type marker gene is intended to be within the scope of the wild type version of a marker described herein. - 20 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0064] A “marker protein” is a protein encoded by a marker nucleic acid or corresponding to a marker, e.g., a mutant nucleic acid, of the invention. For example, a marker protein can be generated by translation of mRNA, e.g., mature or spliced RNA, and includes proteins both before and after cleavage of normally cleaved regions such as transmembrane signal sequences and pro-sequences. The terms “protein” and “polypeptide” are used interchangeably. A protein marker specifically can be referred to by its name or amino acid sequence, e.g., SEQ ID NO:1 or SEQ ID NO:2. It is understood by those skilled in the art, that cancer-associated genetic alterations such as mutations, deletions and / or translocations of marker nucleic acids, e.g., as described for marker genes (e.g., listed in the disclosure above and also in Table 2) can affect protein structure, appearance, cellular location and / or behavior and result in mutant proteins.

[0065] As used herein, a “characteristic” of a marker includes a size, sequence, composition or amount whose value or difference is correlated with prognosis or outcome. The characteristic, e.g., size, sequence, composition or amount of a marker can be obtained by analyzing either nucleic acid, e.g., DNA or RNA, or protein corresponding to the marker gene. In some embodiments, a characteristic size of a marker is length or molecular weight. In some embodiments, a characteristic sequence of a marker is a nucleic acid sequence or protein sequence. In some embodiments, a characteristic composition of a marker is nucleotide base or amino acid composition or peptide digest or gene fragment pattern. In some embodiments, a characteristic amount of a marker is copy number and / or expression level. In some embodiments, a characteristic of a marker, e.g., in a sample from a patient, can indicate outcome of treatment if it is different than the characteristic of the wild type or allelic variant of the marker gene. In some embodiments, a characteristic of a marker can indicate outcome if it is wild type. In an embodiment where the amount of a marker is being measured, an amount can indicate outcome if it is greater than or less than a reference amount by a degree greater than the standard error of the assay employed to assess expression. The relative expression level of a marker can be determined upon statistical correlation of the measured expression level and the outcome, e.g., response, time-to-progression, progression-free survival, minimal residual disease or overall survival. The result of the statistical analysis can establish a threshold for selecting markers or marker sets to use in the methods described herein. Alternatively, a marker, e.g., a chromosome locus marker, or a marker gene that has differential characteristic, e.g., size, sequence, composition or amount will have typical ranges that are predictive of outcome, depending on whether the characteristic, e.g., size, sequence, composition or amount falls within the range determined for the outcome. Still further, a set of markers may indicate outcome if the - 21 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 combination of their characteristics, e.g., sizes, sequences, compositions or amounts either meets or is above or below a pre-determined score as determined by methods provided herein. Genetic alterations including, but not limited to, gene translocation, transcript splice variation, deletion and truncation are examples of alterations which can change marker size, sequence or composition, in addition to point mutations, for example, substitutions, which can change marker sequence or composition. Measurement of only one characteristic of a marker gene, e.g., of a marker nucleic acid (i.e., DNA, RNA) or protein can provide a prognosis, i.e., indicate likely outcome. Measurement of more than one characteristic of a marker gene can provide a prognosis, i.e., indicate outcome when the amounts of the two characteristics are consistent with each other, e.g., the biologies of the results are not contradictory. Examples of consistent results from measurement of multiple characteristics of a marker gene can be identification of a nonsense alteration in a DNA or RNA and a low amount or low molecular weight of encoded protein, or an alteration in a region which encodes a binding pocket or active site of a protein and low activity of the encoded protein. A different example can occur when a protein is in a pathway with a feedback loop controlling its synthesis based on its activity level. In this example, a low amount or activity of protein can be associated with a high amount of its altered mRNA as a tissue, due to the marker gene alteration, thus is starved for the protein activity and repeatedly signals the production of the protein.

[0066] As used herein, “gene deletion” refers to an amount of DNA copy number less than 2 and “amplification” refers to an amount of DNA copy number greater than 2. A “diploid” amount refers to a copy number equal to 2. The term “diploid or amplification” can be interpreted as “not deletion” of a gene copy. Conversely, the term “diploid or deletion” can be interpreted as “not amplification” of copy number. For the sake of clarity, sequence deletion can occur within a gene as a result of marker gene alteration and can result in absence of transcribed protein or a shortened mRNA or protein. Such a deletion may not affect copy number.

[0067] The terms “long term survival,” “long overall survival,” “short term survival” and “short overall survival” refer to the length of time after receiving a first dose of treatment that a cancer patient is predicted to live. A “long term survivor” refers to a patient expected have a slower rate of progression or later death from the tumor than those patients identified as short term survivors. “Enhanced survival” or “a slower rate of death” are estimated life span determinations based upon characteristic, e.g., size, sequence, composition or amount of one or more of marker genes described herein, e.g., as compared to a reference standard such that 70%, 80%, 90% or more of the population will be alive a sufficient time period after receiving a first dose of treatment. A “faster rate of death” or “shorter survival time” refer to estimated life span - 22 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 determinations based upon characteristic, e.g., size, sequence, composition or amount of one or more of marker genes described herein, e.g., as compared to a reference standard such that 50%, 40%, 30%, 20%, 10% or fewer of the population will not live a sufficient time period after receiving a first dose of treatment. In some embodiments, the sufficient time period is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, 12, 18, 24 or 30 months, or 3 years, 4 years or 5 years as measured from the first day of receiving a cancer therapy.

[0068] In some embodiments, a cancer is “responsive” to a therapeutic agent, or combination of therapeutic agents, or there is a “good response” to a therapeutic regimen if its rate of growth is inhibited as a result of contact with a therapeutic agent, or combination of therapeutic agents compared to its growth in the absence of contact with the therapeutic agent, or combination of therapeutic agents, or one or more symptoms of the cancer are ameliorated. Growth of a cancer can be measured in a variety of ways, for example, the size of a tumor or the expression of tumor markers appropriate for that tumor type may be measured. International Working Groups convene periodically to set, update and publish disease and response criteria for various types of cancers. Such published reports can be followed to support the identification of markers of the subject tumors and their response to NAE inhibitors. Examples are criteria for Acute Myelogenous Leukemia (AML, Cheson et al. (2003) J.Clin. Oncol.21:4642-4649), chronic myelomonocytic leukemia (CML, Swerdlow et al. eds. (2008) in WHO Classification of Tumours of Haemotopoietic and Lymphoid Tissues (4th edition, IARC Press), lymphomas, e.g., non-Hodgkin’s and Hodgkin’s lymphoma (Cheson et al. (2007) J.Clin. Oncol.25:579-596). For example in AML, the percentage of blasts in the bone marrow or the presence of blasts with Auer rods can be a measure of response to treatment. Criteria can take into account analysis methods such as Positron Emission Tomography (PET), e.g., for identifying sites with measurable altered metabolic activity (e.g., at tumor sites) or to trace specific markers into tumors in vivo, immunohistochemistry, e.g., to identify tumor cells by detecting binding of antibodies to specific tumor markers, and flow cytometry, e.g., to characterize cell types by differential markers and fluorescent stains, in addition to traditional methods such as histology to identify cell composition (e.g., blast counts in a blood smear or a bone marrow biopsy, presence and number of mitotic figures) or tissue structure (e.g., disordered tissue architecture or cell infiltration of basement membrane). In some embodiments, the quality of being responsive to a therapy comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof can be a variable one, with different cancers exhibiting different levels of “responsiveness” to a given therapeutic agent, under different conditions. Still further, measures - 23 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 of responsiveness can be assessed using additional criteria beyond growth size of a tumor, including, but not limited to, patient quality of life, degree of metastases.

[0069] In some embodiments, a cancer is “non-responsive” or has a “poor response” to a therapeutic agent or therapeutic regimen if its rate of growth is not inhibited, or inhibited to a very low degree, as a result of contact with the therapeutic agent when compared to its growth in the absence of contact with the therapeutic agent. As stated above, growth of a cancer can be measured in a variety of ways, for instance, the size of a tumor or the expression of tumor markers appropriate for that tumor type may be measured. For example, the response definitions used to support the identification of markers associated with non- response of tumors to therapeutic agents, guidelines such as those described above can be used. In some embodiments, the quality of being non-responsive to a therapeutic agent can be a highly variable one, with different cancers exhibiting different levels of “non-responsiveness” to a given therapeutic agent, under different conditions. Still further, measures of non-responsiveness can be assessed using additional criteria beyond growth size of a tumor, including, but not limited to, patient quality of life, degree of metastases.

[0070] As used herein, “long time-to-progression, “long TTP” and “short time-to- progression,” “short TTP” refer to the amount of time until when the stable disease brought by treatment converts into an active disease. On occasion, a treatment results in stable disease which is neither a good nor a poor response, e.g., MR, the disease merely does not get worse, e.g., become a progressive disease, for a period of time. This period of time can be at least 4-8 weeks, at least 3-6 months or more than 6 months.

[0071] As used herein, “progression free survival” or “PFS” refers to the time elapsed between treatment initiation and tumor progression or death from any cause.

[0072] As used herein, “complete response” or “CR” refers to the fraction of patients in a treated population who experience a complete response following treatment.

[0073] As used herein, “complete response with incomplete bone marrow recovery” or “CRi” refers to the fraction of patients in a treated population who experience a complete response with incomplete bone marrow recovery following treatment.

[0074] As used herein, “minimal residual disease” or “MRD” refers to the result of an assay to detect residual malignant cancer or tumor cells in a patient, e.g., after at least some treatment with a therapeutic regimen. MRD negative refers to a result when no residual tumor cells can be found in a sample from the patient. MRD positive refers to a result when a small number of tumor cells can be found in a sample from the patient. MRD can be qualified by an - 24 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 assay threshold, e.g., a 10-4, a 10-5 or a 10-6 threshold, i.e., related to a limit of detection of the assay, such as flow cytometry for detecting tumor cells.

[0075] "Treatment" as used herein in the context of cancer shall mean the use of a therapy to prevent or inhibit further tumor growth, to cause shrinkage of a tumor, alleviate tumor burden, and / or to provide longer survival times. Treatment is also intended to include prevention of metastasis of tumor. A tumor is "inhibited" or “treated” (e.g., as determined by responsiveness, time to progression, progression-free survival, minimal residual disease or indicators known in the art and described herein) if at least one symptom of the cancer or tumor is alleviated, terminated, slowed, minimized, or prevented. Any amelioration of any symptom, physical or otherwise, of a tumor pursuant to treatment using a therapeutic regimen (e.g., comprising an NAE inhibitor, such as comprising pevonedistat or a pharmaceutically acceptable salt thereof) as further described herein, is within the scope of the invention.

[0076] As used herein, the term “agent” is defined broadly as anything that cancer cells, including tumor cells, may be exposed to in a therapeutic regimen. In the context of the present invention, such agents include, but are not limited to, an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, as well as chemotherapeutic agents used in combination with the NAE inhibitor, as known in the art and described in further detail herein.

[0077] The term "probe" refers to any molecule, e.g., an isolated molecule, which is capable of selectively binding to a specifically intended target molecule, for example a marker of the invention. Probes can be either synthesized by one skilled in the art or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic monomers.

[0078] A “normal” or “reference” characteristic, e.g., size, sequence, composition or amount of a marker may refer to the characteristic, e.g., size, sequence, composition or amount in a “reference sample.” A reference sample can be a matched normal or control, e.g., germline, sample from the same patient from whom the cancer sample is derived. A reference sample can be a sample from a healthy subject not having the marker-associated disease or having a reference characteristic e.g., the average characteristic, e.g., size, sequence, composition or amount of the wild type marker in several healthy subjects. A reference sample characteristic, e.g., size, sequence, composition or amount may be comprised of a characteristic, e.g., size, sequence, composition or amount of one or more markers from a reference database. - 25 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 Alternatively, a “normal” characteristic, e.g., size, sequence, composition or amount of a marker is the characteristic, e.g., size, sequence, composition or amount of the marker, e.g., marker gene in non-tumor cells in a similar environment or response situation from the same patient from whom the tumor is derived. The normal amount of DNA copy number is 2 or diploid, with the exception of X-linked genes in males, where the normal DNA copy number is 1.

[0079] "Over-expression" or “upregulation” and "under-expression" or “downregulation” of a marker gene, refer to expression of the marker gene of a patient at a greater or lesser level (e.g. more than three-halves-fold, at least two-fold, at least three-fold, greater or lesser level etc.), respectively, than normal level of expression of the marker gene, e.g., as measured by mRNA or protein, in a test sample that is greater than the standard error of the assay employed to assess expression. A “significant” expression level may refer to a level which either meets or is above or below a pre-determined score for a marker gene set as determined by methods provided herein.

[0080] "Complementary" in the context of nucleic acids, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In an embodiment, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% or all of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0081] "Homologous" as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide - 26 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 residue positions of the two regions that are occupied by the same nucleotide residue (i.e., by percent identity). By way of example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share homology with 50% identity. In one embodiment, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. In an embodiment of 100% identity, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

[0082] Unless otherwise specified herein, the terms “antibody” and “antibodies” broadly encompass naturally-occurring forms of antibodies, e.g., polyclonal antibodies (e.g., IgG, IgA, IgM, IgE) and monoclonal and recombinant antibodies such as IgG, single-chain antibodies, two-chain and multi-chain proteins, chimeric, CDR-grafted, human and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments (e.g., dAbs, scFv, Fab, F(ab)′2, Fab′) and derivatives having at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody. The term “antibody” also includes synthetic and genetically engineered variants.

[0083] A “kit” is any article of manufacture (e.g., a package or container) comprising at least one reagent, e.g. a probe, for specifically detecting a marker or marker set of the invention. The article of manufacture may be promoted, distributed, sold or offered for sale as a unit for performing, e.g., in vitro, the methods of the present invention, e.g., on a sample having been obtained from a patient, e.g., a human patient. The reagents included in such a kit can comprise at least one probe, such as a nucleic acid probe and, optionally, one or more primers and / or at least one antibody probe, for use in detecting marker characteristics, e.g., size, sequence composition or amount, e.g., expression. In addition, a kit of the present invention can contain instructions which describe a suitable detection assay. Such a kit can be conveniently used, e.g., in a clinical or a contract testing setting, to generate results, e.g., on characteristic, e.g., size, sequence, composition or amount of one or more marker, to be recorded, stored, transmitted or received to allow for diagnosis, evaluation or treatment of patients exhibiting symptoms of cancer, in particular patients exhibiting the possible presence of a cancer capable of treatment with a regimen comprising NAE inhibition therapy, including, e.g., hematological cancers e.g., myelomas (e.g., multiple myeloma), lymphomas (e.g., non-Hodgkin’s lymphoma), leukemias (e.g., acute myelogenous leukemia, chronic myelomonocytic leukemia, myelodysplastic syndrome), or solid tumors (e.g., tumors of skin, lung, breast, ovary). - 27 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0084] The present methods and compositions are designed for use in diagnostics and therapeutics for a patient suffering from cancer. A cancer or tumor is treated or diagnosed according to the present methods. “Cancer” or “tumor” is intended to include any neoplastic growth in a patient, including an initial tumor and any metastases. The cancer can be of the hematological or solid tumor type. Hematological tumors include tumors of hematological origin, including, e.g., myelomas (e.g., monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma, smoldering myeloma, multiple myeloma), leukemias (e.g., Waldenstrom’s syndrome, chronic lymphocytic leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia, chronic myelomonocytic leukemia, myelodysplastic syndrome, other leukemias), lymphomas (e.g., B-cell lymphomas, e.g., diffuse large B-cell lymphoma, non- Hodgkin’s lymphoma) and myelodysplastic syndrome. In some embodiments, the MDS is high risk MDS, typically characterized by more than 5% of the bone marrow comprised of immature blast cells. In some embodiments, the AML is low-blast acute AML, de novo AML or secondary AML. The AML patient can have blasts that can express CD33, CD123 and / or CLEC12a. Solid tumors can originate in organs or can metastasize from other tumors, and include cancers such as, but not limited to, in skin, lung, brain, breast, prostate, ovary, colon, kidney, pancreas, liver, esophagus, stomach, intestine, bladder, uterus, cervix, head and neck, central nervous system, bone, testis, and adrenal gland. The cancer can comprise a cell in which a marker gene has an alteration. As used herein, cancer cells, including tumor cells, refer to cells that divide at an abnormal (increased) rate or whose control of growth or survival is different than for cells in the same tissue where the cancer cell arises or lives. Cancer cells include, but are not limited to, cells in carcinomas, such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synoviosarcoma and mesotheliosarcoma; cells in hematologic cancers, such as myelomas, leukemias (e.g., acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelomonocytic leukemia, granulocytic leukemia, monocytic leukemia, lymphocytic leukemia), and cells in lymphomas (e.g., follicular lymphoma, mantle cell lymphoma, diffuse large B-cell - 28 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 lymphoma, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkins disease); and cells in tumors of the nervous system including glioma, meningoma, medulloblastoma, schwannoma or epidymoma.

[0085] As used herein, the term “noninvasive” refers to a procedure which inflicts minimal harm to a subject. In the case of clinical applications, a noninvasive sampling procedure can be performed quickly, e.g., in a walk-in setting, typically without anaesthesia and / or without surgical implements or suturing. Examples of noninvasive samples include, but are not limited to, blood, serum, saliva, urine, buccal swabs, throat cultures, stool samples and cervical smears. Noninvasive diagnostic analyses include x-rays, magnetic resonance imaging, positron emission tomography.

[0086] Described herein is the assessment of outcome for treatment of a tumor through measurement of one or more characteristics of a marker gene. Also described are assessing the outcome by noninvasive, convenient or low-cost means, for example, from blood samples. Typical methods to determine extent of cancer or outcome of a cancer, e.g., a hematological cancer, e.g., lymphoma, leukemia, e.g., AML, CMML, MDS, myeloma (e.g., multiple myeloma) can employ bone marrow biopsy to collect tissue for genotype or phenotype, e.g., histological analysis. The invention provides methods for determining, assessing, advising or providing an appropriate therapy regimen for treating a tumor or managing cancer in a patient. Monitoring a treatment using the kits and methods disclosed herein can identify the potential for unfavorable outcome and allow their prevention, and thus a savings in morbidity, mortality and treatment costs through adjustment in the therapeutic regimen, cessation of therapy or use of alternative therapy.

