Use of inhibitors of the n-acetylaspartate synthetase for the treatment of acute myeloid leukemias

Abstract

OF THE INVENTION USE OF INHIBITORS OF THE N-ACETYLASPARTATE SYNTHETASE FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIAS Acute Myeloid Leukemias (AML) are characterized by the proliferation of immature blood cells, leading to suppression of normal hematopoiesis. Despite existing treatments, resistance and relapse are common. A metabolomic analysis revealed increased N-Acetyl-Aspartate (NAA) levels in leukemic cells compared to normal HSCs, with high NAA levels linked to poor prognosis. The inventors studied the gene NAT8L, responsible for NAA synthesis, and found correlations with patient survival. Using CRISPR-cas9, the inventors targeted NAT8L in AML cell lines and observed that NAA depletion improved response to treatments and increased survival in vivo. Inhibiting NAA synthetase thus represents a therapeutic strategy to enhance chemotherapy efficacy and prevent relapse in AML patients.

Description

[0001] USE OF INHIBITORS OF THE N-ACETYLASPARTATE SYNTHETASE FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIAS

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of oncology and haematology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Acute Myeloid Leukemias (AML) are complex and aggressive hematologic malignancies marked by the clonal proliferation of abnormal myeloid precursor cells. These malignant cells are characterized by their inability to differentiate and mature properly, resulting in the accumulation of immature, dysfunctional blasts in the bone marrow and peripheral blood. The proliferation of these immature cells impairs normal hematopoietic function, leading to pancytopenia, which manifests as anemia, neutropenia, and thrombocytopenia, consequently increasing the risk of infections, bleeding, and fatigue in affected individuals.

[0006] AML is not a single disease but rather a heterogeneous group of disorders, each defined by distinct genetic and molecular abnormalities, which contribute to varied clinical presentations, prognostic implications, and therapeutic responses. The advent of high-throughput genomic technologies has unraveled a myriad of genetic alterations in AML, including mutations in genes such as FLT3, NPM1... These molecular insights have been instrumental in refining risk stratification and guiding personalized treatment approaches.

[0007] Despite advances in understanding the pathophysiology of AML and the development of novel therapeutic agents, the prognosis for many patients remains dismal. The standard treatment regimen for AML typically involves intensive chemotherapy, which can induce remission in a substantial proportion of patients. However, treatment resistance and disease relapse are common challenges, with relapse occurring in approximately 50% of cases. The underlying mechanisms of chemoresistance are multifaceted, involving genetic heterogeneity, clonal evolution, and the protective microenvironment of the bone marrow niche.

[0008] In summary, AML represents a formidable clinical challenge with its diverse genetic landscape and high propensity for relapse and resistance. Continued research efforts are essential to uncover new therapeutic targets and develop more effective treatments to improve the survival and quality of life for patients battling this devastating disease.

[0009] The NAT8L gene encodes N-acetylaspartate synthetase (NAA synthetase), which crucially synthesizes N- Acetyl -Aspartate (NAA) by acetylating aspartate. This process is vital for cellular balance and metabolism. High levels of NAA, linked to NAA synthetase activity, are associated with poor prognosis in cancers like melanoma, breast cancer, and cervical cancer. However, its role in AML has not been studied.

[0010] SUMMARY OF THE INVENTION:

[0011] The present invention is defined by the claims. In particular, the present invention relates to the use of inhibitors of the N-acetylaspartate synthetase for the treatment of acute myeloid leukemias (AML).

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] A first object of the present invention relates to a method of treating acute myeloid leukemia (AML) in patient in need thereof comprising administering to the patient a therapeutically effective amount of an inhibitor of the N-acetylaspartate synthetase.

[0014] As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and may refer to a patient to be treated by the methods disclosed herein. In particular, the patient suffers from an AML. In some embodiments, the patient is a human infant. In some embodiments, the patient is a human child. In some embodiments, the patient is a human adult. In some embodiments, the patient is an elderly.

[0015] As used herein, the term "acute myeloid leukemia" or "acute myelogenous leukemia" or "AML" has its general meaning in the art and refers to a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells.

