Suppressing the pi3kgamma / AKT signalling pathway for the treatment of acute myeloid leukemia

Abstract

Dose-limiting toxicity poses a major limitation to the clinical utility of targeted cancer therapies, often arising from target engagement in non-malignant tissues. This obstacle can be minimized by targeting cancer dependencies driven by proteins with tissue- and / or tumor-restricted expression. Here, the inventors show that in acute myeloid leukemia (AML), suppression of the myeloid-restricted PIK3CG / p110γ-PIK3R5 / p101 axis blocks AKT signaling, compromises cell fitness, and sensitizes to established AML therapies. Importantly, the inventors find that existing small molecule inhibitors against PIK3CG are insufficient to achieve a sustained longterm anti-leukemic effect. To address this concern, the inventors developed a proteolysis- targeting chimera (PROTAC) heterobifunctional molecule that specifically degrades PIK3CG and potently suppresses AML progression alone and in combination with venetoclax in human AML cell lines, primary AML patient samples, and syngeneic mouse models.

Description

[0001] SUPPRESSING THE PI3Kgamma / AKT SIGNALLING PATHWAY FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular oncology and hematology.

[0004] BACKGROUND OF THE INVENTION:

[0005] A tumor’s tissue-of-origin templates key features of its biology, implying that certain lineagespecific programs may be co-opted in neoplastic precursor cells to support tumorigenesis and eventual tumor progression (28753430). Consistent with this concept, a number of successful anticancer therapies function by targeting lineage-specific survival factors (32726532, 17893378, 26563462, 20879881). This approach stands in contrast with most traditional targeted therapies, which engage targets that are ubiquitously expressed and are thus vulnerable to broad side effect profiles. Targeting nodes whose expression and / or function are unique to the cell lineage giving rise to malignancy provides an opportunity to effectively curb tumor survival while minimizing collateral damage in other tissues.

[0006] The effectiveness and tolerability of agents targeting lineage-restricted survival factors, which have ultimately been approved as anticancer therapies, are well typified in hormone-responsive cancers such as receptor-positive breast and prostate cancers. These cancers have long been recognized as susceptible to hormone-inhibiting and hormone-depriving therapies (17893378 , 26563462). In hematological malignancies, the potential of lineage-directed therapies is also well established in the treatment of chronic lymphocytic leukemia (CLL), which has been transformed in the last two decades by the introduction of multiple agents targeting dependencies specific to the B-cell lineage, turning a once-fatal disease into a chronic illness (32726532). For instance, inhibitors of Bruton’s tyrosine kinase (BTK), a B-cell restricted enzyme that relays essential cell survival and migration signals in CLL and other B-cell malignancies, induce durable remissions in CLL patients (26639149, 31628428). Importantly, BTK inhibitors like ibrutinib are widely tolerable, with a narrow side effect profile that is consistent with BTK’s restricted expression, therefore permitting chronic use. Related principles explain the activity of dinutuximab, a monoclonal antibody against GD2 (a disialoganglioside expressed exclusively on tissues of neuro-ectodermal origin), that marks high-risk neuroblastoma cells for immune-mediated destruction (20879881), as well as the activity of rituximab, a monoclonal antibody against the B-cell-specific cell surface protein CD20, which has clinical activity against B-cell malignancies based on the same concept (1130476).

[0007] One type of ideal lineage-restricted cancer therapeutic target would be a druggable protein that fulfills two main criteria. First, it should have pleiotropic regulatory functions, governing essential pro-survival processes in the target cancer tissue. Second, it should display specific expression patterns that are confined to a narrow range of cell types. Enzymes that play critical regulatory roles in the survival of diverse tissues, and that are encoded by tissue-selective isoforms, are an example of such a lineage-restricted target class. In this scenario, the presence of these specific isoforms would render the enzyme a preferential vulnerability only in the tissues that express it. Consequently, inhibitors designed to selectively modulate these isoforms could effectively impede the growth of cancer cells in these specific tissues without causing widespread toxicity.

[0008] Phosphoinositide 3-kinase (PI3K) is a regulatory enzyme with pleiotropic, essential functions that is a frequently altered driver of malignant progression. Common oncogenic events encompass gain-of-function mutations and amplifications in two of the four PI3K isoforms, namely PIK3CA and PIK3CP, which encode catalytic isoforms of PI3K, as well as deletions in PTEN, a prominent negative regulator of the pathway (29508857, 27388585). These events are prevalent in various tumor types, prompting extensive efforts to develop pan-PI3K inhibitors and inhibitors of PI3K effectors (22188813, 18606717, 0571069). Nevertheless, these endeavors have encountered challenges due to dose-limiting toxicities arising from the widespread pattern of PI3K expression across numerous non-malignant tissues and the toxicities that emerge secondary to its inhibition in these tissues (29223745, 27155741, 30051890). Subsequent studies have pinpointed the presence of specific PI3K isoforms in distinct malignant tissue types, sparking the idea that modifying the function of a pivotal enzyme like PI3K might be achievable in a tissue-selective manner. A prime example is the delta isoform of PI3K, PIK3CD / pl 105 (encoded by PIK3CD), which exhibits distinctive tissue expression patterns including notable abundance in lymphoid cells. Subsequent clinical trials have provided substantial evidence that inhibitors of PIK3CD exhibit clinically significant activity with an acceptable toxicity profile in patients with relapsed or refractory CLL (24450857). This compelling success underscores the promise of targeting an enzyme with essential activity, yet tissue-specific isoform expression, as a viable therapeutic strategy. In the wake of the therapeutic benefit of PIK3CD targeting in CLL, further investigations are now needed to identify tissue-specific PI3K vulnerabilities and fully harness the potential for tissue- selective targeting of the PI3K pathway.

[0009] SUMMARY OF THE INVENTION:

[0010] The present invention is defined by the claims. In particular, the present invention relates to the targeting the PI3Ky / AKT signalling pathway for the treatment of Acute Myeloid Leukemia (AML).

[0011] DETAILED DESCRIPTION OF THE INVENTION:

[0012] Dose-limiting toxicity poses a major limitation to the clinical utility of targeted cancer therapies, often arising from target engagement in non-malignant tissues. This obstacle can be minimized by targeting cancer dependencies driven by proteins with tissue- and / or tumor-restricted expression. Here, the inventors show that in acute myeloid leukemia (AML), suppression of the myeloid-restricted PIK3CG / pl 10y-PIK3R5 / pl01 axis blocks AKT signaling, compromises cell fitness, and sensitizes to established AML therapies. Importantly, the inventors find that existing small molecule inhibitors against PIK3CG are insufficient to achieve a sustained longterm anti-leukemic effect. To address this concern, the inventors developed a proteolysistargeting chimera (PROTAC) heterobifunctional molecule that specifically degrades PIK3CG and potently suppresses AML progression alone and in combination with venetoclax in human AML cell lines, primary AML patient samples, and syngeneic mouse models.

[0013] 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 agent that suppresses the PI3Ky / AKT signalling pathway.

[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

[0019] 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 agent that suppresses the PI3Ky / AKT signalling pathway.

[0020] 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.

[0021] In some embodiments the chemotherapeutic agent is cytarabine (cytosine arabinoside, Ara-C, Cytosar-U), quizartinib (AC220), sorafenib (BAY 43-9006), lestaurtinib (CEP-701), midostaurin (PKC412), 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.

[0022] In some embodiments, the chemotherapy consists in a combination of cytarabine and an anthracycline such as daunorubicin or idarubicin.

[0023] 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 agent that suppresses the PI3Ky / AKT signalling pathway.

[0024] 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 agent that suppresses the PI3Ky / AKT signalling pathway. 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).

[0025] As used herein, the term “PI3K” has its general meaning in the art and refers to a phosphoinositide 3 -kinase. PI3Ks belong to a large family of lipid signaling kinases that phosphorylate phosphoinositides at the D3 position of the inositol ring (Cantley, Science, 2002, 296(5573): 1655-7). PI3Ks are divided into three classes (class I, II, and III) according to their structure, regulation and substrate specificity. Class I PI3Ks, which include PI3Ka, PI3KP, PI3Ky, and PI3K5, are a family of dual specificity lipid and protein kinases that catalyze the phosphorylation of phosphatidylinosito-4,5-bisphosphate (PIP2) giving rise to phosphatidylinosito-3,4,5-trisphosphate (PIP3). PIP3 functions as a second messenger that controls a number of cellular processes, including growth, survival, adhesion and migration. All four class I PI3K isoforms exist as heterodimers composed of a catalytic subunit (pl 10) and a tightly associated regulatory subunit that controls their expression, activation, and subcellular localization. PI3Ka, PI3KP, and PI3K5 associate with a regulatory subunit known as p85 and are activated by growth factors and cytokines through a tyrosine kinase-dependent mechanism (Jimenez, et al., J Biol Chem., 2002, 277(44):41556-62) whereas PI3Ky associates with two regulatory subunits (plOl and p84) and its activation is driven by the activation of G-protein- coupled receptors (Brock, et al., J Cell Biol., 2003, 160(l):89-99).

[0026] As used herein, the term “PK3CG” or “pllOy” has its general meaning in the art and refers to the phosphatidylinositol 4, 5 -bisphosphate 3-kinase catalytic subunit gamma isoform. The term is also known as PI3-kinase subunit gamma; PI3K-gamma; PI3Kgamma; PtdIns-3 -kinase subunit gamma, pr phosphatidylinositol 4, 5 -bisphosphate 3-kinase 110 kDa catalytic subunit gamma (PtdIns-3 -kinase subunit pl 10-gamma; pl lOgamma).

[0027] As used herein, the term “PIK3R5” has its general meaning in the art and refers to the phosphoinositide 3-kinase regulatory subunit 5. The term is also known as PI3-kinase regulatory subunit 5, PI3-kinase plOl subunit, phosphatidylinositol 4,5-bisphosphate 3-kinase regulatory subunit (PtdIns-3 -kinase regulatory subunit), protein FOAP-2, PtdIns-3 -kinase plOl or pl01-PI3K.

[0028] In some embodiments, the agent of the present invention is a small molecule. 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.

[0029] One type of small molecule applicable to the present invention is a degrader molecule that degrades the PI3Ky isoform (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; andLai 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 462 108-112) 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. Thus, in some embodiments, the agent of the present invention 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 pl lOy targeting moiety.

[0030] 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.

[0031] 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).

