WO2023144235A1

WO2023144235A1 - Methods for monitoring and treating warburg effect in patients with pi3k-related disorders

Info

Publication number: WO2023144235A1
Application number: PCT/EP2023/051851
Authority: WO
Inventors: Guillaume CANAUD; Sophia LADRAA
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Assistance Publique-Hôpitaux De Paris (Aphp); Centre National De La Recherche Scientifique; Université Paris Cité
Priority date: 2022-01-27
Filing date: 2023-01-26
Publication date: 2023-08-03

Abstract

Using a unique tool of PROS, they demonstrate that PIK3CA mutation leads to GLUT4 membrane accumulation with a negative feedback loop on insulin secretion, a burst of liver IGFBP1 synthesis with IGF1 sequestration and low circulating levels. They further show that AKT2 drives a large part of the phenotype. In addition, they demonstrate for the first time that a single PIK3CA mutation induces metabolic reprogramming with the Warburg effect and protein and lipid synthesis—hallmarks of cancer cells—in vitro, in vivo and in patients. They finally show that alpelisib, an approved PIK3CA inhibitor in oncology, is efficient at preventing and improving PIK3CA-adipose tissue overgrowth and reversing metabolomic anomalies in both animal models and patients. Accordingly, the present invention relates to an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the step of determining the level of at least one metabolite selected in the group consisting of cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject.

Description

METHODS FOR MONITORING AND TREATING WARBURG EFFECT IN PATIENTS WITH PI3K-RELATED DISORDERS FIELD OF THE INVENTION: The present invention is in the field of medicine and relates to methods for monitoring and treating Warburg effect in patients with PI3K-related disorders. BACKGROUND OF THE INVENTION: PIK3CA-related overgrowth syndrome (PROS) is a rare genetic disorder caused by gain- of-function mutations in the PIK3CA gene1. These mutations occur most frequently during embryogenesis and lead to somatic mosaicism2. PIK3CA is a ubiquitously expressed lipid kinase that controls signaling pathways participating in cell proliferation, motility, survival and metabolism3. At the cellular level, PIK3CA is mainly recruited through tyrosine kinase receptors. PIK3CA encodes the 110-kDa catalytic alpha subunit of PI3K (p110α), which converts, at the plasma membrane, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3; or PIP3) and subsequently recruits PDK1, which in turn phosphorylates AKT on the Thr308 residue to initiate downstream cellular effects. PIK3CA also regulates many other pathways, including the Rho/Rac1 signaling cascade4. The clinical presentation of patients with PROS is extremely broad owing to mosaicism and the characteristics of the tissue involved5,6. Patients usually have complex tissue malformations, including abnormal vessels, muscle hypertrophy and/or bone deformation1,7-10. Adipose tissue is frequently involved11. In addition to adipose tissue overgrowth (also known as fibroadipose hyperplasia, fibroadipose vascular anomaly, facial infiltrating lipomatosis or lipomatosis of nerve), these patients usually present with unexplained hormonal dysregulation and metabolic anomalies such as low circulating IGF-1 level and chronic hypoglycemia12,13. We recently generated a mouse model of PROS using ubiquitously expressed inducible Cre recombinase (CAGG CreER)14. However, this mouse model did not allow us to determine the role specifically played by adipose tissue in PROS physiopathology. We therefore decided to explore the consequence of PIK3CA gain-of-function mutations, specifically in adipocytes. SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment. DETAILED DESCRIPTION OF THE INVENTION: In the present study, the Inventors created the first mouse model of PIK3CA-related adipose tissue overgrowth that recapitulates the patient phenotype. Using this unique tool, they demonstrate that PIK3CA mutation leads to GLUT4 membrane accumulation with a negative feedback loop on insulin secretion, a burst of liver IGFBP1 synthesis with IGF1 sequestration and low circulating levels. They further show that AKT2 drives a large part of the phenotype. In addition, they demonstrate for the first time that a single PI3KCA mutation induces metabolic reprogramming with the Warburg effect and protein and lipid synthesis—hallmarks of cancer cells—in vitro, in vivo and in patients. They finally show that alpelisib, an approved PI3KCA inhibitor in oncology, is efficient at preventing and improving PIK3CA-adipose tissue overgrowth and reversing metabolomic anomalies in both animal models and patients. In conclusion, they decipher the endocrine anomalies associated with PROS and show in noncancerous circumstances the presence of reversible metabolomic changes associated with PIK3CA mutations. Methods for monitoring the efficiency of a PI3K inhibitor treatment In a first aspect, the present invention relates to an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising a step of determining the level of at least one metabolite of tricarboxylic acid cycle (TCA cycle). As used herein, the term “TCA cycle” refers to a central driver of cellular respiration, taking as starting metabolite acetyl-CoA produced by the oxidation of pyruvate and originally derived from glucose. In a series of redox reactions, TCA cycle generate bond energy in the form of NADH, FADH2 and ATP molecules. The reduced electron carriers (NADH, FADH2) generated in the TCA cycle pass their electrons into the electron transport chain and, through oxidative phosphorylation, generate most of the ATP produced in cellular respiration. In some embodiments, the at least one metabolite is a TCA cycle metabolite selected in the group consisting of alpha-ketoglutarate, cis-aconitate, succinic acid, citrate, isocitrate, succinyl-CoA, fumarate, malate, oxaloacetate. In some embodiments, the at least one metabolite is a TCA cycle metabolite selected in the group consisting of cis-aconitate, succinic acid, citrate, isocitrate, succinyl-CoA, fumarate, malate, oxaloacetate. In some embodiments, the present invention relates to an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the steps of: i) determining the level of at least one metabolite of TCA cycle in a biological sample obtained from the subject before the treatment; ii) determining the level of the at least one metabolite of the TCA cycle in a biological sample obtained from the subject after the treatment; iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the PI3K inhibitor treatment is efficient when the level of at least one metabolite of TCA cycle determined at step ii) is higher than the level determined at step i) and when the level of at least one other metabolite of TCA cycle is lower than the level determined at step i). In some embodiments, the least one metabolite of TCA cycle at step iv) is alpha- ketoglutarate level and cis-aconitate. In some embodiments, the least one metabolite of TCA cycle at step iv) is cis-aconitate. Accordingly, in some embodiments, the step iv) is to conclude that the PI3K inhibitor treatment is efficient when the alpha-ketoglutarate level determined at step ii) is higher than the level determined at step i) and when the cis-aconitate level determined at step ii) is lower than the level determined at step i). In some embodiments, the step iv) is to conclude that the PI3K inhibitor treatment is efficient when the alpha-ketoglutarate level and the cis-aconitate level determined at step ii) are lower than the levels determined at step i). More particularly, the present invention relates to an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the step of determining the level of at least one metabolite selected in the group consisting of alpha- ketoglutarate (aKG), cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl- lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl- carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject. In some embodiment, the present invention relates to an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the step of determining the level of at least one metabolite selected in the group consisting of cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl- lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl- carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject. Even more particularly, the present invention relates to an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the steps of: i) determining the level of at least one metabolite selected in the group consisting of alpha-ketoglutarate, cis-aconitate, succinic acid, 5-methylcytosine, acetyl- carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject before the treatment; ii) determining the level of the at least one metabolite in a biological sample obtained from the subject after the treatment; iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the PI3K inhibitor treatment is efficient when the alpha- ketoglutarate and/or betain levels determined at step ii) are higher than the levels determined at step i) and/or when the cis-aconitate, succinic acid, 5- methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L- dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan and/or urate levels determined at step ii) are lower than the levels determined at step i). In some embodiments, the present invention relates to an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the steps of: i) determining the level of at least one metabolite selected in the group consisting of alpha-ketoglutarate, cis-aconitate, succinic acid, 5-methylcytosine, acetyl- carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject before the treatment; ii) determining the level of the at least one metabolite in a biological sample obtained from the subject after the treatment; iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the PI3K inhibitor treatment is efficient when the betain level determined at step ii) is higher than the level determined at step i) and/or when the alpha-ketoglutarate, cis-aconitate, succinic acid, 5-methylcytosine, acetyl- carnitine, acetyl-lysine, argininosuccinate, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan and/or urate levels determined at step ii) are lower than the levels determined at step i). In some embodiments, the present invention relates to an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the steps of: i) determining the level of at least one metabolite selected in the group consisting of cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject before the treatment; ii) determining the level of the at least