WO2018067834A1 - Nt5c2 inhibitors useful for the treatment of chemotherapy resistant acute lymphoblastic leukemia - Google Patents

Abstract

Relapse and resistance to therapy is the most significant challenge in the treatment of lymphoblastic leukemia. Mutations in NT5C2 (a cytosolic nucleotidase activated by gain of function mutations in about 20% of relapse ALL cases) characteristically are associated with early relapse and progression under therapy and confer resistance to 6-mercaptopurine chemotherapy in vitro and in vivo. Activating mutations in NT5C2 disrupt intramolecular switch-off mechanisms responsible for returning the enzyme to its resting inactive state after activation and lock the NT5C2 protein in an active state similar to that induced by allosteric activators. Based on structure, NT5C2 inhibitors were developed for specific therapies for preventing and reversing 6-MP resistance in ALL.

Description

NT5C2 INHIBITORS USEFUL FOR THE TREATMENT OF CHEMOTHERAPY

RESISTANT ACUTE LYMPHOBLASTIC LEUKEMIA

CROSS-REFERENCE TO PRIOR APPLICATION

[0001] This application claims benefit to prior application United States provisional application serial no. 62/404,402, filed October 5, 2016. The contents of this application are hereby incorporated by reference in its entirety.

BACKGROUND

[0002] The current treatment of newly diagnosed acute lymphoblastic leukemia (ALL) consists of an initial course of high dose chemotherapy combining glucocorticoids, DNA-damaging agents, mitotic poisons and L-aparaginase, which together obtain clinical and hematologic remission with clearance of leukemic cells from the bone marrow and reconstitution of hematopoietic function in over 90% of cases. Typically, this is followed by additional rounds of highly intensive therapy aimed to further reduce disease burden and then by a 2-year-long lower intensity maintenance therapy with oral 6-mercaptopurine that is essential to prevent the occurrence of relapse. The rationale behind maintenance therapy is that continuous exposure to 6-mercaptopurine may help eradicate residual quiescent leukemia initiating cells persisting at the end of induction and consolidation as they become activated and leave their protective

microenvironment niches.

[0003] 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are thiopurine nucleoside prodrugs activated in leukemia cells via their incorporation into the salvage pathway of purine

biosynthesis by the hypoxanthine-guanine phosphoribosyl transferase enzyme (HGPRT). In this activation step, HGPRT converts 6-MP into thio-IMP, which is subsequently converted to thio- XMP and then to thio-GMP; while 6-TG is directly converted to thio-GMP. See FIG. 1.

Metabolism of thio-GMP by kinases and reductases ultimately produces thio-dGTP, which, upon incorporation to the DNA, triggers cell-cycle arrest and apoptosis by a process that involves the mismatch repair pathway. In addition, thio-IMP and its methylated metabolite methyl-thio-IMP (MeTIMP) function as inhibitors of glutamine-5-phosphoribosylpyrophosphate amidotransferase, a key enzyme in the de novo pathway for purine ribonucleotide synthesis. See FIG. 1.

[0004] A number of mechanisms have been involved as drivers of thiopurine resistance in ALL. Early work associated increased clearance of intracellular thiopurines in diagnostic ALL samples with increased risk of relapse. In addition, alterations in enzymes involved in mismatch DNA repair can interfere with the activity of 6-thioguanine, and decreased levels of MSH2 protein have been described in about 10% of newly diagnosed ALL cases. However, the specific mechanisms driving thiopurine resistance in relapsed leukemia had remained unknown. This scenario has rapidly changed with the identification of thiopurine resistance driving mutations in NT5C2 in about 20% of relapsed ALL cases. See Tzoneva et al., Nat. Med, 2013. The central role of NT5C2 as driver of resistance to 6-MP is highlighted by the rarity of other genetic lesions in this pathway with activating mutations in PRPSl , also linked to 6-MP resistance, accounting for only 2% of relapsed ALL cases. There remains a need for methods and compounds to treat chemotherapy-resistant ALL.

SUMMARY OF THE INVENTION

[0005] The invention therefore relates to chemical compounds containing an aminopyridine moiety and methods for their use to modify or inhibit the activity of the NT5C2 gene product, which are useful for the treatment of ALL. Preferred chemical compounds according to the following structures are contemplated as part of the invention:

Formula 1 Formula 2 wherein Ri is a saturated five- to twelve-membered heterocyclic or heterobicyclic (bycyclic heterocycle) ring having one to four hetero atoms selected from the group consisting of N, O, and S; wherein R₂ is H or Ci_₄ alkyl; wherein R₃ is

or a Ci-4 alkyl ester thereof; wherein R₄ is NH₂, NHCH₃, N(CH₃)₂, or a five- to six-membered heterocyclic ring having one or two N hetero atoms; wherein R5 is H or Ci_₄ alkyl; wherein R6 is H or C_1-4 alkyl; or R5 and R6 are joined to form a cyclohexyl moiety; wherein R7 is selected from the group consisting of

and

and wherein R8 is NH₂, NHCH₃, or N(CH₃)₂; or a salt or hydrate thereof, with the proviso that the compound is not

or

[0006] Additional specific compounds are selected from the group consisting of:

[0008] In addition, pharmaceutical compositions comprising a pharmaceutically acceptable carrier and any of the compounds discussed herein are contemplated as embodiments of the invention, for example pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound of the structure according to Formula 1 or Formula 2:

Formula 1 Formula 2 wherein Ri is a saturated five- to twelve-membered heterocyclic or heterobicyclic ring having one to four hetero atoms selected from the group consisting of N, O, and S; wherein R₂ is H or C_1-4 alkyl; wherein R₃ is

or a Ci-4 alkyl ester thereof; wherein R₄ is NH₂, NHCH₃, N(CH₃)₂, or a five- to six-membered heterocyclic ring having one or two N hetero atoms; wherein R5 is H or Ci_₄ alkyl; wherein R6 is H or C_1-4 alkyl; or R5 and R6 are joined to form a cyclohexyl moiety; wherein R7 is

and

and wherein R8 is NH₂, NHCH₃, or N(CH₃)₂; or a salt or hydrate thereof.

[0009] Further embodiments of the invention include methods of treating chemotherapy- resistant acute lymphoblastic leukemia comprising administering to a subject in need any of the compounds or pharmaceutical compositions disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0011] FIG. 1 is a diagram showing the pathways involved in NT5C2 inactivation of the cytotoxic metabolites of 6-mercaptopurine and 6-thioguanine.

[0012] FIG. 2 is a diagram showing NT5C2 mutations in relapsed ALL.

[0013] FIG. 3 is a Circos plot depiction of mutations in matched diagnosis and relapsed leukemias identified by exome sequencing.

[0014] FIG. 4 is a graph showing that NT5C2 mutations induce increased nucleotidase activity.

[0015] FIG. 5 is a graph showing specific activity versus ATP concentration for the indicated NT5C2 mutations.

[0016] FIG. 6 shows the specific activity of NT5C2 mutant proteins of the indicated classes.

[0017] FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D are dose response viability curves of JURKAT cells with inducible expression of NT5C2 wild type or relapse associated NT5C2 mutations.

[0018] FIG. 8 is a diagram of inactive (Apo; FIG. 8A) and active (ATP-bound; FIG. 8B) NT5C2.

[0019] FIG. 9 A and FIG. 9B are diagrams showing a protein modeling prediction of NT5C2 K359Q structure, with a helical configuration of the helix A domain in the absence of ATP.

[0020] FIG. 10A and FIG. 10B show the crystal structures of wild type NT5C2 bound to the ATP allosteric activator (10A) and of NT5C2 K359Q (10B) in the absence of ATP. The helix A domain region is shown. The K361-D459 interaction responsible for stabilization of the helix A helical structure is circled in red.

[0021] FIG. 11 provides the crystal structures of NT5C2 mutant L375F. FIG. 11 A: model and structure of the L375F. FIG. 1 IB: increased dimer interface surface. FIG. 11C: active NT5C2 WT (ATP bound) and NT5C2 L375F structures focused on Helix A. A salt bridge stabilizing helix A is circled in red. [0022] FIG. 12 shows structure modeling of pocket and loop mutations in the NT5C2 dimer. The position of inter subunit pocket mutations is highlighted in red. The helix-unstructured loop-helix domain is highlighted in blue. Loop mutations are indicated with red circles.

