AXL inhibitors for the treatment of mastocytosis
AXL inhibitors combined with TKIs and BCL-2 inhibitors address the challenge of TKI resistance in mastocytosis by reducing mast cell proliferation and enhancing treatment efficacy.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM)
- Filing Date
- 2025-12-18
- Publication Date
- 2026-06-25
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Abstract
Description
[0001] AXL INHIBITORS FOR THE TREATMENT OF MASTOCYTOSIS
[0002] FIELD OF THE INVENTION:
[0003] The present invention is in the field of medicine.
[0004] BACKGROUND OF THE INVENTION:
[0005] Mastocytosis is a rare and heterogeneous disorder marked by the abnormal accumulation of mast cells (MCs) in various tissues. In approximately 85% of cases, mastocytosis is associated with somatic mutations in the KIT gene, leading to constitutive activation of the receptor and promoting MC proliferation independent of ligand binding. The KIT D816V mutation is particularly prevalent, found in 90% of adult cases and 50% of pediatric cases. Mastocytosis is classified into two major subtypes: cutaneous mastocytosis (CM), which is confined to the skin, and systemic mastocytosis (SM), which affects multiple organs. SM ranges from indolent forms (ISM), characterized by a near-normal life expectancy but reduced quality of life, to aggressive variants (AdvSM), which are associated with significantly reduced life expectancy, particularly in cases of mast cell leukemia or mast cell sarcoma. Patients with AdvSM often harbor the KIT D816V mutation alongside additional mutations in genes such as TET2, SRSF2, ASXL1, RUNX1, JAK2, NRAS, CBL, IDH1 / 2, SF3B1, and EZH2, all of which are linked to a poor prognosis. Although KIT mutations are highly prevalent in mastocytosis, there is growing evidence that other factors also play a critical role in the disease’s pathogenesis and progression. Notably, tyrosine kinase inhibitors have shown limited efficacy, suggesting that additional molecular mechanisms are involved.
[0006] The AXL gene belongs to the TAM family (Tyro3, Axl, and MerTK) and encodes a receptor tyrosine kinase. AXL plays crucial roles across various biological systems, including the immune, hematopoietic, vascular, and nervous systems. TAM receptors, along with their ligands Gas6 and Protein S, are essential for the efficient clearance of apoptotic cells through phagocytosis. In the immune system, TAM receptors act as important regulators, curbing innate inflammatory responses to pathogens. While deficiencies in TAM signaling are associated with chronic inflammatory and autoimmune diseases, excessive activation of these receptors has been strongly linked to cancer progression, metastasis, and resistance to targeted therapies. AXL is highly expressed in a variety of cell types, including myeloid cells such as monocytes, macrophages, and dendritic cells, as well as fibroblasts, epithelial cells, endothelial cells, and neurons. Canonical activation of AXL is mediated by its binding to the Gas6 ligand, followed by receptor dimerization. Gas6 is found in human plasma at concentrations of 13 to 23 ng / mL (0.16 to 0.28 nM). Alternatively, AXL can be activated independently of ligands through homodimerization or heterodimerization with other receptors such as FGFR, PDGFR, EGFR, and MET. Upon activation, AXL undergoes phosphorylation, triggering a cascade of downstream signaling pathways. These pathways play critical roles in processes such as cell proliferation, immune suppression, apoptosis, stem cell maintenance, and epithelial-to- mesenchymal transition (EMT). Importantly, AXL has been implicated in various malignancies, including cutaneous melanoma, squamous cell carcinoma, renal carcinoma, chronic lymphoblastic leukemia, and acute myeloid leukemia. It is particularly notable for its role in driving resistance to tyrosine kinase inhibitors (TKIs), presenting a significant challenge in the treatment of these cancers.
[0007] SUMMARY OF THE INVENTION:
[0008] The present invention is defined by the claims. In particular, the present invention relates to the use of AXL inhibitors for the treatment of mastocytosis.
[0009] DETAILED DESCRIPTION OF THE INVENTION:
[0010] Main definitions:
[0011] As used herein, the term “mastocytosis” has its general and describes a group of disorders in which pathologic mast cells accumulate in tissues. In particular, the term includes cutaneous mastocytosis and systemic mastocytosis (indolent or advanced). The pathogenesis of mastocytosis has been attributed to constitutive activation of the receptor tyrosine kinase KIT. In a large majority of mastocytosis patients, the deregulated tyrosine kinase activity of KIT is due to a mutation within the amino acid 816 of the protein (D816V). In particular, the method of the present invention is particularly suitable for the treatment of systemic mastocytosis. As used herein, the term "systemic mastocytosis” or “SM" encompasses the 5 categories of SM defined by the World Health Organization according to their location and aggressiveness: indolent SM (ISM), smoldering SM (SSM), SM with an associated hematological neoplasm (SM-AHN), aggressive SM (ASM), and mast cell leukemia (MCL). The prognosis of patients with ASM and MCL is poor due to an aggressive nature of the cells and their tendency to detach from the main tumor. Many of these tumors, but not all, carry mutations in the tyrosine kinase receptor: KIT (stem cell factor receptor) that renders it constitutively activated leading to uncontrolled growth of the malignant mast cells (MCs). Clinical presentation in adult SM is heterogenous and includes skin disease (usually urticaria pigmentosa), mast cell mediatorrelease symptoms (headache, flushing, lightheadedness, syncope, anaphylaxis, etc), and direct or indirect organ damage (bone pain from lytic bone lesions, osteoporosis or bone fractures, hepatosplenomegaly, cytopenia from bone marrow involvement). In addition, around 20% of patients with SM may display significant and sometimes isolated blood eosinophilia. In particular, the method of the present invention is particularly suitable for the treatment of patients harbouring a KIT mutation (e.g. D816V or Delta 417-419 insY mutations). In some embodiments, the patient harbors the D816V mutation.
