Use of JAK inhibitors for enhancing the potency of immunotherapy in the treament of cancer

Abstract

Immunotherapy leverages the body's immune system to target cancer cells, but some tumors resist this treatment due to low infiltration of T CD8+ cells and high infiltration of regulatory T cells in the tumor microenvironment. The inventors showed that a JAK inhibitor (i.e. ruxolitinib) reshapes the tumor immune microenvironment resulting in T regulatory cells reduction and T CD8+ cells increase. Ruxolitinib-mediated immune microenvironment effects create new therapeutic opportunities by enhancing anti-PD1 treatment efficacy in cSCC and melanoma and other immunotherapy resistant cancer types such as pancreatic cancer and acute myeloid leukemia (AML). Cell-based immunotherapy response is also improved in AML. Accordingly, the present invention relates to the use of JAK inhibitors for enhancing the potency of immunotherapy.

Description

[0001] USE OF JAK INHIBITORS FOR ENHANCING THE POTENCY OF

[0002] IMMUNOTHERAPY

[0003] FIELD OF THE INVENTION:

[0004] The present invention is in the field of medicine, in particular oncology and immunology.

[0005] BACKGROUND OF THE INVENTION:

[0006] Immunotherapy represents a revolutionary avenue in the treatment of cancers, leveraging the body's immune system to target and destroy malignant cells. This therapeutic approach encompasses various modalities, including cell-based immunotherapy and the use of immune checkpoint blockers. One prominent example of cell-based immunotherapy is the application of chimeric antigen receptor T (CAR-T) cells. These genetically engineered T cells are designed to recognize and attack specific cancer cells, demonstrating remarkable efficacy in certain hematologic malignancies. Similarly, immune checkpoint blockers, such as PD-1 and CTLA-4 inhibitors, have shown significant promise in freeing the immune system from the inhibitory signals that cancers exploit to evade detection. Despite these advancements, a subset of patients remains refractory to immunotherapy, particularly those with "cold tumors" that lack substantial immune cell infiltration. Such tumors present a formidable challenge as they do not elicit a robust immune response, rendering conventional immunotherapeutic strategies less effective. Additionally, the presence of regulatory T cells (Tregs) within the tumor microenvironment can further contribute to the resistance to immunotherapy. Tregs play a critical role in maintaining immune homeostasis by suppressing excessive immune responses, but their accumulation in the tumor microenvironment can inhibit the anti-tumor activity of effector T cells, ultimately facilitating cancer progression. Addressing the mechanisms by which Tregs mediate immunotherapy resistance is essential for enhancing the efficacy of these treatments and developing new strategies to overcome this barrier.

[0007] SUMMARY OF THE INVENTION:

[0008] The present invention is defined by the claims. In particular, the present invention relates to the use of JAK inhibitors for enhancing the potency of immunotherapy.

[0009] DETAILED DESCRIPTION OF THE INVENTION: Main definitions:

[0010] As used herein, the term "patient" or “subject” refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject may be a mammal, preferably a human.

[0011] As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be 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 tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In some embodiments, the subject suffers from a cancer selected from the group consisting of Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal- like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast- ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant, Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplasia, Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, non-small cell lung cancer (NSCLC) which coexists with chronic obstructive pulmonary disease (COPD), Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath, meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastema, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute, lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T- cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, or any combination thereof.

[0012] As used herein, the term "cutaneous squamous cell carcinoma" refers to a type of cancer that begins in the squamous cells, which are thin, flat cells that make up the outer layer of the skin. This form of carcinoma is characterized by the abnormal and uncontrollable growth of these squamous cells, often resulting in lesions or tumors on the skin. Cutaneous squamous cell carcinoma can occur on any part of the body, but it is most commonly found in areas that are frequently exposed to the sun, such as the face, ears, neck, hands, and arms. Early detection and treatment are crucial, as this type of cancer can metastasize to other parts of the body if left untreated.

[0013] As used herein, the term "pancreatic cancer" refers to a malignant neoplasm originating in the tissues of the pancreas, an organ that lies behind the lower part of the stomach. Pancreatic cancer occurs when cells in the pancreas grow uncontrollably, forming a tumor that can interfere with normal pancreatic functions, such as aiding in digestion and regulating blood sugar. This type of cancer is characterized by its aggressive nature and tendency to spread rapidly to nearby organs and lymph nodes. Symptoms may include jaundice, weight loss, diabetes, and abdominal pain. Early detection is challenging, often leading to a diagnosis at advanced stages.

[0014] As used herein, the term "acute myeloid leukemia" refers to a type of cancer that originates in the bone marrow and is characterized by the rapid growth of abnormal immature white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. AML progresses quickly and can be fatal within a few months if not treated. It is most common in adults but can also occur in children. Symptoms may include fatigue, shortness of breath, easy bruising and bleeding, and increased risk of infection. Diagnosis typically involves blood tests, bone marrow examination, and genetic testing, with treatment options including chemotherapy, targeted therapy, and stem cell transplant.

[0015] As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patients 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 subject 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 subject 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., disease manifestation, etc.]).

[0016] As used herein, the term “immunotherapy” has its general meaning in the art and refers to treatments that consist of administering an immunogenic agent capable of inducing, enhancing, or otherwise modifying an immune response. This includes cell-based immunotherapy, particularly hematopoietic stem cell transplantation, CAR-T, CAR-NK, and CAR-macrophage therapies, as well as the use of immune checkpoint blockers.

[0017] As used herein, the term "immune checkpoint blocker" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. As used herein the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. , 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD- 1, LAG-3, TIM-3 and VISTA. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint blockers are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint blockers are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint blockers may be developed in the (near) future. The immune checkpoint blockers include peptides, antibodies, nucleic acid molecules and small molecules.

[0018] As used herein, the expression “enhancing the potency of immunotherapy” refers to the ability of the JAK inhibitor to enhance the therapeutic efficacy of immunotherapy against cancer cells, potentially leading to better clinical outcomes. As used herein, the expression "enhanced therapeutic efficacy," relative to cancer refers to a slowing or diminution of the growth of cancer cells or a solid tumor, or a reduction in the total number of cancer cells or total tumor burden. An "improved therapeutic outcome" or "enhanced therapeutic efficacy" therefore means there is an improvement in the condition of the patient according to any clinically acceptable criteria, including, for example, decreased tumor size, an increase in time to tumor progression, increased progression-free survival, increased overall survival time, an increase in life expectancy, a decrease of immune-adverse effects or an improvement in quality of life. In particular, "improved" or "enhanced" refers to an improvement or enhancement of 1%, 5%, 10%, 25% 50%, 75%, 100%, or greater than 100% of any clinically acceptable indicator of therapeutic outcome or efficacy. As used herein, the expression "relative to" when used in the context of comparing the activity and / or efficacy of a therapeutical combination comprising immunotherapy with a JAK inhibitor to the activity and / or efficacy of the immunotherapy alone, refers to a comparison using amounts known to be comparable according to one of skill in the art.

[0019] As used herein, the term “immunotherapy resistance” refers to the resistance of a cancer to the immune response induced by the immunotherapy. Therefore, a resistant tumor or tumor cell is more likely to escape and survive humoral and / or cellular immune defense mechanisms in a subject receiving the immunotherapy. The phrase “overcoming immunotherapy resistance” in context of the invention shall be effective if compared to a non-treated control, the tumor or tumor cell becomes more sensitive to an immune response induced by immunotherapy. In particular, the patient become a responder. As used herein the term “responder” in the context of the present disclosure refers to a patient that will achieve a response, i.e. a patient where the cancer is eradicated, reduced or improved after immunotherapy. According to the invention, the responders have an objective response and therefore the term does not encompass patients having a stabilized cancer such that the disease is not progressing after immunotherapy. A “non-responder” or “refractory patient” includes patients for whom the cancer does not show reduction or improvement after immunotherapy. The term “non responder” also includes patients having a stabilized cancer. 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. More particularly, the method of the present invention is particularly suitable for preventing tumor escape in a patient treated with immunotherapy. As used herein, the term “tumor escape” refers to any mechanism by which tumors escape the host's immune system.

[0020] As used herein, the term "regulatory T cells” or “Treg cells" refers to cells that suppress, inhibit or prevent T cells activity, in particular cytotoxic activity of T CD8+ cells. Regulatory T cells include i) thymus-derived Treg cells (tTreg, previously referred as "natural Treg cells") and ii) peripherally-derived Treg cells (pTreg, previously referred as "induced Treg cells"). tTregs have the following phenotype at rest CD4+CD25+FoxP3+. pTreg cells include, for example, Tri cells, TGF-P secreting Th3 cells, regulatory NKT cells, regulatory y5 T cells, regulatory CD8+ T cells, and double negative regulatory T cells. The term "Tri cells" as used herein refers to cells having the following phenotype at rest: CD4+CD25- CD127-, and the following phenotype when activated: CD4+CD25+ CD127-. Tri cells, Type 1 T regulatory cells (Type 1 Treg) and IL- 10 producing Treg are used herein with the same meaning.

[0021] As used herein, the term “CD8+ T cell” has its general meaning in the art and refers to a subset of T cells which express CD8 on their surface. They are MHC class I-restricted, and function as cytotoxic T cells. “CD8+ T cells” are also called cytotoxic T lymphocytes (CTL), T-killer cells, cytolytic T cells, or killer T cells. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions. As used herein, the term “tumor infiltrating CD8+ T cell” refers to the pool of CD8+ T cells of the patient that have left the blood stream and have migrated into a tumor.