[0032] In some embodiments, the PTM is a pl lOy inhibitor, and more particularly a pl lOy selective inhibitor. As used herein, the term “pllOy inhibitor” has its general meaning in the art and refers to an inhibitor of the catalytic subunit pl 10 of PI3Ky. As used herein, the term “selective inhibitor” generally refers to a compound that inhibits the activity or expression of the more effectively than at least one other isozyme(s) of the PI3K family. A selective inhibitor compound is therefore more selective than conventional PI3K inhibitors such as wortmannin and LY294002, which are “nonselective PI3K inhibitors. Suitable pl lOy selective inhibitors have been described in U.S. Patent Publication Nos. 2004 / 0092561 Al, 2005 / 004195 Al, 2005 / 020631 Al, 2005 / 020630 Al, 2004 / 248954 Al, 2004 / 259926 Al, 2004 / 0138199 Al, 2004 / 01219996 Al, and 2004 / 0248953 Al, and International Patent Publication No. WO 04 / 029055 Al, the entire disclosures of which are hereby incorporated herein by reference. Further examples of inhibitors include 2-amino-N-[l-(4-chloro-7-ethoxy-2-methyl-2H- indazol-6-yl)ethyl]pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-[l -(4-chl oro-7- ethoxy-2-ethyl-2H-indazol-6-yl)ethyl]pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N- [l-(4-chloro-7-ethoxy-lH-indazol-6-yl)ethyl]pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2- amino-N-[l -(4-chl oro-7-ethoxy-l-methyl-lH-indazol-6-yl)ethyl]pyrazolo[l,5-a]pyrimidine- 3 -carboxamide; 2-amino-N-{l-[4-chloro-7-ethoxy-l-(2-methoxyethyl)-lH-indazol-6- yl]ethyl}pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(4-chloro-7-ethoxy-l-(2- hydroxyethyl)-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N- (1 -(4-chl oro-1 -(cyanomethyl)-7-ethoxy-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3- carboxamide; 2-amino-N-(l-(l-benzyl-4-chloro-7-ethoxy-lH-indazol-6- yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(4-chloro-7-ethoxy-l- isobutyl-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(4- chloro-l-cyclobutyl-7-ethoxy-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3- carboxamide; 2-amino-N-(l -(4-chl oro-7-ethoxy-l -isopropyl-lH-indazol-6- yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(4-chloro-7-ethoxy-2-(2- methoxyethyl)-2H-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N- (1 -(4-chl oro-7-ethoxy -2-(2 -hydroxy ethyl)-2H-indazol-6-yl)ethyl)pyrazolo[l, 5-a]pyrimidine- 3 -carboxamide; 2-amino-N-(l -(4-chl oro-2-(cyanomethyl)-7-ethoxy -2H-indazol-6- yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(2-benzyl-4-chloro-7- ethoxy-2H-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(4- chloro-7-ethoxy-2-isobutyl-2H-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(4-chloro-2-cyclobutyl-7-ethoxy-2H-indazol-6-yl)ethyl)pyrazolo[l,5- a]pyrimidine-3 -carboxamide; 2-amino-N-(l-(4-chloro-7-ethoxy-2-isopropyl-2H-indazol-6- yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(2-(2-amino-2-oxoethyl)-4- chloro-7-ethoxy-2H-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino- N-(l-(l-(2-amino-2-oxoethyl)-4-chloro-7-ethoxy-lH-indazol-6-yl)ethyl)pyrazolo[l,5- a]pyrimidine-3 -carboxamide; 2-amino-N-(l-(2-(but-2-ynyl)-4-chloro-7-ethoxy-2H-indazol-6- yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(l-(but-2-yn-l-yl)-4- chloro-7-ethoxy-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino- N-(l-(4-chloro-7-phenyl-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2- amino-N-(l -(4-chl oro-1 -methyl-7-phenyl-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine- 3 -carboxamide; 2-amino-N-(l -(4-chl oro-2 -methyl -7-phenyl-2H-indazol -6- yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(4-chloro-l-((l-methyl-lH- pyrazol-3-yl)methyl)-7-phenyl-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3- carboxamide; 2-amino-N-(l -(4-chl oro-2-((l-methyl-lH-pyrazol -3-yl)methyl)-7-phenyl-2H- indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(4-chloro-l-(2- morpholinoethyl)-7-phenyl-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l -(4-chl oro-2-(2 -morpholino-2-oxoethyl)-7-phenyl -2H-indazol-6- yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(2-(2-aminoethyl)-4- chloro-7-phenyl-2H-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino- N-(l-(l-(2-aminoethyl)-4-chloro-7-phenyl-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine- 3 -carboxamide; 2-amino-N-( 1 -(3 -bromo-4-chl oro-7 -phenyl- lH-indazol-6- yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l -(4-chl oro-3 -methyl-7- phenyl-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-amino-N-(l-(3,4- dimethyl-7-phenyl-lH-indazol-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2- Amino-N-{l-[8-chloro-5-(3-fluorophenyl)-3-methylimidazo[l,5-a]pyridin-6- yl]ethyl}pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-Amino-N-(l-(8-chloro-5- phenylimidazo[l,5-a]pyridin-6-yl)ethyl)pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-Amino- N-[l-(8-chloro-5-phenylimidazo[l,5-a]pyridin-6-yl)ethyl]pyrazolo[l,5-a]pyrimidine-3- carboxamide; 2-Amino-N-{l-[5-(3-fluorophenyl)-3,8-dimethylimidazo[l,5-a]pyri din-6- yl]ethyl}pyrazolo[l,5-a]pyrimidine-3-carboxamide; 2-Amino-N-[l-(8-cyano-5- phenylimidazo[l,5-a]pyridin-6-yl)ethyl]pyrazolo[l,5-a]pyrimidine-3-carboxamide; or a pharmaceutically acceptable salt thereof.

[0033] In some embodiments, the PTM is AZ2 that has the general formula of : In some embodiments, the a degrader molecule of the present invention is ARM165 that has the formula of:

[0034] O O ARM-165

[0035] In some embodiments, the agent of the present invention is an inhibitor of PIK3CG expression. In some embodiments, the agent of the present invention is an inhibitor of PIK3R5 expression.

[0036] 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 PIK3CG or PIK3R5 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of PIK3CG or PIK3R5, 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 PIK3CG or PIK3R5 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 PIK3CG or PIK3R5 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 PIK3CG or PIK3R5 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 PIK3CG or PIK3R5. 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.

[0037] A further object of the present invention relates to a method of treating AML in patient in need thereof comprising administering to the patient a therapeutically effective combination comprising an agent that suppresses the PI3Ky / AKT signalling pathway and a BCL-2 inhibitor.

[0038] 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.

[0039] 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.

[0040] In some embodiments, a BCL-2 inhibitor interferes with the interaction between the BCL-2 anti-apoptotic protein family member and one or more ligands or receptors to which the BCL- 2 anti-apoptotic protein family member would bind in the absence of the inhibitor. In some embodiments, an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least one BCL-2 protein specifically, binds only to one or more of BCL-xL, BCL-2, BCL-w and not to other Bel -2 anti-apoptotic Bel -2 family members, such as Mcl-1 and BCL2Al. Binding affinity of a BCL-2 inhibitor for BCL-2 family proteins may be measured. By way of example, binding affinity of a BCL-xL inhibitor may be determined using a competition fluorescence polarization assay in which a fluorescent BAK BI 13 domain peptide is incubated with BCL-xL protein (or other BCL-2 family protein) in the presence or absence of increasing concentrations of the BCL-XL inhibitor as previously described (see, e.g., U.S. Patent Publication 20140005190; Park et al. Cancer Res. 73 :5485-96 (2013); Wang et al., Proc. Natl. Acad Sci USA 97:7124-9 (2000); Zhang et al., Anal. Biochem. 307:70-5 (2002); Bruncko et al., J. Med. Chem. 50:641 -62 (2007)). Percent inhibition may be determined by the equation: 1 - [(mP value of well - negative control) / range)] x 100%. Inhibitor}' constant 014) value is determined by the formula: Kj = [I]5o / ([h]5o / Ki+[P]o / KN V) as described in Bruncko et al ., J. Med. Chem. 50:641-62 (2007) (see, also, Wang, FEBS Lett. 360: 111-1 14 (1995)).

[0041] Examples of BCL-2 inhibitors include ABT-263 (4-[4-[[2-(4-chlorophenyl)-5,5- dimethylcyclohexen-l-yl]methyl]piperazin-l-yl]-N-[4-[[(2R)-4-mo holin-4-yl-l- phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide or IUPAC, (R)-4-(4-((4'-chl oro-4, 4-dimethyl-3, 4,5, 6-tetrahydro-[l, r-biphenyl]-2- yl)methyl)piperazin-l-yl)-N-((4-((4-morpholino-l-(phenylthio)butan-2-yl)amino)-3- ((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide) {see, e.g., Park et al., 2008, J. Med. Chem. 51 :6902; Tse et al., Cancer Res., 2008, 68:3421; International Patent Appl. Pub. No. WO2009 / 155386; U.S. Patent Nos. 7390799, 7709467, 7906505, 8624027) and ABT-737 (4- [4-[(4'- Chloro[l,r-biphenyl]-2-yl)methyl]-l-piperazinyl]-N-[[4-[[(lR)-3-(dimethylamino)-l- [(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide, Benzamide, 4-[4-[(4'- chloro[l,l'-biphenyl]-2-yl)methyl]-l-piperazinyl]-N-[[4-[[(lR)-3-(dimethylamino)-l- [(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]- or 4-[4-[[2-(4- chlorophenyl)phenyl]methyl]piperazin-l-yl]-N-[4-[[(2R)-4-(dimethylamino)-l- phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide) {see, e.g., Oltersdorf et al., Nature, 2005, 435:677; U.S. Pat. No. 7973161; U.S. Pat. No. 7642260).

[0042] In some embodiments, the BCL-2 inhibitor is a quinazoline sulfonamide compound {see, e.g., Sleebs et al., 2011, J. Med. Chem. 54: 1914). In some embodiments, the BCL- inhibitor is a small molecule compound as described in Zhou et al., J Med. Chem., 2012, 55:4664 {see, e.g., Compound 21 (R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-l- (phenylthio)butan- 2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-l-yl)phenyl)-5- ethyl-1 -methyl- 1H- pyrrole-2-carboxylic acid) and Zhou et al., J Med. Chem., 2012, 55:6149 {see, e.g., Compound 14 (R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-l- (phenylthio)butan-2- yl)amino)-3 -nitrophenylsulfonamido)phenyl)piperazin- 1 -yl)phenyl)- 1 - ethyl-2-methyl-lH- pyrrole-3 -carboxylic acid; Compound 15 (R)-5-(4-Chlorophenyl)-4-(3-(4-(4- (4-((4- (dimethylamino)-l-(phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl)phenyl)-l-isopropyl-2-methyl-lH-pyrrole-3- carboxylic acid).

