BRAFV600-mutant Melanoma Patients Research Articles

BRAF inhibition for advanced locoregional BRAF V600E mutant melanoma: a potential neoadjuvant strategy.

Selective BRAF inhibitors (BRAFi) yield objective responses in 50% of patients with metastatic BRAF V600E mutant melanoma. Adding an MEK inhibitor increases this response rate to 70%. Limited data are available on the outcomes of unresectable stage III patients, and it remains unclear whether BRAF-targeted therapy can be utilized as a neoadjuvant strategy. Data on patients with advanced locoregional BRAF V600E mutant melanoma treated with BRAF-targeted therapy at Moffitt Cancer Center were analyzed to determine response rates, subsequent resection rates after tumor downsizing, pathologic responses, and patient survival. Fifteen patients with locoregional disease treated with BRAF-targeted therapy, either BRAFi alone (vemurafenib; 11 patients) or a combination of BRAFi and an MEK inhibitor (dabrafenib plus trametinib or placebo; four patients), were identified. The median age was 50 years; the median follow-up was 25.4 months. The median BRAF-targeted therapy treatment duration was 6.0 months (range 1.2-29.4 months). Response Evaluation Criteria In Solid Tumors-based evaluation demonstrated objective response in 11 patients (73.3%). Six patients underwent resection of the remaining disease after therapy. Pathological analysis showed complete pathologic response (n=2), partial pathologic response (n=2), or no pathologic response (n=2). Four of six patients undergoing surgery have been alive for more than 2 years, including three patients currently free from active disease. No complications attributable to BRAF-targeted therapy were observed in the perioperative period. Dose reduction or discontinuation because of toxicities occurred in 10/15 patients. Neoadjuvant BRAF-targeted therapy may be effective in advanced locoregional BRAF V600E mutant melanoma patients in increasing resectability, yielding pathological responses, and achieving prolonged survival.

Eradicating tumor drug resistance at its YAP-biomechanical roots.

Treatment with BRAF kinase inhibitors leads to rapid resistance and tumor regression in BRAF V600E mutant melanoma patients. However, the underlying mechanism of the developed tumor resistance is not fully clear. In this issue of The EMBO Journal, Kim and colleagues show that melanoma cells acquire resistance to BRAF inhibitors by changing cell shape, modifying their cytoskeleton and, in turn, activating the YAP/TAZ mechanotransduction pathway (Kim et al, 2016).

Abstract B50: Actin remodeling confers BRAF inhibitor resistance to melanoma cells through YAP/TAZ activation

Abstract Background The acquired resistance to BRAF inhibitor poses a critical hurdle for the treatment of BRAF V600E mutant melanoma patients. Recent evidences suggest that the Hippo pathway effectors, YAP and its paralog TAZ, promote resistance to anti-cancer therapies. YAP/TAZ activity is tightly coupled to actin cytoskeleton dynamics and architecture. However, the influence of actin cytoskeletal remodeling on cancer drug resistance remains largely uncovered. Here, we report that actin remodeling in response to BRAF inhibitor treatment plays a pivotal role in YAP/TAZ-dependent resistance development in melanoma cells. Materials and Method Two vemurafenib resistant melanoma cell lines (SKMEL28 and WM3248) were established by two months of vemurafenib treatment. We inspected actin cytoskeletal structure changes in resistant cells. YAP/TAZ localization and target gene transcription were compared between parental and resistant melanoma cells. In addition, changes in the sensitivity to vemurafenib upon YAP/TAZ knockdown or overexpression were measured, and transcriptome-wide signatures supported by YAP/TAZ were investigated by microarray analysis. Lastly, a kinome siRNA library screening was performed in resistant cells to identify synthetic lethal targets to suppress resistant cell growth combined with BRAF inhibition. Results Vemurafenib resistant melanoma cells exhibit a substantial increase in both actin stress fiber formation and cell spreading force compared with the parental cells, which appears to promote the nuclear accumulation of YAP/TAZ and the upregulation of their transcriptional activity. Depletion of YAP/TAZ significantly restores vemurafenib sensitivity in resistant cells, whereas overexpression of constitutively active YAP induces vemurafenib resistance in parental cells, suggesting BRAF inhibitor resistance in melanoma cells is highly dependent on YAP/TAZ activity. The microarray analysis comparing control siRNA and YAP/TAZ siRNA knockdown in resistance cells suggests that E2F-related cell cycle progression accompanied by EGFR and c-MYC pathway activation is the major driver of YAP/TAZ-dependent cell proliferation and vemurafenib resistance. Remarkably, pharmacologic inhibition of actin polymerization or actomyosin contraction significantly promotes YAP/TAZ cytoplasmic retention and transcriptional downregulation. Moreover, resistant cell viability is suppressed by pharmacologic inhibition of actin polymerization and cytoskeletal tension. Consistent with the importance of the actin cytoskeleton, our kinome-wide RNAi screening indentifies TESK1, a kinase inhibiting cofilin activity, as a promising synthetic lethal target for BRAF inhibitor resistance. TESK1 knockdown suppresses both enhanced actin stress fiber formation and YAP/TAZ nuclear translocation, provoking growth arrest of vemurafenib-resistant melanoma cells. Conclusion Our results implicate the actin cytoskeleton in the induction of YAP/TAZ-dependent resistance to vemurafenib, and inhibition of actin remodeling might be a promising synthetic lethal strategy to suppress resistance in BRAF inhibitor therapies. Citation Format: Min Hwan Kim, Joon Kim. Actin remodeling confers BRAF inhibitor resistance to melanoma cells through YAP/TAZ activation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B50.

