Key role of glutamine metabolism in persistence of leukemic cells upon exposition to FLT3 tyrosine kinase inhibitors.

Acute myeloid leukemias are a group of hematological malignancies characterized by a poor prognosis for survival. The discovery of oncogenic mutations in the FLT3 gene has led to the development of tyrosine kinase inhibitors such as Quizartinib. However, achieving complete remission in patients remains challenging because these new TKIs are unable to completely eradicate all leukemic cells. Residual leukemic cells persist during Quizartinib treatment, leading to the rapid emergence of drug-resistant leukemia. Given that mitochondrial oxidative metabolism promotes the survival of leukemic cells after exposure to multiple anticancer drugs, we characterized the metabolism of leukemic cells that persisted during Quizartinib treatment and developed metabolic strategies to eradicate them. In our study, employing biochemical and metabolomics approaches, we confirmed that the survival of leukemic cells treated with FLT3 inhibitors critically depends on maintaining mitochondrial metabolism, specifically through glutamine oxidation. We uncovered a synergistic interaction between the FLT3 inhibitor Quizartinib and L-Asparaginase, operating through anti-metabolic mechanisms. Utilizing various models of persistent leukemia, we demonstrated that leukemic cells resistant to Quizartinib are susceptible to L-Asparaginase. This combined therapeutic strategy shows promise in reducing the development of resistance to FLT3 inhibitors, offering a potential strategy to enhance treatment outcomes.

The troglitazone derivative EP13 disrupts energy metabolism through respiratory chain complex I inhibition in breast cancer cells and potentiates the antiproliferative effect of glycolysis inhibitors.

BACKGROUND: The metabolism of cancer cells generally differs from that of normal cells. Indeed, most cancer cells have a high rate of glycolysis, even at normal oxygen concentrations. These metabolic properties can potentially be exploited for therapeutic intervention. In this context, we have developed troglitazone derivatives to treat hormone-sensitive and triple-negative breast cancers, which currently lack therapeutic targets, have an aggressive phenotype, and often have a worse prognosis than other subtypes. Here, we studied the metabolic impact of the EP13 compound, a desulfured derivative of Δ2-troglitazone that we synthetized and is more potent than its parent compounds. METHODS: EP13 was tested on two triple-negative breast cancer cell lines, MDA-MB-231 and Hs578T, and on the luminal cell line MCF-7. The oxygen consumption rate (OCR) of the treated cell lines, Hs578T mammospheres and isolated mitochondria was measured using the XFe24 Seahorse analyser. ROS production was quantified using the MitoSOX fluorescent probe. Glycolytic activity was evaluated through measurement of the extracellular acidification rate (ECAR), glucose consumption and lactate production in extracellular medium. The synergistic effect of EP13 with glycolysis inhibitors (oxamate and 2-deoxyglucose) on cell cytotoxicity was established using the Chou-Talalay method. RESULTS: After exposure to EP13, we observed a decrease in the mitochondrial oxygen consumption rate in MCF7, MDA-MB-231 and Hs578T cells. EP13 also modified the maximal OCR of Hs578T spheroids. EP13 reduced the OCR through inhibition of respiratory chain complex I. After 24 h, ATP levels in EP13-treated cells were not altered compared with those in untreated cells, suggesting compensation by glycolysis activity, as shown by the increase in ECAR, the glucose consumption and lactate production. Finally, we performed co-treatments with EP13 and glycolysis inhibitors (oxamate and 2-DG) and observed that EP13 potentiated their cytotoxic effects. CONCLUSION: This study demonstrates that EP13 inhibits OXPHOS in breast cancer cells and potentiates the effect of glycolysis inhibitors.

MYC dependency in GLS1 and NAMPT is a therapeutic vulnerability in multiple myeloma.