one metabolite in a biological sample obtained from the subject after the treatment; iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the PI3K inhibitor treatment is efficient when the betain level determined at step ii) is higher than the level determined at step i) and/or when the cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl- lysine, argininosuccinate, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan and/or urate levels determined at step ii) are lower than the levels determined at step i). In some embodiments, the at least one metabolite is alpha-ketoglutarate, cis aconitate acetyl-carnitine, argininosuccinate, butyric acid, carnitine, creatine, glycine, hexanoyl- carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan and urate. In some embodiments, the at least one metabolite is cis aconitate acetyl-carnitine, argininosuccinate, butyric acid, carnitine, creatine, glycine, hexanoyl-carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan and urate. According to this embodiment, the method comprises a step iv) concluding that the PI3K inhibitor treatment is efficient when the alpha-ketoglutarate level determined at step ii) is higher than the level determined at step i) and when the cis-aconitate, acetyl-carnitine, argininosuccinate, butyric acid, carnitine, creatine, glycine, hexanoyl- carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan and urate levels determined at step ii) are lower than the levels determined at step i). In some embodiment, the method comprises a step iv) concluding that the PI3K inhibitor treatment is efficient when the alpha-ketoglutarate, cis-aconitate, acetyl-carnitine, argininosuccinate, butyric acid, carnitine, creatine, glycine, hexanoyl-carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan and urate levels determined at step ii) are lower than the levels determined at step i). In some embodiment, the method comprises a step iv) concluding that the PI3K inhibitor treatment is efficient when the cis-aconitate, acetyl-carnitine, argininosuccinate, butyric acid, carnitine, creatine, glycine, hexanoyl-carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan and urate levels determined at step ii) are lower than the levels determined at step i). In some embodiments, the at least one metabolite is a TCA cycle metabolite selected in the group consisting of alpha-ketoglutarate, cis-aconitate, succinic acid. In some embodiments, the at least one metabolite is a TCA cycle metabolite selected in the group consisting of cis- aconitate and succinic acid. Thus, in some embodiments, the method comprises a step iv) concluding that the PI3K inhibitor treatment is efficient when the alpha-ketoglutarate level determined at step ii) is higher than the level determined at step i) and when the cis-aconitate level determined at step ii) is lower than the level determined at step i). In some embodiments, the method comprises a step iv) concluding that the PI3K inhibitor treatment is efficient when the alpha-ketoglutarate level and cis-aconitate level determined at step ii) are lower than the levels determined at step i). In some embodiments, the method comprises a step iv) concluding that the PI3K inhibitor treatment is efficient when the cis-aconitate level determined at step ii) is lower than the level determined at step i). As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine or a primate. In some embodiments, the subject is a human afflicted with or susceptible to be afflicted with PROS. In some embodiments, the subject is a human afflicted or susceptible to be afflicted with a PIK3CA-related fibroadipose overgrowth. In some embodiments, the subject is a human afflicted or susceptible to be afflicted with CLOVES syndrome. In some embodiments, the subject is a human afflicted with or susceptible to be afflicted with a pathology associated with Warburg effect. In some embodiments, the subject is a human afflicted with or susceptible to be afflicted with a cancer. As used herein, the term “Warburg effect” denotes a state when glucose uptake and fermentation of glucose to lactate are increased. More particularly, the Warburg effect is characterized when cells produce energy through a high rate of glycolysis followed by lactic acid fermentation in the cytosol, while normal cells have a comparatively low rate of glycolysis followed by oxidation of pyruvate in the mitochondria. The rate of glucose uptake dramatically increases and lactate is produced, even in the presence of oxygen and fully functioning mitochondria. So far, the Warburg effect was associated with cancer (Liberti MV, Locasale JW. The Warburg Effect: How Does it Benefit Cancer Cells?. Trends Biochem Sci. 2016;41(3):211-218). In some embodiments, the subject suffers from a pathology associated with Warburg effect. In some embodiments, the pathology associated with Warburg effect is PIK3CA-Related Overgrowth Syndrome (PROS). In a particular embodiment, PROS is a PIK3CA-related fibroadipose overgrowth. In a particular embodiment, PROS is a CLOVES syndrome. In some embodiments, the pathology associated with Warburg effect is cancer. As used herein the term “PROS” or “PIK3CA-Related Overgrowth Spectrum” relates to a group of disorders such as fibroadipose overgrowth (FAO), megalencephaly- capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome, Hemihyperplasia Multiple Lipomatosis (HHML) and Klippel–Trenaunay syndrome. As used herein, the term “fibroadipose overgrowth (FAO)” refers to a syndrome, which is characterized by the major findings of segmental progressive overgrowth of subcutaneous, muscular, and visceral fibroadipose tissue with skeletal overgrowth (Lindhurst, Marjorie J et al., Nature genetics vol.44,8928-33.24 Jun.2012). As used herein, the term “megalencephaly-capillary malformation (MCAP) syndrome” refers to a syndrome which is characterized by the major findings of (1) megalencephaly (MEG) or hemimegalencephaly (HMEG) associated with neurologic findings of hypotonia, seizures, and mild to severe intellectual disability; and (2) cutaneous capillary malformations with focal or generalized somatic overgrowth (Mirzaa, Ghayda M et al. American journal of medical genetics. Part C, Seminars in medical genetics vol.163C,2 (2013): 122-30). As used herein, the term “CLOVES” refers to “Congenital, Lipomatous, Overgrowth, Vascular Malformations, Epidermal Nevi and Spinal/Skeletal Anomalies and/or Scoliosis”. This syndrome is characterised by lipomatous tissues showing complex congenital overgrowth (typically appearing as a truncal lipomatous mass) and a combination of vascular and lymphatic malformations. As used herein, the term “Hemihyperplasia Multiple Lipomatosis (HHML)” refers to a condition characterized by asymmetric nonprogressive overgrowth, multiple lipomas, and superficial vascular malformations (Craiglow, Brittany G et al. Pediatric dermatology vol.31,4 (2014): 507-10). As used herein, the term “Klippel–Trénaunay syndrome” refers to a rare congenital medical condition in which blood vessels and/or lymph vessels fail to form properly. Thus, the method according to the present invention can be supplied to a subject, who has been diagnosed as presenting one of the disorders in PROS. 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 include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. As used herein, the term “PI3K” refers to phosphoinositide 3-kinases also called phophatidylinositide 3-kinases. PI3K belongs to a family of enzymes which phosphorylate the 3’hydroxyl group of the inositol ring of the phosphatidylinositol (PtdIns). PIK3CA encodes the 110-kDa catalytic alpha subunit of PI3K (p110α), which converts, at the plasma membrane, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to phosphatidylinositol 3,4,5- trisphosphate (PtdIns(3,4,5)P3; or PIP3) and subsequently recruits PDK1, which in turn phosphorylates AKT on the Thr308 residue to initiate downstream cellular effects. The PI3K family is divided into four different classes. The classifications are based on primary structure, regulation, and in vitro lipid substrate specificity. Class IA and IB encompass: PIK3CA (p110 α), PIK3CB (p110β), PIK3CG (p110-γ), PIK3CD (p110-δ), PIK3R1 (p85-α), PIK3R2 (p85-β), PIK3R3 (p55-γ), PIK3R4 (p150), PIK3R5 (p101), PIK3R6 (p87). Class II encompass: PIK3C2A (PI3K-C2α), PIK3C2B (PI3K-C2β), PIK3C2G (PI3K-C2γ). Class III encompass PIK3C3 (Vps34). As used herein, the term “PI3K inhibitor” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of PI3K. More particularly, such compound is able to inhibit the kinase activity of at least one member of PI3K family, for example, at least one member of Class I PI3K. In a particular embodiment, said PI3K inhibitor may be a pan-inhibitor of Class I PI3K (known as p110) or isoform specific of Class I PI3K isoforms (among the four types of isoforms, p110 ^, p110 ^, p110 ^ or p110 ^). In a particular embodiment, the PI3K inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide. In a particular embodiment, the inhibitor of PI3K is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. In a particular embodiment, the PI3K inhibitor is a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. In a particular embodiment, the PI3K inhibitor is a small molecule selected among the following compounds: BYL719 (Alpelisib, Novartis), GDC-0032 (Taselisib, Genentech/Roche), BKM120 (Buparlisib), INK1117 (Millenium), A66 (University of Auckland), GSK260301 (Glaxosmithkline), KIN-193 (Astra-Zeneca), TGX221 (Monash University), TG1202, CAL101 (Idelalisib, Gilead Sciences), GS-9820 (Gilead Sciences), AMG319 (Amgen), IC87114 (Icos Corporation), BAY80-6946 (Copanlisib, Bayer Healthcare), GDC0941 (Pictlisib, Genentech), IPI145 (Duvelisib, Infinity), SAR405 (Sanofi), PX-866 (Oncothyreon), perifosine, Umbralisib (TGR 1202), Dactolisib, CUDC-907, Voxtalisib (SAR245409, XL765), Pilaralisib, GDC-0077, TAK-117, AZD-8186, IPI-549 or their pharmaceutically acceptable salts. Such PI3K inhibitors are well-known in the art and described for example in Wang, X. et al (Wang, X., Ding, J. & Meng, Lh. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies. Acta Pharmacol Sin 36, 1170–1176 (2015)). In a particular embodiment, the PI3K inhibitor is BYL719. As used herein, the term “BYL719” is an ATP-competitive oral PI3K inhibitor selective for the p110α isoform that is activated by a mutant PIK3CA gene (Furet, Pascal et al. Bioorganic & medicinal chemistry letters vol. 23,13 (2013): 3741-8; Fritsch, Christine et al. Molecular cancer therapeutics vol. 13,5 (2014): 1117-29). This molecule is also called Alpelisib and has the following formula and structure in the art C19H22F3N5O2S:

