BRAF-mutant Melanoma Cell Lines Research Articles

TGF‐β1 and TNF‐α differentially regulate Twist1 mediated resistance towards BRAF/MEK inhibition in melanoma

Resistance to BRAF and MEK inhibition is a common phenomenon in melanoma. Cytokines and transcription factors have been attributed to contribute to the loss of sensitivity towards these inhibitors. Here, we show that transforming growth factor (TGF)-β1 if combined with PLX4032, a BRAF inhibitor, or GSK1120212, a MEK inhibitor, substantially increased cell death in BRAF-mutant melanoma cell lines. This increase was based on the combined regulatory decrease in Twist1, an antiapoptotic protein. Overexpression or silencing of Twist1 attenuated or aggravated induction of apoptosis through PLX4032 or GSK1120212, respectively. Exposure to tumour necrosis factor (TNF)-α, however, led to increased Twist1 levels and oppositional decrease in cell death if exposed to PLX4032 or GSK1120212. This increase in drug resistance again depended on Twist1 levels. Our studies suggest that Twist1 as a common downstream target of multiple signalling cascades plays a crucial role in mediating drug resistance to BRAF- and MEK-targeted molecular inhibitors.

Use of combination treatment with the investigational RAF kinase inhibitor MLN2480 and the investigational MEK kinase inhibitor TAK-733 on the growth of BRAF-mutant and RAS-mutant preclinical models of melanoma and CRC.

e13529 Background: RAS mutant melanoma and colorectal cancer represent areas of significant unmet medical need. MLN2480 is an investigational class II RAF kinase inhibitor and TAK-733 is an investigational allosteric MEK kinase inhibitor; each of which is the subject of a single agent phase I clinical trial. The present studies have characterized the combination activity of these agents in BRAF mutant and RAS mutant preclinical models of melanoma and colorectal cancer. Methods: Combination effects of MLN2480 and TAK-733 on cell viability were studied using an ATP-based cell viability assay across a panel of BRAF and RAS mutant melanoma and CRC cell lines. Western blot analysis was used to compare effects on MAPK pathway signaling and response markers in cell lines showing a range of sensitivity to this combination. Pharmacodynamic responses and growth inhibitory effects of the combination were studied in xenografts of the same cell lines, as well as in primary human tumor xenografts, of RAS mutant melanoma and CRC. Results: MLN2480 inhibits MAPK pathway signaling in BRAF mutant and some RAS mutant preclinical cancer models at concentrations that are tolerated in vivo. MLN2480 is most potent in BRAF mutant melanoma models but also has single agent activity in some RAS mutant models. The combination of MLN2480 with TAK-733 inhibits the growth of a broader range of RAS mutant tumor models than single agent MLN2480, including primary human tumor xenograft models of melanoma and CRC. In vitroanalysis of this drug combination in cell proliferation assays demonstrates synergistic activity. Western blot analysis demonstrated the effect of MLN2480 in reversing feedback activation of MEK in response to TAK-733, leading to more concerted MAPK pathway inhibition. Conclusions: The activity of the RAF kinase inhibitor MLN2480 in preclinical models of BRAF and RAS mutant melanoma and CRC provides a rationale for clinical testing. The combination of MLN2480 with the MEK inhibitor TAK-733 represents an additional strategy for clinical research within these tumor types.

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 3401: Vemurafenib-induced RhoB expression leads to melanoma cell resistance.