Multiple myeloma (MM) is an incurable hematological malignancy in which MYC alterations contribute to the malignant phenotype. Nevertheless, MYC lacks therapeutic druggability. Here, we leveraged large-scale loss-of-function screens and conducted a small molecule screen to identify genes and pathways with enhanced essentiality correlated with MYC expression. We reported a specific gene dependency in glutaminase (GLS1), essential for the viability and proliferation of MYC overexpressing cells. Conversely, the analysis of isogenic models, as well as cell lines dataset (CCLE) and patient datasets, revealed GLS1 as a non-oncogenic dependency in MYC-driven cells. We functionally delineated the differential modulation of glutamine to maintain mitochondrial function and cellular biosynthesis in MYC overexpressing cells. Furthermore, we observed that pharmaceutical inhibition of NAMPT selectively affects MYC upregulated cells. We demonstrate the effectiveness of combining GLS1 and NAMPT inhibitors, suggesting that targeting glutaminolysis and NAD synthesis may be a promising strategy to target MYC-driven MM.

Implication of Rac1 GTPase in molecular and cellular mitochondrial functions.

Rac1 is a member of the Rho GTPase family which plays major roles in cell mobility, polarity and migration, as a fundamental regulator of actin cytoskeleton. Signal transduction by Rac1 occurs through interaction with multiple effector proteins, and its activity is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The small protein is mainly anchored to the inner side of the plasma membrane but it can be found in endocellular compartments, notably endosomes and cell nuclei. The protein localizes also into mitochondria where it contributes to the regulation of mitochondrial dynamics, including both mitobiogenesis and mitophagy, in addition to signaling processes via different protein partners, such as the proapoptotic protein Bcl-2 and chaperone sigma-1 receptor (σ-1R). The mitochondrial form of Rac1 (mtRac1) has been understudied thus far, but it is as essential as the nuclear or plasma membrane forms, via its implication in regulation of oxidative stress and DNA damages. Rac1 is subject to diverse post-translational modifications, notably to a geranylgeranylation which contributes importantly to its mitochondrial import and its anchorage to mitochondrial membranes. In addition, Rac1 contributes to the mitochondrial translocation of other proteins, such as p53. The mitochondrial localization and functions of Rac1 are discussed here, notably in the context of human diseases such as cancers. Inhibitors of Rac1 have been identified (NSC-23766, EHT-1864) and some are being developed for the treatment of cancer (MBQ-167) or central nervous system diseases (JK-50561). Their effects on mtRac1 warrant further investigations. An overview of mtRac1 is provided here.

Extracellular vesicles derived from nasopharyngeal carcinoma induce the emergence of mature regulatory dendritic cells using a galectin-9 dependent mechanism.

Nasopharyngeal carcinoma-derived small extracellular vesicles (NPCSEVs) have an immunosuppressive impact on the tumour microenvironment. In this study, we investigated their influence on the generation of tolerogenic dendritic cells and the potential involvement of the galectin-9 (Gal9) they carry in this process. We analysed the phenotype and immunosuppressive properties of NPCSEVs and explored the ability of DCs exposed to NPCSEVs (NPCSEV-DCs) to regulate T cell proliferation. To assess their impact at the pathophysiological level, we performed real-time fluorescent chemoattraction assays. Finally, we analysed phenotype and immunosuppressive functions of NPCSEV-DCs using a proprietary anti-Gal9 neutralising antibody to assess the role of Gal9 in this effect. We described that NPCSEV-DCs were able to inhibit T cell proliferation despite their mature phenotype. These mature regulatory DCs (mregDCs) have a specific oxidative metabolism and secrete high levels of IL-4. Chemoattraction assays revealed that NPCSEVs could preferentially recruit NPCSEV-DCs. Finally, and very interestingly, the reduction of the immunosuppressive function of NPCSEV-DCs using an anti-Gal9 antibody clearly suggested an important role for vesicular Gal9 in the induction of mregDCs. These results revealed for the first time that NPCSEVs promote the emergence of mregDCs using a galectin-9 dependent mechanism and open new perspectives for antitumour immunotherapy targeting NPCSEVs.

Mitochondrial adaptation decreases drug sensitivity of persistent triple negative breast cancer cells surviving combinatory and sequential chemotherapy.

