WO2025087879A2 - Suppression de la voie de signalisation pi3kgamma/akt pour le traitement de leucémie myéloïde aiguë - Google Patents

Suppression de la voie de signalisation pi3kgamma/akt pour le traitement de leucémie myéloïde aiguë Download PDF

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WO2025087879A2
WO2025087879A2 PCT/EP2024/079767 EP2024079767W WO2025087879A2 WO 2025087879 A2 WO2025087879 A2 WO 2025087879A2 EP 2024079767 W EP2024079767 W EP 2024079767W WO 2025087879 A2 WO2025087879 A2 WO 2025087879A2
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aml
pik3cg
patient
cells
inhibitor
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WO2025087879A3 (fr
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Alexandre PUISSANT
Kris Wood
Anthony Martin
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Ecole Nationale Superieure de Chimie de Montpellier ENSCM
Universite de Montpellier
Universite Paris Cite
Duke University
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Ecole Nationale Superieure de Chimie de Montpellier ENSCM
Universite de Montpellier
Universite Paris Cite
Duke University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention is in the field of medicine, in particular oncology and hematology.
  • 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.
  • CLL chronic lymphocytic leukemia
  • BTK tyrosine kinase
  • ibrutinib a B-cell restricted enzyme that relays essential cell survival and migration signals in CLL and other B-cell malignancies
  • 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.
  • 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).
  • 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.
  • Phosphoinositide 3-kinase 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).
  • the present invention is defined by the claims.
  • the present invention relates to the targeting the PI3Ky/AKT signalling pathway for the treatment of Acute Myeloid Leukemia (AML).
  • AML Acute Myeloid Leukemia
  • 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.
  • AML acute myeloid leukemia
  • suppression of the myeloid-restricted PIK3CG/pl 10y-PIK3R5/pl01 axis blocks AKT signaling, compromises cell fitness, and sensitizes to established AML therapies.
  • the inventors find that existing small molecule inhibitors against PIK3CG are insufficient to achieve a sustained longterm anti-leukemic effect.
  • 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.
  • PROTAC proteolysistargeting chimera
  • 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.
  • AML acute myeloid leukemia
  • 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.
  • the patient is a human infant.
  • the patient is a human child.
  • the patient is a human adult.
  • the patient is an elderly.
  • 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.
  • treatment refers 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.
  • 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.
  • maintenance regimen 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.]).
  • the method of the present invention is suitable for the treatment of the chemoresistant AML.
  • chemoresistant acute myeloid leukemia As used herein; the term “chemoresistant acute myeloid leukemia” or “chemoresistant
  • AML refers to the clinical situation in a patient suffering from an AML when the proliferation of leukemic cells cannot be prevented or inhibited by means of a chemotherapeutic agent or a combination of chemotherapeutic agents usually used to treat AML, at an acceptable dose to the patient.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, imatin
  • the chemotherapy consists in a combination of cytarabine and an anthracycline such as daunorubicin or idarubicin.
  • 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.
  • AML acute myeloid leukemia
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • the agent of the present invention is a small molecule.
  • small molecule refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals.
  • 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.
  • degrader that degrades the PI3Ky isoform
  • 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.
  • Proteolysis Targeting Chimeras 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.
  • 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).
  • the BCL-2 inhibitor is an isoxazolidine compound (see, e.g., Int. Patent Appl. Pub. No. WO 2008/060569, U.S. Pat.
  • 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.
  • the BCL-2 inhibitor is a small molecule heterocyclic compounds (see, e.g., XIS. Pat. No. 9018381).
  • 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.
  • Drugs administered in combination have biological activity in the subject to which the drugs are delivered.
  • 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.
  • the combination of the present invention results in the synthetic lethality of the leukemic cells, in particular DTC.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1,3 -butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • 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.
  • 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.
  • useful diluents include, e.g., lactose.
  • the active ingredient is combined with emulsifying and suspending agents.
  • certain sweetening, flavoring or coloring agents may also be added.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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/m 2 and 500 mg/m 2 .
  • 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.
  • a further object relates to the compound having the formula of:
  • 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.
  • 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.
  • 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.
  • 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; acid
  • 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.
  • FIG. 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.
  • 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.
  • 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
  • AZ2 AZ2
  • 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).
  • 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).
  • 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.
  • shRNAs 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.
  • 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).
  • Cq cycle threshold
  • ACTB housekeeping gene
  • 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.
  • OCI-AML2, MV4-11 and N0M0-1 AML cell were transduced with a construct allowing constitutive expression of 3xFlag-HA-PIK3R5.
  • 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.
  • 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).
  • 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).
  • TCLV2 backbone plasmid 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.
  • 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 * 10 4 cells 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.
  • PDX Patient-Derived Xenograft
  • 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).
  • 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.
  • 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 (W7M 2 -sr) using a standardized region of interest (ROI) encompassing the entire mouse.
  • 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 xlO 6 cells per mouse by intravenous injection.
  • 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 x
  • 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).
  • SAFE nutrition service #E8200P01R
  • 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 10 6 Cbfb-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.
  • PDX Patient-Derived Xenograft
  • 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], IxlO 6 mononucleated 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).
  • 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).
  • 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).
  • CIS-reverse phase DeltaPack Waters, 15 pm, 100A, 100 x 40 mm
  • GILSON HPLC system - SKID LC 009SK equipped with a C18 reversed-phase Luna column
  • 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.
  • TFA trifluoroacetic acid
  • 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).
  • 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).
  • 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.
  • PIK3CG and its Regulatory Subunit PIK3R5 Exhibit a Myeloid-Biased Expression Profile and Are Required for AML Cell Survival
  • TCGA Cancer Genome Atlas
  • GTEx Genotype-Tissue Expression Project
  • 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.
  • PIK3CG was most essential in AML (and secondarily in ALL) with minimal essentiality in malignancies of non-hematopoietic origin (data not shown).
  • PIK3CA an PIK3CR were essential across malignancies of diverse tissue types.
  • PIK3R5 signaling may act as a lineage-restricted driver of disease progression, and a selective survival dependency, in AML.
  • doxycycline (dox)-inducible short-hairpin RNAs shRNAs
  • sgRNA single-guide RNA
  • 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).
  • mice 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.
  • GPCR G- coupled cytokine receptor signaling
  • GPCR G protein- coupled receptors
  • 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).
  • PIK3CG-PIK3R5 is activated via GPCRs, whereas other isoforms are predominantly under the control of receptor tyrosine kinases (RTKs)
  • RTKs receptor tyrosine kinases
  • MITF melanocyte function
  • C/EBPa CCAAT/enhancer-binding protein alpha, encoded by CEBPA
  • 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.
  • 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.
  • 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, ).
  • PIK3CG binds to and regulates the activity of phosphodiesterase 3B (PDE3B) independently of its catalytic activity, likely as part of a protein complex (15294162).
  • PDE3B phosphodiesterase 3B
  • PIK3CG functions are intertwined with the kinase activity in diet-induced obesity, while PIK3CB plays a role in insulin signaling independently (25512233, 20201884, 28179187, 22859670).

