BRAF-mutated Melanoma Patients Research Articles

Correlation between BRAF mutational status and clinical response to pembrolizumab in advanced melanoma patients

Meeting abstracts About 50% of melanoma (MM) have BRAF V600 mutation and BRAF inhibitors, such as vemurafenib and dabrafenib, have shown an high response rate (RR) and overall survival (OS) improvement in BRAF-mutated MM patients (pts). Pembrolizumab, a monoclonal antibody anti-PD-1, has shown a

Increased MAPK reactivation in early resistance to dabrafenib/trametinib combination therapy of BRAF-mutant metastatic melanoma.

One-third of BRAF-mutant metastatic melanoma patients treated with combined BRAF and MEK inhibition progress within 6 months. Treatment options for these patients remain limited. Here we analyse 20 BRAF(V600)-mutant melanoma metastases derived from 10 patients treated with the combination of dabrafenib and trametinib for resistance mechanisms and genetic correlates of response. Resistance mechanisms are identified in 9/11 progressing tumours and MAPK reactivation occurred in 9/10 tumours, commonly via BRAF amplification and mutations activating NRAS and MEK2. Our data confirming that MEK2(C125S), but not the synonymous MEK1(C121S) protein, confers resistance to combination therapy highlight the functional differences between these kinases and the preponderance of MEK2 mutations in combination therapy-resistant melanomas. Exome sequencing did not identify additional progression-specific resistance candidates. Nevertheless, most melanomas carried additional oncogenic mutations at baseline (for example, RAC1 and AKT3) that activate the MAPK and PI3K pathways and are thus predicted to diminish response to MAPK inhibitors.

BM-30 * INTRA CEREBROPSINAL FLUID CONCENTRATIONS OF VEMURAFENIB IN MELANOMA

The prognostic of BRAF mutated melanoma has been improved by Vemurafenib. Nevertheless, the cerebral and extracerebral responses to Vemurafenib may differ. Melanoma are one the most frequent causes of central nervous system metastases among solid tumors in adults. Our aim was to compare the serum and cerebrospinal fluid (CSF) concentrations of Vemurafenib in BRAF mutated melanoma patients presenting with brain metastases (BM) and a suspicion of leptomeningeal metastases (LM). A total of 6 patients were enrolled between February 2012 and March 2013. All patients had provided their written consent. The BRAF mutations were V600E in 5 patients and V600K in one patient. All patients had BM (>3 in 5 patients). Malignant cells were observed in the CSF in only one patient. Serum and CSF samples were performed at the same time while patients received Vemurafenib 960 mg twice a day. The concentrations were determined using liquid chromatography. Serum concentrations varied from 28.9 to 96.1 mg/L and CSF concentrations from 0.12 to 1 mg/L. The mean CSF serum ratio of Vemurafenib concentrations was 0.98% (range, 0.28–2.5%). No clear correlation was observed between the serum and CSF values. The higher concentrations of Vemurafenib in the CSF were observed in the 2 patients previously treated with stereotactic surgery. The great variability observed between patients may be explained by different level of expression and activity of efflux proteins (P-gp, BCRP), but also by the CNS metastases and their treatments.

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.

Dynamics of Chemokine, Cytokine, and Growth Factor Serum Levels in BRAF-Mutant Melanoma Patients during BRAF Inhibitor Treatment

The purpose of this study is to profile the changes in the serum levels of a range of chemokines, cytokines, and growth and angiogenic factors in MAPK inhibitor-treated metastatic melanoma patients and to correlate these changes with clinical outcome and changes in melanoma tissue biopsies taken from the same patients. Forty-two chemokine, cytokine, angiogenic, and growth factors were measured in the sera of 20 BRAF inhibitor-treated and four combination BRAF and MEK inhibitor-treated metastatic melanoma patients using a multiplex chemokine assay. The changes were correlated with Ki-67 and CD8(+) tumor-infiltrating lymphocytes in the tumor biopsies taken at the same time points, as well as clinical outcome, including response rate, progression-free survival, and overall survival. Serum levels of IFN-γ, CCL4, and TNF-α were significantly increased, whereas CXCL8 significantly decreased from pretreatment (PRE) to early during treatment (EDT) serum samples. The decrease in serum CXCL8 levels from PRE to EDT significantly correlated with decreases in markers of melanoma proliferation (Ki-67) and increases in cytotoxic tumor-infiltrating T cells in corresponding tumor biopsies. In addition, a greater fold reduction in CXCL8 serum levels from PRE to EDT serum samples was associated with decreased overall survival. These results suggest that BRAF inhibition causes decreased CXCL8 secretion from melanoma cells and induce an immune response against the tumor associated with increased IFN-γ, CCL4, and TNF-α. Further studies are needed to determine if CXCL8 is predictive of response and to confirm the functions of these chemokine and cytokine in BRAF-mutant melanoma under BRAF inhibition.