[0016] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0017] In particular, the method of the present invention is suitable for the treatment of the chemoresistant AML.

[0018] As used herein; the term "chemoresistant acute myeloid leukemia" or “chemoresistant AML” refers to the clinical situation in a patient suffering from an AML when the proliferation of leukemic cells cannot be prevented or inhibited by means of a chemotherapeutic agent or a combination of chemotherapeutic agents usually used to treat AML, at an acceptable dose to the patient. Thus, the expression "resistance to chemotherapy" is used in its broadest context to refer to the reduced effectiveness of chemotherapy to inhibit the growth of a leukemic cell, kill a leukemic cell or inhibit one or more cellular functions, and to the ability of a cell to survive exposure to an agent designed to inhibit the growth of the leukemic cell, kill the leukemic cell or inhibit one or more cellular functions. The leukemia can be intrinsically resistant prior to chemotherapy, or resistance may be acquired during treatment of leukemia that is initially sensitive to chemotherapy. The resistance displayed by a leukemic cell may be complete in that the chemotherapy is rendered completely ineffective against the leukemic cell, or may be partial in that the effectiveness of the chemotherapy is reduced. The phrase “preventing resistance to chemotherapy” or “overcoming resistance to chemotherapy” in context of the invention shall be effective if compared to a non-treated control, the leukemic cells become more sensitive to chemotherapy. In particular, the patient become a responder. As used herein the term “responder” in the context of the present disclosure refers to a patient that will achieve a response, i.e. a patient where the leukemia is eradicated, reduced or improved after immunotherapy. According to the invention, the responders have an objective response and therefore the term does not encompass patients having a stabilized cancer such that the disease is not progressing after immunotherapy. A “non-responder” or “refractory patient” includes patients for whom the leukemia does not show reduction or improvement after chemotherapy. The term “non responder” also includes patients having a stabilized leukemia. Typically, the characterization of the patient as a responder or non-responder can be performed by reference to a standard or a training set. The standard may be the profile of a patient who is known to be a responder or non-responder or alternatively may be a numerical value. Such predetermined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media. When it is concluded that the patient is a non-responder, the physician could take the decision to administer the patient with a therapeutically effective amount of the inhibitor of the N-acetylaspartate synthetase.

[0019] In other words, the methods of the invention is suitable to enhance the efficacy of chemotherapeutic agents.

[0020] Thus, in particular, the method of the present invention is suitable for the treatment of the chemoresistant AML, wherein the inhibitor of the N-acetylaspartate synthetase is administered in combination of a chemotherapeutic agents to enhance the efficacy of said chemotherapeutic agents.

[0021] As used herein, the term “chemotherapy” has its general meaning in the art and refers to the treatment that consists in administering to the patient a chemotherapeutic agent. As used herein, the term "chemotherapeutic agent" refers to any chemical agent with therapeutic usefulness in the treatment of cancer. Chemotherapeutic agents as used herein encompass both chemical and biological agents. These agents function to inhibit a cellular activity upon which the leukemic cell depends for continued survival. Categories of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most if not all of these drugs are directly toxic to leukemic cells and do not require immune stimulation. Suitable chemotherapeutic agents are described, for example, in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal medicine, 14th edition; Perry et at , Chemotherapeutic, Ch 17 in Abel off, Clinical Oncology 2nd ed., 2000 ChrchillLivingstone, Inc.; Baltzer L. and Berkery R. (eds): Oncology Pocket Guide to Chemotherapeutic, 2nd ed. St. Louis, mosby-Year Book, 1995; Fischer D. S., Knobf M. F., Durivage HJ. (eds): The Cancer Chemotherapeutic Handbook, 4th ed. St. Louis, Mosby-Year Handbook.