[0022] As used herein, the term “tumor sample” means any tissue tumor sample derived from the patient. Said tissue sample is obtained for the purpose of the ex vivo evaluation. In some embodiments, the tumor sample may result from the tumor resected from the patient. In some embodiments, the tumor sample may result from a biopsy performed in the primary tumor of the patient or performed in metastatic sample distant from the primary tumor of the patient. For example, an endoscopical biopsy performed in the bowel of the patient affected by a colorectal cancer. In some embodiments, the tumor sample encompasses (i) a global primary tumor (as a whole), (ii) a tissue sample from the center of the tumor, (iii) a tissue sample from the tissue directly surrounding the tumor which tissue may be more specifically named the “invasive margin” of the tumor, (iv) lymphoid islets in close proximity with the tumor, (v) the lymph nodes located at the closest proximity of the tumor, (vi) a tumor sample collected prior surgery (for follow-up of patients after treatment for example), and (vii) a distant metastasis. As used herein the “invasive margin” has its general meaning in the art and refers to the cellular environment surrounding the tumor. In some embodiments, the tumor sample, irrespective of whether it is derived from the center of the tumor, from the invasive margin of the tumor, or from the closest lymph nodes, encompasses pieces or slices of tissue that have been removed from the tumor center or from the invasive margin surrounding the tumor, including following a surgical tumor resection or following the collection of a tissue sample for biopsy, for further quantification of one or several biological markers, notably through histology or immunohistochemistry methods, through flow cytometry methods and through methods of gene or protein expression analysis, including genomic and proteomic analysis. The tumor sample can, of course, be submitted to a variety of well-known post-collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.). The sample can be fresh, frozen, fixed (e.g., formalin fixed), or embedded (e.g., paraffin embedded).

[0023] As used herein the term “ JAK” has its general meaning in the art and refers to the family of Janus kinases (JAKs) which are cytoplasmic tyrosine kinases that transduce cytokine signaling from membrane receptors to STAT transcription factors. Four JAK family members are described, JAK1, JAK2, JAK3 and TYK2 and the term JAK may refer to all the JAK family members collectively or one or more of the JAK family members as the context indicates.

[0024] As used herein the term “JAK inhibitor” is intended to mean compounds inhibiting the activity or expression of at least one JAK. In some embodiments, the JAK inhibitor is a JAK1 inhibitor. In some embodiments, the JAK inhibitor is a JAK2 inhibitor. In some embodiments, the JAK inhibitor is a JAK3 inhibitor. JAK inhibitors down-regulate the quantity or activity of JAK molecules. One activity of JAK proteins is to phosphorylate a STAT protein. Therefore, an example of an effect of a JAK inhibitor is to decrease the phosphorylation of one or more STAT proteins. The inhibitor may inhibit the phosphorylated form of JAK or the nonphosphorylated form of JAK. In some embodiments, the JAK inhibitor is a selective JAK2 inhibitor. By “selective” is meant that the compound binds to or inhibits JAK2 with greater affinity or potency, respectively, compared to at least one other JAK (e.g., JAK1, JAK3 and / or TYK2). Selectivity can be at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold or at least about 1000-fold. Selectivity can be measured by methods routine in the art. In some embodiments, selectivity can be tested at the Km of each enzyme. In some embodiments, selectivity of compounds for JAK2 can be determined by the cellular ATP concentration.

[0025] In some embodiments, the JAK inhibitor is a selective JAK1 inhibitor. By “selective” is meant that the compound binds to or inhibits JAK1 with greater affinity or potency, respectively, compared to at least one other JAK (e.g., JAK2, JAK3 and / or TYK2). Selectivity can be at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold or at least about 1000- fold. Selectivity can be measured by methods routine in the art. In some embodiments, selectivity can be tested at the Km of each enzyme. In some embodiments, selectivity of compounds for JAK1 can be determined by the cellular ATP concentration.

[0026] As used herein, the term "therapeutically effective amount" is meant a sufficient amount of the drug for treating or reducing the symptoms at reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day. Methods of the present invention:

[0027] An object of the present invention relates to a method for enhancing the potency of immunotherapy administered to a patient as part of a treatment regimen for cancer, the method comprising: administering a pharmaceutically effective amount of a JAK inhibitor to the patient in combination with immunotherapy.

[0028] A further object of the present invention relates to a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective combination of immunotherapy with a JAK inhibitor, wherein administration of the combination results in enhanced therapeutic efficacy relative to the administration of immunotherapy alone.

[0029] A further object of the present invention relates to a method of overcoming immunotherapy resistance in patient suffering from cancer thereof comprising administering to the patient a therapeutically effective amount of a JAK inhibitor.

[0030] Cancers:

[0031] In some embodiments, the patient suffers from a cutaneous squamous cell carcinoma, a melanoma, a pancreatic cancer or an acute myeloid leukemia.

[0032] In some embodiments, the patient does not suffer from a Hodgking Lymphoma or nonsmall cell lung cancer.

[0033] In particular, the method of the present invention is particularly suitable for the treatment of cancer characterized by a high tumor infiltration of Treg cells. More particularly, the method of the present invention is suitable for the treatment of cancer characterized by a low tumor infiltration of CD8+ T cells and a high tumor infiltration of Treg cells.

[0034] Accordingly a further object of the present invention relates to a method of treating cancer in a patient in need thereof comprising i) quantifying the density of Treg cells in a tumor sample obtained from the patient ii) comparing the density quantified at step i) with a predetermined reference value and iii) administering to the patient a therapeutically effective combination of immunotherapy with a JAK inhibitor when the density determined at step i) is higher than the predetermined reference value.

[0035] A further object of the present invention relates to a method of treating cancer in a patient in need thereof comprising i) quantifying the density of Treg cells and CD8+ T cells in a tumor sample obtained from the patient ii) comparing the densities quantified at step i) with their predetermined reference values and iii) administering to the patient a therapeutically effective combination of immunotherapy with a JAK inhibitor when the density for Tregs quantified at step i) is higher than its corresponding predetermined reference value and / or the density quantified for CD8+ T cells quantified at step i) lower that its corresponding predetermined reference value.

[0036] The tumor infiltration of Treg cells and CD8+ T cells is determined by any convention method in the art. For example, said determination comprises quantifying the density of Treg cells and / or CD8+ T cells in a tumor sample obtained from the patient.

[0037] In some embodiments, the quantification of density of Treg cells and / or CD8+ T cells is determined by any method well known in the art. Typically, the quantification of density of Treg cells and / or CD8+ T cells can be determined by any method well known in the art, such as an immunoassay like immunohistochemistry (IHC) or flow cytometry. For example, the quantification of the density of Treg cells and / or CD8+ T cells is performed by contacting the tumor sample with a binding partner (e.g. an antibody) specific for a cell surface marker of said cells. Typically, the quantification of density of Treg cells is performed by contacting the tumor sample with one or more binding partners (e.g. an antibodies) specific for CD4, CD25 and / or FoxP3. Typically, the quantification of density of Treg cells and / or CD8+ T cells is performed by contacting the tumor sample with a binding partner (e.g. an antibody) specific for CD8. Typically, the density of Treg cells and / or CD8+ T cells can expressed as the number of these cells that are counted per one unit of volume or surface area of tissue sample, e.g. as the number of cells that are counted per cm2or mm2of surface area of tumor sample. In some embodiments, the density of cells may also be expressed as the number of cells per one volume unit of sample, e.g. as the number of cells per cm3of tumor sample. In some embodiments, the density of cells may also consist of the percentage of the specific cells per total cells (set at 100%).

[0038] Immunohistochemistry typically includes the following steps i) fixing the tumor sample with formalin, ii) embedding said tumor sample in paraffin, iii) cutting said tumor sample into sections for staining, iv) incubating said sections with the binding partner specific for the marker, v) rinsing said sections, vi) incubating said section with a secondary antibody typically biotinylated and vii) revealing the antigen-antibody complex typically with avidin-biotin- peroxidase complex. Accordingly, the tumor sample is firstly incubated with the binding partners. After washing, the labeled antibodies that are bound to marker of interest are revealed by the appropriate technique, depending of the kind of label is borne by the labeled antibody, e.g. radioactive, fluorescent or enzyme label. Multiple labelling can be performed simultaneously. Alternatively, the method of the present invention may use a secondary antibody coupled to an amplification system (to intensify staining signal) and enzymatic molecules. Such coupled secondary antibodies are commercially available, e.g. from Dako, EnVision system. Counterstaining may be used, e.g. H&E, DAPI, Hoechst. Other staining methods may be accomplished using any suitable method or system as would be apparent to one of skill in the art, including automated, semi-automated or manual systems. For example, one or more labels can be attached to the antibody, thereby permitting detection of the target protein (i.e the marker). Exemplary labels include radioactive isotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof. In some embodiments, the label is a quantum dot. Non-limiting examples of labels that can be conjugated to primary and / or secondary affinity ligands include fluorescent dyes or metals (e.g. fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g. rhodopsin), chemiluminescent compounds (e.g. luminal, imidazole) and bioluminescent proteins (e.g. luciferin, luciferase), haptens (e.g. biotin). A variety of other useful fluorescers and chromophores are described in Stryer L (1968) Science 162:526-533 and Brand L and Gohlke JR (1972) Annu. Rev. Biochem. 41 :843-868. Affinity ligands can also be labeled with enzymes (e.g. horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g.3H,14C,32P,35S or125I) and particles (e.g. gold). The different types of labels can be conjugated to an affinity ligand using various chemistries, e.g. the amine reaction or the thiol reaction. However, other reactive groups than amines and thiols can be used, e.g. aldehydes, carboxylic acids and glutamine. Various enzymatic staining methods are known in the art for detecting a protein of interest. For example, enzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC or Fast Red. In other examples, the antibody can be conjugated to peptides or proteins that can be detected via a labeled binding partner or antibody. In an indirect IHC assay, a secondary antibody or second binding partner is necessary to detect the binding of the first binding partner, as it is not labeled.