Inter- and Intra-Patient Heterogeneity of Response and Progression to Targeted Therapy in Metastatic Melanoma

BackgroundMAPK inhibitors (MAPKi) are active in BRAF-mutant metastatic melanoma patients, but the extent of response and progression-free survival (PFS) is variable, and complete responses are rare. We sought to examine the patterns of response and progression in patients treated with targeted therapy.MethodsMAPKi-naïve patients treated with combined dabrafenib and trametinib had all metastases ≥5 mm (lymph nodes ≥15 mm in short axis) visible on computed tomography measured at baseline and throughout treatment.Results24 patients had 135 measured metastases (median 4.5/patient, median diameter 16 mm). Time to best response (median 5.5 mo, range 1.7–20.1 mo), and the degree of best response (median −70%, range +9 to −100%) varied amongst patients. 17% of patients achieved complete response (CR), whereas 53% of metastases underwent CR, including 42% ≥10 mm. Metastases that underwent CR were smaller than non-CR metastases (median 11 vs 20 mm, P<0.001). PFS was variable among patients (median 8.2 mo, range 2.6–18.3 mo), and 50% of patients had disease progression in new metastases only. Only 1% (1/71) of CR-metastases subsequently progressed. Twelve-month overall survival was poorer in those with a more heterogeneous initial response to therapy than less heterogeneous (67% vs 93%, P = 0.009).ConclusionMelanoma response and progression with MAPKi displays marked inter- and intra-patient heterogeneity. Most metastases undergo complete response, yet only a small proportion of patients achieve an overall complete response. Similarly, disease progression often occurs only in a subset of the tumor burden, and often in new metastases alone. Clinical heterogeneity, likely reflecting molecular heterogeneity, remains a barrier to the effective treatment of melanoma patients.

Abstract C137: Rapid assessment of TORC1 suppression as a functional biomarker predicting responsiveness to RAF and MEK inhibitors in BRAF-mutant melanoma patients.

Abstract The clinical development of selective RAF and MEK inhibitors has transformed the treatment of the ∼50% of melanoma patients whose tumors harbor BRAF mutations. However, a substantial percentage of these patients fail to respond to therapy, and most responses are partial and short-lived. Although multiple mechanisms of resistance have been identified in BRAF-mutant melanoma, no clinically useful biomarkers have been established to predict which patients are most likely to demonstrate sensitivity or resistance to RAF or MEK inhibitors. We found that suppression of TORC1 activity in response to RAF or MEK inhibitors, as measured by decreased phosphorylation of ribosomal protein-S6 (P-S6), was a functional biomarker that effectively predicted sensitivity in BRAF-mutant melanoma cell lines in vitro and in mouse tumor xenografts. In sensitive melanomas, TORC1 and P-S6 were suppressed in response to RAF or MEK inhibitors, but in resistant melanomas, TORC1 activity was maintained, in some cases despite robust suppression of MAPK signaling by these inhibitors. In mouse models, suppression of TORC1 after MAPK inhibition was necessary for induction of apoptosis and tumor response in vivo. Notably, in paired biopsies obtained from patients with BRAF-mutant melanoma before treatment and after initiation of RAF inhibitor therapy, P-S6 suppression was associated with significantly improved progression-free survival [HR 0.19, 95% CI 0.01-0.84, p=0.03]. Finally, we found that changes in P-S6 in patients’ tumor cells could be readily monitored in real-time by multiplexed, quantitative immunofluorescence microscopy of serial fine-needle aspiration biopsies obtained from patients before and during the first 2 weeks of RAF inhibitor therapy. This approach provides a minimally-invasive means of rapidly monitoring the efficacy of treatment, before changes in tumor volume are apparent by traditional radiographic imaging. Together, these results establish suppression of P-S6 after initiation of RAF inhibitor therapy as a robust potential functional biomarker to guide the treatment of patients with BRAF-mutant melanoma, and present a powerful methodology for monitoring changes in potentially any signaling pathway in response to targeted therapies in patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C137. Citation Format: Ryan B. Corcoran, S. Michael Rothenberg, Aaron N. Hata, Anthony C. Faber, Adriano Piris, Rosalynn M. Nazarian, Ronald D. Brown, Jason T. Godfrey, Daniel Winokur, John Walsh, Mari Mino-Kenudson, Shyamala Maheswaran, Jeffrey Settleman, Jennifer A. Wargo, Keith T. Flaherty, Daniel A. Haber, Jeffrey A. Engelman. Rapid assessment of TORC1 suppression as a functional biomarker predicting responsiveness to RAF and MEK inhibitors in BRAF-mutant melanoma patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C137.