[0022] In some embodiments the chemotherapeutic agent is cytarabine (cytosine arabinoside, Ara-C, Cytosar-U), carboplatin, carmustine, chlorambucil, dacarbazine, ifosfamide, lomustine, mechlorethamine, procarbazine, pentostatin, (2'deoxycoformycin), etoposide, teniposide, topotecan, vinblastine, vincristine, paclitaxel, dexamethasone, methylprednisolone, prednisone, all- trans retinoic acid, arsenic trioxide, interferon-alpha, rituximab (Rituxan®), gemtuzumab ozogamicin, imatinib mesylate, Cytosar-U), melphalan, busulfan (Myleran®), thiotepa, bleomycin, platinum (cisplatin), cyclophosphamide, Cytoxan®)., daunorubicin, doxorubicin, idarubicin, mitoxantrone, 5-azacytidine, cladribine, fludarabine, hydroxyurea, 6- mercaptopurine, methotrexate, 6-thioguanine, or any combination thereof.

[0023] In some embodiments, the chemotherapy consists in a combination of cytarabine and an anthracycline such as daunorubicin or idarubicin.

[0024] In some embodiments, the chemotherapy is 5-azacytidine.

[0025] In some embodiments, the chemotherapy consists in administering the patient with tyrosine kinase inhibitor (TKI). Tyrosine kinase inhibitors (TKIs) are a class of targeted therapy drugs that work by inhibiting the activity of specific enzymes known as tyrosine kinases. These enzymes play a vital role in the signaling pathways that control cell division and growth. In the context of AML, certain TKIs have shown promise in treating the disease by targeting abnormal tyrosine kinases that drive the proliferation of leukemic cells. Examples of TKIs suitable for the treatment of AML include midostaurin (PKC412), quizartinib (AC220), sorafenib (BAY 43-9006), and lestaurtinib (CEP-701). These inhibitors specifically target mutations in the FLT3 gene, which is often implicated in AML, and have demonstrated efficacy in clinical trials by reducing leukemic cell counts and improving patient outcomes.

[0026] In some embodiments, the chemotherapy consists in administering the patient with a BCL-2 inhibitor.

[0027] As used herein, the term "BCL-2 inhibitor" refers to an agent that is capable of inhibiting one or more proteins in the BCL-2 family of anti-apoptotic proteins, e.g., BCL-2, BCL-xL, and BCL -w. In some embodiments, a BCL-2 inhibitor of the disclosure inhibits one protein of the BCL-2 family selectively, e.g., a BCL-2 inhibitor may selectively inhibit BCL-2 and not BCL- xl or BCL-w. The BCL-2 inhibitor described herein may inhibit one or more of BCL-2, BCL- xL, and BCL-w. In some embodiments, the inhibitor of BCL-2 anti-apoptotic family of proteins inhibits BCL-2. In some embodiments, the inhibitor of BCL-2 anti-apoptotic family of proteins inhibits BCL-2 and does not inhibit other members of the BCL-2 family of proteins, e.g., does not inhibit BCL-xL or BCL-w. In some embodiments, the BCL-2 inhibitor is a BH3-mimetic.

[0028] In some embodiments, the BCL-2 inhibitor is selected from the group consisting of navitoclax, venetoclax, A-l 155463, A-1331852, ABT-737, obatoclax, S44563, TW-37, A-1210477, AT101, HA14-1, BAM7, sabutoclax, UML77, gambogic acid, maritoclax, MIMI, methylprednisolone, iMAC2, Bax inhibitor peptide V5, Bax inhibitor peptide P5, Bax channel blocker, and ARRY 520 trifluoroacetate.

[0029] In some embodiments, the BCL2 inhibitor is venetoclax (4-(4-((2-(4-chlorophenyl)-4,4- dimethylcyclohex- 1 -en- 1 -yl)methyl)piperazin- 1 -yl)-N-((3 -nitro-4-((tetrahydro-2H-pyran-4- ylmethyl)amino)phenyl)sulfonyl)-2-(lH-pyrrolo(2,3-b)pyridin-5-yloxy)benzamide).

[0030] In some embodiments, the chemotherapy consists in a combination of BCL2 inhibitor and 5- azacytidine.

[0031] In some embodiments, the chemotherapy consists in a combination of venetoclax and 5- azacytidine.