[0039] In particular, the resulting stained specimens can be imaged using a system for viewing the detectable signal and acquiring an image, such as a digital image of the staining. Methods for image acquisition are well known to one of skill in the art. For example, once the sample has been stained, any optical or non-optical imaging device can be used to detect the stain or biomarker label, such as, for example, upright or inverted optical microscopes, scanning confocal microscopes, cameras, scanning or tunneling electron microscopes, canning probe microscopes and imaging infrared detectors. In some examples, the image can be captured digitally. The obtained images can then be used for quantitatively or semi-quantitatively determining the amount of the marker in the sample. Various automated sample processing, scanning and analysis systems suitable for use with immunohistochemistry are available in the art. Such systems can include automated staining and microscopic scanning, computerized image analysis, serial section comparison (to control for variation in the orientation and size of a sample), digital report generation, and archiving and tracking of samples (such as slides on which tissue sections are placed). Cellular imaging systems are commercially available that combine conventional light microscopes with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples. See, e.g., the CAS- 200 system (Becton, Dickinson & Co.). In particular, detection can be made manually or by image processing techniques involving computer processors and software. Using such software, for example, the images can be configured, calibrated, standardized and / or validated based on factors including, for example, stain quality or stain intensity, using procedures known to one of skill in the art (see e.g., published U.S. Patent Publication No. US20100136549). The image can be quantitatively or semi-quantitatively analyzed and scored based on staining intensity of the sample. Quantitative or semi-quantitative histochemistry refers to method of scanning and scoring samples that have undergone histochemistry, to identify and quantitate the presence of the specified biomarker (i.e. the marker). Quantitative or semi -quantitative methods can employ imaging software to detect staining densities or amount of staining or methods of detecting staining by the human eye, where a trained operator ranks results numerically. For example, images can be quantitatively analyzed using a pixel count algorithms (e.g., Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUA® platform), and other standard methods that measure or quantitate or semi-quantitate the degree of staining; see e.g., U.S. Pat. No. 8,023,714; U.S. Pat. No. 7,257,268; U.S. Pat. No. 7,219,016; U.S. Pat. No. 7,646,905; published U.S. Patent Publication No. US20100136549 and 20110111435; Camp et al. (2002) Nature Medicine, 8: 1323-1327; Bacus et al. (1997) Analyt Quant Cytol Histol, 19:316-328). A ratio of strong positive stain (such as brown stain) to the sum of total stained area can be calculated and scored. The amount of the detected biomarker (i.e. the marker) is quantified and given as a percentage of positive pixels and / or a score. For example, the amount can be quantified as a percentage of positive pixels. In some examples, the amount is quantified as the percentage of area stained, e.g., the percentage of positive pixels. For example, a sample can have at least or about at least or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,

[0040] 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,

[0041] 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,

[0042] 80%, 85%, 90%, 95% or more positive pixels as compared to the total staining area. In some embodiments, a score is given to the sample that is a numerical representation of the intensity or amount of the histochemical staining of the sample, and represents the amount of target biomarker (e.g., the marker) present in the sample. Optical density or percentage area values can be given a scaled score, for example on an integer scale. Thus, in some embodiments, the method of the present invention comprises the steps consisting in i) providing one or more immunostained slices of tissue section obtained by an automated slide-staining system by using a binding partner capable of selectively interacting with the marker (e.g. an antibody as above described), ii) proceeding to digitalisation of the slides of step a. by high resolution scan capture, iii) detecting the slice of tissue section on the digital picture iv) providing a size reference grid with uniformly distributed units having a same surface, said grid being adapted to the size of the tissue section to be analyzed, and v) detecting, quantifying and measuring intensity of stained cells in each unit whereby the number or the density of cells stained of each unit is assessed.

[0043] In some embodiments, the cell density of Treg cells and / or CD8+ T cells is determined in the whole tumor sample, is determined in the invasive margin or centre of the tumor sample or is determined both in the centre and the invasive margin of the tumor sample.

[0044] In some embodiments, the predetermined value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of cell densities in properly banked historical patient samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after quantifying the density of Treg cells and / or CD8+ T cells in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured densities in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1- specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0045] Immunotherapy:

[0046] In some embodiments, the immunotherapy is a cell-based immunotherapy. An example of cell-based immunotherapy is hematopoietic stem cell transplantation (HSCT), which typically consists in injecting allogeneic hematopoietic cells to generate an immune response against cancer cells. Cell-based immunotherapy can also involve the genetic modification of a patient's own immune cells to enhance their ability to recognize and attack cancer cells. Among the most prominent forms of cell-based immunotherapy are Chimeric Antigen Receptor (CAR) therapies, which include CAR-T cells, CAR-NK cells, and CAR macrophages. CAR-T cell therapy, perhaps the most well-known, involves engineering T cells to express receptors specific to antigens on the surface of cancer cells, thus directing a potent cytotoxic response. CAR-NK cells, on the other hand, utilize natural killer cells, which provide a rapid and robust attack against cancerous targets, potentially with fewer side effects. CAR macrophages are another innovative approach, modifying macrophages to not only clear cancer cells but also to alter the tumor microenvironment to promote anti-tumor immunity. These therapies are part of a broader movement towards personalized medicine, aiming to tailor treatment strategies to the unique genetic and molecular profile of each patient's cancer.

[0047] In some embodiments, the immunotherapy consists in administering the patient with at least one immune checkpoint blocker. Examples of immune checkpoint blocker includes PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist CTLA-4 antagonist, VISTA antagonist, TIM- 3 antagonist, LAG-3 antagonist, IDO antagonist, KIR2D antagonist, A2AR antagonist, B7-H3 antagonist, B7-H4 antagonist, and BTLA antagonist. In some embodiments, PD-1 (Programmed Death-1) axis antagonists include PD-1 antagonist (for example anti-PD-1 antibody), PD-L1 (Programmed Death Ligand-1) antagonist (for example anti-PD-Ll antibody) and PD-L2 (Programmed Death Ligand-2) antagonist (for example anti-PD-L2 antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, Keytruda®, and SCH-900475), CT-011 (also known as Pidilizumab, hBAT, and hBAT-1), and Cemiplimab (also known as, Libtayo®). In some embodiments, the PD-1 binding antagonist is AMP -224 (also known as B7-DCIg). In some embodiments, the anti-PD- Ll antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX- 1105, and MEDI4736. MDX-1105, also known as BMS-936559, is an anti-PD-Ll antibody described in W02007 / 005874. Antibody YW243.55. S70 is an anti-PD-Ll described in WO 2010 / 077634 AL MEDI4736 is an anti-PD-Ll antibody described in WO2011 / 066389 and US2013 / 034559. MDX-1106, also known as MDX-1106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in U.S. Pat. No. 8,008,449 and W02006 / 121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in U.S. Pat. No. 8,345,509 and W02009 / 114335. CT-011 (Pidizilumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PD- L2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Atezolimumab is an anti-PD-Ll antibody described in U.S. Pat. No. 8,217,149. Avelumab is an anti-PD-Ll antibody described in US 20140341917. CA-170 is a PD-1 antagonist described in W02015033301 & WO2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and / or US 20120114649. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab. In some embodiments, PD-L1 antagonist is selected from the group comprising of Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, A110, KY1003 and Atezolimumab and the preferred one is Avelumab, Durvalumab or Atezolimumab.

[0048] In some embodiments, CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) antagonists are selected from the group consisting of anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA- 4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (Ipilimumab), Tremelimumab, anti-CD28 antibodies, anti-CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA- 4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors of CTLA-4 that agonize the co-stimulatory pathway, the antibodies disclosed in PCT Publication No. WO 2001 / 014424, the antibodies disclosed in PCT Publication No. WO 2004 / 035607, the antibodies disclosed in U.S. Publication No. 2005 / 0201994, and the antibodies disclosed in granted European Patent No. EP 1212422 B. Additional CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; in PCT Publication Nos. WO 01 / 14424 and WO 00 / 37504; and in U.S. Publication Nos. 2002 / 0039581 and 2002 / 086014. Other anti-CTLA-4 antibodies that can be used in a method of the present invention include, for example, those disclosed in: WO 98 / 42752; U.S. Pat. Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., J. Clin: Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP- 675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281. A preferred clinical CTLA-4 antibody is human monoclonal antibody (also referred to as MDX-010 and Ipilimumab with CAS No. 477202-00- 9 and available from Medarex, Inc., Bloomsbury, N.J.) is disclosed in WO 01 / 14424. With regard to CTLA-4 antagonist (antibodies), these are known and include Tremelimumab (CP- 675,206) and Ipilimumab.

[0049] In some embodiments, the immunotherapy consists in administering to the patient a combination of a CTLA-4 antagonist and a PD-1 antagonist.

[0050] Other immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211). Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM-3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94).

[0051] In particular, the immune checkpoint blocker of the present invention is administered for enhancing the proliferation, migration, persistence and / or cytotoxic activity of CD8+ T cells in the subject and in particular the tumor-infiltrating of CD8+ T cells of the subject. For instance, the ability of the immune checkpoint blocker to enhance CD8+ T cell killing activity may be determined by any assay well known in the art. Typically said assay is an in vitro assay wherein CD8+ T cells are brought into contact with target cells (e.g. target cells that are recognized and / or lysed by CD8+ T cells). For example, the immune checkpoint blocker of the present invention can be selected for the ability to increase specific lysis by CD8+ T cells by more than about 20%, preferably with at least about 30%, at least about 40%, at least about 50%, or more of the specific lysis obtained at the same effector: target cell ratio with CD8+ T cells or CD8 T cell lines that are contacted by the immune checkpoint blocker of the present invention. Examples of protocols for classical cytotoxicity assays are conventional.