Abstract Most of melanoma harbour mutations leading to the constitutive activation of the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway (i.e: activating mutations of BRAF, NRAS, c-KIT). Addiction of melanoma to the MAPK pathways led to the development of inhibitors targeting either mutant BRAF, such as Vemurafenib (PLX4032), or MEK. Melanoma treatment recently met a breakthrough with the clinical evaluation of Vemurafenib that demonstrated an unprecedented 80% response rate among patients with BRAF V600E melanomas treated with Vemurafenib, but rapidly limited by drug resistance, which still requires to be elucidated. The MAPK pathway regulates the expression of many genes which can mediate therapeutic response but also resistance, such as RhoGTPases. We analysed the mRNA expression of the RhoGTPases in two BRAF mutated melanoma cell lines, WM266-4 and A375, in response to inhibition of the MAPK pathway by PLX4032. We observed that the mRNA of RhoB was increased by PLX4032 treatment. This result was confirmed in many other BRAF mutated melanoma cell lines by inhibition of the MAPK pathway with BRAF inhibitors (PLX4032 or SB-590885) as well as MEK inhibitors (AZD6244 or AS703026). MEK inhibitors (AZD6244 or AS703026) upregulate RhoB in BRAF wild-type melanoma cells harbouring N-Ras, K-Ras or c-KIT mutation suggesting that inhibiting MAPK pathway is critical for RhoB expression modulation. It is noteworthy that this modulation is cell type dependent, while it was not found in BRAF mutated non-melanoma cells as colon carcinoma cells (Colo205 and RKO). To investigate the role of RhoB induction in cellular responses to BRAF and MEK inhibitors, the RhoB induction was suppressed using RNA interference. The cells treated with siRNA targeting RhoB, WM266-4 and A375 cells, were more sensitive to PLX4032 and AZD6244. Moreover the blockade of RhoB induction led to a significant increase of PLX4032-induced apoptosis as shown by the increase of the subG1 population and PARP cleavage. Our findings show that RhoB upregulation is involved in PLX4032 response and both inhibitions of MAPK pathway and RhoB expression trigger cell death and should prompt a search for agents. Indeed co-targeting MAPK and RhoB pathways, could lead to a synthetic lethality and prevent resistance to either BRAF or MEK inhibitors. Ongoing work focuses on confirming this role in tumour-xenografted mouse models. Citation Format: Audrey Delmas, Julia Cherier, Claire Medale-Giamarchi, Anne Pradines, Gilles Favre. Vemurafenib-induced RhoB expression leads to melanoma cell 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 3401. doi:10.1158/1538-7445.AM2013-3401

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 5605: Pharmacologic inhibition of EphA2 in NRAS and BRAF mutant melanomas.

Abstract The Eph receptors are the largest known family of receptor tyrosine kinases (RTKs). Research studies have shown that Eph receptors and ligands may be involved in many diseases including cancer. EphA2 is a member of the Eph family of receptor tyrosine kinases and is highly expressed in many aggressive cancer types, including melanoma. Our previous studies showed that EphA2 levels are elevated in melanoma cells and EphA2 plays critical roles in melanoma cell survival, proliferation, colony formation and migration in vitro and melanoma tumor growth in vivo. Together, these data suggest that EphA2 is a critical oncogene in melanoma and a potential pharmacologic target in the treatment of metastatic disease. Herein we present early evidence that EphA2 inhibitors (ALW-II-41-27 and HG-6-64-1) are effective in inhibiting proliferation of both NRAS and BRAF mutant melanoma cell lines in vitro through suppressing a series of EphA2 downstream signaling pathways. At the same time, EphA2 inhibitors are also effective at suppressing migration and invasion of melanoma cells with a reduction in actin cytoskeleton reorganization and lamellipodia formation. Animal studies are underway to determine the effectiveness of these agents in suppressing melanoma tumor growth in vivo. These results point to EphA2 inhibition as a promising therapeutic strategy for both NRAS and BRAF mutant melanomas either singly or in combination with BRAF or MEK inhibitors currently in trial. Citation Format: Benchun Miao, Jianming Zhang, Zhenyu Ji, Ching Ni Njauw, Michael Taylor, Raj Kumar, Nathanael Gray, Hensin Tsao. Pharmacologic inhibition of EphA2 in NRAS and BRAF mutant melanomas. [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 5605. doi:10.1158/1538-7445.AM2013-5605

Abstract 917: High antitumor activity of the ERK inhibitor SCH722984 against BRAF-mutant, NRAS-mutant and wild-type melanoma cell lines.