[0032] A further object of the present invention relates to a method of preventing resistance to chemotherapy in a patient suffering from an acute myeloid leukemia (AML) comprising administering to the patient a therapeutically effective amount of an inhibitor of the N- acetylaspartate synthetase.

[0033] A further object of the present invention relates to a method of preventing relapse in a patient suffering from an AML and who was treated by chemotherapy comprising administering to the patient a therapeutically effective amount of an inhibitor of the N-acetylaspartate synthetase.

[0034] As used herein, the term “relapse” refers to reappearance of the leukemia after an initial period of responsiveness (e.g., complete response or partial response). The initial period of responsiveness may involve the level of leukemic cells falling below a certain threshold, e.g., below 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. The reappearance may involve the level of leukemic cells rising above a certain threshold, e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. More generally, a response (e.g., complete response or partial response) can involve the absence of detectable MRD (minimal residual disease).

[0035] As used herein, the term “N-acetylaspartate synthetase” or “NAA synthetase” refers to the enzyme that plays a critical role in the synthesis of N-acetylaspartate. The term is also known as N-acetyltransferase 8-like protein. The EC number is EC :2.3.1.17. This enzyme is implicated in various cellular functions, including cellular metabolism and energy production. In the context of the present invention, the NAA synthetase has been identified as a potential therapeutic target for combating chemoresistant acute myeloid leukemia (AML). By inhibiting the NAA synthetase, the therapeutic strategy aims to enhance the efficacy of chemotherapeutic agents and prevent relapse in patients suffering from AML.

[0036] As used herein, the term “inhibitor of the N-acetylaspartate synthetase” has its general meaning in the art and refers to a compound that inhibits the activity or expression of the enzyme N-acetylaspartate synthetase. In some embodiments, the inhibitor is a selective inhibitor. A “selective inhibitor” specifically refers to a compound that more effectively inhibits the activity or expression of the NAA synthetase compared to at least one other enzyme involved in cellular metabolism.

[0037] As used herein, the term “small molecule” refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, peptides, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500 Da.

[0038] Small molecules that are inhibitors of the NAA synthetase are well known in the art and includes those described in:

[0039] Nesuta, Ondrej, et al. "High throughput screening cascade to identify human aspartate N-acetyltransferase (AN AT) inhibitors for Canavan disease." ACS Chemical Neuroscience 12.18 (2021): 3445-3455,

[0040] Thangavelu, Bharani, et al. "Design and optimization of aspartate N-acetyltransferase inhibitors for the potential treatment of Canavan disease. " Bioorganic & medicinal chemistry 25.3 (2017): 870-885, and

[0041] - Mutthamsetty, Vinay, et al. "Development of bisubstrate analog inhibitors of aspartate N ~ acetyltransferase, a critical brain enzyme. " Chemical Biology & Drug Design 95.1 (2020): 48-57.

[0042] Inhibitors of the NAA synthetase are also disclosed in the international patent application. WO2017172476

[0043] In some embodiments, the inhibitor of the present invention is selected from the group consisting of N-carbobenzyloxy-L-glutamic acid, N-(l-oxo-3-phenylpropyl)-L-aspartic acid, N-[(benzyloxy)carbonyl]-L-aspartic acid, N-chloroacetyl-L-aspartic acid, N-(t- butoxycarbonyl)-L-aspartic acid, N-[(4-methylphenyl)sulfonyl]-L-proline, N-methyl-DL- aspartic acid, N-alanyl-L-aspartic acid, 2-(3-chloro-6-oxopyridazin-l(6H)-yl)acetic acid, isoxazole-3-carboxylic acid, and benzo[d]isoxazole-3-carboxylic acid.