[0087] The term "biological sample" is intended to include a material, e.g., tissue, cells, biological fluids and isolates thereof, obtained, e.g., isolated or collected, from a subject, e.g., a human, such as a patient or a normal subject. A tumor sample from a cancer patient can comprise tumor cells or contents or products thereof. In hematological cancers of the bone marrow, e.g., leukemias or myeloma, a sample for primary analysis of the tumor can be a bone marrow sample. However, some tumor cells, (e.g., clonotypic tumor cells, circulating endothelial cells), are a percentage of the cell population in whole blood. Bone marrow cells also can be mobilized into the blood during treatment of the patient with granulocyte-colony stimulating factor (G-CSF) in preparation for a bone marrow transplant, a standard treatment for hematological cancers, e.g., leukemias, lymphomas and myelomas. Examples of circulating tumor cells in multiple myeloma have been studied e.g., by Pilarski et al. (2000) Blood 95:1056- 65 and Rigolin et al. (2006) Blood 107:2531-5. Thus, noninvasive samples, e.g., for in vitro - 29 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 measurement of markers to determine outcome of treatment, can include peripheral blood samples. Accordingly, cells within peripheral blood can be tested for marker characteristics. For patients with cancer, e.g., hematological cancer, a control, reference sample for normal characteristic, e.g., size, sequence, composition or amount can be obtained from skin or a buccal swab of the patient. For solid tumors, a typical tumor sample is a biopsy of the tumor and thus comprises solid tumor cells. Alternatively, a sample of tumor cells shed or scraped from the tumor site can be collected noninvasively, such as, but not limited to, in blood, sputum, a nipple aspirate, urine, stool, cervical smear. In some embodiments nucleic acid markers described herein can be identified or measured in cell-free portions of blood. For example, nucleic acids released from tumor cells can become circulating tumor DNA. Circulating tumor DNA (ctDNA) can be analyzed in samples of plasma or serum. For solid tumors, a control reference sample for normal characteristic, e.g., size, sequence, composition or amount can be obtained from blood of the patient.

[0088] Blood or bone marrow collection containers can comprise an anti-coagulant, e.g., heparin or ethylene-diaminetetraacetic acid (EDTA), sodium citrate or citrate solutions with additives to preserve blood integrity, such as dextrose or albumin or buffers, e.g., phosphate. If the amount of marker is being measured by measuring the level of its DNA in the sample, a DNA stabilizer, e.g., an agent that inhibits DNase, can be added to the sample. If the amount of marker is being measured by measuring the level of its RNA in the sample, an RNA stabilizer, e.g., an agent that inhibits RNase, can be added to the sample. If the amount of marker is being measured by measuring the level of its protein in the sample, a protein stabilizer, e.g., an agent that inhibits proteases, can be added to the sample. An example of a blood collection container is PAXGENE® tubes (PREANALYTIX, Valencia, CA), useful for RNA stabilization upon blood collection. Peripheral blood samples can be modified, e.g., fractionated, clotted for serum, centrifuged for plasma, sorted or concentrated (e.g., to result in samples enriched with tumor or depleted of tumor (e.g., for a reference sample)). Examples of modified samples include clonotypic myeloma cells, which can be collected by e.g., negative selection, e.g., separation of white blood cells from red blood cells (e.g., differential centrifugation through a dense sugar or polymer solution (e.g., FICOLL® solution (Amersham Biosciences division of GE healthcare, Piscataway, NJ) or HISTOPAQUE®-1077 solution, Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co., St. Louis, MO)) and / or positive selection by binding B cells to a selection agent (e.g., a reagent which binds to a tumor cell or myeloid progenitor marker, such as CD34, CD38, CD138, or CD133, for direct isolation (e.g., the application of a magnetic field to - 30 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 solutions of cells comprising magnetic beads (e.g., from Miltenyi Biotec, Auburn, CA) which bind to the B cell markers) or fluorescent-activated cell sorting).

[0089] Alternatively, a tumor cell line, e.g., OCI-Ly3, OCI-Ly10 cell (Alizadeh et al. (2000) Nature 403:503-511), a RPMI 6666 cell, a SUP-B15 cell, a KG-1 cell, a CCRF-SB cell, an 8ES cell, a Kasumi-1 cell, a Kasumi-3 cell, a BDCM cell, an HL-60 cell, a Mo-B cell, a JM1 cell, a GA-10 cell or a B-cell lymphoma (e.g., BC-3) or a cell line or a collection of tumor cell lines (see e.g., McDermott et al. (2007) PNAS 104:19936-19941 or ONCOPANEL™ anti-cancer tumor cell profiling screen (Ricerca Biosciences, Bothell, WA)) can be assayed. A skilled artisan readily can select and obtain the appropriate cells (e.g., from American Type Culture Collection (ATCC®), Manassas, VA) that are used in the present method. If the compositions or methods are being used to predict outcome of treatment in a patient or monitor the effectiveness of a therapeutic protocol, then a tissue or blood sample having been obtained from the patient being treated is a useful source of cells or marker gene or gene products for an assay.

[0090] The biological sample, e.g., comprising tumor tissue, cells or contents or products thereof, e.g., biopsy or bone marrow, blood or modified blood, (e.g., comprising tumor cells or ctDNA) and / or the reference, e.g., matched control (e.g., germline), sample can be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., nucleic acid and / or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation) prior to analyzing the characteristic of the marker in the sample.

[0091] Assay Methods

[0092] In an embodiment, mutational status of a marker gene, e.g., whether wild type or comprising a gene alteration in a marker can be identified by sequencing a nucleic acid, e.g., a DNA, RNA, cDNA or a protein correlated with the marker gene. There are several sequencing methods known in the art to sequence nucleic acids. A nucleic acid primer can be designed to bind to a region comprising a potential alteration site or can be designed to complement the altered sequence rather than the wild type sequence. For example, SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, and / or 16 can be a primer or probe or be used to design a primer to detect a mutation in an IDH1 and / or IDH2 nucleic acid marker. Primer pairs can be designed to bracket a region comprising a potential alteration in a marker gene. A primer or primer pair can be used for sequencing one or both strands of DNA corresponding to the marker gene. A primer can be used in conjunction with a probe, e.g., a nucleic acid probe, e.g., a hybridization probe, to amplify a region of interest prior to sequencing to boost sequence amounts for detection of an alteration in a marker gene. Examples of regions which can be sequenced include an entire gene, transcripts of the gene and a fragment of the gene or the transcript, e.g., one or more of exons or - 31 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 untranslated regions. Examples of alterations to target for primer selection and sequence or composition analysis can be found in public databases which collect alteration information, such as COSMIC and dbGaP. Some alterations of marker genes such as IDH1 and IDH2 can be associated with sensitivity to an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof. In some embodiments, alterations in IDH1 and IDH2 markers are listed in the above disclosure, in Table 2 and the Examples.

[0093] Sequencing methods are known to one skilled in the art. Examples of methods include the Sanger method, the SEQUENOM™ method and Next Generation Sequencing (NGS) methods. The Sanger method, comprising the use of electrophoresis, e.g., capillary electrophoresis to separate primer-elongated labeled DNA fragments, can be automated for high- throughput applications. The primer extension sequencing can be performed after PCR amplification of regions of interest. Software can assist with sequence base calling and with alteration identification. SEQUENOM™ MASSARRAY® sequencing analysis (San Diego, CA) is a mass-spectrometry method which compares actual mass to expected mass of particular fragments of interest to identify alterations. NGS technology (also called “massively parallel sequencing” and “second generation sequencing”) in general provides for much higher throughput than previous methods and uses a variety of approaches (reviewed in Zhang et al. (2011) J. Genet. Genomics 38:95-109 and Shendure and Hanlee (2008) Nature Biotech.26:1135- 1145). NGS methods can identify low frequency alterations in a marker in a sample. Some NGS methods (see, e.g., GS-FLX Genome Sequencer (Roche Applied Science, Branford, CT), Genome analyzer (Illumina, Inc. San Diego, CA), SOLID™ analyzer (Applied Biosystems, Carlsbad, CA), Polonator G.007 (Dover Systems, Salem, NH), HELISCOPE™ (Helicos Biosciences Corp., Cambridge, MA)) use cyclic array sequencing, with or without clonal amplification of PCR products spatially separated in a flow cell and various schemes to detect the labeled modified nucleotide that is incorporated by the sequencing enzyme (e.g., polymerase or ligase). In one NGS method, primer pairs can be used in PCR reactions to amplify regions of interest. Amplified regions can be ligated into a concatenated product. Clonal libraries are generated in the flow cell from the PCR or ligated products and further amplified (“bridge” or “cluster” PCR) for single-end sequencing as the polymerase adds a labeled, reversibly terminated base that is imaged in one of four channels, depending on the identity of the labeled base and then removed for the next cycle. Software can aid in the comparison to genomic sequences to identify alterations.

[0094] Composition of proteins and nucleic acids can be determined by many ways known in the art, such as by treating them in ways that cleave, degrade or digest them and then - 32 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 analyzing the components. Mass spectrometry, electrophoresis and chromatography can separate and define components for comparison. Alterations which cause deletions or insertions can be identified by size or charge differences in these methods. Protein digestion or restriction enzyme nucleic acid digestion can reveal different fragment patterns after some alterations. Antibodies that recognize particular mutant amino acids, e.g., the mutant amino acids (IDH1132C, 132G, 132H, or 132S; IDH286P, 316L, 140G, 140Q, 172K or SEQ ID NO:17) listed in the above disclosure, in Table 2 and the Examples in their structural contexts, can identify and detect these alterations in samples.

[0095] In an embodiment, DNA, e.g., genomic DNA corresponding to the wild type or altered marker gene, can be analyzed both by in situ and by in vitro formats in a biological sample using methods known in the art. DNA can be directly isolated from the sample or isolated after isolating another cellular component, e.g., RNA or protein. Kits are available for DNA isolation, e.g., QIAAMP® DNA Micro Kit (Qiagen, Valencia, CA). DNA also can be amplified using such kits.

[0096] In another embodiment, mRNA corresponding to the marker gene can be analyzed both by in situ and by in vitro formats in a biological sample using methods known in the art. An example of a method for measuring expression level is included in the Examples. For example, a nucleic acid probe can be used to hybridize to a marker and the amount of probe hybridized can be measured. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from tumor cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Patent No.4,843,155). RNA can be isolated using standard procedures (see e.g., Chomczynski and Sacchi (1987) Anal. Biochem.162:156-159), solutions (e.g., trizol, TRI REAGENT® (Molecular Research Center, Inc., Cincinnati, OH; see U.S. Patent No.5,346,994) or kits (e.g., a QIAGEN® Group RNEASY® isolation kit (Valencia, CA) or LEUKOLOCK™ Total RNA Isolation System, Ambion division of Applied Biosystems, Austin, TX).

[0097] Additional steps may be employed to remove DNA from RNA samples. Cell lysis can be accomplished with a nonionic detergent, followed by microcentrifugation to remove the nuclei and hence the bulk of the cellular DNA. DNA subsequently can be isolated from the nuclei for DNA analysis. In one embodiment, RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the - 33 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 RNA from DNA (Chirgwin et al. (1979) Biochemistry 18:5294-99). Alternatively, separation of RNA from DNA can be accomplished by organic extraction, for example, with hot phenol or phenol / chloroform / isoamyl alcohol. If desired, RNAse inhibitors may be added to the lysis buffer. Likewise, for certain cell types, it may be desirable to add a protein denaturation / digestion step to the protocol. For many applications, it is desirable to enrich mRNA with respect to other cellular RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA). Most mRNAs contain a poly(A) tail at their 3' end. This allows them to be enriched by affinity chromatography, for example, using oligo(dT) or poly(U) coupled to a solid support, such as cellulose or SEPHADEX.R™ medium (see Ausubel et al. (1994) Current Protocols In Molecular Biology, vol.2, Current Protocols Publishing, New York). Once bound, poly(A)+mRNA is eluted from the affinity column using 2 mM EDTA / 0.1% SDS. (see Sambrook et al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

[0098] The characteristic of a marker of the invention in a biological sample, e.g., after obtaining a biological sample (e.g., a bone marrow sample, a whole blood sample, a plasma sample, a serum sample, a tumor biopsy, a sample comprising tumor cells, tumor cell products or tumor cell components, e.g., cytoplasm or mitochondria, or a reference sample) from a subject, may be detected or measured by any of a wide variety of well-known methods for a nucleic acid (e.g., RNA, mRNA, genomic DNA, or cDNA) and / or translated protein. Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, mitochondrial or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods. These methods include gene array / chip technology, RT- PCR, TAQMAN® gene expression assays (Applied Biosystems, Foster City, CA), e.g., under GLP approved laboratory conditions, in situ hybridization, immunohistochemistry, immunoblotting, FISH (fluorescence in situ hybridization), FACS analyses, northern blot, southern blot, INFINIUM® DNA analysis Bead Chips (Illumina, Inc., San Diego, CA), quantitative PCR, bacterial artificial chromosome arrays, single nucleotide polymorphism (SNP) arrays (Affymetrix, Santa Clara, CA) or cytogenetic analyses. The detection methods of the invention can thus be used to detect mutational status in RNA, mRNA, protein, cDNA, ctDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo. Furthermore, in vivo techniques for detection of a polypeptide or nucleic acid corresponding to a marker of the invention include introducing into a subject a labeled probe to detect the biomarker, e.g., a nucleic acid complementary to the transcript of a biomarker or a labeled antibody, Fc receptor or - 34 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 antigen directed against the polypeptide, e.g., wild type or mutant marker. For example, the antibody can be labeled with a radioactive isotope whose presence and location in a subject can be detected by standard imaging techniques. These assays can be conducted in a variety of ways. A skilled artisan can select from these or other appropriate and available methods based on the nature of the marker(s), tissue sample and alteration in question. Different methods or combinations of methods could be appropriate in different cases or, for instance in different types of tumors or patient populations.

[0099] In some embodiments, detection assays involve preparing a sample or reaction mixture that may contain a marker, and a probe, under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and / or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker / probe complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, a sample from a subject, which is to be assayed for presence and / or concentration of marker, can be anchored onto a carrier or solid phase support. In another embodiment, the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay. One example of such an embodiment includes use of an array or chip which contains a marker or marker set anchored for expression analysis of the sample.

[0100] There are many established methods for anchoring assay components to a solid phase, e.g., glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. These include, without limitation, marker or probe molecules which are immobilized through conjugation of biotin and streptavidin. Such biotinylated assay components can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Protein Biology, Thermo Scientific), and immobilized in the wells of streptavidin-coated 96 well plates. In certain embodiments, the surfaces with immobilized assay components can be prepared in advance and stored.

[0101] In order to conduct assays with the above-mentioned approaches, the non- immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the - 35 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 solid phase. The detection of marker / probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.

[0102] In an embodiment, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art. The term "labeled", with regard to the probe (e.g., nucleic acid or antibody), is intended to encompass direct labeling of the probe by coupling (i.e., physically linking) a detectable substance to the probe, as well as indirect labeling of the probe by reactivity with another reagent that is directly labeled. The label can be a radioisotope, a fluorescent compound, an enzyme, an enzyme co- factor, a hapten, a sequence tag, a protein or an antibody. An example of indirect labeling includes detection of a primary antibody using a fluorescently labeled secondary antibody. It is also possible to directly detect marker / probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (FET, see, for example, Lakowicz et al., U.S. Patent No.5,631,169; Stavrianopoulos, et al., U.S. Patent No.4,868,103).

[0103] In another embodiment, determination of the ability of a probe to recognize a marker can be accomplished without labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem.63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.5:699-705). As used herein, “BIA” or “surface plasmon resonance” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIACORE™). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[0104] Alternatively, in another embodiment, analogous detection assays can be conducted with marker and probe as solutes in a liquid phase. In such an assay, the complexed marker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.

[0105] Nucleic acid probes of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, may be produced by recombinant technology, or may be derived from a biological sample, for example, by restriction digestion. The nucleic - 36 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 acids can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone. An example of a nucleic acid label is incorporated using SUPER™ Modified Base Technology (Nanogen, Bothell, WA, see U.S. Patent No.7,045,610). The level of expression can be measured as general nucleic acid levels, e.g., after measuring the amplified DNA levels (e.g. using a DNA intercalating dye, e.g., the SYBR green dye (Qiagen Inc., Valencia, CA) or as specific nucleic acids, e.g., using a probe-based design, with the probes labeled. TAQMAN® assay formats can use the probe-based design to increase specificity and signal-to-noise ratio.

[0106] Hybridization of an RNA, ctDNA or a cDNA with the nucleic acid probe can indicate that the marker in question is being expressed. As used herein, the term “hybridizes” is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other. In some embodiments, the conditions are such that sequences at least about 70%, at least about 80%, at least about 85%, 90% or 95% identical to each other remain hybridized to each other for subsequent amplification and / or detection. Stringent conditions vary according to the length of the involved nucleotide sequence but are known to those skilled in the art and can be found or determined based on teachings in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions and formulas for determining such conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), chapters 7, 9 and 11. A non-limiting example of stringent hybridization conditions for hybrids that are at least 10 basepairs in length includes hybridization in 4X sodium chloride / sodium citrate (SSC), at about 65-70°C (or hybridization in 4X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 1X SSC, at about 65-70°C. A non-limiting example of highly stringent hybridization conditions for such hybrids includes hybridization in 1X SSC, at about 65-70°C (or hybridization in 1X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 0.3X SSC, at about 65-70°C. A non-limiting example of reduced stringency hybridization conditions for such hybrids includes hybridization in 4X SSC, at about 50-60°C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45°C) followed by one or more washes in 2X SSC, at about 50-60°C. It will also be recognized by the skilled practitioner that additional reagents may be added to hybridization and / or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, polyvinylpyrrolidone (PVP) and the like. Nucleic acid probes of the invention can refer to - 37 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 nucleic acids which hybridize to the region of interest and which are not further extended. For example, it specifically hybridizes to a mutant region of a biomarker, and which by hybridization or absence of hybridization to the DNA of a patient or the type of hybrid formed can be indicative of the presence or identity of the alteration of the biomarker or the amount of marker activity.