Treatment patterns and outcomes in BRAF V600E-mutant melanoma patients with brain metastases receiving vemurafenib in the real-world setting.

Brain metastases are a common and serious complication among patients with metastatic melanoma. The selective BRAF inhibitor vemurafenib has demonstrated clinical efficacy in patients with BRAF V600E-mutant melanoma brain metastases (MBM). We examined the real-world application and clinical outcomes of vemurafenib in this patient population. Demographic, treatment patterns, response, and survival data were collected from medical charts. Clinical data on 283 patients with active BRAF V600E-mutant MBM treated with vemurafenib were provided by 70 US oncologists. Mean age was 57.2 years, 60.8% were male, 67.5% had ECOG performance status of 0–1, and 43.1% used corticosteroids at vemurafenib initiation. Median follow-up was 5.7 months. Following vemurafenib initiation, 48.1% of patients experienced intracranial response and 45.6% experienced extracranial response. The Kaplan–Meier estimate for overall survival was 59% at 12 months. Multivariate analyses showed associations between intracranial response and both corticosteroid use and vemurafenib as initial therapy after MBM diagnosis. Larger size (5–10 mm vs. <5 mm) and number of brain metastases (≥5 vs. <2) and progressive extracranial disease at treatment initiation were associated with decreased intracranial response and increased risk of disease progression. Multiple extracranial sites (2 vs. <2) and the absence of local treatments were also associated with increased risk of progression. Increased risk of death was associated with ≥2 extracranial disease sites, progressive extracranial disease, and ≥5 brain metastases. Subgroups of MBM patients may derive more benefit with vemurafenib, warranting prospective investigation.

Abstract PR04: A melanoma transcriptional state distinction influences sensitivity to MAPK pathway inhibitors

Abstract The deployment of cancer therapeutics that exploit oncogenic dependencies has yielded remarkable advances in patient treatment. However, the therapeutic benefit of these approaches is transient and the majority of patients develop resistance within several months. BRAFV600E-mutant malignant melanoma provides an illustrative example of this phenomenon: treatment with RAF and MEK inhibitors yields clinical responses in 50-80% of patients. However, 10-20% fail to respond to treatment (intrinsic resistance) and patients that do respond become drug resistant within ∼9 months (acquired resistance), presenting a formidable and unsolved clinical challenge. It remains incompletely understood why a subset of BRAFV600-mutant melanoma patients (10-20%) fail to respond to MAPK-pathway inhibition. Here, we show that RAF inhibitor sensitive and resistant BRAFV600-mutant melanomas display distinct transcriptional profiles. RAF-inhibitor sensitive cell lines are distinguishable by expression and activity of the melanocytic lineage transcription factor MITF, whereas intrinsically drug-resistant cell lines are defined by expression of the receptor tyrosine kinase AXL and elevated levels of NF-κB signaling. In vitro, these signatures were sufficient to predict MAPK-pathway inhibitor responsiveness in independent panels of melanoma cell lines. MITF-low, AXL/NF-κB high melanomas were resistant to single-agent RAF, MEK and ERK and combined RAF/MEK inhibition. In treatment-naïve patient biopsies, markers of the drug sensitive transcriptional states were associated with improved therapeutic responses to combined RAF/MEK inhibitors in BRAFV600-mutant melanoma. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF and AXL/NF-κB activity can influence intrinsic resistance to MAPK pathway inhibitors in BRAFV600-mutant melanoma. More broadly, these data suggest that the transcriptional context in which an oncogenic event arises can have a profound impact on the establishment of oncogene-dependencies and associated drug susceptibilities. Citation Format: Cory Johannessen, David Konieczkowski, Omar Abudayyeh, Jong Wook Kim, Zachary Cooper, Adriano Piris, Dennie Frederick, Michal Barzily-Rokni, Ravid Straussman, Rizwan Haq, David Fisher, Jill Mesirov, William Hahn, Keith Flaherty, Jennifer Wargo, Pablo Tamayo, Levi Garraway. A melanoma transcriptional state distinction influences sensitivity to MAPK pathway inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR04.