MC13-0075 Rapid assessment of TORC1 suppression as a functional biomarker predicting responsiveness to RAF and MEK inhibitors in BRAF-mutant melanoma patients

MC13-0075 Rapid assessment of TORC1 suppression as a functional biomarker predicting responsiveness to RAF and MEK inhibitors in BRAF-mutant melanoma patients

Abstract C41: BRAFV600 serum/plasma analysis: Predictive value of survival in melanoma treated with BRAF inhibitors.

Abstract Background: BRAF mutation is present in 50% of metastatic melanoma patients. Treatment with BRAF inhibitors improves survival of BRAF mutant melanoma patients, with a median progression free survival time of 6 months. Twenty percent of patients are refractory to BRAF inhibitors, and others have early resistance development. Pretreatment analysis of BRAFV600 mutation in cell free DNA from serum or plasma could help to select patients for BRAF inhibitor treatments. We have investigated the sensitivity of the assay and its predictive value. Methods: Analysis of BRAFV600 mutation was performed in cell free DNA (cf DNA) from serum and plasma of 22 metastatic melanoma patients before starting a BRAF inhibitor treatment. Results: Six patients were women, median age was 62 years old (58-81). Four patients (18%) had complete response to treatment. Eleven patients (50%) had partial response to treatment. Two patients (9%) had progression disease as their best response. Median progression free survival time (PFS) for patients with complete response, partial response, stable disease and progression disease were: not reached, 4.6 months, 3 months and one month, respectively (p&amp;lt;0.00001). Twelve patients (54.5%) had BRAF positive analysis pretreatment in serum/plasma. From BRAF serum/plasma positive patients 1 had CR (8%) and 7 PR (58%). From BRAF serum/plasma negative patients: 3 (30%) CR and 4 (40%) PR. Median PFS for patients treated with BRAF inhibitors was 3.5 months for BRAF serum/plasma positive patients and 13.57 months for BRAF serum/plasma negative patients (p=0.026) Conclusion: BRAFV600 serum/plasma analysis is a good predictor of survival for patients treated with BRAF inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C41. Citation Format: Maria Gonzalez Cao, Salvador Martin Algarra, Eva Muñoz, Clara Mayo de las Casas, Jose Luis Manzano, Javier Cortes, Miguel Angel Molina-Vila, Jose Pablo Berros, Leticia De Mattos-Arruda, Miguel Sanmamed, Alvaro Gonzalez, Carlos Alvarez, Niki Karachaliou, Rafael Rosell. BRAFV600 serum/plasma analysis: Predictive value of survival in melanoma treated with BRAF inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C41.

Mind the IQGAP

The scaffold protein IQGAP1 regulates cell signaling through the RAF/MEK/ERK pathway. Recent data show that cancer cells in which the RAF/MEK/ERK pathway is activated are particularly sensitive to the disruption of IQGAP1 function. IQGAP drugs may be particularly effective in tumors that develop resistance to existing pathway drugs.

Clinical characteristics and survival of BRAF-mutant (BRAF+) metastatic melanoma patients (pts) treated with BRAF inhibitor (BRAFi) dabrafenib or vemurafenib beyond disease progression (PD).