[0012] As used herein, the term "KIT" has its general meaning in the art and refers to the human KIT. KIT is also known as "kit", "c-kit", "CD117" or "stem cell factor receptor". An exemplary native KIT amino acid sequence is provided in the UniProtKB / Swiss-Prot under accession number P10721). Methods of detecting KIT mutations are well known in the art and typically involves PCR assays.
[0013] As used herein, the term “inhibitor” or “antagonist” refers to a compound that decreases the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. In some instances, an inhibitor will substantially decrease the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity or expression observed in the absence of the inhibitor. In some instances, an inhibitor will completely diminish the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. Certain exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, aptamers, antisense oligonucleotides, interfering RNA, carbohydrates or small organic molecules.
[0014] As used herein, the term “AXL” has its general meaning in the art and refers to a receptor tyrosine kinase that is part of the TAM family (Tyro3, Axl, and MerTK). AXL is involved in various cellular processes such as cell survival, proliferation, migration, and immune regulation. It is activated by its ligand Gas6, leading to receptor dimerization and subsequent phosphorylation, which triggers downstream signaling pathways. As used herein, the term “tyrosine kinase inhibitor” or “TKI” has its general meaning in the art and refers to a pharmacological compound that inhibits the action of tyrosine kinases, which are enzymes responsible for the activation of various proteins by signal transduction through the process of phosphorylation. Typically, TKIs as contemplated herein may be categorized to four groups: (1) ATP-competitive inhibitors, which bind predominantly to the ATP -binding site of the kinase when this site is in the active conformation; (2) inhibitors that recognize and bind to the non-active conformation of the ATP -binding site of the kinase, thus making activation energetically unfavorable; (3) allosteric inhibitors, that bind outside of the ATP -binding site, modifying the tridimensional structure of the receptor and disrupting the interaction between the ATP and the kinase pocket; and (4) covalent inhibitors, that bind irreversibly by covalently bonding to the ATP -binding site of the target kinase.
[0015] As used herein the term "resistance to TKI" is used in its broadest context to refer to the reduced effectiveness of TKI to inhibit the proliferation of mast cells, to inhibit the accumulation of mast cells in tissue, kill a mast cell or inhibit one or more cell survival functions, and to the ability of a mast cell to survive exposure to TKI designed to inhibit the growth of the mast cell, kill the mast cell or inhibit one or more cellular functions. The resistance displayed by a mast cell may be complete in that TKI is rendered completely ineffective against the mast cell, or may be partial in that the effectiveness of TKI is reduced. Accordingly, the term "resistant" refers to the progression of mastocytosis independently of whether the disease was cured before said outbreak or progression. The phrase “preventing resistance to TKI” in context of the invention shall be effective if compared to a non-treated control, the mastocytosis or mast cells becomes more sensitive to TKI. In particular, the patient will 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 mastocytosis is eradicated, reduced or improved after TKI. According to the invention, the responders have an objective response and therefore the term does not encompass patients having a stabilized mastocytosis such that the disease is not progressing after TKI. A “non-responder” or “refractory patient” includes patients for whom the mastocytosis does not show reduction but also patients having a stabilized mastocytosis following TKI. The term “non-responder” also includes patients having a stabilized mastocytosis. 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 AXL inhibitor of the present invention.
[0016] As used herein, the term "relapse" refers to the return of the mastocytosis after a period of improvement in which the accumulation of mast cells in tissues was inhibited.
[0017] As used herein, the term "BCL-2 inhibitor" refers to an agent that is capable of inhibiting one or more proteins in the BCL-2 family of anti-apoptotic proteins, e.g., BCL-2, BCL-xL, and BCL -w. In some embodiments, a BCL-2 inhibitor of the disclosure inhibits one protein of the BCL-2 family selectively, e.g., a BCL-2 inhibitor may selectively inhibit BCL-2 and not BCL- xl or BCL-w. The BCL-2 inhibitor described herein may inhibit one or more of BCL-2, BCL- xL, and BCL-w. In some embodiments, the inhibitor of BCL-2 anti-apoptotic family of proteins inhibits BCL-2. In some embodiments, the inhibitor of BCL-2 anti-apoptotic family of proteins inhibits BCL-2 and does not inhibit other members of the BCL-2 family of proteins, e.g., does not inhibit BCL-xL or BCL-w. In some embodiments, the BCL-2 inhibitor is a BH3-mimetic.
