WO2020229300A1 - Method for measuring and targeting an oxidative phosphorylation metabolism - Google Patents

Abstract

The invention relates to the implementation of a low-temperature cell test and to a method for determining whether a cell uses for its energy production the oxidative phosphorylation (OxPhos) or glycolysis, wherein the low-temperature sensitivity is indicative of the OxPhos metabolism.

Description

METHOD OF MEASURING AND TARGETING METABOLISM DEPENDING ON OXIDATIVE PHOSPHORYLATION

Technical area

[0001] The present invention provides a novel method for detecting cellular energy metabolism by measuring cold sensitivity of cells relying on oxidative phosphorylation compared to cells using glycolysis to produce their energy.

Background of the invention

[0002] There are two main metabolic pathways that provide vital energy for a cell: glycolysis and oxidative phosphorylation (OxPhos) also called mitochondrial metabolism.

[0003] Although glycolysis leads to a lower energy yield (2 ATP produced by glycolysis against 38 ATP by oxidative phosphorylation), it has been formally shown over the past five decades that the majority of tumor cells adopt glycolysis to produce their energy, independently of the oxygen supply to the tumor - Warburg effect (Warburg 1956) -. However, since 2012, the number of studies demonstrating the existence of subgroups of cancers dependent on OxPhos metabolism has steadily increased (diffuse large B-cell lymphoma, lung cancer, pancreas, acute myeloid leukemia, chronic myeloid leukemia and cancer breast) (Caro et al. 2012; Viale et al. 2014, Hensley et al. 2016, Kuntz et al. 2017; Farge et al. 2017). The discovery of distinct metabolic fingerprints within the same tumor entity has reignited the debate on the exclusive use of glycolysis by tumor cells. In addition, in 2017, three studies showed that OxPhos metabolic status positively influences resistance to treatment and negatively affects patient survival (Kuntz et al. 2017; Farge et al. 2017; Lee et al. 2017). Thus, targeting OxPhos metabolism offers a significant new opportunity to block drug resistance in various cancers, including leukemia (B cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia). [0004] Different methods and apparatus are widely used in research, medicine and industry to measure cellular energy metabolism (described in the example of patent US80975207). No single device or method is able today to determine cellular energy metabolism. Those skilled in the art know that several often independent approaches and several measurement checks are necessary. All current approaches quantify the appearance or disappearance of components involved in metabolic pathways, often directly in the extracellular environment of the cell, such as oxygen consumption-consumption or lactate production. Examples of publications regarding existing metabolic assays include the following references: (Choi, Gerencser, and Nicholls 2009; Pike et al. 2011; Hill et al. 2009; Dranka et al. 2011). Examples regarding glycolysis measurement assays include (Ibrahim-Hashim et al. 2011). The combined measurement of the two pathways is described in (Ferrick, Neilson, and Beeson 2008).

[0005] The methods and embodiments of the invention presented here have a number of significant advantages over the methods of the prior art. For example, the present invention provides a new method which makes it possible to simultaneously distinguish whether a mammalian cell has an OxPhos or glycolytic metabolism, to measure the amplitude of F OxPhos and to kill said cells using F OxPhos. Unlike all other approaches, the method of the present invention can be used at the single cell level, is unaffected by cell density, and can be implemented in many existing measuring devices to provide new metabolic information not obvious to those skilled in the art. In the context of cellular tests, the method makes it possible to determine the metabolic stability of a cell line or to demonstrate a possible deviation over time from the original cell. In the process of discovering new drugs, the present invention makes it possible to identify a new treatment interfering with the OxPhos metabolism and possibly being synergistic when it is combined with the current treatment given in the first line to treat such or such cancer. Finally, since the present invention makes it possible to specifically kill OxPhos cells while sparing glycolytic cells, this approach can be used as an enrichment / purification method to treat a sample composed of a mixture of cells with different energy metabolisms. Summary of the invention

[0006]. The invention relates to the establishment of a low temperature cell test and method for determining whether a cell uses oxidative phosphorylation (OxPhos) or glycolysis for its energy production, the low temperature sensitivity being. indicative of OxPhos metabolism (FIGURE 1).

[0007] Low temperature means thermal energy below a physiological thermal energy level. Preferably, the temperature is in the range of non-cryogenic storage temperatures, with cooling between 2 and 10 degrees Celsius.

[0008] The present invention relates to a method allowing the qualitative and quantitative detection of the energy metabolism used by the cell (s) tested. The present invention relates to a method for high throughput screening of compounds capable of influencing this metabolism. The present invention relates to a method for identifying adjuvant compounds capable of interfering with the metabolic state of cancer cells to improve the therapeutic efficacy of conventional chemotherapeutic treatment.

[0009] In a certain embodiment of the invention, the step of measuring cell sensitivity to low temperature is performed by measuring cell viability.

The measurement can be single or intermittent or continuous over time, it can be expressed as a variation in cell viability relative to a control sample described here.

[0011] Typically, the OxPhos metabolism of a cell sample tested is demonstrated by measuring a loss of viability over a period of time of exposure to the low temperature compared to a control sample described here.

Preferably, the measurement of the loss of 20 to 30% of cell viability relative to a control sample is carried out during the first 48 hours (h) of exposure to the low temperature.

The method can comprise maintaining the tested cell sample at a constant or variable low temperature (s).

The invention relates to a method of classifying the level of OxPhos metabolism of one or more cells by comparison with an appropriate control consisting of either to control cells tested in parallel, or to a control value stored in a database. As shown in FIGURE 2, different controls are suitable such as:

(i) the same cells tested which do not follow the low temperature survival test at 4 degrees Celsius and which are maintained in a condition of optimal physiological survival, for example at 37 degrees Celsius.

(ii) other cells which follow the low temperature survival test at 4 degrees Celsius, cells possibly exhibiting a differentiation profile different from the cells tested and with glycolytic metabolism, and resistance to predefined cold exposure. Optionally, if the sample of cells tested is not healthy, e.g. if the cell sample is from a cancer patient, the control sample can also consist of cells showing a comparable differentiation profile but from a healthy patient, e.g. normal B cells might be an appropriate control if the cell sample tested is a B cell leukemia sample.

(iii) other cells which follow the low temperature survival test at 4 degrees Celsius but which are also in the tested cell sample, with a different differentiation profile, with non-pathological glycolytic metabolism, and resistance to l 'pre-defined cold exposure, eg. normal T cells in a blood sample from a patient with acute myeloid leukemia.

(iv) no other control processed in parallel with the cell sample tested, but a reference level calculated by the average of the responses obtained from a large number of control samples previously tested under the same conditions and stored in a database of data.

[0015] The invention also relates to a method using sensitivity to low temperature to develop methods of identifying compounds which affect the mitochondrial cellular metabolism of cells, for example compounds which affect the metabolism of cancer cells, with the examination of a relevant biomarker, such as sensitivity to low temperatures, related to OxPhos.

[0016] The invention also relates to a method of screening for antidiabetic agents modifying cellular mitochondrial metabolism, e.g. agents targeting respiratory chain complexes, e.g. agents capable of treating type 2 diabetes, e.g. agents with activity similar to metformin and thiazolidinediones. [0017] An object of the present invention is to provide a practical screening system for obtaining a substance useful as an agent for treating a metabolic disease including cancer (in particular an agent capable of sensitizing cancer cells by influencing their metabolism. OxPhos) and diabetes (particularly an agent capable of controlling blood sugar levels).

The invention relates to an in vitro method of evaluating the effectiveness of a treatment modifying the cellular mitochondrial metabolism.

[0019] The method provides a novel way to longitudinally monitor the metabolic state of a mammalian cell in a standard manner, for example, to monitor in vitro the metabolic state of the primary cancer cells of a patient during the evolution of his illness. Alternatively the method provides a new way of monitoring an immortalized cancer cell line to monitor its metabolic stability or its deviation over time.

The invention relates to a method for in vitro measurement of OxPhos or of the glycolysis of normal cells or of cancer cells in a cellular biological sample.

The invention relates to an in vitro method of evaluating the effectiveness of a treatment supposed to modify the cellular mitochondrial metabolism.

The present invention comprises the step of measuring the sensitivity to cold of a sample of cancer cells obtained from a patient, and relates to an in vitro method for diagnosing or prognosing the pathology of said patient.

[0023] The invention also relates to an in vitro method of monitoring a patient for his resistance to drugs, for example chemoresistance. Therefore, the invention can be used to predict and to anticipate the therapeutic response of a patient with cancer, to receive therapy known to be influenced by the energy metabolism of the patient's cancer cells.

[0024] The invention also relates to a method of identifying patients suffering from cancer, likely to respond to a candidate agent modifying cellular mitochondrial energy metabolism, eg. to select patients who might benefit from a particular treatment or clinical trial using a therapeutic agent targeting OxPhos. Finally, the invention relates to a method for enriching and / or purifying cells according to their energy metabolic profile in a sample composed of a mixture of cells with different energy metabolisms, for example by increasing the percentage of cells glycosolytics and decreasing the percentage of OxPhos cells after incubation at low temperature for an appropriate period of time.

[0026] Thus, the invention relates to an in vitro method for eliminating, within a population of mammalian cells, the cells which follow the OxPhos metabolic pathway, by subjecting the population of cells to a temperature of between 2 and 10 ° C. .

Description of figures

Fig. 1

[0027] [Fig. 1] Cells whose energy metabolism relies on mitochondrial oxidative phosphorylation (OxPhos) are more sensitive to low temperature exposure than cells which depend on glycolysis. Dead cells (closed circles) appear faster and accumulate with a greater amplitude in the OxPhos cell population (double lines) compared to glyco lytic cells (single lines).

Fig. 2

[0028] [Fig. 2] Different controls possible for the cellular biological sample of cells tested. An appropriate control cell biological sample can be composed of the same cells as those of the tested cell sample but which does not follow the 4 degree Celsius low temperature survival test, and can be maintained under optimal survival conditions e.g. at 37 degrees Celsius (a). A suitable control cell biological sample can be other cells that follow the 4 degree Celsius low temperature survival test, but with known glycolytic metabolism and resistant to cold exposure (b). A suitable control cell biological sample can be another population of cells within the cell samples tested with a distinct differentiation profile, but with known glycolytic metabolism and resistant to cold exposure (c). A suitable control can be a baseline obtained from the average responses of a large number of control cell samples tested. previously under the same conditions (d). Compared to the control, the sensitivity of the tested cell sample demonstrated by the low temperature survival test at 4 degrees Celsius may be either more sensitive (dotted rectangle) indicative of an OxPhos metabolism or less or equally sensitive (rectangle linear) indicating glycol lytic metabolism.

Fig. 3

[0029] [Fig. 3] A high throughput screening approach for chemical compound libraries to identify compounds for inhibiting or increasing oxidative phosphorylation. Schematic of the 5-step screening design consisting of (i) providing one or more samples of test cells and control cells; (ii) put them in contact with several doses of the library of compounds and incubate at 37 degrees Celsius for a sufficient time determined by a positive control. At this stage, an additional step determining the median toxic dose (TD50) and the median inhibitory concentration (IC50) is a mandatory step in order to censor the false positive conditions increasing cell death without increasing 1 OxPhos; (iii) measuring the viability of the cellular biological sample 6 to 36 hours after incubation at 4 degrees Celsius; (iv) compare the cold sensitivity of the test cell sample with or without incubation with the test compounds; (v) selecting a test compound for which a decrease or an increase in cold sensitivity of the cellular biological sample indicates that the test compound is likely to decrease or increase OxPhos respectively.