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Abstract

La toxicité limitant la dose pose une limitation majeure à l'utilité clinique de thérapies anticancéreuses ciblées, souvent due à un engagement de la cible dans des tissus non malins. Cet obstacle peut être réduit au minimum en ciblant des dépendances cancéreuses entraînées par des protéines avec une expression restreinte par un tissu et/ou une tumeur. Ici, les inventeurs ont montré que dans la leucémie myéloïde aiguë (LMA), la suppression de l'axe PIK3CG/p110γ-PIK3R5/p101 à restriction myéloïde bloque la signalisation AKT, compromet l'aptitude cellulaire, et sensibilise à des thérapies anti-LMA établies. De manière importante, les inventeurs trouvent que des inhibiteurs à petites molécules existants contre le PIK3CG sont insuffisants pour obtenir un effet anti-leucémique à long terme soutenu. Pour résoudre ce problème, les inventeurs ont développé une molécule hétérobifonctionnelle chimère ciblant la protéolyse (PROTAC) qui dégrade spécifiquement le PIK3CG et supprime puissamment la progression de la LMA, seule et en combinaison avec du vénétoclax dans des lignées cellulaires humaines de LMA, des échantillons primaires de patients atteints de LMA et des modèles de souris syngéniques.
PCT/EP2024/079767 2023-10-23 2024-10-22 Suppression de la voie de signalisation pi3kgamma/akt pour le traitement de leucémie myéloïde aiguë Pending WO2025087879A2 (fr)

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