9062 Background: Accelerated tumor growth has been demonstrated on BRAFi cessation in BRAFi-resistant melanoma cell lines. In BRAFi-treated pts initial response rates are high but most have PD at 6+ months. The benefit of ongoing BRAFi after PD is unknown. We sought to describe the characteristics of pts treated beyond progression (TBP) vs those not TBP, and whether TBP prolongs survival. Methods: Clinicopathologic data were collected on 112 pts enrolled in phase I-IV clinical trials at Westmead Hospital and Melanoma Institute Australia from July 2009 to Sept 2012 with unresectable stage IIIC or IV BRAF+ melanoma treated with single agent dabrafenib or vemurafenib. TBP was defined as ongoing BRAFi &gt;28 days beyond RECIST PD. Pt and disease characteristics at baseline and at PD were examined, as well as survival data. Results: 92/112 (82%) pts had RECIST PD. 36/92 (39%) pts were TBP (mean 144 days, median 93 days, range 29–572 days). Pts TBP were significantly more likely to have achieved a RECIST response (CR/PR) prior to PD, have a lower ECOG at PD, presence of brain metastases at PD, have PD treated locally and a smaller RECIST sum of diameters (SoD) at PD, compared with those not TBP (all p&lt;0.05). Median OS from commencement of BRAFi in those TBP was longer than those not TBP (15.0 vs 6.5 months, p&lt;0.001), as was OS from RECIST PD (7.4 vs 1.9 mo, p=0.001). In multivariate analysis of all pts, TBP improved OS from RECIST PD (HR 0.32, p=0.012) even after adjusting for other potential prognostic factors at PD (see table). Within the TBP cohort, RECIST SoD at PD was the only factor that influenced OS from PD (p=0.009), and presence of brain metastases did not. Conclusions: Treatment with BRAFi may be continued after RECIST PD in selected pts, and is associated with a prolonged OS compared with ceasing treatment at PD. [Table: see text]

Gamma knife stereotactic radio-surgery and BRAF inhibition in metastatic melanoma.

9088 Background: Gamma knife radio-surgery (GK) is an effective approach to treating brain metastases in patients with metastatic melanoma. BRAF inhibition with vemurafenib produces a median progression free survival (PFS) of 7 months, but low central spinal fluid penetration may limit its effectiveness in patients who develop brain metastases. We report long term follow-up of patients with BRAF mutant melanoma treated with vemurafenib and GK. Methods: Demographics and clinical outcomes were characterized for 18 BRAF mutant melanoma patients with brain metastases treated between 2007 and 2012 with GK and with vemurafenib (vem) for &gt;1 month. Results: The median age at starting vem was 51 yrs (range 34-76). 61% of the patients were women. Patients were treated with a median of 1 prior therapy (range 0-3). 7/18 patients (39%) had brain involvement prior to starting vem. 16/18 patients (89%) had stage M1c disease at the time of starting vem. Patients were treated with vem for a median of 8.4 months (range 2.1 to 27 months), had a median survival of 15.7 months after starting vem (range 4.2-29.4), and a median survival after GK of 7.8 months (range 1.2-21.1). Patients underwent GK to a median of 3 lesions (range 1-6). In total, 8/18 patients (44%) were treated with WBRT. 7/18 patients underwent craniotomies, 2 of which were for progression in lesions treated with GK, and 4 of which were also among the patients treated with WBRT. In 7/18 patients treated with GK for new brain progression after starting vem, vem was continued after GK for a median duration of 4.1 months (range 1.3 to 15.8). In these 7 patients that had vem and GK and continued vem, the median overall survival after starting vem was 19.9 months (range 6.7 to 29.3). Of these 7 patients, 4 required whole brain radiation therapy (WBRT); 3 have not required any additional brain directed therapy, including 2 patients who continue on vemurafenib at the time of analysis. 5/18 patients were treated with more than 1 round of GK, 3 of which were subsequently treated with WBRT. Conclusions: Despite the brain being a common site of progression on vemurafenib therapy, long term survival can be achieved in some cases by continuing vem after GK therapy.

Abstract 4568: Rapid assessment of TORC1 suppression predicts responsiveness to RAF and MEK inhibition in BRAF mutant melanoma.