[0043] In some embodiments, the BCL- inhibitor is a BCL-2 / BCL-xL inhibitor such as BM-1074 {see, e.g., Aguilar et al., 2013, J. Med. Chem. 56:3048); BM-957 {see, e.g., Chen et al., 2012, J. Med. Chem. 55:8502); BM-1197 {see, e.g., Bai et al., PLoS One 2014 Jun 5;9(6):e99404. Doi: 10.1371 / joumal.pone. 009904);ven U.S. Patent Appl. No. 2014 / 0199234; N- acylsufonamide compounds (see, e.g., Int. Patent Appl. Pub. No. WO 2002 / 024636, Int. Patent Appl. Pub. No. WO 2005 / 049593, Int. Patent Appl. Pub. No. WO 2005 / 049594, U.S. Pat. No. 7767684, U.S. Pat. No. 7906505). In some embodiments, the BCL-2 inhibitor is a small molecule macrocyclic compound (see, e.g., Int. Patent Appl. Pub. No. WO 2006 / 127364, U.S. Pat. No. 7777076). In some embodiments, the BCL-2 inhibitor is an isoxazolidine compound (see, e.g., Int. Patent Appl. Pub. No. WO 2008 / 060569, U.S. Pat. No. 7851637, U.S. Pat. No. 7842815). In some embodiments, the BCL-2 inhibitor is S44563 (see, e.g., Loriot et. al., Cell Death and Disease, 2014, 5, el423). In some embodiments, the BCL-2 inhibitor is (R)- 3-((4'-chloro-[l,r-biphenyl]- 2-yl)methyl)-N-((4-(((R)-4-(dimethylamino)-l-(phenylthio)butan- 2-yl)amino)-3- nitrophenyl)sulfonyl)-2,3,4,4a,5,6-hexahydro-lH-pyrazino[l,2-a]quinoline-8- carboxamide. In another embodiment, the BCL-2 inhibitor is a small molecule heterocyclic compounds (see, e.g., XIS. Pat. No. 9018381).

[0044] 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.

[0045] In some embodiments, the BCL2 inhibitor is venetoclax (4-(4-((2-(4-chlorophenyl)-4,4- dimethylcyclohex-l-en-l-yl)methyl)piperazin-l-yl)-N-((3-nitro-4-((tetrahydro-2H-pyran-4- ylmethyl)amino)phenyl)sulfonyl)-2-(lH-pyrrolo(2,3-b)pyridin-5-yloxy)benzamide). As used herein, the term “combination” is intended to refer to all forms of administration that provide a first drug together with a further (second, third...) drug. The drugs may be administered simultaneous, separate or sequential and in any order. Drugs administered in combination have biological activity in the subject to which the drugs are delivered. Within the context of the invention, a combination thus comprises at least two different drugs, and wherein one drug is at least an agent that suppresses the PI3Ky / AKT signalling pathway and wherein the other drug is a BCL-2 inhibitor. In some instance, the combination of the present invention results in the synthetic lethality of the leukemic cells, in particular DTC.

[0046] 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 agent 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.

[0047] Typically, the agent 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, di sodium 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 corn 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.

[0048] A further object relates to the compound having the formula of:

[0049] O O ARM-165

[0050] A further object relates to a pharmaceutical composition comprising the compound ARM-165. A further object relates to a method of therapy in a patient in need thereof comprising administering to the patient a therapeutically effective amount of ARM-165.

[0051] A further object relates to a method of cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of ARM-165.

[0052] As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malign melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0053] 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.

[0054] FIGURES: Figure 1: Synthetic scheme for the preparation of PIK3CG degrader, ARM165. Reagents and conditions: i) LiOH monohydrate, MeOH, H2O, 60°C, 40 h (94%) ; ii) Boc-AOc-OH, T3P 50% in ethyl acetate, pyridine, N,N-dimethylformamide, 80°C, 16 h ; iii) TFA, CH2C12, r.t. 2 h ; iv) Glutaric anhydride, N,N-diisopropylethylamine, toluene, N,N-dimethylformamide, 110°C, 2 h (62% over 3 steps, ii-iv) ; v) Compound I, HATU, N,N-diisopropylethylamine, N,N- dimethylformamide, r.t. 16 h (55%).

[0055] Figure 2: PROTAC-Based PIK3CG Degradation Exhibits Greater Potency than PIK3CG-Targeting Small-Molecule Inhibitors in Affecting AML Growth, Both Alone and in Combination with Venetoclax.

[0056] A. Growth inhibition of two representative human AML cell lines treated with increasing AZ2 and ARM 165. Error bars represent mean ± SD of seven technical replicates after three days of seeding.

[0057] B. AUCs reflecting viability of multiple indicated human AML and non-AML cell lines treated with increasing AZ2 and ARM 165. Error bars represent mean ± SD of seven technical replicates after three days of seeding. P-values calculated using Mann-Whitney test.

[0058] C. Growth inhibition of four primary samples from patients with AML treated with increasing AZ2 and ARM 165. Error bars represent mean ± SD of eight technical replicates after five days of seeding.

[0059] D. Normalized AUCs reflecting venetoclax sensitization effect of AZ2 and ARM165 across indicated human AML cell lines. Error bars represent mean ± SD of 7 technical replicates after three days of seeding.

[0060] E. Colony formation from three human AML cell lines treated with AZ2 or ARM165 in combination with venetoclax (MOLM-14: IpM AZ2 or ARM165, and 375nM venetoclax; MV4-11: 0.5pM AZ2 or ARM, and 75nM venectoclax; OCI-AML2: IpM AZ2 or ARM165, and 500nM venetoclax). Error bars represent mean ± SD of four technical replicates after seven days of seeding.

[0061] F. Colony formation from two human AML primary samples treated with indicated AZ2 or ARM165 concentrations in combination with 375nM venetoclax. Error bars represent mean ± SD of four technical replicates after seven days of seeding.

[0062] D-F. P-values calculated using one-way ANOVA.

[0063] G. Bliss synergy plots for two primary patient samples with AML and treated with ARM165 and venetoclax across a drug-dilution matrix for five days. Delta scores from high synergy (lighter blue) to no synergy (dark blue). H. Bliss synergy scores for 9 primary patient samples with AML and treated with a drugdilution matrix of AZ2 and venetoclax, or ARM165 and venetoclax. Bars represent median and violin plot the range. P-values calculated using Mann-Whitney test.

[0064] I. In vivo limiting dilution assay performed with primary murine Cbfb-MYHl 1 leukemic cells treated for 24h with 0.5pM AZ2 or ARM 165 and injected into sublethally-irradiated recipient animals at decreasing cell concentrations. Determination of Leukemia-Initiating Cell (LIC) frequency with a 95% confidence interval in each group using Extreme Limiting Dilution Analysis (ELD A). Two-sided chi-squared test used for statistics.

[0065] J. Proportion of Cbfb-MYHl 1 -driven GFP-positive leukemic blasts in bone marrow of euthanized animals treated by intravenous injection with vehicle (Veh), 0.051mg / kg AZ2, or 0.051mg / kg ARM165 for five consecutive days (n=9 mice per group). Error bars represent mean ± SD.

[0066] K. Proportion of Cbfb-MYHl 1 -driven GFP-positive leukemic blasts in spleen (left panel) and corresponding spleen weight (right panel) of euthanized animals treated by intravenous injection with vehicle (Veh) or 0.051mg / kg ARM165 for five consecutive days (n=l 1 mice in vehicle and n=10 mice in ARM165 group). Error bars represent mean ± SD.

[0067] L. Proportion of blood circulating Cbfb-MYHl 1 -driven GFP-positive blasts in animals treated by intravenous injection with vehicle (Veh), 0.051mg / kg AZ2, or 0.051mg / kg ARM165 in combination with lOOmg / kg venetoclax for five consecutive days (n=5 mice per group). Error bars represent mean ± SD.

[0068] M. Proportion of blood circulating CD45-positive human AML primary blasts in animals transplanted with a PDX for a month and then treated by intravenous injection with vehicle (Veh) or 0.051mg / kg ARM165 in combination with lOOmg / kg venetoclax for sept consecutive days (n=5 mice per group). Error bars represent mean ± SD.

[0069] J-M. P-values calculated using Mann-Whitney test.

[0070] EXAMPLE:

[0071] Methods:

[0072] Cell Culture and Reagents

[0073] Cell lines were either purchased from the American Type Culture Collection (ATCC) or the Duke University Cell Culture Facility (CCF), and MOLM-14 cells were provided by Dr. Scott Armstrong (Dana-Farber Cancer Institute, Boston, Massachusetts). Identity of all cell lines was confirmed by short tandem repeat loci profiling. All cell lines were tested negative for Mycoplasma using MycoAlert PLUS Mycoplasma Detection Kit (Lonza #LT07-705). All cell lines were maintained in RPMI 1640 (Sigma #R2405) supplemented with 1% penicillinstreptomycin and 10% FBS (Sigma # F2442) in a humidified incubator at 37°C with 5% CO2. HEK293T cells were maintained in DMEM (Sigma #D6429) supplemented with 10% FBS and lOOU / mL penicillin-streptomycin (Sigma #P4333). Cells infected with shRNA and sgRNA constructs were maintained in culture with 1 pg / ml Puromycin and 1 pg / ml Doxycycline three days prior to fluorescence activated cell sorting and thereafter. Venetoclax (HY-15531), IPI- 549 (HY-100716), MK-2206 (HY-10358), and AZ2 (HY-111570) were purchased from MedChemExpress.

[0074] Patient Profiling and Primary Patient Sample Preparation

[0075] Patient samples were collected from AML patients, from whom informed consent had been given as part of an ongoing clinical registry at St Louis Hospital (THEMA, IRB approval #IDRCB2021-A00940-41). Samples were anonymized and stored at the St Louis Hospital Tumor biobank, as declared to the ministry of Higher Education, Research and Innovation. The use of these primary patient cells for experimental procedures derived from clinical practice approved by the INSERM IRB. In accordance with the declaration of Helsinki and French protection of personal data law, only anonymized clinical data were made available to research teams. Cytogenetic analyses required karyotyping and fluorescence in situ hybridization studies guided by karyotype. Genetic profiling consisted of targeted sequencing of 94 recurrently mutated genes in AML (>500X coverage; Agilent SureSelect Illumina) plus fragment analysis for NPM1, FLT3 and IDH1 / 2 mutational status.

[0076] Mononuclear cells from patients with AML were isolated using Ficoll-Paque PLUS (GE Healthcare #17-1440-02) and red blood cells were lysed (Sigma # R7757). For methylcellulose and synergy assays, cells were maintained in RPMI 1640 medium supplemented with 20% FBS, 20ng / mL IL3 (Peprotech, #200-03), 20ng / mL IL6 (Peprotech, #200-06), 20ng / mL GM-CSF (Peprotech, #300-03), lOng / ml G-CSF (PeproTech, #300-23), lOng / mL EPO (PeproTech, #100-64), 50ng / mL TPO (PeproTech, #300-18), lOOng / mL FLT3-Ligand (PeproTech, #300- 19), and lOOng / mL SCF (PeproTech, #300-07).