[0023] FIG. 13 shows the D407 loop interaction with the inter subunit pocket of NT5C2.

Following activation by allosteric regulators, the D407 loop invades the intersubunit pocket (the 20 most favorable energy configurations are shown) accessing the helix A domain located at the bottom.

[0024] FIG. 14 is a schematic representation of the dynamic interaction between the D407 loop and K361 in the helix A domain of NT5C2.

[0025] FIG. 15 shows the results of nucleotidase assays for wild type NT5C2 protein, incubated with IgG (control) and two purified antibodies (A and B) against D407 loop synthetic peptide.

[0026] FIG. 16 shows the Class II structures of the indicated mutations of NT5C2.

[0027] FIG. 17 is a bar graph showing the relative 5 '-NT activity for NT5C2, C-term peptide, C- term Ab 1, and C-term Ab 2.

[0028] FIG. 18 shows the structure of NT5C2 inhibitor compounds 11 and 20. The pyrimidine ring is marked in red.

[0029] FIG. 19 provides data showing that ATP pocket binding compounds 11 and 20 inhibit NT5C2 nucleotidase activity in basal conditions and in the presence of 0.3 mM ATP allosteric activation.

[0030] FIG. 20 shows modeling of inhibitor compounds 11 and 20 binding to the ATP allosteric pocket of NT5C2.

[0031] FIG. 21 shows the structure of a chimeric small molecule (Chi), which combines the core pyrimidine ring (marked in red) and the side chains of compounds 11 and 20 involved in interaction with the allosteric pocket of NT5C2.

[0032] FIG. 22 shows molecular docking-based NT5C2 interactions of compounds 11 and 20 and of the chimeric compound (Chi), which combines the common pyrimidine ring with the side chains of these two inhibitors. [0033] FIG. 23 provides the structures of morpholine ring substituted compounds based on Compound 11 with predicted improved NT5C2 binding based on their molecular docking GlideScores.

[0034] FIG. 24 provides the structure of lead compound related molecules with inhibitory activity against NT5C2 in malachite green assays.

[0035] FIG. 25 A, FIG. 25B, FIG. 25C, and FIG. 25D provide data on the NT5C2 inhibitory activity of molecules chemically similar to Compound 20 in in vitro malachite green assays.

[0036] FIG. 26A and FIG. 26B provide data on the NT5C2 inhibitory activity of molecules chemically similar to Compound 11 in in vitro malachite green assays.

[0037] FIG. 27A, FIG. 27B and FIG 27C provide data on the design of Nt5c2 R367Q knockin mice (Fig.27 A), effective expression of the Nt5c2 R367Q allele upon tamoxifen treatment (FIG. 27B) and induction of resistance to 6-MP (FIG. 27C).

[0038] FIG. 28A and FIG. 28B provide data demonstrating resistant to 6-MP in vivo in leukemias from Nt5c2 R367Q knockin mice after activation of Nt5c2 R367Q.

DETAILED DESCRIPTION

1. Definitions

[0039] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood in the art to which this invention pertains and at the time of its filing. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. However, the skilled should understand that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, it should also be understood that as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration and any other measurements, quantities or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary. Hence, where appropriate to the invention and as understood by those of skill in the art, it is proper to describe the various aspects of the invention using approximate or relative terms and terms of degree commonly employed in patent applications, such as: so dimensioned, about, approximately, substantially, essentially, consisting essentially of, comprising, and effective amount. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0040] Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein, and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention generally are performed according to conventional methods well known in the art and as described in various general and more specific references, unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et ah, Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lan, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Principles of Neural Science, 4th ed., Eric R. Kandel, James H. Schwartz, Thomas M. Jessell editors. McGraw-Hill/ Appleton & Lange: New York, N. Y. (2000). Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0041] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0042] The terms "administering" or "administer" or "administration" as used herein with respect to an agent means providing the agent to a subject using any of the various methods or delivery systems for administering agents or pharmaceutical compositions known to those skilled in the art. Modes of administering include, but are not limited to oral administration, parenteral administration such as intravenous, subcutaneous, intramuscular or intraperitoneal injections, rectal administration by way of suppositories, transdermal administration, intraocular

administration or administration by any route or method that delivers a therapeutically effective amount of the drug or composition to the cells or tissue to which it is targeted. Alternatively, routine experimentation will determine other acceptable routes of administration.

[0043] The term "alkyl" refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 4 or fewer carbon atoms in its backbone (e.g., Ci-C₄ for straight chains.

[0044] The term "chemotherapy-resistant acute lymphoblastic leukemia" refers to acute lymphoblastic leukemia (ALL) in which cancer cells are resistant to thio-purine (e.g. 6- mercaptopurine) chemotherapy. Alternatively, or additionally, "Chemotherapy-resistant acute lymphoblastic leukemia" includes ALL in which cancer cells harbor one or more mutations of NT5C2 that disrupt intramolecular switch off mechanisms responsible for returning the enzyme to its resting inactive state after activation and lock the NT5C2 protein in an active state similar to that induced by allosteric activators.

[0045] The term "compound(s) of the invention" as used herein encompass(es), for example, any NT5C2 inhibitor compound disclosed herein including any pharmaceutically acceptable salt or solvate thereof. Specific examples of compounds of the invention include those of formula (I) or (II) and any subgenera and/or species, or a pharmaceutically acceptable salt or solvate thereof. A compound of the invention, includes the disclosed structure or a stereoisomer thereof.

[0046] The terms "heterocyclyl", "heterocycle", "heterocyclic", and the like refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 8-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclyl," "heterocyclic," and the like also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0047] The term "solvate" as used herein means a compound formed by solvation (the combination of solvent molecules with molecules or ions of the solute), or an aggregate that consists of a solute ion or molecule, i.e., a compound of the invention, with one or more solvent molecules. When water is the solvent, the corresponding solvate is a "hydrate." Examples of hydrate include, but are not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, hexahydrate, etc. It should be understood by one of ordinary skill in the art that the present compound and/or the pharmaceutically acceptable salt of the present compound may also exist in a solvate form. The solvate is typically formed via hydration which is either part of the preparation of the present compound or through natural absorption of moisture by the anhydrous compound of the present invention.

[0048] The terms "subject," "individual," "host," and "patient," are used interchangeably herein to refer to an animal being treated with one or more enumerated agents as taught herein, including, but not limited to, simians, humans, avians, felines, canines, equines, rodents, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets. A suitable subject for the invention can be any animal, preferably a human, that is suspected of having, has been diagnosed as having, or is at risk of developing a disease that can be ameliorated, treated or prevented by administration of one or more enumerated agents.

[0049] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non- aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible

substituents of organic compounds described herein which satisfy the valences of the

heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a

thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be

understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. [0050] As used herein, the term "substituent," means H, cyano, oxo, nitro, acyl, acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate, ester, ether, carboxylic acid, thio, thioalkyl, thioester, thioether, Ci_₈ alkyl, Ci_₈alkoxy, Ci_₈alkenyl, Ci_₈aralkyl, 3- to 8-membered carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone, alkylsulfonyl, or arylsulfonyl.

[0051] The term "treating" or "treatment of as used herein refers to providing any type of medical management to a subject. Treating includes, but is not limited to, administering a composition comprising one or more active agents to a subject using any known method for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition or one or more symptoms or manifestations of a disease, disorder or condition.

[0052] A "therapeutically effective amount" refers to an amount which, when administered in a proper dosing regimen, is sufficient to reduce or ameliorate the severity, duration, or progression of the disorder being treated (e.g., cancer), prevent the advancement of the disorder being treated (e.g., cancer), cause the regression of the disorder being treated (e.g., cancer), or enhance or improve the prophylactic or therapeutic effects(s) of another therapy. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations per day for successive days.