Abstract The clinical development and potent efficacy of selective RAF and MEK inhibitors has transformed the treatment of the ∼50% of melanoma patients whose tumors harbor BRAF mutations. However, a substantial percentage of these patients fail to respond to therapy, and all who respond eventually develop resistance. We found that suppression of TORC1 activity, as measured by decreases in ribosomal protein S6 phosphorylation (P-S6) in response to RAF or MEK inhibitors, was a highly effective predictor of sensitivity in BRAF mutant melanoma cell lines in vitro and in mouse xenografts. TORC1 and P-S6 were suppressed in response to RAF or MEK inhibitors in sensitive but not resistant melanoma cells, despite robust suppression of the MAPK pathway by these inhibitors. Notably, suppression of P-S6 levels, as assessed by immunohistochemistry in paired biopsies obtained from BRAF mutant melanoma patients before and during treatment, was associated with significantly improved progression-free survival. Finally, we show that changes in P-S6 levels specifically in tumor cells can be readily monitored by multiplexed, quantitative immunofluorescence microscopy of fine needle aspiration biopsies obtained from patients before and during the first two weeks of BRAF inhibitor therapy. This provides a minimally invasive means for monitoring the efficacy of treatment in real time, before changes in tumor volume are apparent on traditional radiographs. Together, these results establish changes in P-S6 levels with therapy as a robust biomarker to guide the treatment of patients with BRAF mutant melanoma, and present a powerful methodology for monitoring changes in potentially any signaling pathway in response to therapy in patients. Citation Format: Ryan B. Corcoran, Stephen M. Rothenberg, Aaron N. Hata, Anthony C. Faber, Daniel Winokur, Adriano Piris, Rosalynn M. Nazarian, Ronald D. Brown, Jason T. Godfrey, Mari Mino-Kenudson, Jeffrey Settleman, Jennifer A. Wargo, Keith T. Flaherty, Daniel A. Haber, Jeffrey A. Engelman. Rapid assessment of TORC1 suppression predicts responsiveness to RAF and MEK inhibition in BRAF mutant melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4568. doi:10.1158/1538-7445.AM2013-4568

Abstract 3404: Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma.

Abstract We generated melanoma cell lines resistant to BRAF inhibitors and show that the EGF receptor (EGFR)-SRC family kinase (SFK)-STAT3 signaling pathway was upregulated in these cells. In addition to driving proliferation of resistant cells, this pathway also stimulated invasion and metastasis. EGFR inhibitors cooperated with BRAF inhibitors to block the growth of the resistant cells in vitro and in vivo, and monotherapy with the broad specificity tyrosine kinase inhibitor dasatinib blocked growth and metastasis in vivo. We analyzed a tumor from a BRAF mutant patient with intrinsic resistance to vemurafenib. Vemurafenib treatment induced SFK activation in this tumor and we show that dasatinib blocked its growth and metastasis in immunocompromised mice. Our data shows that BRAF inhibitor-mediated activation of EFGR/SFK/STAT3 signaling can mediate resistance in BRAF mutant melanoma patients. We describe two treatments that appear to overcome this resistance and could deliver therapeutic efficacy in drug-resistant BRAF mutant melanoma patients. Citation Format: Maria R. Girotti, Malin Pedersen, Berta Sanchez-Laorden, Amaya Viros, Samra Turajlic, Dan Niculescu-Duvaz, Alfonso Zambon, John Sinclair, Andrew Hayes, Martin Gore, Caroline Springer, James Larkin, Claus Jorgensen, Richard Marais. Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3404. doi:10.1158/1538-7445.AM2013-3404

Abstract LB-144: Modeling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance.