[0107] In one format, the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the nucleic acid probe(s) are immobilized on a solid surface and the RNA is contacted with the probe(s), for example, in an AFFYMETRIX® gene chip array or a SNP chip (Santa Clara, CA) or customized array using a marker set comprising at least one marker indicative of treatment outcome. A skilled artisan can readily adapt known RNA and DNA detection methods for use in detecting the amount of the markers of the present invention. For example, the high density microarray or branched DNA assay can benefit from a higher concentration of tumor cell in the sample, such as a sample which had been modified to isolate tumor cells as described in earlier sections. In a related embodiment, a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate having fixed thereto a polynucleotide complementary to or homologous with at least a portion (e.g., at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or more nucleotide residues) of a marker nucleic acid. If polynucleotides complementary to or homologous with the marker are differentially detectable on the substrate (e.g., detectable using different chromophores or fluorophores, or fixed to different selected positions), then the levels of expression of a plurality of markers can be assessed simultaneously using a single substrate (e.g., a "gene chip" microarray of polynucleotides fixed at selected positions). In an embodiment when a method of assessing marker expression is used which involves hybridization of one nucleic acid with another, the hybridization can be performed under stringent hybridization conditions.

[0108] In the present examples, a bait set can have a higher relative concentration for more specifically desired sequences of interest that comprise one or more IDH1 and / or IDH2 gene mutations as provided herein. Such bait sets are combined with a sample under conditions that allow hybridization of the target molecules with the baits. Then, captured molecules are isolated using the capture moiety. For example, a biotin capture moiety is bead-based streptavidin.

[0109] For in situ methods, RNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample is prepared / processed using known - 38 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to RNA that encodes the marker.

[0110] In vitro techniques for detection of a polypeptide corresponding to a marker of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, protein array, immunoprecipitations and immunofluorescence. In such examples, expression of a marker is assessed using an antibody (e.g., a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain) which binds specifically with a marker protein or fragment thereof, e.g., a protein or fragment comprising a region which can be altered, e.g., comprising an active site amino acid (such as R100 or R132 of IDH1 or R140 or R172 of IDH2) or a portion comprising an altered, e.g., mutant amino acid, sequence, or an altered residue in its structural context, including a marker protein which has undergone all or a portion of its normal post-translational modification. An antibody can detect a marker gene protein described herein, e.g., a protein corresponding to IDH1 or IDH2. Alternatively, an antibody can detect a genetically altered IDH1 or IDH2 protein, e.g., a protein with an amino acid sequence comprising a mutant of SEQ ID NO:1 or 2. Examples of IDH1 or IDH2 alterations that can be detected by antibodies include the mutations described above as they pertain to IDH1 and IDH2 (IDH1132C, 132G, 132H, or 132S; IDH286P, 316L, 140G, 140Q, 172K or SEQ ID NO:17) listed in Table 2 and in the Examples. Residues translated from altered codons, for example, codons described in Table 2, can be prepared in immunogenic compositions for generation of antibodies that will specifically recognize and bind to the mutant residues. Another method can employ pairs of antibodies, wherein one of the pair would bind a marker protein upstream, i.e., N-terminal to the region of expected alteration, e.g., nonsense or deletion and the other of the pair would bind the protein downstream. Wild type protein would bind both antibodies of the pair, but a protein with a nonsense or deletion alteration would bind only the N-terminal antibody of the pair. An assay such as a sandwich ELISA assay could detect a loss or gain of quantity of the wild type protein in the tumor sample, e.g., in comparison to the reference sample, or a standard ELISA would permit comparison of the levels of binding of the antibodies to infer that an alteration is present in a tumor sample.

[0111] In some embodiments, indirect methods for determining the amount or functionality of a protein marker also include measurement of the activity of the protein. For example, a sample, or a protein isolated from the sample or expressed from nucleic acid isolated, cloned or amplified from the sample can be assessed for marker protein activity. The enzymatic - 39 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 activity of IDH1 or IDH2 can be measured (Murugan et al. (2011) Biochem. Biophys. Res. Comm.393:555-559), e.g., in a tumor cell sample.

[0112] Another method for determining the level of a polypeptide corresponding to a marker is mass spectrometry. For example, intact proteins or peptides, e.g., tryptic peptides can be analyzed from a biological sample, e.g., a bone marrow sample, a blood sample, a lymph sample or other sample, containing one or more polypeptide markers. The method can further include treating the sample to lower the amounts of abundant proteins, e.g., serum albumin, to increase the sensitivity of the method. For example, liquid chromatography can be used to fractionate the sample so portions of the sample can be analyzed separately by mass spectrometry. The steps can be performed in separate systems or in a combined liquid chromatography / mass spectrometry system (LC / MS, see for example, Liao, et al. (2004) Arthritis Rheum.50:3792-3803). The mass spectrometry system also can be in tandem (MS / MS) mode. The charge state distribution of the protein or peptide mixture can be acquired over one or multiple scans and analyzed by statistical methods, e.g. using the retention time and mass-to- charge ratio (m / z) in the LC / MS system, to identify proteins expressed at statistically significant levels differentially in samples from patients responsive or non-responsive to NAE inhibition therapy. Examples of mass spectrometers which can be used are an ion trap system (ThermoFinnigan, San Jose, CA) or a quadrupole time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA). The method can further include the step of peptide mass fingerprinting, e.g. in a matrix-assisted laser desorption ionization with time-of-flight (MALDI- TOF) mass spectrometry method. The method can further include the step of sequencing one or more of the tryptic peptides. Results of this method can be used to identify proteins from primary sequence databases, e.g., maintained by the National Center for Biotechnology Information, Bethesda, MD, or the Swiss Institute for Bioinformatics, Geneva, Switzerland, and based on mass spectrometry tryptic peptide m / z base peaks.

[0113] In one embodiment, expression of a marker is assessed by preparing mRNA / cDNA (i.e., a transcribed polynucleotide) from cells in a biological sample, and by hybridizing the mRNA / cDNA with a reference polynucleotide, e.g., an isolated nucleic acid probe, e.g., a hybridization probe, which is a complement of a marker nucleic acid, or a fragment thereof. cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide. Expression of one or more markers likewise can be detected using quantitative PCR to assess the level of expression of the marker(s). An example of the use of measuring mRNA levels is that an inactivating alteration in a marker gene can result in an altered level of mRNA in a cell. The level can be upregulated due - 40 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 to feedback signaling protein production in view of nonfunctional or absent protein or downregulated due to instability of an altered mRNA sequence. Alternatively, any of the many known methods of detecting alterations (e.g. single nucleotide polymorphisms, deletions, discussed above) of a marker of the invention may be used to detect occurrence of an alteration in a marker gene in a patient.

[0114] In some embodiments direct measurement of nucleic acid amount is quantification of transcripts. As used herein, the level or amount of expression refers to the absolute amount of expression of an mRNA encoded by the marker or the absolute amount of expression of the protein encoded by the marker. As an alternative to making determinations based on the absolute expression amount of selected markers, determinations may be based on normalized expression amounts. Expression amount can be normalized by correcting the absolute expression level of a marker upon comparing its expression to the expression of a control marker that is not a marker, e.g., in a housekeeping role that is constitutively expressed. Suitable markers for normalization also include housekeeping genes, such as the actin gene or beta-2 microglobulin. Reference markers for data normalization purposes include markers which are ubiquitously expressed and / or whose expression is not regulated by oncogenes. Constitutively expressed genes are known in the art and can be identified and selected according to the relevant tissue and / or situation of the patient and the analysis methods. Such normalization allows one to compare the expression level in one biological sample, to another biological sample, e.g., between biological samples from different times or different subjects. Further, the expression level can be provided as a relative expression level. The baseline of a genomic DNA sample, e.g., diploid copy number, can be determined by measuring amounts in cells from subjects without a tumor or in non-tumor cells from the patient. To determine a relative amount of a marker or marker set, the amount of the marker or marker set is determined for at least 1, or 2, 3, 4, 5, or more samples, e.g., 7, 10, 15, 20 or 50 or more samples in order to establish a baseline, prior to the determination of the expression level for the sample in question. To establish a baseline measurement, the mean amount or level of each of the markers or marker sets assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker or marker sets in question. The amount of the marker or marker set determined for the test sample (e.g., absolute level of expression) is then divided by the baseline value obtained for that marker or marker set. This provides a relative amount and aids in identifying abnormal levels of marker protein activity.

[0115] Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences corresponding to one or more markers of the - 41 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 invention. The probe can comprise a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Probes can be used as part of a diagnostic test kit for identifying cells or tissues which express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been altered.

[0116] Primers or nucleic acid probes comprise a nucleotide sequence complementary to a specific marker or an altered region thereof and are of sufficient length to selectively bind or hybridize with a marker gene or nucleic acid associated with a marker gene, e.g., they can bind to the nucleic acid with base sequence specificity and remain bound, after washing. Primers and probes can be used to aid in the isolation and sequencing of marker nucleic acids. In one embodiment, the primer or nucleic acid probe, e.g., a substantially purified oligonucleotide, an isolated nucleic acid, comprises a region having a nucleotide sequence which binds, e.g., hybridizes, e.g., under stringent conditions, to about 5 to 15, 10 to 25, 15 to 50, 20 to 100, 50 to 350 or 500 or more consecutive nucleotides of a marker gene or a region comprising an alteration in a marker gene or transcript therefrom, e.g., a mutated codon described in the disclosure above, in Table 2, and the Examples, or a complement thereof. In another embodiment, the primer or nucleic acid probe is capable of hybridizing to a marker nucleic acid corresponding to a marker gene described herein, e.g., IDH1 or IDH2, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 3, 4, 5 or 6, or a complement of any of the foregoing. For example, a primer or nucleic acid probe comprising a nucleotide sequence of at least about 10 consecutive nucleotides, at least about 15 consecutive nucleotides, about 10 to 25 consecutive nucleotides, about 20 to 40 consecutive nucleotides, about 30 to 60 consecutive nucleotides, or having from about 15 to about 30 nucleotides set forth in any of SEQ ID NOS: 3 or 5, an open reading frame of IDH1 or IDH2, e.g. SEQ ID NOs:4 or 6, or a complement of any of the foregoing are provided by the invention. Primers or nucleic acid probes having a sequence of more than about 25, 40 or 50 nucleotides of SEQ ID NOs:3, 4, 5 or 6, e.g., targeting a IDH1 or IDH2 sequence portion comprising a mutated codon described in the disclosure above, in Table 2, and in the Examples, or having a sequence comprising SEQ ID NOs:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or a complement of any of the foregoing are also within the scope of the invention. In another embodiment, a primer or nucleic acid probe can have a sequence at least 70%, at least 75%, 80% or 85%, or at least, 90%, 95% or 97% identical to the nucleotide sequence of IDH1, IDH2, or a complement of any of the foregoing. Nucleic acid analogs can be used as binding sites for hybridization. An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al., Nature 363:566568 (1993); U.S. Pat. No.5,539,083). - 42 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0117] In some embodiments, a nucleic acid probe can be designed to bind to the wild type sequence, so the presence of an alteration in that region can cause a decrease, e.g., measurable decrease, in binding or hybridization by that probe. In another embodiment, a nucleic acid probe can be designed to bind to a mutant sequence, e.g., a mutated codon described in the disclosure above, in Table 2, and in the Examples, so the presence of an alteration in that region can cause an increase in binding or hybridization by that probe. In other embodiments, a probe and primer set or a primer pair can be designed to bracket a region in a marker that can have an alteration so amplification based on that set or pair can result in nucleic acids which can be sequenced to identify the alteration, as described above.

[0118] Primers or nucleic acid probes can be selected using an algorithm that takes into account binding energies, base composition, sequence complexity, cross-hybridization binding energies, and secondary structure (see Friend et al., International Patent Publication WO 01 / 05935, published Jan.25, 2001; Hughes et al., Nat. Biotech.19:342-7 (2001). Useful primers or nucleic acid probes of the invention bind sequences which are unique for each transcript, e.g., target altered regions and can be used in PCR for amplifying, detecting and sequencing only that particular nucleic acid, e.g., transcript or altered transcript. Examples of some marker genes, e.g., IDH1 and / or IDH2, which may be altered in cancer, e.g., hematological cancer, e.g., AML, CMML or MDS, are found in Table 1. Examples of alterations of IDH1 and / or IDH2 associated with favorable outcome to treatment comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof, are described in the Examples and disclosure above, and Table 2. Other alterations are described in reference articles cited herein and in public databases described herein. One of skill in the art can design primers and nucleic acid probes for the markers disclosed herein or related markers with similar characteristics, e.g., markers on the chromosome loci, or alterations in different regions of the same marker gene described herein, using the skill in the art, e.g., adjusting the potential for primer or nucleic acid probe binding to standard sequences, mutants or allelic variants by manipulating degeneracy or GC content in the primer or nucleic acid probe. Computer programs that are well known in the art are useful in the design of primers with the required specificity and optimal amplification properties, such as Oligo version 5.0 (National Biosciences, Plymouth, MN). While perfectly complementary nucleic acid probes and primers can be used for detecting the markers described herein and mutants, polymorphisms or alleles thereof, departures from complete complementarity are contemplated where such departures do not prevent the molecule from specifically hybridizing to the target region. For example, an oligonucleotide primer may have a non-complementary fragment at its 5' end, with the remainder of the primer being complementary to the target region. - 43 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 Alternatively, non-complementary nucleotides may be interspersed into the nucleic acid probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.

[0119] An indication of treatment outcome can be assessed by studying a characteristic of 1 marker, characteristics of markers in a marker set comprising 2 markers, 3 markers or 4 markers, or more, e.g., 5, 6, 7, 8, 9, 10, 15, 20, or 25 markers, or altered portions thereof e.g., marker genes which interact with DNA, signaling pathways or are involved in AML tumorigenesis. In some embodiments, the marker set includes a plurality of chromosome loci, a plurality of marker genes, and / or a plurality of markers of one or more marker genes (e.g., nucleic acid and protein, genomic DNA and mRNA, or various combinations of markers described herein). Markers can be studied in combination with another measure of treatment outcome, e.g., biochemical markers (e.g., M protein level in myeloma, kidney health marker such as proteinuria, serum levels of C-reactive protein or cytokeratin 19, cytokeratin fragment 21-1 (CYFRA21-1) for NSCLC, urine levels of fibrinogen / fibrinogen degradation products for bladder cancer, urine or blood levels of catecholamines for neuroblastoma, serum levels of carbohydrate antigen 19-9 (CA 19-9) or metabolic profiling for pancreatic cancer or blood levels of soluble mesothelin-related peptides (SMRP) in mesothelioma) or histology assessment (e.g., fewer than 5% blast cells in the bone marrow of a leukemia or myeloma patient, number of mitotic figures per unit area, depth measurement of invasion of melanoma tumors, esophageal tumors or bladder tumors).

[0120] Statistical methods can assist in the determination of treatment outcome upon measurement of a characteristic such as an amount of a marker, e.g., measurement of DNA, RNA or protein. The amount of one marker can be measured at multiple timepoints, e.g., before treatment, during treatment, after treatment with an agent, e.g., an NAE inhibitor. To determine the progression of change in expression of a marker from a baseline, e.g., over time, the expression results can be analyzed by a repeated measures linear regression model (Littell, Miliken, Stroup, Wolfinger, Schabenberger (2006) SAS for Mixed Models, 2nd edition. SAS Institute, Inc., Cary, NC)): Equation 1 Yijk^Yij0^Yij0^treatmenit ^dayk^(treatmen*t day )ik^ ^ijkwhere Yijkis the log2 transformed expression (normalized to the housekeeping genes) on the kth day of the jth animal in the ith treatment, Yij0 is the defined baseline log2 transformed expression (normalized to the housekeeping genes) of thejth animal in theith treatment, daykis treated as a - 44 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 categorical variable, and εijkis the residual error term. A covariance matrix (e.g., first-order autoregressive, compound symmetry, spatial power law) can be specified to model the repeated measurements on each animal over time. Furthermore, each treatment time point can be compared back to the same time point in the vehicle group to test whether the treatment value was significantly different from vehicle.

[0121] A number of other methods can be used to analyze the data. For instance, the relative expression values could be analyzed instead of the cycle number. These values could be examined as either a fold change or as an absolute difference from baseline. Additionally, a repeated-measures analysis of variance (ANOVA) could be used if the variances are equal across all groups and time points. The observed change from baseline at the last (or other) time point could be analyzed using a paired t-test, a Fisher exact test (p-value = Σ P(X=x) from x=1 to the number of situations, e.g., alterations, tested that show sensitivity to NAE inhibition) for testing significance of data of small sample sizes, or a Wilcoxon signed rank test if the data is not normally distributed, to compare whether a cancer patient was significantly different from a normal subject.

[0122] A difference in amount from one timepoint to the next or from the tumor sample to the normal sample can indicate prognosis or treatment outcome. A baseline level can be determined by measuring expression at 1, 2, 3, 4, or more times prior to treatment, e.g., at time zero, one day, three days, one week and / or two weeks or more before treatment. Alternatively, a baseline level can be determined from a number of subjects, e.g., normal subjects or patients with the same health status or disorder, who do not undergo or have not yet undergone the treatment, as discussed above. Alternatively, one can use expression values deposited with the Gene Expression Omnibus (GEO) program at the National Center for Biotechnology Information (NCBI, Bethesda, MD). For example, datasets of myeloma mRNA expression amounts sampled prior to proteasome inhibition therapy include GEO Accession number GSE9782, also analyzed in Mulligan, et al. (2006) Blood 109:3177-88 and GSE6477, also analyzed by Chang et al. (2007) Cancer Res.67:292-9. In another example, a dataset of SNP genotype profiling of paired tumor and normal samples from AML patients has the GEO Accession number GSE61323 and can be used to query additional patient populations from the tumor sample data or to establish reference characteristics from the normal sample data. To test the effect of the treatment on the tumor, the expression of the marker can be measured at any time or multiple times after some treatment, e.g., after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months and / or 6 or more months of treatment. For example, the amount of a marker can be measured once after some treatment, or at multiple - 45 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 intervals, e.g., 1-week, 2-week, 4-week or 2-month, 3-month or longer intervals during treatment. In some embodiments, the measurement of a marker during treatment can be compared to the same marker measurement at baseline. In other embodiments, the measurement of a marker during treatment can be compared to the same marker measurement at an earlier timepoint. Conversely, to determine onset of progressive disease after stopping the administration of a therapeutic regimen, the amount of the marker can be measured at any time or multiple times after, e.g., 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months and / or 6 or more months after the last treatment. The measurement of a marker after treatment can be compared to the same marker measurement at the end of treatment. One of skill in the art would determine the timepoint or timepoints to assess the amount of the marker depending on various factors, e.g., the pharmacokinetics of the treatment, the treatment duration, pharmacodynamics of the treatment, age of the patient, the nature of the disorder or mechanism of action of the treatment. A trend toward an unfavorable outcome relative to baseline or a pre-determined standard of expression of a marker of sensitivity to NAE inhibition therapy can indicate a decrease in response of the tumor to the therapy, e.g., increase in resistance. A trend toward a favorable outcome relative to the baseline or a pre-determined standard of expression of a marker of treatment outcome indicates usefulness of the therapeutic regimen or continued benefit of the therapy, and accordingly, treatment with the therapy would be continued.