In a particular embodiment, the PI3K inhibitor is GDC-0032, developed by Roche. This molecule also called Taselisib has the following formula and structure in the art C₂₄H₂₈N₈O₂:

In some embodiments, the PI3K inhibitor is an antibody. As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/11161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each 25 of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No.4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos.6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0368684, WO 06/030220 and WO 06/003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B- cell hybridoma technique and the EBV-hybridoma technique. In a particular embodiment, the PI3K inhibitor is an intrabody having specificity for PI3K. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. In some embodiments, the PI3K inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of PI3K. In a particular embodiment, the inhibitor of PI3K expression is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide- long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted 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). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. 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. In some embodiments, the inhibitor of PI3K expression is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR). In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiments, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol.339 : 823–826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol.8:e2671.), plants (Mali et al., 2013, Science, Vol.339 : 823–826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707–714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol.41 : 4336–4343.), Drosophila (Gratz et al., 2014 Genetics, doi:10.1534/genetics.113.160713), monkeys (Niu et al., 2014, Cell, Vol.156 : 836– 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol.6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol.24 : 122–125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol.56 : 122–129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci. In some embodiments, the endonuclease is CRISPR-Cpf1 which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpf1) in Zetsche et al. (“Cpf1 is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13). Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity of PI3K. In some embodiments, the assay first comprises determining the ability of the test compound to bind to PI3K. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to inhibit the activity of PI3K. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of inhibiting the activity of PI3K, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules. As used herein, the terms “treating”, “treatment” or “therapy” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject 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 subject 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 subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]). As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor of PI3K) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. As used herein, the term “before the treatment” denotes before a first PI3K inhibitor administration. In some embodiments, the first PI3K inhibitor administration is the first administration under the control of the in vitro method for monitoring the efficiency of the PI3K inhibitor treatment. As used herein, the term “after the treatment” denotes after a first PI3K inhibitor administration, after an administration of a PI3K inhibitor or after a last administration of a PI3K inhibitor. In some embodiments, after the treatment is at least 1 hour after an administration of a PI3K inhibitor. In some embodiments, after the treatment is at least 2 hours after an administration of a PI3K inhibitor. In some embodiments, after the treatment is at least 12 hours after an administration of a PI3K inhibitor. In some embodiments, after the treatment is at least 5 days after a first administration of a PI3K inhibitor. In some embodiments, after the treatment is at least 6 weeks after a first administration of a PI3K inhibitor. In some embodiments, after the treatment is at least 7 weeks after a first administration of a PI3K inhibitor. In some embodiments, after the treatment is at least 12 weeks after a first administration of a PI3K inhibitor. In some embodiments, after the treatment is at least 6 months after a first administration of a PI3K inhibitor. As used herein, the term “biological sample” refers to any biological sample obtained from the subject for the purpose of evaluation in vitro. In some embodiments, the biological sample is a body fluid sample. Examples of body fluids are blood, serum, plasma, amniotic fluid, brain/spinal cord fluid, liquor, cerebrospinal fluid, sputum, throat and pharynx secretions and other mucous membrane secretions, synovial fluids, ascites, tear fluid, lymph fluid and urine. As used herein, the term “blood sample” means a whole blood sample obtained from the subject. In some embodiments, the sample is a tissue sample. As used herein, the term "tissue", when used in reference to a part of a body or of an organ, generally refers to an aggregation or collection of morphologically similar cells and associated accessory and support cells and intercellular matter, including extracellular matrix material, vascular supply, and fluids, acting together to perform specific functions in the body. There are generally four basic types of tissue in animals and humans including muscle, nerve, epithelial, and connective tissues. In some embodiments, the tissue sample is a biopsy sample. In some embodiments, when the subject suffers from a cancer, the tissue sample is a tumor tissue sample. As used herein, the term “tumor tissue sample” means any tissue tumor sample derived from the patient. In the context of the invention, the term “a biological sample” may be understood as a single biological sample for one metabolite, one biological sample per metabolite when the level of two or more metabolites is determined or at least two biological sample when the level of two or more metabolites is determined. In some embodiments, the sample is a plasma sample. As example, the sample is a plasma sample when the at least one metabolite is acetyl-carnitine, argininosuccinate, carnitine, creatine, glycine, hexanoyl-carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan and/or urate. In some embodiments, the sample is a urine sample. As example, the sample is a urine sample wherein the at least one metabolite is alpha-ketoglutarate, butyric acid and/or cis aconitate. In some embodiment, the sample is a urine sample wherein the at least one metabolite is butyric acid and/or cis aconitate. In some embodiments, the level of at least one metabolite as described above is determined by immunoassay. As used herein, the term “Immunoassays” encompass any assay wherein a capture reagent (i.e binding partner) is immobilized on a support and wherein detection of an analyte of interest (i.e at least one metabolite) is performed through the use of antibodies directed against the said analyte of interest (i.e at least one metabolite). Such assays include, but are not limited to agglutination tests; enzyme-labeled and mediated immunoassays, such as enzyme- linked immunosorbent assays (ELISAs); biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis-mass spectroscopy technique (CE-MS) etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. Immunoassays includes competition, direct reaction, or sandwich type assays. Typically, the antibody against at least one metabolite is labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labelled", with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer may be labelled with a radioactive molecule by any method known in the art. For example, radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as I<123>, I<124>, In<111>, Re<186>, Re<188>. Preferably, the antibodies against at least one metabolite are already conjugated to a fluorophore (e.g. FITC- conjugated and/or PE-conjugated). In a particular embodiment, the antibody according to the invention is conjugated with a detectable label. In a particular embodiment, the detectable label is a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label or a bioluminescent label. In a particular embodiment, the label is selected from the group consisting of β-galactosidase, glucose oxidase, peroxidase (e.g. horseradish perodixase) and alkaline phosphatase. In some embodiments, the level of at least one metabolite is determined by enzyme- labeled and mediated immunoassays (ELISA). In some embodiments, the level of at least one metabolite is determined by direct ELISA. The at least one metabolite is directly immobilized to a surface of a multi-well plate and detected with a biotin-conjugated detection antibody specific for the at least one metabolite. This antibody is directly conjugated to a detection system (horseradish peroxidase (HRP)-conjugated Streptavidin or other detection molecules). In some embodiments, the level of at least one metabolite is determined by indirect ELISA. The at least one metabolite is directly immobilized to a surface of a multi-well plate and detected with an unconjugated primary detection antibody specific for the at least one metabolite. A conjugated secondary antibody directed against the host species of the primary antibody is then added. Substrate then produces a signal proportional to the amount of the at least one metabolite bound in the well. In some embodiments, the level of the at least one metabolite is determined by sandwich ELISA. According to the invention, “sandwich” ELISA refers to an immunoassay wherein free at least one metabolite may be sandwiched between two antibodies that specifically bind to free at least one metabolite. In some embodiments, the level of at least one metabolite is determined by Immunohistochemistry (IHC). In some embodiments, the level of at least one metabolite is determined by Immunohistochemistry (IHC) when the sample is a tissue sample. For example, the quantification of the level of at least one metabolite is performed by contacting a tissue sample with binding partners (e.g. antibodies) specific for the at least one metabolite. Immunohistochemistry typically includes the following steps i) fixing the tissue sample with formalin, ii) embedding said tissue sample in paraffin, iii) cutting said tissue 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 tissue sample is firstly incubated with the binding partners, such as antibodies. 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. Hematoxylin & Eosin, 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 metabolite (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 J R (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 or 125I) 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. The resulting stained specimens are each 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, or the absolute number of cells positive for the maker of interest, or the surface of cells positive for the maker of interest. Various automated sample processing, scanning and analysis systems suitable for use with IHC 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 metabolite (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. 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 metabolite (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%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more positive pixels as compared to the total staining area. For example, the amount can be quantified as an absolute number of cells positive for the maker of interest. 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 metabolite (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 metabolite (e.g. an antibody as above described), ii) proceeding to digitalisation of the slides of step i).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 or the absolute number of stained cells in each unit whereby the number or the density of cells stained of each unit is assessed. In some embodiments, the level of at least one metabolite is determined by mass spectrometry. In some embodiments, the level of at least one metabolite is determined by mass spectrometry when the sample is a blood sample. Mass spectrometry (MS) is an analytical technique used to measure a mass-to-charge ratio of ions in pure samples as well as complex mixtures. Results are depicted in a spectrum. The spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical identity or structure of molecules and other chemical compounds. In a typical MS procedure, the sample may be solid, liquid, or gaseous. The sample is ionized, for example by bombarding it with a beam of electrons. Some of the sample's molecules break up into positively charged fragments or simply become positively charged without fragmenting. These ions (fragments) are then separated according to their mass-to-charge ratio, for example by accelerating them and subjecting them to an electric or magnetic field. The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. Results are displayed as spectra of the signal intensity of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern. As used herein, the term “efficient” denotes a state wherein the administration of one or more drugs to a subject permit to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or to prolong the survival of a subject beyond that expected in the absence of such treatment. A "therapeutically effective amount" is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds 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 or coincidential with the specific compound employed; 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, especially from 1 mg to about 100 mg of the active ingredient. In some embodiments, the daily dosage of the drug is varied over a range from 25 to 250 mg per day. An effective amount of the drug is 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. In some embodiments, the daily dosage of the drug is varied over a range from 0.5 to 6 mg/kg. In some embodiments, the starting dose of the drug is at least 250 mg per day if the subject is an adult. In some embodiments, the starting dose of the drug is at least 50 mg per day if the subject is a child. In some embodiments, the starting dose of the drug is at least 25 mg per day if the subject is an infant. Method of treating PROS In a second aspect, the present invention relates to a method of treating a subject in need thereof with a PI3K inhibitor comprising a step of performing the in vitro method for monitoring the efficiency of a PI3K inhibitor treatment and a step of continuing the treatment with the PI3K inhibitor if the treatment is efficient. In some embodiments, the present invention relates to a method of treating PROS in a subject in need thereof comprising a step of performing the in vitro method for monitoring the efficiency of a PI3K inhibitor treatment and a step of continuing the treatment with the PI3K inhibitor if the treatment is efficient. In some embodiments, the present invention relates to a method of treating cancer in a subject in need thereof comprising a step of performing the in vitro method for monitoring the efficiency of a PI3K inhibitor treatment and a step of continuing the treatment with the PI3K inhibitor if the treatment is efficient. In some embodiments, the PI3K inhibitor is BYL719 (alpelisib) as described above. As used herein, the term “continuing” means not permanently discontinuing the treatment, in particular the administration of a PI3K inhibitor. Continuing the treatment encompass maintaining the treatment at the same posology or adjusting the posology (e.g. increasing or decreasing the dosage, increasing or decreasing the administration interval of the drug). Drugs targeting Warburg effect for use in PROS In a third aspect, the present invention relates to at least one drug targeting Warburg effect for use in a method of treating a subject suffering from PROS. In some embodiments, the at least one drug targeting Warburg effect is a drug targeting cancer metabolism. In particular, the drug targeting cancer metabolism is a drug used to alleviate or eradicate Warburg effect in PROS. In some embodiments, the present invention relates to i) at least one drug targeting cancer metabolism and ii) a PI3K inhibitor, as a combined preparation for use in the treatment of PROS in a subject in need thereof. In some embodiments, the PI3K inhibitor is BYL719 (alpelisib). As used herein, “a drug targeting cancer metabolism” denotes a molecule able to inhibit metabolic pathways or interfere with metabolites necessary for cancer progression. In some embodiments, the drug targeting cancer metabolism is gemcitabine, fludarabine, 6-mercaptopurine, enasidenib, AG-221, ivosidenib, AG-120, AG-881, IDH305, BAY1436032, FT-2102, metformin, leflunomide, bempedoic acid, chloroquine, hydroxycholoroquine, 5-(N-ethyl-N-isopropyl) amiloride, CPI-613, IM156, IACS-010759, IPN60090, DRP-104, AZD-3965, AG-270, SM-88, indoximod, apacadostat, epacadostat, oxythiamine, 6-aminonicotinamide, dehydroepiandrosterone, kongic acid, AZD3965, STF-31, BAY-876, glutor, 2-deoxyglucose, benitrobenrazide, NCT-503, PH-755, AGF347, SHIN2, GNE-140, NCI-006, GSK28387808A, silybin, phloretin, cytochalasin B, fasentin, 3- bromopyruvate, 2-deoxy-D-glucose, lonidamine, phosphonoacetohydroxamate, SF2312 or 3- (3-pyridinyl)- 1-(4-pyridinyl)-2-propen-1-one, sulfasalazine, 6-diazo-5-oxo-L-norleucine (DON), CB-839, IPN60091 or bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3, GSK1940029, C75, TVB-2640, TOFA, ND-646, VY-3-135, bempedoic acid or NDI-091143, NCT-503, PH-755, AGF347, SHIN2, methotrexate, 6-mercaptopurinol, 6-mercaptopurine, 6- thioguanine, mycophenolic acid, hydroxyurea, purine analogues, leflunomide, brequinar, capecitabine, aminopterin, AG-636, 5-fluorouracil or pemetrexed. Many examples of drugs targeting cancer metabolism and their mode of action are also described in Stine, Zachary E et al. or in Lv, Jing et al. These drugs include inhibitors of glucose metabolism, inhibitors of glutamine