Abstract MAPK pathway dysregulation via mutations in proteins such as BRAFV600E and NRASQ61 is a key feature in melanomas, leading to constitutive ERK signaling. Currently, there are no FDA-approved targeted therapies effective in vemurafenib-resistant BRAFV600E mutant, NRAS mutant, or wild-type melanoma. The effect of SCH722984, an allosteric inhibitor of ERK 1/2, was determined in a panel of 53 melanoma cell lines, including wild-type, NRAS-mutant and vemurafenib-sensitive and resistant BRAF-mutant melanoma. 54% (13 of 24) of BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH722984 with an IC50 <1μM. 100% (3 of 3) of BRAF/NRAS double mutants, 71% (10 of 14) of NRAS mutants and 71% (5 of 7) of wild-type melanomas were sensitive. 13% (5 of 24) of BRAF mutants, 14% (2 of 14) of NRAS mutants and 29% (2 of 7) of wild-type melanomas were intermediately sensitive (IC50 1-1.5μM). Among BRAFV600E mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH722984. Combining vemurafenib and SCH722984 in BRAF mutant melanoma was synergistic in a majority of cell lines, resulting in up to 10-fold decrease in the 50% inhibitory concentration (IC50) and with combination indices (CI) below 1. SCH722984 inhibited phosphorylation of RSK, an ERK downstream target. Finally, long term cultures of two BRAFV600E-mutant cell lines sensitive to SCH722984 showed that treatment with SCH722984 alone or in combination with vemurafenib was more potent than vemurafenib alone and resulted in a significant delay in developing acquired resistance. SCH722984 may therefore be clinically applicable as a treatment for non-BRAF mutant melanoma or in BRAF-mutant melanoma with innate or acquired resistance, alone or in combination with BRAF inhibitors. Citation Format: Deborah J. Wong, Lidia Robert, Mohammad S. Atefi, David Foulad, Earl V. Avramis, Begonya Comin-Anduix, Ahmed A. Samatar, Antoni Ribas. High antitumor activity of the ERK inhibitor SCH722984 against BRAF-mutant, NRAS-mutant and wild-type 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 917. doi:10.1158/1538-7445.AM2013-917

Abstract PR8: Insensitivity to RAF inhibition by vemurafenib in BRAF mutant colorectal cancer by EGFR-mediated reactivation of MAPK signaling

Abstract BRAF mutations occur in 10–15% of colorectal cancers (CRCs) and confer adverse outcome in the metastatic setting. While RAF inhibitors such as vemurafenib (PLX4032) have proven effective in BRAF mutant melanoma (∼60–80% response rates), they are surprisingly ineffective in BRAF mutant CRCs (∼5% response rate), and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRC cell lines and patient tumor specimens expressed significantly higher levels of phospho-EGFR than BRAF mutant melanoma cell lines and primary tumor specimens, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells and markedly improved efficacy in vitro. In vivo, combined RAF and EGFR inhibition led to sustained suppression of MAPK signaling and to robust tumor regressions in BRAF mutant colorectal xenografts. These findings support evaluation of combined RAF and EGFR inhibition in clinical trials in patients with BRAF mutant CRC. This proffered talk is also presented as Poster B11.

Abstract LB-350: EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition by vemurafenib

Abstract Oncogenic BRAF mutations occur in 10-15% of colorectal cancers (CRCs) and confer adverse outcome. While RAF inhibitors such as vemurafenib (PLX4032) have produced dramatic response rates in BRAF mutant melanoma (∼60-80%), they are surprisingly ineffective in BRAF mutant CRCs (∼5% response rate), and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRC cell lines and patient tumor specimens expressed higher levels of phospho-EGFR than BRAF mutant melanoma cell lines and primary tumor specimens, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells, markedly improved efficacy in vitro, and led to robust tumor regressions in vivo in BRAF mutant xenografts models. These findings support combined RAF and EGFR inhibition as a novel and promising therapeutic strategy for evaluation in clinical trials in patients with BRAF mutant CRC. 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 LB-350. doi:1538-7445.AM2012-LB-350

Abstract 1065: Growth factor-mediated resistance to BRAF/MEK inhibitors in BRAF mutant melanoma