[0123] Any marker, e.g., nucleic acid or protein corresponding to a marker gene or combination of markers of the invention, or alterations thereof as well as any known markers in combination with the markers of the invention, may be used in the compositions, kits, and methods of the present invention. In general, markers are selected for as great as possible ability to judge mutational status of a marker gene to predict outcome of treatment with a therapeutic regimen comprising an NAE inhibitor, more preferably a combination comprising an NAE inhibitor for example, pevonedistat, or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and a hypomethylating agent, for example azacitidine. For example, the choice of markers are selected for as great as possible difference between the characteristic, e.g., size, sequence, composition or amount of the marker in samples comprising tumor cells and the characteristic, e.g., size, sequence, composition or amount of the same marker in control cells. Although this difference can be as small as the limit of detection of the method for assessing the amount of the marker, in another embodiment, the difference can be at least greater than the standard error of the assessment method. In the case of RNA or protein amount, a difference can be at least 1.5-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 100-, 500-, 1000-fold or greater. - 46 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 “Low” RNA or protein amount can be that expression relative to the overall mean across tumor samples (e.g., from a hematological tumor, e.g., leukemia or myeloma) is low. In the case of amount of DNA, e.g., copy number, the amount is 0, 1, 2, 3, 4, 5, 6, or more copies. A deletion causes the copy number to be 0 or 1; an amplification causes the copy number to be greater than 2. The difference can be qualified by a confidence level, e.g., p < 0.05, p < 0.02, p < 0.01 or lower p-value.

[0124] Selected marker sets can be assembled from the markers provided herein or selected from among markers using methods provided herein and analogous methods known in the art. A way to qualify a new marker for use in an assay of the invention is to correlate a characteristic, e.g., size, sequence, composition or amount in a sample comprising tumor cells with differences in characteristic, e.g., size, sequence, composition or amount (e.g., fold-change from baseline) of a marker, e.g., a marker gene. A useful way to judge the relationship is to calculate the coefficient of determination r2, after solving for r, the Pearson product moment correlation coefficient and / or preparing a least squares plot, using standard statistical methods. For example, a correlation can analyze DNA copy number versus the level of expression of marker, e.g., a marker gene. A gene product can be selected as a marker if the result of the correlation (r2, e.g., the linear slope of the data in this analysis), is at least 0.1- 0.2, at least 0.3- 0.5, or at least 0.6-0.8 or more. Markers can vary with a positive correlation to response, PFS or survival (i.e., change expression levels in the same manner as copy number, e.g., decrease when copy number is decreased). Markers which vary with a negative correlation to copy number (i.e., change expression levels in the opposite manner as copy number levels, e.g., increase when copy number is decreased) provide inconsistent determination of outcome. In another embodiment, marker set can be prepared using a scoring method known in the art (e.g., weighted voting, combination of threshold features (CTF), Cox proportional hazards analysis, principal components scoring, linear predictive score, K-nearest neighbor, etc.), e.g., using expression values deposited with the Gene Expression Omnibus (GEO) program at the National Center for Biotechnology Information (NCBI, Bethesda, MD).

[0125] In embodiments when the compositions, kits, and methods of the invention are used for characterizing treatment outcome in a patient, the marker or set of markers is selected such that a significant result is obtained in at least about 20%, at least about 40%, 60%, or 80%, or in substantially all patients treated with the test agent. The marker or set of markers can be selected such that a positive predictive value (PPV) of greater than about 10% is obtained for the general population and additional confidence in a marker can be inferred when the PPV is coupled with an assay specificity greater than 80%. - 47 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0126] In an example, treatment with a regimen comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof, comprises determining the presence or absence of mutations in IDH1 and / or IDH2 in cancer cells, e.g., one or more mutations described in the disclosure above, in Table 2, and in the Examples. Evaluation of the mutation status of IDH1 and / or IDH2 is then used to determine whether the patient is expected to have a favorable outcome and would benefit from treatment, e.g., treatment with a NAE inhibitor , for example, pevonedistat, or a pharmaceutically acceptable salt thereof, a BCL2 inhibitor, such as venetoclax (ABT-199), and a hypomethylating agent, e.g., azacitidine; or treatment with a BCL2 inhibitor, a hypomethylating agent, and the NAE inhibitor; or other agent(s) or treatments expected to have a similar effect on survival. Evaluation of the mutation status of IDH1 and / or IDH2 can also be used to determine whether a patient is expected to have an unfavorable outcome and would benefit from a cancer therapy other than NAE inhibition therapy or would benefit from an altered NAE inhibition therapy regimen. A patient whose cancer is characterized as having a marker profile indicative of favorable outcome to NAE inhibition therapy, e.g., pevonedistat or a pharmaceutically acceptable salt thereof, will undergo treatment with the therapy and a more aggressive therapy regimen will be identified for a patient with an expected unfavorable outcome.

[0127] Some illustrative methods of treating a hematological cancer, or hematological cancer patient, may include, without limitation, a) measuring at least one characteristic of at least one marker corresponding to the IDH1 gene or the IDH2 gene in a biological sample obtained from the patient; b) identifying at least one mutation in the IDH1 gene and / or the IDH2 gene in the biological sample from the at least one characteristic measured in step a); and c) administering the combination to the patient, wherein the combination comprises treatment with a NAE inhibitor , for example, pevonedistat, or a pharmaceutically acceptable salt thereof, a BCL2 inhibitor, such as venetoclax (ABT-199), and a hypomethylating agent, e.g., azacitidine.

[0128] Some illustrative methods of preventing progression or treating a hematological cancer, or hematological cancer patient, may include, administering a therapeutically effective amount of a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and azacitidine to the hematological cancer patient. In various embodiments, the patient has been identified as having a gain of function mutation in the IDH1 gene and / or the IDH2 gene, for example, in at least a portion of the hematological cancer. The mutation in the IDH1 gene results in the mutation of the IDH1 protein, wherein the mutation is position R132, of SEQ ID NO: 1, and the mutation in the IDH2 gene results in the mutation of the IDH2 protein, selected from the group consisting of position - 48 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 L86, position R140, positions L143-T146, position R172 and position Q316, of SEQ ID NO: 2. In some embodiments, one of the nucleotides at a position selected from the group consisting of 689, 690, and 691of SEQ ID NO:3 is mutated. In other embodiments, one of the nucleotides at a position selected from the group consisting of 394, 395, and 396 of SEQ ID NO:4 is mutated. In some embodiments, one of the nucleotides at a position selected from the group consisting of 420, 421, 422, 582, 583, 584, 678, 679, 680, 1110, 1111, 1112, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601 and 602 of SEQ ID NO:5 is mutated. In other embodiments, one of the nucleotides at a position selected from the group consisting of 256, 257, 258, 418, 419, 420, 514, 515, 516, 946, 947,948, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437 and 438 of SEQ ID NO:6 is mutated. In an embodiment a deletion begins at nucleotide 591, 592 or 593 of SEQ ID NO:5. In another embodiment, a deletion begins at nucleotide 427, 428 or 429 of SEQ ID NO:6. In various embodiments, the hematological tumor cancer is selected from the group consisting of leukemia, lymphoma, myeloma, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML). In related embodiments, the method also includes the steps of:

[0129] a) measuring at least one characteristic of at least one marker corresponding to IDH1 or IDH2 in a biological sample obtained from the patient;

[0130] b) identifying at least one characteristic selected from: (i) a mutation of the IDH1 gene, or the IDH2 gene, (ii) a mutation of an IDH1 nucleotide selected from base 689, 690, and 691of SEQ ID NO:3 or selected from base 394, 395, and 396, of SEQ ID NO:4, (iii) an IDH1 protein having an amino acid mutation selected from R132C, R132G, R132H, or R132S or (iv) a mutation of an IDH2 nucleotide selected from base 420, 421, 422, 582, 583, 584, 678, 679, 680, 1110, 1111, and 1112 of SEQ ID NO:5 or selected from base 256, 257, 258, 418, 419, 420, 514, 515, 516, 946, 947 and 948 of SEQ ID NO:6, (v) a deletion beginning at nucleotide 591, 592 or 593 of SEQ ID NO:5 or beginning at nucleotide 427, 428 or 429 of SEQ ID NO:6, and (vi) an IDH2 protein having an amino acid mutation selected from L86P, Q316L, R140G, R140Q, R172K, and in-frame deletion of L143-T146, resulting in an amino acid sequence comprising SEQ ID NO:17, in the at least one marker measured in step a); and

[0131] c) administering the combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and azacitidine to the patient.

[0132] In practicing the present methods described above, the at least one marker is a nucleic acid or a protein, for example, the at least one marker is a nucleic acid, and also wherein the at least one characteristic is protein or nucleic acid sequence. As described above, in one illustrative embodiment, the at least one characteristic sequence related to the marker genes - 49 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 IDH1 and / or IDH2 is a nucleic acid sequence of the subject’s tumor, ctDNA or cancer cell IDH1 gene or IDH2 gene, which may be determined by any method known to a person skilled in the art, for example, performed using Sanger sequencing or next-generation sequencing (NGS).

[0133] In related embodiments, the hematological cancer comprises mutations in the IDH1 protein having substitutions selected from one or more of: R132C, R132G, R132H, and R132S relative to SEQ. ID NO: 1. In some embodiments, the hematological cancer comprises mutations in the IDH1 protein having substitutions selected from the R132C, R132G, R132H, and R132S relative to SEQ. ID NO: 1, and the hematological tumor is a cancer selected from leukemia, lymphoma and myeloma.

[0134] In related embodiments, the hematological cancer comprises mutations in the IDH2 protein having substitutions selected from one or more of L86P, Q316L, R140G, R140Q, and R172K, or an in-frame deletion of L143-T146 relative to SEQ. ID NO: 2. In some embodiments, the hematological cancer comprises mutations in the IDH2 protein having substitutions selected from the L86P, Q316L, R140G, R140Q, R172K, or an in-frame deletion of L143-T146 relative to SEQ ID NO: 2. and the hematological tumor is a cancer selected from leukemia, lymphoma and myeloma.

[0135] In some illustrative embodiments, the present disclosure provides a method for continuing to treat hematological cancer in a patient with a therapeutic regimen comprising a therapeutically effective amount of a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and a hypomethylating agent, the method comprising: a) obtaining a first biological sample from the patient and a second biological sample from the patient, wherein both samples comprise hematological cancer cells or ctDNA, and wherein the first sample is obtained prior to the second sample and the patient is treated with the therapeutic regimen prior to the second sample; b) measuring at least one characteristic of at least one marker corresponding to an IDH1 gene and / or an IDH2 gene in the two samples; c) comparing the results of the measurements in b); and d) continuing treatment with the therapeutic regimen if the comparison indicates that the hematological cancer cells or ctDNA in the second sample comprises at least one marker gene whose mutational status indicates a favorable outcome, wherein the at least one marker gene is an IDH1 gene and / or an IDH2 gene.

[0136] In some embodiments, practicing the above method the at least one characteristic is selected from the group consisting of size, sequence, composition and amount, - 50 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 and wherein the mutational status of the at least one marker is mutant. In some embodiments, practicing the above method the at least one marker is selected from the group consisting of nucleic acid and protein corresponding to the at least one marker, wherein in some embodiments, the at least one characteristic is sequence of the at least one marker. In various embodiments, the at least one marker is selected from the group consisting of nucleic acid and protein. In illustrative example, of the present methods, the nucleic acid is selected from the group consisting of DNA, ctDNA, mRNA and cDNA and any portion of any of the foregoing, wherein the nucleic acid corresponds to at least one mutation site of the at least one marker gene.

[0137] In some embodiments, the present application provides a use of a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and a hypomethylating agent, for example azacitidine, for the manufacture of a medicament for treating a hematological cancer patient, said treating comprises a) using a mutational status of at least one marker gene in a patient sample comprising hematological cancer cells or ctDNA to select a hematological cancer patient who is expected to have a favorable outcome of treatment with the combination, wherein the mutational status is indicative of the favorable outcome, wherein the at least one marker gene is an IDH1 gene and / or an IDH2 gene; and b) treating the hematological cancer patient expected to have the favorable outcome with the combination. In related embodiments, the mutational status is determined by measuring at least one characteristic of at least one marker associated with the at least one marker gene.

[0138] For example, in some examples, the at least one characteristic is selected from the group consisting of size, sequence, composition, and amount. In practicing the methods described herein, the at least one marker is selected from the group consisting of a nucleic acid, and a protein associated with the at least one marker gene, for example, the nucleic acid is genomic DNA, RNA, mRNA, ctDNA or cDNA of the at least one marker gene. In still further embodiments, the at least one characteristic is a sequence and the at least one marker is a nucleic acid. The present methods encompass determination of the mutational status of the at least one marker gene, wherein the marker gene is mutant. In various embodiments, the use described above can be further executed using the following steps: a) measuring at least one characteristic of at least one marker corresponding to the IDH1 gene and / or the IDH2 gene in a biological sample obtained from the patient; b) identifying at least one mutation in the IDH1 gene and / or the IDH2 gene in the biological sample from the at least one characteristic measured in step a); and - 51 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 c) administering the combination to the patient. In various embodiments, the at least one marker is selected from the group consisting of nucleic acid and protein corresponding to the IDH1 gene and / or the IDH2 gene. Preferably, the at least one marker is nucleic acid, such that, the at least one characteristic is selected from the group consisting of: size, sequence, composition, activity and amount of the nucleic acid or protein. In some embodiments, the the at least one characteristic is sequence, and when the sequence is a sequence of nucleic acid, the nucleic acid is selected from the group consisting of DNA, ctDNA, mRNA and cDNA and a portion of any of the foregoing, and wherein the nucleic acid comprises at least one mutation of the IDH1 gene and / or the IDH2 gene as exemplified in the tables and examples disclosed herein.

[0139] In some embodiments, exemplary mutant marker genes for use in the present methods can include a mutant marker gene that results in the mutation of the codon encoding amino acid R132 of the IDH1 gene. In various embodiments, the mutations to the IDH1 gene can include a mutation of the codon relating to R132, wherein the mutated sequence of the IDH1 results in one or more of the following mutations: R132C, R132G, R132H, and R132S. Exemplary mutant marker genes for use in the present methods can include a mutant marker gene that results in the mutation of the codon encoding amino acid L86, R140, L143-T146, R172, or Q316 of the IDH2 gene. In various embodiments, the mutations to the IDH2 gene can include a mutation of the codon relating to L86, R140, L143-T146, R172, or Q316, wherein the mutated sequence of the IDH2 results in one or more of the following mutations: L86P, Q316L, R140G, R140Q, and R172K,. In the above recited method steps, the methods of use and treatment of a hematological cancer patient contemplates the treatment of a hematological cancer selected from the group consisting of leukemia, lymphoma, myeloma, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).

[0140] Therapeutic Agents

[0141] The markers and marker sets described herein assess the likelihood of favorable outcome of therapy (e.g., sensitivity to a therapeutic agent) in patients, e.g., patients having cancer, e.g., hematological cancer, such as leukemia, lymphoma or myeloma (e.g., acute myelogenous leukemia, myelodysplastic syndrome or chronic myelomonocytic leukemia), based on a characteristic, e.g., size, sequence, composition or amount of a marker or markers of the invention. Using this prediction, the patient can be treated with a therapy regimen best suitable for a favorable outcome.

[0142] In particular, the methods characterize patient cancer treatment outcome with a regimen comprising an NAE inhibitor as described in earlier sections. The agents provided in the - 52 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 present methods can be a single agent or a combination of agents. The methods include combination of NAE inhibition therapy, for example, pevonedistat, or a pharmaceutically acceptable salt thereof, with a BCL2 inhibitor, for example, venetoclax (ABT-199), and a hypomethylating agent, for example, azacitidine. For example, the present methods can be used to determine whether a hematological cancer (e.g., AML, MDS, or CMML) is more likely to benefit from treatment with (a) a regimen that includes a BCL2 inhibitor, a hypomethylating agent, and an NAE inhibitor; or (b) a regimen that includes a BCL2 inhibitor and a hypomethylating agent, but does not include an NAE inhibitor. Useful combinations can include agents that have other mechanisms of action, e.g., an anti-mitotic agent, an alkylating agent, an antimetabolite, or a proteasome inhibitor. In some embodiments, the methods provide for a therapeutic regimen comprising pevonedistat or a pharmaceutically acceptable salt thereof, a BCL2 inhibitor, e.g. venetoclax (ABT-199), and a hypomethylating agent, e.g. azacitidine. In some embodiments, the methods provide for a therapeutic regimen comprising pevonedistat or a pharmaceutically salt thereof, venetoclax (ABT-199), and azacitidine. Illustrative hematological cancers treatable using the methods of the present disclosure may include, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).Other hematological cancers can include, without limitation, myeloma, leukemia, lymphoma, and myelodysplastic syndrome, acute myelogenous leukemia, chronic myelomonocytic leukemia, myelodysplastic syndrome, low blast acute myelogenous leukemia, high risk myelodysplastic syndrome, and chronic myelomonocytic leukemia. In some embodiments, some AML patients may express blasts expressing CD33, CD123 and / or CLEC12a.

[0143] As used herein, the term "proteasome inhibitor" refers to any substance which directly inhibits enzymatic activity of the 20S or 26S proteasome in vitro or in vivo. Examples of proteasome inhibitors are bortezomib, carfilzomib, ixazomib, disulfiram, epigallocatechin-3- gallate, salinosporamid A, ONX0912, CEP-18770, and epoxomicin.