metabolism, inhibitors of fatty acid synthesis or inhibitor of nucleotide synthesis (Stine, Zachary E et al. “Targeting cancer metabolism in the era of precision oncology.” Nature reviews. Drug discovery, 1–22.3 Dec.2021; Lv, Jing et al. “The greedy nature of mutant RAS: a boon for drug discovery targeting cancer metabolism?.” Acta biochimica et biophysica Sinica vol.48,1 (2016): 17-26). Thus, in some embodiments, the at least one drug targeting cancer metabolism is an inhibitor of glucose metabolism, an inhibitor of glutamine metabolism, an inhibitor of fatty acid synthesis or an inhibitor of nucleotide synthesis. As used herein, the term “an inhibitor of glucose metabolism” refers to a compound able to inhibits a step in the biochemical process responsible for the metabolic formation, breakdown, and interconversion of carbohydrates in an organism. As example, an inhibitor of glucose metabolism may be kongic acid, AZD3965, STF-31, BAY- 876, glutor, 2-deoxyglucose, benitrobenrazide, NCT-503, PH-755, AGF347, SHIN2, GNE- 140, NCI-006, GSK28387808A, silybin, phloretin, cytochalasin B, fasentin, 3-bromopyruvate, 2-deoxy-D-glucose, lonidamine, phosphonoacetohydroxamate, SF2312 or 3-(3-pyridinyl)- 1- (4-pyridinyl)-2-propen-1-one. As used herein, the term “an inhibitor of glutamine metabolism” refers to a compound able to inhibits a step in the biochemical process responsible for the metabolic formation, breakdown, and interconversion of glutamine in an organism. As example, an inhibitor of glutamine metabolism may be sulfasalazine, 6-diazo-5-oxo-L- norleucine (DON), CB-839, IPN60091 or bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2- yl)ethyl sulfide 3. As used herein, the term “an inhibitor of fatty acid synthesis” refers to a compound able to inhibits a step in the biochemical process responsible for fatty acid synthesis. As example, an inhibitor of fatty acid synthesis may be GSK1940029, C75, TVB-2640, TOFA, ND-646, VY-3-135, bempedoic acid or NDI-091143. As used herein, the term “an inhibitor of nucleotide synthesis” refers to a compound able to inhibits a step in the biochemical process responsible for nucleotide synthesis. As example, an inhibitor of nucleotide synthesis may be NCT-503, PH-755, AGF347, SHIN2, methotrexate, 6-mercaptopurinol, 6-mercaptopurine, 6-thioguanine, mycophenolic acid, hydroxyurea, purine analogues, leflunomide, brequinar, capecitabine, aminopterin, AG-636, 5- fluorouracil or pemetrexed. In some embodiments, the PI3K inhibitor is BYL719 and the drug targeting cancer is an inhibitor of glucose metabolism, an inhibitor of glutamine metabolism, an inhibitor of fatty acid synthesis or an inhibitor of nucleotide synthesis. As used herein, the term “combined preparation” also called as “combined therapy” or “therapy combination” refers to a treatment that uses more than one medication. The combined therapy may be dual therapy or bitherapy. In the context of the invention, the term “combined preparation” denotes the use of PI3K and another compound, such as a drug targeting cancer metabolism, for simultaneous, separate or sequential use. As used herein, the term “administration simultaneously” refers to administration of at least two or three active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of at least two or three active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of at least two or three active ingredients at different times, the administration route being identical or different. In the context of the invention, the term “simultaneous use” denotes the use of a PI3K inhibitor and at least one drug targeting cancer metabolism occurring at the same time. In the context of the invention, the term “separate use” denotes the use of a PI3K inhibitor and at least one drug targeting cancer metabolism not occurring at the same time. In the context of the invention, the term “sequential use” denotes the use of a PI3K inhibitor and at least one drug targeting cancer metabolism occurring by following an order. The PI3K inhibitor and the drug targeting cancer metabolism as described above can be used as part of a multi-therapy for the treatment of PROS in a subject in need thereof. The PI3K inhibitor can be used alone as a single inhibitor or in combination with other drugs like drugs targeting cancer metabolism. When several drugs are used, a mixture of drugs is obtained. In the case of multi-therapy (for example, bi-, tri- or quadritherapy), at least one other drug can accompany the PI3K inhibitor. In a particular embodiment, the PI3K inhibitor and the drug targeting cancer metabolism can be combined as a bi-therapy for use in the treatment of PROS in a subject in need thereof. In a particular embodiment, the PI3K and the drug targeting cancer metabolism can be combined for use as a bi-therapy, wherein the PI3K inhibitor is BYL719 and the drug targeting cancer metabolism is an inhibitor of glucose metabolism, an inhibitor of glutamine metabolism, an inhibitor of fatty acid synthesis or an inhibitor of nucleotide synthesis. The PI3K inhibitor as described above may also be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer 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, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. 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. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. 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. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Kits In a fourth aspect, the present invention relates to a kit for use in an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof, said kit comprising: - a solid support, - a binding partner against at least one metabolite, and - instructions for use. As used herein, the term “solid support” denotes a firm support able to contain reactants. The reactants specifically allow for the determination of the level of at least one metabolite. As example, the solid support can be made of polystyrene, polyethylene, polyacrylamide, agarose, glass or silicone rubber. The solid support can be, as example, a microtiter plate, a bead or a small tube. In some embodiments, one reactant is immobilized on the solid support. In some embodiments, at least one reactant is labelled in order to be detected and quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive, fluorescent labelling chemioluminescent, enzymatic labels or dye molecules. As used herein, the term “binding partner against at least one metabolite” denotes an antibody, an immunoglobulin, an aptamer, a nucleic acid sequence, a ligand or a receptor protein addable in or immobilized on the solid support in order to measures the presence or concentration of the at least one metabolite. In some embodiments, the at least one metabolite is a TCA cycle metabolite. In some embodiments, the at least one metabolite is a TCA cycle metabolite selected in the group consisting of alpha-ketoglutarate, cis-aconitate, succinic acid, citrate, isocitrate, succinyl-CoA, fumarate, malate, oxaloacetate. In some embodiments, the at least one metabolite is a TCA cycle metabolite selected in the group consisting of cis-aconitate, succinic acid, citrate, isocitrate, succinyl-CoA, fumarate, malate, oxaloacetate. In some embodiments, the at least one metabolite is selected in the group consisting of alpha-ketoglutarate, cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl- lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl- carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate. In some embodiments, the at least one metabolite is selected in the group consisting of cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L- dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate. In some embodiments, the at least one metabolite is alpha-ketoglutarate, cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L- dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan and urate. In some embodiments, the at least one metabolite is cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl- carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan and urate. In some embodiments, the at least one metabolite is selected in the group consisting of alpha-ketoglutarate, cis aconitate, acetyl-carnitine, argininosuccinate, carnitine, creatine, glycine, hexanoyl-carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan, urate, butyric acid. In some embodiments, the at least one metabolite is selected in the group consisting of cis aconitate, acetyl-carnitine, argininosuccinate, carnitine, creatine, glycine, hexanoyl-carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan, urate, butyric acid. In some embodiments, the at least one metabolite is alpha-ketoglutarate, cis aconitate, acetyl-carnitine, argininosuccinate, butyric acid, carnitine, creatine, glycine, hexanoyl- carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan and urate. In some embodiments, the at least one metabolite is cis aconitate, acetyl-carnitine, argininosuccinate, butyric acid, carnitine, creatine, glycine, hexanoyl-carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan and urate. In some embodiments, the kit according to the invention may comprise instructions for: i) determining whether a PI3K inhibitor treatment is efficient or not; ii) determining whether a PI3K inhibitor treatment is required or not; iii) determining whether a continuation of a PI3K inhibitor treatment is required or not; The instructions for this purpose may include at least one reference expression profile. In a particular embodiment, the at least one reference expression profile is a responder (i.e. a subject with an efficient therapy) expression profile. Alternatively, at least one reference expression profile may be a non-responder expression profile. In a further embodiment, the reference expression profile is a reference expression level of at least one metabolite. Said reference expression profile can be obtained from a subject: who does not have the medical disorder, for whom the administration of the compound has prevented, cured, delayed the onset of, reduced the severity of, or improved one or more symptoms of the disorder or recurrent disorder, or for which it has prolonged the subject's survival beyond that expected in the absence of such treatment. In some embodiments, the expression level of the at least one metabolite can be determined by any technology known in the art consisting of but not limited to: ELISA, Ella® (automated microfluidic immunoassay, ProteinSimple), Luminex™ technology, high- performance liquid chromatography (HPLC), electrochemiluminescence. In some embodiments, the level of the at least one metabolite as described above is determined by immunoassay. In some embodiments, the level of the at least one metabolite as described above is determined by an enzyme-linked immunoassay (ELISA). Computer-implemented methods In some embodiments, the invention relates to a computer-implemented method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof. In some embodiments, the method as described above is implemented by a computer executing code instructions stored on a memory. In a particular embodiment, the invention relates to a computer-implemented method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the following steps: i) obtaining a biological sample from said subject; ii) determining the level of at least one metabolite selected in the group consisting of alpha-ketoglutarate (aKG), cis-aconitate, succinic acid, 5-methylcytosine, acetyl- carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject iii) incorporating the level(s) determined at step ii) in a software; and iv) concluding that the PI3K inhibitor treatment is efficient when the alpha- ketoglutarate and/or betain levels determined at step ii) are higher than the levels determined at step i) and/or when the cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan and/or urate levels determined at step ii) are lower than the levels determined at step i). In another particular embodiment, the invention relates to a computer-implemented method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the following steps: i) obtaining a biological sample from said subject; ii) determining the level of at least one metabolite selected in the group consisting of alpha-ketoglutarate (aKG), cis-aconitate, succinic acid, 5-methylcytosine, acetyl- carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject iii) incorporating the level(s) determined at step ii) in a software; and iv) concluding that the PI3K inhibitor treatment is efficient when the betain level determined at step ii) is higher than the level determined at step i) and/or when the alpha-ketoglutarate, cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan and/or urate levels determined at step ii) are lower than the levels determined at step i). In another particular embodiment, the invention relates to a computer-implemented method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the following steps: i) obtaining a biological sample from said subject; ii) determining the level of at least one metabolite selected in the group consisting of cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject iii) incorporating the level(s) determined at step ii) in a software; and iv) concluding that the PI3K inhibitor treatment is efficient when the betain level determined at step ii) is higher than the level determined at step i) and/or when the cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl- carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N- oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan and/or urate levels determined at step ii) are lower than the levels determined at step i). In another particular embodiment, the invention relates to a computer-implemented method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the following steps: i) obtaining a biological sample from said subject; ii) determining the level of at least one metabolite selected in the group consisting of alpha-ketoglutarate (aKG), cis-aconitate, succinic acid, 5-methylcytosine, acetyl- carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject iii) incorporating the level(s) determined at step ii) in a software; and iv) concluding that the PI3K inhibitor treatment is efficient when the alpha- ketoglutarate level determined at step ii) is higher than the level determined at step i) and when the cis-aconitate level determined at step ii) is lower than the level determined at step i). In another particular embodiment, the invention relates to a computer-implemented method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the following steps: i) obtaining a biological sample from said subject; ii) determining the level of at least one metabolite selected in the group consisting of alpha-ketoglutarate (aKG), cis-aconitate, succinic acid, 5-methylcytosine, acetyl- carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject iii) incorporating the level(s) determined at step ii) in a software; and iv) concluding that the PI3K inhibitor treatment is efficient when the alpha- ketoglutarate and cis-aconitate levels determined at step ii) are lower than the levels determined at step i). In another particular embodiment, the invention relates to a computer-implemented method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the following steps: i) obtaining a biological sample from said subject; ii) determining the level of at least one metabolite selected in the group consisting of cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject iii) incorporating the level(s) determined at step ii) in a software; and iv) concluding that the PI3K inhibitor treatment is efficient when the cis-aconitate level determined at step ii) is lower than the level determined at step i). 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. FIGURES: Figure 1: PIK3CA gain-of-function mutation is associated in vitro and in vivo with aerobic glycolysis, lactate production and macromolecule synthesis. A. Graphic example of metabolite modification observed in a heatmap of the 50 top metabolite changes observed in PIK3CAWT and PIK3CACAGG-CreER fibroblasts (n=3 per condition) cultured in low glucose conditions. AU: Arbitrary units. B. Graphic example of metabolite modification observed in a heatmap of the 50 top metabolite changes observed in the plasma from PIK3CAWT (n= 16) and PIK3CAAdipo-CreER mice (n= 24). C. 18F-fluorodeoxyglucose uptake in PIK3CAWT (n= 4) and PIK3CAAdipo-CreER mice (n= 8). Figure 2: PIK3CA^Adipo-CreER mainly signals through AKT2. A. Male body weights of PIK3CAWT, PIK3CAAdipo-CreER and PIK3CAAkt2ko mice (n= 15 per group) following Cre recombination. B. Adipose tissue volume quantification in a whole-body T2-weighted magnetic resonance images (MRI) of PIK3CAWT (n= 12), PIK3CAAdipo-CreER mice (n= 9) and PIK3CAAkt2ko (n= 16) mice. C. Amnis ImageStream analysis of WAT adipocytes isolated from PIK3CAWT, PIK3CAAdipo-CreER and PIK3CAAkt2ko mice (n = 5 mice per group). D. Representative H&E staining of WAT of PIK3CAWT, PIK3CAAdipo-CreER mice and PIK3CAAkt2ko mice. Scale bar: 10 ^m. E. Western blot quantification of p110, P-AKTser473, P-S6RP and adiponectin in WAT of PIK3CAWT (n =9), PIK3CAAdipo-CreER (n=9) and PIK3CAAkt2ko (n=10) mice. AU: Arbitrary units. F. Twelve-hour fasted glycemia in PIK3CAWT (n= 13), PIK3CAAdipo-CreER mice (n= 9) and PIK3CAAkt2ko (n= 10) mice. G. Insulin circulating levels in PIK3CAWT (n= 8), PIK3CAAdipo- CreER (n= 7) and PIK3CAAkt2ko (n= 6) mice. H. Western blot quantification of adiponectin in WAT of PIK3CAWT (n =9), PIK3CAAdipo-CreER (n=9) and PIK3CAAkt2ko (n=10) mice. I. Circulating IGF-1 levels in PIK3CAWT (n= 13), PIK3CAAdipo-CreER (n= 14) and PIK3CAAkt2ko (n= 8) mice. J. IGFBP1 mRNA expression in the livers of PIK3CAWT, PIK3CAAdipo-CreER and PIK3CAAkt2ko mice (n= 8 per group). K. Graphic example of metabolite modification observed in a heatmap of the 50 top metabolite changes observed in the plasma from PIK3CAWT (n= 16) and PIK3CAAdipo-CreER mice (n= 16). Figure 3: Alpelisib prevents and reverses adipose tissue overgrowth in PIK3CA^Adipo- ^CreER mice and only partially corrects endocrine disruption. A. Male body weights of PIK3CAWT (n=