Braf Mutation Analysis in Cell Free Tumoral Dna (Cfdna) of Melanoma Patients: Preliminary Results from the Spanish Melanoma Group Prospective Study Gem1304

ABSTRACT Aim: Our data in BRAFV600 mutant melanoma patients treated with BRAF inhibitors showed a significant difference in median PFS by cfDNA status (3.5 months vs 13.57 months for BRAFV600 positive and negative, respectively p = 0.026)1. The Spanish Melanoma Group initiated a translational study to assess BRAFV600 mutation in cfDNA of stage IV BRAF mutant melanoma patients to determine the prognostic value of BRAFV600 mutation and its role in monitoring disease evolution (GEM1304) (ClinicalTrials.gov Identifier: NCT01960634). Methods: A quantitative 5'-nuclease PCR based assay for determination of BRAFV600 mutation in cfDNA was developed and validated in 92 previously genotyped cancer patients. Planned accrual for GEM1304 study was 58 BRAFV600 mutated melanoma patients. Serum and plasma samples are collected before and during treatment until disease progression. Results: The assay detected 2.5 pg mutated DNA/µL and a ratio of V600E versus wild type allele of 1:20000 with clinical sensitivity of 58% and specificity of 100%. Twenty-six patients, 47 samples, have been included to date. Pretreatment analysis was positive in serum in 16 (64%) and in plasma in 15 (60%) patients. Eleven patients have more than one sample; in four patients no mutation was detected in pretreatment and follow up samples (2 m+. 5 m+. 5 m+. 7 m+). BRAFV600 was detected in pretreatment samples in 6 patients.: in 4 responding patients BRAFV600 mutation was not detectable in subsequent analysis until disease progression (3m + , 6m, 8m + , 9m+). In two primary resistant patients, BRAFV600 persisted in follow up samples and time to death was less than 3 months. Discordant results between clinical response and cfDNA were found in one single patient: radiologic partial response was diagnosed but BRAFV600 mutation cfDNA load increased and patient died after 3 months on treatment. Re-evaluation of the previously performed scan showed new bone metastasis that had been inadvertent. Conclusions: Noninvasive genotyping of ctDNA by PCR assay could allow effective translation to the clinical setting. BRAFV600 mutation in pretreatment samples and persistence of BRAFV600 mutation at first evaluation could be prognostic markers of survival. Updated results will be presented during the meeting. Disclosure: M. Gonzalez-Cao: Work on this study was supported by a grant from Roche Pharmaceuticals. All other authors have declared no conflicts of interest.