[0044] In some embodiments, the inhibitor of the present invention is ANAT inhibitor-1 (CAS No. : 331751-78-1) having the formula of: In some embodiments, the inhibitor of the present invention is ANAT inhibitor-2 (CAS No. : 1048244-34-3) having the formula of:

[0045] One type of small molecule applicable to the present invention is a degrader molecule that degrades NAT8L (see, e.g., Ding, et al., Emerging New Concepts of Degrader Technologies, Trends Pharmacol Sci. 2020 July; 41(7): 464-474). As used herein, the terms “degrader” and “degrader molecule” refer to all compounds capable of specifically targeting a protein for degradation (e.g., ATTEC, AUTAC, LYTAC, or PROTAC). Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome (see, e.g., Zhou et al., Discovery of a Small-Molecule Degrader of Bromodomain and Extra-Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable of Achieving Tumor Regression. J. Med. Chem. 2018, 61, 462-481; Bondeson and Crews, Targeted Protein Degradation by Small Molecules, Annu Rev Pharmacol Toxicol. 2017 Jan. 6; 57: 107-123; and Lai et al., Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL Angew Chem Int Ed Engl. 2016 Jan. 11; 55(2): 807-810). In particular, Proteolysis Targeting Chimeras (PROTACs) are a class of bifunctional molecules that live in the “beyond rule of 5” (bRo5) (Barbie, D. A.; Tamayo, P.; Boehm, J. S.; Kim, S. Y.; Moody, S. E.; Dunn, I. F.; Schinzel, A. C.; Sandy, P.; Meylan, E.; Scholl, C.; et al. Nature 2009 462 1 OS- 112) space that hijack the endogenous protein homeostasis machinery via recruitment of an E3 ubiquitin ligase via one component ligand and associating it with a target protein of interest (Pol) through another component ligand to mediate ubiquitin transfer to, and degradation of, the latter via the proteasome.

[0046] Thus, in some embodiments, the degrader molecule is a bifunctional compound having the chemical structure ULM-PTM, wherein the ULM is a small molecule E3 ubiquitin ligase binding moiety that binds an E3 ubiquitin ligase; and the PTM is a small molecule comprising the NAT8L targeting moiety.

[0047] As used herein, the term “ubiquitin ligase” refers to a family of proteins that facilitate the transfer of ubiquitin to a specific substrate protein, targeting the substrate protein for degradation. For example, IAP an E3 ubiquitin ligase protein that alone or in combination with an E2 ubiquitin-conjugating enzyme causes the attachment of ubiquitin to a lysine on a target protein, and subsequently targets the specific protein substrates for degradation by the proteasome. Thus, E3 ubiquitin ligase alone or in complex with an E2 ubiquitin conjugating enzyme is responsible for the transfer of ubiquitin to targeted proteins. In general, the ubiquitin ligase is involved in polyubiquitination such that a second ubiquitin is attached to the first; a third is attached to the second, and so forth. Polyubiquitination marks proteins for degradation by the proteasome. However, there are some ubiquitination events that are limited to mono- ubiquitination, in which only a single ubiquitin is added by the ubiquitin ligase to a substrate molecule. Mono-ubiquitinated proteins are not targeted to the proteasome for degradation, but may instead be altered in their cellular location or function, for example, via binding other proteins that have domains capable of binding ubiquitin. Further complicating matters, different lysines on ubiquitin can be targeted by an E3 to make chains. The most common lysine is Lys48 on the ubiquitin chain. This is the lysine used to make polyubiquitin, which is recognized by the proteasome.

[0048] In some embodiments, the present invention provides compounds comprising an E3 ubiquitin ligase binding moiety (“ULM”) that is an IAP E3 ubiquitin ligase binding moiety (an “ILM”), a cereblon E3 ubiquitin ligase binding moiety (a “CLM”), a Von Hippel-Lindae E3 ubiquitin ligase (VHL) binding moiety (VLM), and / or a mouse bould minute 2 homologue (MDM2) E3 ubiquitin ligase binding moiety (MLM). In some embodiments, the ILM / VLM / CLM / MLM and PTM are joined or coupled via a chemical linker (L).