Triple negative breast cancer (TNBC) is an aggressive malignancy for which chemotherapy remains the standard treatment. However, between 3 and 5 years after chemotherapy, about half patients will relapse and it is essential to identify vulnerabilities of cancer cells surviving neoadujuvant therapy. In this study, we established persistent TNBC cell models after treating MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide, and then with paclitaxel, for a total of 18 weeks. The resulting chemo-persistent cell lines were more proliferative, both in vitro and in xenografted mice. Interestingly, MDA-MB-231 persistent cells became less sensitive to chemotherapeutic drugs, whereas SUM159-PT persistent cells kept similar sensitivity compared to control cells. The reduced sensitivity to chemotherapy in MDA-MB-231 persistent cells was found to be associated with an increased oxidative phosphorylation (OXPHOS) and modified levels of tricarboxylic acid cycle (TCA) intermediates. Integration of data from proteomics and metabolomics demonstrated TCA cycle among the most upregulated pathways in MDA-MB-231 persistent cells. The absence of glucose and pyruvate impeded OXPHOS in persistent cells, while the absence of glutamine did not. In contrast, OXPHOS was not modified in control cells independently of TCA substrates, indicating that MDA-MB-231 persistent cells evolved towards a more pyruvate dependent profile. Finally, the inhibition of pyruvate entry into mitochondria with UK-5099 reduced OXPHOS and re-sensitized persistent cells to therapeutic agents. Together, these findings suggest that targeting mitochondrial pyruvate metabolism may help to overcome mitochondrial adaptation of chemo-persistent TNBC.

NOD2 in monocytes negatively regulates macrophage development through TNFalpha.

Objective: It is believed that intestinal recruitment of monocytes from Crohn's Disease (CD) patients who carry NOD2 risk alleles may repeatedly give rise to recruitment of pathogenic macrophages. We investigated an alternative possibility that NOD2 may rather inhibit their differentiation from intravasating monocytes. Design: The monocyte fate decision was examined by using germ-free mice, mixed bone marrow chimeras and a culture system yielding macrophages and monocyte-derived dendritic cells (mo-DCs). Results: We observed a decrease in the frequency of mo-DCs in the colon of Nod2-deficient mice, despite a similar abundance of monocytes. This decrease was independent of the changes in the gut microbiota and dysbiosis caused by Nod2 deficiency. Similarly, the pool of mo-DCs was poorly reconstituted in a Nod2-deficient mixed bone marrow (BM) chimera. The use of pharmacological inhibitors revealed that activation of NOD2 during monocyte-derived cell development, dominantly inhibits mTOR-mediated macrophage differentiation in a TNFα-dependent manner. These observations were supported by the identification of a TNFα-dependent response to muramyl dipeptide (MDP) that is specifically lost when CD14-expressing blood cells bear a frameshift mutation in NOD2. Conclusion: NOD2 negatively regulates a macrophage developmental program through a feed-forward loop that could be exploited for overcoming resistance to anti-TNF therapy in CD.

TRPC3 shapes the ER-mitochondria Ca2+ transfer characterizing tumour-promoting senescence.

Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca2+ load via negative regulation of IP3 receptor-mediated Ca2+ release, is down regulated in senescence regardless of the type of senescence inducer. This remodelling promotes cytosolic/mitochondrial Ca2+ oscillations and elevates mitochondrial Ca2+ load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca2+ load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.

Comparison of the in vivo genotoxicity of electronic and conventional cigarettes aerosols after subacute, subchronic and chronic exposures.