[0077] AML patients H3K27ac ChlP-seq Analysis

[0078] H3K SRA database was accessible under accession number SRP 103200. Sequencing reads were aligned to the human hg 19 version of the genome using Bowtie2 (19261174) and duplicate reads were marked using Picard tools MarkDuplicates. Normalized bigwig files for gene track representations were generated using Deeptools (27079975) with the — normalizeUsing RPKM — extendReads 200 — smoothLength 150 — ignoreDuplicates options. Normalized density signals from PIK3CG and PIK3R5 gene were extracted using bwtool (24489365) and density heatmaps with average signal bar plots were generated in R using iheatmatpr package.

[0079] In vitro Drug Sensitivity Assays

[0080] Cells were seeded in 384-well plates, with between 5 and 7 replicates (Coming, #3570). A range of 10 descending concentrations plus a control vehicle-treated well were plated per drug tested. ATP content was measured using CellTiter-Glo® (Promega, #G7573) per manufacturer’s instructions. Relative cell viability was determined by normalizing raw luminescence values to either DMSO or the indicated background drug. AUC values were approximated from dose-response curves plotted using the GraphPad / Prism 8 software.

[0081] In vitro Synergy Assays

[0082] Cells were seeded in 384-well plates, with 4 replicates per concentration combination of venetoclax, and AZ2 or ARM165. Drugs were diluted 1 :2 across, with 10 descending concentrations of venetoclax and 7 descending concentrations of ARM 165. Maximum applied concentration of venetoclax and ARM165 were 5pM and 5pM, respectively. Synergy matrix was determined using Bliss additive synergy analysis using the following formula: C=A+B- A*B. (A= effect of agent 1. B= effect of agent 2. C= an expected effect of the combined response).

[0083] Western Immunoblotting

[0084] Western immunoblotting was performed as previously described (32826232) using cell lysates normalized for total protein content; cells were resuspended in lysis buffer (Cell Signaling Technologies, #9803 S) supplemented with Halt protease and Phosphatase Inhibitor cocktail, EDTA-free (Thermo Fisher Scientific, #78443). Membranes were probed with primary antibodies overnight (16 hours):

[0085] CXCL12 Cytokine Experiment

[0086] Cells were incubated in serum free RPMI-1640 medium for one hour prior to spiking with 200ng / ml CXCL12 / SDF-la (PeproTech, #300-28A). After 30 minutes, cells were harvested, washed twice, and pellets produced for western blotting.

[0087] Generation of Stably-Expressing Doxycycline-Inducible shRNA Cell Lines

[0088] Inducible expression of shRNAs was achieved as previously described using a doxycycline- inducible pLKO-Tet-On lentiviral system (35668193). Lentivirus was produced and cells were transduced as previously described (32826232). Following selection with puromycin, shRNA- transduced cells were treated with doxycycline (75ng / mL) for 72 hours prior to analysis or experimentation.

[0089] RT-qPCR

[0090] RNA was isolated from cells using QIAshredder Homogenizers and the RNEasy Mini kit (Qiagen) and reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (BioRad) with Ipg of RNA template. qRT-PCR was then performed using iQ SYBR Green Supermix run on a CFX384 Touch Real-Time PCR Detection System. To quantify fold expression change, the AACq method was used to quantify fold expression change by normalizing cycle threshold (Cq) values to housekeeping gene (ACTB) and normalized to control sample (no doxycycline).

[0091] Generation of Stably Expressing Doxycycline-Inducible sgRNA Cell Lines

[0092] Inducible expression of sgRNAs was achieved using a modified version of the plasmid LentiCRISPR v2 to form a modified all-in-one dox inducible system, namely TLCV2 (Addgene, #87360). Lentivirus particles were produced and cells were transduced as previously described (32826232). Following selection with lug / mL puromycin for a minimum of seven days, cells were treated with lug / mL doxycycline for 72 hours prior to flow cytometry-based sorting of the GFP+population. The sorted fraction containing a bulk population of sgRNA- transduced cells was then reintroduced into culture supplemented in media containing lug / ml puromycin and lug / ml doxycycline for a minimum of 24 hours and a maximum of seven days, during which time cells were used for experimentation.

[0093] Tandem Affinity Purification of PIK3R5 / pl01 and Proteomics Analysis

[0094] OCI-AML2, MV4-11 and N0M0-1 AML cell were transduced with a construct allowing constitutive expression of 3xFlag-HA-PIK3R5. After harvesting, cell pellets were lysed in lysis buffer (lOOmM KC1, 5mM MgC12, 20mM Tris-HCl pH 8, 0.1% Tween 20, 0.1% NP40, 10% glycerol + protease inhibitors) and Img lysate for each four replicates per conditions was used to perform tandem affinity purification. Briefly, lysates were first incubated with anti-flag agarose beads (Sigma-Aldrich, #A2220) for four hours, washed four times with lysis buffer and eluted with 3xflag peptides (Sigma-Aldrich, #F4799). Eluates were then incubated overnight with anti-HA agarose beads and wash three times with lysis buffer and 2x with water before digesting them for four hours with Ipg of trypsin prior desalting and MS acquisition. Top scoring PIK3R5 interactors were defined based on the fact that the hit was observed in at least one of the three AML cell lines. Biological pathways were assigned to the whole list of hits using an over-representation analysis package genekitr available on R (37221491) and the network was designed using the Cytoscape software and the STRING database (36370105).

[0095] Whole-Genome CRISPR / Cas9 Screen shRNA library was amplified and prepared as previously described (35668193) using the Toronto Knockout CRISPR Library - Version 3 (TKOv3) obtained from Addgene (Pooled Libraries #90294, #125517). OCI-AML2 cells were transduced at 1000X coverage of the library (72 x 106cells transduced) and cultured for a minimum of 1000X coverage for the duration of the screen. After 7 days of puromycin selection, cells were divided into three treatment arms - cells treated with DMSO or MK-2206 (IpM) - for two weeks. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen). Amplification of sgRNA barcodes and indexing of each sample was performed via 2-step PCR as described previously (32203462). Identification of sensitizing or resistor genes was performed as previously described by comparing the final drug treated populations to DMSO treated (32203462). Plasmids and sgRNA Constructs sgRNA constructs directed against human and murine PIK3CG or PIK3R5 (sequences listed below) were cloned into TCLV2 vector (addgene #87360-ref). 5pg of TCLV2 backbone plasmid was digested and dephosphorylated in 60pl reaction containing IX Fast digest Buffer (Fermentas), Fast digested BsmBI (Fermentas) and ImM DTT, FastAP (Fermentas) for 30 min at 37 °C. Plasmids were gel purified using QIAquick Gel Extraction Kit. lOOpM of sense and antisense of sgRNA oligo were phosphorylated and hybridized in a lOpl reaction containing IX T4 ligation buffer and T4 PNK (NEB M0201S) at 37°C for 10 minutes; 95 °C for 5 minutes and then ramped down from 25 °C to 5 °C / min. The phosphorylated and digested BsmBI sgRNA were ligated into BsmBI digested TCLV2 plasmid in a lOpl reaction containing IX Quick ligase buffer (NEB), diluted oligo duplex and Quick ligase (NEB M2200S) for 10 minutes at room temperature.

[0096] Methylcellulose

[0097] OCI-AML2, MV4-11 or MOLM-14 cell lines were infected with control or PIK3CG- rected shRNAs. Cells were selected with puromycin two days after infection and Cas9 expression was then induced with Ipg / mL doxycycline for three days before the sorting of GFP -positive cells. Sorted cells were then counted by Trypan blue exclusion, and l * 104cells were plated 1 : 10 (vol / vol) in methylcellulose (Clona-Cell-TCS Medium #03814) with Ipg / ml doxycycline, Ipg / ml puromycin and treated with indicated drug concentrations. Primary viable cells (l * 105cells / ml) were plated onto semi-solid methylcellulose medium (MethoCult; StemCell Technologies, #04435) and treated with indicated drug concentrations. Cell colonies were evaluated after a minimum of 10 days after plating.

[0098] Flow Cytometry

[0099] For the detection of GFP -positive Gbfb-MYH 1 I-AVW' QW or luciferase-expressing OCI-AML2 leukemic cells, bone marrow and spleen were crushed and smashed, respectively, prior to be washed and resuspended in PBS 2mM EDTA, and analyzed on a BD FACSCanto II Instrument (BD Biosciences). For the detection of primary Patient-Derived Xenograft (PDX) cells, peripheral blood was collected from NOG-EXL mice prior to lysis of red blood cells for 10 minutes (Sigma-Aldrich, #R7757), washing, and resuspension of the leukemic cells into PBS 0.1% BSA 2mM EDTA. PDX cells were then stained for 25 minutes at four degrees with PE- Vio770-coupled anti-hCD45 antibody (Miltenyi Biotec, # 130-110-634). Cells were then washed twice with PBS 0.1% BSA 2mM EDTA, and analyzed on a BD FACSCanto II Instrument (BD Biosciences).

[0100] Generation of Luciferase-Expressing OCI-AML2 Cells

[0101] OCI-AML2 cells were transduced with pMMP-LucNeo retrovirus and selected with Img / mL neomycin. These cells were then transduced with lentiviral particles encoding either a nontargeting control or PIK3CG-directed sgRNAs, and then selected for seven days with Ipg / mL puromycin and then used for subsequent in vivo studies.

[0102] In Vivo Imaging

[0103] After a minimum of six days post-injection of bioluminescent cells, mice were injected with 150mg / kg luciferin (Xenolight, #12799) 15 minutes prior to imaging, anesthetized with 2-4% isoflurane, and imaged, using the IVIS Lumina III according to the manufacturer’s protocol. Bioluminescent signal was quantified in radiance (W7M2-sr) using a standardized region of interest (ROI) encompassing the entire mouse.

[0104] In Vivo Genetic Studies

[0105] Luciferase-expressing OCI-AML2 cells were infected with non-targeting control or PIK3CG- directed sgRNAs, selected with puromycin, the GFP-positive cell fraction was sorted as described above. Following sorting, cells were allowed to recover in media supplemented with Ipg / mL doxycycline and Ipg / m puromycin for four days. 6 week old male NSG mice (NOD.Cg.Prkdc «scid> I12rg<tmlSug>Tg(SV40 / HTLV-IL-3, CSF2)10-7jic Taconic) were then irradiated at 1.25 Gy and injected with 1 xlO6cells per mouse by intravenous injection. These mice were supplemented with doxycycline-containing food (SAFE nutrition service, #E8200P01R) for the whole duration of the experiment. Imaging of the mice was performed every three days to follow the disease progression. Peripheral blood was collected by submandibular bleeding and bone marrow biopsies were performed on mice femurs to collect bone marrow mononuclear cells. At the end point, mice were euthanized, and spleen and bone marrow harvested. The GFP-positive AML cell fraction was quantified in both bone marrow and spleen on a BD FACSCanto II Instrument (BD Biosciences).