[0144] Other therapeutic agents for use in combination with NAE inhibition therapy include chemotherapeutic agents. A "chemotherapeutic agent" is intended to include chemical reagents which inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable. Chemotherapeutic agents used in the treatment of hematological cancer, such as leukemia, e.g., AML, MDS or CMML include antibiotics, e.g., daunorubicin, adriamycin or idarubicin, or antimetabolites, e.g., pyrimidine analogs, e.g., cytarabine or gemcitabine, and are well known in the art (see e.g., Gilman A.G., et al., The Pharmacological - 53 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 Basis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)), and are typically used to treat neoplastic diseases.

[0145] In some embodiments, the patient with cancer, e.g., a hematological cancer, for example, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML), is administered a combination of pevonedistat or a pharmaceutically acceptable salt thereof and venetoclax (ABT-199), and a hypomethylating agent, for example, azacitidine, when that hematological cancer is characterized by harboring a mutation in the isocitrate dehydrogenase 1 (IDH1) gene, and / or the isocitrate dehydrogenase 2 (IDH2) gene. In some embodiments, patients with AML are ineligible for intensive chemotherapy due to pre-existing comorbidities, such as severe cardiac or pulmonary disorder or disorders identified by clinical laboratory values that make them ineligible for standard-of-care, and / or older age, e.g., ≥75 years old.

[0146] In some embodiments, pevonedistat or a pharmaceutically acceptable salt thereof is administered on each of days 1, 3, and 5 of a 28 day cycle. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is less than or equal to 50 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 37 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 25 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 20 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 15 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 10 mg / m2 to about 30 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 15 mg / m2 to about 30 mg / m2.

[0147] In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 20 mg / m2, which can be given as a 60-minute intravenous infusion.

[0148] In some embodiments, pevonedistat or a pharmaceutically acceptable salt thereof is administered on each of days 1, 3, and 5 of a 21 day cycle. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each - 54 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 of days 1, 3, and 5 of a 21 day cycle is less than or equal to 50 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 37 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 25 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 20 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 15 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 10 mg / m2 to about 30 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 15 mg / m2 to about 35 mg / m2.

[0149] In some embodiments, pevonedistat or a pharmaceutically acceptable salt thereof is administered on each of days 1, 8, and 15 of a 28 day cycle. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 8, and 15 of a 28 day cycle is less than or equal to 100 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 8, and 15 of a 28 day cycle is about 25 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 8, and 15 of a 28 day cycle is about 20 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 8, and 15 of a 28 day cycle is about 15 mg / m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 8, and 15 of a 28 day cycle is about 15 mg / m2 to about 40 mg / m2.

[0150] In some embodiments, the number of cycles of pevonedistat or a pharmaceutically acceptable salt thereof administered to patients is at least two cycles, at least three cycles, at least four cycles, at least five cycles, at least six cycles, at least seven cycles, at least eight cycles, at least nine cycles or at least ten cycles. In some embodiments, a patient is administered two to twenty-five cycles of pevonedistat or a pharmaceutically acceptable salt thereof. In some embodiments, a patient is administered four to twelve cycles of pevonedistat or a pharmaceutically acceptable salt thereof. In an embodiment, pevonedistat or a pharmaceutically acceptable salt thereof is administered on each of days 1, 3, and 5 of a 28 day cycle at about 20 mg / m2 for at least six cycles. - 55 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0151] The agents disclosed herein may be administered by any route, including intradermally, subcutaneously, orally, intraarterially or intravenously. In one embodiment, administration will be by the intravenous route. Parenteral administration can be provided in a bolus or by infusion.

[0152] In some embodiments, pevonedistat or a pharmaceutically acceptable salt thereof is administered in combination with venetoclax. In some embodiments, a 100 mg venetoclax tablet is administered orally on day 1; a 200 mg venetoclax tablet is administered orally on day 2; and thereafter a 400 mg venetoclax tablet is administered daily on days 3-28 in a first 28-day cycle; and in subsequent cycle(s), a 400 mg venetoclax tablet is administered daily on days 1-28, if tolerated.

[0153] In some embodiments, pevonedistat or a pharmaceutically acceptable salt thereof, and venetoclax, is administered in combination with azacitidine. In some embodiments, azacitidine is administered intravenously or subcutaneously at about 75 mg / m2 on days 1 through 7 of a 28-day cycle; or, at about 75 mg / m2 on days 1 through 5, day 8, and day 9 of a 28-day cycle. In some embodiments, azacitidine is administered intravenously or subcutaneously at about 75 mg / m2 on days 1 through 5, day 8, and day 9 of a 28-day cycle.

[0154] In some embodiments, a patient with a confirmed mutation in IDH1 and / or IDH2 is treated with a combination of pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax, and azacitidine. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day schedule is about 20 mg / m2. In some embodiments, a 100 mg venetoclax tablet is administered orally on day 1; a 200 mg venetoclax tablet is administered orally on day 2; and thereafter a 400 mg venetoclax tablet is administered daily on days 3-28 in a first 28-day cycle; and in subsequent cycle(s), a 400 mg venetoclax tablet is administered daily on days 1-28, if tolerated. In some embodiments, azacitidine is administered intravenously or subcutaneously at about 75 mg / m2 on days 1 through 5, day 8, and day 9 of a 28-day cycle.

[0155] In some embodiments, a hematological cancer patient with a confirmed mutation in IDH1 and / or IDH2 is treated with a combination of pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax, and azacitidine, according to the following regimen: about 20 mg / m2 pevonedistat or a pharmaceutically acceptable salt thereof is administered intravenously on each of days 1, 3, and 5 of a 28 day cycle; about 100 mg venetoclax is administered orally on day 1, about 200 mg venetoclax is administered orally on day 2, and about 400 mg venetoclax is administered on days - 56 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 3 through 28 in a first 28-day cycle, and if tolerated in subsequent cycle(s), about 400 mg venetoclax is administered on days 1-28; and about 75 mg / m2 azacitidine is administered intravenously or subcutaneously on days 1 through 5, day 8, and day 9 of a 28-day cycle.

[0156] The concentration of a disclosed compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic characteristics of the compound(s) employed, and the route of administration. The agent may be administered in a single dose or in repeat doses. Treatments may be administered daily or more frequently depending upon a number of factors, including the overall health of a patient, and the formulation and route of administration of the selected compound(s).

[0157] Electronic Apparatus Readable Arrays

[0158] Electronic apparatus, including readable arrays comprising at least one predictive marker of the present invention is also contemplated for use in conjunction with the methods of the invention. As used herein, “electronic apparatus readable media” refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus. As used herein, the term “electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention and monitoring of the recorded information include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems. As used herein, “recorded” refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the markers of the present invention.

[0159] For example, microarray systems are well known and used in the art for assessment of samples, whether by assessment of gene expression (e.g., DNA detection, RNA detection, protein detection), or metabolite production, for example. Microarrays for use according to the invention include one or more probes of predictive marker(s) of the invention characteristic of response and / or non-response to a therapeutic regimen as described herein. In one embodiment, the microarray comprises one or more probes corresponding to one or more of markers selected from the group consisting of markers which demonstrate increased expression - 57 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 in short term survivors, and genes which demonstrate increased expression in long term survivors in patients. A number of different microarray configurations and methods for their production are known to those of skill in the art.

[0160] A microarray thus comprises one or more probes corresponding to one or more markers identified herein, e.g., those indicative of treatment outcome, e.g., to identify wild type marker genes, normal allelic variants and alterations of marker genes. The microarray can comprise probes corresponding to, for example, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, or at least 100, markers and / or alterations thereof indicative of treatment outcome. The microarray can comprise probes corresponding to one or more markers as set forth herein. Still further, the microarray may comprise complete marker sets as set forth herein and which may be selected and compiled according to the methods set forth herein. The microarray can be used to assay expression of one or more predictive markers or predictive marker sets in the array. In one example, the array can be used to assay more than one predictive marker or marker set expression in a sample to ascertain an expression profile of markers in the array. In this manner, up to about 44,000 markers can be simultaneously assayed for expression. This allows an expression profile to be developed showing a battery of markers specifically expressed in one or more samples. Still further, this allows an expression profile to be developed to assess treatment outcome.

[0161] The array is also useful for ascertaining differential expression patterns of one or more markers in normal and abnormal (e.g., sample, e.g., tumor) cells. This provides a battery of markers that could serve as a tool for ease of identification of treatment outcome of patients. Further, the array is useful for ascertaining expression of reference markers for reference expression levels. In another example, the array can be used to monitor the time course of expression of one or more markers in the array.

[0162] In addition to such qualitative determination, the invention allows the quantification of marker expression. Thus, predictive markers can be grouped on the basis of marker sets or outcome indications by the amount of the marker in the sample. This is useful, for example, in ascertaining the outcome of the sample by virtue of scoring the amounts according to the methods provided herein.

[0163] Reagents and Kits

[0164] The invention also encompasses kits for assaying a characteristic, e.g., size, sequence, composition or amount, of a marker, e.g., polypeptide or nucleic acid corresponding to a marker gene of the invention in a biological sample (e.g., a bone marrow or blood sample, - 58 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 tumor biopsy or a reference sample). Such kits can be used, e.g., in the methods described herein, to determine mutational status of at least one marker gene for treating a patient who will experience a favorable outcome, e.g., after a treatment regimen comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof. For example, the kit can comprise a probe or reagent capable of detecting a genomic DNA segment, ctDNA, a polypeptide or a transcribed RNA corresponding to a marker of the invention or an alteration of a marker gene in a biological sample and means for determining the amount of the genomic DNA segment, the ctDNA, the polypeptide or the mRNA in the sample. Suitable reagents for binding with a marker protein include antibodies, antibody derivatives, antibody fragments, and the like. Suitable reagents for binding with a marker nucleic acid (e.g., a genomic DNA, a ctDNA, an mRNA, a spliced mRNA, a cDNA, or the like) include complementary nucleic acids. A label can be directly attached to the marker binding agent, e.g., probe, e.g., nucleic acid reagent such as a probe or primer or protein reagent, such as a specific binding agent or antibody, or a secondary reagent can comprise a label for indirect labeling. The kit can also contain a control or reference sample or a series of control or reference samples which can be assayed and compared to the test sample. For example, the kit may have a positive control sample, e.g., including one or more markers or alterations described herein, or reference markers, e.g. housekeeping markers to standardize the assay among samples or timepoints or reference genomes, e.g., from subjects without tumor e.g., to establish diploid copy number baseline or reference expression level of a marker. By way of example, the kit may comprise fluids (e.g., buffer) suitable for annealing complementary nucleic acids or for binding an antibody with a protein with which it specifically binds and one or more sample compartments. The kit of the invention may optionally comprise additional components useful for performing the methods of the invention, e.g., a sample collection vessel, e.g., a tube, and optionally, means for optimizing the amount of marker detected, for example if there may be time or adverse storage and handling conditions between the time of sampling and the time of analysis. For example, the kit can contain means for increasing the number of tumor cells in the sample, as described above, a buffering agent, a preservative, a stabilizing agent or additional reagents for preparation of cellular material or probes for use in the methods provided; and detectable label, alone or conjugated to or incorporated within the provided probe(s). In one exemplary embodiment, a kit comprising a sample collection vessel can comprise e.g., a tube comprising anti-coagulant and / or stabilizer, e.g., an RNA stabilizer, as described above, or known to those skilled in the art. The kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate). For marker sets, the kit can comprise a marker set array or chip for use in detecting - 59 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 the biomarkers. Kits also can include instructions for interpreting the results obtained using the kit. The kit can contain reagents for detecting one or more markers, e.g., 2, 3, 4, 5, or more markers described herein.

[0165] In one embodiment, the kit comprises a probe to detect at least one nucleic acid marker, e.g., a marker indicative of treatment outcome (e.g., upon NAE inhibitor treatment). In an exemplary embodiment, the kit comprises a nucleic acid probe to detect a marker nucleic acid corresponding to a marker gene described herein. The marker nucleic acid can be selected from the group consisting of SEQ ID NO: 3 or 5 and SEQ ID NO: 4 or 6 listed in Table 1, a portion of a marker nucleic acid comprising a sequence or mutated codon listed in in the disclosure above, Table 2, and in the Examples, and a complement of any of the foregoing. In some embodiments, the kit comprises a probe to detect a nucleic acid corresponding to a marker gene selected from the group consisting of IDH1 and / or IDH2. In another embodiment, a kit comprises a probe to detect wild type IDH1 and a probe to detect a genetic alteration, e.g., a mutation or truncation, in IDH2. In another embodiment, a kit comprises a probe to detect wild type IDH2 and a probe to detect a genetic alteration, e.g., a mutation or truncation, in IDH1. In another embodiment, a kit comprises a probe to detect a genetic alteration, e.g., a mutation or truncation, in IDH1 and a probe to detect a genetic alteration, e.g., a mutation or truncation, in IDH2. For kits comprising nucleic acid probes, e.g., oligonucleotide-based kits, the kit can comprise, for example: one or more nucleic acid reagents such as an oligonucleotide (labeled or non-labeled) which hybridizes to a nucleic acid sequence corresponding to a marker of the invention, optionally fixed to a substrate; and can optionally further comprise labeled oligonucleotides not bound with a substrate, a primer, a pair of PCR primers, e.g., useful for amplifying a nucleic acid molecule corresponding to a marker of the invention, molecular beacon probes, a marker set comprising oligonucleotides which hybridize to at least two nucleic acid sequences corresponding to markers of the invention, and the like. The kit can contain an RNA- stabilizing agent.

[0166] PCR-based kits to measure IDH1 and IDH2 mutations in blood and bone marrow samples from AML patients are available as companions to some AML treatments. The Abbott REALTIME IDH1 assay measures five single nucleotide variants at R132 of IDH1 for TIBSOVO® (ivosidenib) or with REZLIDHIATM(olutasidenib). Abbott REALTIME IDH2 assay measures four single nucleotide variants at R140 and five single nucleotide variants at R172 of IDH2.

[0167] In another embodiment, a kit to assay mutational status of a protein marker of the invention can comprise one or more reagents to measure the activity of the protein. For - 60 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 example, the kit may comprise a reporter gene for use in the methods described herein. In another example, the kit may comprise a substrate of an enzyme, e.g., isocitrate.

[0168] In another embodiment, the kit comprises a probe to assay for a characteristic, e.g., size, sequence, composition or amount, of a protein marker of the invention. For kits comprising protein probes, e.g., ligand or antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention, e.g., having a sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO:2 or to a genetically altered form(s) thereof, e.g., a mutation in the sequence or a truncation, e.g., comprising SEQ ID NO:17 or as described in the disclosure above, Table 2, and the Examples; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label. The kit can contain a protein stabilizing agent. The kit can contain reagents to reduce the amount of non- specific binding of non-marker material from the sample to the probe. Examples of reagents to reduce non-specific binding include nonioinic detergents, non-specific protein containing solutions, such as those containing albumin or casein, or other substances known to those skilled in the art.

[0169] To generate antibodies for use in the methods described herein or for providing in the kits and compositions described herein or for performing the methods described herein, an isolated polypeptide corresponding to a marker gene of the invention, or a fragment or mutant thereof, can be used as an immunogen. For example, an immunogen typically is used to prepare antibodies by immunizing a suitable (i.e., immunocompetent) host such as a rabbit, sheep, goat, mouse, or other mammal, chicken or invertebrate. In still a further aspect, the invention provides monoclonal antibodies or antigen binding fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, an amino acid sequence encoded by a cDNA of the present invention, a fragment of at least 8, 10, 12, 15, 20 or 25 consecutive amino acid residues of an amino acid sequence of the present invention, e.g., a fragment comprising the mutant amino acid sequence, e.g., SEQ ID NO:17 or as described in the disclosure above, Table 2, and the Examples, or comprising a region beyond a truncation in a marker protein, an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4) and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the - 61 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 present invention, or a complement thereof, under conditions of hybridization of 6X SSC at 45 ^C and washing in 0.2 X SSC, 0.1% SDS at 65 ^C. The monoclonal antibodies can be human, humanized, chimeric and / or non-human antibodies. An appropriate immunogenic preparation can contain, for example, recombinantly-expressed or chemically-synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.

[0170] Methods for making human antibodies are known in the art. One method for making human antibodies employs the use of transgenic animals, such as a transgenic mouse. These transgenic animals contain a substantial portion of the human antibody producing genome inserted into their own genome and the animal’s own endogenous antibody production is rendered deficient in the production of antibodies. Methods for making such transgenic animals are known in the art. Such transgenic animals can be made using XENOMOUSETMtechnology or by using a "minilocus" approach. Methods for making XENOMICETMare described in U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598 and 6,075,181, which are incorporated herein by reference. Methods for making transgenic animals using the "minilocus" approach are described in U.S. Pat. Nos.5,545,807, 5,545,806 and 5,625,825; also see International Publication No. WO93 / 12227, which are each incorporated herein by reference.

[0171] Antibodies include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention or a genetically altered form thereof, e.g., a mutated amino acid or a truncation of the protein expressed by a marker gene of the invention. A molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Polyclonal and monoclonal antibodies can be produced by a variety of techniques, including conventional murine monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975) the human B cell hybridoma technique (see Kozbor et al., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see Cole et al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985) or trioma techniques. See generally, Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Current Protocols in Immunology, Coligan et al. ed., John Wiley & Sons, New York, 1994. Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma - 62 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.

[0172] If desired, the antibody molecules can be harvested or isolated from the host (e.g., from the blood or serum of the host) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. Alternatively, antibodies specific for a protein or polypeptide of the invention can be selected or (e.g., partially purified) or purified by, e.g., affinity chromatography to obtain substantially purified and purified antibody. By a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those of the desired protein or polypeptide of the invention, at most 20%, at most 10%, or at most 5% (by dry weight) of the sample is contaminating antibodies. A purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein or polypeptide of the invention.

[0173] An antibody directed against a polypeptide corresponding to a marker of the invention (e.g., a monoclonal antibody) can be used to detect the marker (e.g., in a cellular sample) in order to evaluate the level and pattern of expression of the marker. The antibodies can also be used diagnostically to monitor protein levels in tissues or body fluids (e.g. in a blood sample or urine) as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ^-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include125I,131I,35S or3H.