Tobacco smoking is classified as a human carcinogen. A wide variety of new products, in particular electronic cigarettes (e-cigs), have recently appeared on the market as an alternative to smoking. Although the in vitro toxicity of e-cigs is relatively well known, there is currently a lack of data on their long-term health effects. In this context, the aim of our study was to compare, on a mouse model and using a nose-only exposure system, the in vivo genotoxic and mutagenic potential of e-cig aerosols tested at two power settings (18 W and 30 W) and conventional cigarette (3R4F) smoke. The standard comet assay, micronucleus test and Pig-a gene mutation assay were performed after subacute (4 days), subchronic (3 months) and chronic (6 months) exposure. The generation of oxidative stress was also assessed by measuring the 8-hydroxy-2'-deoxyguanosine and by using the hOGG1-modified comet assay. Our results show that only the high-power e-cig and the 3R4F cigarette induced oxidative DNA damage in the lung and the liver of exposed mice. In return, no significant increase in chromosomal aberrations or gene mutations were noted whatever the type of product. This study demonstrates that e-cigs, at high-power setting, should be considered, contrary to popular belief, as hazardous products in terms of genotoxicity in mouse model.

Antimetabolic cooperativity with the clinically approved l-asparaginase and tyrosine kinase inhibitors to eradicate CML stem cells.

OBJECTIVE: Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS: Using complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS: Although TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION: Targeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells.

Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I.

SMIP004-7 is a small molecule inhibitor of mitochondrial respiration with selective in vivo anti-cancer activity through an as-yet unknown molecular target. We demonstrate here that SMIP004-7 targets drug-resistant cancer cells with stem-like features by inhibiting mitochondrial respiration complex I (NADH:ubiquinone oxidoreductase, complex I [CI]). Instead of affecting the quinone-binding site targeted by most CI inhibitors, SMIP004-7 and its cytochrome P450-dependent activated metabolite(s) have an uncompetitive mechanism of inhibition involving a distinct N-terminal region of catalytic subunit NDUFS2 that leads to rapid disassembly of CI. SMIP004-7 and an improved chemical analog selectively engage NDUFS2 in vivo to inhibit the growth of triple-negative breast cancer transplants, a response mediated at least in part by boosting CD4+ and CD8+ T cell-mediated immune surveillance. Thus, SMIP004-7 defines an emerging class of ubiquinone uncompetitive CI inhibitors for cell autonomous and microenvironmental metabolic targeting of mitochondrial respiration in cancer.

p65/RelA NF-κB fragments generated by RIPK3 activity regulate tumorigenicity, cell metabolism, and stemness characteristics.

Receptor-interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces caspase-mediated p65/RelA cleavage, resulting in N-terminal 1-361 and C-terminal 362-549 fragments. We show here that a noncleavable p65/RelA D361E mutant expressed in DA1-3b leukemia cells decreases mouse survival times and that coexpression of p65/RelA fragments increases the tumorigenicity of B16F1 melanoma cells. This aggressiveness in vivo did not correlate with NF-κB activity measured in vitro. The fragments and p65/RelA D361E mutant induced different expression profiles in DA1-3b and B16F1 cells. Stemness markers were affected: p65/RelA D361E increased ALDH activity in DA1-3b cells, and fragment expression increased melanoma sphere formation in B16/F1 cells. p65/RelA fragments and the D361E noncleavable mutant decreased oxidative or glycolytic cell metabolism, with differences observed between models. Thus, p65/RelA cleavage initiated by kinase-independent RIPK3 activity in cancer cells is not neutral and induces pleiotropic effects in vitro and in vivo that may vary across tumor types.

Clinically Relevant Oxygraphic Assay to Assess Mitochondrial Energy Metabolism in Acute Myeloid Leukemia Patients.

Resistant acute myeloid leukemia (AML) exhibits mitochondrial energy metabolism changes compared to newly diagnosed AML. This phenotype is often observed by evaluating the mitochondrial oxygen consumption of blasts, but most of the oximetry protocols were established from leukemia cell lines without validation on primary leukemia cells. Moreover, the cultures and storage conditions of blasts freshly extracted from patient blood or bone marrow cause stress, which must be evaluated before determining oxidative phosphorylation (OXPHOS). Herein, we evaluated different conditions to measure the oxygen consumption of blasts using extracellular flow analyzers. We first determined the minimum number of blasts required to measure OXPHOS. Next, we compared the OXPHOS of blasts cultured for 3 h and 18 h after collection and found that to maintain metabolic organization for 18 h, cytokine supplementation is necessary. Cytokines are also needed when measuring OXPHOS in cryopreserved, thawed and recultured blasts. Next, the concentrations of respiratory chain inhibitors and uncoupler FCCP were established. We found that the FCCP concentration required to reach the maximal respiration of blasts varied depending on the patient sample analyzed. These protocols provided can be used in future clinical studies to evaluate OXPHOS as a biomarker and assess the efficacy of treatments targeting mitochondria.