Abstract Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥50% of tumors expressing the BRAF (V600E) oncoprotein. Moreover, the impressive tumor regression and increased survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance. Although vemurafenib therapy has transformed the treatment of late-stage melanoma patients, the durability of patient response to this drug is limited by the emergence of lethal drug resistant disease. Thus, understanding mechanisms and designing approaches to prevent the emergence of resistance are critical to providing improved therapy. Here we model the emergence of vemurafenib resistance using two early passage primary human-patient-derived xenograft (PDX) BRAF (T1799A)-mutated melanoma models, which were continuously treated with vemurafinib in immunocompromised mice. In one of these models vemurafenib resistance is due to selection of cells with elevated expression of the BRAF (V600E) oncoprotein. Remarkably, vemurafenib resistant tumors are not only drug resistant but also develop drug dependency such that tumors require exposure to vemurafenib for their continued proliferation. Indeed, cessation of drug administration in these PDX models leads to inhibition of tumor growth and, in many cases, tumor regression. This fitness deficit upon cessation of drug administration is based on an adaptive response to levels of MAPK pathway signaling output. Melanoma cells appear to proliferate best at a defined intermediate level of RAF/MEK/ERK signaling output. Hence, when drug resistant tumor cells are deprived of drug, the level of signaling output is increased leading to decreased proliferation. We sought to determine whether vemurafenib-resistant tumors in melanoma patients displayed similar evidence of drug dependency. In a small cohort of patients (n=42) with vemurafenib-resistant tumors for which CT scans were available following cessation of treatment (n=19), 14 out of 19 patients demonstrated evidence of reduced tumor growth velocity. Taken together, these data support the hypothesis that continuous treatment with vemurafenib can select for drug-resistant tumor cells that show a fitness deficit in the absence of the drug. Furthermore, we demonstrate in the preclinical setting that these observations can be leveraged therapeutically by using a discontinuous treatment regimen to forestall the emergence of resistance in two different PDX models. Consequently, these data may have important implications for the treatment of BRAF mutated melanoma patients with BRAF (V600E) inhibitors and suggest that altered dosing might delay or prevent the emergence of lethal drug resistant disease. Citation Format: Meghna Das Thakur, Rosalie Fisher, Fernando Salangsang, Allison Landman, William Sellers, Nancy Pryer, Mitchell Paul Levesque, Reinhard Dummer, Martin Gore, James Larkin, Martin McMahon, Darrin Stuart. Modeling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-144. doi:10.1158/1538-7445.AM2013-LB-144

Abstract 3405: Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines.

Abstract Background: Resistance to targeted therapies represents a key hurdle in the treatment of cancer. Preclinical data suggest that resistance is frequently driven by redundant growth factor signaling. For example, HGF has been reported to mediate resistance to BRAF inhibitors in V600E BRAF mutant melanoma. Furthermore, clinical data support these findings as circulating and tumor HGF levels have been shown to correlate with outcome in BRAF mutant melanoma patients treated with vemurafenib. In this study, we evaluated the ability of HGF / MET signaling to attenuate the effects of BRAF and MEK inhibitors in the context of V600E BRAF and NRAS mutant cell lines. Our data suggest that combined blockade of MET and MAPK pathway signaling may be required for a robust and durable clinical response in melanoma. Methods: To confirm the ability of HGF to rescue V600E BRAF mutant melanoma cell lines from the effects of vemurafenib, G361 and SKMEL5 cells were treated with vemurafenib alone or in combination with HGF. Time lapse imaging, viability assays and cell cycle analysis were used to measure treatment effects. Analogous experiments were performed with the selective MEK inhibitor PD0325901. Combinations of a potent and selective MET inhibitor (Compound A) with vemurafenib or PD0325901 were tested to evaluate their ability to attenuate the HGF-mediated resistance. To identify other cell lines rescued by HGF, thirteen V600E BRAF mutant melanoma cell lines were treated with a dose response matrix of vemurafenib and HGF. The ability of six additional growth factors to rescue cells from the effects of vemurafenib was also tested. Three NRAS mutant melanoma cell lines were also screened for HGF-mediated resistance to PD0325901. Results: Nine of thirteen V600E BRAF mutant melanoma cell lines were rescued from the effects of vemurafenib by co-treatment with HGF. Detailed analysis of G361 and SKMEL5 cells revealed that HGF was also capable of rescuing the cells from the effects of PD0325901. This HGF-mediated rescue was attenuated by treatment with combinations of vemurafenib + compound A or PD0325901 + compound A. Mechanistic cell cycle analysis of the HGF-mediated rescue of vemurafenib in G361 cells revealed an increase in proliferating cells and a reduction in growth arrested cells, whereas rescue of PD0325901 treatment occurred through a reduction in the percentage of dead or dying cells. Of the seven growth factors tested, HGF was the predominate growth factor capable of conferring resistance to vemurafenib. Furthermore, HGF rescued one of three NRAS mutant melanoma cell lines from the effects of PD0325901, highlighting the breadth of HGF-mediated resistance in melanoma cell lines. Conclusion: These findings demonstrate the potential role for HGF / MET signaling in mediating resistance to BRAF and MEK inhibitors in melanoma, and support the clinical evaluation of MET kinase inhibitors and neutralizing antibodies to HGF in this setting. Citation Format: Sean Caenepeel, Elaina Cajulis, Rick Kendall, Angela Coxon, Paul Hughes. Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2013-3405

Abstract SY17-03: Targeting BRAF and PI3′-kinase signaling for therapy of melanoma.