Abstract BRAF and MEK inhibitors induce striking tumor regressions in BRAF V600E melanoma patients. However, relapse occurs in the majority of patients within several months. Reported resistance mechanisms include acquisition of NRAS mutations, PDGFR or IGF1R activation, Cot amplification, or truncated BRAF upregulation. We report here that hepatocyte growth factor (HGF), neuregulin-1 (NRG1), and fibroblast growth factor-2 (FGF2) cause robust resistance in vitro to BRAF or MEK inhibitors in BRAF mutant melanoma cell lines. NRAS mutant lines were similarly resistant to MEK inhibition in the presence of these growth factors. By contrast other mitogenic factors such as PDGF, EGF, NGF, BDNF, MSP, MDK, GRO, and IGF1 did not maintain cell viability or growth. HGF, NRG1 and FGF2 prevented growth arrest and cell death and in some cell lines maintained cells in progressive growth. In addition, HGF and NRG1 could prevent growth arrest after dual treatment with BRAF and MEK inhibitors. Western analysis revealed that HGF and NRG1 reactivated Erk signaling and activated S6RP and AKT phosphorylation. Combined pharmacological inhibition mTor signaling and Akt signaling was required to fully block growth factor-mediated proliferation. In addition, small molecule Met inhibitors and an anti-HGF antibody blocked HGF-mediated resistance, while lapatinib and an anti-NRG1 antibody blocked NRG1-mediated resistance. While combined Akt and mTor inhibition potently inhibited cell line growth independent of BRAF or MEK inhibition, lapatinib and Met inhibitors had minimal effect on growth, suggesting potential for less adverse events with receptor inhibitors. In vitro co-culture experiments revealed that primary cells present in normal skin can cause resistance, and we have identified growth factors produced by these cells that contribute to resistance. We are analyzing receptor activation (Met and Erbb3) and FGF2 expression in wildtype, NRAS, and BRAF mutant primary melanoma samples. In future experiments we plan to test clinical samples from patients treated with BRAF/MEK inhibitors, and we hypothesize that HGF, NRG1, and FGF2 signaling is elevated in patients with poor initial responses or relapse to BRAF or MEK inhibitors. 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 1065. doi:1538-7445.AM2012-1065

Abstract 5601: Overcoming acquired resistance to BRAF inhibitors in melanoma with the HSP90 inhibitor ganetespib

Abstract Activating BRAF(V600E) kinase mutations occur in 50% of melanoma, 45% of papillary thyroid cancer and 10% of colon cancer. BRAF inhibitors have elicited significant clinical response in melanomas but acquired resistance frequently develops after initial responses. Therefore, consideration of new treatment strategies to block activity of mutant BRAF and deter therapeutic resistance is warranted. Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability of hundreds of client proteins, including the RAF family of kinases, and has emerged as a highly relevant anticancer target. Ganetespib is a second generation, small molecule HSP90 inhibitor currently being evaluated in multiple clinical trials. To investigate the potential for ganetespib in the treatment of melanoma we evaluated its preclinical activity as monotherapy, and in combination with targeted agents, in cells sensitive or resistant to BRAF inhibitors. Ganetespib displayed potent anticancer activity in a large panel of BRAF(V600E) mutant melanoma, thyroid and colon cancer cell lines, with IC50 between 4 and 40 nM. Further analysis showed that ganetespib disrupted oncogenic signaling by inducing the destabilization of mutant BRAF and subsequent inactivation of MEK and ERK. Levels of wild-type BRAF were only slightly reduced in response to ganetespib treatment, implying that the stability of activated BRAF is more reliant on HSP90 and that ganetespib may selectively deplete oncogenic forms of BRAF. Notably, ganetespib treatment also led to depletion of CRAF, which is implicated in driving therapeutic resistance to BRAF inhibitors. In an effort to maximize antitumor activity, we combined low levels of ganetespib with BRAF or MEK inhibitors in vitro and observed increased cytotoxicity compared to single agent treatment. Both the BRAF and MEK inhibitors were cytostatic on their own, which may be due to continued PI3K/mTOR signaling. We subsequently assayed the combination of ganetespib with a PI3K/mTOR inhibitor and observed synergistic increases in cell death. Importantly, ganetespib was capable of blocking the feedback activation on MAPKK induced by a MEK inhibitor as well as the activation of ERK by PI3K/mTOR inhibitors. To determine if these results translated in vivo, similar combinations were performed in BRAF mutant human melanoma xenografts. Co-administration of ganetespib with either a BRAF or PI3K/mTOR inhibitor suppressed in vivo tumor growth to a greater extent compared to monotherapy. Finally, we show that ganetespib effectively blocked MAPK signaling and killed RPMI-7951 melanoma cells (IC50 = 4 nM), which are resistant to BRAF and MEK inhibitors due to the overexpression of COT. In conclusion, targeting HSP90 with ganetespib represents a promising approach for the treatment of BRAF(V600E)-driven cancers and may be especially relevant in the context of acquired resistance to BRAF inhibitors. 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 5601. doi:1538-7445.AM2012-5601

