Inhibition of mTORC1/2 overcomes resistance to MAPK pathway inhibitors mediated by PGC1? and oxidative phosphorylation in melanoma.
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 PGC1?. 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 PGC1?. In contrast, mTORC1/2 inhibition triggered cytoplasmic localization of MITF, decreasing PGC1? expression and inhibiting OxPhos. Analysis of tumor biopsies from patients with BRAF-mutant melanoma progressing on BRAF inhibitor ± MEK inhibitor revealed that PGC1? 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.
Project description:BACKGROUND:Genotyping of melanomas is used to identify patients for treatment with BRAF and MEK inhibitors, but clinical responses are highly variable. This study investigated the utility of protein expression phenotyping to provide an integrated assessment of gene expression programs in BRAF/NRAS melanoma which would be useful for prognosis and may predict response to MEK inhibition. METHODS:Mass spectrometry profiling of early passage cell lines established from Stage III cutaneous melanomas was conducted. Basal protein expression was correlated with in vitro response to the MEK inhibitor, selumetinib. Protein expression in a cohort of 32 drug naïve BRAF/NRAS metastatic melanoma specimens was examined. The prognostic utility of a subset of these proteins and mRNA transcripts from a separate cohort was determined. RESULTS:Unsupervised analysis of basal cell line protein abundances delineated response to selumetinib, but BRAF/NRAS genotype did not. Resistance was associated with functions including cell motility, cell adhesion and cytoskeletal organization. Several of these response biomarkers were observed in lymph node biospecimens and correlated with melanoma-specific survival. Loss of ICAM-1 protein and mRNA expression was a strong prognosticator of diminished survival in BRAF/NRAS mutant melanoma. CONCLUSIONS:These results demonstrate the utility of proteomic phenotyping to identify both putative biomarkers of response to MEK inhibition and prognostication associated with metastatic melanoma.
Project description:The mitogen-activated protein-kinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas. MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy.We assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth. Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression. Human melanoma samples (n = 77) from various stages were analyzed for SMURF2 and PAX3 expression. RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish. All statistical tests were two-sided.Activation of transforming growth factor ? (TGF-?) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition. Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-? signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF. High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity. Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitor-induced apoptosis. Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells. Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval = 38.65% to 155.50%, P = .005).Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.
Project description:BRAF mutations are detected in >50% of all melanomas. These mutations impair the LKB1-AMPK signaling, an important metabolic pathway associated with cell growth, proliferation and survival. Melanoma patients with BRAF mutations are usually treated with BRAF inhibitors such as vemurafenib, but responses are short-lived as drug resistant tumors metabolically switch to mitochondrial oxidative phosphorylation (OXPHOS) to escape metabolic stress-induced BRAF inhibition. Additionally, a large subset of melanoma utilizes OXPHOS in their metabolism, which can confer de novo resistance to BRAF inhibitors. Therefore, uncoupling of OXPHOS to perturb energy homeostasis and to indirectly stimulate AMPK could be a novel treatment for melanoma and to overcome intrinsic and acquired resistance to BRAF inhibitors. Here, we investigated the effects of SR4 and niclosamide, two small molecule mitochondria uncouplers, on the growth and proliferation of treatment-naïve and vemurafenib-resistant melanomas in vitro and in vivo. SR4 and niclosamide inhibited melanoma proliferation irrespective of BRAF/NRAS status. Melanomas with greater OXPHOS phenotype (higher OCR/ECAR), with LKB1 mutation, or with acquired resistance to vemurafenib displayed greater sensitivity to both uncouplers. More importantly, SR4 and niclosamide inhibited tumor growth in both treatment-naïve and vemurafenib-resistant xenograft mice models. Mechanistic studies indicate both uncouplers induced energetic stress, modulated the AMPK-mTOR pathway, and promoted apoptosis without affecting MEK-ERK MAPK signaling. These results suggest that uncouplers such as SR4 and niclosamide may be useful as first line treatment against melanoma regardless of BRAF/NRAS status, and as an adjuvant therapy for patients failing MAPK inhibitors.
Project description:Increased expression of the Microphthalmia-associated transcription factor (MITF) contributes to melanoma progression and resistance to BRAF pathway inhibition. Here we show that the lack of MITF is associated with more severe resistance to a range of inhibitors, while its presence is required for robust drug responses. Both in primary and acquired resistance, MITF levels inversely correlate with the expression of several activated receptor tyrosine kinases, most frequently AXL. The MITF-low/AXL-high/drug-resistance phenotype is common among mutant BRAF and NRAS melanoma cell lines. The dichotomous behaviour of MITF in drug response is corroborated in vemurafenib-resistant biopsies, including MITF-high and -low clones in a relapsed patient. Furthermore, drug cocktails containing AXL inhibitor enhance melanoma cell elimination by BRAF or ERK inhibition. Our results demonstrate that a low MITF/AXL ratio predicts early resistance to multiple targeted drugs, and warrant clinical validation of AXL inhibitors to combat resistance of BRAF and NRAS mutant MITF-low melanomas.