[0053] Unless specifically stated as "unsubstituted," references to chemical moieties herein are understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

2. Overview

[0054] The 5 '-nucleotidase, cytosolic II (NT5C2) gene (a protein coding gene) encodes cytosolic purine 5-nucleotidase, a 64,970 Da (561 amino acids) hydrolase thai is located in the cytoplasmic matrix of cells and acts primarily on inosine 5'-monophosphate (IMP) and other purine nucleotides as a homotetramer, Purine 5-prime-nucleotidase is allosterically activated by various compounds, including ATP. It therefore has a role in purine metabolism, including maintaining proper ratios and quantities of intracellular purine and pyriniidine nucleotides, acting in cooperation with other nucleotidases. This gene also is responsible for the inactivalion of nucleoside- analog chemotherapy drugs in about 19% of relapse T cell acute lymphoblastic leukemia (ALL) cells and about 3% of relapse B-precursor ALLs. Additional information on this gene is available in Tzoneva et al., "Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL." Nat. Med. 1 (3):368-371 , 2013, which is hereby incorporated by reference in its entirety.

[0055] Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors, which requires treatment with intensive chemotherapy. Despite much effort over the last decades, cure rates remain suboptimal, making relapsed ALL the fourth most frequent malignancy in children. Moreover, relapsed ALL is frequently associated with chemotherapy resistance and, despite salvage therapy with intensified treatment, cure rates are still unsatisfactory low. This is particularly the case in patients with relapsed T-ALL and in cases with primary resistance or early relapse, which is associated with higher risk of failure to achieve a second complete remission, shorter duration of chemotherapy response and poor survival. As result, relapsed ALL is still the leading cause of pediatric cancer associated death.

[0056] Multiple mechanisms have been implicated as drivers of relapse and resistance in ALL. Thus, leukemia initiating cells with stem cell properties including intrinsic self-renewal capacity and increased resistance to chemotherapy have been proposed to function as drivers of disease progression and relapse. In addition, protective niches in the bone marrow microenvironment may allow ALL cells to escape the cytotoxic effects of chemotherapy. Finally, clonal heterogeneity at diagnosis and genetic Darwinian evolution driven by the selective pressure of chemotherapy has been proposed to result in the emergence of leukemia clones harboring specific mutations driving chemotherapy resistance at the time of relapse. Thus, mapping the genetic landscape of relapse leukemias, defining the mechanism of action of relapse-associated mutations and developing effective strategies to reverse chemotherapy resistance have become major research imperatives in the field.

[0057] The NT5C2 gene product is a 5 '-nucleotidase enzyme responsible for the

dephosphorylation of metabolic intermediates in the salvage pathway of purine biosynthesis (IMP, XMP, GMP) and catalyzes a critical step for their export out of the cell in the form of inosine, xanthosine and guanosine. The nucleotidase activity of NT5C2 is tightly regulated. In basal conditions (the absence of allosteric activators), this enzyme adopts an inactive configuration. It is only activated upon interaction with positive allosteric regulators (e.g., ATP, ADP, Ap₄A), which induce conformational changes that make the active center accessible to its substrates and competent for catalysis. However, the specific molecular mechanisms by which relapse-associated NT5C2 mutations trigger constitutively active NT5C2 nucleotidase activity remain to be elucidated.

[0058] Mechanistically, relapse- associated NT5C2 mutant proteins show increased nucleotidase activity in vitro and increased sensitivity to allosteric activators. In addition, and of utmost importance in the context of ALL therapy, NT5C2 mutations enhance the capacity of leukemic lymphoblasts to dephosphorylate and clear thio-IMP, thio-XMP and thio-GMP, the active cytotoxic metabolites of the thiopurine nucleoside analogs 6-mercaptopurine (6-MP) and 6- thioguanine (6-TG), two drugs broadly used in the treatment of ALL. Consistently, relapse- associated NT5C2 mutations confer selective resistance to 6-MP and 6-TG chemotherapy in ALL lymphoblasts.

[0059] NT5C2 mutant proteins show increased nucleotidase activity in vitro and confer resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

[0060] NT5C2 mutations result in increased enzymatic activity and drive resistance to thiopurine nucleoside analogs by decreasing the intracellular levels of active cytotoxic metabolites mediating the antileukemic effects of these drugs. Identifying activating mutations in NT5C2 as major drivers of chemotherapy resistance in 20% of relapsed ALLs (NT5C2 mutations are the single most recurrent genetic alteration acquired in relapsed lymphoblastic leukemia and are specifically associated with early relapse and progression under treatment) have provided a method to locate sites for interaction with NT5C2 inhibitors. NT5C2 inhibitors that modulate or reduce the activity of NT5C2 mutations can be used for the reversal of thiopurine resistance in relapsed ALL, including the preferred compounds described herein.

[0061] Enzymatic and structural analyses of NT5C2 mutant proteins, identified three distinct functional classes of relapse associated NT5C2 mutations. Each of these groups of mutations lock the NT5C2 protein in an active state, either by forcing a constitutively active configuration similar to that induced by allosteric activators, or via disruption of intramolecular switch-off mechanisms responsible for terminating activation and returning the enzyme to its resting inactive state. In this context, structure-function analysis of NT5C2 mutant proteins provided important new mechanistic insights on the mode of action of these mutations and support that small molecules interfering with allosteric activation can be active against the majority of NT5C2 mutant proteins. Two families of active small molecule NT5C2 inhibitors have been identified, as well as a core structure for binding to NT5C2.

3. Detailed Description of Embodiments

Compositions and Formulations

[0062] The NT5C2 inhibitors and related compounds discussed herein are contemplated for use as pharmaceutical compositions, which are useful for treatment of acute lymphoblastic leukemia. Therefore, the compounds of the invention are formulated into pharmaceutical compositions, including a carrier, for administration to human subjects in a biologically compatible form suitable for administration in vivo. The present invention thus provides a pharmaceutical composition comprising compounds of the invention in admixture with a pharmaceutically acceptable diluent and/or carrier. The pharmaceutically-acceptable carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not

deleterious to the recipient thereof.

[0063] Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21 ^stEdition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.

[0064] The pharmaceutical formulations of the present invention are prepared by methods well- known in the pharmaceutical arts. For example, the compounds of the invention are brought into association with a carrier and/or diluent, as a suspension or solution. Optionally, one or more accessory ingredients (e.g., buffers, flavoring agents, surface active agents, and the like) also are added. The choice of carrier is well within the prevue of the person of ordinary skill in the relevant art and is determined by the solubility and chemical nature of the compounds, chosen route of administration and standard pharmaceutical practice. These accessory ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (1 1 ) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monosterate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21 ) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.

[0065] For administration to a suitable subject, preferably to a human patient suffering from or suspected of suffering from acute lymphoblastic leukemia, the compounds described here are prepared according to methods known in the art into suitable formulations for any route of administration and suitable doses. Suitable subjects for administration and treatment can be any mammal, including rats, mice, dogs, cats, farm animals such as cattle, sheep, horses and the like or any mammal.

Dosing and Dosage Forms

[0066] The appropriate dose of compound of the invention depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher for example, the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, the frequency of administration, the severity of the disease, and the effect which the practitioner desires the an active agent to have. Furthermore, appropriate doses of an active agent depend upon the potency with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein or which are convenient to the practitioner and know in the art. When one or more of these active agents are to be administered to an animal (e.g., a human), a relatively low dose may be prescribed at first, with the dose subsequently increased until an appropriate response is obtained. In addition, the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0067] Dosages and regimens for administration are determined by the person of skill, including physicians. Administration of compositions, including the compounds described here, can be performed a single time, or repeated at intervals, such as by continuous infusion or repeated oral doses, over a period of time, four times daily, twice daily, daily, every other day, weekly, monthly, or any interval to be determined by the skilled artisan based on the subject involved. Treatment can involve administration over a period of one day only, a week, a month, several months, years, or over a lifetime. Regimens and duration can vary according to any system known in the art, as is known to the skilled person.

[0068] Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.

[0069] Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type maybe employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter.

These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.

[0070] Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.