Abstract 1914: Potent anticancer activity against both BRAF-mutant and BRAF wild-type melanoma cell lines using a novel CRM1 nuclear export inhibitor

Abstract INTRODUCTION: BRAF and NRAS oncogenic mutations occur in over half of all human melanomas. BRAF-mutant tumors are highly sensitive to small molecule inhibitors directed against the oncogenic BRAF protein. However, in tumors that lack the specific mutation, patients have few effective therapeutic options. To address the need for new therapies for patients with BRAF-wild-type (wt) melanoma, we hypothesized that the inhibition of the major nuclear export receptor CRM1 would result in nuclear accumulation of nuclear proteins, increase apoptosis and induce cell cycle arrest independent of BRAF mutational status. To test our hypothesis, we examined the effects of a novel CRM1 inhibitor on melanoma cell lines with various BRAF and NRAS mutational backgrounds. METHODS: We used human malignant melanoma cell lines with BRAF mutation (Sk-Mel-28, A375, Mel-624, UACC903, MalMe-3M), BRAF-wt (MeWo) and BRAF-wt and NRAS mutation (Hs940T) to assess the effects of CRM1 inhibition, BRAF inhibition (PLX4032) or MEK inhibition (U0126) using both mono- or combination therapy. MTT assays were used to evaluate the effects of treatment on cell proliferation. FACS analysis with annexin/PI staining was performed to assess effects on cell cycle and cell death. The Chou-Talalay method was used to assess synergistic drug combination effects. Treatment effects on cleaved caspase-3, PARP cleavage, P53, and FOXO3 were analyzed by western blot analysis. RESULTS: As mono therapy, CRM1 inhibition led to a significant decrease in cell proliferation, cell cycle arrest and an increase in apoptosis in both BRAF wt and mutant cell lines. The degree of inhibition and cell death achieved by CRM1 inhibition across all melanoma cell lines is comparable to the results achieved by BRAF inhibitor PLX4032 in BRAF mutant cell lines. Furthermore, the combination of CRM1 inhibition and BRAF inhibition results in a strongly synergistic combination against BRAF mutant cell lines with a statistically significant decrease in cell proliferation and increased apoptotic cell death. CONCLUSION: We found that relative to other cancer cell types, both BRAF-mutant and BRAF-wt melanoma cells are exquisitely sensitive to CRM1 inhibition. In addition, CRM1 inhibition significantly enhanced the effect of PLX4032 in BRAF-mutant cell lines, suggesting that the combination of these two drugs may address the emergence of acquired resistance to BRAF monotherapy. Based on these results, CRM1 inhibition warrants further investigation to determine its potential benefit in treating melanoma patients independent of BRAF mutation status. 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 1914. doi:1538-7445.AM2012-1914

Abstract 4837: Tumor microenvironment contributes to RAF-inhibitor resistance through HGF secretion

Abstract Drug resistance remains a vexing problem in the treatment of cancer patients. While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy. We employed a novel co-culture assay to systematically assay the ability of 23 stromal cell types to influence the sensitivity of 45 cancer cell lines to 35 anti-cancer drugs testing more than 15,000 cancer-stroma-drug combinations. We found that stroma-mediated resistance is surprisingly common - particularly to targeted chemotherapies. We decided to further characterize the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most patients exhibit some degree of innate resistance. Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition. Immunohistochemistry confirmed tumor stromal HGF expression in 11 out of 15 patients with BRAF-mutant melanoma. Among these patients, only one experienced a complete response, and that patient was negative for stromal HGF, consistent with our prediction of HGF-mediated RAF-inhibitor resistance. In contrast, of 6 evaluable patients with stable disease 4 had particularly high levels of stromal HGF expression. Dual inhibition of RAF and MET in BRAF-mutant melanoma cell lines resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. Interestingly, the activation of MET in these cell lines was cell autonomous, caused, for example, by autocrine secretion of HGF and resulting in complete resistance to RAF inhibitors even in the absence of stromal cells. Treatment with a MET inhibitor re-sensitized these cell lines to RAF inhibitors suggesting the same RAF/MET combination therapy for non-melanoma BRAF mutant cancers as well. More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance. 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 4837. doi:1538-7445.AM2012-4837

Abstract A27: The combination of navitoclax and PLX4720 in melanoma in vitro and in vivo.