[0106] In vivo Drug Treatment Studies

[0107] Cbfb-MYHll Syngeneic Mouse Model'. The Cbfb-MYHl 7-driven leukemic cells were kindly provided by Dr. Lucio U. Castilla’s team. Those were isolated from the offspring of floxed Cbfb-MYHll knock-in mice which were crossed with the Mxl-Cv transgenic mice (16413472). 0.5 x 106Cbfb-MYHll cells were injected into 5 to 8 weeks old male C57BL / 6J mice (Envigo). Seven days after injection, a biopsy of the bone marrow was performed to confirm disease engraftment and mice were randomized to ensure homogenous disease onset across animals prior to the start of the treatment.

[0108] Patient-Derived Xenograft (PDX) Mouse Model'. The PDX sample was derived from a 69-year- old female who was diagnosed with secondary AML with MDS-related changes; the genetic profiling of this patient revealed mutations in CEBPAI ASXLH RUNXH EZH2I JAK2I TET2 patient karyotype is 46, XX , t(6;7)(q23;ql 1.2)[l] / 46,XX[cpl9], IxlO6mononucleated cells were injected via tail vein into sublethally-irradiated (1.25Gy) 8-week-old NOG-EXL mice (Taconic Biosciences). Blasts engraftment was confirmed by flow cytometry one month after injection using the PE-Vio770-coupled CD45 (hCD45)-based staining protocol detailed in the flow cytometry section to confirm disease engraftment and mice were randomized accordingly. Venetoclax was administered daily at a dosage of lOOmg / kg by oral gavage (in 5% DMSO; 40% PEG300, 5% TWEEN80, 50% Saline), AZ2 and ARM165 were administered daily until mice demise at 0.051mg / kg by IV injection (in 20% A-methylpyrrolidone EMPLU, # 8060721000, Sigma-Aldrich; 16% PEG400, 64% saline solution). At indicated time points response to treatment was tracked using either peripheral mandibular bleeding and bone marrow biopsy. 14 days after cell injection, mice were euthanized, bone marrow and spleen were harvested, weighted and the GFP-positive leukemic cell fraction was quantified using a BD FACSCanto II Instrument (BD Biosciences).

[0109] Chemistry

[0110] General considerations:

[0111] All organic solvents were purchased from commercial sources and used as received. All chemicals were purchased from Merck, IRIS Biotech, BLDPharm and used without further purification.JH andl3C { 'H } NMR spectra were recorded on a Bruker 400 or 500 MHz spectrometer; chemical shifts are given in ppm and referenced to the solvent residual peak (CDC13: 7.16 ppm for 'H and 77.16 for13C{JH}; DMSO-t / 6: 2.50 ppm for 1H and 39.52 for13C{JH}), coupling constants are given in Hertz (Hz). The purity of the final compound (ARM 165) was verified to be > 95 % purity by UPLC analysis at 214 nm on a Waters Acquity H-Class system equipped with a UV detector, SQD2 mass spectrometer detector and an acquity HSS T3 column (100A, 1.8 pm, 2.1 mm x 50 mm; gradient water / acetonitrile (l%o formic acid): 100 / 0 to 0 / 100 in 5 min; flow: 0.8 mL / min). Preparative HPLC were performed either on Gilson PLC2250 using a CIS-reverse phase (DeltaPack Waters, 15 pm, 100A, 100 x 40 mm) or a GILSON HPLC system - SKID LC 009SK equipped with a C18 reversed-phase Luna column (Phenomenex 15 pm, 100A, 250 x 50 mm). On the Gilson PLC2250 equipped with DeltaPack, elution was performed using gradients of acetonitrile in water, and a constant concentration 0.1 vol.% of trifluoroacetic acid (TFA) and a 50 mL / min flow rate. On the GILSON HPLC system - SKID LC 009 SK equipped with a Cl 8 reversed-phase Luna column, elution was performed using gradients of acetonitrile in water, and a constant concentration 0.1 vol.% of Formic acid and a 120 mL / min flow rate. Docking of AZ2 in PIK3CG was performed on the Alphafold structure of human PIK3CG (Uniprot P48736) using Autodock Vina and the following grid parameters on a rigid receptor: centre x, y, z: 18.701, 19.536, -3.725 and size x, y, z: 21.75, 21.75, 21.75. (19499576)

[0112] Synthesis ofNI-(5-(2-((S)-l-cyclopropylethyl)-7-methyl-l-oxoisoindolin-5-yl)-4-methylthiazol- 2-yD-N5-(8-( (2-(2, 6-dioxopiperidin-3-yl)-l , 3-dioxoisoindolin-4-yl)amino)-8- oxooctyDslutaramide (ARM 165, Figure 1):

[0113] 1- Preparation of Compound I ((5)-5-(2-amino-4-methylthiazol-5-yl)-2-(l-cyclopropylethyl)- 7 -methyli soindolin- 1 -one)

[0114] Synthesis: To a solution AZ2 (cas # 2231760-33-9; 1.0 g, 2.71 mmol, 1.0 equiv.), in methanol (10 mL) at room temperature, was added a solution of lithium hydroxide monohydrate (1.14 g, 27.1 mmol, 10 equiv.) in water (10 mL). Thereafter, the reaction mixture was stirred at 60°C complete consumption of the starting material (LC monitoring, ca. 40 h). The reaction mixture was then concentrated to remove the methanol and diluted with ethyl acetate (100 mL). This organic layer was successively washed with a saturated aqueous solution of sodium bicarbonate and brine, before being dried over magnesium sulphate, filtered, and concentrated to dryness. The title compound was obtained as a beige solid in a 94% yield (834 mg) without further purification.

[0115] Characterization: 'H NMR (CDC13, 400 MHz) 5 7.25 (s, 1H), 7.17 (s, 1H), 5.02 (s, 2H), 4.49 (d, J= 17.0 Hz, 1H), 4.38 (d, J= 17.0 Hz, 1H), 3.75 (dq, J= 9.4, 6.8 Hz, 1H), 2.73 (s, 3H), 2.33 (s, 3H), 1.33 (d, J= 6.8 Hz, 3H), 1.04 - 0.98 (m, 1H), 0.64 - 0.60 (m, 1H), 0.44 - 0.37 (m, 1H);13C{JH} NMR (CDCI3, 101 MHz) 5 168.5, 165.4, 144.5, 142.6, 137.9, 135.4, 130.5, 129.0, 121.0, 120.4, 51.9, 45.5, 18.6, 17.4, 16.5, 16.1, 4.7, 3.6; MS (ESI+): m / z 328.3 [M+H]+(100%). 2- Preparation of Compound IV (5-((8-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)-8-oxooctyl)amino)-5-oxopentanoic acid):

[0116] Synthesis: A) To a solution of pomalidomide (cas # 19171-19-8, 1.0 g, 3.65 mmol, 1.0 equiv.), and Boc-AOc-OH (8-[(tert-butoxycarbonyl)amino]octanoic acid, cas # 30100-16-4, 0.95 g, 3.65 mmol, 1.0 equiv.) in NA -di methyl form am ide (25 mL) and pyridine (3.0 mL, 36.5 mmol, 10 equiv.) at room temperature, was added a solution of T3P (2,4,6-tripropyl-l,3,5,2,4,6- trioxatriphosphorinane) 50 wt.% in ethyl acetate (cas # 68957-94-8, 10.8 mL, 18.25 mmol, 5.0 equiv.). Then, the reaction mixture was allowed to stir at 80°C during 16 h. Next, the volatiles were evaporated off and the oily residue dissolved in 100 mL of ethyl acetate. This organic layer was successively washed with a saturated aqueous solution of sodium bicarbonate and brine, before being dried over magnesium sulphate, filtered, and concentrated to dryness. The crude Compound II was directly engaged in the subsequent step. B) Crude Compound II (3.65 mmol) was dissolved in 15 mL of di chloromethane and treated with trifluoroacetic acid (8 mL) at room temperature during 2 h. After concentration to dryness and lyophilisation from a 1 / 1 mixture of acetonitrile / water, Compound III was obtained as a trifluoroacetate salt in a light orange solid form and directly engaged in the next step. C) Crude Compound III (3.65 mmol, 1.0 equiv.) was dissolved in 20 mL of N -di methyl form am ide and 40 mL of toluene. To this mixture, N,N-diisopropylethylamine (1.91 mL, 10.95 mmol, 3.0 equiv.) and glutaric anhydride (458 mg, 4.02 mmol, 1.1 equiv.) were successively added and the media was allowed to stir at 110°C for 2 h. Thereafter, the volatiles were evaporated off and the residue resuspended in a 1 / 1 mixture of acetonitrile / water prior to its lyophilization. The crude residue (2.8g) was solubilized in water / acetonitrile 35 / 65 (v / v) mixture with l%o Formic acid (FA) (20ml), filtered on a 0,45 pm syringe filter and purified by RP -preparative HPLC. The purification was performed on a GILSON HPLC system - SKID LC 009SK - equipped with a Cl 8 reversed- phase Luna column (Phenomenex 15 pm, 100 A, 250 x 50 mm) with a flow rate of 120 mL / min. Eluents were water l%o FA (A) and acetonitrile l%o FA (B). The purification is carried out according to the following gradient: From 0% to 20% of B in 5 minutes then from 20% to 30% of B in 5 minutes and finally, from 30% to 45% of B in 15 minutes. The compound of interest is eluted in 19 minutes. UV detection was performed at 214 nm. Collected fractions were concentrated and freeze-dried to obtain 1,2 g of pure Compound IV as a colourless solid in a 62% overall yield (3 steps).

[0117] Characterization: 'H NMR (DMSO-t / d, 500 MHz) 5 12.01 (s, 1H), 11.14 (s, 1H), 9.69 (s, 1H), 8.49 - 8.46 (m, 1H), 7.82 (dd, J= 8.4, 7.3 Hz, 1H), 7.74 (t, J= 5.6 Hz, 1H), 7.61 (dd, J= 7.3, 0.7 Hz, 1H), 5.15 (dd, 7 = 12.9, 5.4 Hz, 1H), 3.01 (q, 7 = 6.9 Hz, 2H), 2.90 (ddd, J= 17.1, 13.9, 5.5 Hz, 1H), 2.64 - 2.51 (m, 2H), 2.46 (t, J= 7.5 Hz, 2H), 2.18 (t, J= 7.4 Hz, 2H), 2.09 - 2.04 (m, 3H), 1.69 (p, J= 7.5 Hz, 2H), 1.65 - 1.59 (m, 2H), 1.40 - 1.23 (m, 3H);13C{JH} NMR (DMSO-t / 6, 126 MHz) 5 174.2, 172.8, 172.0, 171.3, 169.8, 167.7, 166.7, 136.6, 136.1, 131.5, 126.3, 118.3, 117.0, 48.9, 38.4, 36.5, 34.5, 33.0, 30.9, 29.1, 28.5, 28.5, 26.3, 24.7, 22.0, 20.7; MS (ESI+): m / z 529.3 [M+H]+(100%).