[0018] 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 simultaneously, separately or sequentially 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 2 different drugs, and wherein the first drug is the AXL inhibitor, the second drug is the drug selected from the group consisting of tyrosine kinase inhibitors and BCL-2 inhibitors. In some instance, the combination of the present invention results in the synthetic lethality of the mast cells. In some embodiments, the AXL inhibitor is administered to the patient before the tyrosine kinase inhibitor, thereby preventing the resistance to said tyrosine kinase inhibitors. In some embodiments, the patient is first administered with at least one cycle (Cl) of administering the drug selected from the group consisting of tyrosine kinase inhibitors and BCL-2 inhibitors followed by administration of at least one cycle (C2) of administering the AXL inhibitor. As used herein, the term “cycle” refers to a period of time during the treatment is administered to the patient. Typically, a cycle of therapy is followed by a rest period during which no treatment is given. Following the rest period, one or more further cycles of therapy may be administered, each followed by additional rest periods. In some embodiments, cycle (Cl) comprises administering a dose of the drug selected from the group consisting of tyrosine kinase inhibitors and BCL-2 inhibitors daily or every 2, 3, 4, or 5 days. In some embodiments, the drug selected from the group consisting of tyrosine kinase inhibitors and BCL-2 inhibitors is administered continuously (i.e. every day) during cycle (Cl). In some embodiments the administration of a dose of the drug selected from the group consisting of tyrosine kinase inhibitors and BCL-2 inhibitors is alternated with the administration of a dose of the AXL inhibitor. Typically cycle (Cl) can last one or more days, but is usually one, two, three or four weeks long. In some embodiments cycle (Cl) is repeated at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times before administering cycle (C2).
[0019] 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 a regular interval, 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., pain, disease manifestation, etc.]).
[0020] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.
[0021] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.
[0022] As used herein, the term “Therapeutically effective amount” refers to the level or amount of an antibody or antigen-binding fragment thereof as described herein that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of the disease, disorder, or condition; (4) reducing the severity or incidence of the disease, disorder, or condition; or (5) curing the disease, disorder, or condition. A therapeutically effective amount may be administered prior to the onset of the disease, disorder, or condition, for a prophylactic or preventive action. Alternatively or additionally, the therapeutically effective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action.
[0023] Methods of the present invention:
[0024] Accordingly, the present invention provides: a method of treating an mastocytosis in a patient in need thereof comprising administering to the patient a therapeutically effective combination comprising a TKI and an AXL inhibitor a method of preventing resistance to TKI in a patient suffering from mastocytosis comprising administering to the patient a therapeutically effective amount of an AXL inhibitor, a method of treating an mastocytosis resistant to TKI in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an AXL inhibitor, a method for enhancing the potency of TKI administered to a patient suffering from an mastocytosis as part of a treatment regimen, the method comprising administering to the patient a pharmaceutically effective amount of an AXL inhibitor in combination with the TKI, a method for preventing relapse of a patient suffering from an mastocytosis who was treated with a TKI comprising administering to the patient a therapeutically effective amount of an AXL inhibitor, a method of overcoming resistance to TKI in a patient suffering from an mastocytosis comprising administering to the patient a therapeutically effective amount of an AXL inhibitor a method of treating an mastocytosis in a patient in need thereof comprising administering to the patient a therapeutically effective combination comprising an AXL inhibitor and a BCL-2 inhibitor.
[0025] In some embodiments, the patient according to the invention is a human. In some embodiments, the patient according to the invention is a girl or a boy. In some embodiments, the patient according to the invention is an adult. In some embodiments, the patient according to the invention is a child (human being between the stages of birth and puberty), a teenager (human being between the stages of puberty to adulthood) or an elderly person (human being after the puberty). In some embodiments, the patient is less than 15 years old. In some embodiments, the patient is less than 10 years old. In some embodiments, the patient is less than 7 years old. In some embodiments, the patient is less than 5 years old. In some embodiments, the patient is less than 3 years old.
[0026] AXL inhibitors:
[0027] Without wishing to be bound by a theory, an AXL inhibitor can act by a number of different pathways. For example, an AXL inhibitor can bind to a ligand bind site on AXL and interfere with binding of the ligand to AXL, bind to a nonligand binding site on AXL and interfere with binding of the ligand to AXL, bind with a AXL receptor ligand and interfere with binding of the ligand to AXL, or inhibit the expression of a polynucleotide (e.g., mRNA) expressing AXL. The AXL inhibitors can encompass numerous classes of chemical molecules, e.g., small organic or inorganic molecules, polysaccharides, biological macromolecules, e.g., peptides, proteins, peptide analogs and derivatives, peptidomimetics, antibodies, antibody fragments, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, naturally occurring or synthetic compositions. Thus, an AXL inhibitor can be a molecule of any type that interferes with the activity or expression of AXL, for example, either by decreasing transcription or translation of AXL encoding nucleic acid, or by inhibiting or blocking AXL activity, or both. Examples of AXL inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, AXL -specific aptamers, anti-AXL antibodies, AXL- binding fragments of anti-AXL antibodies, AXL-binding small molecules, AXL-binding peptides, and other polypeptides that specifically bind AXL (including, but not limited to, AXL- binding fragments of one or more AXL ligands, optionally fused to one or more additional domains), such that the interaction between the AXL inhibitor and AXL results in a reduction or cessation of AXL activity or expression. It will be appreciated that AXL inhibitors described herein may be strong inhibitors of AXL. In some embodiments, an AXL inhibitor inhibits the biological activity of AXL by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative to a control. In some embodiments, an AXL inhibitor completely abrogates the biological activity of AXL relative to a control. A control can comprise a sample that is not treated with an AXL inhibitor.