Project description:The BT-40 low-grade childhood astrocytoma xenograft model expresses mutated BRAF(V600E) and is highly sensitive to the MEK inhibitor selumetinib (AZD6244). In this study, we developed and characterized selumetinib resistance and explored approaches to circumventing the mechanisms of acquired resistance.BT-40 xenografts were selected in vivo for selumetinib resistance. Resistant tumors were obtained and characterized, as were tumors that reverted to sensitivity. Characterization included expression profiling, assessment of MEK signature and compensatory pathways, MEK inhibition, BRAF expression, and cytokine levels. Combination treatment of BT-40/AZD-resistant tumors with the MEK inhibitor and a STAT3 inhibitor (LLL12) was assessed.Resistance was unstable, tumors reverting to selumetinib sensitivity when passaged in untreated mice, and MEK was equally inhibited in sensitive and resistant tumors by selumetinib. Drug resistance was associated with an enhanced MEK signature and increased interleukin (IL)-6 and IL-8 expression. Selumetinib treatment induced phosphorylation of STAT3 (Y705) only in resistant xenografts, and similar results were observed in BRAF(V600E) astrocytic cell lines intrinsically resistant to selumetinib. Treatment of BT-40-resistant tumors with selumetinib or LLL12 had no significant effect, whereas combined treatment induced complete regressions of BT-40/AZD-resistant xenografts.Resistance to selumetinib selected in vivo in BT-40 tumor xenografts was unstable. In resistant tumors, selumetinib activated STAT3, and combined treatment with selumetinib and LLL12 induced complete responses in resistant BT-40 tumors. These results suggest dual targeting BRAF (V600E) signaling and STAT3 signaling may be effective in selumetinib-resistant tumors or may retard or prevent onset of resistance.
Project description:Once melanomas have progressed with acquired resistance to mitogen-activated protein kinase (MAPK)-targeted therapy, mutational heterogeneity presents a major challenge. We therefore examined the therapy phase before acquired resistance had developed and discovered the melanoma survival oncogene MITF as a driver of an early non-mutational and reversible drug-tolerance state, which is induced by PAX3-mediated upregulation of MITF. A drug-repositioning screen identified the HIV1-protease inhibitor nelfinavir as potent suppressor of PAX3 and MITF expression. Nelfinavir profoundly sensitizes BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors. Moreover, nelfinavir is effective in BRAF and NRAS mutant melanoma cells isolated from patients progressed on MAPK inhibitor (MAPKi) therapy and in BRAF/NRAS/PTEN mutant tumors. We demonstrate that inhibiting a driver of MAPKi-induced drug tolerance could improve current approaches of targeted melanoma therapy.
Project description:Although melanoma is the most aggressive skin cancer, recent advances in BRAF and/or MEK inhibitors against BRAF-mutated melanoma have improved survival rates. Despite these advances, a treatment strategy targeting NRAS-mutated melanoma has not yet been elucidated. We discovered CH5126766/RO5126766 as a potent and selective dual RAF/MEK inhibitor currently under early clinical trials. We examined the activity of CH5126766/RO5126766 in a panel of malignant tumor cell lines including melanoma with a BRAF or NRAS mutation. Eight cell lines including melanoma were assessed for their sensitivity to the BRAF, MEK, or RAF/MEK inhibitor using in vitro growth assays. CH5126766/RO5126766 induced G1 cell cycle arrest in two melanoma cell lines with the BRAF V600E or NRAS mutation. In these cells, the G1 cell cycle arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27 and down-regulation of cyclinD1. CH5126766/RO5126766 was more effective at reducing colony formation than a MEK inhibitor in NRAS- or KRAS-mutated cells. In the RAS-mutated cells, CH5126766/RO5126766 suppressed the MEK reactivation caused by a MEK inhibitor. In addition, CH5126766/RO5126766 suppressed the tumor growth in SK-MEL-2 xenograft model. The present study indicates that CH5126766/RO5126766 is an attractive RAF/MEK inhibitor in RAS-mutated malignant tumor cells including melanoma.
Project description:Most melanomas harbor oncogenic BRAF(V600) mutations, which constitutively activate the MAPK pathway. Although MAPK pathway inhibitors show clinical benefit in BRAF(V600)-mutant melanoma, it remains incompletely understood why 10% to 20% of patients fail to respond. Here, we show that RAF inhibitor-sensitive and inhibitor-resistant BRAF(V600)-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-?B signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-?B-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-?B activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-?B activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAF(V600)-mutant melanoma.Although most BRAF(V600)-mutant melanomas are sensitive to RAF and/or MEK inhibitors, a subset fails to respond to such treatment. This study characterizes a transcriptional cell state distinction linked to MITF and NF-?B that may modulate intrinsic sensitivity of melanomas to MAPK pathway inhibitors.
Project description:To compare the efficacy and tolerability of the mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) 1/2 inhibitor selumetinib versus temozolomide in chemotherapy-naive patients with unresectable stage III/IV melanoma.This phase II, open-label, multicenter, randomized, parallel-group study examined the effect of 100 mg oral selumetinib twice daily in 28-day cycles versus oral temozolomide (200 mg/m(2)/d for 5 days, then 23 days off-treatment). The primary endpoint was progression-free survival.Two hundred patients were randomized. Progression-free survival did not differ significantly between selumetinib and temozolomide (median time to event 78 and 80 days, respectively; hazard ratio, 1.07; 80% confidence interval, 0.86-1.32). Objective response was observed in six (5.8%) patients receiving selumetinib and nine (9.4%) patients in the temozolomide group. Among patients with BRAF mutations, objective responses were similar between selumetinib and temozolomide groups (11.1% and 10.7%, respectively). However, five of the six selumetinib partial responders were BRAF mutated. Frequently reported adverse events with selumetinib were dermatitis acneiform (papular pustular rash; 59.6%), diarrhea (56.6%), nausea (50.5%), and peripheral edema (40.4%), whereas nausea (64.2%), constipation (47.4%), and vomiting (44.2%) were reported with temozolomide.No significant difference in progression-free survival was observed between patients with unresectable stage III/IV melanoma unselected for BRAF/NRAS mutations, who received therapy with selumetinib or temozolomide. Five of six patients with partial response to selumetinib had BRAF mutant tumors.