Fig. 4

[0030] [Fig. 4] A method of identifying an agent influencing OxPhos having an additive or synergistic effect with conventional chemotherapy for the treatment of cancer. The method comprises two parts, the first part consisting of steps i) to v) described in FIGURE 3 consisting of screening it using the low temperature survival test at 4 degrees Celsius and leading to a selection of anti-OxPhos candidates. The second part of the method consists of a combinatorial study and a selectivity analysis to determine whether these anti-OxPhos candidates could improve the standard anti-cancer treatment of specific cancer.

Fig. 5 [0031] [Fig. 5] Experimental distinction between glycolytic leukemia cells and OxPhos. A: The images show the color change of phenol red based on the acidification of the medium due to the release of lactate from the glycolytic cells for the indicated acute myeloid leukemia (AML) cell lines (for HL-60, NB-4 and U937) compared to OxPhos cells (Kasumi-1, MOLM-14, THP -1). The images displayed well represent the culture with a similar cell density 24 hours after the initial seeding. B: Data shows glucose consumption and lactate production rate at different time points in the extracellular medium of the indicated AML cell lines using the YSI2950 Chemistry Analyzer. AML cells based on the energy metabolism of glycolysis (HL-60, KG-1, NB-4 and U937) have faster glucose consumption and lactate production kinetics compared to AML cells based on energy metabolism from OxPhos.

Fig. 6

[0032] [Fig. 6] Human acute myeloid leukemia (AML) using OxPhos may increase their glycolysis, AML already glycolytic cannot. The glycolytic cells (filled histograms) and OxPhos (squared histograms) were cultured at different concentrations of oxygen 20-3-1% 02 for 72 h then the concentrations of glucose (upper histograms) and lactate (lower histograms) were determined in the different conditioned media and analyzed using the YSI2950 metabolite analyzer. The data show that OxPhos cells sequentially increase their glucose consumption and lactate release at 3 and 1% O2 versus 20% O2 while the glycolytic cells are not impacted.

Fig. 7

[0033] [Fig. 7] Cold exposure distinguishes human acute myeloid leukemia (AML) cells using OxPhos or glycolysis. A: Human AML cells were incubated at 4 ° C for the time indicated and then the incorporation of DAPI was measured by flow cytometry to determine cell viability. Values representative of 8 experiments. Data show that OxPhos cells (OCI-AML2, MV4-11, THP-1, MOLM-14, OCI-AML3, KASUMI-1; light dashed lines) are more sensitive to the 4 degree low temperature survival test Celsius than glycolytic cells (U937, NB-4, KG-1; solid dark lines). B: Average time in hours observed for the 25% loss of cell viability at low temperature for the indicated AML (n = 3 to 5). Cell lines were classified as low or high lactate producer and comparison was made using Mann-Whitney test. **** p <0.0001. C: Sensitivity to cold correlates with lactate production. Average time in hours observed for the 25% loss of cell viability at 4 ° C for the indicated AML as a function of the respective average lactate production rate in femto moles per cell per minute. The parametric Spearman correlation test was applied.

Fig. 8

[0034] [Fig. 8] Resistance to the 4 degree Celsius low temperature survival test is indicative of a persistent glycolytic energy metabolism independent of mitochondrial metabolism. A: The flowchart illustrates the experimental procedure for B-D. Acute myeloid leukemia (AML) cells were treated for 72 h at 37 ° C with rotenone, an electronic transport chain complex I inhibitor, and then subjected to the low temperature survival test. B: Rotenone blocks the sensitivity to cold exposure of OxPhos AML but has no effect on glycolytic AML. C: The images show for similar cell densities, at 72 hours, the color of phenol red dependent on the acidification of the medium for cells cultured without (top) or with (bottom) 0.1 mM of rotenone for 72 h. D: Lactate concentrations of conditioned media of OxPhos cells and glycolytic cells evaluated by the YSI 2950 chemistry analyzer. The Mann-Whitney unpaired test was applied to compare the condition with or without rotenone. * p <0.05. E: The flowchart illustrates the experimental procedure for F. The OxPhos THP-1 cells were treated for 0.16 or 24 or 72 hours at 37 ° C with rotenone and then subjected to the low temperature survival test for 24 hours. F: Only a prolonged inhibition of mitochondrial metabolism is able to block the sensitivity of THP-1.

Fig. 9

[0035] [Fig. 9] OxPhos cells sensitive to the low temperature survival test are also sensitive to inhibition of the cholesterol synthesis pathway. A: Dose-response curve of OxPhos Kasumi-1, MV4-11, MOLM-14, THP-1, SKM-1 cells (lines dotted lines) and glycol lytic cells HL60, KG-1, NB-4, U937 (dark solid lines) treated with Atorvastatin. Cell growth was measured using the WST-1 assay and was plotted as the average percentage of the control (cells not exposed to drugs). AML cells were continuously exposed for 72 h to the indicated concentration of torvastatin. B: mean time in hours observed for the 25% loss of cell viability at 4 ° C for the indicated AML as a function of the 50% inhibitory concentration (IC50) of atorvastatin. IC50 values were calculated from (A) using Graphpad Prism software. The IC50s for HL60, KG-1, NB-4, U937 being above the highest dose tested, could not be determined and indicated by the upper statement out of range (OOR).

Fig. 10

[0036] [Fig. 10] The low temperature survival test enriches glycolytic cells and reduces OxPhos cells from a cell mixture. A: The flowchart illustrates the experimental procedure for B. OxPhos and glycolytic LAM cells were mixed at T0 at the initial ratio of 1: 1 (50% 50%). To distinguish each cell type, OxPhos cells were labeled with the non-diffusible cell proliferation dye eFluor 670 before mixing the two populations. Then the cell mixture was incubated at 4 degrees Celsius for 24 hours and then labeled with propidium iodide before the FACS analysis. B: Glycolytic NB-4 cells were mixed with THP-1 OxPhos cells (left panel group) or with Kasumi-1 OxPhos (right panel group). The frequencies of cells determined at 24 h in each population and subpopulation are shown. Doublets and debris are excluded from the first parental population of the upper panels which show lateral scatter (SSC) as a function of fluorescence intensity of eFluor 670. Lower panels show fluorescence intensity of propidium iodide based on the fluorescence intensity of eFluor 670 to question the viability of distinct populations.

Detailed description of the invention

[0037] The inventors have demonstrated the causal correlation between energy metabolism and the survival of a cell subjected to a low temperature. Cells with cellular energy metabolism based on oxidative phosphorylation mitochondrial die after a few hours of incubation at low temperature. Conversely, cells with cellular energy metabolism based on glycolysis have a much higher viability, or are not impacted when they are placed under the same conditions.

This observation is potentially applicable to all mammalian cells, non-cancerous or cancerous, including leukemia.

[0039] 1. Definitions of terms

[0040] In the present invention, it should be understood that the terms "mitochondrial oxidative phosphorylation" and "mitochondrial metabolism" and "oxidative phosphorylation" and "OxPhos metabolism" and "OxPhos" are equivalent terms. As used herein, the term "OxPhos" refers to the system that provides cellular energy through mitochondria. It is the last stage of cellular respiration consisting of four stages and in which only the first stage occurs in the cytoplasm, while the next three stages occur in and require the mitochondria. The first step in cellular respiration is glycolysis ending with the production of 2 molecules of pyruvic acid. The second stage consists of the formation of 2 molecules of acetyl-coenzyme A. In the third stage, this acetyl-coenzyme A is further degraded (catabolism) during eight stages of enzymatic reactions of the Krebs cycle, also called the cycle of tricarboxylic acid. The gain in steps two and three is the production of high energy compounds, through the reduction of coenzymes. 8 Nicotinamide adenine dinucleotides (NAD) are transformed into 8 NADH and 2 flavin adenine dinucleotides (F AD) are transformed into 2 FADH2. The fourth and final step is oxidative phosphorylation by itself. This step couples the oxidation of these coenzymes with the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). This is the step that produces cellular energy in the mitochondrial ridges. The electron transport chain creates a proton chemiosmotic potential and consists of a gradient of protons across the inner mitochondrial membrane by oxidizing the above coenzymes in the Krebs cycle step. This process regenerates the reduced coenzymes to an oxidized state allowing them to be reused in steps 2 and 3 described above.

[0041] As used herein, the term "anaerobic glycolysis" or simply "glycolysis" refers to the process taking place without the consumption of oxygen leading to the degradation of the oxygen. glucose sugar. In this system, the breakdown of glucose (catabolism) provides energy coupled with the production of GATR. When sugar is catabolized it is only partially broken down and one of its byproducts which is lactic acid. Glycolysis occurs in the cytoplasm independently of the mitochondria. The Warburg effect consists of an increase in the conversion of glucose to lactate. Otto Heinrich Warburg in 1924 observed this phenomenon in tumor cells, even in the presence of normal oxygen levels. Cancer cells show an increased dependence on glycolysis to meet their energy needs, whether they are well oxygenated or not. Glycolysis is less efficient than oxidative phosphorylation because 19 times less ATP is generated per unit of glucose metabolized. Therefore, a higher rate of glucose uptake is required and is observed to meet the same energy needs for cells relying only on glycolysis. Aerobic glycolysis supports various pathways of cancer biosynthesis. The PI3K pathway via AKT1 and mTOR signaling is a major determinant of the glycolytic phenotype (DeBerardinis et al. 2008).

The term "sensitivity" can be defined as a significant and measurable modification of the homeostatic parameters comparing the tested cell sample to the control cell sample or "control" defined here.

[0043] The term "cell viability" or "sample viability" or "cell population survival rate" as used herein can be defined as the number or percentage of cells which are not dead and not distressed in the cell sample tested by the present invention. Measurements of viability can be made by measuring viable cells or by subtracting the measurement of dead cells from the total number of cells. Therefore, the term "viability" used herein refers to the number of total cells tested minus the number of cells undergoing any kind of cell death. In addition, all stages of cell death, individually or at once, can be detected and used to determine viability in the method of the present invention.

The term "the sample of cells tested" or "sample" or "biological sample" is used interchangeably herein and refers to a cell sample from either a subject, preferably a patient, or from a cell line derived from collections of immortalized cell lines bank. Thus, the cellular biological sample can be either of primary origin, which should be understood as originating directly from the patient or of secondary origin. The present invention can be tested with any biological tissue. Such cellular biological samples include, but are not limited to, blood, bone marrow aspirates, tumor biopsies, leukapheresis. In some embodiments, one or more genes in the sample is / are genetically altered qualitatively or quantitatively, e.g. by downregulation or overexpression of certain genes. In some embodiments, the cellular biological sample is genetically engineered to suppress expression of one or more genes. In some embodiments, the sample is genetically engineered to express a mutated form of a gene protein, e.g. an enzyme. In another embodiment, the sample is genetically engineered to express a detectable fluorescent label or a luminescent label. In some embodiments, the genetically modified sample is injected into an immunodeficient recipient mouse for research.