Dabrafenib for the treatment of BRAF V600-positive melanoma: a safety evaluation

Introduction: V-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors are emerging as the standard of care for treating advanced melanomas harboring the BRAF V600 oncogenic mutation. Dabrafenib is the second approved selective BRAF inhibitor (after vemurafenib) for the treatment of unresectable or metastatic BRAF V600-positive melanoma.Areas covered: This review covers the current data on the efficacy and safety of the selective BRAF inhibitor dabrafenib in patients with metastatic BRAF V600 positive melanoma. The pharmacological, safety and efficacy data are discussed from Phase I, II, and III studies of dabrafenib monotherapy as well as in combination with the MEK inhibitor trametinib.Expert opinion: Dabrafenib has demonstrated comparable efficacy to vemurafenib in BRAF V600E mutant melanoma patients. Dabrafenib is well tolerated in patients with metastatic melanoma, including patients with brain metastases. Nevertheless side effects are common, but usually manageable. In the Phase III study testing dabrafenib, 53% of patients reported grade 2 or higher adverse events (AEs). Toxicities were similar to those seen in the early-phase trials, with the most common being cutaneous manifestations (hyperkeratosis, papillomas, palmar-plantar erythrodysesthesia), pyrexia, fatigue, headache, and arthralgia. Combining a BRAF inhibitor with a MEK inhibitor, which may block paradoxical MAPK activation in BRAF wild type (skin) cells, may lower the incidence of squamoproliferative eruptions.

Treatment patterns and outcomes in BRAF V600E mutant melanoma patients with brain metastases receiving vemurafenib in the real-world setting.

9035 Background: Metastatic Melanoma (mM) patients (pts) with brain metastases (BM) have a poor prognosis and median survival of <6 months. Selective BRAF inhibitors may play a role in the treatmen...

A phase I trial of BKM120 combined with vemurafenib in BRAFV600E/k mutant advanced melanoma.

9101 Background: Vemurafenib induces transient objective responses in half of BRAFV600Emutant melanoma patients and a median PFS of 5.3 months. PTEN loss and PI3K activation are common in BRAF mutant metastatic melanoma, and PI3K activation has been implicated as a cause acquired resistance to BRAF inhibitors. This phase I study tested the safety of combining the BRAF inhibitor, vemurafenib, with the PI3K inhibitor, BKM120, in patients with metastatic BRAF mutant melanoma. Methods: Vemurafenib-naive patients receive a single dose of oral BKM120 (d -7) then vemurafenib twice daily with BKM120 daily (starting on c1d1). Patients with prior progression on vemurafenib received both vemurafenib and BKM120 starting on c1d1 after a vemurafenib washout of at least 14 days. Serial biopsies prior to treatment, on cycle 1 day 15, and at progression were obtained for pharmacodynamics analysis in patients with visible or palpable tumors. 3 + 3 dose escalation was planned starting at vemurafenib 720 mg PO twice daily wi...

A phase I/II trial of BKM120 combined with vemurafenib (PLX4032) in BRAFV600E/k mutant advanced melanoma.

TPS8602 Background: Vemurafenib induces transient objective responses in half of BRAFV600E mutant melanoma patients and a median PFS of 5.3 months (NEJM. 2011;364:2507-2516). BRAF mutations are not sufficient to cause melanoma, but the combination of BRAFV600E mutation and PTEN loss is sufficient to recapitulate the malignant melanoma phenotype in vivo (Nat. Genet. 2009;41:544-552). PTEN loss and PI3K activation are common in BRAF mutant metastatic melanoma, and PI3K activation has been implicated as a cause acquired resistance to BRAF inhibitors (Cancer Cell. 2010;18:683-695). This phase I/II study is the first trial to test the safety and efficacy of combining a potent BRAF inhibitor, vemurafenib, with a potent PI3K inhibitor, BKM120, in patients with metastatic BRAF mutant melanoma. Methods: Design: Phase I patients receive a single dose of oral BKM120 (d -7) then vemurafenib twice daily with BKM120 daily (starting on c1d1). PK analysis is performed for BKM120 alone and for both drugs in combination. Doses of both drugs will be escalated in 3+3 scheme. Phase II patients receive continuous dosing of vemurafenib twice daily and BKM120 daily. Serial biopsies for PD and mRNA expression analyses are required for patients with visible or palpable tumors. Reimaging will be performed every 8 weeks.Eligibility: This study is enrolling BRAFV600E/K mutant metastatic melanoma patients with no prior exposure to BRAF inhibitors or PI3K inhibitors, ECOG PS ≥ 2 and adequate organ function. Endpoints: The primary endpoint for phase 1 is the recommended phase 2 dose of the combination. The primary endpoint for phase II is the 6 month PFS rate. Secondary endpoints include median PFS and OS. Baseline PTEN expression and changes is pS6 protein levels will be examined as predictors of efficacy, and changes in gene expression profiles will be assessed. Summary: This is the first trial examining the safety and efficacy of combined BRAF/PI3K inhibition in BRAF mutant melanoma patients.