Abstract Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with greater than 50% of tumors expressing the BRAF(V600E) oncoprotein. BRAF(V600E) is a constitutively active protein kinase that promotes sustained activation of BRAF→MEK→ERK signaling in the melanoma cell. Importantly, the marked tumor regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF(V600E) in melanoma maintenance. However, since most patients relapse with lethal vemurafenib resistant disease, understanding and preventing mechanism(s) of drug resistance will be critical to extending the durability of patients' responses. Although mutational activation of BRAF is often the initiating event, progression of BRAF mutated melanomas is frequently associated with silencing of the PTEN tumor suppressor, a negative regulator of PI3′-kinase signaling. Whilst PTEN silencing is common in human melanoma, mutational activation of PIK3CA (encoding the alpha catalytic subunit of PI3′-kinase) is extremely rare, even though PIK3CA mutations are common in other human cancers. Since PTEN is reported to have tumor suppressor functions independent of its PI3′-lipid phosphatase activity, PTEN silencing may have more profound tumor promoting activity than mutational activation of PIK3CA. To test this, we generated mice carrying a conditional knock-in allele of Pik3ca that permits Cre-induced expression of PIK3CA(H1047R), a mutationally activated form of PIK3CA detected in many cancer types. Expression of PIK3CA(H0147R) in melanocytes elicited no obvious phenotype, consistent with the absence of phenotype in PTEN(Null) melanocytes. As observed previously, melanocyte-specific expression of BRAF(V600E) combined with PTEN silencing led to metastatic melanoma. Interestingly, metastatic melanoma also arose from melanocytes expressing both BRAF(V600E) and PIK3CA(H1047R), although these tumors grew more slowly than their BRAF(V600E)/PTEN(Null) counterparts. Both BRAF(V600E)/PTEN(Null) and BRAF(V600E)/PIK3CA(H1047R) melanomas were sensitive to the tumor prevention effects of pharmacological blockade of class 1 PI3′-kinases. However, the clinically relevant subset of BRAF(V600E)/PTEN(Null) melanoma was resistant to pharmacological blockade of AKT. Using genetically engineered mouse models and melanoma-derived cell lines, we demonstrate cooperative anti-tumor effects of combining RAF and PI3′-kinase inhibitors, suggesting that combined pathway-targeted chemotherapy may maximize melanoma patients' therapeutic response while delaying the onset of lethal drug resistant disease. Using patient derived xenografts propagated in immunocompromised mice we explored the cause and consequences of drug resistance, which was selected by continuous vemurafenib administration. As observed previously, resistant tumors displayed continued dependence on BRAF(V600E)→MEK→ERK signaling due to elevated BRAF(V600E) expression. However, remarkably, we observed that vemurafenib-resistant melanomas displayed a striking drug dependency for their continued proliferation, such that cessation of drug administration led to regression of established drug-resistant tumors. We further demonstrated that a discontinuous dosing strategy, which exploits the fitness deficit displayed by drug-resistant cells in the absence of the vemurafenib, forestalls the onset of lethal drug resistant disease. These data highlight the concept that drug-resistant cells may also show dependency, such that altered drug dosing may prevent the emergence of lethal drug resistance. Citation Format: Martin McMahon, Meghna Das Thakur, Victoria Marsh, Jillian Silva, Allison Landman, Marian Deuker, Fernando Salangsang, Nancy Pryer, Wayne Phillips, Mitchell Levesque, Reinhard Dummer, Marcus Bosenberg, William R. Sellers, Darrin Stuart. Targeting BRAF and PI3′-kinase signaling for therapy of melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY17-03. doi:10.1158/1538-7445.AM2013-SY17-03

Abstract 1724: BRAF inhibition is enhanced by BH3-mimetic through BIM induction.