As used herein, the term "patient" refers to a human or other mammalian subject (eg, primate, dog, cat, goat, horse, pig, mouse, rat, rabbit) which may be. affected by a metabolic disorder or cancer. The cancer could be a solid tumor or leukemia. The cancer could be, for example, and not limited to, acute lymphoblastic leukemia (ALL); Acute myeloid leukemia (AML); Adrenocortical carcinoma; Lymphoma related to AIDS; Anal cancer; Astrocytoma; Atypical teratoid / rhabdoid tumor; Basal cell carcinoma of the skin; Cancer of the biliary tract; Bladder cancer; Bone cancer; Breast cancer; Bronchial tumors; Burkitt's lymphoma; Carcinoid tumor; Cardiac tumors; Cancer of the cervix Cholangiocarcinoma; Chordoma; Chronic eosinophilic leukemia; Chronic neutrophilic leukemia; Chronic lymphocytic leukemia (CLL); Chronic myelogenous leukemia (CML); Chronic myeloproliferative neoplasms; Colorectal cancer; Craniopharyngioma; Cutaneous T cell lymphoma; Diffuse large B-cell lymphoma; Ductal carcinoma in situ (DCIS); Embryonic tumors; Germ cell tumors; Endometrial cancer (cancer of the uterus); Ependymoma; Esophageal cancer; Esthesioneuroblastoma (Cancer of the head and neck); Ewing's sarcoma; Ewing's sarcoma (bone cancer); Extracranial germ cell tumor; Extragonadal germ cell tumor; Eye cancer; Fallopian tube cancer; Fibrous histiocytoma of the bone; Cancer of the gallbladder; Cancer of the stomach (stomach); Gastrointestinal carcinoid tumors; Gastrointestinal stromal tumors (GIST); Gestational trophoblastic disease; Tricho leukocyte leukemia; Cancers of the head and neck; Tumors of the heart; Hepatocellular cancer (liver); Hodgkin lymphoma; Hypopharyngeal cancer (cancer of the head and neck); Intraocular melanoma; Islet cell tumors; Kaposi's sarcoma; Kidney (renal cells) Cancer; Langerhans cell histiocytosis; Laryngeal cancer (cancer of the head and neck); Lip and cancer of the oral cavity (cancer of the head and neck); Liver cancer; Lung cancer (non-small cell and small cell); Lymphoma; Male breast cancer; Malignant fibrous histiocytoma; Malignant fibrous histiocytoma of bone and osteosarcoma; Melanoma; Merkel cell carcinoma (skin cancer); Mesothelioma; Metastatic cancer Carcinoma of the midline with changes in the NUT gene; Oral cancer (cancer of the head and neck); Multiple endocrine neoplastic syndromes; Multiple Myeloma Cell Neoplasms / Plasma Neoplasms; Mycosis fungoides (lymphoma); Myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms; Myeloproliferative neoplasms, chronic; The nasal cavity and cancer of the paranasal sinuses (cancer of the head and neck); Nasopharyngeal cancer (cancer of the head and neck); Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Cancer of the oral cavity, cancer of the lips and oral cavity and cancer of the oropharynx (cancer of the head and neck); Oropharyngeal cancer; Osteosarcoma; Osteosarcoma and Malignant Fibrous Histiocytoma of Bone; Ovarian cancer; Pancreatic cancer; Pancreatic neuroendocrine tumors; Papillomatosis; Paraganglioma; Paranasal sinus and cancer of the nasal cavity (cancer of the head and neck); Parathyroid cancer; Penile cancer; Peritoneal cancer; Cancer of the pharynx (cancer of the head and neck); Pheochromocytoma; Pituitary tumor; Plasma cell neoplasm / multiple myeloma; Pleuropulmonary blastoma; Primary CNS lymphoma (lymphoma); Prostate cancer; Rectal cancer; Renal cell cancer (kidney); Retinoblastoma; Rhabdomyosarcoma; Cancer of the salivary glands (cancer of the head and neck); Small intestine cancer; Squamous cell carcinoma of the skin; Squamous cancer of the neck; T cell lymphoma; Testicular cancer; Throat cancer (cancer of the head and neck); Thymoma and thymic carcinoma; Thyroid cancer; Transitional cell cancer of the renal pelvis and ureter (kidney cancer (renal cells)); Primary cancer unknown; Urethral cancer; Cancer of the uterus, endometrium; Vaginal cancer; Vascular tumors (soft tissue sarcoma); Vulvar cancer; Wilms Tumor. [0046] In some embodiments, said patient relapses from a previous medical condition, e.g. acute myeloid leukemia, and said patient has the same leukemia on relapse.

[0047] In another embodiment, said patient relapses from a previous medical condition, e.g. a myelodysplastic syndrome, and said patient has different leukemia at relapse, e.g. acute myeloid leukemia.

The term "treatment" or "therapeutic advantage" should be understood as an act performed on a patient reducing or mitigating at least one side effect or symptom associated with the medical condition of said patient suffering from a metabolic disorder including diabetes and diabetes. Cancer. The term treatment also includes a new adjuvant therapy improving the current average efficiency of standard care according to the most recent landmark publication reporting the results of standard care treatment on a cohort of patients.

[0049] The sample of cells tested by the method of the present invention can come from a patient suffering from cancer, in particular from a patient suffering from leukemia. Said patient may be at different stages of the disease or at different stages of clinical care, e.g. at diagnosis or defined remission phase e.g. by the return to a normal blood cell formula or the disappearance of abundant abnormal cell types. In another embodiment, a cellular biological sample is taken from said patient at the relapse phase of the disease. In another embodiment, a cellular biological sample is from a patient who has participated in a clinical trial.

[0050] In some embodiments, the cell biological sample tested may have been exposed to known methods of treating cancer, including leukemia, for example, and may be treated with chemotherapy or radiation or with cytokines and drugs. growth factors for supportive therapy or with inhibitors targeting kinases. Cell samples tested by the method described here may also have been pre-exposed to pharmacologically active agents, for example, but not limited to the following agents: AMD3100, abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arnifostine , arsenic trioxide, asparaginase, azacitidin, bethamethasone, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, CD44 agonism, cetuximab, chlorambucil, cisplatin, cladabospinazin, cytarofibazin, cytarofarabazin, cytarofarabine dactinomycin, sodium dalteparin, dasatinib, daunorubicin, decitabine, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, epoetin alfa, erlotinibidinibrate, etgrosinibidin, estramuroside, exgrosimidiniboxide, estramustanyrate, etgroxemanyrate, estramustanyidine, estramustanyidine , fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, hydroxyurea, ibritumomab, tiuxetan, idarubicin, ifosfamide, imatinibide 2 mesylate, lenibinfa-alfa, inotuzabidumab, imatinibecanida 2, lenibinfa-alfa, or inotuzabidum , letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalene, mitomycin, mitotane, mitoxantrone, nandrolone, nandrolabetumaromelpropionate, pepamidumparumelase, peparumaromitinate, pepamidumparumelpropionate, pegasidium, peparumaromelpropionate , pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, pl icamycin, prednisolone, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thiuznei-tomino, topecanicin, tricaminoid, toepecan, topecan, toepecan, toepecan, topotecan, thiuzemotin, topecan, topecan, toepecanic valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, zoledronate.

"Low temperature survival test", "cold exposure", "cold challenge", "4 degree Celsius low temperature survival test" are used interchangeably here and relate to sample incubation of Cells tested at a cooling temperature excluding freezing temperatures, and including all aspects and embodiments described herein.

The term "temperature" is defined as a level of thermal energy expressed in degrees Celsius. "Low temperature" means lower than a physiological thermal energy level, at least 10 degrees Celsius lower or at least 20 degrees Celsius lower or at least 30 degrees Celsius lower than the reference physiological mean internal temperature of the specimen of which comes from the cellular biological sample. The physiological reference of 37 degrees Celsius is heard if the cell sample is from a human specimen. Preferably, the temperature is in the range of cooling preservation temperatures between 2 and 10 degrees Celsius. Thus, the term "low temperature" is also intended to mean "cooling preservation conditions". The invention relates to cells of homeothermic mammals and is unrelated to cells of non-homeothermic mammalian species and possibly heterothermic such as e.g. the arctic ground squirrel, Spermophilus parryii or the duck-billed platypus Ornithorhynchus anatinus.

[0053] Terms such as "compound", "candidate compound", "candidate agent", "candidate bioactive agent" or grammatical equivalents herein refer to any molecule, e.g. proteins (i.e., peptides and polypeptides), polysaccharides, small organic or inorganic molecules or polynucleotides. Preferably, the candidate agent is small in size with a molecular weight of less than 3000 Daltons.

[0054] 2. OxPhos measurement method

The inventors have identified a new, non-obvious method to determine whether a cell uses an oxidative phosphorylation metabolism (OxPhos) or a glyco lytic metabolism (GLYCO).

The invention relates to an in vitro method for determining the metabolic pathway followed by a population of mammalian cells to generate ATP, the method comprising measuring the survival rate of the population of cells when subjected to a temperature between 2 and 10 ° C, a high survival rate being associated with the glycolysis pathway and a low survival rate being associated with the oxidative phosphorylation pathway (OxPhos pathway).

[0057] In one embodiment, exposure to the low temperature has no effect on the cell or cells tested.

[0058] In one embodiment, the cell or cells tested is or are sensitive to exposure to the low temperature.

[0059] In one embodiment, the test cell or cells is or are sensitive to exposure to low temperature due to its or their OxPhos energy metabolism (FIGURE 1).

[0060] In one embodiment, the cell or cells tested is or are insensitive to low temperature exposure due to its or their glyco lytic energy metabolism (FIGURE 1).

The method includes maintaining the tested cell sample at low temperature over a period of time. The method can include maintaining the tested cell sample over a period of time at a constant or variable low temperature.

[0063] The set low temperature can be 10 ° C or less, 9 ° C or less, 8 ° C or less, 7 ° C or less, 6 ° C or less, 5 ° C or less, 4 ° C or less, 3 ° C or less, 2 ° C and not lower.

Preferably, the temperature applied is between 3 ° C and 5 ° C inclusive.

[0065] According to a preferred embodiment, the cell population is subjected to a temperature of about 4 ° C.

[0066] In one embodiment, the OxPhos energy metabolism of the cells tested is modified by methods well known in the art (for example pharmacologically or genetically) and results in a modification of their sensitivity to exposure to low temperature. compared to unmodified cells.

In one embodiment, the OxPhos energy metabolism of the cells tested is modified with a different amplitude, which leads to a different amplitude of sensitivity of the exposure to the low temperature compared to the unmodified cells.

In one embodiment, a linear or non-linear regression between the amplitude of the energy metabolism of OxPhos and the amplitude of the sensitivity to exposure to low temperature is established and makes it possible to establish a standard curve. .

[0069] In one embodiment, said sensitivity is demonstrated by measuring cell viability.

[0070] In one embodiment, the method of the invention measuring OxPhos comprises the step of monitoring the sensitivity of the exposure to low temperature through the measurement of the variation in cell viability.

[0071] In some embodiments, the change in cell viability measured during or after exposure to the low temperature is a reduction in viability. The reduction in viability should be understood here as a loss in viability compared to the viability of the control. OxPhos metabolism revealed by sensitivity to low temperature may be admitted for loss of viability of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35 % or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more by compared to a control cell sample.

Preferably, the measurement of at least 20% to 30% variation in cell viability, relative to a control cell sample, is a marker of OxPhos metabolism.

The variation in cell viability can be determined after 5 hours (h), after 8 h, after 16 h, 24 h, 32 h, 40 h, 48 h, 72 h, or 96 h of incubation at low temperature of the tested cell sample.

[0074] According to a preferred embodiment, the measurement of the survival rate is carried out within 48 hours.

Preferably, the measurement of at least 20% to 30% variation in cell viability should be carried out during the first 36 hours of exposure to a constant low temperature of between 3 and 5 degrees Celsius.

The approach to estimate the loss of cell viability can be carried out while maintaining the cell sample at low temperature if the approach and the chosen reading parameter (s) are compatible with the low temperature.