with either vehicle (n= 14) or preventive (n= 3) or curative alpelisib (n= 5). B. Adipose tissue volume quantification in a whole-body T2- weighted magnetic resonance images (MRI) of PIK3CAWT (n= 14), PIK3CAAdipo-CreER vehicle- treated (n= 14), PIK3CAAdipo-CreER-treated with preventive alpelisib (n= 3) and PIK3CAAdipo- CreER-treated therapeutic alpelisib (n= 5) mice. C. Representative H&E staining of WAT of PIK3CAWT and PIK3CAAdipo-CreER mice treated with either vehicle or preventive or curative alpelisib. Scale bar: 10 ^m. D. Amnis ImageStream analysis of WAT adipocytes isolated from PIK3CAWT and PIK3CAAdipo-CreER mice treated with vehicle, preventive or curative alpelisib (n= 5 per group). E. Twelve-hour fasted glycemia in PIK3CAWT (n= 23) and PIK3CAAdipo-CreER mice treated with vehicle (n= 23), preventive (n= 12) or curative (n= 11) alpelisib. F. Insulin circulating levels in PIK3CAWT (n= 8) and PIK3CAAdipo-CreER mice treated with either vehicle (n= 8), preventive (n= 8) or curative (n= 8) alpelisib. G. Circulating IGF-1 levels in PIK3CAWT (n= 7) and PIK3CAAdipo-CreER mice treated with vehicle (n= 8), preventive (n= 8) or curative (n= 8) alpelisib. H. Graphic example of metabolite modification observed in a heatmap of the 55 top metabolite changes observed in the plasma from PIK3CAWT (n= 16), PIK3CAWT treated with alpelisib (n= 7) and PIK3CAAdipo-CreER mice treated with vehicle (n= 24), preventive (n= 8) or curative (n= 8) alpelisib. AU: Arbitrary units. Figure 4: Alpelisib improves adipose tissue overgrowth and metabolic changes in patients with PROS. A. Representative H&E staining in skin biopsies performed in controls (n= 3) and in patients with PROS (n= 6). B. Immunofluorescence quantification. AU: Arbitrary units. C. Circulating IGF-1 levels in patients with PROS before and after alpelisib introduction (n= 12 patients). D. Percentage change of the volume of the preselected lesion in 7 patients. E. Graphic example of metabolite changes observed in serum from patients with PROS before and after alpelisib initiation. Figure 5: Metabolic changes observed in the serum of patients with PROS treated with alpelisib. AU: Arbitrary units. EXAMPLE: MATERIALS AND METHODS Animals For this study, we interbred homozygous R26StopFLP110* (Stock# 012343) mice with Adiponectin Cre-ER mice (Stock# 025124), both obtained from The Jackson Laboratory. We obtained R26StopFLP110*+/- x Adipo Cre-ER+ (henceforth PIK3CAAdipo-CreER) and R26StopFLP110*+/- x Adipo Cre-ER- (henceforth PIK3CAWT) mice. We also generated Adipo Cre-ER+ mice with a homozygous R26StopFLP110* mutation (henceforth PIK3CAAdipo-HO). The p110* protein expressed by R26StopFLP110* mice is a constitutively active chimera that contains the iSH2 domain of p85 fused to the NH2- terminus of p110 via a flexible glycine linker15. To generate tissue-specific p110*-transgenic mice, a cloned loxP-flanked neoR-stop cassette was inserted into a modified version of pROSA26-1, followed by cDNA encoding p110* and then a frt-flanked IRES-EGFP cassette and a bovine polyadenylation sequence (R26StopFLP110*)16. To follow Cre recombination, PIK3CAWT and PIK3CAAdipo-CreER mice were then interbred with Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/Jmice17,18. These mice express a cell membrane-localized tdTomato fluorescent protein in all tissues that is replaced by GFP after Cre recombination. Animals were fed ad libitum and housed at a constant ambient temperature in a 12-h light cycle. Animal procedures were approved by the Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation (APAFIS N°20439-2018121913526398). All appropriate procedures were followed to ensure animal welfare. At the age of 6 weeks, PIK3CAWT and PIK3CAAdipo-CreER mice received a daily dose of 40 mg.kg-1 tamoxifen for 5 consecutive days. Tamoxifen was administered through oral gavage. PIK3CAAdipo-CreERmice were interbred with AKT2-/- mice obtained from The Jackson Laboratory (Stock# 006966) to generate mice with PIK3CA gain-of-function mutations in adipocytes but lacking AKT2 (PIK3CAAdipo-CreER-Akt2ko mice; henceforth PIK3CAAkt2ko). PIK3CAWT and PIK3CAAdipo-CreERmice were treated with the PI3KCA inhibitor alpelisib (MedChem Express, Germany; 50 mg.kg-1 in 0.5% carboxymethylcellulose (Sigma Aldrich), daily p.o.) or vehicle (0.5% carboxymethylcellulose (Sigma Aldrich), daily p.o.). Treatment was started either 1 (preventive study) or 6 weeks (therapeutic study) following Cre induction. The last dose of alpelisib or vehicle was administered approximately 3 hours before sacrifice. All mice were fasted for 12 hours before blood glucose measurement (Accuchek Performa, Roche Diagnostic), MRI and sacrifice. A previously described PIK3CACAGG-CreERmouse model14 was used to isolate fibroblasts for metabolic in vitro experiments. Magnetic resonance imaging (MRI) evaluation All images were acquired with a 4.7-T small-animal MRI system (BioSpec USR47/40; Bruker BioSpin, Billerica, Mass) on the platform “IRM, INSERM U970, Centre de recherche cardiovasculaire de Paris.” Mice underwent whole-body magnetic resonance imaging using 3D T2-weighted sequences with and without fat saturation. Volumetric evaluation by MRI was performed with 3D Slicer software19 and determined by thresholding on T2-weighted sequences. A T2-weighted sequence with fat saturation was used to remove hypersignals induced by water. Volume was calculated by summing images based on the 2D contours and slice thickness. FDG PET-CT Imaging Mice were fasted overnight with free access to water. Mice were then anesthetized (2±0.5% isoflurane in dioxygen) and weighed, and glycemia was measured in blood drawn from the caudal ventral artery using an Accu-Chek® Aviva Nano A (Accu-Chek, France). A 29G needle catheter (Fischer Scientific, France) connected to 5 cm polyethylene tubing (Tygon Microbore Tubing, 0.010" x 0.030"OD; Fisher Scientific, France) was inserted into the caudal vein for radiotracer injection. 9.2±1.5 MBq of 2'-deoxy-2'-[18F]fluoro-D-glucose (FDG; Advanced Applied Applications, France) in 0.2 mL saline was injected via the catheter. Mice were left awake in their cage for 45 min and then installed into the PET-CT dedicated bed. Respiration and body temperature were registered. Body temperature was maintained at 34±2 °C, and anesthesia was controlled according to the breathing rate throughout the entire PET- CT examination. CT was acquired in a PET-CT scanner (nanoScan PET-CT; Mediso Medical Imaging Systems, Hungary) using the following acquisition parameters: semicircular mode, 50 kV tension, 720 projections full scan, 300 ms per projection, and binning 1:4. CT projections were reconstructed by filtered retroprojection (filter: cosine; cutoff: 100%) using Nucline 3.00.010.0000 software (Mediso Medical Imaging Systems, Hungary). Fifty-five minutes post tracer injection, PET data were collected for 10 min in list mode and binned using a 5 ns time window, with a 400-600 keV energy window and a 1:5 coincidence mode. Data were reconstructed using the Tera-Tomo reconstruction engine (3D-OSEM-based manufactured customized algorithm) with expectation maximization iterations, scatter, and attenuation correction. Volumes of interest (VOIs) were delineated on the organs or anatomical structure of interest on PET/CT fusion slices using the PMOD software package (PMOD Technologies Ltd, Zürich, Switzerland). FDG accumulation was quantified as the standard uptake value (SUV), which measures the ratio of the radioactivity concentration in VOI to the whole-body concentration of the injected radioactivity. Blood and Plasma analysis At the end of each experiment, blood samples were collected from the mice in EDTA- coated tubes. To measure blood count, fresh blood samples were analyzed on a hematology analyzer (ProCyte Dx; IDEXX Laboratories) and centrifuged at 500×g for 15 min. The collected plasma concentration was used to determine leptin, (Novus Biologicals, ref#M0B00), adiponectin (Novus Biologicals, ref#MRP300), insulin (Novus Biologicals, ref#NBP2-62853), IGF-1 (Novus Biologicals, ref#MG100) and GH (Ozyme, ref#MOFI00849) circulating levels using enzymatic methods from commercially available kits. Nonesterified fatty acids (NEFAs) were detected with an enzymatic colorimetric assay (Fujifilm; Wako). Total cholesterol (TC), triglycerides and creatinine were measured by IDEXX Laboratories. Oral glucose tolerance test (OGTT) Mice were fasted overnight (15 h) with access to drinking water. All body weights were measured, and tails were carefully cut for blood glucose determination (time point 0). Fresh prepared glucose solution was administered by oral gavage directly in the stomach (1 g glucose/kg body weight of 20% glucose solution in water, (Sigma, ref#G8270)). After 15, 30, 45, 60, 90 and 120 minutes, blood glucose was measured again at all time points. Morphological analysis Mouse tissues were fixed in 4% paraformaldehyde and paraffin embedded. Tissue sections (4 µm thick) were stained with hematoxylin and eosin (H&E). All experiments were performed in gonadal fat pads for white adipose tissues (WAT) and in the interscapular region for brown adipose tissues (BAT). Measurement of adipocyte area HE WAT slides were scanned with a NanoZoomer 2.0HT (Hamamatsu) and analyzed with Qupath-0.2.320. Adipocytes were classified using machine learning, and their area was measured with Fiji21. Immunohistochemistry and immunofluorescence Paraffin-embedded tissue sections (4 μm) were submitted to antigen retrieval protocols using high temperature (120 °C) and high pressure in citrate buffer and a pressure cooker. Sections were then incubated with primary antibodies (Supplementary Table 1). For the immunofluorescence procedure, appropriate Alexafluor-conjugated secondary antibodies (Thermo Fischer Scientific) were incubated on the samples and analyzed using an LSM 700 confocal microscope (Zeiss) or Eclipse Ni-E (Nikon). Immunohistochemistry revelation was performed with appropriate horseradish peroxidase (HRP) linked secondary antibodies and analyzed with E800 (Nikon). STochastic Optical Reconstruction Microscopy (STORM) Paraffin-embedded WAT sections were submitted to antigen retrieval protocols using high temperature (120 °C) and high pressure in citrate buffer and a pressure cooker. Sections were then incubated with Glut 4 primary antibody (Supplementary Table 1), and AlexaFluor 647 antibody was incubated on samples. An oxygen-poor mounting medium was used (50% Vectashield (Vector laboratories; #H1000), 30% glycerol (Sigma) + 2% nPropyl gallate (Sigma; #02370), 20% Tris 1 M pH 8) to activate photoswitchable molecules. Acquisitions were performed using Nikon TiE and NIS-Element (Nikon), and images were reconstructed using the UNLOC plugin for ImageJ software (ImageJ, NHI)22. mRNA analysis mRNAs were quantified in mouse tissues by quantitative PCR with reverse transcriptase (RT–PCR) using CFX Connect (Bio–Rad). Western blot Tissues were crushed and then lysed in RIPA lysis buffer supplemented with phosphatase and protease inhibitors. Protein concentrations were determined through the bicinchoninic acid method (Pierce). Then, protein extracts were resolved by SDS–PAGE before being transferred onto the appropriate membrane and incubated with the primary antibody followed by the appropriate peroxidase-conjugated secondary antibody (dilution 1:10,000). Chemiluminescence was acquired using Chemidoc MP, and bands were quantitated using Image Lab Software (Bio–Rad Laboratories). Imaging Flow Cytometry (ImageStream) During centrifugation of digested WAT, the adipocytes formed a layer at the top of the liquid. The cells were transferred to microtubes, and samples were run on an Imagestream ISX mkII (Amnis) that combines flow cytometry with detailed cell imaging and functional studies. A 40× magnification was used for all acquisitions. Data were acquired with INSPIRE software (Amnis) and analyzed with IDEAS software (v.6.2, Amnis). Glucose Uptake Assay Fibroblasts were collected from PIK3CAWT and PIK3CACAGG-CreER mice using standard methods14. To generate dermal fibroblast cultures, skin samples were minced and incubated at room temperature in 0.05% trypsin-EDTA (Thermo Fisher) solution for 30 min with gentle shaking. Cells were collected by centrifugation at 500 g for 10 min, resuspended in cell culture medium containing 20% FBS, and plated in a T25 flask to establish lines. Fibroblast cultures were grown and maintained in 1×DMEM (Gibco) supplemented with 20% FBS and penicillin/streptomycin (Gibco), with a final concentration of 100 IU penicillin and 500 μg ml−1 streptomycin. Cells at similar population doublings were plated 1:4 from confluent cultures and allowed to grow until ~80% confluence was achieved. The cells were seeded in 24-well tissue culture plates at 5x104 cells/well. Then, the medium was replaced with medium containing 4-OH tamoxifen (1 μM) (Sigma) for 48 h to activate Cre recombinase. The glucose incorporation test was performed by flow cytometry with an AssayGenie BN00905 kit. After 15 hours, the cells were treated with alpelisib or vehicle control with 0.5% FBS for 3 hours. A fluorescent glucose analog was added to the cells for 30 min, and samples were analyzed using Sony SP6800 and Sony SP6800 software. Targeted LC–MS metabolites analyses Blood samples were obtained in EDTA tubes for plasma analysis and EDTA-free tubes for serum analysis. Plasma and serum were obtained after centrifugation of the blood at 500 g for 10 min. Cell, plasma and serum samples were immediately snap-frozen in liquid nitrogen. For the LC–MS analyses, metabolites were extracted as follows. The extraction solution was composed of 50% methanol, 30% ACN, and 20% water. The volume of the added extraction solution was adjusted to the cell number (1 ml per 1 million cells) or