[0049] In some embodiments, the PTM is selected among small molecules that inhibit the activity of the NAA synthetase. In some embodiments, the PTM is selected from the group consisting of N-carbobenzyloxy-L-glutamic acid, N-(l-oxo-3-phenylpropyl)-L-aspartic acid, N- [(benzyloxy)carbonyl]-L-aspartic acid, N-chloroacetyl-L-aspartic acid, N-(t-butoxycarbonyl)- L-aspartic acid, N-[(4-methylphenyl)sulfonyl]-L-proline, N-methyl-DL-aspartic acid, N- alanyl-L-aspartic acid, 2-(3-chloro-6-oxopyridazin-l(6H)-yl)acetic acid, isoxazole-3- carboxylic acid, and benzo[d]isoxazole-3-carboxylic acid. In some embodiments, the PTM is ANAT inhibitor- 1 or ANAT inhibitor-2 as disclosed above.

[0050] In some embodiments, the inhibitor of the present invention is an inhibitor of NAT8L gene expression.

[0051] An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In some embodiments, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of NAT8L mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of NAT8L, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding NAT8L can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. The NAT8L gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that NAT8L gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing NAT8L. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0052] As used herein, the expression "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of drug to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for drug depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of drug employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, for example about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg or 0.1-3 mg / kg, for example about 0.5-2 mg / kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the inhibitor of the present invention in an amount of about 0.1-100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0053] Typically, the inhibitor of the present invention is administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a subject, the composition will be formulated for administration to the subject. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. For example, an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg / mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg / m2and 500 mg / m2. However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials. A pharmaceutical composition of the invention for injection (e.g., intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g. 1 ml for intramuscular), and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of the inhibitor of the invention.

[0054] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0055] FIGURES:

[0056] Figure 1. NAA is highly express in leukemic blast and is implicated in leukemogenesis in a mouse model. A. NAA in 3 times higher in murin bone marrow MLL-AF9 leukemic cells (BM-LC) than in normal bone marrow HSC (BM-HSC) (n=6 and 7 respectively; **p<0;01). B. Meis lb and HoxA9 transformed murin HSPC expressing or lacking Nat8l proliferation over 72h (n=5 per genotype; ****p<0;001). C. Meislb and HoxA9 transformed murin HSPC expressing or lacking Nat8l in vitro apoptosis (n=5 per genotype; *p<0;05 and ***p<0;005). D. Colonies forming assay of Meislb and HoxA9 transformed murin HSPC expressing or lacking Nat8l (n=4 per genotype; *p<0;05). E. Kaplan-Meier survival curve of recipients transplanted with 200,000 Meislb and HoxA9 transformed murin HSPC expressing or lacking Nat8l (n = 41 per genotype).

[0057] Figure 2. CRISPR-Cas9-mediated NAT8L deletion in human OCI-AML3 and MOLM-14 AML compromise their leukemic potential. A. NAA levels quantified by conductimetry in OCI-AML3 CRISPR / Cas9 clones. B. NAA levels quantified by conductimetry in MOLM-14 CRISPR / Cas9 clones. C. Reduction and abolition of NAA levels in OCI-AML3 reduces cell cycle proliferative phases (n=3; *p=0,05). D. Reduction and abolition of NAA levels in MOLM-14 increases sensitivity to cytarabine (Ara C) in vitro (n=3; **p=0,01 and ***p<0;005). E. Kaplan-Meier survival curve of recipients xenotransplanted with 25,000 CRISPR / Cas9 OCI- AML3 clones (n = 4 to 10 per clone).

[0058] Figure 3. Low NAT8L expression is associated with better overall survival in AML patients. A. Kaplan -Mei er survival curve of 105 patients treated with intensive chemotherapy (iCT) from DAT AML -Bordeaux (CHUBX 2024 / 45) cohort based on NAT8L expression levels. B. Kaplan-Meier survival curve of 74 patients treated 5-azacytidine and venetoclax (AZA+VEN) from DATAML-Bordeaux (CHUBX 2024 / 45) cohort based on NAT8L expression levels.