[0052] JAK inhibitors:

[0053] JAK inhibitors are well known in the art. For example, JAK inhibitors include phenylaminopyrimidine compounds (W02009 / 029998), substituted tricyclic heteroaryl compounds (W02008 / 079965), cyclopentyl-propanenitrile compounds (W02008 / 157208 and W02008 / 157207), indazole derivative compounds (W02008 / 114812), substituted ammo- thiophene carboxylic acid amide compounds (W02008 / 156726), naphthyridine derivative compounds (W02008 / 112217), quinoxaline derivative compounds (WO2008 / 148867), pyrrolopyrimidine derivative compounds (W02008 / 119792), purinone and imidazopyridinone derivative compounds (W02008 / 060301 ), 2,4-pyrimidinediamine derivative compounds (W02008 / 118823), deazapurine compounds (W02007 / 117494) and tricyclic heteroaryl compounds (W02008 / 079521). Examples of JAK inhibitors include compounds disclosed in the following publications: US2004 / 176601, US2004 / 038992, US2007 / 135466, US2004 / 102455, W02009 / 054941, US2007 / 134259, US2004 / 265963, US2008 / 194603,

[0054] US2007 / 207995, US2008 / 260754, US2006 / 063756, US2008 / 261973, US2007 / 142402, US2005 / 159385, US2006 / 293361, US2004 / 205835, WO2008 / 148867, US2008 / 207613, US2008 / 279867, US2004 / 09799, US2002 / 055514, US2003 / 236244, US2004 / 097504, US2004 / 147507, US2004 / 176271, US2006 / 217379, US2008 / 092199, US2007 / 043063, US2008 / 021013, US2004 / 152625, W02008 / 079521, US2009 / 186815, US2007 / 203142, W02008 / 144011, US2006 / 270694 and US2001 / 044442. JAK inhibitors further include compounds disclosed in the following publications: W02003 / 011285, WO2007 / 145957, W02008 / 156726, W02009 / 035575, W02009 / 054941, and W02009 / 075830. JAK inhibitors further include compounds disclosed in the following patent applications: US Serial Nos. 61 / 137475 and 61 / 134338.

[0055] Specific JAK inhibitors include AG490, AUB-6-96, AZ960, AZD1480, baricitinib (LY3009104, INCB28050), BMS-911543, CEP-701, CMP6, CP352,664, CP690,550, momelotinib (CYT-387), INCB20, Jak2-IA, lestaurtinib (CEP-701), LS104, LY2784544, NS018, pacritinib (SB1518), Pyridone 6, ruxolitinib (INCB018424), SB1518, TG101209, fedratinib (TG101348, SAR302503), TG101348, tofacitinib (CP-690,550), WHI-PI 54, WP1066, XL019, and XLOI 9. Ruxolitinib (Jakafi™, INCB018424; (3R)-3-cyclopentyl-3-[4- (7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-l-yl]propanenitrile) is a potent, orally available, selective inhibitor of both JAK1 and JAK2 of the JAK-STAT signaling pathway. CYT387 is an inhibitor of Janus kinases JAK1 and JAK2, acting as an ATP competitor with IC50 values of 11 and 18 nM, respectively. TG101348 (SAR302503) is an orally available inhibitor of Janus kinase 2 (JAK-2). TGI 01348 acts as a competitive inhibitor of protein kinase JAK-2 with IC50=6 nM; related kinases FLT3 and RET are also sensitive, with IC50=25 nM and IC50=17 nM, respectively. AZD1480 is an orally bioavailable inhibitor of Janus-associated kinase 2 (JAK2) with potential antineoplastic activity. JAK2 inhibitor AZD 1480 inhibits JAK2 activation, leading to the inhibition of the JAK / STAT (signal transducer and activator of transcription) signaling including activation of STAT3. Lestaurtinib (CEP-701) is a tyrosine kinase inhibitor structurally related to staurosporine. Pacritinib (SB 1815) is an orally bioavailable inhibitor of JAK2 and the JAK2 mutant JAK2V617F. Pacritinib competes with JAK2 for ATP binding, which may result in inhibition of JAK2 activation, inhibition of the JAK-STAT signaling pathway, and therefore caspase-dependent apoptosis. Baricitinib (LY3009104, INCB28050) is an orally bioavailable inhibitor of JAK1 and JAK2 with IC50=5.9 nm and IC50=5.7, nm respectively. Baricitinib preferentially inhibits JAK1 and JAK2, with 10- fold selectivity over Tyk2 and 100-fold over JAK3. XL019 is an orally bioavailable inhibitor of Janus-associated kinase 2 (JAK2). XL019 inhibits the activation of JAK2 as well as the mutated form JAK2V617F. NS018 is a potent JAK2 inhibitor with some inhibition of Src- family kinases. NS018 has been shown to be highly active against JAK2 with a 50% inhibition (IC50) of <1 nM, and had 30-50-fold greater selectivity for JAK2 over other JAK-family kinases.

[0056] Selective JAK2 inhibitors are well known in the art and are typically described in the following publications:

[0057] - 1 : Lin TE, HuangFu WC, Chao MW, Sung TY, Chang CD, Chen YY, Hsieh JH, Tu HJ, Huang HL, Pan SL, Hsu KC. A Novel Selective JAK2 Inhibitor Identified Using Pharmacological Interactions. Front Pharmacol. 2018 Dec 4;9: 1379. doi: 10.3389 / fphar.2018.01379. eCollection 2018. PubMed PMID: 30564118; PubMed Central PMCID: PMC6288363.

[0058] 2: Wan H, Schroeder GM, Hart AC, Inghrim J, Grebinski J, Tokarski JS, Lorenzi MV, You D, Mcdevitt T, Penhallow B, Vuppugalla R, Zhang Y, Gu X, Iyer R, Lombardo LJ, Trainor GL, Ruepp S, Lippy J, Blat Y, Sack JS, Khan JA, Stefanski K, Sleczka B, Mathur A, Sun JH, Wong MK, Wu DR, Li P, Gupta A, Arunachalam PN, Pragalathan B, Narayanan S, K C N, Kuppusamy P, Purandare AV. Discovery of a Highly Selective JAK2 Inhibitor, BMS-911543, for the Treatment of Myeloproliferative Neoplasms. ACS Med Chem Lett. 2015 Jul 12;6(8):850-5. doi: 10.1021 / acsmedchemlett.5b00226. eCollection 2015 Aug 13. PubMed PMID: 26288683; PubMed Central PMCID: PMC4538448.

[0059] 3: Kuroda J, Kodama A, Chinen Y, Shimura Y, Mizutani S, Nagoshi H, Kobayashi T, Matsumoto Y, Nakaya Y, Tamura A, Kobayashi Y, Naito H, Taniwaki M. NS-018, a selective JAK2 inhibitor, preferentially inhibits CFU- GM colony formation by bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients. Leuk Res. 2014 May;38(5):619-24. doi: 10.1016 / j.leukres.2014.03.001. Epub 2014 Mar 11. PubMed PMID: 24679585.

[0060] 4: Verstovsek S, Tam CS, Wadleigh M, Sokol L, Smith CC, Bui LA, Song C, Clary DO, Olszynski P, Cortes J, Kantarjian H, Shah NP. Phase I evaluation of XL019, an oral, potent, and selective JAK2 inhibitor. Leuk Res. 2014 Mar;38(3):316-22. doi: 10.1016 / j.leukres.2013.12.006. Epub 2013 Dec 11. PubMed PMID: 24374145; PubMed Central PMCID: PMC4414320.

[0061] 5: Dugan BJ, Gingrich DE, Mesaros EF, Milkiewicz KL, Curry MA, Zulli AL, Dobrzanski P, Serdikoff C, Jan M, Angeles TS, Albom MS, Mason JL, Aimone LD, Meyer SL, Huang Z, Wells-Knecht KJ, Ator MA, Ruggeri BA, Dorsey BD. A selective, orally bioavailable l,2,4-triazolo[l,5-a]pyridine-based inhibitor of Janus kinase 2 for use in anticancer therapy: discovery of CEP-33779. J Med Chem. 2012 Jun 14;55(11):5243-54. doi: 10.1021 / jm300248q. Epub 2012 May 18. PubMed PMID: 22594690.

[0062] 6: Seavey MM, Lu LD, Stump KL, Wallace NH, Hockeimer W, O'Kane TM, Ruggeri BA, Dobrzanski P. Therapeutic efficacy of CEP-33779, a novel selective JAK2 inhibitor, in a mouse model of colitis-induced colorectal cancer. Mol Cancer Ther. 2012 Apr; 11(4): 984-93. doi: 10.1158 / 1535-7163.MCT-11- 0951. Epub 2012 Feb 14. PubMed PMID: 22334590.

[0063] 7: Purandare AV, McDevitt TM, Wan H, You D, Penhallow B, Han X, Vuppugalla R, Zhang Y, Ruepp SU, Trainor GL, Lombardo L, Pedicord D, Gottardis MM, Ross-Macdonald P, de Silva H, Hosbach J, Emanuel SL, Blat Y, Fitzpatrick E, Taylor TL, McIntyre KW, Michaud E, Mulligan C, Lee FY, Woolfson A, Lasho TL, Pardanani A, Tefferi A, Lorenzi MV. Characterization of BMS-911543, a functionally selective small-molecule inhibitor of JAK2. Leukemia. 2012 Feb;26(2):280-8. doi: 10.1038 / leu.2011.292. Epub 2011 Oct 21. PubMed PMID:22015772.