[0071] Pharmaceutical compositions for rectal or vaginal administration may be presented as a suppository, which maybe prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

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

[0072] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically- acceptable carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.

[0073] Pharmaceutical compositions suitable for parenteral administrations comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.

[0074] In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.

[0075] The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally- administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

[0076] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

Salts

[0077] Salts of the enumerated compounds of the invention disclosed herein include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2- ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p- chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p- toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-l -carboxylic acid, glucoheptonic acid, 4,4'-methylenebis(3-hydroxy-2-ene-l -carboxylic acid), 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like. Salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide.

Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylgluc amine and the like.

Stereoisomers

[0078] It is understood that the disclosure of a compound herein encompasses all stereoisomers of that compound. As used herein, the term "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures which are not interchangeable. The three-dimensional structures are called configurations.

Stereoisomers include enantiomers, optical isomers, and diastereomers.

[0079] The terms "racemate" or "racemic mixture" refer to a mixture of equal parts of enantiomers. The term "chiral center" refers to a carbon atom to which four different groups are attached. The term "enantiomeric enrichment" as used herein refers to the increase in the amount of one enantiomer as compared to the other.

[0080] It is appreciated that compounds of the present invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, diastereomeric, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

[0081] As noted, the compounds of the invention may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double -bond isomers (i.e., geometric isomers such as E and Z), enantiomers or diastereomers. Thus, reference to a compound of the invention includes each of the isolated stereoisomeric forms (such as the enantiomerically pure isomers, the E and Z isomers, and etc.) as well as mixtures of

stereoisomers in varying degrees of chiral purity or percetange of E and Z, including racemic mixtures, mixtures of diastereomers, and mixtures of E and Z isomers of the compound.

Accordingly, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan. The invention includes each of the isolated stereoisomeric forms as well as mixtures of stereoisomers in varying degrees of chiral purity, including racemic mixtures. It also encompasses the various diastereomers. Other structures may appear to depict a specific isomer, but that is merely for convenience, and is not intended to limit the invention to the depicted olefin isomer. When the chemical name does not specify the isomeric form of the compound, it denotes any one of the possible isomeric forms or a mixture of those isomeric forms of the compound.

[0082] In certain embodiments, "optically active" and "enantiomerically active" refer to a collection of molecules, which has an enantiomeric excess of no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, no less than about 91%, no less than about 92%, no less than about 93%, no less than about 94%, no less than about 95%, no less than about 96%, no less than about 97%, no less than about 98%, no less than about 99%, no less than about 99.5%, or no less than about 99.8%. In certain embodiments, the compound comprises about 95% or more of one enantiomer and about 5% or less of the other enantiomer based on the total weight of the racemate in question. [0083] The compounds described herein may also exist in several tautomeric forms. The term "tautomer" as used herein refers to isomers that change into one another with great ease by a proton or an alkyl shift from one atom of a molecule to another atom of the same molecule so that they can exist together in equilibrium. For example, ketone and enol are two tautomeric forms of one compound.

4. Summary of Supporting Results

[0084] Results presented here have demonstrated surprising allelic diversity in NT5C2 mutant proteins, with three distinct groups of mutations based on their intrinsic enzymatic activity and response to allosteric regulation. New insights also were gained from in-silico structure modeling and crystallography studies presented here on the specific molecular mechanisms regulating the activity of NT5C2. Lead compounds having a structure that binds to the active sites of NT5C2 (and structurally related compounds) were discovered and shown to have inhibiting activity. Compounds were tested for NT5C2 inhibitory activity using malachite green assays and shown to be active.

[0085] Experimental approaches described here include (i) protein structure crystallographic analysis of mutant NT5C2 proteins, (ii) antibody based functional analysis of NT5C2 regulatory domains, (iii) structure-based drug screens for the development of NT5C2 inhibitors and (iv) advanced therapeutic platforms, including inducible models of mutant NT5C2 expression driving thiopurine resistance in ALL cell lines.

Recurrent NT5C2 mutations in relapse ALL

[0086] To address the genetic mechanisms contributing to chemotherapy resistance in ALL, exome sequencing analysis was performed of matched diagnostic, remission (normal) and relapse DNA samples from leukemia patients and interrogated the role of relapsed-associated mutations in drug resistance. This analysis revealed as the most notable finding the presence of recurrent, heterozygous, relapse associated mutations in the cytosolic 5 '-nucleotidase II (NT5C2) at relapse. See FIG. 2.

[0087] Following on these results, whole exome sequencing analysis was extended to a series of 57 matched diagnostic, remission and relapse ALL DNA samples including 33 T-cell ALLs and 24 B-cell precursor ALLs. These studies confirmed the presence of NT5C2 mutations in in 10/57 relapse ALL samples and identified NT5C2 as the only gene selectively mutated exclusively at the time of relapse. See FIG. 3. Extended NT5C2 targeted resequencing analysis of 103 relapse T-ALL and 265 relapse B-precursor ALL samples was completed. NT5C2 mutations found in relapse ALL are characteristically point mutations frequently involving recurrent residues (R39, R238, R367, L375 and D407) and resulting in recurrent amino acid substitutions (NT5C2 R367Q n=39, NT5C2 R238W n=17, R39Q n=5, NT5C2 L375F n=2, D407A n=2). FIG. 2. The only nonsense NT5C2 allele in our series (NT5C2 Q523*) encodes a C-terminal truncated NT5C2 protein which retains the catalytic domain. See FIG. 2.

Relapse-associated NT5C2 mutations are gain of function alleles.

[0088] The NT5C2 gene encodes a ubiquitously expressed cytosolic nucleotidase responsible for the final dephosphorylation of 6-hydroxypurine nucleotide monophosphates such as IMP, XMP and GMP before they can be exported out of the cell in the form of inosine, xanthosine and guanosine. In addition, NT5C2 also can dephosphorylate and inactivate 6-thioinositol monophosphate (thio-IMP) and 6-thioguanosine monophosphate (thio-GMP) which mediates the cytotoxic effects of 6-MP and 6-TG. Given the described role of NT5C2 in the metabolism and inactivation of nucleoside analog drugs, the recurrent finding of the NT5C2 R367Q, NT5C2 R238W, NT5C2 R39Q and NT5C2 L375F alleles and the reported association between increased levels of nucleotidase activity with thiopurine resistance and worse clinical outcome in ALL, without wishing to be bound by theory, the relapse-associated NT5C2 mutations represent gain of function alleles with increased enzymatic activity. Consistent with this prediction, mutant NT5C2 recombinant proteins (including the highly recurrent NT5C2 R367Q) have a 15-50 fold increase in 5 '-nucleotidase activity compared to wild type NT5C2. See FIG. 4, which shows the relative 5 '-NT activity of the indicated mutants. The Q523X mutation show basal levels of activity, but is hyperresponsive to ATP. See FIG. 5.

[0089] Analysis of response to ATP demonstrated three classes of activating NT5C2 alleles based on their response to this allosteric regulator: (i) Class I mutants (e.g. K359Q) which show high levels of basal activity (hyperactive and ATP independent); (ii) class II mutants (e.g.

R367Q) which show increased basal activity together with increased sensitivity to allosteric activation (hyperactive and ATP hyperresponsive) and (iii) class III mutants (e.g. Q523*) which show limited activity in basal conditions, but aberrantly high enzymatic activity in response to allosteric activation (ATP hyperresponsive). The data in FIG. 5 indicate that NT5C2 mutations show increased activity and allosteric activation by ATP. Similar results were obtained for other allosteric regulators (ADP and Ap₄A). See also FIG. 6, which demonstrates a differential response to allosteric activation by ATP of NT5C2 mutant proteins. Similar results were obtained for other allosteric regulators (ADP and Ap₄A). These results support at least three different mechanisms of action for activating NT5C2 mutations. Most NT5C2 mutants are hyperactive and ATP hyperresponsive (Class II). It is likely that, despite lying in different regions of the NT5C2 protein, they share a common underlying molecular mechanism.

Relapse associated NT5C2 mutations induce thiopurine resistance in ALL.