[0077] Some viability tests cannot be performed at low temperature, eg. enzymatic measurement. Thus, in a certain embodiment, the tested cell sample can be transferred after exposure to cold at a permissive conventional temperature, for example 37 degrees Celsius to perform the chosen viability test, for example and not limited to, a colorimetric test of an enzyme involved in cell viability.

[0078] Therefore, all viability tests to measure morphological and / or biochemical characteristics could be applied to determine the sensitivity of the cell sample to exposure to cold.

The invention relates to a method for classifying the level of OxPhos metabolism of one or more cells. Preferably, the level of OxPhos metabolism is established by comparing the tested cell sample response determined by the method of the invention, with a response standard curve generated with multiple control cell samples.

The loss of viability is normalized relative to a control cell sample. The control cell sample can be:

i) a duplicate of the cell sample tested, not following the low temperature survival test at 4 degrees Celsius, and whose viability is determined in vitro shortly after collection from the patient or is determined at the same time from the 'cell sample tested considering that in this case it was maintained under optimized conditions obvious to those skilled in the art (for example at 37 degrees Celsius).

ii) other cells which follow the low temperature survival test at 4 degrees Celsius in parallel with the tested cell sample, other cells possibly showing a differentiation profile different from the cells tested and having a known glyco lytic metabolism and a predefined resistance to cold exposure. Optionally, if the tested cell sample is not healthy, e.g. if the cell sample is from a cancer patient, the control cell sample can also consist of cells showing a comparable differentiation profile but from a healthy patient, e.g. normal B cells could be an appropriate control if the cell sample tested is a B cell leukemia sample or for example a myeloblast or monocyte could be an appropriate control cell sample for myeloid leukemia. Alternatively, healthy cells exhibiting the same extracellular markers could be used as an appropriate control for example normal CD45 + CD34 + cells from a healthy individual without pathology could compose the appropriate control cell sample for the sample tested for acute myeloid leukemia compound of CD34 + cells. In one embodiment, if immunohistochemistry (IHC) is the method chosen to measure the change in cell viability after the low temperature survival test at 4 degrees Celsius, the control population could be the non-tumor cells in section tissue surrounding the tumor population.

iii) other cells which follow the low temperature survival test at 4 degrees Celsius but which are also in the tested cell sample, with a different differentiation profile, with non-pathological glyco lytic metabolism, and resistance to 'pre-defined cold exposure, eg. normal T cells in a blood sample from a patient with acute myeloid leukemia. iv) no other control processed in parallel with the cell sample tested, but a cutoff value determined by the median response of a large cohort of healthy cell samples subjected to the same conditions.

Preferably, the control cell sample is measured at the same time as the samples the tested cell sample.

[0082] The only parameter change between the cell samples subjected to cold and the control cell samples should be the temperature difference. For example and without limitation, the control cell sample must be maintained in the same medium, at the same cell density, in the same plastic culture system, with the same pressure and gas atmosphere.

The viability tests applied can measure morphological parameters such as the loss of impermeability of cell or organelle membranes. The loss of viability induced by the low temperature survival test can be quantified by measuring cellular biological parameters such as the loss of impermeability of cell membranes or that of organelles. Imaging could be done by microscopy. The microscopy could be carried out on living cells or after fixation, for example during an incubation period with a solution containing paraformaldehyde. An immunohistochemistry (IHC) method could be used for imaging in the context of a solid tumor. The IHC specifically provides a method of in situ detection of one or more markers in a tissue sample. The overall cellular organization of the tissue sample is maintained during the IHC allowing identification of the tumor and non-tumor area. Typically, a cell sample is fixed with formalin, embedded in paraffin, and cut into sections for staining and subsequent inspection by light microscopy. For IHC, the appropriate staining procedure to choose to distinguish CKC-induced living / dead cells cannot be affected by the fixation step or must be performed before the fixation step.

[0084] Living cell imaging may be a preferred approach using known simultaneous imaging systems including, for example and without limitation, the EVOS ™ FLoid ™ Cell Imaging Station - Thermo Fisher Scientific, the InCellis cell imager - Bertin Instruments, the BioStation IM-Q - NIKON, the Cytation 5 - BioTek Instruments or the Incucyte - Sartorious. In another embodiment, high throughput cell imaging or HCS (high density) cell screening systems could be used such as Opera, Operetta - PerkinElmer, Celllnsight CX7 - Thermo Scientific. In another embodiment, flow cytometry systems could be chosen such as FACSCalibur FACSCanto Fortessa - BD Biosciences, or MACSQuant® - Miltenyi Biotec or FlowSight® Imaging Flow Cytometer - MilliporeSigma.

In another embodiment, automated cell counters capable of measuring viability and average cell size could be used such as Countess II -ThermoFisher Scientific, Vi-CELL Cell Analyzer - Beckman Coulter or Muse ™ Cell Analyzer - MilliporeSigma .

[0085] In one embodiment, the invention provides a method, performed by a single analyzer, for determining the sensitivity to cold exposure of a cell sample.

[0086] In another embodiment, the sensitivity to cold exposure of a cell sample is determined by more than one testing device.

[0087] Those skilled in the art will further recognize a wide variety of distinct procedures and assay devices for measuring cold sensitivity which may be developed based on the description provided herein.

In some embodiments, the tested cells can be stained with a fluorescent dye prior to analysis.

[0089] In some embodiments, cell viability reagents are used to measure the cold sensitivity of the cell sample being tested.

Cell viability reagents can be DNA stains impermeable to the cell membrane and / or a dye sensitive to mitochondrial metabolic activity and / or probes sensitive to mitochondrial potential. Alternatively, one skilled in the art will further choose a wide variety of distinct viability reagents.

[0091] In some embodiments, cell-impermeable DNA stains can be used to determine the viability of the cell sample being tested, e.g. but not limited to, using PicoGreen® -Invitrogen; Helixyte Green (TM) -AAT Bioquest; 7-AAD (7-aminoactinomycin D); Propidium iodide; SYTOX® AADvanced ™, SYTOX® Blue, SYTOX® Orange, SYTOX® Red, SYTOX ™ Green, Scientific ThermoFisher; DRAQ7 ™, BioLegend; Trypan Blue; DAPI (4 6-diamidino-2-phenylindole).

In some embodiments, the dye sensitive to mitochondrial metabolic activity can be used to determine the cold sensitivity of the cell sample tested, eg, but not limited to, Resazurin; The tetrazolium salts MTT, MTS, XTT and WST-1.

In some embodiments, mitochondrial potential sensitive probes can be used to determine the cold sensitivity of the tested cell sample, e.g. but not limited to, TMRE (tetramethylrhodamine, ethyl ester, perchlorate), JC-1 (5,5 ', 6,6'-tetrachloro-1, r, 3,3'-tetraethylbenzimidazolyl-carbo-cyanine iodide) and JC- 9 (3,3'-dimethyl iodide - [: S-naphthoxazolium) MitoTracker Orange CMTMRos and MitoTracker Red CMXRos ThermoFisher scientific.

[0094] Those skilled in the art will further recognize a wide variety of distinct reagents and protocols for measuring cell viability achievable after the low temperature 4 degree Celsius survival test which can be developed based on the description. provided here.

In certain embodiments, the reagent and the protocol chosen to measure cell viability are not used after the low temperature survival test but during the low temperature survival test, for example if a measurement continues "live. "is performed during the low temperature survival test using the device described above (eg Incucyte - Sartorious).

The cell samples tested can be directly isolated from the patients and then tested directly in vitro in the method described herein without prior culture or other manipulation.

[0097] In another embodiment, a chemical modification could be applied to the cell sample comprising rinsing with an osmotic medium such as phosphate buffered saline or replacing the serum from the original cell sample with a culture centre. In addition, before the replacement of the medium, for example, with RPMI medium, the tested cell sample could also be manipulated in vitro and follow different possible procedures such as density gradient centrifugation, magnetic selection, cell sorting by flow cytometry.

In another embodiment, the cell sample tested could be cells in suspension, or adherent cells. The cell sample can be stored in a single sterile container such as culture flasks or petri dishes or multi-well cultures, 4-well, 6-well, 12-well, 24-well, 48-well, 96-well, 384- well, using a conventional method described.

The adherent cells can be maintained adherent both for the period of cold exposure and for the measurement of viability. Alternatively, the adherent cells can be kept adherent for exposure to cold and then suspended by various methods well known in the art, for example after treatment with trypsin, before analysis of cell viability. Alternatively, the adherent cells are suspended both for cold exposure and for cell viability analysis.

[0100] For the purposes of a further application of the invention claimed herein, the cell sample may need to remain in culture in vitro for a short time, e.g. a few hours or for a longer period, eg. a few days or weeks before initiating the low temperature survival test. Much research, including ours, has reported the benefit of culturing primary cancer cells with adherent feeder stromal cells. This procedure is called a co-culture. In some embodiments, the low temperature survival test is performed in coculture.

[0101] Suitable stromal cells are known to those skilled in the art.

[0102] The cells tested from the collected cell sample could be placed in an artificial culture medium used as standard in laboratories after centrifugation. Said test cells can be placed in a medium such as e.g. BME; CMRL; Medium independent of C02; DMEM Media; Ham F-12 F-12 Nutrient Blend; Glasgow's Minimum Essential Medium (GMEM); Improved MEM; Iscove (IMDM); L-15 from Leibovitz; McCoy's 5 A; MCDB 131; Minimum Essential Media (MEM); Modified Eagle's Medium (MEM); The middle of Waymouth; Williams' Media E; Myelocult® Long-Term Culture Medium (StemCell Technologies), including solution Phosphate buffered saline (PBS). The medium may or may not be supplemented with phenol red and / or L-glutamine and / or HEPES buffer and / or sodium pyruvate and / or antibiotics or alternatively modified for example with different concentrations of glucose for example and not limited to 0 g / L of glucose or about 0.25 g / L or less or about 0.5 g / L or less or about 1 g / L or less or about 2 g / L or less or about 3 g / L or less or about 4 g / L or less or about 5 g / L or less or about 6 g / L or less. In some embodiments, the media are either serum-free or supplemented with a different percentage of one or more fetal calf, horse or human sera, or sera, typically with 5% or 10% or 15% or 20%. .

[0103] Alternatively, the cells tested can be encapsulated in three-dimensional (3D) semi-solid media by standard methods known in the art such as semi-solid media, e.g. methylcellulose, a Matrigel-based matrix, or an alginate-based hydrogel, semi-solid media formed from natural molecules and / or synthetic polymers such as fibronectin, type I, II, IV collagen , laminin.

[0104] The cell sample tested in semi-solid media may have been incubated under conventional culture conditions to form colonies prior to exposure to low temperature in one embodiment of the present invention.

[0105] According to a preferred embodiment, the population of cells is a liquid cell suspension or a solid section of tissue.

[0106] According to a preferred embodiment, the cell population is composed of cancer cells from a human patient.

[0107] 3. Identify compounds for inhibiting or improving oxidative phosphorylation

[0108] The invention also relates to a method for determining the effect of a compound on the pathway followed by a population of mammalian cells to generate ATP, in which the compound is contacted with the population of cells and the method according to the invention described above is carried out.

[0109] The inventors have demonstrated the causal correlation between cellular energy metabolism and cell viability at low temperature. Cells producing their energy through aerobic oxidative mitochondrial phosphorylation die after a few hours of incubation at low temperature. Conversely, cells relying on glycolysis either have a much higher viability or are not affected at all when placed under the same conditions. The present invention is based, at least in part, on the development of methods of identifying compounds which affect energy metabolism, with examination of a relevant biological reading, e.g. sensitivity to cold exposure associated with OxPhos metabolism.