[0064] 8: Shide K, Kameda T, Markovtsov V, Shimoda HK, Tonkin E, Fang S, Liu C, Gelman M, Lang W, Romero J, McLaughlin J, Bhamidipati S, Clough J, Low C, Reitsma A, Siu S, Pine P, Park G, Torneros A, Duan M, Singh R, Payan DG, Matsunaga T, Hitoshi Y, Shimoda K. R723, a selective JAK2 inhibitor, effectively treats JAK2V617F-induced murine myeloproliferative neoplasm. Blood. 2011 Jun 23;117(25):6866-75. doi: 10.1182 / blood-2010-01-262535. Epub 2011 Apr 29. PubMed PMID: 21531978.

[0065] 9: Pardanani A, Gotlib JR, Jamieson C, Cortes JE, Talpaz M, Stone RM, Silverman MH, Gilliland DG, Shorr J, Tefferi A. Safety and efficacy of TG101348, a selective JAK2 inhibitor, in myelofibrosis. J Clin Oncol. 2011 Mar l;29(7):789-96. doi: 10.1200 / JC0.2010.32.8021. Epub 2011 Jan 10. PubMed PMID: 21220608; PubMed Central PMCID: PMC4979099.

[0066] In some embodiments, the selective JAK2 inhibitor is selected from the group consisting of Ruxolitinib, Fedratinib, Gandotinib, Lestaurtinib, Momelotinib and Pacritinib.

[0067] In some embodiments, the selective JAK1 inhibitor is selected from the group consisting of Ruxolitinib, Oclacitinib (APOQUEL®), Filgotinib (Jyseleca®), Golidocitinib and Itacitinib. In some embodiments, the JAK inhibitor is an inhibitor of expression of JAK. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In some embodiments, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. Gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that JAK2 gene expression or JAK1 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cell. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0068] Typically, the JAK inhibitor is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semisolid 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.

[0069] 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.

[0070] FIGURES:

[0071] Figure 1. Ruxolitinib treatment reshapes the tumor immune microenvironment resulting in Tregs reduction and T CD8+cells increase. (A) Quantification of cutaneous carcinoma Tumor Infiltrating Lymphocytes (TILs) in control (n=5 tumors) versus ruxolitinib (n=l 1 tumors) treated mice. High (scores of 2 and 3) or low (scores of 0 and 1) percentage of TILs infiltration in the intra-tumoral and peritumoral areas are represented in white and black respectively. (B) CD8+T cells proportion within cutaneous carcinomas from control (n=5) versus ruxolitinib (n=5) treated mice. Statistical significance determined using Mann- Whitney test. Error bars represent the mean of five mice per group ± SEM. * p<0.05. (C) CD8+T cells number relative to lymphocytes monoculture DMSO condition, at day 3 of DMSO or ruxolitinib treatment, in lymphocytes monoculture, lymphocytes / cSCC (cutaneous squamous cell carcinoma) co-culture or lymphocytes monoculture within supernatant obtained from cSCC culture. Statistical significance determined using Mann-Whitney test in comparison to (*) DMSO lymphocytes monoculture or (#) DMSO co-culture / supernatant conditions. Error bars represent the mean of four technical replicates ± SD. Presented data is representative of two biological replicates. * p<0.05, # p<0.05 (D) Tregs (CD4+CD25+Foxp3+) number relative to lymphocytes monoculture DMSO condition, at day 3 of DMSO or ruxolitinib treatment, in lymphocytes monoculture and lymphocytes / cSCC co-culture conditions. Statistical significance determined using Mann-Whitney test in comparison to (*) DMSO lymphocytes monoculture or (#) DMSO co-culture conditions. Error bars represent the mean of four technical replicates ± SD. Presented data is representative of two biological replicates. * p<0.05, # p<0.05. (E) Quantification of Foxp3 positive area in aggressive cSCC developed by ruxolitinib treated MPN patients (n=5) in comparison to matched spontaneously developed cSCC (n=5). Statistical significance determined using Mann-Whitney test in comparison to spontaneously developed cSCC. Error bars represent the mean of five different tumors ± SD. ** p<0.01. (F) Apoptosis (Annexin V / Propidium iodide staining) analysis of CD8+murine lymphocytes in cSCC co-culture model treated with ruxolitinib (3.125pM) for 3 days. Statistical significance determined using Mann-Whitney test in comparison to DMSO condition. Error bars represent the mean of four technical replicates ± SD. Presented data is representative of two biological replicates. (*) in comparison to DMSO condition for viable cells, (#) in comparison to DMSO condition for apoptotic cells (early and late apoptosis). * p<0.05, # p<0.05. (G, H) CD8+activated T cells (CD8+CD25+PD1+TIM3 ) number relative to DMSO in cSCC co-culture at H+36 (G) and H+72 (H). Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four technical replicates ± SD. Presented data is representative of two biological replicates. * p<0.05. (I) PDL1+tumor cells in cSCC co-culture treated with DMSO, ruxolitinib (3.125pM), CI-1040 (MEKi, 2pM) or CI-1040 and ruxolitinib. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four technical replicates ± SD. Presented data is representative of two biological replicates. (*) in comparison to DMSO condition, (#) in comparison to ruxolitinib condition. * p<0.05, # p<0.05.

[0072] Figure 2. Ruxolitinib synergizes with anti-PDl therapy to increase immunotherapy response in cSCC, pancreatic cancer and acute myeloid leukemia. (A) Cell growth of GFP+primary cSCC murine cells in co-culture with activated lymphocytes and treated with DMSO, ruxolitinib (3.125pM), pembrolizumab (lOOpg / ml) or a combination of ruxolitinib and pembrolizumab. Pembrolizumab was added two days after ruxolitinib initiation to allow for visualization of the intrinsic proliferative effects of ruxolitinib prior to anti-PDl treatment. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of eight technical replicates ± SD. Presented data is representative of two biological replicates. (*) Ruxolitinib compared to DMSO condition. (#) Ruxolitinib + pembrolizumab condition compared to ruxolitinib condition. *** p<0.001 ### p<0.001. (B, C) Waterfall plot (B) and bar graph (C) depicting tumor volume changes from baseline of mice with cutaneous carcinoma developed under vehicle or ruxolitinib (45mg / Kg bid) treatment. Pembrolizumab (12.5 mg / kg) was added in both groups one and eight days after tumor volume reached 150-200 mm3. Mice treated with anti-PDl only (n=6) and mice treated with both ruxolitinib and anti-PDl (n=8) are represented in gray and back respectively. Tumor volume was measured 11 days after anti-PDl treatment initiation according to international immunotherapy response criteria (iRECIST). Response, stability and progression were respectively defined as a tumor volume decrease of at least 30%, a tumor volume decrease lower than 30% or a tumor volume increase lower than 20%, and a tumor volume increase higher than 20%, in comparison to baseline. (D) Treatment received (left) by the six MPN patients diagnosed with an aggressive JAKi-induced cSCC and response (right) to the association between anti-PDl and JAKi for the four patients who received this combination. (E) Cell growth of GFP+primary cSCC murine cells in co-culture with activated lymphocytes and treated with DMSO (in black), ruxolitinib (3.125pM), pembrolizumab (lOOpg / mL) or a combination of ruxolitinib and pembrolizumab. Pembrolizumab was added simultaneously to ruxolitinib to allow for visualization of the effect of anti-PDl therapy in spontaneously developed cSCC. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four technical replicates ± SD. Presented data is representative of two biological replicates. (*) in comparison to DMSO condition (#) in comparison to anti-PDl condition. * p<0.05, # p<0.05. (F, G) Tumor volume changes of mice treated with anti-PDl (n=4, 12.5 mg / kg at days 1 and 8) or a combination of anti-PDl and ruxolitinib (n=4, 45mg / kg bid). Mean tumor volume variation from time of anti- PDl treatment initiation is indicated for each group in the corresponding color (F). Statistical significance determined using Mann-Whitney test in comparison to anti-PDl monotherapy at day 11. Error bars represent the mean of four mice per group ± SEM. * p< 0.05. Response to treatment, stability and progression are respectively represented (G). (H) Cell growth of GFP+KPC pancreatic cancer cells in co-culture with activated lymphocytes and treated with DMSO, ruxolitinib (3.125pM), pembrolizumab (lOOpg / mL) or a combination of ruxolitinib and pembrolizumab at day 6. Pembrolizumab was added simultaneously to ruxolitinib. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four technical replicates ± SD. Presented data is representative of two biological replicates. (*) in comparison to anti-PDl condition. * p<0.05. (I) Cell growth of CBFb-MYHll (left panel) and MLL-AF9 (right panel) leukemic cells in co-culture with activated lymphocytes and treated with DMSO, ruxolitinib (3.125pM), pembrolizumab (lOOpg / mL) or a combination of ruxolitinib and pembrolizumab at day 3. Pembrolizumab was added simultaneously to ruxolitinib. Statistical significance determined using Mann-Whitney test in comparison to anti-PDl monotherapy. Error bars represent the mean of four technical replicates ± SD. Presented data is representative of two biological replicates. * p< 0.05. (J) Bone marrow leukemic cells infiltration thirteen days after leukemic cells injection in the graft- versus-leukemia MLL-AF9 murine model. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of five mice per group ± SD. (*) in comparison to OT1 vehicle. (#) in comparison to MH vehicle. ** p< 0.01, ## p<0.01. (K) Overall survival leukemic cells injection in the graft-versus-leukemia MLL-AF9 murine model. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of five mice per group ± SD. * p<0.05. (L) Bone marrow Tregs infiltration thirteen days after leukemic cells injection in the graft-versus- leukemia MLL-AF9 murine model. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of five mice per group ± SD. ** p<0.01. (M) Bone marrow T CD8+infiltration thirteen days after leukemic cells injection in the graft-versus-leukemia MLL-AF9 murine model. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of five mice per group ± SD. * p<0.05.