[0090] To formally test the role of NT5C2 mutations in chemotherapy resistance, the effects of relapse-associated mutant NT5C2 expression in the response of ALL cells to 6-MP and 6-TG was analyzed. Expression of mutant NT5C2 in ALL cell lines resulted in marked and specific resistance to 6-MP and 6-TG while expression of wild type NT5C2 did not alter the therapeutic activity of these drugs. See FIG. 7, which shows that NT5C2 mutations induce resistance to 6- MP.

Structure-function analyses of class I NT5C2 mutant proteins.

[0091] NT5C2 is a homo-tetrameric protein in which the dimers represent the minimally active form of the enzyme. In basal conditions, the NT5C2 enzyme sits in a primarily inactive configuration with residues from an unstructured domain (amino acids 355 to 364) preventing substrate access to the catalytic center. Physiologic activation of NT5C2 is mediated by binding of allosteric regulators (ATP, ADP, Ap₄A) to an allosteric pocket proximal to the NT5C2 active site. See FIG. 8. Consequently, allosteric activation results in increased ordering of G355-E364 into an alpha helix configuration (Helix A), which in turn displaces F354 from the catalytic center (making it accessible for substrate binding) and moves D356 into the active site of the protein (making it competent for catalysis). See FIG. 8. This active configuration is further stabilized by the interaction of K361, in the helix A of one monomer, with the negatively charged D459 residue from the other subunit. ATP binding induces NT5C2 activation via helical configuration of the helix A domain.

[0092] Mapping of relapse-associated NT5C2 mutations in the crystal structure of NT5C2 showed that the NT5C2 K359Q mutation is located in the regulatory helix A domain. In-silico analysis of the effect of NT5C2 K359Q using energy minimization and rotamer library analysis in Chimera and prediction of protein stability changes with the SDM potential energy statistical algorithm, showed that this mutation potentially can mimic the effect of ATP binding by inducing increased NT5C2 helix A stability, resulting in an active configuration with

displacement of inhibitory F354 out of the NT5C2 catalytic center and positioning of catalytic D356 into the active site of the enzyme. FIG. 9.

[0093] To confirm this model, active NT5C2 K359Q (mutant) recombinant protein was generated, purified and crystallized, and the crystal structure of this mutant was solved. These analyses demonstrated that NT5C2 K359Q does adopt a characteristic NT5C2 active

configuration with an accessible catalytic center and organized helix A, despite the absence of ATP. FIG. 10. These results demonstrate that NT5C2 K359Q induces activation of NT5C2 by forcing an active configuration in the absence of allosteric activators and is consistent with the enzymatic assays which show high levels of NT5C2 activity in absence of ATP and limited response to allosteric activation (class I) for this mutant. The pattern of enzymatic activity and response to allosteric activation of the NT5C2 K359Q mutation is highly similar to that of the recurrent NT5C2 L375F mutation, suggesting a common mechanism of action.

[0094] Structural mapping and molecular environment analysis indicate that L375 is present within a dedicated hydrophobic pocket responsible for assisting NT5C2 dimerization. Mutation of residue L375 to a phenylalanine residue introduces an additional hydrophobic ringed structure in this region, resulting in a more stable NT5C2 dimer and increased helix A stabilization. An active NT5C2 L375F recombinant protein was generated, purified and crystallized and the crystals structure solved to test the effects of this mutation. These analyses demonstrate that NT5C2 L375F is inserted in a dedicated hydrophobic pocket formed by residues A31, Y32, F36, 1372, L375, L379, W382, L440, F441, Y461, A463 and H486 of the opposing monomer.

Increased dimerization forces a conformational change that fixes the regulatory helix A in an organized active configuration. See FIG. 11. Close inspection of the structures of K359Q and L375F and comparison with the structure of wild type NT5C2 bound to ATP revealed that the active structure induced by these relapse-associated mutations differs from that of the allosterically activated wild type protein. See FIG. 10 and FIG. 11. K359Q and L375F alter the configuration of helix AK361 and its activating interaction with D459 in the neighbor subunit, which results in a neomorphic and more stable configuration of helix A, distinct from that present in the wild type enzyme. [0095] The convergent enzymatic and structural features of class I NT5C2 mutations define this group as neomorphic constitutively active alleles and strongly supports the relevance of crystal structure analyses for the characterization of NT5C2 mutations with distinct mechanisms of action.

Structure-function analyses of class II NT5C2 mutant proteins.

[0096] Given the insight on the mechanisms of NT5C2 regulation, and given that

homodimerization is strictly required for enzymatic activity, additional activating NT5C2 mutations can specifically target regulatory elements in the quaternary structure of the enzyme. The monomer-monomer interface in the active NT5C2 dimer creates a positively charged pocket, which connects with the helix A domain at the bottom. Several of the relapse-associated NT5C2 mutations involve positively charged amino acids (R39, R367, R446 and R478) within this inter- monomeric cavity, (see FIG. 12) and the recurrently mutated R238 residue is located at the surface of the protein facing the entrance of this pocket. In addition, the entrance of this cavity sits right below a previously uncharacterized domain formed by two amphipathic alpha helices (AA 375-400 and AA 420-433) connected by an unstructured loop (AA 401-419) (see FIG. 12). This unstructured loop is 100% conserved in vertebrates, shows a highly specific ordering of polar, negatively charged, and hydrophobic residues, and is recurrently targeted by relapsed associated NT5C2 mutations (K404N, D4G7A, D407Y, D407E, D407H, S408R) in relapsed ALL.

[0097] To test if these two domains (flexible loop and dimer pocket) can engage in electrostatic interactions, in-silico analysis of the configuration of these regions was performed in the context of inactive/resting (Apo) and allosterically activated (ATP-bound) NT5C2 structures. In the absence of ATP, NT5C2 showed a short loop (L(405)DSSSNERPD) (SEQ ID NO: l) with limited capacity to interact with the inter-monomer pocket of the NT5C2 dimer. However, modeling of the active (ATP-bound) NT5C2 structure revealed a longer disordered loop

(E(399)LYKHLDSSSNERPD) (SEQ ID NO:2) allowing a broader range of interactions and highly dynamic, yet restricted, movement of this loop within the positively charged inter- monomer cavity of the NT5C2 dimer subunits. See FIG. 13.

[0098] In this model, the interactions between the flexible loop and positively charged residues in the inter- monomer pocket are driven primarily by the loop residue D407. In the most favorable (lowest potential energy ) loop-pocket interaction model, D407 engages in hydrogen bonding with residue K361, an interaction that disengages this residue from D459, destabilizing the helix A domain and inactivating NT5C2. Disruption of the helix A stabilizing interaction between K361 and D459 returns NT5C2 to a basal inactive configuration. See FIG. 14.

Together, these analyses suggest that activation of NT5C2 is followed by a conformation change that allows the loop containing D407 to interact with positively charged residues in the pocket cavity formed in the interface between two NT5C2 monomers and that this loop-pocket interaction functions as a switch-off mechanism triggering the destabilization of helix A as D407 "attacks" the interaction between K361 and D459. FIG. 14.

[0099] Two experimental lines of evidence support this model: first, both pocket and loop NT5C2 mutations show similar enzymatic properties with increased basal nucleotidase activity and increased sensitivity to ATP (Class II mutations) (see FIG. 5 and FIG. 6), and second, antibodies raised against the D407 loop phenocopy the effects of loop mutations and induce increased nucleotidase activity in response to ATP in in vitro nucleotidase assays. FIG. 15 shows that antibodies against the D407 loop induce NT5C2 hyperactivation in response to ATP. In this test, nucleotidase assays for wild type NT5C2 protein were incubated with IgG (control) and two purified antibodies (A and B) against a D407 loop synthetic peptide.

[0100] Based on these observations, it is believed that these domains (D4Q7 loop and inter- subunit pocket) may represent a functional unit involved in the regulation of the enzymatic activity of NT5C2.