[0110] Thus, in one aspect, the invention provides methods for identifying a candidate compound for influencing cellular energy metabolism, e.g. a compound influencing OxPhos metabolism. The present method is suitable for screening any library of candidate agents, including the wide variety of combinatorial chemistry libraries. The present method is particularly suitable for an automated high throughput screening type cell assay.

[0111] The method comprises two steps. The first part is the screening which identifies "hits" (eg, compounds that increase or decrease sensitivity to low temperature). This first step consists of:

(i) provide one or more test cell sample and control cell sample comprising one or more cells, of mammalian origin, non-cancerous or cancerous, including leukemic, a homogeneous population of cells or a heterogeneous population of cells, e.g. . a heterogeneous population of cells composed of 10 different types of cells, e.g. mammalian blood, an immortalized cell line or primary cells directly isolated from a patient, which are directly tested by the method of the invention or which have been cultured in vitro for several days or weeks before the test;

(ii) contacting the test cell sample with a test compound and maintaining treatment under conditions for a sufficient period of time determined by known control compounds, e.g. for one to three days at 37 degrees Celsius;

(iii) measure the sensitivity of the test cell sample to cold exposure, e.g. cell viability measured 8 to 48 hours after incubation at 4 degrees Celsius;

(iv) comparing the cold sensitivity of the test cell sample versus the test sample in the absence of the test compound, and / or control samples;

(v) and select a test compound which modifies the sensitivity to cold exposure, such as a decrease in the cold sensitivity of the cell sample tested after step (iv) indicates that the test compound is likely to reduce OxPhos; such that an increase in the cold sensitivity of the cell sample tested after step (iv) indicates that the test compound is likely to increase OxPhos.

[0112] In one embodiment, the cell sample tested depends largely on OxPhos metabolism and is sensitive to exposure to cold.

[0113] In another embodiment, step (ii) consisting in contacting the sample tested with the candidate compounds is carried out with different doses of candidate compounds and the median toxic dose (TD50) and the median inhibitory concentration (IC50) are determined at the end of (ii) eg after the incubation period at 37 degrees Celsius. Depending on the toxicity observed, certain compounds or doses are excluded before proceeding to step (iii) or censored at the end of step (iii) (FIGURE 3). This is to greatly decrease false positive candidate compounds mistakenly assigned to increase OxPhos.

The decrease or increase in sensitivity to cold is understood to be compared to a control cell sample. As noted here, different controls may be suitable. In some embodiments, proper control is only control. In some embodiments, the appropriate controls are multiple controls. In some embodiments, a suitable control is the tested cell sample which has not been contacted with the test compounds. Preferably, the control is composed of the same cells as the test sample and is also contacted with the test compounds, but does not follow the low temperature survival test.

[0115] Positive and negative controls are also included in the method. For example, a controlling condition known to increase or decrease OxPhos metabolism and increase or decrease sensitivity to cold respectively. In some embodiments, different doses of positive and negative control can be applied to the test cell sample to generate an increasing and decreasing cold sensitivity standard curve.

[0116] Those skilled in the art will further recognize a wide variety of distinct screens which may be developed based on the description provided herein.

The second step of the method consists in confirming the impact of the selected compounds on OxPhos by a variety of techniques known in the art or described here. such as measuring oxygen consumption or quantifying GATR produced by OxPhos or comparing the proportion of ATP produced either by glycolysis or by OxPhos by the method disclosed in Moschoi et al. 2016.

[0118] The effectiveness of the candidate agent, or combined candidate agents, can be compared to the effectiveness of an agent already known to interfere with cellular energy metabolism, for example an agent known to interfere with POxPhos. The efficacy of the candidate agent, or the combined candidate agents, can be compared to the standard curve generated with different positive and negative controls.

[0119] In certain embodiments, the candidate compounds are rinsed before proceeding with the low temperature survival test. Preferably, the compound is maintained during the low temperature survival test, however in this case further tests should rule out interference of said compound with the final technique of cold sensitivity measurement, for example verifying that the compounds do not alter the fluorescence of the cell if the final parameter is assessed by fluorescence flow cytometry technology.

[0120] The test sample can be contacted with a single candidate agent or with a combination of one or more candidate agents. The test sample can be contacted with the different agents simultaneously or sequentially. In addition to being contacted with a single candidate agent or with a combination of one or more candidate agents, the test cell sample can also be contacted with an agent already known for the treatment of cancer stated above. high.

[0121] Candidate agents encompass many classes of chemicals. In a preferred embodiment, the candidate agents are organic molecules, small in size, having a molecular weight less than 3000 Daltons. Candidate agents often include cyclic or heterocyclic carbon structures or aromatic or polyaromatic structures.

[0122] Candidate bioactive agents are obtained from a wide variety of sources and will be appreciated by those skilled in the art, including libraries of natural compounds. In this way, libraries of prokaryotic and eukaryotic compounds can be prepared for screening. Other embodiments would instead use libraries of bacterial, fungal, viral and mammalian proteins (eg, human proteins). Other embodiments would instead use synthetic libraries. The present invention provides a quick and easy method for screening any library for candidate agents, including the wide variety of known combinatorial chemistry libraries.

[0123] In a preferred embodiment, the candidate agents are synthetic compounds. A number of techniques are available for the random and direct synthesis of organic compounds and biomolecules, and include the expression of randomized oligonucleotides. See, e.g., WO 94/24314, specifically cited herein by reference, which describes methods for generating new compounds, including random chemical and enzymatic methods, e.g. candidate agents are synthesized from a series of substrates which can be chemically modified. "Chemically modified" herein includes traditional chemical reactions as well as enzymatic reactions. These substrates generally include acids; alcohols; aldehydes; alkaloids; alkyl; amides; amine; amino acids; aryl; cyclic compounds; esters; ethers; compounds bearing heteroatoms; heterocyclic compounds; ketones; nucleosides; organometallic compounds; steroids; chemical reactions can be performed on the fragments to form new substrates or candidate agents which can then be tested using the present invention. One of the advantages of the present method is that it is not necessary to characterize the candidate bioactive agents before the test. Only candidate agents influencing sensitivity to cold need to be identified. In one embodiment, candidate agents can be selected from among 22,021 Type II compounds found on the approved Food and Drug Administration list for the purpose of repositioning drugs already in use.

[0124] The method of the invention can be implemented on a high throughput semi-automated scale and can provide an innovative framework for a promising new agent with multiple uses in medicine and biotechnology.

[0125] In some embodiments, the identified candidate compound inhibits one or more complexes of the mitochondrial respiratory chain.

[0126] In certain embodiments, the identified candidate compound inhibits one or more steps of the enzymatic cycle of tricarbocylic acid upstream of the electron transport chain. In one embodiment, the identified candidate compound inhibits the entry of the pyruvate transport shuttle into the mitochondria. [0127] 4. Method for identifying an agent influencing oxidative phosphorylation having an additive or synergistic effect with standard chemotherapy for the treatment of cancer.

[0128] The method can be used for screening agents exhibiting activity against OxPhos metabolism. Based on our current knowledge of the importance of OxPhos metabolism for cancer cells, the method identifies an agent to treat certain cancers and leukaemias (Coller 2014).

[0129] The identified candidate compounds influencing cellular energy metabolism, e.g. influencing OxPhos metabolism, could be characterized to determine their effectiveness in improving the anti-cancer action of other agents already used routinely for the treatment of the cancer listed above.

[0130] The present invention relates to methods of screening compound libraries to obtain useful substances having sensitization action in cancer cells to their conventional treatment by influencing their OxPhos metabolism (FIGURE 4). The method may comprise steps i) to v) described above relating to the procedure for screening candidate agent libraries to identify agents interfering with OxPhos metabolism.

[0131] The method further comprises the step of combinatorial analysis aimed at determining whether the selected agents decreasing F OxPhos can potentiate the effect of conventional treatments for a type of cancer, e.g. combined with standard chemotherapeutic agents presented here. Mathematical equations or software dedicated to the analysis of drug combinations or to any other known method in order to provide proof of significant superiority over single agents, could be used for this purpose for example (Chou 2006, 2010). Further testing may further demonstrate the benefit of the combination therapy over the standard anti-cancer treatment, e.g. with assays including, but not limited to, measuring viability in vitro in short-term or long-term culture, in liquid or semi-solid culture, in single cell suspension or in co-culture with stromal cells described here, using a preclinical in vivo model has a mouse model. The effectiveness of the compounds identified to improve the conventional chemotherapeutic agent can be tested by other methods known in the art, for example, described in the patent application WO2014132032A1 or WO2012112956A2. [0132] The invention also relates to a method of identifying a suitable compound for the treatment or prevention of diabetes. In one embodiment, the bioactive agent identified by the method of this invention targets one or more complexes of the respiratory chain and has near or superior efficacy to metformin and thiazolidinediones used for the treatment of type 2 diabetes. Thus, the method identifies an agent for treating metabolic disease, including cancer and diabetes.

[0133] 5. Quality control to demonstrate the metabolic deviance over time of a cell type

[0134] The invention relates to an in vitro method for monitoring the energy metabolism of a mammalian cell line over time to confirm its maintenance or deviation from a previous time comprising the step of determining the time required for exposure to cold to lose a precise percentage of cell viability, compare said time with the value obtained previously under the same conditions and stored in a database.

[0135] This maintenance or deviation monitoring could be tested on mammalian cells, primary or immortalized cell lines. In the context of a cancer patient, said monitoring could interrogate the metabolic state during the disease for scientific purposes.

[0136] The immortalized cell line has been the standard cell model in biology for more than 5 decades which requires regulated tissue culture equipment, reagents and procedures. Quality control is important in all aspects of tissue culture to ensure the authenticity of the cell model used. In addition, quality controls, implemented as routine tests, are a compulsory practice to verify the accuracy of the results obtained and to guarantee their reproducibility. The inventors have demonstrated that sensitivity to cold is an intrinsic characteristic of a cell which reflects its metabolic identity. The authors showed that each cell line has its own kinetics of cell death when exposed to 4 degrees Celsius in a way that correlates with its energy metabolic activity. In the absence of external disturbances, for example under stable conditions of temperature, medium and stable nutrients, the proportion of energy produced either by glycolysis or by OxPhos is a real characteristic property of the cell line. Recently Research groups have used an alternative methodology to classify glycolytic cell lines or OxPhos (Farge et al. 2017; Bosc, Selak, and Sarry 2017). However, it is well known that cell lines can spontaneously lose their integrity over time in culture in the absence of improper handling. (Spees et al. 2006).

[0137] The invention relates to a method for monitoring the maintenance or deviation of the energy metabolism of an immortalized cell line maintained in culture or for standardizing a cell model used throughout the world.

[0138] The invention also relates to a method for monitoring the state of the energy metabolism of a cell sample over time.

[0139] The method consists of maintaining the tested cell sample at low temperature over a period of time by measuring the sensitivity of the cell to exposure to cold, e.g. by measuring the loss of cell viability by well known methods, to determine the percentage of cell viability after a specific period of cold exposure or to determine the time of cold exposure required to achieve a specific percentage of cell viability, to compare the values with the reference value obtained previously or with the reference cell sample.

[0140] In another embodiment, the control values come from a database listing the mean response value of the same cell line tested previously under the same conditions.