[0073] Figure 3. Ruxolitinib synergizes with anti-PDl therapy to increase immune checkpoint inhibitors (CPI) response in melanomas. (A) Cell growth of GFP+ YUMMER1 .7 melanoma cancer cells in co-culture with activated lymphocytes and treated with DMSO (in black), ruxolitinib (in red, 3.125pM), pembrolizumab (in gray, lOOpg / mL) or a combination of ruxolitinib and pembrolizumab (in orange). Pembrolizumab was added simultaneously to ruxolitinib. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four replicates ± SD. (B-D) Regulatory T cells (B), CD8+ T cells (C) and activated CD8+ T cells (D) number relative to DMSO in YUMMER1.7 co-culture at day 3. Regulatory T cells and activated CD8+ T cells were defined as CD4+ CD25high Foxp3+ and CD8+ CD25+ cells respectively. Statistical significance determined using Mann- Whitney test in comparison to DMSO condition. Error bars represent the mean of four replicates ± SD. (E) Cell growth of GFP+ B16-OVA melanoma cancer cells in co-culture with activated lymphocytes and treated with DMSO (in black), ruxolitinib (in red, 3.125pM), pembrolizumab (in gray, lOOpg / mL) or a combination of ruxolitinib and pembrolizumab (in orange). Pembrolizumab was added simultaneously to ruxolitinib. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four replicates ± SD. (F-H) Regulatory T cells (F), CD8+ T cells (G) and activated CD8+ T cells (H) number relative to DMSO in B16-OVA co-culture at day 3. T regulatory T cells and activated CD8+ T cells were defined as CD4+ CD25high Foxp3+and CD8+ CD25+ cells respectively. Statistical significance determined using Mann-Whitney test in comparison to DMSO condition. Error bars represent the mean of four replicates ± SD.

[0074] Figure 4. Ruxolitinib-induced immune TME remodeling is mediated by JAK1 inhibition. (A-B) Regulatory T cell (A) and CD8+ T cells (B) number relative to lymphocytes monoculture DMSO condition, at day 3 of DMSO or itacitinib (JAK1 inhibitor, O. lOpM), fedratinib (JAK2 inhibitor, 0.02pM) or momelotinib (JAK1 / 2 inhibitor, O. lOpM) treatments in lymphocytes / Cscc co-culture. Regulatory T cells were defined as CD4+ CD8- CD25high Foxp3+ cells. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four replicates ± SD. (C) Activated CD8+ T cells number relative to DMSO in cSCC co-culture at day 3. Activated CD8+ T cells were defined as CD8+ CD25+ T cells. Statistical significance determined using Mann-Whitney test in comparison to DMSO condition. Error bars represent the mean of four replicates ± SD. (D) Cell growth decrease of GFP+ primary cSCC murine cells in co-culture with activated lymphocytes and treated with the indicated JAK inhibitors, pembrolizumab (lOOpg / ml) or a combination of JAK inhibitors (0. lOpM for itacitinib, 0.02pM for fedratinib and 0. lOpM for momelotinib) and pembrolizumab at day 5. Pembrolizumab was added two days after JAK inhibitor initiation. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four replicates ± SD. (E-F) T cells (E) and CD8+T cells (F) proportion among viable cells within cutaneous carcinomas from control versus fedratinib treated mice. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four mice per group ± SD. (G) Regulatory T cells proportion among CD3+ T cells within cutaneous carcinomas from control versus fedratinib treated mice. Regulatory T cells were defined as CD4+CD8' CD25+Foxp3+ cells. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four mice per group ± SD. (H) MDSC-M cells proportion among viable cells within peripheral blood and cutaneous carcinomas from control versus fedratinib treated mice. MDSC- M were defined as CDl lb+L6Clow+F4 / 80lowcells. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of four mice per group ± SD. (I) Bar graph depicting tumor volume changes from baseline of mice with cutaneous carcinoma treated by pembrolizumab (12.5mg / kg, day 1 and day 8) alone (n=5) or fedratinib (60mg / kg bid) and pembrolizumab (n=5). Tumor volume was measured 11 days after anti-PDl treatment initiation according to international immunotherapy response criteria (iRECIST). Response, stability and progression were respectively defined as a tumor volume decrease of at least 30%, a tumor volume decrease lower than 30% or a tumor volume increase lower than 20%, and a tumor volume increase higher than 20%, in comparison to baseline. Response to treatment, stability and progression are respectively represented in red, in hatched gray and in gray in the bar graphs. (J-K) Activated (J) and degranulated (K) CD8+T cells proportion among CD3+ T cells from pembrolizumab versus fedratinib and pembrolizumab treated mice. Activated and degranulated CD8+T cells were defined as CD8+CD25+and CD8+CD25+CD107a+cells respectively. Statistical significance determined using Mann-Whitney test. Error bars represent the mean of five mice per group ± SD.

[0075] EXAMPLE:

[0076] Methods: Ethical approval

[0077] The French National Ethics Committee on Animal Care reviewed and approved all mouse experiments described in this study (authorization number APAFIS #23169- 2019120416018976 v4). Mice were euthanized using cervical dislocation if animals showed limit points (weight loss > 20% or major tumor progression signs).

[0078] Chemically induced cSCC murine model

[0079] Three steps model - This model described by Abel et al. (Abel, E. L., Angel, J. M., Kiguchi, K. & DiGiovanni, J. Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications. NatProtoc 4, 1350- 1362 (2009)) is composed of three phases: i) an initiation phase in which an initiating oncogenic agent is applied to mice shaved skin to initiate cutaneous carcinogenesis, ii) a promotion phase in which a proinflammatory agent is applied to mice shaved skin twice weekly leading to epidermal hyperplasia and pre-cancerous lesions, named papillomas, and iii) a progression phase in which a subset of papillomas transform into overt cSCC.

[0080] Mice strain - Seven weeks-old female FVB / N mice purchased from Janvier lab were used for this model.

[0081] Initiating agent - 200nM DMBA (7, 12-dimethylbenz[a]-anthracene, #D3254, Sigma- Aldrich) in 0.2mL of acetone solution (0.2uL per mouse) was applied in the shaved back skin of the mice at weeks 0 and 3.

[0082] Promoting agent - 5nM TP A (12-O-tetradecanoylphorbol-13-Acetate, #P-1680, LC Laboratories) in 0.2mL of acetone solution (0.2uL per mouse) was applied in the shaved back skin of the mice twice weekly from week 4 to 27.

[0083] Clinical evaluation - Papilloma and cSCC number and volume were evaluated weekly.

[0084] Drug administration - Ruxolitinib (HY-50856, MedChemExpress) was diluted in a solvent composed by 5% of N,N-dimethylacetamide (#8032351000, merckmillipore) and 0.5% of methocellulose (#43147, ThermoFisher) diluted in water. Ruxolitinib was used at 45mg / kg twice daily (bid) by gavage during two consecutive weeks followed by three weeks of interruption and then restarted one week prior to the end of the experiment. Anti-PDl intraperitoneal injection was started when tumor volume reached 150-200mm3with two injections at days 1 and 8.

[0085] Response to anti-PDl treatment - cSCC tumor volume was evaluated at days 1, 5, 8 and 11. Treatment response criteria were defined according to international immunotherapy response criteria (iRECIST) (Seymour, L. et al. iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol 18, el 43 el 52 (2017)). Response, stability and progression were defined as a tumor volume decrease of at least 30%, a tumor volume decrease lower than 30% or a tumor volume increase lower than 20%, or a tumor volume increase higher than 20% respectively, in comparison to baseline.

[0086] Graft-versus-leukemia MLL-AF9 murine model

[0087] 6-week-old wild-type C57BL / 6 female mice (Envigo) were irradiated at 3.5Gy prior to transplantation with 2 / I 05DsRed Af / . / .-d / ’'9-driven leukemic cells according to the protocol previously reported in Su et al12. MataHari and OT-1 T cells were isolated from lymph nodes and spleens of 6 to 16-week-old Ragl ^ anti-H-Y TCR transgenic (kindly provided by Dr.

[0088] Philippe Bousso, Pasteur Institute, Paris, France) and C57BL / 6-Tg(TcraTcrb)1100Mjb / J female mice (The Jackson Laboratory), respectively. Lymph nodes and spleens were crushed and UTY or SL8 peptides were used to activate the MataHari or OT-1 CD8+T cells, respectively. Activated T cells were isolated using a Ficoll (Ficoll-Paque, Fisher scientific) and expanded in lOng / ml mouse IL2-supplemented RPMI media. 2xl06MataHari or OT-1 T cells were injected intravenously four days after the transplantation of the mice with the DsRed+MLL-AF9 leukemic cells. Mice were treated by oral gavage twice daily with 90mg / kg ruxolitinib from day 3 post-transplantation until the end of the experiment.

[0089] Results:

[0090] Ruxolitinib treatment reshapes the tumor immune microenvironment resulting in Tregs reduction and T CD8+cells increase.

[0091] MAPK pathway activation is known to induce an immunosuppressive tumor microenvironment (TME) with immunosuppressive cytokines and infiltration by Tregs, both resulting in decreased T CD8+cytotoxic lymphocytes activity (Molina-Arcas, M. & Downward, J. Exploiting the therapeutic implications of KRAS inhibition on tumor immunity. Cancer Cell 42, 338-357 (2024)). Moreover, ruxolitinib is associated with T lymphopenia in MPN patients’ blood and a reduction of T cells activation (Harrison, C. N. et al. Long-term findings from COMFORT-II, a phase 3 study of ruxolitinib vs best available therapy for myelofibrosis. Leukemia 30, 1701-1707 (2016)). Based on this knowledge, we hypothesized that both ruxolitinib exposure and its subsequent MAPK pathway activation could result in a reduced anti-tumor immunity in ruxolitinib-induced cSCC. This extrinsic effect could further increase the aggressiveness of cSCC secondary to ruxolitinib exposure.