[0118] 3- Preparation of ARM165 Nl-(5-(2-((S)-l-cyclopropylethyl)-7-methyl-l-oxoisoindolin-5-yl)-

[0119] 4-methylthiazol-2-yl)-N5-(8-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)-8- oxooctyl) glutar amide :

[0120] Synthesis:

[0121] Compound I, (120 mg, 0.37 mmol, 1.0 equiv.), and Compound IV (194 mg, 0.37 mmol, 1.0 equiv.) were dissolved in 1 mL of VW-dim ethyl form am ide and V,7V-diisopropylethylamine (320 pL, 1.84 mmol, 5.0 equiv.). Then, HATU (Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium, 140 mg, 0.37 mmol, 1.0 equiv.) was added and the mixture was allowed to stir at room temperature for 6 h before an additional portion of 0.2 equiv. (39 mg) of HATU was introduced. After 16 h, without any treatment, the reaction media was purified on reverse phase preparative HPLC - PLC2250 Gilson (DeltaPack Waters, 15 pm, 100A, 100 * 40 mm). Elution was performed using gradients of acetonitrile (B) in water (A), at a constant concentration 0.1 vol.% of TFA, and a flow rate of 50 mL / min. The following stepwise gradient was applied: 100% water (A), 0% acetonitrile (B) during 3 min., 100% A to 65 / 35 A / B in 7 min., 65 / 35 to 55 / 45 A / B in 5 min., and 55 / 45 to 35 / 65 A / B in 20 min. The product is eluted out at 55% of B. The title compound ARM 165 was obtained as colourless solid in a 55% yield (168 mg).

[0122] Characterization: 'HNMR (CDC13, 400 MHz) 5 9.42 (s, 1H), 8.79 (d, J= 8.5 Hz, 1H), 8.38 (s, 1H), 7.70 (dd, J= 8.5, 7.3 Hz, 1H), 7.53 (dd, J = 7.3, 0.7 Hz, 1H), 7.30 (s, 1H), 7.22 (s, 1H), 6.28 (t, J= 5.8 Hz, 1H), 4.98 - 4.93 (m, 1H), 4.56 (d, J= 17.5 Hz, 1H), 4.45 (d, J= 17.4 Hz, 1H), 3.76 (dq, J= 9.4, 6.8 Hz, 1H), 3.29 (q, J= 6.7 Hz, 2H), 2.94 - 2.75 (m, 6H), 2.69 (t, J = 7.2 Hz, 2H), 2.51 (s, 3H), 2.48 (t, J= 7.3 Hz, 2H), 2.34 (t, J= 7.2 Hz, 2H), 2.20 - 2.17 (m, 1H), 2.11 (p, 7= 7.1 Hz, 2H), 1.77 (p, 7= 7.3 Hz, 2H), 1.58 (p, J= 7.2 Hz, 2H), 1.47 - 1.41 (m, 2H), 1.35 (d, 7= 6.8 Hz, 3H), 1.07 - 0.98 (m, 1H), 0.69 - 0.63 (m, 1H), 0.50 - 0.34 (m, 3H);13C{JH} NMR (CDC13, 126 MHz) 5 172.6, 172.3, 172.2, 171.2, 169.4, 169.3, 168.3, 166.9, 157.0, 142.7, 138.1, 137.9, 136.6, 134.3, 131.3, 130.8, 129.8, 125.6, 125.1, 120.7, 118.7, 115.5, 52.0, 49.4, 45.5, 39.6, 38.0, 35.1, 35.0, 31.6, 29.7, 28.7, 28.6, 26.5, 25.3, 23.0, 21.2, 18.7, 17.5, 16.0, 15.6, 4.7, 3.6; MS (ESI+): m / z 838.5 [M+H]+(87%), 419.9 [M+2H]2+(100%); HPLC (X2i4) 97 % purity.

[0123] External Data Analysis

[0124] Gene expression data for normal and malignant tissues was obtained from GeTex gene expression dataset and accessed via the Gepia portal. Gene dependency data was obtained from the DepMap dependency dataset. Data analyses were performed using R or GraphPad / Prism 8.

[0125] Statistical Analysis

[0126] Determination of statistical significance using Microsoft Excel and Prism 8.0.1 (GraphPad); unpaired students T-test were applied where data was normally distributed. Where data variance was high, we applied Welch’s correction to analyze significance, and pairwise comparison of data which was not normally distributed was analyzed using non-parametric Mann-Whitney test. Multiple comparison analysis testing was achieved using the One-way ANOVA test and the mean of each condition was compared with the mean of every other column. The level of significance was set at 0.05.

[0127] Results:

[0128] PIK3CG and its Regulatory Subunit PIK3R5 Exhibit a Myeloid-Biased Expression Profile and Are Required for AML Cell Survival

[0129] To identify lineage-specific genetic dependencies in AML, we used publicly available data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression Project (GTEx) to compare gene expression in tumor samples and normal samples across different tissue types. We identified upregulation of PIK3CG expression (encoding PIK3CG / pl l0y) in the myeloid compartment relative to other tissue types. By contrast, PIK3CA, PIK3CR, and PIK3CD, encoding the PI3K isoforms PIK3CA / pl 10a, PIK3CB / pl l0 and PIK3CD / pl 105, respectively, displayed widespread expression across many tissue types, suggesting a specific role for PIK3CG in the myeloid compartment (data not shown). The limited expression of PIK3CG is complemented by the expression of its exclusive, cognate regulatory subunit, PIK3R5 (encoding PIK3R5 / pl01), which exhibit limited expression in normal tissues and upregulation in AML. We then mined publically-available ChlP-seq data for establishing the binding pattern of the H3K27ac histone mark, which is associated with active transcription, at the promoter and gene body regions of PIK3CG and PIK3R5 in primary samples from AML patients and cord-blood-derived CD34+cells from healthy donors (data not shown). This analysis revealed that several genomic regions in PIK3CG and PIK3R5 were marked by H3K27ac in a significant proportion of AML patients and healthy donors. Notably, the increased signal of H3K27ac across the PIK3R5 gene was more pronounced in the majority of AML patient-derived primary cells compared to normal CD34+cells. This suggests heightened PIK3R5 transcriptional activity in the malignant context, which is consistent with the substantial RNA upregulation of PIK3R5 observed in AML relative to its healthy counterpart (data not shown).

[0130] To explore whether the increased expression of PIK3CG and PIK3R5 in myeloid cells might be linked to a potential dependence on their suppression in myeloid leukemias, we then examined the DepMap dataset to establish the dependency profile on the catalytic and regulatory subunits of PI3K across a wide range of cancer types. Of the four PI3K isoforms, PIK3CG was most essential in AML (and secondarily in ALL) with minimal essentiality in malignancies of non-hematopoietic origin (data not shown). By contrast, PIK3CA an PIK3CR were essential across malignancies of diverse tissue types. A selective dependency on PIK3R5 was also observed in AML and Chronic Myelogenous Leukemia (CML), both related myeloid malignancies, while the regulatory subunits associated with PIK3CA, PIK3CB, and PIK3CD exhibited widespread essentiality across cancers of other tissues. All together, these data suggest that PIK3CG-PIK3R5 signaling may act as a lineage-restricted driver of disease progression, and a selective survival dependency, in AML.

[0131] To validate the isoform-selective essentiality of PIK3CG in AML, we designed doxycycline (dox)-inducible short-hairpin RNAs (shRNAs) to knockdown PIK3CA, PIK3CB, PIK3CD, and PIK3CG. AML cell viability was reduced following shRNA-mediated depletion of PIK3CG but not following suppression of the other catalytic members of the PI3K family, suggesting an isoform-specific role for PIK3CG in AML cell survival (data not shown). We then introduced a dox-inducible CRISPR-Cas9 single-guide RNA (sgRNA) system targeting the catalytic subunit of PIK3CG in various AML cell lines. Knockout of PIK3CG resulted in a significant growth impairment of all AML cell lines tested, further evidencing a dependency on PIK3CG for the growth of AML cells (data not shown). A similar CRISPR-Cas9 approach was then used to knockout the expression of PIK3R5 and yielded a marked reduction in AML cell viability (data not shown). Using a colony formation assay, we further evidenced that both PIK3CG and PIK3R5 gene suppression significantly dampened the colony-forming ability of multiple AML cell lines (data not shown). To interrogate whether PIK3CG knockout could affect AML progression in vivo, we finally assessed the bioluminescence of NOD-SCID gamma (NSG) mice injected with luciferase-expressing OCI-AML2 cells infected with either a nontargeting control (sgControl) or a PIK3CG-directed (sgPIK3CG) sgRNA. Mice transplanted with AML cells expressing s PIK3CG demonstrated a significantly lower disease burden in comparison with animals injected with sgControl OCI-AML2 cells, indicating that AML cell dependency on PIK3CG signaling is conserved in vivo (data not shown).

[0132] PIK3CG / PIK3R5 Repression Potentiates the Effect of Venetoclax in AML

[0133] We next examined whether suppression of the PIK3CG-PIK3R5 signaling axis could modulate AML cell response to the front-line targeted small molecules and chemotherapies used in this disease, including azacytidine, cytarabine, daunorubicin, decitabine, and venetoclax. Consistently upon the suppression of PIK3CG and PIK3R5, AML cells exhibited a significant increase in sensitivity to the BCL-2 inhibitor, venetoclax, but not to other chemotherapies (data not shown). The potentiating effect of PIK3CG or PIK3R5 loss on venetoclax sensitivity was further validated across multiple AML cell lines (data not shown). Venetoclax combined with PIK3CG or PIK3R5 targeting sgRNAs also reduced the colony-forming capacity more than either perturbation alone, in multiple AML cell lines (data not shown).

[0134] To explore the importance of the PIK3CG-PIK3R5 signaling axis in AML progression and sensitivity to venetoclax treatment in an in vivo system, we injected a pool of luciferaseexpressing GFP / Cas9-positive OCI-AML2 cells infected with either a non-targeting control or a PIK3CG-directed sgRNA into NSG mice (data not shown). Following injection, disease burden was quantified through the measurement of a bioluminescence signal. Mice injected with PIK3CG knockout cells exhibited a lower overall disease burden compared to recipient animals injected with cells carrying unaltered PIK3CG levels. This decreased disease burden was potentiated when mice were treated with venetoclax combined with PIK3CG knockout (data not shown). In addition, at the experimental endpoint (defined by the control cohort), we observed a profound decrease in the proportion of GFP -positive leukemic cells in bone marrow of mice transplanted with PIK3CG knockout cells along with a significant sensitization to venetoclax treatment, compared to mice treated with venetoclax alone (data not shown). Overall, these data suggest that specific targeting the PIK3CG-PIK3R5 signaling axis can potentiate the effect of venetoclax both in vitro and in vivo. AKT Signaling is Exquisitely Dependent on the PIK3CG / PIK3R5 Signaling Module in

[0135] AML Cells

[0136] To characterize the PIK3CG-PIK3R5 signaling network, we performed tandem purification and mass spectrometry-based interactomic profiling of the two known PIK3R5 isoforms in three different AML cell lines: MV4-11, 0CI-AML2, and NOMO-1. We successfully identified 534 proteins that interact with the two PIK3R5 isoforms in at least one of the three AML cell lines (data not shown). Notably, our findings confirmed the interaction between PIK3CG and PIK3R5 in all three cell lines. Leveraging this comprehensive list of interactions, we constructed a network of proteins that interact with PIK3R5, categorized into 17 distinct biological nodes significantly enriched based on the MsigDB C2 collection database (data not shown). Within these nodes, we detected multiple upstream pathways linked to integrin and G- coupled cytokine receptor signaling (GPCR). This observation aligns with prior reports indicating that PIK3CG-PIK3R5 signaling becomes active upon engagement with certain of these receptors. Numerous downstream signaling pathways were found to be linked to PIK3CG-PIK3R5, encompassing JAK / STAT, SRC, MAPK, mTOR, and AKT signaling. Additionally, we observed less expected pathways related to cell sternness features, such as Hedgehog, TGFB signaling, and to response to DNA damage.