[0028] In some embodiments, the AXL inhibitor is an antibody, more particularly an anti-AXL antibody. The antibodies can be selected from pre-existing antibodies (e.g., publicly available hybridomas or recombinant antibody libraries, further described herein below) or from newly generated antibodies produced according to methods which are well-known in the art and further described herein. Examples of AXL specific antibodies are disclosed in US Patent Application No. 20210070869, 20190275149 and 20190352407, the contents of which are incorporated herein by reference. In some embodiments, the AXL inhibitor is a nucleic acid. Exemplary AXL nucleic acid inhibitors include, but are not limited to, antisense oligonucleotides, siRNAs, shRNAs, microRNAs, aptamers, ribozymes and decoy oligonucleotides. A AXL nucleic acid inhibitor can inhibit the expression of a AXL gene.
[0029] In some embodiments, the AXL inhibitor is a small molecule inhibitor. Examples of such inhibitors include, but are not limited to R428, bemcentinib, YW327.652, GL2I.T, TP-0903, LY2801653, amuvatinib, bosutinib, MGCD 265, ASP2215, cabozantinib, foretinib, SGL7079, MGCD516, ASLAN002, and gilteritinib. According to a particular embodiment, the AXL inhibitor is bemcetinib.
[0030] Tyrosine kinase inhibitors:
[0031] In some embodiments, the TKI is selected from the group consisting of trametinib, afatanib, afatinib dimaleate, masitinib, erlotinib, gefitinib, lorlatinib, alectinib, crizotinib, sotorasib, adagrasib, osimertinib, ceritinib, brigatinib, dacomitinib, mobocertinib, mobocertinib succinate, entrectinib, capmatinib, capmatinib hydrochloride, tepotinib, tepotinib hydrochloride, selpercatinib, pralsetinib, dabrafenib, vemurafenib, encorafenib, cetuximab, panitumumab, necitumumab, amivantamab-vmjw, ramucirumab, erdafitinib (Balversa), enfortumab vedotin-ejfv (Padcev), sacituzumab govitecan-hziy (Trodelvy), everolimus (Afinitor), belzutifan (Welireg), tamoxifen (Nolvadex), toremifene (Fareston), trastuzumab (Herceptin), fulvestrant (Faslodex), anastrozole (Arimidex), exemestane (Aromasin), lapatinib (Tykerb), letrozole (Femara), ado-trastuzumab emtansine (Kadcyla), palbociclib (Ibrance), ribociclib (Kisqali), neratinib maleate (Nerlynx), abemaciclib (Verzenio), olaparib (Lynparza), talazoparib tosylate (Talzenna), alpelisib (Piqray), fam-trastuzumab deruxtecan-nxki (Enhertu), tucatinib (Tukysa), sacituzumab govitecan-hziy (Trodelvy), pertuzumab, trastuzumab, margetuximab-cmkb (Margenza), tisotumab vedotin-tftv (Tivdak), Cetuximab (Erbitux), panitumumab (Vectibix), regorafenib (Stivarga), ramucirumab (Cyramza), encorafenib (Braftovi), Imatinib mesylate (Gleevec), Lanreotide acetate (Somatuline Depot), lenvatinib mesylate (Lenvima), Trastuzumab (Herceptin), ramucirumab (Cyramza), fam- trastuzumab deruxtecan-nxki (Enhertu), Cetuximab (Erbitux), pembrolizumab (Keytruda), Imatinib mesylate (Gleevec), sunitinib (Sutent), regorafenib (Stivarga), avapritinib (Ayvakit), ripretinib (Qinlock), pexidartinib hydrochloride (Turalio), sorafenib (Nexavar), sunitinib (Sutent), pazopanib (Votrient), temsirolimus (Torisel), everolimus (Afinitor), axitinib (Inlyta), cabozantinib (Cabometyx), lenvatinib mesylate (Lenvima), tivozanib hydrochloride (Fotivda), belzutifan (Welireg), Tretinoin (Vesanoid), imatinib mesylate (Gleevec), dasatinib (Sprycel), nilotinib (Tasigna), bosutinib (Bosulif), ibrutinib (Imbruvica), idelalisib (Zydelig), venetoclax (Venclexta), ponatinib hydrochloride (Iclusig), midostaurin (Rydapt), enasidenib mesylate (Idhifa), inotuzumab ozogamicin (Besponsa), ivosidenib (Tibsovo), duvelisib (Copiktra), glasdegib maleate (Daurismo), gilteritinib (Xospata), tagraxofusp-erzs (Elzonris), acalabrutinib (Calquence), avapritinib (Ayvakit), asciminib hydrochloride (Scemblix), Sorafenib (Nexavar), regorafenib (Stivarga), lenvatinib mesylate (Lenvima), cabozantinib (Cabometyx), ramucirumab (Cyramza), pemigatinib (Pemazyre), infigratinib phosphate (Truseltiq), ivosidenib (Tibsovo), crizotinib (Xalkori), erlotinib (Tarceva), gefitinib (Iressa), afatinib dimaleate (Gilotrif), ceritinib (LDK378 / Zykadia), ramucirumab (Cyramza), osimertinib (Tagrisso), necitumumab (Portrazza), alectinib (Alecensa), brigatinib (Alunbrig), trametinib (Mekinist), dabrafenib (Tafinlar), dacomitinib (Vizimpro), lorlatinib (Lorbrena), entrectinib (Rozlytrek), capmatinib hydrochloride (Tabrecta), selpercatinib (Retevmo), pralsetinib (Gavreto), tepotinib hydrochloride (Tepmetko), sotorasib (Lumakras), amivantamab-vmjw (Rybrevant), mobocertinib succinate (Exkivity), brentuximab vedotin (Adcetris), vorinostat (Zolinza), romidepsin (Istodax), bexarotene (Targretin), bortezomib (Velcade), pralatrexate (Folotyn), ibrutinib (Imbruvica), siltuximab (Sylvant), belinostat (Beleodaq), copanlisib hydrochloride (Aliqopa), acalabrutinib (Calquence), venetoclax (Venclexta), duvelisib (Copiktra), polatuzumab vedotin-piiq (Polivy), zanubrutinib (Brukinsa), tazemetostat hydrobromide (Tazverik), selinexor (Xpovio), tafasitamab-cxix (Monjuvi), crizotinib (Xalkori), umbralisib tosylate (Ukoniq), loncastuximab tesirine-lpyl (Zynlonta), Bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib citrate (Ninlaro), selinexor (Xpovio), Imatinib mesylate (Gleevec), ruxolitinib phosphate (Jakafi), fedratinib hydrochloride (Inrebic), olaparib (Lynparza), rucaparib camsylate (Rubraca), niraparib tosylate monohydrate (Zejula), Erlotinib (Tarceva), everolimus (Afinitor), sunitinib (Sutent), olaparib (Lynparza), belzutifan (Welireg), Selumetinib sulfate (Koselugo), Cabazitaxel (Jevtana), enzalutamide (Xtandi), abiraterone acetate (Zytiga), apalutamide (Erleada), darolutamide (Nubeqa), rucaparib camsylate (Rubraca), olaparib (Lynparza), Vismodegib (Erivedge), sonidegib (Odomzo), vemurafenib (Zelboraf), trametinib (Mekinist), dabrafenib (Tafinlar), cobimetinib (Cotellic), alitretinoin (Panretin), encorafenib (Braftovi), binimetinib (Mektovi), Pazopanib (Votrient), alitretinoin (Panretin), tazemetostat hydrobromide (Tazverik), sirolimus protein-bound particles (Fyarro), Larotrectinib sulfate (Vitrakvi), entrectinib (Rozlytrek), trastuzumab (Herceptin), ramucirumab (Cyramza), fam- trastuzumab deruxtecan-nxki (Enhertu), Imatinib mesylate (Gleevec), midostaurin (Rydapt), avapritinib (Ayvakit), Cabozantinib (Cometriq), vandetanib (Caprelsa), sorafenib (Nexavar), lenvatinib mesylate (Lenvima), trametinib (Mekinist), dabrafenib (Tafinlar), selpercatinib (Retevmo), and pralsetinib (Gavreto). According to a particular embodiment, the TKI is midostaurin (PKC412) or avapritinib.
[0032] BCL-2 inhibitors:
[0033] 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, UMI-77, gambogic acid, maritoclax, MIMI, methylprednisolone, iMAC2, Bax inhibitor peptide V5, Bax inhibitor peptide P5, Bax channel blocker, and ARRY 520 trifluoroacetate.
[0034] 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).
[0035] Pharmaceutical compositions:
[0036] Typically, the drugs herein disclosed are combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
[0037] 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.
[0038] FIGURES:
[0039] Figure 1: Effects of single and combination treatments of PKC412 or R428 on abnormal mast cells. Fresh PBMCs from patient MCL3 were treated with DMSO (vehicule), 400 nM PKC412, 1 pM R428, or their combination. MCs viability (CD117+, FcsRI+, Sytox Blue ) was monitored by FACS over 21-days. Due to limited cell availability, only days 6 and 14 were analyzed in duplicate. Statistical significance was assessed by one-way ANOVA. *p < 0.05.
[0040] Figure 2: Effects of single and combination treatments of PKC412, R428 or Navitoclax on abnormal mast cells. Fresh PBMCs from patient MCL3 were treated with DMSO (vehicule), 200 nMPKC412, 500nMR428, 1 pM Navitoclax or their combination. MCs viability (CD 117+, FcsRI+, Sytox Blue ) was monitored by FACS over 6-days. Due to limited cell availability, experiment was conducted without technical duplicates.
[0041] EXAMPLE:
[0042] Unexpected expression of AXL in neoplastic Mast Cells
[0043] We identified six patients with ASM, ISM, CM or mastocytoma, all carrying mutations in the AXL gene, in addition to KIT mutations. Given the presence of AXL mutations in several patients, we hypothesized a role for this receptor in mastocytosis. In addition to studying the wild-type form of AXL, we focused on the mutation carried by a child diagnosed with ASM affecting the skin, bone marrow, liver, and spleen. This patient represented the most aggressive case among the cohort, carrying both the KIT D816V mutation and a germline AXL L197M mutation (Fig. 1). PolyPhen and SIFT analyses suggest that this mutation may have a potentially damaging effect. The substitution of CTG (leucine) with ATG (methionine) at position 197 of the AXL protein has not been associated with a specific tumor type, unlike the CTG to GTG (valine) substitution, which has been identified in a pleural tumor.