[0092] GSEA of ruxolitinib versus control murine tumors transcriptomic data confirmed the down-regulation of T cell activation and interferon gamma production / response pathways in ruxolitinib induced carcinomas (data not shown). To evaluate intratumoral T cell infiltration, we first quantified Tumor Infiltrating Lymphocytes (TILs) in ruxolitinib induced mice tumors (n=l l) compared to control tumors (n=5), using HE (hematoxylin eosin) stained histological images, as described by the International Immuno-Oncology Biomarkers Working Group (Hendry, S. et al. Assessing Tumor-Infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method from the International Immuno-Oncology Biomarkers Working Group: Part 2: TILs in Melanoma, Gastrointestinal Tract Carcinomas, Non-Small Cell Lung Carcinoma and Mesothelioma, Endometrial and Ovarian Carcinomas, Squamous Cell Carcinoma of the Head and Neck, Genitourinary Carcinomas, and Primary Brain Tumors. Adv Anat Pathol 24, 311 335 (2017)). Contrary to what we expected, TILs were increased in ruxolitinib induced cSCC, both in intra-tumoral and peritumoral areas (Figure 1A). This observation was confirmed using flow cytometry analysis which showed an increase in CD8+T lymphocytes in ruxolitinib induced tumors (Figure IB, p=0.0159). To further validate these in vivo findings and understand their underlying mechanism, we next developed an autologous in vitro 2D co-culture model. Activated T lymphocytes extracted from FVB / N mice spleens were seeded in co-culture with primary murine tumor cells derived from cSCC obtained from the previously described chemically- induced murine model (not shown). Ruxolitinib dose (3.125pM) was selected to be non toxic for T lymphocytes (data not shown) while able to increase cSCC murine cells proliferation (data not shown). When lymphocytes are cultured alone, ruxolitinib treatment reduces T CD8+cells number (Figure 1C, p=0.0286), in line with the T cell lymphopenia observed in MPN patients’ peripheral blood under ruxolitinib (Harrison, C. N. et al. Long-term findings from COMFORT-II, a phase 3 study of ruxolitinib vs best available therapy for myelofibrosis. Leukemia 30, 1701-1707 (2016)). When lymphocytes are co-cultured with cSCC tumor cells, T CD8+number is reduced (p=0.0286), suggesting a tumor-mediated immunosuppressive effect (Figure 1C). Interestingly, ruxolitinib treatment rescues this immunosuppressive effect, resulting in an increased T CD8+number (p=0.0286), in line with our in vivo results (Figure 1C). To explore whether this effect was driven by cell contact or a secreted factor, we applied supernatants obtained from cSCC cell culture to activated autologous spleen derived lymphocytes. We observed a similar increase in T CD8+cells number after exposure to supernatant from ruxolitinib treated cSCC (Figure 1C, p=0.0286), suggesting a paracrine mediated effect. To characterize the impact of ruxolitinib on cSCC secretome, we next performed a proteomic screening of cytokines and chemokines expression in lymphocytes monoculture and in co-culture conditions. Among statistically dysregulated cytokines and chemokines, immunosuppressive cytokines (1110 and Tgf-b), myeloid cells growth factors (Gm-csf, G-csf and M-csf) and attracting myeloid chemokines (Ccl4, Ccll2) were up-regulated in presence of tumor cells (data not shown). Ruxolitinib exposure decreased the level of these cytokines / chemokines (data not shown). Expression of the majority of these cytokines / chemokines and their respective receptors was also reduced in ruxolitinib mice tumors using transcriptomic differential gene expression analysis (data not shown). 1110 and Tgf-b concentration reduction was confirmed in blood serums of mice harboring ruxolitinib- induced carcinomas (data not shown). Importantly, IL 10 and TGF-b concentrations also decreased in serum obtained from a patient with ruxolitinib-induced aggressive cSCC after ruxolitinib treatment was restarted (data not shown).

[0093] Within the TME, the increase of myeloid growth factors and attracting chemokines is known to be associated with the presence of myeloid-derived suppressor cells (MDSCs) (Veglia, F., Sanseviero, E. & Gabrilovich, D. I. Myeloid-derived suppressor cells in the era of increasing myeloid cell diversity. Nat Rev Immunol 21, 485- -498 (2021) and Bronte, V. et al. Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nat Commun 7, 12150 (2016)). Accordingly, CDl lb+Grl+cell infiltration was decreased in vivo under ruxolitinib (data not shown, p=0.0286). Moreover, murine cSCC single cell RNA sequencing data showed that previously described MDSCs markers, including Cd84, Csfl, Statl an IHORA were down-regulated in myeloid cells of ruxolitinib tumors (data not shown). GSEA analysis within the myeloid compartment, confirmed that myeloid cells expressed less immunosuppressive signatures and were enriched in immune activation signatures in ruxolitinib mice tumors (data not shown).

[0094] Both MDSCs and ILlO / TGF-b accumulation are known to induce Tregs infiltration within the TME. Interestingly, while tumor cells contact resulted in an increased Tregs number (CD4+CD25+Foxp3+) (Figure ID, p=0.0286), ruxolitinib treatment reduced the number of Tregs within our in vitro co-culture model, thus rescuing this tumor-mediated immunosuppressive phenotype (Figure ID, p=0.0286). This finding was further validated using Foxp3 immunohistochemistry staining of ruxolitinib-induced cSCC obtained from MPN patients in comparison to spontaneously developed matched tumors (Figure IE). Tregs are known to decrease CD8+cytotoxic cells activity, in particular by inducing their apoptosis (S, S. et al. Regulatory T Cells and Human Disease. Annual review of immunology 38, (2020)). Accordingly, Propidium Iodide / Annexin V staining of CD8+cells within our co-culture model showed a decrease in CD8+cells apoptosis under ruxolitinib (Figure IF), offering an explanation for the observed increase of CD8+cells number. Intriguingly, this positive immune microenvironment reshaping did not result in efficient antitumor activity, thus enabling aggressive cSCC development under ruxolitinib. To understand this counter intuitive observation, we further explored the phenotype of CD8+subpopulations by flow cytometry within our co-culture model. Interestingly, after 36 hours of treatment, ruxolitinib increased all CD8+T cells subpopulations (data not shown), including activated T CD8+cells (CD25+PD1+) (Figure 1G). After 3 days of treatment, only exhausted (CD25+PD1+TIM3+) and inactivated (CD25 ) CD8+T cells remained increased (Figure 1H). Inactivation / exhaustion of T CD8+cells was maintained by tumor antigen exposure increase (data not shown) and exposure to tumor cells expressing high levels of PDL1 (Figure II). Additionally, MEKi treatment rescued PDL1 expression increase, suggesting that ruxolitinib- mediated MAPK pathway activation is responsible for such overexpression (Figure II).

[0095] Altogether, our findings suggest that ruxolitinib reshapes cSCC TME through downregulation of the tumor-induced immunosuppressive cytokinome and reduction of immunosuppressive cellular populations infiltration, especially Tregs. Ultimately, CD8+T cells apoptosis is decreased leading to an increase in CD8+T cells in the TME. Ruxolitinib mediated MAPK pathway activation resulting in increased tumor antigen and PDL1 exposure is ultimately responsible for inactivation / exhaustion of these CD8+cells (not shown).

[0096] Ruxolitinib synergizes with anti-PDl therapy to increase immunotherapy response in cSCC, pancreatic cancer and acute myeloid leukemia.

[0097] PDL1 expression, Tregs reduction and T CD8+infiltration are associated with increased immune checkpoint inhibitors (CPI) response (Tanaka, A. & Sakaguchi, S. Targeting Treg cells in cancer immunotherapy. Eur J Immunol 49, 1140 1146 (2019); Garcia-Corbacho, J. et al. Determinants of activity and efficacy of anti-PDl / PD-Ll therapy in patients with advanced solid tumors recruited in a clinical trials unit: a longitudinal prospective biomarker -based study. Cancer Immunol Immunother 72, 1709- -1723 (2023)). suggesting a potential synergistic effect between JAKi and anti-PDl treatment. We therefore set to evaluate whether ruxolitinib- induced TME remodeling could result in the creation of novel therapeutic opportunities. We first used our in vitro co-culture model where autologous lymphocytes were seeded with GFP+ cSCC tumor cells and treated with ruxolitinib, until ruxolitinib-induced tumor cells growth was observed (2 days). We then evaluated response to the anti-PDl treatment pembrolizumab, showing an improved reduction of tumor cells growth when pembrolizumab was combined with ruxolitinib (-22% versus 0%, p<0.001) (Figure 2A). Mechanistically, ruxolitinib increased CD8+T cells number, and anti-PDl treatment maintained their activation (data not shown). The synergistic effect between ruxolitinib and pembrolizumab was next evaluated in vivo. Mice were randomized into ruxolitinib or vehicle groups and pembrolizumab treatment was started when tumors reached 150-200mm3. Eleven days after pembrolizumab treatment, seven mice (88%) showed a clinical response in the anti-PDl / ruxolitinib group, while only one mouse (17%) had a clinical response in the anti-PDl monotherapy group (Figures 2B-C). Tumor volume mean percentage change at day 11 was -40% range [-67; -22] with the anti-PDl / ruxolitinib treatment combination versus +37% range[-69;+172] when mice were treated with pembrolizumab alone (p=0.0426) (data not shown). Importantly, our in vitro and in vivo findings were confirmed in MPN patients. Among the six MPN patients who developed an aggressive cSCC under JAKi treatment within our patients’ cohort, one patient was treated with radiotherapy, one with surgery and four received anti-PDl while JAKi was maintained to stabilize their hematological disorder (data not shown). Anti-PDl treatment was administered to these patients after failure of first line regimens composed of chemotherapy and / or targeted therapy. All patients presented a response to the anti-PDl / JAKi treatment combination; with three patients (75%) achieving a complete response (Figure 2D). Interestingly, three patients for which anti-PDl treatment was discontinued after initial response while pursuing ruxolitinib, relapsed with aggressive disease or developed a second cSCC (data not shown). Two of these patients for which immunotherapy was restarted at time of relapse had developed refractory disease (data not shown).