[0101] Without wishing to be bound by theory, the model on the mechanisms of action of class II NT5C2 mutations (hyperactive and hyperresponsive to allosteric activation by ATP) is that these alleles disrupt an intramolecular switch off mechanism mediated by the interplay between a flexible loop containing D407 and a positively charged pocket cavity formed in the interface between two NT5C2 monomers (FIG. 12, FIG. 13, FIG. 14, and FIG. 16). Consistently, both pocket and loop NT5C2 mutations showed similar enzymatic properties with increased basal nucleotidase activity and increased sensitivity to allosteric activation by ATP (FIG. 5).

Moreover, antibodies directed against the flexible loop phenocopied the effects of these mutations and induced increased NT5C2 nucleotidase activity in response to ATP presumably by interfering with this loop-pocket interaction mediated switch off mechanism (FIG. 15). To verify this model experimentally, the crystal structures of full length loop (NT5C2 D407A) and selected pocket mutations (NT5C2 R39Q, NT5C2 R367Q and NT5C2 R238W) were solved following the same protocol detailed above for the crystallization and analysis of the NT5C2 K359Q and L375F mutant. This information can determine (i) that class II mutants result in a similar helical configuration of the helix A domain, (ii) that this configuration overlaps with the ATP-bound active structure of wild type NT5C2.

[0102] The C-terminal region of NT5C2 consists of a highly mobile disordered linker region and a striking and highly conserved 13 residue long C-terminal acidic stretch

(D(549)EDDDEEEEEEEE; SEQ ID NO: l). Enzymatic analysis of NT5C2 wild type and NT5C2 Q523* mutations demonstrate that this allele shows low levels of nucleotidase activity in basal conditions but is hyperresponsive to allosteric activation by ATP. See FIG. 5. This result sheds new light into the function of the C-terminal domains of NT5C2 and supports the presence of an as yet poorly characterized negative regulatory domain in this region. In the absence of allosteric activators, the C-terminal acidic stretch of NT5C2 may interact with the positive surface of the NT5C2 intermonomeric pocket forcing the NT5C2 dimer into a closed inactive configuration. Upon activation, opening of the dimer interface would release the C-terminal domain to overcome this inhibition. Alternatively, it is possible that the C-terminal acidic tail of NT5C2 binds to the positively charged helix A domain (K(359)SKKRKQ; SEQ ID NO:2), preventing it from adopting an helical active configuration. Allosteric activation induced helical configuration of the helix A domain would release the C-terminal domain and overcome this inhibition. In both scenarios, the C-terminal tail of NT5C2 can be predicted to regulate the threshold and rate of NT5C2 activation in response to ATP adopting a structured inhibitory configuration in basal (apo) conditions and a flexible unstructured conformation in the active (ATP-bound) NT5C2 protein. This is consistent with the structures of the constitutively active class I NT5C2 mutants K359Q and L375F in which the C-terminal domain of NT5C2 adopts a flexible configuration.

[0103] NT5C2 crystallization of full length and the relapsed associated NT5C2 Q523* mutant proteins was performed as detailed above. This showed that NT5C2 Q523* adopts the active helical configuration similar to that of NT5C2 class II mutants and wild type NT5C2 activated in the presence of ATP.

[0104] These structural studies are complemented with the functional characterization of the C- terminal acidic stretch of NT5C2 using antibodies targeting this regulatory region. Following an approach similar to that employed to define the inhibitory function of the flexible D407 loop (see FIG. 15), peptide antibodies against the 13 amino acid acidic tail of NT5C2 were generated and tested for their ability to induce increased nucleotidase activity in response to ATP. Briefly, rabbit polyclonal antibodies were generated against a synthetic DEDDDEEEEEEEE (SEQ ID NO:3) peptide conjugated with the Keyhole Limpet Hemocyanin (KLH) carrier protein. Specific immunoglobulins recognizing DEDDDEEEEEEEE (SEQ ID NO:3) are purified by peptide affinity purification and evaluated for their capacity to recognize recombinant NT5C2 protein by western blot analysis. The activity of these antibodies was tested in nucleotidase assays against recombinant wild type NT5C2 protein in basal conditions and in presence of ATP. Non-specific immunoglobulins and neutralized C-terminal antibody (preincubated with the NT5C2 C-terminal peptide) are used as negative controls. C-terminal acidic tail specific antibodies induced ATP dependent increased nucleotidase activity in wild type full length NT5C2. (FIG. 17).

[0105] Given the dependency of class II and class III NT5C2 mutants on allosteric factors for activation, it is proposed that small molecules that interfere with this mechanism can be used to abrogate NT5C2 activity in leukemia cells harboring these mutations. Moreover we propose that inhibition of allosteric activation may be readily achieved by small molecules that occupy the allosteric ATP binding pocket of NT5C2.

5. Examples

[0106] This invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein, are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Example 1. Methods.

[0107] General Methods: Nucleotidase activity against wild type and mutant recombinant NT5C2 proteins is performed using a malachite green assay in the presence and absence of ATP as an allosteric activator. Cellular toxicity is evaluated by alamarBlue cell viability in ALL wild type and mutant cells treated with increased concentrations of NT5C2 inhibitors in the presence and absence of 6-MP. Cell permeability is evaluated using Caco-2 permeability and the parallel artificial membrane permeability assay (PAMPA). Cellular systems available for in vitro drug testing include: ALL lines with inducible expression of NT5C2 mutants; isogenic primary wild type and mutant mouse ALL lines; matched diagnosis and relapse human ALL samples and xenografts with wild type and mutant NT5C2, respectively. Animal models available for in vivo drug testing include: isogenic primary wild type and mutant mouse ALL lines and matched diagnosis and relapse human ALL xenografts with wild type and mutant NT5C2, respectively.

[0108] Highly active NT5C2 inhibitors are evaluated for membrane and cell permeability using parallel artificial membrane permeability (PAMPA) and Caco-2 permeability assays, respectively and further modified to optimize their bioavailability. In the case of compound 11 related molecules, where the acidic moiety implicated in inhibition may interfere with cell permeability, acetoxymethyl ester prodrugs can be generated.

[0109] Library Screen/Docking Analysis: Potential docking sites in the NT5C2 structure were analyzed using Schrodinger Suite SiteMap software applied to the crystal structures of wild type and the resolved mutant NT5C2 proteins. Four different pockets with Sitemap SiteScores above 0.8, indicating sites targetable with small molecule binders, were identified. Candidate small molecules targeting NT5C2 were identified via in-silico virtual screening of chemical databases containing 1.2 million drug-like, commercially available compounds for their ability to interact with and potentially inhibit NT5C2 at these sites. Briefly, wild type and mutant NT5C2 crystal structures were used as a rigid computational receptor, into which virtual databases of small molecules were docked and scored for binding affinity. This virtual screening identified roughly 1,000 molecules predicted to potentially interact at the NT5C2 catalytic site, ATP allosteric binding site or one of the additional pockets with a cutoff GlideScore of < -9.5 after clustering with Lead Follower and K-means using Canvas in order to maximize structural diversity. Of these, 68 compounds showed GlideScores of < -11, predicting high affinity binding, and 7 of them even scored below the -12 threshold. Biochemical evaluation of the inhibitory activity of the top 40 scoring compounds (10 top scoring compounds at catalytic site, 10 top scoring compounds at the Site 4 and top 10 acidic and top 10 non-acidic compounds at the ATP allosteric binding site) were analyzed using a malachite green-based NT5C2 nucleotidase assay (see below).

[0110] Malachite Green Assay with Inhibitors: Nucleotidase activity was measured with the abeam phosphate assay kit (ab65622). Enzymatic reactions were set up at final concentrations: 0.5 μΜ WT NT5C2, 1 mM compound in DMSO, 25 μΜ IMP (+/- 0.3 mM ATP). Briefly, WT NT5C2 protein and compound were incubated on ice for 10 minutes. Then, substrate (IMP) was added and the reaction was incubated at 37°C for 5 minutes. Following the enzymatic reaction, phosphate reagent was added and the plate was incubated for 15 minutes in the dark at room temperature. The plate was then read at 650nm on a BioTek plate reader.

Example 2. Structure -based identification of NT5C2 small molecule inhibitors.