plasma and serum volume (200 µl per 10 µl of plasma or serum). After the addition of extraction solution, samples were vortexed for 5 min at 4 C and then centrifuged at 16,000 g for 15 min at 4 C. The supernatants were collected and stored at −80 C until the analyses were performed. LC–MS analyses were conducted using a QExactive Plus Orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe coupled to a Dionex UltiMate 3000 UPLC system (Thermo). External mass calibration was performed using the standard calibration mixture every 7 d as recommended by the manufacturer. Five microliters of each sample was injected onto Zic‐ pHilic (150 mm × 2.1 mm i.d. 5 μm) with the guard column (20 mm × 2.1 mm i.d. 5 μm) (Millipore) for liquid chromatography separation. Buffer A was 20 mM ammonium carbonate and 0.1% ammonium hydroxide (pH 9.2); buffer B was acetonitrile. The chromatographic gradient was run at a flow rate of 0.200 μl/min as follows: for 0–20 min, linear gradient from 80 to 20% B; for 20–20.5 min, linear gradient from 20 to 80% B; for 20.5–28 min, hold at 80% B. The mass spectrometer was operated in full-scan polarity switching mode with the spray voltage set to 2.5 kV and the heated capillary held at 320 C. The sheath gas flow was set to 20 units, the auxiliary gas flow was set to 5 units, and the sweep gas flow was set to 0 units. The metabolites were detected across a mass range of 75–1000 m/z at a resolution of 35,000 (at 200 m/z) with the AGC target set to 106 and a maximum injection time of 250 ms. Lock masses were used to ensure mass accuracy below 5 ppm. Data were acquired with Thermo Xcalibur 4.0.27.13 software (Thermo). The peak areas of metabolites were determined using Thermo TraceFinder 3.3 SP1 software (Thermo) and identified by the exact mass of each singly charged ion and by known retention time in the HPLC column. Patients The study was conducted on 12 patients, including 8 children and 6 females who were followed at Hôpital Necker Enfants Malades. This protocol was approved by the Agence Nationale de Sécurité du Médicament et des Produits de Santé. Written informed consent was obtained from adult patients and from the parents of pediatric patients. Alpelisib was compassionately offered by Novartis. Adult patients received 250 mg/day, and pediatric patients received 50 mg/day14. Alpelisib was taken orally every morning during breakfast. Patients were assessed at regular intervals as previously reported14. When possible, we assessed the volume of adipose tissue overgrowth using MRI for each patient. MR examination was performed using T1, T2 and fat suppression, and T2-weighted imaging sequences were performed before alpelisib (Day 0) introduction and again 6 months after. Volumetric evaluation of adipose tissue malformation was determined by thresholding and manually delineating hypersignal T2 lesions. Volume was calculated by summing images based on the 2D contours and slice thickness. Data analysis and statistics Data were expressed as the means ± s.e.m. Survival curves were analyzed with the Mantel–Cox (log-rank) test. Differences between the experimental groups were evaluated using ANOVA, followed by the Tukey–Kramer post hoc test when the results were significant (P < 0.05). When only two groups were compared, Mann–Whitney tests were used. The statistical analysis was performed using GraphPad Prism software (version 7.0a). RESULTS Mouse model of adipose tissue overgrowth We started by breeding the R26StopFLP110* mouse strain with Adiponectin Cre mice to generate PIK3CAAdipo-CreER animals that express a constitutively overactivated form of PIK3CA upon tamoxifen administration. To follow Cre recombination, PIK3CAAdipo-CreER mice were then interbred with Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP)Luo/J mice17. In all tissues, these mice express a cell membrane-localized tdTomato fluorescent protein that is replaced by GFP after Cre recombination. To overcome developmental issues, we used six-week-old mice that were treated with a daily dose of 40 mg.kg-1 tamoxifen for five days to induce Cre recombination. We observed that starting two weeks after Cre recombination, PIK3CAAdipo-CreER mice progressively gained weight compared to their wild-type littermates (PIK3CAWT^ for ^up to 24 weeks (i.e, the latest follow-up) (data not shown). This was the case for both males and females. We first decided to study mice 6 weeks after tamoxifen administration (i.e., at 12 weeks of age) when the phenotype was well established. Whole-body MRI revealed a subcutaneous and perivisceral adipose tissue content in PIK3CAAdipo-CreER mice approximately double that in controls (data not shown). We sacrificed 20 controls and 20 PIK3CAAdipo-CreER mice 6 weeks after tamoxifen administration. Necropsy examination showed severe and diffuse adipose tissue infiltration but also a reduction in the size of different organs in PIK3CAAdipo-CreER mice compared to control littermates (data not shown). We confirmed that mutant p110 ^ (p110*) and GFP were expressed in the adipose tissue of PIK3CAAdipo-CreER mice (data not shown). As expected, western blot and immunofluorescence studies showed AKT/mTOR pathway activation in WAT (data not shown) and BAT (data not shown) of PIK3CAAdipo-CreER mice. Histological examination revealed anomalies in WAT and BAT with hyperplastic adipocytes (data not shown). We did not observe any other histological anomalies with the exception of the presence of adipose tissue dissociating striated muscles (data not shown). Mechanistically, PIK3CA is involved in cell growth. Using the Amnis ImageStream® system, we confirmed that adipocytes isolated from WAT of PIK3CAAdipo-CreER mice were hypertrophic compared to controls (data not shown). Immunostaining and flow cytometry analysis showed increased adipose tissue infiltration by F4/80+ CD11b+ cells in PIK3CAAdipo-CreER mice compared to controls (data not shown). Blood sampling examination revealed nonregenerative anemia with a low white blood cell count in PIK3CAAdipo-CreER mice. Both lymphocyte and neutrophil counts were reduced (data not shown). Blood and bone marrow flow cytometry analysis revealed peripheral T- CD8+ and B cell reductions (data not shown). We thus created a mouse model that recapitulates adipose tissue overgrowth that we observed in a subset of patients with PROS. Hormonal dysregulation Adipose tissue is a metabolically dynamic organ that synthetizes a certain number of hormones and participates in metabolic homeostasis. PROS patients with adipose tissue involvement often present with metabolic and hormonal dysregulation, such as hypoglycemia and low circulating levels of IGF-112. Hence, we decided to explore the consequences of PIK3CA gain-of-function mutations in adipose tissue on hormones and metabolism. We first examined the expression of adiponectin and leptin, two hormones extremely sensitive to adipose tissue content. We observed in PIK3CAAdipo-CreER mice low adiponectin expression in adipose tissue (data not shown) with increased levels of circulating leptin (data not shown), as observed during obesity. PIK3CAAdipo-CreERmice exhibited lower cholesterol, triglyceride and nonesterified fatty acid (NEFA) levels than controls (data not shown). Excess adipose tissue is usually associated with insulin resistance; thus, we next investigated glycemic control in our PIK3CAAdipo-CreER mouse model. We observed that compared to controls, PIK3CAAdipo-CreER mice were hypoglycemic after fasting (data not shown). Hypoglycemia was even more severe in the homozygous mutant of PIK3CA (referred to as PIK3CAHO), which died rapidly after Cre recombination from severe spontaneous hypoglycemia even without fasting (data not shown). Pancreas histological examination of the PIK3CAAdipo-CreER mice revealed no difference in ^ islet size compared to controls (data not shown). However, circulating insulin levels were reduced (data not shown), with consistent decreased expression of insulin receptors in WAT (data not shown). The oral glucose tolerance test showed a normal insulin response (data not shown). At the cellular level, glucose uptake from the blood into adipocytes is mainly mediated by the GLUT4 transporter after insulin stimulation23. PIK3CA recruits AKT, which in turn controls GLUT4 trafficking to the plasma membrane24-26. We hypothesized that PIK3CA gain- of-function mutation activates AKT and facilitates GLUT4 migration to the cell surface. Since adipocyte culture is challenging, we decided to use the primary culture of fibroblasts derived from our previous model for PIK3CACAGG-CreER mice14 to test our hypothesis. We observed that fibroblasts derived from PIK3CACAGG-CreER mice had an increased uptake of tagged glucose compared to fibroblasts derived from control littermate mice (data not shown). Using STORM technology, we then confirmed in vivo that GLUT4 accumulated at the cell membrane of adipocytes in PIK3CAAdipo-CreER mice compared to controls (data not shown). Indeed, we concluded that PIK3CA gain-of-function mutation enhances GLUT4 trafficking to the plasma membrane through AKT activation, which leads to chronic hypoglycemia with negative feedback on the ^ pancreatic islets and insulin secretion. Insulin levels correlate with IGF-1, so we decided to measure circulating IGF-1 levels in the PIK3CAAdipo-CreER mouse model. We observed that IGF-1 levels were reduced in PIK3CAAdipo-CreER mice compared to controls (data not shown), which is the most likely explanation for the size reduction observed in organs of PIK3CAAdipo-CreER mice (data not shown). However, circulating growth hormone levels were not different between mutant and control mice (data not shown). Liver examination revealed that IGF-1 mRNA synthesis was similar between PIK3CAAdipo-CreER and control mice (data not shown), but IGF-1 protein was significantly reduced in mutant mice (data not shown). Most IGF-1 molecules are bound by one of the members of the IGF-binding protein (IGFBP) family, of which 6 distinct types exist. These proteins bind to IGF-1 with an equal or greater affinity than the IGF-1 receptor and are thus in a key position to regulate IGF signaling globally and locally. We therefore measured IGFBP mRNA levels in mouse livers. Whereas IGFBP2 to 6 mRNA levels did not or only modestly change (data not shown), we observed a 30-fold increase in the IGFBP1 mRNA level (data not shown), which was then confirmed at the protein level (data not shown) in PIK3CAAdipo-CreER mice compared to controls. This finding was consistent with IGFBP1 regulation by insulin, but the fold increase was dramatically above IGFBP1 levels observed during starvation27. Similar observations were made in WAT (data not shown). Low circulating levels of IGF-1 and IGFBP1 evoked liver degradation (data not shown). However, we did not observe any evidence of endoplasmic reticulum stress (data not shown) or an increase in liver ubiquitination (data not shown), which suggests that there were other mechanisms of IGF-1/IGFBP1 degradation. Indeed, PIK3CA gain-of-function mutation in adipose tissue recruits AKT, which addresses GLUT-4 at the cell membrane and allows permanent cell glucose entry and subsequent hypoglycemia. The latter reduces insulin secretion, which in turn dramatically increases IGFBP1 production followed by liver IGF-1 sequestration and a reduction in IGF-1 circulating levels. Indeed, the PIK3CAAdipo-CreER mouse model recapitulates endocrinologic disorders observed in patients. PIK3CA gain-of-function mutation in adipose tissue induces metabolomic reprogramming An increase in glucose uptake to favor cell growth and proliferation is a hallmark of cancer cells28. We therefore explored whether PIK3CA gain-of-function mutations in our model were associated with metabolic switches, such as those observed in cancer29,30. We first explored in vitro the metabolic changes associated with PIK3CA mutation in fibroblasts derived from PIK3CACAGG-CreER and control mice. In normoglycemic conditions (glucose 5 mmol. L-1), we observed that fibroblasts derived from PIK3CACAGG-CreER mice demonstrated a moderate increase in glycolysis, lactate production and NAD+ production compared to PIK3CAWT (data not shown). However, when mutant fibroblasts were placed in low glucose conditions (glucose <1 mmol. L-1), such as in our mouse model, we observed a major shift toward glycolysis with lactate production (Fig.1A and data not shown). PIK3CA mutant fibroblasts were associated with a significant increase in several tricarboxylic acid cycle intermediates, such as citrate, cis- aconitate or succinate, with aerobic lactate production but also a significant increase in nicotinamide and nicotinamide-N-oxide, which are precursors of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). NAD and its reduced form (NADH) were increased (Fig.1A and data not shown), although the change was not significant. The expression of other markers of mitochondrial respiration, such as FAD (an electron carrier) and its precursor, riboflavin, was also elevated (Fig.1A and data not shown). In addition, aerobic glycolysis was associated with anabolic pathways, as assessed by the important accumulation of essential (leucine, isoleucine, lysine, threonine, phenylalanine, methionine, histidine and tryptophan) and nonessential (arginine and tyrosine) amino acids and the increase in fatty acid products such as myristic acid, palmitoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, butyric acid or linoleic acid as well as carnitine consumption (Fig.1A and data not shown). Taken together, these data suggest that the PIK3CA construct that we used was sufficient to induce the Warburg effect with the biosynthesis of macromolecules in fibroblasts. We next speculated whether these findings could be verified in PIK3CAAdipo-CreER