Abstract Vemurafenib (Vem) is a highly selective inhibitor of mutant BRAF, FDA-approved for the treatment of patients with BRAF-positive advanced melanoma. While response rates to Vem are high, the emergence of resistance is common, placing high priority in the development of strategies to enhance Vem efficiency. Navitoclax is a BH3-mimetic that binds and inactivates the anti-apoptotic proteins BCL-2, BCL-xl, and BCL-w and leads to apoptosis in tumors with low expression of another anti-apoptotic protein, MCL-1. Our data demonstrates that treatment of BRAF-mutant melanoma patients with Vem is associated with decreased expression of MCL-1 in addition to an increase in the pro-apoptotic, BH3-only protein BIM in serial biopsy samples. Therefore, we hypothesize that the combination of PLX-4720 (Vem analog) with navitoclax will result in increased anti-tumor activity. To test our hypothesis, we performed cell viability studies, Annexin:PI and ELISA apoptosis assays in addition to protein and mRNA analysis. In-vivo studies were performed in two BRAF mutant xenograft models using navitoclax and PLX-4720. In-vitro, our cell viability studies demonstrated that concurrent treatment with PLX-4720 and navitoclax results in an additive viability reduction in BRAF mutant but not in BRAF WT cell lines. The decrease in cell viability correlated to downmodulation of MCL-1 and BIM induction after PLX-4720 treatment, with further enhancement of BIM expression and PARP cleavage after adding navitoclax. In vivo, the navitoclax enhanced the efficacy of PLX-4720 in both xenograft models. The combination of a selective BRAF inhibitor with a BH3-mimetic may be a promising therapeutic strategy to enhance the clinical efficacy of BRAF inhibitors. Citation Format: Dennie T. Frederick, Roberto A. Salas Fragomeni, Taylor Hoff, Zachary A. Cooper, James W. Mier, Jennifer A. Wargo, Ryan J. Sullivan. BRAF inhibition is enhanced by BH3-mimetic through BIM induction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2013-1724

Mutant B-RAF-Mcl-1 survival signaling depends on the STAT3 transcription factor

Approximately 50% of melanomas depend on mutant B-RAF for proliferation, metastasis and survival. The inhibition of oncogenic B-RAF with highly targeted compounds has produced remarkable albeit short-lived clinical responses in B-RAF mutant melanoma patients. Reactivation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may provide new strategies for managing melanoma. Oncogenic B-RAF(V600E) is known to promote the stabilizing phosphorylation of the anti-apoptotic protein Mcl-1, implicated in melanoma survival and chemoresistance. We now show that B-RAF(V600E) signaling also induces the transcription of Mcl-1 in melanocytes and melanoma. We demonstrate that activation of STAT3 serine-727 and tyrosine-705 phosphorylations is promoted by B-RAF(V600E) activity and that the Mcl-1 promoter is dependent on a STAT consensus-site for B-RAF-mediated activation. Consequently, suppression of STAT3 activity disrupted B-RAF(V600E)-mediated induction of Mcl-1 and reduced melanoma cell survival. We propose that STAT3 has a central role in the survival and contributes to chemoresistance of B-RAF(V600E) melanoma.

Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance

Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥50% of tumours expressing the BRAF(V600E) oncoprotein. Moreover, the marked tumour regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance. However, as most patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is critical to providing improved therapy. Here we investigate the cause and consequences of vemurafenib resistance using two independently derived primary human melanoma xenograft models in which drug resistance is selected by continuous vemurafenib administration. In one of these models, resistant tumours show continued dependency on BRAF(V600E)→MEK→ERK signalling owing to elevated BRAF(V600E) expression. Most importantly, we demonstrate that vemurafenib-resistant melanomas become drug dependent for their continued proliferation, such that cessation of drug administration leads to regression of established drug-resistant tumours. We further demonstrate that a discontinuous dosing strategy, which exploits the fitness disadvantage displayed by drug-resistant cells in the absence of the drug, forestalls the onset of lethal drug-resistant disease. These data highlight the concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance. Such observations may contribute to sustaining the durability of the vemurafenib response with the ultimate goal of curative therapy for the subset of melanoma patients with BRAF mutations.