[0141] The cell lines tested by the method of this invention could be 1184; 1221; 1306; 1321N1; 142BR; 143B; 149BR; 153BR; 155BR; 161BR; 171BR; 174BR; 175BR; 180BR; 1BR.3.G; 1BR.3.GN; 1BR.3.N; 1BR3; 200D; 208F; 23CLN; 253D; 26 CB1; 293; 293 N3S; 3.354 SC5 / 8; 311; 31F4L; 33B; 3A under E; Clone 3T3 A31; 3T3 Ll; 3T3 Swiss Albino; 3T3; 3T3-MSV; 3T6-Swiss Albino; 4/4 RM-4; 416B; 45.6. TGI.7; 4647; 46Br.lGl; 46BR.1N; 5637; 59M; 45078; 60H9; 707 DAP10; 707B1011C3; 70Z / 3; 745C2; 81.3; 8305C; 84BR; 8505C; 92BR; 94022802; A 172; A72; A.704; A1085; A15; A2; A2058; A2780; A2780cis; A2H; A375; A431; A549; A6; A673; A7r5; A8 / DI; A9; A9HT; AC2; ACHN; Aedes aegypti; Aedes albopictus; AGLCL; AGS; AHL-1; AK-D; AKR-2B; Amdur II; Ampli-GPE; Anr4; Anther cells; AR42J; ARH 77; ARL-6; ARLJ 301-3; At 1; AT-3.1; AT6.1; ATHOS; AtT-20 / D16v-F2; AtT20; AV3; B / C.Sk; B12; B14FAF28-G3; B16 melanoma 4A5; B16-F0; B16-F1; B2.Sp; B3 / AN; B50; B6 / FA; B65; B82; B9; B92; B95-8; BAE-1; BALB / 3T12-3; BAOEC; BB; BC3H1; BCL1 Clone CW13.20-3B3; BE ; BE ; BE10-7; BEIO-Intermediate; BE10-Late; BE11; BeWo; BF-2; BFA; BGM; BHK 21; BHK 21 CL13; BHK 21 S TRAIN 31; BHK 21 S TRAIN 35; BHK 21 S TRAIN 38; BHK TK-; BHK / AC9; BHK21C13-2P; BHK21C13-3P; BL-3; BLO-11; BME / CTVM 4; BP AEC; BRISTOL 8; B RL 3A; BRL-1; BS1-B4; BSC1; BT; Drank; Bu25 TK-; BUD-8; BW1J; BW5147.G.1.4.0UA / R1; BW5147.G.1.4.0UAR.1; BxPC-3; C-4 II; C-4I; C127I; C1300 CLONE NA; C16; C2; C2- Rev 7; C211; C2C12; C32; C3H / MCA clone 16; C57 / B1; C6; C6 / CH; C8166; Ca Ski; CA46; CACO-2; CAKI 2; CALU 1; CAR; CC-1; 13Lu CCD; 16Lu CCD; CCD 18Lu; CCD 19Lu; CCD 25Lu; CCD 8Lu; CCD-lLu; CCD-29Lu; CCD-32Lu; CCD-33Lu; CCD-34Lu; CCD-37Lu; CCD-39Lu; CCF-STTG1; CCO; CCRF S-180 II; CCRF- CEM; CCRF-HSB-2; CCRF-SB; CESS; CF2Th; CFPAC-1; CGR8; Chang Liver; CHH-1; CHL; CHO; CHO-K1; CHO-K1 / SF; CHO / dhFr-; CHP 3; CHP 4; CHSE-214; Eyelash; Citrullinemia; CL-S1; CLC; Clone 1-5c-4; Clone 15 HL-60; The clone 707; Clone 81; Clone 9; Clone C6 / 36; Clone M-3; CMT 93; CMT64 / 61; CMT93 / 69; CNC 1271; COLO 201; COLO 205; COLO 320 DMF; COLO 320DM; COLO 320HSR; COLO 357; COLO 668; COLO 677; COLO 679; COLO 684; COLO 685; COLO 699 N; COLO 720 E; COLO 720 L; COLO 741; COLO 775; COLO 792; COLO 800; COLO 818; COLO 829; COLO 832; COLO 839; COLO 853; COLO 857; COLO 858; COR-L105; COR-L23; COR-L24; COR-L279; COR-L311; COR-L47; COR-L51; COR-L88; COS-1; COS-7; CP 132; CPA; CPAE; CRE BAG2; CRFK; Cry du Chat; CRI-10P; CRI-D11; CRI-D2; CRI-Gl; CRI-G5; CSE-119; CTLL-2; CV-1; CV-1 Clone B5; CYNOM-K1; D12 / DM; D17; D2; D98 / AH2 Clone B; DAUDI; DBS-FCL-2; DBS-FRhL-2; DBTRG.05MG; DDT1-MF2; DEDE; Detroit 510; Detroit 525; Detroit 529; Detroit 532; Detroit 539; Detroit 551; Detroit 562; Detroit 573; DH14; DH15; DH33; DH82; DHD / K12 / TRb; DK; DLD-1; DMS 153; DMS 273; DMS 454; DMS 53; DMS 79; DMS 92; DoCll; DOK; DON; Duck embryo; E.Derm; EHIV; EB; EB176; EB185; EBTr; ECV304; Ehrlich-Letter Ascites strain E; EIII; EJ138; EJG; EL4; EL4.BU.OR6; EL4.NOB-1; EoL-1 cell; Eos-HL-60; EPC; ESK-4; EZZ; F9; Fao; FB2.Sp; FBHE; Fc2Lu; Fc3Tg; FEA4 + PFSC / Cl; FEL; IRON; FGC4; FHM; FL; FLYA13; FLYRD18; FM3A; FM3Ats C1.T85; FoLu; FR; FT; FTC-133; FTC-238; G; G-292 Clone A141B1; G-361; G-401; G-402; G-7; G-8; GEEP; GeLu; GH1; GH3; GIRARDI HEART; GL-1; GP2d; GP5d; GR-M; GRL101; GRL101; Grunt Fin; GS-109-V-20; GS-109-V-34; GS-109-V-63; GS-9L; H-4-II-E; H- EMC-SS; H2.35; H33HJ-JA1; H4-II-E-C3; H4II; H4S; H5; H69; H69V; H9; H9c2; Hak; HaP-Tl; HCT 116; HCT-15; HCT-8; HEL 12469; HEL 299; HEL 92.1.7; HeLa; HeLa B; HeLa Ohio; HeLa S3; HeLa229; Hep 3B; Hep G2; Hep-2C; Hep2; Hep2; Hepa 1-6; Hepa-lclc7; HEPM; HF1; HF1-5; HF19; HF2x653; HFFF2; HFL1; HG261; HGC-27; HL60; HL60 15-12; HL60 Ast.3; HL60 Ast. 4; HL60 M2; HL60 M4; HLF-a; HMVII; HOS; HR5; HR5-CL11; HRT-18; Hs 27; Hs 431; Hs 578T; Hs 633T; Hs 68; Hs 888Lu; HS-Sultan; HSDM1C1; HT 1080; HT 1197; HT 1376; HT115; HT2 Clone A5E; HT29; HT29 carbohydrate Cl; HT29 / 219; HT55; HTC; HTC; HTK-; HtTA-1; HUC-Fm; HuP-T3; HuP-T4; HUT-78; HVS-SILVA 40; 1-10; IA-Xs SBR; ICR-2A; IEC 18; IEC 6; IgH-2; IM 9; IMR 32; IMR-90; Indian muntjac; INT 407; IPI-1; IPI-2I; J.CaM1.6; J45.01; J558L; J774.2; J774A.1; JEG3; Jensen's sarcoma; JH4 clone 1; JIII; JIYOYE; JM; JTC-19; JTC-27; Jurkat E6.1; JVM-13; K-BALB; Kl; K562; K562 cl. 6; K6H6 / B5; KASUMI-1; KATO-III; KB; KC; KCB 85015; KCB 89001; KCB 90008; KELLY; KG-1; KG the; KHOS-240S; KHOS-312H; KHOS / NP; KLN 205; KNA; KNRK; KU-812; KU-812E; KU-812F; KYSE-30; L-2; LM; LM; L1210; L132; L14; L23; L231; L35; L45; L5178Y; L5178Y-R; L5178Y-S; L52; L6.C10; L6.C11; L6.G8; L6.G8.C5; L929; L929; L929S; LA-4; LA7; LBRM-33-1A5; LC540; LL / 2; LL24; LL29; LL47; LL86; LLC-PK1; LLC-RK1; LLC-WRC 256; LLCMK2; LoVo; LS 123; LS174T; LS180; LTK-; LUDLU-1; M-MSV-BALB / 3T3; M. dunni; Ml; M15; M1WT3; M3 Clone M-3; MA104; MAT-Lu; MAT-LyLu; MB-III; MC 116; MCA-RH 7777; McCoy; MCF7; MDA-MB-157; MDA-MB-231; MDA-MB-361; MDBK; MDCK; MEG-01; MES-SA; MES-SA / Dx-5; Meta 10; Meta 15; Meta 7; MEWO; MFE-280; MFE-296; MG-63; MH-22A; MH-S; MH1C1; MIA-Pa-Ca-2; MiCL1; ML-1; MLA144; MLg; MMT-060562; MNNG / HOS; Mo-B; MOG-G-CCM; MOG-G-UVW; MOH; MOLM-14; MOLT-3; MOLT-4; MOP-8; MOPC 315; MOPC 31C; MOUSE L CELLS; MPC-11; Mpf; MPK; MRC-5; MRC-5 SV1 TGI; MRC-5 SV1 TG2; MRC-5 SV2; MRC-7; MRC-9; MS; MSV.B; MSVIF-TK +; MT-2; MT4; MTC SK; Mv.l.Lu; MV4-11; Nl-Sl; N18; N1E-115; Namalwa; Nb2-1 l; NB4 1A3; NBT-II; NC-37; NCI-H292; NCI-H322; NCI-H358; NCI-H727; NCI-H929; NCTC 2071; NCTC 3749; NCTC 4093; NCTC 4206; NCTC cloned 1469; NCTC cloned 2472; NCTC cloned 2555; NCTC cloned 929; ND-E; ND-U1; ND27; ND7 / 23; Neopu; Neuro 2a; NFS-25 C-3; NFS-5 C1; NFS-70 CIO; NG108-15; NG115-401L; NH17; NIH 3T3; NIH-3T3 4-2; NIH-3T3D4; NMuMG; NOR 10; NRK; NRK-49F; NRK-52E; NS0; NTERA-2 clone D1; Nthy-ori 3-1; Nthy-tsl; OAW28; OAW42; OCI-AML2; OCI-AML3; OE19; OE21; OE33; OE47; EO50; OKAY; MKO; UFOs; PI; Pl.HTR; P1.HTR.TK-; P19; PIBbl.l; P2; P22; P3 / NS1 / 1-Ag4.1; P388.D1; P3HR-1; P3U1; P3X63Ag8; P3X63Ag8.653; P3X63Ag8U.l; P4; P815-1-1; PA- 1; PA317; PANC-1; PC-12; PC-14; PC-3; PD5; PEA- 10; PG-4; PG13; Phi 1; PI.l Ut; PLC / PRF / 5; PNT1A; PNT2; PORTHOS; PSA1; PSA1-NG2; Psi-CRE; Psi-CRIP; PSM-B; PSN1; Ptkl; Ptk2; Pu-518; PUTKO; QIMR-WIL; QT 35; QT 6; R2C; R74; R9; R9ab; RAB-9; RAG; RAJI; RAJI TK +; RAJI TK-; RAJPA; RAMOS; RAMOS; RAMOS-AW; RAMOS-EHRB; RAT-2; RAW 264; RAW 264.7; RBL-1; RC-4B / C1; RCE; RD; RED.l; RENE 1; RENE 2; RENT 4; RENT KING; RENT R03; RENT ts3.1; RENT tsSl.l; RFL-6; Ri-1; RIN-5F; RIN-m; RK13; RLC 27; RN 22; R082-W-1; RPMI 1788; RPMI 1846; RPMI 2650; RPMI 6666; RPMI 7666; RPMI 7932; RPMI 8226; RPMI 8866; RR-CHOKI; RR1022; RT 1; RT112 / 84; RT4; RT4-D6P2T; RTG-2; S1814.PB5; SBAC; SC 11; SC-1; SCP; Sf lEp; Sf9; SH-SY5Y; SHOK; SIRC; SJMRF; SK LU 1; SK-HEP-1; SKM-1; SU MES; SK-N-AS; SK-N-BE; SK-N-DZ; SK-N-F1; SK-N-SH; SK-OV-3; SK-PN-DW; SplK; SP2 / 0-Agl4; SR-4987; ST; STO; 707 BUF subclone; Subclone 707 DAP8; Subclone 707 DK 4.8; 707 DKA subclone; 707 DKE subclone; 707 DKH subclone; 707 DK J subclone; 707 TGI subclone; Subclone 707 TG2; SUP-T1; SV-T2; SV3T3B; SW 1116; SW 13; SW 1417; SW 1463; SW 403; SW 48; SW 480; SW 620; SW 837; SW 948; T / G HA-VSMC; T24 / 83; T47D; T84; T98G; T A3 Hauschka; TAK3; TB1 Lu; TCMK-1; TE 671; TE671 Subclone No. 2; TF1; TGP 49; TGP 52; TGP 54; T GP 55; TH-1 B1 subclone; THP-1; TK-TS 13; TK6; TK6TGR; TM3; TM4; Tn5 B 1-4; TOU; TR33B; TR6Bcl; TRA-171; TT; Tufted Deer; U-87 MG; U266B1; U373 MG; U937; UMR-106; UMR-108; UT-1; V79; V79-4; V79-HG04; VA-ES-BJ; Vero; Vero 317; Vero Cl 008; Vero76; VH2; Vx2; WB2054M; WEHI 164; WEHI 3B; WEHI 3BD; WEHI-231; WEHI-274.1; WEHI-279; WES; Wgd5; WI 1003; WI 26 VA4; WI 38VA13 2RA subclone; WiDr; WIL2 NS; WIL2.NS.6TG; WILCL; WISH; WKD; WM 266-4; WM-115; WRC; WRL 68; WS1; WX310; Xl / 5; XC; XrS6; Yl; Y3.AG.1.2.3; Y79; YAC-1; YO; ZR-75-1; ZR-75-30.