[0111] In order to identify new highly active small molecule NT5C2 inhibitors, potential docking sites in the NT5C2 structure were analyzed using Schrodinger Suite SiteMap software applied to crystal structures of wild type and the resolved mutant NT5C2 proteins. Four different pockets with Sitemap SiteScores above 0.8, which indicates sites targetable with small molecule binders, were identified. These include the catalytic site (SiteScore 1.058) and the ATP allosteric binding site (SiteScore 1.058).

[0112] Candidate small molecules targeting NT5C2 were identified via in-silico virtual screening of chemical databases containing 1.2 million drug-like, commercially available compounds for their ability to interact with and potentially inhibit NT5C2 at these sites. Briefly, wild type and mutant NT5C2 crystal structures were used as a rigid computational receptor, into which virtual databases of small molecules were docked and scored for binding affinity. A comprehensive spatial survey on how a flexible ligand fits into regions on a fixed protein crystal structure was performed using Glide (grid-based ligand docking with energetics) in which each docking pose is assigned a score reflecting the ligand' s binding affinity for the designated area of the protein. This approach allows large libraries of small molecules, as well as collections of novel, but synthetically accessible, drug-like molecules to be docked in-silico with high speed and accuracy (1 A),

[0113] This virtual screening identified roughly 1,000 molecules predicted to potentially interact at the NT5C2 catalytic site, ATP allosteric binding site or one of the additional pockets with a cutoff GlideScore of < -9.5 after clustering with Lead Follower and K-means using Canvas in order to maximize structural diversity. Of these, 68 compounds showed GlideScores of < -11, which predicts high affinity binding, and 7 of them scored below the -12 threshold. Biochemical evaluation of the inhibitory activity of the top 40 scoring compounds ( 10 top scoring compounds at catalytic site, 10 top scoring compounds at the Site 4 and top 10 acidic and top 10 non-acidic compounds at the ATP allosteric binding site), analyzed using a malachite green-based NT5C2 nucleotidase assay, identified two small molecule NT5C2 inhibitors predicted to bind the ATP allosteric site (compound 11 , (5-{ l-[2-aniino-6-(morpholin-4-yl)pyrimidine-4-yl]piperidin-4-yl }- lH-pyrazol-3-yl)acetic acid, GlideScore -14.07; and compound 20, N4-[2-(2-amino-l,3-thiazol- 4-yl)ethyl]-5,6,7,8-tetrahydroquinazoline-2,4-diamine, GlideScore -10.52). See Tables 1 and 2, below.

Table 1. Compound Activity.

Inhibitor Activity measured at 0.3 mM ATP.

Table 2. Compound Activity.

Morpholine ATP -12.7 NA

^Inhibitor Activity measured at 0.3 mM ATP; NA indicates Not Available.

[0114] Both compound 1 1 and compound 20 (see FIG. 18) induced almost complete NT5C2 inhibition in basal conditions and effectively abrogated NT5C2 activity 7 fold and 4 fold in the presence of 0.3 mM ATP aliosterie activator, respectively. FIG. 19, which provides data showing that ATP pocket binding compounds 11 and 20 inhibit NT5C2 nucleotidase activity in basal conditions and in the presence of 0.3 mM ATP aliosterie activation. Docking analysis of these small molecules in the NT5C2 crystal structure showed extensive interactions in the aliosterie pocket of the enzyme predicted to interfere with ATP binding. FIG. 20. These two compounds both contain a pyrimidine ring predicted to stabilize their interaction with NT5C2 in docking analyses. See FIG. 18 and FIG. 20. These compounds, therefore, are considered lead compounds, the structure of which can interact productively with NT5C2.

Example 3. NT5C2 inhibitors for the treatment of chemotherapy resistant/relapsed ALL.

[0115] Following the identification of compound 11 and compound 20 as NT5C2 inhibitors (see Example 2 and FIG. 18), related small molecules in their chemical class (structurally similar compounds) were screened to identify new active compounds with increased NT5C2 binding and inhibitory activity. Initial tests include the "masking" of highly related compounds to maximize the chemical diversity of the results. The process can be reversed to recover all commercially- available compound 11- and compound 20- related small molecules in available libraries for testing for NT5C2 binding and inhibition. NT5C2 nucleotidase activity in basal conditions and upon ATP aliosterie activation using malachite green-based enzymatic assay (see FIG. 25A-D and FIG. 26A-B). Binding affinity can be analyzed by isothermal titration calorimetry (ITC) in an autoiTC200 instrument.

[0116] Although compound 11 and compound 20 share common NT5C2 interactions mediated by their pyrimidine moiety, their side chains occupy different regions of the NT5C2 aliosterie pocket (FIG. 20 and FIG. 22). A chimeric compound (Chi; 2-(5-(l-(2-amino-6-((2-(2- aminothiazol-4-yl)ethyl)amino)pyrimidin-4-yl)piperidin-4-yl)-lH-pyrazol-3-yl)acetic acid) containing the side chains of compound 1 1 and compound 20 was shown by docking of Chi in the NT5C2 allosteric pocket, and gave an improved GlideScore over compounds 11 and 20, indicating increased ligand-protein interactions and predicting tighter binding (FIG. 21 and FIG. 22).

Chimeric Compound (Chi)

[0117] This chimeric compound is synthesized and evaluated for its NT5C2 binding and inhibition properties using methods previously described. In addition, additional compound 11- related small molecules with specific substitutions of its morpholine ring also are evaluated, in particular the 8 compounds below, which have predicted increased NT5C2 binding compared to compound 1 1 based on their GlideScore. See FIG. 23, which provides the structures. Highly active NT5C2 inhibitors are evaluated for stability and cell permeability and further modified to optimize their bioavailability.

Morpholine ring substitutions Example 4. Evaluation of the therapeutic activity of NT5C2 inhibitors for the reversal of chemotherapy resistance in ALL.

[0118] To analyze the effect of the most common NT5C2 mutations in chemotherapy resistance and the effect of NT5C2 inhibitors in the treatment of this disease, 4 primary ALL xenograft models of NT5C2 mutant leukemia were established from relapsed ALL samples harboring NT5C2 mutations (R367Q n=3). In addition, a mouse model of Nt5c2 -mutant ALL was established, derived from a newly generated knockin line with conditional expression of the Nt5c2 R367Q allele from the endogenous Nt5c2 locus. In basal conditions, cell from these mice express wild type Nt5c2 as exon 13 is spliced into an exon 14-18 minigene (see FIG. 27 A). This cassette can be removed by expression of Cre recombinase, allowing exon 13 to be spliced into the R367Q mutant exon 14 with consequent expression of R367Q mutant Nt5c2 (FIG. 27B). To make this system inducible, the C57BL6 Nt5c2 R367Q conditional knockin mice were crossed to Rosa26-Cre ERT2 animals, which express a tamoxifen-inducible form of Cre recombinase from the ubiquitously expressed Rosa26 locus. Finally, leukemias from these mice were generated via retroviral infection of hematopoietic progenitors with viral particles expressing a constitutively active form of NOTCH 1, a robust and broadly used model of T-ALL. In vitro analysis of the response to 6-MP demonstrated resistance to 6-MP in this model after tamoxifen-induced expression of Nt5c2 R367Q (FIG. 27C).

[0119] Transplant of these T-ALL lymphoblasts into isogenic, immunocompetent C57BL6 recipients resulted in 6-MP sensitive (Nt5c2 wild type expressing) leukemias first, which effectively became 6-MP resistant upon tamoxifen treatment/Cre-mediated activation of the Nt5c2 R367Q allele (FIG. 28A). In all, these three models facilitated the evaluation of the activity of NT5C2 inhibitors in the context of (i) multiple NT5C2 alleles in ALL cell lines, (ii) primary human tumor xenografts with NT5C2 mutations, and (iii) mouse primary isogenic tumors expressing wild type Nt5c2 and the most common Nt5c2 mutation (R367Q). FIG. 28B provides results showing effective resistance to 6-MP after activation of the NT5C2 R367Q allele via tamoxifen treatment. Example 5. Reversal of 6-MP and 6-TG Resistance.