[0137] Based on this network, we next sought to investigate whether the highly tissue-specific expression pattern of the PIK3CG-PIK3R5 signaling network in AML conferred isoform- specific control of downstream AKT signaling. We reasoned that if PIK3CG controls AKT signaling in AML cells, those cells should exhibit PIK3CG dependence proportional to their AKT dependence. We tested this in a panel of 15 AML cell lines and found that PIK3CG dependency was correlated with dependency on AKT1 and AKT2 in AML cell lines but not in cell lines from other tissues (data not shown). Given the correlation between PIK3CG dependency and AKT1 / 2, we used a CRISPR / Cas9 drug-modifier screen to identify a common set of genes that significantly altered sensitivity to MK-2206, a pan-AKT inhibitor. Cells harboring sgRNAs targeting BCL2 were most depleted in replicate MK-2206 modifier screens, while cells harboring sgRNAs targeting negative regulators of mTOR signaling (TSC1, TSC2, NPR 2) were enriched (data not shown). These findings are consistent with our data and others’ positioning the venetoclax target, BCL2, and mTOR as protein candidates whose inhibition synergistically enhances PI3K pathway ablation (29357370, 29666304). Collectively, these data nominated AKT as a potential downstream signaling pathway that might be selectively activated by PIK3CG-PIK3R5, possibly accounting for the increased sensitivity of AML cells to venetoclax.

[0138] To further confirm the connection between PIK3CG-PIK3R5 with AKT signaling, we sought to evaluate the consequence of CRISPR-Cas9-mediated PIK3CG knockout on AKT signaling. We demonstrated by western blot that PIK3CG and PIK3R5 knockouts in OCI-AML3 and OCL AML2 cells diminish the phosphorylation of AKT and several of its previously reported downstream substrates, PRAS40 and TSC2 (data not shown). To determine whether AKT signaling relies selectively on PIK3CG in AML cells, we employed doxycycline-inducible shRNAs to knock down the expression of PIK3CA, PIK3CB, and PIK3C1), and revealed that only the suppression of PIK3CG yielded a marked decrease in the activation of AKT, affecting both T308 and S473 phosphorylation sites, in contrast to the other PIK3 catalytic isoforms (data not shown). A similar approach employed to selectively reduce the expression of the PI3K regulatory subunits enabled us to confirm that only shRNAs directed against PIK3R5 significantly dampens AKT phosphorylation compared to the inhibition of the other regulatory subunits (data not shown). Conversely, overexpression of wild-type PIK3CG under a constitutive EFla promotor resulted in hyperactivation of AKT signaling. We then introduced an shRNA directed against the 3’ UTR region of PIK3CG which was capable of knocking down endogenous PIK3CG without affecting exogenously expressed PIK3CG cDNA lacking the 3’ UTR. Expression of wild-type PIK3CG cDNA was able to fully rescue AKT suppression secondary to the suppression of endogenous PIK3CG (data not shown). These data indicate that PIK3CG is both necessary and sufficient for the activation of AKT signaling in AML.

[0139] Because PIK3CG-PIK3R5 activity was reported to be predominantly controlled by G protein- coupled receptors (GPCR) (9094719 , 24014027), we sought to investigate whether the modulation of GPCR functions could affect PIK3CG-mediated AKT activation. CXCL12 (also known as SDFla) is the ligand for CXCR4, a GPCR that signals to downstream pathways including the PI3K / AKT pathway. The CXCL12 / CXCR4 signaling axis has been implicated in maintenance of the leukemic niche (15087398), and both the receptor and ligand are upregulated in AML cells relative to normal hematopoietic cells (data not shown). Pertussis toxin targets G-proteins and broadly impairs the ability of GPCRs to activate PI3K / AKT and other signaling pathways (data not shown). The addition of pertussis toxin to AML cell lines reduced phosphorylation of AKT, demonstrating the predominant role of GPCRs over other ligand-binding receptors across AML cell lines to modulate downstream AKT signaling (Figure 3J). Because PIK3CG-PIK3R5 is activated via GPCRs, whereas other isoforms are predominantly under the control of receptor tyrosine kinases (RTKs), we interrogated the effect of PIK3CG suppression in presence or absence of the CXCR4 GPCR-activating ligand, CXCL12 (data not shown). We determined that, following PIK3CG knockout, CXCL12 failed to activate downstream AKT signaling. These data provide evidence that PIK3CG mediates downstream AKT signaling in AML cells by engaging GPCRs, including CXCR4.

[0140] Small-Molecule Inhibitors of PIK3CG Do Not Sustain Long-Term AKT Inactivation and AML Cell Growth Impairment

[0141] Given the strong dependence we observed on PIK3CG in several AML models, we anticipated that small molecule inhibitors targeting PIK3CG would efficiently alter AML cell growth. Surprisingly, however, the pharmacological inhibition of PIK3CG using IPL549 and AZ2 did not result in a significant reduction in AML cell viability and colony-forming capacity when compared to the genetic ablation of PIK3CG and PIK3R5 using PIK3CG- an PIK3R5- Qcte< sgRNAs (data not shown). We, therefore, postulated that these small molecule inhibitors might inadequately inhibit PIK3CG-mediated AKT signaling. We substantiated our hypothesis by showing that the long-term suppression of AKT phosphorylation was more effective when using sgRNAs targeting PIK3CG or PIK3R5 compared to treatment with IPL549 and AZ2 (data not shown). In contrast, the AKT inhibitor MK-2206, used here as a positive control for AKT signaling inhibition, repressed durably AKT phosphorylation. Taken as whole, these findings imply that existing small molecule inhibitors of PIK3CG are inadequate in achieving a durable reduction in downstream AKT signaling, and consequently, they do not attain the same level of cytotoxicity as PIK3CG and PIK3R5 knockout. Conversely, the genetic suppression of PIK3CG and PIK3R5 effectively prevents feedback reactivation, leading to a sustained and effective blockade of AKT phosphorylation. Therefore, a novel therapeutic approach for targeting PIK3CG, one that ensures a sustained disruption of PIK3CG / PIK3R5- mediated AKT signaling in AML cells, was needed.

[0142] As an alternative strategy for pharmacologically targeting PIK3CG, we opted to harness recent advancements in targeted protein degradation. We employed a PROteolysis TArgeting Chimera (PROTAC) system, in which AZ2, a PIK3CG-isoform selective inhibitor, was conjugated to a cereblon-targeting moiety. We capitalized upon the presence of an acetyl moiety on the AZ2 parental compound that is orientated towards the outside of the kinase pocket to attach a linker and a subsequent cereblon-recruiting moiety (data not shown). We discovered a lead compound, designated as ARM165, which was synthesized from AZ2 through a 5-stepwise chemical reaction (Figure 1). To confirm that ARM165 targets PIK3CG, we treated OCI- AML2 cells with increasing doses of ARM165 and assessed PIK3CG protein levels through western blot analysis. Within 24 hours, we achieved 50% degradation of PIK3CG, but not PIK3R5, at a concentration of less than IpM ARM165 (data not shown). Co-treatment of cells with the proteasome inhibitor bortezomib fully rescued the loss of PIK3CG protein induced by ARM165, suggesting that ARM165 reduces PIK3CG levels through proteasome-mediated degradation. Additionally, co-treatment of cells with ARM165 and lenalidomide, which binds to the same site on cereblon, competed with ARM165 in inducing PIK3CG degradation (data not shown). To determine if ARM165 could effectively inhibit downstream AKT signaling to a similar extent as sgRNAs targeting PIK3CG and PIK3R5, we compared the level of AKT phosphorylation in OCI-AML2 cells infected with CRISPR-Cas9 sgRNAs targeting PIK3CG and PIK3R5 or treated with IpM ARM165. Cells treated with ARM165 demonstrated a reduction in PIK3CG comparable to that achieved by CRISPR-Cas9 targeting sgRNAs, resulting in a similar level of disruption in downstream AKT signaling (data not shown). Ultimately, we confirmed that ARM165 significantly diminished the colony -forming capacity of OCI-AML2 cells through a selective degradation of PIK3CG because cells lacking PIK3CG were resistant to the PROTAC degrader (data not shown). Collectively, these findings support ARM165 as a newly developed selective heterobifunctional degrader of PIK3CG which durably alters AKT signaling in AML cells.

[0143] Degradation of PIK3CG Demonstrated Superior Cytotoxic Abilities Over Existing Small- Molecule Inhibitors of PIK3CG

[0144] We then questioned how the PROTAC -mediated degradation of PIK3CG and the subsequent disruption of AKT signaling could affect the viability of AML cells. In order to investigate this, we compared the impact of both ARM165 and the parental AZ2 on seven AML cell lines. Increasing concentrations of ARM165 significantly affected the growth of these cell lines, whereas AZ2 had minimal anti-leukemia effects, consistent with our previous results (Figures 2A and 2B). Since the dependency on PIK3CG and its regulatory subunit PIK3R5 was primarily observed in myeloid cells, we hypothesized that the degrader's efficacy would be more specific to this cell type. Consequently, we assessed the ARM165 degrader's effect on a range of non-AML cell lines. Our results demonstrated unambiguously that these cell lines were mostly insensitive to ARM165, even at doses exceeding IpM (Figure 2B). These data support the notion that ARM165 could be used as a lineage-specific treatment in AML to avoid the dose-limiting effects of targeting PI3K in non-hematopoietic tissues. To expand the applicability of our results to a wider range of AML cell models, we exposed primary cells isolated from AML patients to AZ2 and ARM165 (data not shown). ARM165 significantly compromised the viability of primary cells when compared to its parental compound, AZ2, thereby confirming the enhanced potency of the PIK3CG degrader in affecting AML cell growth (Figure 2C).