Mitochondrial inhibitors circumvent adaptive resistance to venetoclax and cytarabine combination therapy in acute myeloid leukemia.

Therapy resistance represents a major clinical challenge in acute myeloid leukemia (AML). Here we define a 'MitoScore' signature, which identifies high mitochondrial oxidative phosphorylation in vivo and in patients with AML. Primary AML cells with cytarabine (AraC) resistance and a high MitoScore relied on mitochondrial Bcl2 and were highly sensitive to venetoclax (VEN) + AraC (but not to VEN + azacytidine). Single-cell transcriptomics of VEN + AraC-residual cell populations revealed adaptive resistance associated with changes in oxidative phosphorylation, electron transport chain complex and the TP53 pathway. Accordingly, treatment of VEN + AraC-resistant AML cells with electron transport chain complex inhibitors, pyruvate dehydrogenase inhibitors or mitochondrial ClpP protease agonists substantially delayed relapse following VEN + AraC. These findings highlight the central role of mitochondrial adaptation during AML therapy and provide a scientific rationale for alternating VEN + azacytidine with VEN + AraC in patients with a high MitoScore and to target mitochondrial metabolism to enhance the sensitivity of AML cells to currently approved therapies.

A New Strategy to Preserve and Assess Oxygen Consumption in Murine Tissues.

Mitochondrial dysfunctions are implicated in several pathologies, such as metabolic, cardiovascular, respiratory, and neurological diseases, as well as in cancer and aging. These metabolic alterations are usually assessed in human or murine samples by mitochondrial respiratory chain enzymatic assays, by measuring the oxygen consumption of intact mitochondria isolated from tissues, or from cells obtained after physical or enzymatic disruption of the tissues. However, these methodologies do not maintain tissue multicellular organization and cell-cell interactions, known to influence mitochondrial metabolism. Here, we develop an optimal model to measure mitochondrial oxygen consumption in heart and lung tissue samples using the XF24 Extracellular Flux Analyzer (Seahorse) and discuss the advantages and limitations of this technological approach. Our results demonstrate that tissue organization, as well as mitochondrial ultrastructure and respiratory function, are preserved in heart and lung tissues freshly processed or after overnight conservation at 4 °C. Using this method, we confirmed the repeatedly reported obesity-associated mitochondrial dysfunction in the heart and extended it to the lungs. We set up and validated a new strategy to optimally assess mitochondrial function in murine tissues. As such, this method is of great potential interest for monitoring mitochondrial function in cohort samples.

Lipid Metabolism and Resistance to Anticancer Treatment.

Metabolic reprogramming is crucial to respond to cancer cell requirements during tumor development. In the last decade, metabolic alterations have been shown to modulate cancer cells' sensitivity to chemotherapeutic agents including conventional and targeted therapies. Recently, it became apparent that changes in lipid metabolism represent important mediators of resistance to anticancer agents. In this review, we highlight changes in lipid metabolism associated with therapy resistance, their significance and how dysregulated lipid metabolism could be exploited to overcome anticancer drug resistance.

Mitochondrial spare respiratory capacity: Mechanisms, regulation, and significance in non-transformed and cancer cells.

Mitochondrial metabolism must constantly adapt to stress conditions in order to maintain bioenergetic levels related to cellular functions. This absence of proper adaptation can be seen in a wide array of conditions, including cancer. Metabolic adaptation calls on mitochondrial function and draws on the mitochondrial reserve to meet increasing needs. Among mitochondrial respiratory parameters, the spare respiratory capacity (SRC) represents a particularly robust functional parameter to evaluate mitochondrial reserve. We provide an overview of potential SRC mechanisms and regulation with a focus on its particular significance in cancer cells.