[0044] We first investigated the expression of AXL in neoplastic mast cells (MCs) from patients with mastocytosis. To achieve this, immunohistochemical staining (H4C) was performed on skin and bone marrow (BM) biopsies from patients with various forms of mastocytosis and differing degrees of MCs infiltration, including the patient with the AXL L197M mutation (Pat-A¥L 197M). None of the other patients analyzed carried a mutation in the AXL gene. Hematoxylin and eosin (H&E) staining, along with KIT staining, were used as indicators of MC infiltration. KIT staining revealed significant MC infiltration in the skin of Pat- 4 7. I 97M, all strongly expressing AXL. Skin biopsies from two other patients, one with CM and one with ISM, showed moderate and weak MC infiltration, respectively, with AXL expression observed in both cases.
[0045] IHC staining of BM biopsies from patients with ISM, SSM, MCL, and the ASM patient (Pat- AXL197M) revealed high AXL expression in myeloid cells, consistent with literature data, and heterogeneous AXL expression levels in neoplastic MCs across patients. Notably, strong AXL expression was observed in SSM-1 and MCL-1 patients, while it was weakly positive in Pat- AXL197M and ISM-2 patients, and intermediate in the MCL-2 patient.
[0046] To further confirm AXL expression in BM neoplastic MCs, we performed flow cytometry staining on five available DMSO-frozen BM samples: 2 ISM, 2 SM-HNMD, and 1 SSM. In healthy individuals, the frequency of MCs (CD117+ / FcsRI+) in BM is typically below 10A-5. In the patient samples, MC frequencies ranged from 0.02% to 5%, with all MCs expressing AXL, albeit at varying levels depending on the individual. For instance, in the ISM-4 and SM- AHN-1 samples, 0.3% and 5% of MCs were detected, respectively, with both showing low AXL expression. In contrast, in the ISM-3, SM-AHN-2, and SSM-2 samples, 1.2%, 0.02%, and 3% of MCs were detected, respectively, all expressing AXL predominantly at high levels. This variability in AXL expression is consistent with the patterns observed by IHC in BM biopsies.
[0047] We analyzed RNA-seq data from an unpublished study conducted by our collaborators, which involved isolating MCs through microdissection from skin lesion biopsies of 15 mastocytosis patients (12 with ISM and 3 with AdvSM). AXL was detected in all patients, with notably higher expression in the small group of AdvSM patients. To evaluate whether Gas6, the ligand of AXL, is expressed in patients with mastocytosis at levels comparable to those reported in healthy individuals (PMID: 15790929), we measured plasma Gas6 levels using a Luminex assay. Gas6 was detected in all individuals, with no significant differences observed between healthy controls (n=10) and patients (n=37), consistent with previously published findings (PMID: 15790929).
[0048] AXL is barely detectable in normal mast cells
[0049] Determining whether MCs express AXL in normal skin or BM is challenging due to their low abundance in these tissues and the broad expression of AXL across non-mast cell populations. Single-cell RNA sequencing (scRNA-seq) offers a potential solution to this issue; however, obtaining fresh biopsies from healthy individuals for cell isolation and sequencing presents ethical challenges. To overcome this limitation, we used publicly available scRNA-seq datasets, which enabled the identification of MCs and the evaluation of AXL expression patterns. Specifically, we analyzed data from the Immune Single Cell Consortium, focusing on the dataset "disco mast basophil vl.O.rds," which includes mast cells and basophils from various tissues. We first focused on MCs embeded in normal skin and identified 2,400 cells, of which only 21 cells expressed AXL (-0.875%). Subsequently, we examined MCs from the same dataset in primary skin tumors, including basal and cutaneous squamous cell carcinoma. Among the 536 MCs identified in these tumors, only 1 MC expressed AXL (-0.187%). We also collected data from the (disco bone marrow vl.O) dataset and analyzed the MCs of 68 normal BM samples. We identified 1,086 KIT+ FcERI+MCs, among which only 74 expressed AXL (-6%) belonging to 6 healthy individuals.
[0050] Overall, AXL is expressed by very few, if any, mast cells in normal skin or bone marrow, whereas it is consistently expressed, by neoplastic mast cells in mastocytosis. However, the rarity of mast cells and their localization within tissues make isolating them from both patients and healthy individuals particularly challenging, thereby limiting their use for in-depth studies on AXL's role. To address this limitation and better understand the expression and function of AXL in mast cells, we utilized ROSA KIT WT, ROSA XZZ D816V, and HMC1.2 mast cell lines, generously provided by Prof. Michel Arock, France.