[0142] 6. In vitro method of diagnosis and prognosis

[0143] Questioning the nature of the energy metabolism of tumor cells, including leukemia, is a central concern in medicine today. Glycolytic status or OxPhos has recently been shown to be a determinant for the therapeutic response and the survival of the patient.

[0144] However, standard metabolic analysis approaches, available in health centers (hospitals or clinics for example), have been developed for the diagnosis of systemic metabolic disorders. These methods are not suitable for establishing the metabolism of the tumor. Indeed, such approaches are in fact optimized for metabolic disorders or metabolic dysfunctions of the organs leading to affect the whole body. These approaches measure, for example, the concentration of amino acid or organic acid in the patient's plasma. However, in the context of cancer, the abnormal cancerous metabolic signature is diluted in the bloodstream the metabolic signature of non-cancerous cells. Most cancers actually show a normal full metabolic picture at the time of diagnosis despite the abnormal metabolic signature of cancer cells which can only be revealed if a sample of cancer cells is analyzed in vitro. For example, the blood sugar in the leukemic patient is normal before myeloablative treatment despite an exceptional tumor burden of immature cells with a completely different metabolism from a normal blood composition in a healthy individual. Plasma blood is therefore not informative for various pathologies including cancer. Cellular analysis is therefore necessary in oncology to determine the metabolism of cancer cells.

[0145] It has been shown previously that cancer cells can directly take up functional mitochondria from their environment to support the change in their metabolism during chemotherapy (Moschoi et al. 2016). Analyzing the metabolism of a tumor currently requires hospital departments with large, highly technological equipment such as a positron emission tomograph (PET) or a multiparametric flow cytometry analyzer and associated qualified personnel. Current techniques used to measure cell metabolism require specific instruments as well as the iterative calibration of these instruments. In current commercial protocols, a freshly buffered medium should be prepared, before pre-inoculating the tested cell sample into the culture well at a precise cell density determined empirically in a series of experiments dedicated to parameter development. . Experimenters should also carefully follow the guidelines of the protocols and optimize the various parameters that are decisive for the quality and interpretation of the final results. Each current approach has its advantages and disadvantages, such as the low or high number of cells required or the economical or expensive price or such unnecessary or mandatory know-how. In view of all this, the method described here represents a major advance in the metabolic diagnosis of cancer cells directly isolated from a patient cell sample. For example, the in vitro method of the present invention can be easily performed by diagnostic laboratories in health centers, in experimental laboratories and does not require tedious counting of cells or training of special instrumentation.

[0146] The glycolytic or OxPhos metabolism of cancer cells is decisive for certain responses to cancer treatment. Since the method of the invention makes it possible to distinguish between glycolytic metabolism and OxPhos metabolism, the method of this invention would also make it possible to detect cancer cells resistant to standard therapy and to determine the magnitude of this resistance.

[0147] Thus, the invention also relates to a method for diagnosing cancer with a poor prognosis or for predicting treatment failure for a patient suffering from cancer, comprising the following step:

performing the method according to the invention as described above on a population of cancer cells of the patient, a low survival rate of cancer cells being associated with cancer with a poor prognosis or with resistance to treatment.

Typically, this in vitro method of diagnosis and prognosis in vitro comprises the step:

(i) obtaining a cell sample from a patient, eg, a blood sample, a biopsy and sometimes aspiration of bone marrow from a patient with cancer, including leukemia;

(ii) measuring the sensitivity of the test cell sample to exposure to cold, e.g. by measuring the loss of cell viability by methods well known in the art, for example determining either the loss of cell viability after a specific period of exposure to cold, or determining the time required of exposure to cold for achieve a precise percentage of cell viability;

(iii) comparison of these values with a reference control value possibly from a control cell sample. Suitable control samples are described above. In another embodiment, the control sample can also be a cancer cell sample with a predefined, known OxPhos metabolism sensitive to exposure to cold. In one embodiment, a copy of the tested cell sample is contacted with an OxPhos influencing test compound. In one embodiment, the reference value is obtained from the average responses of a large number of control samples previously tested under the same conditions and stored in a database such as high sensitivity to cold compared to at the reference value is indicative of a high OxPhos energy metabolism predicting resistance to treatment.

[0149] In another embodiment, the method of the present invention serves to detect in vitro whether tumor cells including leukemia cells have acquired a pattern of drug resistance during the disease.

[0150] Thus, in a particular embodiment, the method comprises the steps of:

- measurement of the in vitro sensitivity to cold exposure of different test cell samples from the same patient and taken at different times during the disease, e.g. against the background of leukemia, at the time of diagnosis and then at the time of relapse;

- comparison of said cold sensitivity with a previous analysis or with the reference level, such as the cold sensitivity of the control cell sample (s) described here, in which a maintenance of the cold resistance between the two times is indicative of a maintenance of glycol lytic metabolism and predictive of maintenance of drug or therapy sensitivity during disease, where the increase in cold resistance between the two stages indicates an increase in OxPhos metabolism and a prediction of drug or therapy resistance during illness.

[0151] New strategies targeting the energy metabolism of cancer, in particular the OxPhos metabolism, are under development (Caro et al. 2012; Viale et al. 2014; Hensley et al. 2016; Kuntz et al. 2017; Farge et al. 2017). the present invention could be useful clinically for two additional aspects. The first aspect is to use the in vitro method of this invention to select cancer patients likely to respond to a candidate metabolism modifying agent. cellular mitochondrial energy, eg. select patients who may benefit from a clinical trial affecting mitochondria, in which detection of sensitivity to cold above a control baseline indicates OxPhos cancer cells likely to benefit from novel anti-OxPhos therapy in a clinical trial.

[0152] In a second aspect, the invention relates to a method for evaluating the effectiveness of a treatment modifying the energy metabolism in a patient suffering from cancer, comprising the following step:

perform the method according to the invention as described above on a population of cancer cells of the patient under treatment.

Typically, this method comprises the steps of measuring the sensitivity to cold exposure of different test cell samples from the same patient at different times, for example before and during treatment.

[0154] Compare said sensitivity to cold between an untreated cell sample and a treated cell sample, in which maintaining the sensitivity to cold between the two untreated and treated cell samples is indicative of a failure of the treatment to have an impact on OxPhos metabolism; wherein a loss of cold sensitivity between the two untreated and treated cell samples indicates that treatment success negatively impacts OxPhos metabolism.

Examples

[0155] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0156] 1. Methods

[0157] The following methods were used in the studies described here.

[0158] Cells. LAM HL-60, Kasumi-1, KG-1, KG- la, MOLM-14, MV-4-11, NB-4, OCI-AML2, OCI-AML3, SKM-1, THP-1, U -937 were provided by Dr Collette (CRCM, Marseille, France) and Dr Sarry (CRCT, Toulouse, France). The cells were maintained in RPMI1640, with 10% fetal calf serum (FCS), 2 mM L-glutamine, penicillin (50 U / ml) and streptomycin (50 mg / ml). Low oxygen concentration cultures were performed in a double HERACell incubator gas injector MCO-5M SANYO as well as in a hypoxia chamber (STEMCELL Technologies) using a special mixture of gases allowing to achieve in parallel the conditions of 20%; 3% or 1% Oxygen.

[0159] Low temperature survival test at 4 degrees Celsius. Liebherr FK2642 refrigerated cabinet was used for cold exposure. The temperature setting of 4 degrees Celsius was checked with a refrigerator thermometer (Fisherbrand ™). Using a hypoxia chamber (STEMCELL Technologies) and a special mixture of gases composed of O2 (21%) N2 (74%) and C02 (5%), we have demonstrated that the lack of atmosphere of C02 does not was not responsible for the sensitivity of the OxPhos cells. Maintaining or stopping at a low temperature to determine impact on viability is detailed in the next section.

[0160] Flow cytometry. Cytometric analyzes were performed using the MACSQuant or MACSQuant® VYB analyzers (Miltenyi Biotec). For some analyzes, human hematopoietic cells were stained as specified with antiCD20-PE from Miltenyi. The viability markings were carried out either with DAPI (4,6 diamidino-2-phenylindole) or with propidium iodide. LAM cells were stained with 5 mM eFluor ™ 670 proliferation dye (Thermo fisher) for 10 minutes at 37 ° C in PBS. The cells were washed twice and incubated for 18 h at 37 ° C to allow stabilization of the eFluor ™ 670 label. The cytometry analyzes were carried out with the FlowJo software (Tree Star).

[0161] Metabolite measurements. Glucose and lactate, consumption and production were measured using a YSI 2950 analyzer calibrated before each run. For this, the cells tested were seeded in their respective medium in a series of initial dilutions, each cell line having a doubling time of its own, we decided to compare only at each time point analyzed, the data obtained at from equivalent cell density at the time of analysis. All data presented in grams per liter (g / l) were measured for an equivalent number of cells. Conditioned media were sampled and metabolic assay performed in triplicate and compared with fresh media. The rate of lactate production expressed in femto moles per cell per minute was calculated by the average of 3 to 6 independent experiments.