[0120] Following drug screens and lead optimization, NT5C2 inhibitors with nanomolar potency in in vitro assays are tested against wild type and mutant NT5C2 ALL cells for their capacity to reverse 6-MP (and 6-TG) resistance in vitro. Briefly, dose response curves are performed for 6-MP (and 6-TG) in the presence and absence of NT5C2 inhibitors in CUTLL1 and JURKAT cells infected with lentiviruses driving doxycycline inducible expression of wild type and our array of mutant NT5C2 proteins using methods previously described. See FIG. 7.

[0121] Highly active NT5C2 inhibitors then are evaluated for their capacity to reverse 6-MP resistance in vivo. Towards this goal, first the maximum tolerated dose and pharmacokinetic profiles is established for each of these drugs. Following these preliminary analyses, their efficacy is evaluated alone and in combination with 6-MP in C57BL6 mice transplanted

(intravenous injection) luciferised, GFP positive, Nt5c2 wild type (n=40) and isogenic Nt5c2 R367Q NOTCH 1 -induced T-ALLs (n=40). Two independent isogenic Nt5c2 wild type (n=40) and matched Nt5c2 R367Q NOTCH 1 -induced T-ALLs are tested. Upon tumor engraftment, as detected by luciferase in vivo biomaging and assessment of circulating GFP positive tumor cells by flow cytometry, leukemia bearing animals are segregated into homogeneous cohorts (10 mice/group) according to tumor load and treated with vehicle only, 6-MP (50 mg/kg IP), NT5C2 inhibitor, or NT5C2 inhibitor plus 6-MP for 5 days. The therapeutic response to 6-MP, NT5C2 inhibitor and the drug combination are evaluated at the end of therapy by luciferase in vivo bioimaging, histology and flow cytometry analysis of GFP positive ALL lymphoblast infiltration in bone marrow and spleen. A sample size of 10 animals per group has >80% power to detect a difference in tumor burden >1.2 s.d. in a one-sided t-test with a=0.05.

[0122] Finally, and following on the results of these analyses, the effects of the top NT5C2 inhibitors in the response of primary human ALL xenografts to 6-MP are analyzed in vivo.

Three independent primary patient-derived ALL xenografts harboring relapse activating mutations in NT5C2 are injected intravenously into 40 NRG immunodeficient mice each (n=80) and tumor engraftment is monitored by flow cytometry analysis of human CD45 expressing cells. Animals with tumor loads of about 15% human lymphoblasts in peripheral blood are randomized into groups (10 mice/group), treated daily with vehicle, 6-MP (80 mg/kg via IP daily), NT5C2 inhibitor, or the combination of 6-MP plus NT5C2-inhibitor for 5 days, and evaluated for disease progression and therapy response at the end of therapy by histology and flow cytometry analysis of human CD45-positive ALL lymphoblast infiltration in bone marrow and spleen. Changes in tumor load in each therapeutic arm are compared with vehicle treated controls. A sample size of 10 animals per group has >80% power to detect a difference in tumor burden >1.2 s.d. in a onesided t-test with a=0.05.

[0123] Inhibitors with strong activity in combination with 6-MP in vivo are further evaluated for their capacity to extend survival in this model. Briefly, NT5C2-mutant ALL primary xenograft cells are transplanted via intravenous injection as before (n=76) and segregated into vehicle only (n=19), 6-MP (80 mg/kg IP; n=19), NT5C2 inhibitor (n=19) or NT5C2 inhibitor plus 6-MP (n=19) treatment groups. Following 5 days of treatment, the therapeutic response is assessed as before and the colony monitored for survival analysis. Differences between control and treatment groups are represented using Kaplan-Meier curves and statistical significance is determined using Fisher's exact test. A sample size of 19 mice per group has over 80% power to detect a 40% difference in survival at 0.05 confidence.

[0124] As will be apparent to one of ordinary skill in the art from a reading of this disclosure, further embodiments of the present invention can be presented in forms other than those specifically disclosed above. The particular embodiments described above are, therefore, to be considered as illustrative and not restrictive. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. Such equivalents are considered to be within the scope of this invention. Although the invention has been described and illustrated in the foregoing illustrative embodiments, it is understood that the present disclosure has been made only by way of example, and that numerous changes in the details of implementation of the invention can be made without departing from the spirit and scope of the invention, which is limited only by the claims that follow. Features of the disclosed embodiments can be combined and rearranged in various ways within the scope and spirit of the invention. The scope of the invention is as set forth in the appended claims and equivalents thereof, rather than being limited to the examples contained in the foregoing description. All publications mentioned herein, are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. REFERENCES

[0125] References listed below and throughout the specification are hereby incorporated by reference in their entirety.

1. Dursun et al., Neurogenetics 10:325-331, 2009.

2. Novarino et al., Science 343:506-511, 2014.

3. Oka et al., Biochem. Biophys. Res. Commun. 205:917-922, 1994.

4. Tzoneva et al., Nat. Med. 19:368-371, 2013.

5. Brouwer et al., Clin. Chim. Acta 361:95-103, 2005.

6. Hunsucker et al., J. Pharmacol. Ther. 107: 1-30, 2005.

7. Galmarini et al., Leuk. Lymphoma 44: 1105-1111, 2003.

8. Gallier et al., PLoS computational biology 7:el002295, 2011.

9. Jordheim et al., Biochem. Pharmacol. 85:497-506, 2013.

10. PCT/US2013/068821 to Ferrando et al.

Claims

CLAIMS What is claimed is:

1. A compound of the structure according to Formula 1 or Formula 2:

Formula 1 Formula 2 wherein Ri is a saturated five- to twelve-membered heterocyclic or heterobicyclic ring having one to four hetero atoms selected from the group consisting of N, O, and S; wherein R₂ is H or Ci_₄ alkyl; wherein R₃ is

or a Ci_₄ alkyl ester thereof; wherein R₄ is NH₂, NHCH₃, N(CH₃)₂, or a five- to six-membered heterocyclic ring having one or two N hetero atoms; wherein R5 is H or Ci_₄ alkyl; wherein R6 is H or Ci_₄ alkyl; or R5 and R6 are joined to form a cyclohexyl moiety; wherein R7 is selected from the group consisting of

and and wherein R8 is NH₂, NHCH₃, or N(CH₃)₂;

or a salt or hydrate thereof, with the proviso that the compound is not

2. A compound of claim 1, which is selected from the group consisting of:

3. A compound which comprises:

4. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to claim 1.

5. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to claim 2.

6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to claim 3.

7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the structure according to Formula 1 or Formula 2:

Formula 1 Formula 2 wherein Ri is a saturated five- to twelve-membered heterocyclic or heterobicyclic ring having one to four hetero atoms selected from the group consisting of N, O, and S; wherein R₂ is H or Ci_₄ alkyl; wherein R₃ is

or a Ci_4 alkyl ester thereof;

wherein R₄ is NH₂, NHCH₃, N(CH₃)₂, or a five- to six-membered heterocyclic ring having one or two N hetero atoms; wherein R5 is H or C_1-4 alkyl; wherein R6 is H or C_1-4 alkyl; or R5 and R6 are joined to form a cyclohexyl moiety; wherein R7 is selected from the group consisting of:

and

and wherein R8 is NH₂, NHCH₃, or N(CH₃)₂; or a salt or hydrate thereof.

8. A method of treating acute lymphoblastic leukemia (ALL) comprising administering to a subject in need a compound of claim 1.

9. A method of treating ALL comprising administering to a subject in need a compound of claim 2.

10. A method of treating ALL comprising administering to a subject in need a compound of claim 3.

11. A method of treating ALL comprising administering to a subject in need a pharmaceutical composition of claim 4.

12. A method of treating ALL comprising administering to a subject in need a pharmaceutical composition of claim 5.

13. A method of treating ALL comprising administering to a subject in need a pharmaceutical composition of claim 6.

14. A method of treating ALL comprising administering to a subject in need a pharmaceutical composition of claim 7.

15. The method of any of claims 8-14 wherein the ALL is chemotherapy-resistant acute lymphoblastic leukemia.