[0051] AXL expression is induced by inflammatory factors in mast cells We assessed the expression of wild type AXL (AXL-WT) by RT-qPCR and found barely detectable expression in ROSA KIT WT and ROSA XZTD816V cells (Cq = 34.72 ± 0.32 and 34.06 ± 0.39, respectively) and very low expression in HMC1.2 cells (Cq = 31.94 ± 0.07) (Fig. 5 A). Based on these findings, we investigated whether environmental factors — such as hypoxia, interferons (IFN-a, IFN-P, IFN-y), TNF-a, GM-CSF, LPS, or poly(I:C) — could induce AXL expression in mast cells, as reported in other cell types (PMID: 30923103), (DOI 10.1016 / j. cell.2007.10.034) (PMID: 29907882) (PMID: 9858144) (PMID: 25603826) (PMID: 36532777). Exposure of MCs to hypoxic conditions (1% or 3% O2), poly(I:C), or GM-CSF did not increase AXL expression (data not shown). In contrast, stimulation with IFN-a (100-300 ng / mL) significantly enhanced AXL expression, with increases of up to 40-fold in ROSA KIT WT and ROSA XZT D816V, and up to 60-fold in HMC1.2 cells. IFN-P induced a striking upregulation of AXL expression, with increases of up to 250-fold at 1 ng / mL and 800-fold at 25 ng / mL in ROSA XZT D816V cells (Fig. 5C). IFN-y (10-400 ng / mL) also induced AXL expression, with increases of up to 5-fold in ROSA KIT D816V and 10-fold in HMC1.2 cells. LPS stimulation resulted in AXL expression increasing up to 15-fold at 100 ng / mL, while TNF- a led to a modest 2-fold increase at 150 ng / mL (data not shown). AXL upregulation in response to IFN-a, IFN-P, and IFN-y was confirmed at the protein level through immunoblotting and Western blot analysis.
[0052] AXL expression was also evaluated in primary MCs derived from CD34+ cells using RT- qPCR, revealing extremely low basal levels (Cq = 38). Stimulation with IFN-a (100-300 ng / mL) significantly increased AXL expression, with levels rising up to 300-fold. This upregulation was further confirmed at the protein level through flow cytometry, following IFN- a stimulation. Given these results, we investigated whether patients with mastocytosis have elevated interferon levels. We analyzed plasma samples from 21 patients using the SIMOA assay and observed significantly higher IFN-a levels in most patients compared to controls. Some patients displayed elevated levels of IFN-P and / or IFN-y.
[0053] AXL WT and AXL L197M enhance proliferation of ROSA A7TD816V but not ROSA KIT WT cells
[0054] To assess the potential role of wild-type AXL and the mutated 4A7.-L I 97M on mast cell proliferation, we generated lentiviral vectors expressing 4 7.-WT-tdTomato, 4A7.-L I 97M- tdTomato, or tdTomato alone (Ctrl) and transduced ROSA cell lines. Proliferation kinetics revealed no effect of AXL expression on ROSA A77-WT cells but showed a significant increase in ROSA A77-D8 I 6V cell proliferation with both AYL-WT and AXL-L 97M compared to controls. Similar results were observed in bone marrow-derived mast cells expressing KIT- D814V (BMMC-A77-D8 I4V), confirming increased proliferation with both AXL forms compared to controls. These results support the oncogenic cooperation between A77-D8 I 6V and both AYL-WT and AYL-L197M. The expression of AXL-WT and AXL-L197M, in transduced cells, was confirmed by immunoblotting. We assessed the impact of AYL-WT and AYL-L197M on colony formation in ROSA 7 -D816V cells. Both resulted in a significant enhancement of colony numbers, with 2.5-fold and 2-fold increases, respectively, compared to control cells. To investigate the molecular mechanisms driving the increased proliferation of ROSA KIT D816V cells in the presence of AYL-WT and AYL-L197M compared to controls, we performed immunoblotting. The analysis revealed a significant upregulation of pFAK and a notable trend toward elevated pSTAT3 levels, while total FAK and STAT3 levels remained unchanged.
[0055] Co-expression of XL-WT or AXL-L197M with KIT D816V in mast cells confers resistance to TKIs.
[0056] Given AXL’s role in inducing resistance to tyrosine kinase inhibitors in various tumors, including CML and AML (PMID: 23684620, PMID: 18502572, PMID: 22141136), we aimed to determine whether it could confer similar effects in mast cell lines treated with midostaurin (PKC412) one drug approved for mastocytosis treatment. Our results show that AYL-WT or AYL-L197M significantly diminished the inhibitory effects of PKC412 (200 nM) on ROSA A77-D8 I 6V cell proliferation, whereas control cells exhibited growth arrest at these drug concentrations.
[0057] We further evaluated cell resistance to death following 96 hours of treatment. In the presence of DMSO (drug diluent), cells expressing AXL-WT or AXL-L197M showed a similar percentage of dead cells as control cells (~2% vs. ~5%, respectively). Notably, treatment with 200 nM PKC412 conferred significant resistance to death in cells expressing AXL-WT or AXL- L197M, with ~7% cell death compared to -18% in control cells. To investigate the difference in cell mortality observed under treatment, we analyzed the expression of anti-apoptotic factors through immunoblotting. Both the wild-type and mutant forms of AXL were associated with elevated levels of BCL-XL, BCL2, and MCL1 compared to control cells. Treatment with PKC412 reduced BCL-XL expression in all conditions. Despite this reduction, BCL-XL levels in AXL-expressing cells remained elevated, comparable to untreated control cells. Notably, BCL2 and MCL1 levels were consistently upregulated in all cells, and treatment with either tyrosine kinase inhibitors did not reduce their expression; instead, levels tended to increase compared to treated control cells. We investigated the potential involvement of Caspase 3 in the observed effects. Immunoblot analyses revealed increased pro-Caspase 3 levels in cells expressing either AXL-WT or AXL-L197M, irrespective of TKI treatment. However, the cleaved Caspase 3 / pro-Caspase 3 ratio was significantly reduced in AXL-WT or AXL-L197M cells, with a 2- to 4-fold decrease compared to control cells. Notably, PKC412 treatment led to a two-fold increase in the cleaved Caspase 3 / pro-Caspase 3 ratio across all conditions, independent of AXL expression.