[0174] Accordingly, in one aspect, the invention provides substantially purified antibodies or fragments thereof, and non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence encoded by a marker identified herein. The substantially purified antibodies of the invention, or fragments thereof, can be human, non-human, chimeric and / or humanized antibodies. - 63 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0175] The invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use. Still another aspect of the invention is a prognostic composition comprising a probe of the invention and a pharmaceutically acceptable carrier. In one embodiment, the prognostic composition contains an antibody of the invention, a detectable moiety, and a pharmaceutically acceptable carrier.

[0176] Use of Information

[0177] In one method, information, e.g., about the mutational status of a patient’s cancer, e.g., the patient’s marker(s) characteristic, e.g., size, sequence, composition or amount (e.g., the result of evaluating a marker or marker set described herein), or about whether a patient is expected to have a favorable outcome, is provided (e.g., communicated, e.g., electronically communicated) to a third party, e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company). For example, choice of medical procedure, whether to pay for a medical procedure, payment by a reimbursing party, or cost for a service or insurance can be function of the information. E.g., the third party receives the information, makes a determination based at least in part on the information, and optionally communicates the information or makes a choice of procedure, payment, level of payment, coverage, etc., based on the information. In the method, the characteristic, e.g., size, sequence, composition or amount, of a marker or a marker set selected from or derived from Table 2and / or described herein above and in the Examples is determined. For example, an entity, e.g., a hospital, care giver, government entity, or an insurance company or other entity which pays for, or reimburses medical expenses, can use the result of a method described herein to determine whether a party, e.g., a party other than the subject patient, will pay for services (e.g., a particular therapy) or treatment provided to the patient. In an embodiment, the method further comprises paying for the procedure wherein the patient has a favorable outcome to therapy comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically active salt thereof. In another embodiment, the method comprises paying for a procedure comprising treatment with pevonedistat, venetoclax and azacitidine.

[0178] In one embodiment, a premium for insurance (e.g., life or medical) is evaluated as a function of information about one or more marker expression or activity levels, e.g., a marker or marker set, e.g., a level of expression or enzymatic activity associated with treatment outcome (e.g., the informative amount). For example, premiums can be increased (e.g., by a certain percentage) if the marker genes of a patient or a patient’s marker set described herein have different characteristic, e.g., size, sequence, composition or amount between an insured candidate (or a candidate seeking insurance coverage) and a reference value (e.g., a non-afflicted person) or a reference sample, e.g., matched control. Premiums can also be scaled depending on - 64 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 the result of evaluating a marker or marker set described herein. For example, premiums can be assessed to distribute risk, e.g., as a function of marker, e.g., the result of evaluating a marker or marker set described herein. In another example, premiums are assessed as a function of actuarial data that is obtained from patients that have known treatment outcomes.

[0179] Information about marker characteristic, e.g., size, sequence, composition or amount, e.g., the result of evaluating a marker or marker set described herein, can be used, e.g., in an underwriting process for life insurance. The information can be incorporated into a profile about a subject. Other information in the profile can include, for example, date of birth, gender, marital status, banking information, credit information, children, and so forth. An insurance policy can be issued as a function of the information on marker characteristic, e.g., size, sequence, composition or amount, e.g., the result of evaluating a marker or marker set described herein, along with one or more other items of information in the profile. For example, a first entity, e.g., an insurance company, can use the outcome of a method described herein to determine whether to continue, discontinue, enroll an individual in an insurance plan or program, e.g., a health insurance or life insurance plan or program.

[0180] In one aspect, the disclosure features a method of providing data. The method includes providing data described herein, e.g., generated by a method described herein, to provide a record, e.g., a record described herein, to proceed a payment. In some embodiments, the data are provided by computer, compact disc, telephone, facsimile, email, or letter. In some embodiments, the data are provided by a first party to a second party. In some embodiments, the first party is selected from a healthcare provider, a treating physician, a health maintenance organization (HMO), a hospital, a governmental entity, or an entity which sells or supplies the drug. In some embodiments, the second party is a third party payor, an insurance company, employer, employer-sponsored health plan, HMO, or governmental entity. In some embodiments, the first party is selected from a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is a governmental entity. In some embodiments, the first party is selected from a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is an insurance company.

[0181] In another aspect, the disclosure features a record (e.g., computer readable record) which includes a list and value of characteristic, e.g., size, sequence, composition or amount for the marker or marker set for a patient. In some embodiments, the record includes more than one value for each marker. - 65 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0182] The present invention will now be illustrated by the following Examples, which are not intended to be limiting in any way. EXAMPLES Example 1. Phase 2 clinical study of Pevonedistat and Venetoclax Combined With Azacitidine.

[0183] The clinical trial (NCT04266795) centered on a study administering pevonedistat (PEV) with venetoclax (VEN) combined with azacitidine (AZA) in adults unable to receive intensive chemotherapy. The main aim was to see how the combination of PEV+ VEN+ AZA compares to VEN + AZA in adults recently diagnosed with AML who are unable to be treated with intensive chemotherapy. The Primary objectives included disease response, measured as event free survival (EFS) up to 36 months. Secondary objectives included (1) Overall Survival (OS) ( Up to 36 months ], (2)Thirty-day Mortality Rate [ Time Frame: Day 30) (3) Sixty-day Mortality Rate (Time Frame: Day 60); (4) Percentage of Participants with Complete Remission (CR), (Time Frame: Up to 36 months); (5) Percentage of Participants with Composite Complete Remission (CCR) (Time Frame: Up to 36 months); (6) Overall Response Rate (ORR) (Time Frame: Up to 36 months); (7) Percentage of Participants with CR + CRh (Time Frame: Up to 36 months); (8) Percentage of Participants with Leukemia Response [ Time Frame: Up to 36 months (9) Duration of CR and CRi (Time Frame: Up to 36 months); (10) Time to First CR, CRi and PR (Time Frame: Up to 36 months); and (11) Plasma Concentration of Pevonedistat. Patients ≥60 years with treatment-naïve AML, unfit for standard induction therapy.

[0184] Introduction. Current therapy in acute myeloid leukemia (AML) is inadequate. Although some progress has been made in this disease, the prognosis for older patients, deemed unfit to receive intensive chemotherapy, remains very poor. The use of hypomethylating agents (HMAs) as alternative induction therapies for these patients has become commonplace. Two large randomized studies reported higher rates of remission for older patients treated with 5- azacitidine (AZA) compared with conventional care approaches, which included supportive care. Considering the widespread use of AZA in older patients who are not candidates for chemotherapy, combination studies with promising new agents are actively enrolling.

[0185] Scientists have previously reported the therapeutic potential of single-agent pevonedistat (PEV) (previously TAK-924 / MLN4924) in patients with AML. PEV is a small molecule inhibitor of the NEDD8 activating enzyme (NAE), which processes Neural Cell Developmentally-Downregulated 8 (NEDD8) for binding to target substrates. The best characterized NAE targets in cells are the Cullin-RING E3 ubiquitin ligases (CRLs), which - 66 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 direct the degradation of specific substrates (e.g., p27, CDT1, Nrf-2) through the proteasome. In response to PEV treatment, impaired NAE activity leads to CRL substrate accumulation, causing anti-proliferative effects in AML. A variety of mechanisms are implicated in driving these effects, including disruption of cellular redox via stabilization of pIKB (a critical mediator of cell killing), DNA replication, and cell cycle arrest. In a phase 1b study of patients with relapsed / refractory AML and MDS (myelodysplastic syndrome), PEV was administered as a 1- hour IV infusion on days 1, 3, and 5 (schedule A, n = 27) or days 1, 4, 8, and 11 (schedule B, n = 26) every 21 days. The maximum tolerated doses (MTDs) for schedules A and B were 59 and 83 mg / m2, respectively. On schedule A, elevation of alanine aminotransferase (ALT) / aspartate aminotransferase (AST) was dose limiting. Multi-organ failure (MOF) was dose limiting on schedule B. Overall response rate (ORR) in patients treated at or below the MTD was 17% (4 / 23, 2 complete remissions [CRs], 2 partial remissions [PRs]) for schedule A and 10% (2 / 19, 2 PRs) for schedule B. To identify clinically effective PEV combinations, a high-throughput viability screen in AML cells confirmed that combined treatment using PEV with either decitabine or AZA was synergistically lethal by Combination Index and Blending Synergy Analysis. In the case of AZA, combined treatment with PEV significantly increased DNA damage and cell death when compared with either agent alone, as measured by immunoblotting and flow cytometry analysis of cell cycle distributions. In vivo studies were performed in AZA-resistant HL-60 and THP-1 xenografts. Although the doses of PEV and AZA would be sub-therapeutic if used as single-agent treatment, the combination led to complete and sustained tumor regression in these models. The mechanisms underlying the observed synergistic effects are currently under investigation. Considering the promising clinical data for PEV as a single agent and its enhanced antitumor activity when combined with AZA in laboratory models, we conducted a phase 2 trial of PEV combined with VEN and AZA for patients with AML, deemed unfit to receive intensive chemotherapy.

[0186] Methods. Pevonedistat 20 mg / m2was administered as a 60-minute intravenous (IV) infusion on Days 1, 3, and 5 in each 28-day cycle plus venetoclax 100 mg tablet orally on Day 1; 200 mg on Day 2; thereafter, at 400 mg on Day 3 through Day 28 in Cycle 1 (cycle length= 28 days) and 400 mg on Days 1 through 28 in Cycle 2 and beyond if tolerated. If remission was confirmed in Cycle 1 or thereafter, venetoclax 400 mg could be administered on Day 1 through 21 or 28 as per Investigator's discretion, plus azacitidine 75 mg / m2IV or subcutaneous (SC) dosing on Day 1 through 7 or Days 1 through 5, Days 8, and 9 in each cycle.

[0187] Participants (Arm A) would receive either PEV+ VEN + AZA or (Arm B) VEN + AZA in 28-day treatment cycles. Bone marrow samples (biopsy) were to be collected - 67 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 throughout the study. PEV was given as an intravenous (IV) infusion and AZA will be given through IV or subcutaneous (under the skin). VEN was to be administered as an oral tablet. Study treatments could continue as long as the participant is receiving benefit from it.

[0188] Patients: Eligible patients were ≥18 years old with untreated AML, considered unlikely to benefit from standard induction defined by ≥1 of the following: Has morphologically confirmed diagnosis of AML (World Health Organization [WHO] criteria 2008). Participants may have newly diagnosed primary de novo AML or secondary AML (sAML), defined as AML after myelodysplastic syndromes (MDS) or myeloproliferative neoplasm (MPN), or therapy- related AML (t-AML) following cytotoxic therapy, and / or radiotherapy for a malignant or nonmalignant disease. Patients may also be unfit for treatment with a standard arabinosylcytosine (Ara-C) and anthracycline induction regimen due to age or co-morbidities defined by 1 of the following:≥75 years of age. OR≥18 to <75 years of age with at least one of the following:

[0189] The patient has a Eastern Cooperative Oncology Group (ECOG) performance status of 2 or 3.

[0190] Severe cardiac disorder (e.g., congestive heart failure requiring treatment, ejection fraction ≤50%, or chronic stable angina).

[0191] Severe pulmonary disorder (e.g., carbon monoxide lung diffusion capacity ≤65% or forced expiratory volume in 1 second ≤65%).

[0192] Creatinine clearance (CrCl) <45 mL / min (but ≥30 mL / min as part of general eligibility criteria).

[0193] Hepatic disorder with total bilirubin >1.5 times the upper limit of the normal range (ULN).

[0194] Has clinical laboratory values within the following parameters (repeat within 3 days before the first dose of study drug if laboratory values used for randomization were obtained more than 3 days before the first dose of study drug): a. Total bilirubin ≤1.5 times the ULN except in participants with Gilbert's syndrome. Participants with Gilbert's syndrome may enroll with direct bilirubin ≤3 times the ULN of the direct bilirubin. Elevated indirect bilirubin due to posttransfusion hemolysis is allowed. b. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤3.0 times the ULN. c. Creatinine clearance (CrCl) ≥30 mL / min (calculated by the Modification of Diet in Renal Disease [MDRD] Study equation). d. Albumin >2.7 g / dL. - 68 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 e. White blood cell (WBC) count <25 × 10^9 / L. Participants who are cytoreduced with leukapheresis or with hydroxyurea may be enrolled if they meet the eligibility criteria before starting therapy.

[0195] Exclusion Criteria:

[0196] Patients were excluded if one or more of the following applied:

[0197] Has history of MPN with BCR-ABL1 translocation or AML with BCR-ABL1 translocation.

[0198] Has genetic diagnosis of acute promyelocytic leukemia.

[0199] Is eligible for intensive chemotherapy and / or allogeneic stem cell transplantation.

[0200] Has extramedullary AML without evidence of bone marrow involvement.

[0201] Had prior treatment with hypomethylating agents for AML (hypomethylating agent treatment for prior MDS is not exclusionary).

[0202] Has clinical evidence of or history of central nervous system involvement by AML.

[0203] Had diagnosed or treated for another malignancy (except for adequately treated carcinoma in situ of any organ or nonmelanoma skin cancer) within 1 year before randomization or previously diagnosed with another malignancy and have any evidence of residual disease that may compromise the administration of pevonedistat, venetoclax or azacitidine. Prior MDS is also allowed, but the participant cannot have received treatment for MDS within 14 days before first dose of any study drug.

[0204] Has a WBC count ≥25 × 109 / L

[0205] Has uncontrolled human immunodeficiency virus (HIV) infection. Note: Known HIV positive participants who meet the following criteria will be considered eligible:

[0206] A. Cluster difference 4 (CD4) count >350 cells / mm^3.

[0207] B. Undetectable viral load.

[0208] C. Maintained on modern therapeutic regimens utilizing non-cytochrome P (CYP)-interactive agents.

[0209] D. No history of acquired immune deficiency syndrome (AIDS)-defining opportunistic infections.

[0210] Participant is known to be positive for hepatitis B or C infection, with the exception of those with an undetectable viral load within 3 months (hepatitis B or C testing is not required for eligibility assessment).

[0211] Has hepatic cirrhosis. - 69 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0212] Has uncontrolled coagulopathy or bleeding disorder.

[0213] Has high blood pressure which cannot be controlled by standard treatments.

[0214] Has prolonged rate QTc interval ≥500 msec, calculated according to institutional guidelines.

[0215] Has left ventricular ejection fraction (LVEF) <40%, based on echocardiogram or multi gated acquisition (MUGA) scan at screening (data to be available within last 3 months of screening).

[0216] As infection is a common feature of AML, participants with active infection are permitted to enroll provided that the infection is under control and no signs of systemic inflammatory response beyond low grade fever that makes participant clinically unstable in the opinion of the investigator. Participants with uncontrolled infection shall not be enrolled until infection is treated and brought under control.

[0217] All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

[0218] Study Design: This open-label, phase 2 study was conducted at 57 sites across the globe. The primary objectives were to see how the combination of pevonedistat + venetoclax + azacitidine compares to venetoclax + azacitidine in adults recently diagnosed with AML who are unable to be treated with intensive chemotherapy. Secondary objectives included descriptions of PEV pharmacokinetics (PK) in whole blood and a preliminary assessment of anti-tumor activity. The study enrolled approximately 164 patients. Participants were to be randomly assigned in 1:1 ratio to one of the two treatment groups in 28-day treatment cycles and which were to remain disclosed to the patient and study doctor during the study:

[0219] Pevonedistat 20 mg / m2+ Venetoclax 400 mg (ramp-up dose, Cycle 1 only: 100-400mg) + Azacitidine 75 mg / m2

[0220] Venetoclax 400 mg (ramp-up dose, Cycle 1 only: 100-400 mg) + Azacitidine 75 mg / m2

[0221] Cycles were repeated every 28 days, Bone marrow samples (biopsy) will be collected throughout the study. Pevonedistat will be given as an intravenous (IV) infusion and Azacitidine will be given through IV or subcutaneous (under the skin). VEN will be administered as an oral tablet. Study treatments may continue as long as the participant is receiving benefit from it. Participants may choose to stop treatment at any time. The study will enroll approximately 164 patients. Participants will be randomly assigned in 1:1 ratio to one of - 70 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 the two treatment groups in 28-day treatment cycles and which will remain disclosed to the patient and study doctor during the study:

[0222] PEV 20 mg / m2+ VEN 400 mg (ramp-up dose, Cycle 1 only: 100-400mg) + AZA 75 mg / m2

[0223] VEN 400 mg (ramp-up dose, Cycle 1 only: 100-400 mg) + AZA 75 mg / m2

[0224] This multi-center trial will be conducted worldwide. The overall time to participate in this study is approximately 3 years. Participants will attend the end-of-treatment visit 30 days after the last dose of study drug or before the start of subsequent anti-neoplastic therapy if that occurs sooner.

[0225] Experimental Arm A (PEV + VEN + AZA):

[0226] PEV dosed at 20 mg / m2will be administered as a 60-minute intravenous (IV) infusion administered on Days 1, 3, and 5 in each 28-day cycle plus VEN 100 mg tablet orally on Day 1; 200 mg on Day 2; and thereafter, at 400 mg on Day 3 through Day 28 in Cycle 1 (cycle length= 28 days) and 400 mg on Days 1 through 28 in Cycle 2 and beyond if tolerated. If remission is confirmed in Cycle 1 or thereafter, VEN 400 mg can be administered on Day 1 through 21 or 28 as per Investigator's discretion, plus AZA at 75 mg / m2intravenous (IV) or subcutaneous (SC) dosing on Day 1 through 7 or Days 1 through 5, Days 8, and 9 in each cycle.

[0227] Experimental Arm B (VEN + AZA):

[0228] VEN will be administered as a 100 mg tablet orally on Day 1; 200 mg on Day 2; thereafter, at 400 mg on Day 3 through Day 28 in Cycle 1 (cycle length= 28 days) and 400 mg on Days 1 through 28 in Cycle 2 and beyond if tolerated. If remission is confirmed in Cycle 1 or thereafter, VEN dosed at 400 mg can be administered on Day 1 through 21 or 28 as per Investigator's discretion, plus AZA 75 mg / m2IV or SC dosing on Days 1 through 7 or Days 1 through 5, Days 8, and 9 in each cycle.

[0229] Safety and efficacy assessments.

