00085472can141392-sup-131377_1_supp_2661062_nbys9d.pptx (11.83 MB)

Supplementary Figures S1-S12 from Inhibition of mTORC1/2 Overcomes Resistance to MAPK Pathway Inhibitors Mediated by PGC1α and Oxidative Phosphorylation in Melanoma

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posted on 2023-03-30, 22:40 authored by Y.N. Vashisht Gopal, Helen Rizos, Guo Chen, Wanleng Deng, Dennie T. Frederick, Zachary A. Cooper, Richard A. Scolyer, Gulietta Pupo, Kakajan Komurov, Vasudha Sehgal, Jiexin Zhang, Lalit Patel, Cristiano G. Pereira, Bradley M. Broom, Gordon B. Mills, Prahlad Ram, Paul D. Smith, Jennifer A. Wargo, Georgina V. Long, Michael A. Davies

Supplementary Figures S1-S12. Figure S1. Ingenuity Pathway Analysis (IPA) and Netwalker analysis of the PLX4720-synthetic lethal genes. Figure S2. Effect of Selumetinib on the mitochondrial gene expression in high OxPhos MEL624 cell lines. Figure S3. Differential energy consumption and release in selumetinib-sensitive and resistant melanoma cell lines. Figure S4. Oxygen consumption in MEKi+mTORC1/2i combination-resistant and Ã¢??sensitive melanoma cell lines. Figure S5. Cellular effects of FDA approved single agent MEK and BRAF inhibitors Trametinib and Dabrafenib and their combinations with AZD8055. Figure S6. RPPA proteomic analysis of protein lysates from three combination-resistant (1) and three Ã¢??sensitive (2) BRAF mutant melanoma cell lines following MEK or mTORC1/2 inhibition. Figure S7. Correlation of MITF expression with selumetinib and AZD8055 combination synergy in 14 melanoma cell lines. Figure S8. Effect of selumetinib and AZD8055 on the cellular localization of MITF and consequence of MITF knockdown Figure S9. Comparative effects of AZD8055 and AZD2014. Figure S10. Drug concentration against percentage of cell growth inhibition. Figure S11. The proliferation of the A375, its resistant clones Ã¢??R1 and -R2, and WM35 and its resistant clones in presence of increasing concentrations of selumetinib, AZD8055 and their combination after 72 h treatment was determined as described in Figure S8. Figure S12. A) Ratio of PGC1ÃŽÂ± and MITF gene expression at the time of disease progression versus pre-treatment in the MGH cohort of patients treated with single-agent vemurafenib (MGH-25) or with dabrafenib + trametinib (all others). B) PGC1ÃŽÂ± expression in 917 cell lines of the Cancer Cell Line Encyclopedia (CCLE) representing 23 cancer types.

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ARTICLE ABSTRACT

Metabolic heterogeneity is a key factor in cancer pathogenesis. We found that a subset of BRAF- and NRAS-mutant human melanomas resistant to the MEK inhibitor selumetinib displayed increased oxidative phosphorylation (OxPhos) mediated by the transcriptional coactivator PGC1α. Notably, all selumetinib-resistant cells with elevated OxPhos could be resensitized by cotreatment with the mTORC1/2 inhibitor AZD8055, whereas this combination was ineffective in resistant cell lines with low OxPhos. In both BRAF- and NRAS-mutant melanoma cells, MEK inhibition increased MITF expression, which in turn elevated levels of PGC1α. In contrast, mTORC1/2 inhibition triggered cytoplasmic localization of MITF, decreasing PGC1α expression and inhibiting OxPhos. Analysis of tumor biopsies from patients with BRAF-mutant melanoma progressing on BRAF inhibitor ± MEK inhibitor revealed that PGC1α levels were elevated in approximately half of the resistant tumors. Overall, our findings highlight the significance of OxPhos in melanoma and suggest that combined targeting of the MAPK and mTORC pathways may offer an effective therapeutic strategy to treat melanomas with this metabolic phenotype. Cancer Res; 74(23); 7037–47. ©2014 AACR.