[0059] EXAMPLE:

[0060] Acute Myeloid Leukemias (AML) are characterized by the malignant proliferation of blood cells arrested at an immature differentiation stage, which ultimately leads to the suppression of normal hematopoietic function. AML are a heterogeneous group of diseases with various genetic and molecular subtypes, each associated with different prognostic outcomes and responses to treatment. Despite treatment availability, resistance and relapse is frequent resulting in low 5-year overall survival hence the need of finding new therapeutic targets.

[0061] To do so, we performed a metabolomic analysis, which revealed significant changed in the levels of N- Acetyl -Aspartate (NAA) in the leukemic cells when compared to normal hematopoietic stem cells (HSCs) (Figure 1A). NAA is the second most abundant metabolite in the brain and is involved in different processes such as histone acetylation, lipogenesis, and TCA cycle. Studies focusing on melanoma, breast cancer, and cervical cancer reported that high levels of NAA were correlated with poor prognosis. To investigate the requirement for NAT8L in AML initiation, we combined genetic Ko mouse model for Nat8l (that encodes the NAA synthase (Asp-NAT)) and a mouse model of AML driven by overexpression of Meislb and HoxA9, oncogenes that are frequently overexpressed in human AML and which drive leukemogenesis. We found that Meislb / HoxA9 transformed Nat8l KO hematopoietic stem and progenitor cells (HSPCs) displayed defective proliferation (Figure IB) compared to control cells, an increased apoptosis (Figure 1C) and generated substantially fewer colonies (Figure ID). Notably, Nat8L -deficient cells significantly prevent AML development when Meislb / HoxA9 transformed were transplanted in vivo in half of the recipients (Figure IE).

[0062] We then established different cell line models using the CRISPR-cas9 technology to target NAT8L and to reduce / suppress NAA levels in different AML lines. We confirmed that NAA levels were directly correlated with NAT8L expression (Figure 2A and 2B). In vitro assays highlighted an impact of NAA depletion on key cellular functions such as cell cycle (Figure 2C) as well as on response towards treatments currently used in the clinics such as Cytarabine, (Figure 2D). In vivo experiments revealed a delay of cell engraftment as well a longer survival when NAT8L expression is decreased (Figure IE).

[0063] To assess the potential role of NAA in the context of AML, we looked into two patient cohorts treated with either intensive chemotherapy and a less-intensive regimen found an association between the expression of the gene (NAT8L) and the patient overall survival in both treatment groups (Figure 3A and 3B).

[0064] In conclusion, inhibiting the NAA synthetase represents a therapeutic strategy to enhance the efficacy of chemotherapeutic agents and prevent relapse in patients suffering from AML.

[0065] REFERENCES:

[0066] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of treating acute myeloid leukemia (AML) in patient in need thereof comprising administering to the patient a therapeutically effective amount of an inhibitor of the N-acetylaspartate synthetase.

2. The method according to claim 1 for the treatment of the chemoresistant AML.

3. A method of preventing resistance to chemotherapy in a patient suffering from an acute myeloid leukemia (AML) comprising administering to the patient a therapeutically effective amount of an inhibitor of the N-acetylaspartate synthetase.

4. A method of preventing relapse in a patient suffering from an AML and who was treated by chemotherapy comprising administering to the patient a therapeutically effective amount of an inhibitor of the N-acetylaspartate synthetase.

5. The method according to any one of claims 2 to 4, wherein the chemotherapy consists in administering cytarabine.

6. The method according to any one of claims 2 to 4, wherein the chemotherapy consists in administering the patient with a combination of cytarabine and an anthracycline such as daunorubicin or idarubicin.

7. The method according to any one of claims 2 to 4, wherein the chemotherapy consists in administering the patient with tyrosine kinase inhibitor (TKI).

8. The method according to any one of claims 2 to 4, wherein the chemotherapy consists in administering the patient with a BCL-2 inhibitor.

9. The method according to any one of claims 2 to 4, wherein the chemotherapy consists in administering the patient with a combination of BCL-2 inhibitor and 5-azacytidine.

10. The method according to claims 8 or 9, wherein the BCL-2 inhibitor is venetoclax.

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