Abstract Introduction: Approximately 40–50% of patients with melanoma have a tumor which harbors a BRAF mutation (BRAF+). Vemurafenib is a highly selective inhibitor of mutant BRAF which has recently been FDA-approved for the treatment of patients with BRAF+ advanced melanoma. While response rates to vemurafenib are high, the vast majority of patients will develop resistance. Identification of therapeutic strategies to enhance the efficacy of vemurafenib is a high priority. Navitoclax is a BH3-only mimetic which has single agent activity in tumors with low Mcl-1 and enhances the toxicity of agents which inhibit or downregulate MCL-1. As recent data suggests that selective BRAF inhibitors inactivate Mcl-1 through upregulation of BIM, we therefore studied the effects of combining PLX4720, a selective BRAF inhibitor very similar to vemurafenib, with navitoclax in vitro and in vivo. Methods: Three BRAF mutant melanoma cell lines (A2058, A375, SKMel5) were selected for in vitro analysis and treated for 6 hours with DMSO, navitoclax (1 uM), PLX4720 (1 uM), or the combination. Cells were then lysed after 6 hours of treatment and western blot analysis was performed probing for vinculin, PARP, total and phosphorylated Erk, Mcl-1, BIM, BID, AKT, pS6, and NOXA. Xenografts were generated from the A375 and A2058 cell lines in nude beige mice and grown to 10 mm in size. Mice were then treated with vehicle, navitoclax (100 mg/kg/day), PLX4720 (100 mg/kg/day), or the combination once daily by gavage for 14 days. Tumor measurements were taken daily. Results: Treatment of melanoma cell lines with PLX4720 resulted in downmodulation of Mcl-1 and induction of BIM both in vitro and in vivo. In vitro, the combination of navitoclax and PLX4720 led to greater PARP cleavage than either agent alone. In vivo, the navitoclax enhanced the efficacy of PLX4720 in both xenograft models. Interestingly, the two xenograft models showed differential sensitivity to treatment with the respective single agents. Conclusion: 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. Efforts are underway to assess such a combination clinically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A27.

Protein Kinase D3 Sensitizes RAF Inhibitor RAF265 in Melanoma Cells by Preventing Reactivation of MAPK Signaling

RAS mutations occur in more than 30% of all human cancers but efforts to directly target mutant RAS signaling as a cancer therapy have yet to succeed. As alternative strategies, RAF and MEK inhibitors have been developed to block oncogenic signaling downstream of RAS. As might be expected, studies of these inhibitors have indicated that tumors with RAS or BRAF mutations display resistance RAF or MEK inhibitors. In order to better understand the mechanistic basis for this resistance, we conducted a RNAi-based screen to identify genes that mediated chemoresistance to the RAF kinase inhibitor RAF265 in a BRAF (V600E) mutant melanoma cell line that is resistant to this drug. In this way, we found that knockdown of protein kinase D3 (PRKD3) could enhance cell killing of RAF and MEK inhibitors across multiple melanoma cell lines of various genotypes and sensitivities to RAF265. PRKD3 blockade cooperated with RAF265 to prevent reactivation of the MAPK signaling pathway, interrupt cell cycle progression, trigger apoptosis, and inhibit colony formation growth. Our findings offer initial proof-of-concept that PRKD3 is a valid target to overcome drug resistance being encountered widely in the clinic with RAF or MEK inhibitors.

Abstract 5111: Integrative phospho-proteomic and genomic analyses identify AXL as a potential biomarker and therapeutic target for NRAS-mutated melanoma

Abstract Currently, few therapeutic options exist for the 15-20% of patients whose melanomas harbor activating mutations in NRAS. Here, we have used comprehensive phospho-proteomic and genomic analysis to characterize the patterns of intracellular signaling in a panel of NRAS and BRAF-mutated melanoma cell lines, with the goal of identifying new biomarkers and therapeutic targets for NRAS mutated melanomas. Our preliminary analyses showed BRAF mutated melanomas to have less diverse intracellular signaling than the NRAS group, which tended to have constitutive phosphorylation in a wide-range of receptor tyrosine kinases (RTKs), such as c-MET, EGFR, HER2 and c-Abl. Although these patterns of RTK activity were heterogeneous across the NRAS mutated melanoma cell line panel, all of the NRAS mutated cell lines examined showed constitutive activity in the receptor tyrosine kinase Axl. A role for Axl in the oncogenic behavior of NRAS mutated melanomas was suggested by array CGH studies showing genomic amplification of Axl in a significant fraction of the NRAS mutated melanoma cell lines and by Western Blot experiments demonstrating increased expression of Axl and its ligand Gas6. Mass spectrometry analysis and immunoprecipitation revealed Axl to be constitutively phosphorylated at the Tyr702 autophosphorylation site in NRAS mutated melanoma cell lines only. Functionally, siRNA knockdown and pharmacological inhibition of Axl impaired the survival and invasion of NRAS mutated melanoma cell lines grown under 3D organotypic cell culture conditions. Studies are ongoing to confirm the role of Axl in the initiation and progression of melanomas harboring NRAS mutations and determine whether Axl constitutes a therapeutic target for this melanoma sub-group. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5111. doi:10.1158/1538-7445.AM2011-5111