[0058] Combined PKC 412 and R428 treatment significantly reduces the viability of AXL- expressing cells in vitro compared to monotherapy:
[0059] Cell viability was assessed following treatment with 200 nM PKC 412, 500 nM R428, their combination, or DMSO as a control. Viability was quantified using the WST-8 assay, with absorbance recorded at 450 nm. The graphs present the results for all conditions after 7 days of treatment. In cells treated with 200 nM PKC 412, 20% of control cells remained viable, compared to 50% and 45% of AXL-WT and AXL-L197M cells, respectively. Treatment with 500 nM R428 resulted in 50% viability in control cells, and increased to 80% and 90% in AXL- WT and AXL-L197M cells, respectively. Notably, the combination of both treatments reduced cell viability to 20% across all conditions, highlighting its efficiency in inducing 80% of cell death.
[0060] Mast cells harboring the KIT D816V mutation and expressing AXL exhibit significant upregulation of anti-apoptotic factors.
[0061] Our previous findings demonstrated that BCL-2 expression remains unchanged in advanced systemic mastocytosis (ASM) patients treated with Midostaurin, as shown by immunohistochemistry staining (Canioni et al., ASH 2019). We now show that mast cells harboring the KIT D816V mutation and expressing AXL exhibit significant upregulation of the anti-apoptotic factors BCL-2, BCL-XL, and MCL-1, and that Midostaurin is unable to decrease this expression. These findings underscore the limitations of Midostaurin as a standalone therapy and strongly support the potential of combination treatments targeting AXL (±KIT) alongside BCL-2 inhibitors, such as Venetoclax, as a promising strategy for treating mastocytosis. R428 alone triggers neoplastic MC death in an MCL patient
[0062] We had the rare opportunity to analyze a fresh peripheral blood sample from a mast cell leukemia (MCL3) patient with 27% circulating MCs carrying KIT F522C, SF3B1 and TERT mutations. The patient was refractory to midostaurin (PKC412). Flow cytometry analysis revealed strong AXL expression in all MCs (Data not shown). PBMCs were immediately treated with 400 nM PKC412, 1 M R428, the combination, or DMSO (vehicule). Limited cell number availability restricted duplicate testing to days 6 and 14. MC viability was monitored by FACS for three weeks, with medium and drug renewal every three days. Consistent with clinical data, PKC412 had no effect on MCs. By contrast, R428 monotherapy induced -50% cell death by day 6, -70% by day 14, and -90 % by day 21. The combination was no more effective than R428 alone (Figure 1). These findings highlight the therapeutic potential of AXL inhibition in AdvSM patients with non-D816V KIT mutations.
[0063] Effect of the combination of R428 and Navitoclax in an MCL patient
[0064] The combination of R428 and Navitoclax induced -100% cell death by day 6 (Figure 2). Navitoclax (ABT-263) is a BH3 mimetic that inhibits multiple anti-apoptotic BCL-2 family proteins (notably BCL-2 and BCL-XL). These findings support the therapeutic potential of combined AXL and BCL-2 / Bcl-XL inhibition in mastocytosis.
[0065] Conclusion:
[0066] In conclusion, our study highlighted significant findings notably the unexpected expression of AXL in neoplastic mast cells from patients with various forms of mastocytosis. This is particularly noteworthy as AXL expression had never been investigated in mast cells, whether neoplastic or normal. This study demonstrates that AXL (WT or L197M), in cooperation with KIT D816V, promotes mast cell proliferation and resistance to therapy. Furthermore, our results suggest that combining AXL inhibitors with inhibitors targeting anti-apoptotic BCL-2 family members (BCL-2 and BCL-XL) may represent a promising therapeutic strategy for mastocytosis. In conclusion, AXL inhibition emerges as a potential therapeutic approach in mastocytosis.
[0067] 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 an mastocytosis in a patient in need thereof comprising administering to the patient a therapeutically effective combination comprising a tyrosine kinase inhibitor (TKI) and an AXL inhibitor.
2. A method of preventing resistance to TKI in a patient suffering from mastocytosis comprising administering to the patient a therapeutically effective amount of an AXL inhibitor.
3. A method of treating an mastocytosis resistant to TKI in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an AXL inhibitor.
4. A method for enhancing the potency of TKI administered to a patient suffering from an mastocytosis as part of a treatment regimen, the method comprising administering to the patient a pharmaceutically effective amount of an AXL inhibitor in combination with the TKI.
5. A method for preventing relapse of a patient suffering from an mastocytosis who was treated with a TKI comprising administering to the patient a therapeutically effective amount of an AXL inhibitor.
6. A method of overcoming resistance to TKI in a patient suffering from an mastocytosis comprising administering to the patient a therapeutically effective amount of an AXL inhibitor.
7. A method of treating an mastocytosis in a patient in need thereof comprising administering to the patient a therapeutically effective combination comprising an AXL inhibitor and a BCL-2 inhibitor.