Glucose Metabolism and NRF2 Coordinate the Antioxidant Response in Melanoma Resistant to MAPK Inhibitors

Khamari et al. Cell Death and Disease (2018) 9:325 DOI 10.1038/s41419-018-0340-4 Cell Death & Disease ARTICLE Open Access Glucose metabolism and NRF2 coordinate the antioxidant response in melanoma resistant to MAPK inhibitors Raeeka Khamari1,AnneTrinh1, Pierre Elliott Gabert1, Paola Corazao-Rozas1,SamuelRiveros-Cruz1, Stephane Balayssac2, Myriam Malet-Martino2, Salim Dekiouk1, Marie Joncquel Chevalier Curt3,PatriceMaboudou4, Guillaume Garçon5, Laura Ravasi6, Pierre Guerreschi1, Laurent Mortier7,BrunoQuesnel1,8, Philippe Marchetti1,9 and Jerome Kluza1 Abstract 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 1234567890():,; 1234567890():,; 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. Introduction of BRAF kinase and drive oncogenic signaling through Activating V600E/K mutations in the BRAF oncogene MAPK activation. Targeted therapies as BRAF and MEK are found in over half of the patients with metastatic inhibitors combination (e.g., the BRAF inhibitors vemur- melanoma. These mutations confer constitutive activation afenib and the MEK inhibitor trametinib) have revolu- tionized the treatment of patients leading to increase progression-free survival and overall survival. Unfortu- Correspondence: Jerome Kluza ([email protected]) nately, disease progression occurs after a median of few 1 – Univ. Lille, INSERM, UMR S1172 Jean Pierre Aubert Research Centre 59045 months and in 80% of patients after 3 years of therapies1. Lille, France 2Laboratoire SPCMIB UMR CNRS 5068 UniversitéPaul Sabatier 118 Route de Immune checkpoint inhibitors (as PD-L1/PD-1 or CTLA- Narbonne, 31062 Toulouse Cedex 9, France 4 monoclonoal antibodies) have also shown activity in Full list of author information is available at the end of the article Raeeka Khamari, Anne Trinh contributed equally to this work. Jerome Kluza, Philippe Marchetti jointly supervised this work. Edited by A. Finazzi-Agrò © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Khamari et al. Cell Death and Disease (2018)9:325 Page 2 of 14 some patients with BRAFV600E-mutant melanoma, but = 5) was treated with vehicle only (PBS). In vemurafenib- most of the patients progressed on this treatment. Clinical treated mice, tumors have drastically reduced until to be trials are actually evaluating combination of both MAP- undetectable at 30 days post treatment (Fig. 1b). At the Kinase inhibitors and PD-1/PD-L1 antagonists2. opposite, tumors have exponentially increased in vehicle- In the context of MAPK constitutive activation, mutant treated mice. As observed in patients who relapsed after BRAF stimulates glycolytic activity and inhibits mito- treatment, cancer cells are finally able to escape to anti- chondrial oxidative phosphorylation3. BRAF inhibition BRAF therapy and tumors have re-initiated growth in two causes a decrease in ERK activation resulting in G1 phase mice. Then, mice have been killed and cancer cells have cell cycle arrest and inducing endoplasmic reticulum been isolated from tumors to generate three cell lines: – (ER)-stress-mediated cell death4 6. We and others have A375-v and A375RIV1/A375RIV2 from vehicle-treated shown that MAPK inhibitors reverse also the metabolic mice and vemurafenib-treated mice, respectively. As phenotype by decreasing glycolytic activity and increasing expected, clonogenic assays confirm that A375RIV1 and mitochondrial oxidative phosphorylation (OXPHOS) of A375RIV2 cell lines were more resistant to vemurafenib BRAF-driven melanoma cells5,7,8. These modifications of than A375 or A375-v (Fig. 1c). To confirm the behavior of mitochondrial metabolism following MAPK inhibition these cells in vivo, cell lines were engrafted in SCID mice could be considered as an adaptive response to compen- and tumor volumes were periodically measured. As seen sate drug-induced glycolytic inhibition. In agreement with in Fig. 1d, A375 and A375-v tumors exhibited the same this observation, drugs repressing directly or indirectly growth rate while A375RIV1 volume increased faster. mitochondrial oxidative metabolism favorized cell death Moreover, unlike A375 or A375-v engrafted mice, mac- – under BRAFi therapy5,9 13. roscopic and histological lung analysis showed the pre- Several mechanisms of acquired resistance to BRAFi sence of many metastases in A375RIV1-engrafted mice have been identified, mostly mediated by MAPK pathway indicating a more aggressive phenotype (Fig. 1e, f). Finally, reactivation including mutations in NRAS and MEK A375RIV1 cell lines were also resistant to other BRAFi oncogenes, BRAFV600E splice variants, and BRAF (dabrafenib) and MEK inhibitors (cobimetinib and tra- amplification or activation of alternative tumorigenic metinib) alone or in combination (Fig. 1g). Thus, we pathway. We and others have also shown that generated in vivo models for relapsed melanoma cells, vemurafenib-resistant melanoma cells maintain an which mimic the clinical process of melanoma in humans increase of mitochondrial oxidative phosphorylation even treated with BRAFi. in absence of BRAFi9,10. Marais and coll have shown In previous work, we and others have observed an BRAFi that resistant melanoma cells support mitochon- increase of mitochondrial oxidative phosphorylation drial respiration and ATP supply by sustaining TCA cycle (OXPHOS) in melanoma cell lines resistant to BRAFi9,10. metabolites levels using glutaminolysis10. Oxygen con- In agreement with our previous results, tumor biopsies sumption increase leads to mitochondrial ROS over- from A375RIV1-engrafted mice also exhibited an increase production through electron transfer chain and to in oxygen consumption compared to those from A375-v enhancement of oxidative stress. As a weakness in spite of or A375-engrafted mice (Fig. 2a). Increase in OXPHOS overall strength, we have demonstrated that BRAFi- activity was not associated with modification of mito- resistant cells with increased endogenous ROS are more chondrial content as judged by mtDNA quantification in sensitive to cell death upon exposure to mitochondrial resistant cells (Fig. 2b). Because the pyruvate dehy- pro-oxidative agents9. drogenase complex is a key modulator of mitochondrial Here, we asked the question how BRAFi-resistant mel- activity through pyruvate oxidation in melanoma14,we anoma cells use metabolism to cope with ROS production studied its regulation by specific kinases, which results in and therefore to survive under oxidative stress. To inhibitory phosphorylation. No differences in the level of understand the mechanisms leading to BRAFi-resistant PDH phosphorylation at Ser293 and in PDK1/PDK3 melanoma cells survival in this context, we have generated expression were seen between A375-v and A375RIV1 mouse models bearing human BRAFV600E melanoma (Fig. 2c). Moreover, the PDK inhibitor, DCA, increased cells that mimic clinical relapse and acquired resistance to OXPHOS to the same extent in both cell lines (Fig.

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