Abstract 976: BRAF inhibitor GSK2118436 - single agent and combination activity with other targeted agents in BRAF V600 mutant melanoma

Abstract Although BRAF inhibitors display promising clinical response in BRAFV600 mutant melanoma patients, not all patients respond to these agents and the durability of the response is limited. To improve BRAF targeted therapy, we characterized a panel of BRAFV600 mutant melanoma cell lines and determined their response to cell growth inhibition, apoptosis induction and cell signaling changes by GSK2118436 alone and in combination with the MEK inhibitor GSK1120212 or PI3K/mTOR inhibitor GSK2126458. Nine of 11 BRAFV600E, 4/5 BRAFV600K and 1/1 BRAFV600D mutant lines were sensitive to GSK2118436 (IC50&amp;lt;0.3µM). These sensitive lines had wild-type MEK1/2 with or without PTEN mutations. Two of the three lines insensitive to GSK2118436 had either MEK1P124S or MEK2K66_K68del mutations. GSK2118436 treatment decreased MEK and ERK phosphorylation and cyclin D1 in both the sensitive and insensitive lines. However, GSK2118436 decreased S6 ribosomal protein phosphorylation (pS6P) only in the sensitive lines. Cell lines sensitive to GSK2118436 were also sensitive to the allosteric MEK inhibitor GSK1120212 (IC50&amp;lt;0.01µM). The combination of GSK2118436 and GSK1120212 had a synergistic to additive effect on cell growth inhibition in the sensitive lines, but had minimal benefit in the three insensitive lines. Although 5/6 PTEN NULL lines were sensitive to GSK2118436 based on their IC50s, growth of these lines was inhibited less effectively by GSK2118436 compared to the PTEN WT lines evaluated by IC70. PTEN NULL cells displayed higher levels of basal AKT phosphorylation (pAKT) than PTEN WT lines. Treatment of PTEN NULL lines with GSK2118436 increased pAKT while similar treatment of PTEN WT lines decreased pAKT and increased PTEN expression. In combination with the PI3K/mTOR inhibitor GSK2126458, GSK2118436 or GSK1120212 effectively inhibited MEK/ERK and PI3K/mTOR signaling, enhanced cell growth inhibition, and induced apoptosis evaluated by caspase 3/7 activation and/or PARP cleavage in both PTEN WT and PTEN NULL lines. In PTEN deficient lines, these combinations were more effective than the combination of GSK2118436 and GSK1120212. These results indicate MEK1/2 mutations associate with lack of sensitivity to single agent BRAF and MEK inhibitors. In addition, PTEN deficiency may reduce the effectiveness of BRAF or MEK inhibitors. The combination of GSK2118436 with GSK1120212 or GSK2126458 may improve the effectiveness of BRAF targeted therapy in BRAFV600 mutant melanoma. Our results support the testing of GSK2118436 with these combinations in the clinic and warrant further investigation of MEK and PTEN mutation status and pS6P as a pharmacodynamic marker to improve treatment outcome in BRAFV600 mutant melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 976. doi:1538-7445.AM2012-976

Using quantitative proteomic analysis to understand genotype specific intrinsic drug resistance in melanoma

The discovery of activating BRAF V600E mutations in 50% of all melanoma patients and the development of small molecule BRAF inhibitors looks set to revolutionize the therapy of disseminated melanoma. However, in the recent clinical trial of the BRAF inhibitor, vemurafenib (PLX4032), a significant percentage of BRAF V600E mutant melanoma patients did not meet the RECIST criteria for a response. Recent work from our lab identified loss of the tumor suppressor phosphatase and tensin homolog (PTEN) as being a possible mediator of intrinsic BRAF inhibitor resistance. In this commentary, we describe the development of a novel mass spectrometry based proteomic screen of Bcl-2 family proteins that was used to delineate the PTEN-dependent differences in apoptosis signaling observed when BRAF was inhibited. We further discuss how use of these sensitive quantitative proteomic methods gives unique insights into the signaling of cancer cells that are not captured through routine biochemical techniques and how this may lead to the development of combination therapy strategies for overcoming intrinsic BRAF inhibitor resistance.