[0145] Considering the potentiation effect that we previously observed with sgRNAs targeting PIK3CG and venetoclax, we investigated whether the ARM 165 degrader would sensitize AML cells to venetoclax treatment to a similar extent. We determined that increasing doses of ARM165 significantly amplified the effect of venetoclax on reducing the growth and colonyforming capacity of various AML cell lines, in contrast to cells treated with the parental AZ2 compound (Figures 2D and 2E). This potentiation of venetoclax's effect was also proven in primary patient cells. ARM165 alone induced a marked decrease in colony formation when compared to parental AZ2, and this effect was further enhanced when combined with venetoclax (Figure 2F). We finally conducted an analysis of the synergistic activity of ARM165 and venetoclax using an excess over Bliss analysis over a wide range of concentrations. The analysis pointed out that ARM165 and venetoclax act synergistically to impair AML cell viability (Figure 2G). Notably, this synergistic activity of ARM165 with venetoclax was significantly greater than that of AZ2 with venetoclax across nine primary patient samples (Figure 2H).

[0146] In light of reports that durable response to therapy in AML necessitates an abrogation of leukemia-initiating cells (LICs), we investigated the impact of ARM165 on the LIC fraction of a Cbfb-MYHl 7-driven mouse model of AML. Cbfb-MYH 17-positive cells were treated ex vivo with either ARM165 or AZ2 prior to reinjection into sublethaly-irradiated secondary recipient mice at various cell concentration. Using extreme limiting dilution analysis, we observed a 4.9- fold decrease in LIC frequency in secondary recipients injected with blasts treated with ARM165 compared to those engrafted with blasts exposed to AZ2 (Figure 21). These promising results led us to evaluate the impact of ARM165 on animals injected with Cbfb- MYHii -driven mouse cells. The large molecular weight of PROTACs and other heterobifunctional molecules is well-known for presenting challenges in terms of solubility and bioavailability for in vivo testing. Nevertheless, we initially evaluated both intraperitoneal (IP) and intravenous (IV) routes of administration for ARM165. We established that IV administration of ARM165 over four consecutive days was the most promising treatment regimen as it slowed down disease progression when compared to IP injection. IP administration of ARM165 was ineffective in affecting leukemic cell progression due to the rapid precipitation of ARM165 in the peritoneal cavity of the animals (not shown). Employing the IV treatment regimen in mice injected with Cbfb-MYHl 7-driven AML cells resulted in a significant reduction in leukemia burden in bone marrow when compared to animals treated with either vehicle or AZ2 (Figure 2J). The assessment of leukemic infiltration in secondary organs, such as the spleen, confirmed the anti-leukemia effect of ARM165, mirroring the results observed in the bone marrow (Figure 2K). Furthermore, we investigated the anti-leukemic effect of ARM165 in combination with venetoclax in i) syngeneic mice transplanted with Cbfb- MYH / / -driven mouse cells and ii) NSG-S mice xenografted with AML primary patient cell material (Figures 2L and 2M). In both mouse models, animal treated with the combination of these two drugs displayed a significantly reduced leukemic burden compared to those treated with ARM165 or venetoclax alone (Figure 2L). These findings confirm the promise of PROTAC -based PIK3CG destabilization, whether used alone or in combination with venetoclax, as a therapeutic approach with potential advantages over existing PIK3CG- targeting small-molecule inhibitors.

[0147] Discussion:

[0148] The effectiveness of targeted therapies in cancer is intricately linked to the capacity to identify molecular subtypes within tumors and discern the critical dependencies responsible for their onset and progression. In this context, the concept of lineage addiction emerged as the idea that certain cancer types are reliant on the abnormal activation of genes or pathways that are generally essential for the development and function of their lineage of origin. The discovery that the master transcription factor regulator of melanocyte function, MITF, acts as a melanoma oncogene constitutes an informative example in this regard (16001072). Melanoma, originating from melanocytes, retains its reliance on MITF. Consequently, MITF serves as a lineagespecific controller, functioning within normal cells and being similarly employed by melanoma cells for progression and propagation. Consistent with this, several melanoma cell lines exhibit increased dosage at a region of chromosome 3p containing MITF (16001072). The transcription factor C / EBPa (CCAAT / enhancer-binding protein alpha, encoded by CEBPA) is another example of strict regulator of myeloid development whose dysregulation is associated with AML pathogenicity. C7EBPa instructs myeloid lineage differentiation to granulocytes and monocytes. Dysregulation of C7EBPa increases proliferation of immature myeloid cells, contributing to the development of leukemia (refs). The genetic targeting of these two lineage addictions has proven to be especially effective in these two cancer types. In our study, we demonstrated that, similar to the exquisite function of MITF in melanocytes and C / EBPcr in myeloid cells, the expression of PIK3CG and its regulatory subunit, PIK3R5, is predominantly restricted to the myeloid compartment. Using a variety of genetic tools, we showed that the suppression of these genes has a significant impact on AKT signaling and the growth of AML cells, an effect which was, in contrast, not so markedly observed upon knockdown of other PI3K isoforms. Despite the expression of other PI3K isoforms in AML cells, our findings thereby suggest that PIK3CG and PIK3R5 constitute AML-specific vulnerabilities, primarily because of their prominent role in regulating AKT signaling in comparison to the other PI3K isoforms. Targeting this 'lineage-specific signaling addiction' may enable therapy to be directed toward the tissue compartment where malignancy is present, creating the potential for a therapeutic window that can spare non-malignant tissues.

[0149] We demonstrated that targeting PIK3CG can enhance the effectiveness of the BCL-2 inhibitor, venetoclax, which is a newly approved standard-of-care therapy for elderly patients with AML (ref). This increased sensitivity to venetoclax is likely a direct result of AKT inhibition. We established this through a whole-genome CRISPR screen, which ranked BCL-2 as one of the top sensitizers to the AKT inhibitor MK-2206. The underlying mechanism of this interrelationship between AKT and BCL-2 may lie in the fact that AKT plays a pivotal role in regulating apoptosis via the inactivation of the pro-apoptotic protein BAD through direct phosphorylation and by mediating the transcriptional upregulation of BCL-2 in response to various stimuli (10753867, 9346240, ...).

[0150] Considering the evidence that merely inhibiting PIK3CG-mediated AKT activation with small molecules was insufficient to eliminate AML cell survival, we developed a comprehensive targeting strategy using a CRBN-based PROTAC system to degrade PIK3CG. This approach demonstrated a significant anti-leukemia effect. Notably, several studies speak to the dual scaffolding and catalytic role of PIK3CG in promoting downstream signaling events. A comparison of knockout versus kinase-dead knock-in mice yielded critical insights into the non- catalytic functions of PIK3CG (15817396). Specifically, the contrasting phenotypes between / VAUCG-deficient versus PIK3CG kinase-dead mice revealed that this enzyme serves as a molecular scaffold orchestrating cellular signaling complexes independently of its lipid kinase activity (22859670, 31480354). For instance, it has been reported that PIK3CG binds to and regulates the activity of phosphodiesterase 3B (PDE3B) independently of its catalytic activity, likely as part of a protein complex (15294162). The disruption of PDE3B activity in PIK3CG knockout mice leads to increased cAMP levels, resulting in heightened heart contractility, a phenomenon seen exclusively in E / GCG-knockout mice, not in PIK3CG kinase-dead animals. These scaffolding functions are notably selective, and the absence of a specific PI3K isoform is unlikely to be compensated for by others. Interestingly, these functions have been well- documented for GPCR-dependent PI3 -kinases like PIK3CG, and they may act in concert or independently from the kinase activity. For example, PIK3CG ’s functions are intertwined with the kinase activity in diet-induced obesity, while PIK3CB plays a role in insulin signaling independently (25512233, 20201884, 28179187, 22859670).

[0151] The findings that PI3K isoform scaffolding and kinase activity functions can be disentangled to affect a broad array of cellular functions highlights how various PROTAC -based degradation approaches, similar to the one we have carried out for targeting PIK3CG, can result in more profound downstream signaling alteration and significant variations in functional outcomes compared to the targeting of its kinase function alone. This extends the potential application of this technology for more effective PI3K signaling inhibition, with possible implications for clinical use. Similar to our findings with PIK3CG degradation, targeted protein degradation can often outperform small molecule inhibitors initially designed for the same protein target. For example, dBETl exhibited a greater apoptotic response in primary AML cells compared to its precursor, JQ1, emphasizing the potential superiority of BET degradation over bromodomain inhibition (25999370). Additionally, PROTAC degraders targeting STAT3 and STAT5 have demonstrated significantly greater potency than small molecule inhibitors, which were reported to lack adequate potency and selectivity (36732620, 31715132). Similarly, the development of a selective FAK kinase degrader exhibited improved activity in downstream signaling, cancer cell viability, and migration compared to FAK kinase inhibitors (37088717).

[0152] The findings presented here argue for cancer therapeutics specifically designed to target dependencies driven by proteins with tissue- and / or tumor-restricted expression. Our results also provide a proof-of-concept that the degradation, rather than inhibition, of signaling molecules, such as PIK3CG, could offer more significant therapeutic benefits for patients, and more sustained response to adjuvant therapy regimens.

[0153] REFERENCES: 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 agent that suppresses the PI3Ky / AKT signalling pathway.

2. The method of 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 agent that suppresses the PI3Ky / AKT signalling pathway.

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 agent that suppresses the PI3Ky / AKT signalling pathway.

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

6. A method of treating AML in patient in need thereof comprising administering to the patient a therapeutically effective combination comprising an agent that suppresses the PI3Ky / AKT signalling pathway and a BCL-2 inhibitor.

7. The method of claim 6 wherein the BCL-2 inhibitor is venetoclax.

8. The method according to any one of claims 1 to 7 wherein the agent is a small molecule.

9. The method of claim 8 wherein the small molecule is a degrader molecule that degrades the PI3Ky isoform.

10. The method of claim 9 wherein the degrader 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 pl 10y targeting moiety.

11. The method of claim 1° wherein the an E3 ubiquitin ligase binding moiety (“ULM”) 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) bindingmoiety (VLM), and / or a mouse bould minute 2 homologue (MDM2) E3 ubiquitin ligase binding moiety (MLM).

12. The method of claim 11 wherein the ILM / VLM / CLM / MLM and PTM are joined or coupled via a chemical linker (L).

13. The method according to any one of claims 10 to 12 wherein the PTM is a pl lOy inhibitor, and more particularly a pl 10y selective inhibitor.

14. The method of claim 13 wherein the PTM is AZ2 that has the general formula of :

15. The method of claim 9 wherein the degrader molecule is ARM165 that the formula of:O O ARM-16516. The method according to any one of claims 1 to 7 wherein the agent is an inhibitor of PIK3CG expression or an inhibitor of PIK3R5 expression.

17. The method of claim 16 wherein the inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme.

18. The compound having the formula of:O O ARM-16519. A pharmaceutical composition comprising the compound of claim 18.

20. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the compound of claim 18.