mice. Plasma from 12- hour-fasted PIK3CAWT and PIK3CAAdipo-CreER mice was then collected and processed for metabolomic analysis. Plasma derived from PIK3CAAdipo-CreER mice showed lactate production with increased levels of nicotinamide N-oxide and riboflavin, both of which are involved in mitochondrial respiration, compared to controls (Fig. 1B and data not shown). Similar to PIK3CA mutant fibroblasts, we observed activation of anabolic pathways with amino acid accumulation (leucine, isoleucine, lysine and oxoadipate its metabolism intermediates, acetyl- lysine, phenylalanine, thymidine, tyrosine, valine, hydroxy-L-proline, arginine and ornithine, its subsequent metabolite that in turn will lead to citrulline) or consumption (tryptophan) (Fig. 1B and data not shown). We also noticed that the methionine/cysteine pathway was altered and profound modification of several metabolites and enzymes involved in their metabolism, such as betaine, L-sarcosine, pyridoxal, cysteine sulfinic acid, S-adenosyl-L-homocysteine and cystathionine (Fig.1B and data not shown). Aside from protein synthesis, several metabolites involved in ribo- and deoxyribonucleic acid synthesis, such as 5-methylcytosine and cytidine, were significantly elevated, which evokes nucleotide biosynthesis (Fig. 1B and data not shown). Fatty acid metabolism (decanoic acid, ac arachidonic, eicosapentaenoic acid, aminoadipate, dodecanoic acid/lauric acid, linoleic acid, linolenic acid, oleic acid and palmitic acid) was also profoundly affected in PIK3CAAdipo-CreER mice, which is evidence of lipid synthesis (Fig. 1B and data not shown). Thus, plasma derived from PIK3CAAdipo-CreER mice showed metabolic evidence of aerobic glycolysis with activated anabolic pathways. To confirm the metabolic switch observed in PIK3CAAdipo-CreER mice, we performed FDG uptake. We consistently observed a significant increase in WAT FDG uptake in PIK3CAAdipo-CreER mice similar to that in patients with PROS14 and adipose tissue overgrowth, thus demonstrating the Warburg effect (Fig.1C). PIK3CA gain-of-function mutations in adipose tissue partially signal through AKT2 In mammals, three distinct genes encode AKT homologs—AKT1, AKT2 and AKT3— and AKT2 is known to be the main isoform in WAT and BAT24. AKT2-/-mice have mild growth deficiency, progressive lipoatrophy and altered glucose homeostasis with moderated hyperglycemia and hyperinsulinemia, which progressively leads to ^ islet failure24. We next sought to investigate whether PIK3CA gain-of-function mutations were signaling mainly through AKT or other downstream targets. To answer this question, we interbred AKT2-/- mice with PIK3CAAdipo-CreER animals to obtain PIK3CAAdipo-CreE-Akt2ko mice (henceforth PIK3CAAkt2ko). PIK3CAAkt2ko mice were indistinguishable from other mice for the first 6 weeks. At the age of 6 weeks, mice were given a daily dose of tamoxifen 40 mg.kg-1 for five days to induce Cre recombination. Following Cre induction, PIK3CAAdipo-CreER mice gained excessive weight but the body weights of PIK3CAAkt2ko mice did not differ from those of PIK3CAWT control littermates (Fig.2A and data not shown). At 13 weeks post tamoxifen administration (i.e., at 19 weeks of age), MRI showed no excess of subcutaneous and perivisceral adipose tissue in PIK3CAAkt2ko mice (Fig.2B). Mice were then sacrificed, and necropsy examination confirmed that PIK3CAAkt2ko mice were indistinguishable from control littermates. Mice had no excess adipose tissue, partial correction in organ weights (data not shown) and F4/80 CD11b+ cell WAT infiltration (data not shown). Deletion of AKT2 was associated with a complete correction of hemoglobin and white blood cell anomalies (data not shown). Histological examination and ImageStream analysis showed that the adipocyte size of PIK3CAAkt2ko mice was partially rescued compared to that of PIK3CAAdipo-CreER mice (Fig. 2C, 2D and data not shown). AKT and S6RP phosphorylation in the WAT of PIK3CAAkt2ko mice was only partially blunted compared to that of PIK3CAAdipo-CreER mice (Fig. 2E). Interestingly, although fasting glycemia was higher in PIK3CAAkt2ko than in PIK3CAAdipo-CreER mice, blood glucose levels remained lower than those of controls (Fig.2F). The oral glucose tolerance test showed a peak of glycemia in PIK3CAAkt2ko mice superposable to control mice at 15 min, followed by a rapid decline in glycemia (data not shown). We then checked GLUT4 expression and observed persistent expression at the adipocyte cell membrane in PIK3CAAkt2ko mice (data not shown). GLUT4 expression has already been shown to be unaffected in AKT2-/- mice24. Consistent with GLUT4 cell membrane expression, circulating levels of insulin (Fig. 2G), adiponectin expression in WAT (Fig. 2H), circulating levels IGF1 (Fig. 2I), and IGFBP1 mRNA in the liver (Fig.2J and data not shown) were not corrected. We then explored the metabolomic consequences of AKT2 deletion in our model. Analysis of the sera derived from PIK3CAAkt2ko mice showed partial correction of several metabolic anomalies, such as lactate production, carnitine and its derivatives (butyryl carnitine, hexanoyl carnitine, octanoyl carnitine) and nicotinamide N-oxide production (both of which are involved in mitochondrial respiration), reduction in amino acid levels (arginine, acetyl lysine, methyl lysine, phenylalanine, thymidine or valine) and the methionine/cysteine pathway (betaine, L-sarcosine, ornithine or pyridoxal) (Fig.2K and data not shown). We interbred mice to ultimately obtain PIK3CAHO deleted for AKT2 (henceforth PIK3CAHO Akt2ko). Importantly, we observed that AKT2 deletion extended the survival of PIK3CAHO mice (data not shown). We concluded that the adipose tissue overgrowth observed in PIK3CAAdipo-CreER mice is driven through AKT2, but since AKT2 deletion did not correct GLUT4 accumulation at the cell membrane or endocrine disorders, it appears likely that these events are regulated through compensatory activation of AKT1. Impact of alpelisib on the mouse model We recently identified alpelisib (BYL719), a PIK3CA inhibitor, as a promising therapeutic option for patients with PROS14. We decided to test whether this molecule was efficient at improving adipose tissue overgrowth in our mouse model. To this end, we used two different approaches. We first administered alpelisib daily starting 48 h after Cre induction to perform a preventive study. We observed that alpelisib-treated PIK3CAAdipo-CreER mice had an overtly normal appearance during the 6 weeks of treatment and a body weight increase similar to that of control mice (Fig. 3A). MRI performed 6 weeks after introduction of alpelisib treatment showed no adipose tissue overgrowth (Fig. 3B). Mice were then sacrificed, and necropsy examination confirmed that alpelisib-treated PIK3CAAdipo-CreER mice had no excess adipose tissue, but organ weights were only partially restored (data not shown). Histology analysis showed that alpelisib-treated PIK3CAAdipo-CreER mice had adipocytes of intermediate size compared to PIK3CAWT (Fig. 3C and 3D) with persistent F4/80+CD11b+ cell infiltration (data not shown). Biologically, hemoglobin and white blood cell counts were not modified by alpelisib (data not shown). Twelve-hour fasting glycemia was not corrected (Fig. 3E), nor were circulating insulin and IGF1 levels (Fig.3F and 3G). Next, we administered alpelisib to PIK3CAAdipo-CreER mice 6 weeks after Cre induction when adipose tissue was already prominent for 6 additional weeks as a therapeutic study. Following alpelisib introduction, we noticed a rapid body weight decrease in alpelisib-treated PIK3CAAdipo-CreER mice (Fig. 3A). T2-weighted MR images performed 7 weeks after the start of treatment (i.e., 12 weeks after Cre induction) did not reveal the presence of adipose tissue overgrowth (Fig.3B). Organ weights were again partially corrected (data not shown), and we noticed a partial recovery in the size of adipocytes (Fig. 3C and 3D). F4/80+CD11b+ cell infiltration in WAT was still persistent (data not shown). Similar to the alpelisib preventive study, blood cell counts were not ameliorated (data not shown), and glycemia partially improved after 12 h of fasting (Fig. 3E). This was consistent with partial rescue of glucose uptake in vitro (data not shown). Both schemes of alpelisib administration were associated with macroscopic improvement of adipose tissue overgrowth but persistent hypoglycemia and endocrine anomalies were less pronounced in the therapeutic scheme. To better decipher the discrepancy between the preventive and therapeutic protocols, we first explored the phosphorylation of AKT and S6RP in WAT at different time points following alpelisib gavage. We observed that phosphorylation of the two proteins was blunted 1.5 h post alpelisib administration and returned to higher values very quickly afterward (data not shown). A peak in glycemia was consistently observed approximately 1.5 h following alpelisib, which thereafter quickly returned to lower values (data not shown). Interestingly, the kinetics of glycemia following alpelisib administration were different between the treatment schemes (data not shown). Blood glycemia rapidly returned to lower values in the preventive group following alpelisib administration, while glycemia improvement was more sustained in the therapeutic protocol. This suggests that the half-life of alpelisib in adipose tissue is relatively short and more sustained when alpelisib is introduced after the disease is already installed. Consistent with what was observed in PIK3CAAdipo-CreER fibroblasts exposed to alpelisib (data not shown), pooled analysis of the sera derived from mutant mice treated with alpelisib was associated with a complete or partial rescue of different metabolites, such as lactates, carnitine and its derivatives (hexanoyl carnitine and octanoyl carnitine), and cytidine (a nucleoside molecule), less accumulation of amino acids such as thymidine and L-sarcosine, and a reduction of pyridoxal (which is involved in the methionine/cysteine pathway) and quinolinic acid (which participates in NAD formation and mitochondrial respiration) (Fig. 3H and data not shown). Indeed, alpelisib was able to correct aerobic glycolysis and reduce the activity of anabolic pathways. Finally, we treated PIK3CAHO with alpelisib and observed extension of mouse survival (data not shown). We concluded that alpelisib improves PIK3CAAdipo-CreER mice and may represent a promising therapeutic for patients with PIK3CA-related adipose tissue overgrowth. Patients with PIK3CA-related adipose tissue overgrowth Adipose tissue overgrowth is a common observation in patients with PROS. We decided to test whether our findings were relevant to patients. Among our cohort, we identified 12 patients with PROS (Table 1) mainly affected by adipose tissue overgrowth who were treated with alpelisib for at least 6 months. We first performed P-AKTSer473, P-S6RP, leptin and GLUT4 immunostaining in skin biopsies from controls and 12 patients with PIK3CA-related adipose tissue overgrowth before alpelisib introduction. Immunofluorescence revealed adipose tissue disorganization with detectable AKT and S6RP phosphorylation in affected patients compared to controls (Figs. 4A and 4B). Importantly, we observed linear GLUT4 accumulation at the cell membrane of adipocytes, as was the case in the PIK3CAAdipo-CreER mouse model, compared to granular cytoplasmic staining in controls (data not shown). Biologically, we observed low circulating levels of IGF-1 in the serum of the 12 patients prior to alpelisib introduction that was fully corrected 6 months following drug introduction (Fig. 4C and data not shown). As previously observed, alpelisib was associated with clinical improvement with aesthetic changes (data not shown). Adipose tissue malformation was accessible to volume measurement using MRI in 6 patients (Table 1). MRI showed a decrease in the mean preselected adipose tissue lesion volume from 4187.1 (interquartile range, 184-14254.8) cm3 prior to alpelisib introduction to 2451.2 (interquartile range, 155-9774) mm3 over 6 months on alpelisib (mean [SD] change -28.6 [18.7] %) (Fig.4D and Table 1). Finally, we explored the metabolomic changes prior to and after alpelisib introduction in this unique cohort. As observed first in vitro and then in vivo, we observed important changes in several serum metabolites. Alpelisib was associated with a reduction in circulating lactate and pyruvate levels, a decrease in the levels of several metabolite intermediates of mitochondrial respiration (carnitine and several metabolites, nicotinamide N-oxide), amino acids (acetyl lysine, glycine, hydroxy- L-proline, tryptophan), TCA intermediates (arginino succinate, aKG, cis aconitate), methionine/cysteine pathway changes (betaine) and fatty acid production (Fig.4E, Fig.5 and data not shown). Thus, plasma derived from patients treated with alpelisib showed metabolic evidence of aerobic glycolysis and anabolic pathway modifications 6 months after drug introduction. CONCLUSION For the present study, we created a mouse model of PIK3CA gain-of-function mutations in adipose tissue. This new model recapitulates the phenotype of a subgroup of patients with PROS and their specific metabolic anomalies, including hypoglycemia with low circulating levels of insulin and IGF-1. We deciphered the mechanism of chronic hypoglycemia observed in this population and demonstrated that these anomalies were mainly driven through AKT2. We showed the efficacy of alpelisib in a mouse model and then confirmed these data in patients. Finally, we showed for the first time that isolated PI3KCA gain-of-function mutations are associated, in preclinical models and in patients, with the Warburg effect and a biosynthesis of macromolecules similar to that in cancer cells.