[0230] Patient demographics and medical history were recorded at baseline. Adverse event (AE) assessments, physical examination, vital signs, and ECOG PS were documented at baseline and on day 1 of subsequent cycles for the duration of the study. Safety was assessed from informed consent to 30 days after final doses of study therapy. Treatment-emergent (all- cause) AEs were graded according to the National Cancer Institute’s Common Terminology Criteria for AEs, version 4.03.20 Patients with AML were assessed for efficacy according to published International Working Group criteria.21

[0231] Primary and Secondary Outcome Measures Primary Outcome Measures: - 71 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0232] Event-Free Survival (EFS) [ Time Frame: Up to 36 months ]. EFS is defined as time from study randomization to date of failure to achieve complete remission (CR) / CR with incomplete blood count recovery (CRi), relapse from CR / CRi, or death. Assessments of disease response based on criteria: European Leukemia Net (ELN) 2017 guidelines. CR is defined as bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; absolute neutrophil count (ANC)≥1.0×109 / L (1000 / µL); platelet count≥100×109 / L (100,000 / µL). CRi is defined as all CR criteria except for residual neutropenia (<1.0×109 / L [1000 / µL]) or thrombocytopenia (<100×109 / L [100,000 / µL]). For participants who achieved CR / CRi, if relapse is not observed by time of analysis, will be censored at the date of last disease assessment. If fail to achieve CR / CRi, date of treatment failure will be set on day of randomization.

[0233] Secondary Measures:

[0234] Overall Survival (OS) [ Time Frame: Up to 36 months ]. OS is defined as time from randomization to death from any cause. Participants without documentation of death at the time of analysis will be censored at the date last known to be alive.

[0235] Thirty-day Mortality Rate [ Time Frame: Day 30 ]

[0236] Mortality rate is defined as percentage of participants who survive at most 30 days from the first dose of study drug.

[0237] Sixty-day Mortality Rate [ Time Frame: Day 60 ].

[0238] Mortality rate is defined as percentage of participants who survive at most 60 days from the first dose of study drug.

[0239] Percentage of Participants with Complete Remission (CR) [ Time Frame: Up to 36 months ].

[0240] CR rate is defined as the percentage of participants who achieve the CR as evaluated by the investigator. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines. CR is defined as bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC≥1.0×109 / L (1000 / µL); platelet count≥100×109 / L (100,000 / µL).

[0241] Percentage of Participants with Composite Complete Remission (CCR) [ Time Frame: Up to 36 months ].

[0242] CCR rate is defined as the percentage of participants who achieve the CR + CRi as evaluated by the investigator. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines. CR is defined as bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC≥1.0×109 / L - 72 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 (1000 / µL); platelet count≥100×109 / L (100,000 / µL). CRi is defined as all CR criteria except for residual neutropenia (<1.0×109 / L [1000 / µL]) or thrombocytopenia (<100×109 / L [100,000 / µL]).

[0243] Overall Response Rate (ORR) [ Time Frame: Up to 36 months ].

[0244] ORR is defined as the percentage of participants who achieve the CR + CRi + Partial Remission (PR) as evaluated by the investigator. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines. CR is defined as bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC≥1.0×109 / L (1000 / µL); platelet count≥100×109 / L (100,000 / µL). CRi is defined as all CR criteria except for residual neutropenia (<1.0×109 / L [1000 / µL]) or thrombocytopenia (<100×109 / L [100,000 / µL]). PR is defined as all hematologic criteria of CR; decrease of bone marrow blast percentage to 5% to 25%; and decrease of pretreatment bone marrow blast percentage by at least 50%.

[0245] Percentage of Participants with CR + CRh [ Time Frame: Up to 36 months ].

[0246] CR + CRh rate is defined as the percentage of participants who achieve the CR + CRh as evaluated by the investigator. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines. CR is defined as bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC≥1.0×109 / L (1000 / µL); platelet count≥100×109 / L (100,000 / µL). CRh is defined as bone marrow with <5% blasts, peripheral blood neutrophil count >0.5×103 / µL and peripheral blood platelet count >0.5×105 / µL.

[0247] Percentage of Participants with Leukemia Response [ Time Frame: Up to 36 months ].

[0248] Leukemia response rate is defined as the percentage of participants who achieve the CR + CRi + PR + morphological leukemia-free state [MLFS, marrow CR (mCR)]) as evaluated by the investigator. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines. CR is defined as bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC≥ 1.0×109 / L (1000 / µL); platelet count≥100×109 / L (100,000 / µL). CRi is defined as all CR criteria except for residual neutropenia (<1.0×109 / L [1000 / µL]) or thrombocytopenia (<100×109 / L [100,000 / µL]). PR is defined as all hematologic criteria of CR; decrease of bone marrow blast percentage to 5% to 25%; and decrease of pretreatment bone marrow blast percentage by at least 50%. MLFS is defined as bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; no hematologic recovery required.

[0249] Duration of CR and CRi [ Time Frame: Up to 36 months ]. - 73 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0250] Duration of CR and CRi is defined as the time from first documentation of CR or CRi to the date of first documentation of PD or relapse from CR or CRi, and will be summarized descriptively using the K-M method based on the responders. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines. CR is defined as bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC≥ 1.0×109 / L (1000 / µL); platelet count≥100×109 / L (100,000 / µL). CRi is defined as all CR criteria except for residual neutropenia (<1.0×109 / L [1000 / µL]) or thrombocytopenia (<100×109 / L [100,000 / µL]).

[0251] Time to First CR, CRi and PR [ Time Frame: Up to 36 months ].

[0252] Time to first CR, CRi, and PR is defined as the time from randomization until the first documented CR, CRi or PR, whichever occurs first, and will be analyzed using the K-M method. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines. CR is defined as bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC≥ 1.0×109 / L (1000 / µL); platelet count≥100×109 / L (100,000 / µL). CRi is defined as all CR criteria except for residual neutropenia (<1.0×109 / L [1000 / µL]) or thrombocytopenia (<100×109 / L [100,000 / µL]). PR is defined as all hematologic criteria of CR; decrease of bone marrow blast percentage to 5% to 25%; and decrease of pretreatment bone marrow blast percentage by at least 50%.

[0253] Plasma Concentration of Pevonedistat [ Time Frame: At multiple time points pre-dose and post-dose on Days 1, 3 and 5 in Cycle 1 and post-dose on Day 1 in Cycles 2 and 4 (cycle length= 28 days) ].

[0254] Trial Summary. Overall, 164 patients from 85 sites globally were randomized to receive pevonedistat + venetoclax + azacitidine (n = 83 (81 patients received at least one dose)) or venetoclax + azacitidine (n = 81 (80 patients received at least one dose)). One hundred forty-five patients were evaluable for response. Twenty-five (29%) patients receiving pevonedistat + venetoclax + azacitidine and 21 (26%) patients receiving venetoclax + azacitidine were not evaluable for response due to having no baseline assessment (13% vs 16%), no post- baseline follow-up assessment (14% vs 9%) or having not received study treatment (2% vs 1%). Example 2. Patient Mutation Analysis

[0255] For mutation analysis, library preparation was performed with DNA isolated from subjects in the trial. DNA was isolated from patient biological samples, e.g., bone marrow aspirates.

[0256] Clinical sites collected bone marrow aspirate and peripheral blood at screening and specified timepoints as per the protocol. A portion of bone marrow aspirate collected at - 74 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 screening was transferred to a sodium heparin tube and shipped directly to Brigham and Women’s Hospital, Boston MA for cytogenetic / karyotyping and / or Fluorescence In Situ Hybridization (FISH) analysis. For the remaining bone marrow aspirate at screening and for other timepoints, a portion was transferred to a sodium heparin tube and another portion to a K2EDTA tube. Both tubes were shipped to Q2Solutions (Durham, NC) for flow cytometry analysis and cell pellet processing, respectively. Peripheral blood samples were collected in sodium heparin tubes and shipped to Q2Solutions for flow cytometry analysis.

[0257] Bone Marrow Mononuclear Cell Processing: Cell pellets were prepared at Q2Solutions from bone marrow aspirates collected in K2EDTA. After mononuclear cell separation using standard Ficoll-Paque gradient centrifugation, the cell interface layer was harvested carefully, followed by cell lysis with RBC lysis buffer. The cells were then washed with PBS. Supernatant was removed, and the cell pellet was frozen.

[0258] DNA and RNA isolation: Bone marrow cell pellets were shipped from Q2Solutions to the Broad institute (Cambridge, MA) for DNA / RNA isolation. The All-Prep DNA / RNA / miRNA Universal Kit (Qiagen Inc.) was used following manufacturer’s protocol. DNA samples were used for further mutational analyses.

[0259] NGS Analyses: Molecular mutational analysis by NGS was performed at Center for Advanced Molecular Diagnostics at Brigham and Women’s Hospital (Boston, MA) using their Rapid Heme Panel (RHP) assay. The Rapid heme panel identifies gene alterations associated with various neoplasms that are frequently driven by recurrent, somatically acquired genetic variants that affect DNA sequence or copy number. The RHPv3 assay identifies somatic single nucleotide variants (SNVs), small insertion / deletions (indels), and copy number variants (CNV) implicated in hematologic neoplasms in a targeted panel consisting of 88 genes (Table 3), using NEBNext Direct technology. Parallel sequencing was performed on an Illumina NextSeq 550Dx.

[0260] Table 3: List of genes in the Rapid Heme Panel (RHP) for NGSBTK EZH2 NFE2 SMC1A - 75 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 CALR FBXW7 NOTCH1 SMC3

[0261] DNA samples were processed in duplicate using the NEBNext Direct hybridization-based target enrichment method (New England Biolabs, Ipswich, MA, USA). As used herein, nucleic acids in a sample can be subject to target enrichment, in which molecules having target sequences are captured for subsequent analysis. Target enrichment can involve use of a bait set comprising oligonucleotide baits labeled with a capture moiety, such as biotin. The probes can have sequences selected to tile across a panel of regions, such as the genes in the RHP v3. Genomic DNA is enzymatically nicked and then hybridized to biotinylated oligonucleotide baits that capture both strands of the target DNA and define the 3' ends of the regions of interest. After hybridization, the bait-target hybrids are bound to streptavidin beads and any 3' off-target sequences are removed enzymatically. This combination of hybridization with enzymatic removal of 3' off-target sequences enables greater sequencing specificity relative to conventional hybridization-based enrichment methods. The trimmed targets are then converted into Illumina-compatible libraries that include a 12 bp unique molecular identifier in the i5 index location and an 8bp sample barcode in the Illumina i7 index location.

[0262] The Flt3-ITD allelic ratio was determined by a PCR-based assay using DNA as input. PCR products were amplified from the juxtamembrane domain (exons 14 and 15) of the FLT3 gene using fluorescently labeled primers. If present, an ITD results in a larger PCR product than normal. PCR products were then visualized by capillary electrophoresis (CE) using ABL 3500xL Dx Genetic Analyzer with a Genescan LIZ size standard. Traces were analyzed on the GeneMapper 6.0 software. Allelic ratio was calculated using the area under the curve (AUC) calculations for the mutant(s) over wild type peaks.

[0263] AML risk assessment was performed by a single pathologist for consistency at BWH by combining cytogenetic abnormalities, mutation data from the RHP panel, and FLT3- - 76 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 ITD results to stratify patients into three outcome groups, i.e., Adverse, Intermediate, and Favorable, using the European Leukemia Net (ELN) 2017 risk stratification system. For nine of 139 samples where cytogenetic results were not available from internal assessments at BWH (e.g., poor sample quality), cytogenetic results from pathology reports from the local sites were used instead.

[0264] Results

[0265] The present study employed the recently revised European LeukemiaNet (ELN-2017) recommendations for diagnosis and management of adult patients with acute myeloid leukemia (AML). This stratification analysis is broadly accepted by physicians as a gold standard and provide guidelines to stratify patients into three outcome groups: favorable, intermediate, and adverse based on cytogenetics and mutation status of commonly mutated and oncogenic genes in AML, such as ASXL1, CEBPA, FLT3, NPM1, RUNX1, DNMT3A, IDH1, IDH2, SRSF2, TET2 and TP53. This stratification scheme provides a simple, yet powerful means to triage patients for appropriate therapies. Previous studies have validated the performance of ELN-2017 guidelines in patients ≤65 years old with AML. ELN risk score and status did not correlate with lower / higher response rate to Pevonedistat + Ven+ Aza. When looking at other variables, mutational status of IDH1 / IDH2 correlates with higher CR / CRi Rates is Unlikely Due to Other Variables (e.g. Mutation Load, ELN Risk). Other markers such as CD33, CD123, or CLEC12a on AML blasts or leukemic stem cells were also analyzed in the present study. Importantly, the present study did not see any significant difference in Median Fluorescence Intensity (MFI) or % expression in any of the three proteins (CD33, CD123, or CLEC12a) after treatment with Ven and Aza (+ / - Pevo). Table 4. IDH1 / IDH2 Correlation with Higher CR / CRi Rates is Unlikely Due to Other Variables (e.g. Mutation Load, ELN Risk)BSA- Body Surface Area. IDH1 or IDH2 patients only: pevo / ven / aza (n=21) vs ven / aza (n=21). p=0.0093 for IDH1 / 2 (no stratification). - 77 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0266] Of the 88 genes that were analyzed, 11 were further analyzed for correlation to response readouts. These were chosen due to prevalence and / or scientific interest as shown in Figure 3.

[0267] FLT3 status (from both R-HP panel and ITD assay) and 8 other commonly mutated AML genes did not correlate with lower / higher response rate to Pevonedistat + Ven+ Aza.

[0268] Common AML Gene Mutations Do Not Correlate with Higher Response Rate for Either Arm.

[0269] Table 5. CCR (CR+CRi) Rate % G N V l A i idi P di V l P V l T D A T R S C N F o F F

[0270] IDH1 and IDH2 and IDH1 / IDH2 Mutation analysis results

[0271] There were 42 response-evaluable patients with IDH1 or IDH2 mutations, 21 in the control arm (Ven + aza) and 21 in the treatment arm (Pevo + Ven + Aza).

[0272] Table 6 lists the mutations found in sam les from the atient o ulation: , R R R RR172K Substitution*9- 78 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 L143-T146 In-frame deletion *m nt-to-treat (ITT) patients that were not RE. One Response Evaluable patient had both an IDH1 and an IDH2 mutation.

[0273] Based on the preliminary analyses above, only IDH1 / 2 status was identified to show a significant correlation with response and as a result, further formal statistical analyses were applied to confirm this observation. Patients with mutations in IDH1 and IDH1 / 2 genes showed higher response rate (100% and 95% CR + CRi, respectively (7 / 7 and 18 / 19 and was adjusted for age and AML subtype based on pre-specified randomization stratification factors. (Note other potential confounding factors including mutation load, age, baseline BSA, AML subtype, and ELN risk category as well as presence of other co-mutations were also informally evaluated and were shown not to correlate with response, further supporting IDH1 / 2 mutation status as the primary reason for this association). As shown in Figure 1, the increase in % CR + CRi rate between ven / aza / pevo and ven / aza for the IDH1 / 2 mutant subpopulation was statistically significant, with a p-value less than 0.05. Although EFS data were more limited, they were consistent with the CR+CRi rates, with HR=0.19 and a p-value of 0.006. In contrast, in patients who did not have mutated IDH1 / 2, the CR+CRi rate was 76% (29 / 38) for the ven+aza arm, and 68% (23 / 34) for the ven+aza+pev arm, demonstrating that this nearly 100% CR + CRi rate was only observed in the IDH1 / 2 mutant population. Analysis of the EFS curves for the population without mutated IDH1 / 2 also did not demonstrate any significant difference between the two treatment arms. This result may reflect a mechanism whereby the combination of PEV+ VEN+ AZA is inhibiting two pathways (MCL1 and BCL2) and patients with mutated IDH1 / 2 are more sensitive to these pathways.

[0274] Mutations in FLT3 are the most common genetic alteration in AML, identified in approximately one third of newly diagnosed patients. FLT3 internal tandem duplication mutations (FLT3-ITD) are associated with increased relapse and inferior overall survival. In addition, AML with a FLT3 internal tandem duplication (FLT3 / ITD) mutation has been previously thought to be an aggressive hematologic malignancy with a generally poor prognosis. It can be successfully treated into remission with intensive chemotherapy, but it routinely relapses. However, in the present study, FLT3 status did not correlate with increased response rate to treatment with Arm A (PEV+ VEN + AZA) as shown in Table 4. The present results therefore suggest that the mutational status of IDH1 and / or IDH2 in patients with AML provide a much better predictor to higher response rates in the PEV+VEN+AZA combination arm compared with treatment with VEN + AZA. - 79 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01

[0275] All patents and publications mentioned in this specification are herein incorporated by reference in their entireties, to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

[0276] Equivalents

[0277] Although embodiments of the invention have been described using specific terms, such description are for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the invention. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. - 80 - 56340669.1

Claims

Attorney Docket No.223266-549182 / PAT27156PCT01 CLAIMS What is claimed:

1. A method for treating a hematological cancer patient comprising a hematological cancer characterized by a mutation in the isocitrate dehydrogenase 1 (IDH1) gene, and / or the isocitrate dehydrogenase 2 (IDH2) gene, the method comprising the step of administering to the patient a therapeutically effective amount of a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and a hypomethylating agent.

2. The method of claim 1, wherein the hypomethylating agent is azacitidine.

3. The method of any one of claims 1 to 2, wherein the hematological cancer is selected from the group consisting of leukemia, lymphoma and myeloma.

4. The method of any one of claims 1 to 3, wherein the hematological cancer is selected from the group consisting of acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).

5. The method of any one of claims 1 to 4, further comprising the steps of: a) measuring at least one characteristic of at least one marker corresponding to the IDH1 gene or the IDH2 gene in a biological sample obtained from the patient; b) identifying at least one mutation in the IDH1 gene and / or the IDH2 gene in the biological sample from the at least one characteristic measured in step a); and c) administering the combination to the patient.

6. The method of claim 5, wherein the at least one marker is selected from the group consisting of nucleic acid and protein corresponding to the IDH1 gene and / or the IDH2 gene.

7. The method of claim 6, wherein the at least one marker is nucleic acid.

8. The method of any one of claims 5 to 7, wherein the at least one characteristic is selected from the group consisting of: size, sequence, composition, activity and amount.