Abstract 251: Kinome-wide RNAi screen identification of synthetic lethal interactions in BRAF-mutant melanoma

Abstract BACKGROUND. Mutations in the BRAF oncogene are prevalent in many types of solid cancers, where they promote cell proliferation and transformation by constitutively activating the RAF-MEK-ERK signaling pathway. Inhibitors of BRAF have been developed as potential cancer therapies but unfortunately many have performed poorly in clinical trials. As an alternative approach, we performed RNA interference (RNAi) screening to identify genes that are synthetically lethal with the BRAF-mutant oncogene in malignant melanoma cell lines. Recent advances in RNAi and deep sequencing technologies have made it possible to systematically interrogate the genome for synthetic lethal interactions. METHODS. Two BRAF-mutant melanoma cell lines (SKMEL28 and Malme3M) and one wild-type BRAF cell line (MeWo) were infected with a subset of The RNAi Consortium (TRC) shRNA library comprising all the kinases in the human genome as well as the genes involved in the tyrosine kinase>RAS>RAF>MEK>ERK pathway. Cells were collected at 4 and 19 days post-infection, genomic DNA was isolated, shRNAs were PCR amplified and subjected to massively parallel sequencing. Statistical analysis identified ∼200 shRNAs that were selectively depleted in BRAF-mutant but not wild-type BRAF cell lines; individual canidate shRNA were tested in a panel of 6 melanoma cell lines 3 BRAF-mutant (UACC62, UACC257, Malme3M) and 3 wild-type BRAF (WM3912, WM3918, MeWo). SUMMARY. The screen identified a diverse number of genes that are synthetically lethal with the BRAF-mutant oncogene in human malignant melanoma. Not surprisingly, a number of the identified synthetic lethal genes are known downstream effectors of BRAF or known scaffold proteins involved in MAPK signaling. Paradoxically, epiregulin (EPGN), a known ligand of the epidermal growth factor receptor (EGFR) and EGFR itself, which are upstream of the BRAF oncogene were also identified in the screen as synthetically lethal with mutant BRAF. CONCLUSIONS. The BRAF oncogene is synthetically lethal with genes involved in diverse cellular processes such as kinase signaling, cell division, nuclear shuttling and cell metabolism. Many of the genes identified in the RNAi screen are known effectors of the MAPK cascade. Paradoxically, two members of the MAPK cascade, EGFR and its ligand EPGN, both of which are upstream of BRAF, were identified in the screen and validated to be synthetically lethal with mutant BRAF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 251. doi:10.1158/1538-7445.AM2011-251

Abstract A66: Integrative phosphoproteomic analysis of signaling pathways in MMS-mutated melanoma