Table 1: Patient characteristics

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Claims

CLAIMS: 1. An in vitro method for monitoring the efficiency of a PI3K inhibitor treatment in a subject in need thereof comprising the step of determining the level of at least one metabolite selected in the group consisting of cis-aconitate, succinic acid, 5- methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L- dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject.

2. The in vitro method for monitoring the efficiency of a PI3K inhibitor treatment according to claim 1 comprising the steps of : i) determining the level of at least one metabolite selected in the group consisting of cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate, in a biological sample obtained from the subject before the treatment; ii) determining the level of the at least one metabolite in a biological sample obtained from the subject after the treatment; iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the PI3K inhibitor treatment is efficient when the betain level determined at step ii) is higher than the level determined at step i) and/or when the cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl-lysine, argininosuccinate, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl- carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N- oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan and/or urate levels determined at step ii) are lower than the levels determined at step i).

3. The in vitro method according to claim 1 or 2, wherein the at least one metabolite is cis aconitate, acetyl-carnitine, argininosuccinate, butyric acid, carnitine, creatine, glycine, hexanoyl-carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan and urate.

4. The in vitro method according to claim 2 or 3, comprising a step iv) concluding that the PI3K inhibitor treatment is efficient when the cis-aconitate, acetyl-carnitine, argininosuccinate, butyric acid, carnitine, creatine, glycine, hexanoyl-carnitine, lactate, palmitoyl-carnitine, pyruvate, tryptophan and urate levels determined at step ii) are lower than the levels determined at step i).

5. The in vitro method according to claim 2, wherein the step iv) is to conclude that the PI3K inhibitor treatment is efficient when the cis-aconitate level determined at step ii) is lower than the level determined at step i).

6. The in vitro method according to claim 1 to 5, wherein the PI3K inhibitor is BYL719 (alpelisib).

7. The in vitro method according to claim 1 to 6, wherein the subject suffers from a pathology associated with Warburg effect.

8. The in vitro method according to claim 7, wherein the subject suffers from PROS.

9. A method of treating a subject in need thereof with a PI3K inhibitor comprising a step of performing the in vitro method for monitoring the efficiency of a PI3K inhibitor treatment according to claim 1 to 8 and a step of continuing the treatment with the PI3K inhibitor if the treatment is efficient.

10. At least one drug targeting Warburg effect for use in a method of treating a subject suffering from PROS.

11. The at least one drug targeting Warburg effect for use according to claim 10, wherein the at least one drug targeting Warburg effect is a drug targeting cancer metabolism.

12. i) At least one drug targeting cancer metabolism and ii) a PI3K inhibitor, as a combined preparation for use in the treatment of PROS in a subject in need thereof.

13. i) At least one drug targeting cancer metabolism and ii) a PI3K inhibitor, as a combined preparation for use according to claim 12, wherein the PI3K inhibitor is BYL719 and the drug targeting cancer is an inhibitor of glucose metabolism, an inhibitor of glutamine metabolism, an inhibitor of fatty acid synthesis or an inhibitor of nucleotide synthesis.

14. A kit for use in an in vitro method for monitoring the efficiency of a PI3K inhibitor treatment according to claim 1 to 8, said kit comprising: - a solid support, - a binding partner against at least one metabolite, and - instructions for use.

15. The kit according to claim 14, wherein the at least one metabolite is selected in the group consisting of cis-aconitate, succinic acid, 5-methylcytosine, acetyl-carnitine, acetyl- lysine, argininosuccinate, betaine, butyric acid, carnitine, creatine, glucose, glycine, hexanoyl-carnitine, L-fucose, lactate, L-dihydroorotic acid, linolenic acid, nicotinamide N-oxide, palmitoyl-carnitine, panthotenate, pyruvate, quinolinic acid, tryptophan, urate.