9. The method of claim 8, wherein the at least one characteristic is sequence. - 81 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 10. The method of claim 9, wherein the nucleic acid is selected from the group consisting of DNA, mRNA cDNA and ctDNA and a portion of any of the foregoing, wherein the nucleic acid comprises at least one mutation site of the IDH1 gene and / or the IDH2 gene.

11. The method of any one of claims 1 to 10, wherein the pharmaceutically acceptable salt of pevonedistat is a hydrochloride salt.

12. An NAE inhibitor or a pharmaceutically acceptable salt thereof for use in a method of treating a hematological cancer in a patient whose hematological cancer is characterized by having a mutation in the IDH1 gene and / or the IDH2 gene.

13. The NAE inhibitor or a pharmaceutically acceptable salt thereof for the use of claim 12, wherein the NAE inhibitor or pharmaceutically acceptable salt thereof is for use in combination with venetoclax (ABT-199) and a hypomethylating agent.

14. The NAE inhibitor or pharmaceutically acceptable salt thereof for the use of claim 13, wherein the hypomethylating agent is azacitidine.

15. The NAE inhibitor or pharmaceutically acceptable salt thereof for the use of any one of claims 12 to 14, wherein the hematological cancer is selected from the group consisting of leukemia, lymphoma and myeloma.

16. The NAE inhibitor or pharmaceutically acceptable salt thereof for the use of any one of claims 12 to 15, wherein the hematological cancer is selected from the group consisting of acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), and chronic myelomonocytic leukemia (CMML).

17. The NAE inhibitor or pharmaceutically acceptable salt thereof for the use of any one of claims 12 to 16, wherein the NAE inhibitor is pevonedistat or a pharmaceutically acceptable salt thereof.

18. The NAE inhibitor or pharmaceutically acceptable salt thereof for the use of claim 17, wherein the pharmaceutically acceptable salt of pevonedistat is a hydrochloride salt. - 82 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 19. A method for preventing or treating a hematological cancer in a patient in need thereof, the method comprising, administering a therapeutically effective combination of pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199) and a hypomethylating agent, wherein the patient’s hematological cancer harbors a mutation of the IDH1 gene.

20. The method of claim 19, wherein the hypomethylating agent is azacitidine.

21. The method of any one of claims 19 to 20, wherein the hematological cancer is selected from the group consisting of leukemia, lymphoma and myeloma.

22. The method of any one of claims 19 to 21, wherein the hematological cancer is selected from the group consisting of acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).

23. The method of any of claims 19 to 22, further comprising the steps of: a) measuring at least one characteristic of at least one marker corresponding to the IDH1 gene or the IDH2 gene in a biological sample obtained from the patient; b) identifying at least one mutation in the IDH1 gene and / or the IDH2 gene in the biological sample from the at least one characteristic measured in step a); and c) administering the combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199) and a hypomethylating agent to the patient.

24. The method of claim 23, wherein the at least one marker is selected from the group consisting of nucleic acid and protein corresponding to the IDH1 gene and / or the IDH2 gene.

25. The method of claim 24, wherein the at least one marker is a nucleic acid.

26. The method of any one of claims 23 to 25, wherein the at least one characteristic is selected from the group consisting of: size, sequence, composition, activity and amount.

27. The method of claim 26, wherein the at least one characteristic is sequence. - 83 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 28. The method of claim 27, wherein the nucleic acid sequence is selected from the group consisting of DNA, mRNA and cDNA or a portion of any of the foregoing, wherein the portion comprises at least a gene coding sequence which encodes the active site of the IDH1 protein and / or the IDH2 protein.

29. The method of any one of claims 19 to 28, wherein the pharmaceutically acceptable salt of pevonedistat is a hydrochloride salt.

30. The method of any one of claims 1 to 11, and 19 to 29, and the use of any one of claims 12 to 18, wherein the IDH1 mutation comprises a mutation of the IDH1 gene, which encodes an amino acid mutation at position R132 in an IDH1 protein.

31. The method of any one of claims 1 to 11, and 19 to 29, wherein the mutation is in an IDH1 protein active site, and confers a gain of function which enables the IDH1 protein to produce the metabolite 2-hydroxyglutarate (2-HG).

32. The method of any one of claims 1 to 11, and 19 to 29, and the use of any one of claims 12 to 18, wherein the IDH2 mutation comprises a mutation of the IDH2 gene, which encodes an amino acid mutation selected from the group consisting of: position L86, position Q316, position R140 and position R172 in an IDH2 protein.

33. The method of any one of claims 1 to 11, and 19 to 29, wherein the mutation is in an IDH2 protein active site, and confers a gain of function which enables the IDH2 protein to produce the metabolite 2-hydroxyglutarate (2-HG).

34. A method for determining whether to treat a patient having a hematological cancer, with a therapeutic regimen comprising a combination of pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199) and a hypomethylating agent comprising: a) measuring at least one characteristic of at least one marker corresponding to the IDH1 gene in a biological sample obtained from the patient; b) identifying a mutational status in the IDH1 gene and / or the IDH2 gene in the biological sample from the at least one characteristic measured in step a); and c) determining to treat the patient with the therapeutic regimen if the mutational status indicates a favorable outcome. - 84 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 35. The method of claim 34, further comprising administering the therapeutic regimen to the patient.

36. The method of claim 34, wherein the at least one marker is selected from the group consisting of nucleic acid and protein corresponding to the IDH1 gene and / or the IDH2 gene.

37. The method of claim 36, wherein the at least one marker is a nucleic acid.

38. The method of any one of claims 34 to 37, wherein the at least one characteristic is selected from the group consisting of: size, sequence, composition, activity and amount.

39. The method of claim 38, wherein the at least one characteristic is sequence.

40. The method of any one of claims 34 to 39, wherein the mutational status in the IDH1 gene is a mutation in the IDH1 gene which results in a mutation of the IDH1 protein at position R132.

41. The method of any one of claims 34 to 37, wherein the mutational status in the IDH2 gene is a mutation in the IDH2 gene which results in a mutation of the IDH2 protein selected from the group consisting of at position L86, at position R140, at positions L143-T146, at position R172, and at position Q316.

42. The method of any one of claims 34 to 41, wherein the hematological cancer is selected from the group consisting of myeloma, leukemia, lymphoma, and myelodysplastic syndrome.

43. The method of any one of claims 34 to 42, wherein the hematological cancer is selected from the group consisting of acute myelogenous leukemia, chronic myelomonocytic leukemia, and myelodysplastic syndrome.

44. The method of claim 43, wherein the hematological cancer is selected from the group consisting of low blast acute myelogenous leukemia, de novo acute myelogenous leukemia, - 85 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 secondary acute myelogenous leukemia, high risk myelodysplastic syndrome, and chronic myelomonocytic leukemia.

45. The method of any one of claims 34 to 44, wherein the biological sample comprises hematological cancer cells, contents from the hematological cancer cells, or products from the hematological cancer cells.

46. A method for preventing cancer progression or treating a hematological cancer in a patient in need thereof, the method comprising, administering a therapeutically effective amount of a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and azacitidine, wherein the patient has been identified as having a gain of function mutation in the IDH1 gene and / or the IDH2 gene, in at least a portion of the hematological cancer, wherein the mutation in the IDH1 gene results in the mutation of the IDH1 protein, at position R132 of SEQ ID NO: 1, and the mutation in the IDH2 gene results in the mutation of the IDH2 protein, selected from the group consisting of position L86, position R140, positions L143-T146, position R172 and position Q316, of SEQ ID NO:

2.

47. The method of claim 46, wherein the hematological tumor cancer is selected from the group consisting of leukemia, lymphoma and myeloma.

48. The method of claim 47, wherein the hematological cancer is selected from the group consisting of acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).

49. The method of any one of claims 46 to 48, further comprising the steps of: a) measuring at least one characteristic of at least one marker corresponding to IDH1 or IDH2 in a biological sample obtained from the patient; b) identifying at least one characteristic selected from: (i) a mutation of the IDH1 gene, or the IDH2 gene, and (ii) an IDH1 protein having an amino acid mutation selected from R132C, R132G, R132H, and R132S or (iii) an IDH2 protein having an amino acid mutation selected from L86P, Q316L, R140G, R140Q, R172K and in-frame deletion of L143-T146, in the at least one marker measured in step a); and - 86 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 c) administering the combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and azacitidine to the patient.

50. The method of claim 49, wherein the mutation of the IDH1 gene or the IDH1 protein corresponds to the mutation R132C, and wherein the mutation of the IDH2 gene or the IDH2 protein corresponds to the mutation R140Q or R172K.

51. The method of claim 49, wherein the at least one marker is a nucleic acid or a protein.

52. The method of claim 51, wherein the at least one marker is a nucleic acid.

53. The method of any one of claims 51 to 52, wherein the at least one characteristic is sequence.

54. The method of claim 53, wherein the at least one characteristic is a nucleic acid sequence of the subject’s IDH1 gene or IDH2 gene performed using Sanger sequencing or next-generation sequencing (NGS).

55. The method of any one of claims 46 to 49, wherein the hematological cancer comprises mutations in the IDH1 protein having substitutions selected from R132C and R132H, and the hematological tumor is a cancer selected from leukemia, lymphoma and myeloma.

56. The method of any one of claims 46 to 49, wherein the hematological cancer comprises mutations in the IDH2 protein having substitutions selected from R140G and R172K, and the hematological tumor is a cancer selected from leukemia, lymphoma and myeloma.

57. The method of any one of claims 55 to 56, wherein the hematological cancer is selected from the group consisting of acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).

58. A method for determining whether to continue treatment of hematological cancer in a patient with a therapeutic regimen comprising a therapeutically effective amount of a - 87 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and a hypomethylating agent, the method comprising: a) obtaining a first biological sample from the patient and a second biological sample from the patient, wherein both samples comprise hematological cancer cells, and wherein the first sample is obtained prior to the second sample and the patient is treated with the therapeutic regimen prior to the second sample; b) measuring at least one marker corresponding to an IDH1 gene and / or an IDH2 gene in the two samples; c) comparing the results of the measurements in b); and d) determining to continue treatment with the therapeutic regimen if the comparison indicates that the hematological cancer cells in the second sample comprises at least one marker gene whose mutational status indicates a favorable outcome, wherein the at least one marker gene is an IDH1 gene and / or an IDH2 gene.

59. The method of claim 58, further comprising continuing to administer the therapeutic regimen to the patient.

60. The method of claim 58, wherein the at least one characteristic is selected from the group consisting of size, sequence, composition and amount.

61. The method of claim 58 or 59, wherein the mutational status of the at least one marker is mutant.

62. The method of claim 58 or 59 wherein the mutational status of the at least one marker is wild type.

63. The method of any one of claims 58 to 62, wherein the at least one marker is selected from the group consisting of nucleic acid and protein corresponding to the at least one marker.

64. The method of any one of claims 58 to 59, wherein the hematological cancer is selected from the group consisting of myeloma, leukemia, lymphoma, and myelodysplastic syndrome. - 88 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 65. The method of any one of claims 58 to 64, wherein the hematological cancer is selected from the group consisting of acute myelogenous leukemia, chronic myelomonocytic leukemia, and myelodysplastic syndrome.

66. The method of any one of claims 58 to 65, wherein the hematological cancer is selected from the group consisting of low blast acute myelogenous leukemia, high risk myelodysplastic syndrome, and chronic myelomonocytic leukemia.

67. The method of any one of claims 58 to 66, wherein the at least one characteristic is sequence of the at least one marker.

68. The method of any one of claims 58 to 67, wherein the at least one marker is selected from the group consisting of nucleic acid and protein.

69. The method of claim 68, wherein the nucleic acid is selected from the group consisting of DNA, mRNA, ctDNA and cDNA and any portion of any of the foregoing, wherein the nucleic acid corresponds to at least one mutation site of the at least one marker gene.

70. The method of any one of claims 58 to 69, wherein the pharmaceutically acceptable salt of pevonedistat is a hydrochloride salt.

71. The method of any one of claims 58 to 70, wherein the hypomethylating agent is azacitidine.

72. Use of a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and a hypomethylating agent, for the manufacture of a medicament for treating a hematological cancer patient, said treating comprises a) using a mutational status of at least one marker gene in a patient sample comprising hematological cancer cells to select a hematological cancer patient who is expected to have a favorable outcome of treatment with the combination, wherein the mutational status is indicative of the favorable outcome, wherein the at least one marker gene is an IDH1 gene and / or an IDH2 gene; and - 89 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 b) treating the hematological cancer patient expected to have the favorable outcome with the combination.

73. The use of claim 72, wherein the hypomethylating agent is azacitidine.

74. The use of any one of claims 72 or 73, wherein the mutational status is determined by measuring at least one characteristic of at least one marker associated with the at least one marker gene.

75. The use of claim 74, wherein the at least one characteristic is selected from the group consisting of size, sequence, composition, and amount.

76. The use of any one of claims 70 or 71, wherein the at least one marker is selected from the group consisting of a nucleic acid, and a protein associated with the at least one marker gene.

77. The use of claim 76, wherein the nucleic acid is genomic DNA, RNA, mRNA, ctDNA or cDNA.

78. The use of claim 76, wherein the at least one characteristic is a sequence and the at least one marker is a nucleic acid.

79. The use of claim 72, wherein the mutational status of the marker gene is mutant.

80. The use of claim 79, wherein the mutant marker gene results in the mutation of the codon encoding amino acid R132 of the IDH1 gene.

81. The use of claim 79, wherein the mutant marker gene results in the mutation of the codon encoding amino acid L86, R140, L143-T146, R172, or Q316 of the IDH2 gene.

82. The use of any one of claims 80 or 81, wherein the mutation of the IDH1 gene corresponds to the codon encoding an amino acid mutation R132C, and wherein the mutation - 90 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 of the IDH2 gene corresponds to a codon encoding an amino acid mutation R140Q or R172K.

83. The use of claim 72, wherein the mutational status of the marker gene is wild type.

84. The use of any one of claims 72 to 83, wherein the hematological cancer is selected from the group consisting of leukemia, lymphoma and myeloma.

85. The use of any one of claims 72 to 84, wherein the hematological cancer is selected from the group consisting of acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).

86. The use of any one of claims 72 to 85, wherein the pharmaceutically acceptable salt of pevonedistat is a hydrochloride salt.

87. Use of a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and a hypomethylating agent for the treatment of a hematological cancer, wherein the hematological cancer is characterized by a mutation in an IDH1 gene and / or an IDH2 gene.

88. The use of claim 87, wherein the hypomethylating agent is azacitidine.

89. The use of any one of claims 84 or 87, wherein the hematological cancer is selected from the group consisting of leukemia, lymphoma and myeloma.

90. The use of any one of claims 87 to 89, wherein the hematological cancer is selected from the group consisting of acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).

91. The use of any one of claims 87 to 90, further comprising the steps of: a) measuring at least one characteristic of at least one marker corresponding to the IDH1 gene and / or the IDH2 gene in a biological sample obtained from the patient; b) identifying at least one mutation in the IDH1 gene and / or the IDH2 gene in the biological sample from the at least one characteristic measured in step a); and - 91 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 c) administering the combination to the patient.

92. The use of any one of claims 87 to 91, wherein the at least one marker is selected from the group consisting of nucleic acid and protein corresponding to the IDH1 gene and / or the IDH2 gene.

93. The use of any one of claims 87 to 92, wherein the at least one marker is nucleic acid.

94. The use of any one of claims 87 to 93, wherein the at least one characteristic is selected from the group consisting of: size, sequence, composition, activity and amount.

95. The use of any one of claims 94, wherein the at least one characteristic is sequence.

96. The use of claim 94, wherein the nucleic acid is selected from the group consisting of DNA, mRNA, ctDNA and cDNA and a portion of any of the foregoing, wherein the nucleic acid comprises at least one mutation site of the IDH1 gene and / or the IDH2 gene.

97. The use of any one of claims 87 to 96, wherein the pharmaceutically acceptable salt of pevonedistat is a hydrochloride salt.

98. The method or use of any one of claims 1, 12, 19, 34, 46, 72, and 87, wherein the administration of the combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199) and a hypomethylating agent to the patient, reduces one or more symptoms of the hematological cancer, or delays or terminates the progression of the hematological cancer.

99. The method or use of claim 98, wherein the hypomethylating agent is azacitidine.

100. The method or use of any one of claims 98 or 99, wherein the administration of the combination reduces growth of hematological cancer cells in the subject.

101. The method or use of any one of claims 98 or 99, wherein the administration of the combination reduces growth for at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20% of the hematological cancer cells in the subject. - 92 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 102. The method or use of any one of claims 98 or 99, wherein the administration is a dose of from about 1 to about 100 mg / m2of pevonedistat or a pharmaceutically acceptable salt thereof, on days 1, 3, and 5 in a 28-day cycle.

103. The method of claim 102, wherein the administration is a dose of about 20 mg / m2of pevonedistat or a pharmaceutically acceptable salt thereof.

104. The method or use of any one of claims 98 or 99, wherein the administration is intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, oral, topical, infusion, or inhalation.

105. A kit comprising a reagent to measure at least one characteristic of at least one marker in a biological sample, wherein the at least one marker corresponds to an IDH1 gene and / or an IDH2 gene.

106. A kit comprising a reagent to measure at least one characteristic of the markers in a biological sample in the method of any of claims 1-11.

107. The kit of claim 105 or 106, wherein the at least one characteristic is selected from the group consisting of size, sequence, composition and amount.

108. The kit of any one of claims 105 to 107, wherein the at least one marker is selected from the group consisting of nucleic acid and protein corresponding to the IDH1 gene and / or the IDH2 gene.

109. The kit of any one of claims 105 to 108, further comprising a stabilizer to add to the sample.

110. The kit of any one of claims 105 to 109, wherein the at least one marker is nucleic acid and the reagent is at least one primer.

111. The kit of any one of claims 105 to 110, further comprising a probe. - 93 - 56340669.1Attorney Docket No.223266-549182 / PAT27156PCT01 112. The kit of any one of claims 105 to 111, wherein the biological sample comprises hematological cancer cells.

113. The kit of any one of claims 105 to 112, wherein the biological sample is blood.

114. The kit of claim 112 or 113, further comprising enriching the biological sample for hematological cancer cells. - 94 - 56340669.1

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