[0162] 2. Examples [0163] Example 1: Sensitivity to exposure to cold is indicative of a sustained energy metabolism based on mitochondrial oxidative phosphorylation

[0164] The main therapeutic barrier for the treatment of solid cancers and leukemia is the persistence of residual disease after the first cycle of chemotherapy. By activating the regenerative properties and the survival processes, chemoresistant cancer stem cells will re-initiate a new version of the pathology which involves metabolic plasticity, a property known for a decade but whose implication during conventional therapies has barely been made. to be revealed in the context of Acute Myeloid Leukemia (AML) (Farge et al. 2017). It has been formally shown that the majority of tumor cells adopt glycolysis to produce their energy, regardless of the oxygen supply to the tumor (Warburg effect (Vander Heiden, Cantley, and Thompson 2009)). However, since 2012, the number of studies demonstrating the existence of subgroups dependent on OxPhos metabolism has steadily increased (diffuse large B-cell lymphoma, lung cancer, pancreas, acute myeloid leukemia, chronic myeloid leukemia and breast cancer. ) and the discovery of metabolic fingerprints within the same tumor entity has revived the debate on the exclusive use of glycolysis by tumor cells. In 2017, the study by Dr. JE Sarry showed that AMLs could be classified according to their metabolic status (glycolytic and OxPhos). In addition, these studies demonstrated that in response to ARA, treatment-resistant cells have an exacerbated OxPhos metabolism, regardless of the metabolic status of the cells of origin Farge et al. (Farge et al. 2017), Kuntz et al. (Kuntz et al. 2017) as well as Lee et al. (Lee et al. 2017) reached similar conclusions in chronic myelogenous leukemia (CML) and a drug-resistant breast cancer subtype, respectively. Our work in 2016 describing a transfer of functional mitochondria from the microenvironment to leukemia cells also supports this finding (Moschoi et al. 2016).

[0165] Thus, targeting the metabolism of cancer cells offers considerable opportunities for blocking the energy production of drug-resistant cancer cells. Finally, set the OxPhos status or glycolytic and target OxPhos metabolism of a cancer cell is of central importance in oncology to our _j. [0166] Glyco lytic cells and OxPhos cells use glucose for energy. However, in OxPhos cells, the end product of glycolysis, pyruvate, will be directed to the mitochondria to fuel the TCA cycle which will produce a high reducing potential, which will be regenerated and transformed into a proton gradient at the chain level. electron transport. As for the glycolysis, it will eventually convert the pyruvate into lactate and will couple this reaction to a regenerative reduction of coenzyme, a step necessary to allow a new cycle of glycolysis. Lactate is one of the sources of acidification of the culture medium responsible for the change in color of phenol red. Because the energy efficiency of glycolysis is almost 20 times lower than that of OxPhos, glycolytic cells use more glucose and acidify their medium faster than OxPhos cells.

[0167] In order to take into account the heterogeneity of leukemia, we first studied a panel of the 12 most commonly used Acute Myeloid Leukemia cell lines which represent a wide range of molecular, clinical, biological and immunological abnormalities. -phenotypic.

[0168] Among this panel, we identified highly lactate-producing lines, HL-60, KG-1, NB-4, U937, which we defined as being glycolytic cells and cell lines that produced low lactate, KASUMI- 1, MOLM-14, MV4-11, SKM-1, THP-1 which we have defined as OxPhos cells.

We were able to verify the faster acidification of the culture media conditioned by the glycolytic cell lines simply by comparing the color variation of the phenol red pH indicator dye (FIGURE 5 A) or by more precisely quantifying the glucose concentration. and lactate in the culture medium (FIGURE 5B) varying over time much more rapidly for glycolytic cells.

[0170] We then asked ourselves if this metabolic difference between these two groups of leukemia cells could be influenced and adapt to external signals. We tested different percentages of oxygen in culture and observed in an interesting way that only OxPhos cells modified their metabolism in hypoxia and could increase their glucose consumption and lactate production. Glycolytic cells did not respond as if they had already reached their maximum rate of glucose catabolism (FIGURE 6). This also suggests that the Mitochondrial oxidative phosphorylation is absolutely not involved in the energy metabolism of glycol lytic cells.

[0171] During routine experimentation, we believed we noticed that a cell line systematically lost viability after short storage on ice before FACS analysis. We performed a more thorough kinetic analysis at 4 degrees Celsius (4 ° C) on several cell lines and observed that the glyco lytic cells maintained their viability at 4 ° C for 36h while the OxPhos cells began to lose up to 25% of their viability during the first 10 hours of cold exposure (FIGURE 7A). The 25% loss of viability was found to be a practical empirical threshold for capturing the difference between glyco lytic cells and OxPhos in a short period of time. We repeated the low temperature survival test at 4 degrees Celsius and confirmed that the time taken to lose 25% of the population was reproducible and consistent for each cell line. Cell lines with low production of OxPhos lactate lose on average 25% of their viability in the first 14 hours (± 2.5h) compared to 39 hours (± 8.8h) for glycolytic cells producing high lactate (FIGURE 7B). The graphical representation of the rate of lactate production as a function of the time required to reach 25% loss of viability, shows the positive correlation (p <0.01) between these two parameters and distinguishes cold-sensitive OxPhos cells from resistant glycolytic cells cold (FIGURE 7C). Finally, we were able to demonstrate that cell density does not influence cell survival during CKC4, suggesting that this cell test could also be applied to solid tissue samples.

[0172] We then examined whether the correlation between OxPhos and sensitivity to cold was a causal relationship by pharmacologically interfering with the electron transport chain (ETC). We selected rotenone which inhibits complex I of the respiratory chain for its non-reversibility combined with its high activity at low concentration and identified a non-toxic treatment of 0.1 mM for 72 h before starting low temperature survival test ( FIGURE 8A). Strikingly, the rotenone pretreatment profoundly reverses the cold sensitivity of OxPhos MOLM-14, MV4-11, OCI-AML2 and THP-1 cells and has no effect on KG-1, NB-4 glycolytic cells. and U937 (FIGURE 8B). Rotenone induced a metabolic change only in OxPhos cells but not in glycolytic cells as evidenced by changes in color of phenol red and as well as variations in lactate concentration (FIGURE 8C-D). These results first confirm previous data generated in hypoxia showing that glycolysis is already at its maximum rate and that mitochondrial oxidative phosphorylation has no contribution to the energy metabolism of glyco lytic cells. On the other hand, OxPhos cells have metabolic plasticity allowing them to increase their glycolysis to compensate for the lack of energy produced by OxPhos and invalidated by rotenone.

[0173] Most importantly, these results demonstrate the causal relationship between OxPhos metabolism and cold sensitivity.

[0174] Previous studies on cellular energy metabolism, including our own, clearly established the immediate metabolic change after pharmacological interference, especially with rotenone. So we wondered if cold sensitivity was also reversed as quickly as OxPhos was inhibited. To answer this question, we compared different incubation periods with rotenone and found that immediate inhibition of the mitochondrial respiratory chain has no direct impact on cold resistance, but requires a certain period of time. 'extinction to trigger the protection. This result suggests that resistance or sensitivity to low temperature expresses a stable and long-lasting metabolism which intrinsically alters cell composition. Considering that enzymatic reactions are profoundly slowed down in both OxPhos and glycolytic cells, we speculated that low temperature survival testing did not reveal any signaling differences but rather a structural difference between the two groups. We wondered if this difference could be due to the lipid composition of cells. To test this hypothesis, we compared cellular sensitivity to atorvastatin, a selective and competitive inhibitor of HMG-CoA reductase, the enzyme responsible for controlling the metabolism of 3-hydroxy-3-methyl-glutaryl-coenzyme A in mevalonate, a precursor of sterols and cholesterol. Consistently we determined that OxPhos cells were sensitive to atorvastatin compared to glycolytic cells. The graphical representation of the respective IC50s of atorvastatin as a function of the time required to lose 25% viability at low temperature, also highlights the relationship between these parameters (FIGURE 9A-B). Overall, these results show that continued mitochondrial metabolism could control the concentration of cellular cholesterol and possibly the composition of the lipid membrane which would influence the survival of cells at low temperature.

These results show that the energy metabolism of cancer cells can be detected qualitatively and quantitatively by measuring the sensitivity to cold. The method presents enormous opportunities for diagnostic approaches and chemical library screens to identify new compounds that inhibit or stimulate oxidative phosphorylation or glycolysis.

[0176] Example 2: The low temperature survival test at 4 degrees Celsius makes it possible to enrich or purify cells according to their energy metabolism

Since the OxPhos cells and the glycolytic cells are respectively sensitive and resistant to low temperature, we proposed to verify that this method could be used to enrich the glycolytic population in a mixed population.

[0178] We mixed in equal proportions OxPhos cells and glycolytic cells with a maximum viability level before initiating exposure to 4 degrees Celsius over 24 hours (FIGURE 10A). For the two mixtures tested, the OxPhos population saw its proportion reduced by 3 to 5 times after 24 hours at 4 degrees Celsius (FIGURE 10B). The differential sensitivity was verified by staining with propidium iodide which demonstrates the higher cell mortality of OxPhos cells.

[0179] Thus, these results demonstrate that low temperature incubation can be used to enrich glycolytic cells in a mixture of cells with different metabolisms.

[0180] The low temperature survival test at 4 degrees Celsius can also be used to monitor which population, in a mixed population sample, has OxPhos metabolism, by comparing said proportion of population before and after the low survival test. temperature, where the population with an undetermined metabolism, the proportion (or absolute number) of which decreases after the low temperature survival test, is a population based on the OxPhos metabolism.

List of documents cited [0181] For all practical purposes, the following publications are cited:

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Claims

Claims

[Claim 1] An in vitro method for determining the metabolic pathway followed by a population of mammalian cells to generate GATR, the method comprising measuring the survival rate of the population of cells when subjected to a temperature between 2 and 10 ° C, a high survival rate being associated with the glycolysis pathway and a low survival rate being associated with the oxidative phosphorylation pathway (OxPhos pathway).

[Claim 2] The in vitro method according to claim 1, wherein the population of cells is subjected to a temperature of about 4 ° C.

[Claim 3] The in vitro method according to claim 1 or 2, wherein the measurement of the survival rate is carried out within 48 hours.

[Claim 4] An in vitro method according to any of claims 1 to 3, wherein the population of cells is a liquid cell suspension or a solid section of tissue.

[Claim 5] An in vitro method according to any one of claims 1 to 4, wherein the cell population consists of cancer cells from a human patient.

[Claim 6] An in vitro method for determining the effect of a compound on the pathway followed by a population of mammalian cells to generate ATP, wherein the compound is contacted with the population of cells and performed. method according to any one of claims 1-5.

[Claim 7] An in vitro method for diagnosing cancer with a poor prognosis or prognosis for treatment failure of a cancer patient, comprising the following step:

performing the method according to any one of claims 1 to 5 on a population of cancer cells of the patient, a poor survival rate of cancer cells being associated with cancer with poor prognosis or resistance to treatment.

[Claim 8] An in vitro method for evaluating the effectiveness of a treatment modifying energy metabolism in a cancer patient, comprising the step of: performing the method according to any one of claims 1 to 5 on a population of cancer cells of the patient under treatment. [Claim 9] An in vitro method for eliminating within a population of mammalian cells the cells which follow the oxPhos metabolic pathway, by subjecting the population of cells to a temperature between 2 and 10 ° C.