First-line Screening of OXPHOS Deficiencies Using Microscale Oxygraphy in Human Skin Fibroblasts: A Preliminary Study.

The diagnosis of mitochondrial diseases is a real challenge because of the vast clinical and genetic heterogeneity. Classically, the clinical examination and genetic analysis must be completed by several biochemical assays to confirm the diagnosis of mitochondrial disease. Here, we tested the validity of microscale XF technology in measuring oxygen consumption in human skin fibroblasts isolated from 5 pediatric patients with heterogeneous mitochondrial disorders. We first set up the protocol conditions to allow the determination of respiratory parameters including respiration associated with ATP production, proton leak, maximal respiration, and spare respiratory capacity with reproducibility and repeatability. Maximum respiration and spare capacity were the only parameters decreased in patients irrespective of the type of OXPHOS deficiency. These results were confirmed by high-resolution oxygraphy, the reference method to measure cellular respiration. Given the fact that microscale XF technology allows fast, automated and standardized measurements, we propose to use microscale oxygraphy among the first-line methods to screen OXPHOS deficiencies.

AMP-Activated Protein Kinase Is Essential for the Maintenance of Energy Levels during Synaptic Activation.

Although the brain accounts for only 2% of the total body mass, it consumes the most energy. Neuronal metabolism is tightly controlled, but it remains poorly understood how neurons meet their energy demands to sustain synaptic transmission. Here we provide evidence that AMP-activated protein kinase (AMPK) is pivotal to sustain neuronal energy levels upon synaptic activation by adapting the rate of glycolysis and mitochondrial respiration. Furthermore, this metabolic plasticity is required for the expression of immediate-early genes, synaptic plasticity, and memory formation. Important in this context, in neurodegenerative disorders such as Alzheimer disease, dysregulation of AMPK impairs the metabolic response to synaptic activation and processes that are central to neuronal plasticity. Altogether, our data provide proof of concept that AMPK is an essential player in the regulation of neuroenergetic metabolic plasticity induced in response to synaptic activation and that its deregulation might lead to cognitive impairments.

Glucose metabolism and NRF2 coordinate the antioxidant response in melanoma resistant to MAPK inhibitors.

Targeted therapies as BRAF and MEK inhibitor combination have been approved as first-line treatment for BRAF-mutant melanoma. However, disease progression occurs in most of the patients within few months of therapy. Metabolic adaptations have been described in the context of acquired resistance to BRAF inhibitors (BRAFi). BRAFi-resistant melanomas are characterized by an increase of mitochondrial oxidative phosphorylation and are more prone to cell death induced by mitochondrial-targeting drugs. BRAFi-resistant melanomas also exhibit an enhancement of oxidative stress due to mitochondrial oxygen consumption increase. To understand the mechanisms responsible for survival of BRAFi-resistant melanoma cells in the context of oxidative stress, we have established a preclinical murine model that accurately recapitulates in vivo the acquisition of resistance to MAPK inhibitors including several BRAF or MEK inhibitors alone and in combination. Using mice model and melanoma cell lines generated from mice tumors, we have confirmed that the acquisition of resistance is associated with an increase in mitochondrial oxidative phosphorylation as well as the importance of glutamine metabolism. Moreover, we have demonstrated that BRAFi-resistant melanoma can adapt mitochondrial metabolism to support glucose-derived glutamate synthesis leading to increase in glutathione content. Besides, BRAFi-resistant melanoma exhibits a strong activation of NRF-2 pathway leading to increase in the pentose phosphate pathway, which is involved in the regeneration of reduced glutathione, and to increase in xCT expression, a component of the xc-amino acid transporter essential for the uptake of cystine required for intracellular glutathione synthesis. All these metabolic modifications sustain glutathione level and contribute to the intracellular redox balance to allow survival of BRAFi-resistant melanoma cells.