[0098] After demonstrating the synergistic effect between anti-PDl and ruxolitinib treatment in ruxolitinib-induced cSCC, we next hypothesized that such synergistic effect could also be observed in spontaneously developed cSCC. In vitro, simultaneous ruxolitinib addition to pembrolizumab treatment reduced cSCC cell growth by 50% while anti-PDl monotherapy resulted in an 8% decrease (p=0.0286) (Figure 2E). This effect was further confirmed in vivo after five days of ruxolitinib pre-treatment. Baseline tumor volume was not impacted by short term ruxolitinib exposure, suggesting that five days of ruxolitinib treatment is not enough to induce its pro-oncogenic effects in vivo (Figure 2F). Mean tumor volume decrease under the ruxolitinib / pembrolizumab combination was -44%, while a mean tumor volume increase of +248% was observed with anti-PDl monotherapy (p=0.0286) (Figure 2F). Overall, three mice (75%) had a response and one mouse had stable disease in the ruxolitinib / pembrolizumab group, while one mouse (25%) had a clinical response, one had stable disease and two had progressive disease when pembrolizumab was administered alone (Figure 2G).

[0099] Finally, we set to evaluate whether the immune-modulating effects of ruxolitinib could also enhance response to immunotherapy in other cancer types. We focused on pancreatic cancer and acute myeloid leukemia (AML), both considered as “cold” tumors, displaying high primary resistance rates to immunotherapy. Indeed, these poor prognosis cancer types evolve within an immunosuppressive microenvironment with high Tregs infiltration, resulting in low efficacy of immunotherapies (Gomez-Llobell, M., Peleteiro Raindo, A., Climent Medina, J., Gomez Centurion, I. & Mosquer a Orgueira, A. Immune Checkpoint Inhibitors in Acute Myeloid Leukemia: A Meta-Analysis. Front Oncol 12, 882531 (2022) and Bian, J. & Almhanna, K. Pancreatic cancer and immune checkpoint inhibitors — still a long way to go. Transl Gastroenterol Hepatol 6, 6 (2021)). We therefore hypothesized that ruxolitinib-mediated downregulation of tumor induced immunosuppression, mainly through cytokines / chemokines remodeling and reduction of infiltrating Tregs and MDSCs, could turn this “cold” tumors into “hot” tumors responsive to immunotherapy. We first developed autologous in vitro coculture models, using activated spleen lymphocytes obtained from C57BL / 6 mice, seeded with the KRASPI2Dmutated murine pancreatic cancer cell line KPC or AML murine primary cells obtained from CBFb-MYH 11 or MLL-AF9 driven leukemic murine models. In line with their high dependency on the MAPK pathway, KPC cells were intrinsically resistant to ruxolitinib treatment when seeded in monoculture, up to 25 pM (data not shown). Interestingly, when pembrolizumab was added simultaneously to ruxolitinib, we observed an increased response to pembrolizumab of KPC cells compared to pembrolizumab alone (-25% vs -8%, p=0.0286) (Figure 2H). Mechanistically, ruxolitinib reduced Tregs and increased CD8+T cells number (data not shown), while anti-PDl treatment maintained CD8+T cells activation (data not shown); in line with our previous results in cSCC. CBFb-MYHl l cells were resistant to ruxolitinib while MLL-AF9 cells were more sensitive (data not shown). Pembrolizumab / ruxolitinib combination enhanced the anti-PDl response in both AML models (-34% v -8% and -63% vs +5% for CBFb-MYHl 1 and MLL-AF9 cells respectively) (Figure 21).

[0100] Finally, we developed a graft-versus-leukemia (GvL) MLL-AF9 murine model (not shown) to evaluate whether ruxolitinib-mediated immune microenvironment reshaping could also result in an enhanced response to hematopoietic stem cell transplantation (HSCT), the most commonly used immunotherapeutic approach for AML treatment. Increasing the GvL effect without increasing graft-versus-host-disease (GvHD) remains one of the main challenges of such therapeutic strategy (Maurer, K. & Soiffer, R. J. The delicate balance of graft versus leukemia and graft versus host disease after allogeneic hematopoietic stem cell transplantation. Expert Rev Hematol 16, 943- -962 (2023)). Ruxolitinib is known to reduce GvHD but its effects on GvL have not yet been explored (Zeiser, R. et al. Ruxolitinib for Glucocorticoid-Refractory Chronic Graft-versus-Host Disease. N Engl J Med 385, 228- -238 (2021)). We thus hypothesized that ruxolitinib could uncouple the GvL / GvHD effects resulting in improved patients’ outcome post-HSCT. To escape to allogeneic T CD8+lymphocytes immunosurveillance, the main mediator of the GvL effect, AML cells develop immunosuppressive mechanisms, notably Tregs induction within the bone marrow microenvironment (Vadakekolathu, J. & Rutella, S. Escape from T-cell-targeting immunotherapies in acute myeloid leukemia. Blood 143, 2689 2700 (2024)). Taking advantage of our MLL-AF9 GvL mouse model, we evaluated if ruxolitinib also mediates Tregs decrease within the bone marrow and if this effect is able to enhance the GvL effect induced by MataHari (MH) CD8+T lymphocytes. MataHari T lymphocytes injection resulted in decreased leukemic bone marrow (BM) infiltration and prolonged overall survival (OS) in comparison to control OT1 T lymphocytes injection (Figures 2J-K). Interestingly, this GvL effect was further increased when mice were treated with ruxolitinib (Figures 2J-K). Mechanistically, Tregs BM infiltration was reduced when mice were treated with ruxolitinib (Figure 2L). Accordingly, T CD8+BM infiltration was increased under ruxolitinib treatment (Figure 2M), resulting in an increased GvL effect in this model.

[0101] We also show that Ruxolitinib synergizes with anti-PDl therapy increase immune checkpoint inhibitors (CPI) response in melanomas (Figures 3 A to 3H).

[0102] Finally, we show that Ruxolitinib-induced immune TME remodeling is mostly mediated by JAK1 inhibition (Figures 4 A to 4K).

[0103] Altogether, these findings highlight the key role of JAK inhibition in positively remodeling the immune microenvironment, resulting in an improved response to different immunotherapeutic interventions in diverse types of cancer.

[0104] Conclusion:

[0105] In summary, combining JAK inhibitors with immunotherapy shows promise in cancer treatment. By reshaping the immune microenvironment, JAK inhibitors can enhance the effectiveness of immunotherapies like immune checkpoint blockers and cell-based immunotherapy. This combination has potential in overcoming resistance in tumors with highly immunosuppressive environments, possibly improving patient survival and quality of life.

[0106] 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

38 -CLAIMS:

1. A method for enhancing the potency of immunotherapy administered to a patient as part of a treatment regimen for cancer, the method comprising: administering a pharmaceutically effective amount of a JAK inhibitor to the patient in combination with immunotherapy.

2. A method of treating cancer in a subj ect in need thereof comprising administering to the subject a therapeutically effective combination of immunotherapy with a JAK inhibitor, wherein administration of the combination results in enhanced therapeutic efficacy relative to the administration of immunotherapy alone.

3. A method of overcoming immunotherapy resistance in patient suffering from cancer thereof comprising administering to the patient a therapeutically effective amount of a JAK inhibitor.

4. The method according to any one of claims 1 to 3 wherein the patient suffers from a cutaneous squamous cell carcinoma, a melanoma, a pancreatic cancer or an acute myeloid leukemia.

5. The method according to any one of claims 1 to 3 wherein the patient does not suffer from a Hodgking Lymphoma or non-small cell lung cancer.

6. The method according to any one of claims 1 to 5 wherein the cancer is characterized by a high tumor infiltration of Treg cells.

7. The method according to any one of claims 1 to 5 wherein the cancer is characterized by a low tumor infiltration of CD8+ T cells.

8. The method according to any one of claims 1 to 7 wherein the immunotherapy is a cellbased immunotherapy.

9. The method according to claim 8 wherein the cell-based immunotherapy is hematopoietic stem cell transplantation (HSCT) or is a Chimeric Antigen Receptor (CAR) therapiy, which include CAR-T cells, CAR-NK cells, and CAR macrophages.

10. The method according to any one of claims 1 to 7 wherein the immunotherapy consists in administering the patient with at least one immune checkpoint blocker.

11. The method according to claim 10 wherein the immune checkpoint blocker is selected from the group consisting of PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists.

12. The method according to any one of claims 1 to 11 wherein the JAK inhibitor is a selective JAK2 inhibitor.

13. The method according to claim 12 wherein the selective JAK2 inhibitor is selected from the group consisting of Ruxolitinib, Fedratinib, Gandotinib, Lestaurtinib, Momelotinib and Pacritinib.

14. The method according to any one of claims 1 to 11 wherein the JAK inhibitor is a selective JAK1 inhibitor.

15. The method according to claim 14 wherein the selective JAK1 inhibitor is selected from the group consisting of Ruxolitinib, Oclacitinib, Filgotinib, Golidocitinib and Itacitinib

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