Abstract Introduction: Recent years have seen great progress in the development of targeted therapy strategies for melanoma, with impressive therapeutic responses now being observed in patients whose melanomas harbor either activating mutations in BRAFor c-KIT. In contrast, few therapeutic options exist for the 15-20% of melanomas with mutations in NRAS. Previous attempts to define the biology of NRAS mutant melanomas have focused upon microarray analysis-an approach that failed to distinguish NRAS from BRAF-mutated melanomas. Despite apparently overlapping RNA expression profiles between the two sub-groups, BRAFand NRAS mutated melanomas were found to respond differently to small molecule signal transduction inhibitors, suggesting important differences in cell signaling. The aim of the current study was to perform comparative phosphoproteomic analysis on a panel of NRAS and BRAF mutated melanoma cell lines and to build intracellular signaling networks of the two groups. Methods: Phosphotyrosine enrichment was performed using the PhosphoScan protocol (Cell Signaling Technology) where phosphotyrosine-containing peptides were immunoprecipitated with immobilized anti-phosphotyrosine antibodies before being analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). These data yielded a catalog of tyrosine phosphorylations across a wide range of signaling proteins and enabled the relative quantification of each site based on exctracted ion chromatograms from high resolution accurate mass measurements of the peptides. Analysis of the phosphorylated proteins and the quantitative data was performed using GeneGO software, so that signaling maps of BRAFand NRAS mutated melanoma cell lines could be constructed. Results: A preliminary analysis of the data revealed BRAFmutated melanomas to be less diverse than the NRAS group, which tended to have constitutive phosphorylation in a wide-range of receptor tyrosine kinases (RTKs), such as c-MET, EGFR, and HER2, as well as non-receptor tyrosine kinases, such as c-Abl. Studies are ongoing to validate the role of these RTKs in the oncogenic behavior of the NRAS-mutated melanoma cell line panel and address whether any of the identified RTKs would be suitable therapeutic targets for this group of melanomas. Conclusions: Phosphoproteomics has proven useful in elucidating cancer biology of melanomas that harbor mutations in BRAF and NRAS. These differences in kinase activation and protein phosphorylation may prove to be useful in selection of targeted therapy regimens for melanoma patients and serve as candidate biomarkers for patient assessment. ∗This presenting student is supported by an ARRA supplement to the NCI Cancer Center Support Grant awarded to Moffitt (3P30 CA076292-11S6 PI WS Dalton) to create a training program for underrepresented undergraduate students in clinical proteomics Project LINK (Leaders In New Knowledge-Emerging Technologies) that also emphasizes education in health disparities and community outreach. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A66.

Novel tricyclic pyrazole BRAF inhibitors with imidazole or furan central scaffolds

V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that is mutated with high frequency in cutaneous melanoma, and many other cancers. Inhibition of mutant BRAF is an attractive therapeutic approach for the treatment of melanoma. A triarylimidazole BRAF inhibitor bearing a phenylpyrazole group (dimethyl-[2-(4-{5-[4-(1H-pyrazol-3-yl)-phenyl]-4-pyridin-4-yl-1H-imidazol-2-yl}-phenoxy)-ethyl]-amine, 1a) was identified as an active BRAF inhibitor. Based on this starting point, we synthesized a series of analogues leading to the discovery of 6-{2-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-2,4-dihydro-indeno[1,2-c]pyrazole (1j), with nanomolar activity in three assays: inhibition of purified mutant BRAF activity in vitro; inhibition of oncogenic BRAF-driven extracellular regulated kinase (ERK) activation in BRAF mutant melanoma cell lines; and inhibition of proliferation in these cells.

Methylthioadenosine (MTA) inhibits melanoma cell proliferation and in vivotumor growth

BackgroundMelanoma is the most deadly form of skin cancer without effective treatment. Methylthioadenosine (MTA) is a naturally occurring nucleoside with differential effects on normal and transformed cells. MTA has been widely demonstrated to promote anti-proliferative and pro-apoptotic responses in different cell types. In this study we have assessed the therapeutic potential of MTA in melanoma treatment.MethodsTo investigate the therapeutic potential of MTA we performed in vitro proliferation and viability assays using six different mouse and human melanoma cell lines wild type for RAS and BRAF or harboring different mutations in RAS pathway. We also have tested its therapeutic capabilities in vivo in a xenograft mouse melanoma model and using variety of molecular techniques and tissue culture we investigated its anti-proliferative and pro-apoptotic properties.ResultsIn vitro experiments showed that MTA treatment inhibited melanoma cell proliferation and viability in a dose dependent manner, where BRAF mutant melanoma cell lines appear to be more sensitive. Importantly, MTA was effective inhibiting in vivo tumor growth. The molecular analysis of tumor samples and in vitro experiments indicated that MTA induces cytostatic rather than pro-apoptotic effects inhibiting the phosphorylation of Akt and S6 ribosomal protein and inducing the down-regulation of cyclin D1.ConclusionsMTA inhibits melanoma cell proliferation and in vivo tumor growth particularly in BRAF mutant melanoma cells. These data reveal a naturally occurring drug potentially useful for melanoma treatment.