BRAF-mutant Cell Lines Research Articles

Abstract 3400: Small molecule biological screens and global analytical approaches reveal the complexity of cancer cell signaling networks and the challenge of personalized medicine.

Abstract BRAF is mutationally activated in 50% of melanomas with a variety of data indicating its role as a driver of malignancy. However, while patients with BRAF mutations respond to vemurafenib, most responses are not long lasting. Moreover, the 25% of melanomas harboring an NRAS activating mutation are resistant de novo to BRAF inhibitors. In order to identify effective drug combinations that increase the effectiveness of BRAF inhibition, we used combinatorial small molecule biological screens and global analytical approaches to identify compensatory and redundant pathways. We screened a drug library for combinations with BRAF or MEK inhibitors that cause synergistic cytotoxicity in melanoma cell lines. Although BRAF mutational status predicted sensitivity to inhibitors of BRAF, each BRAF mutant line differed in the combination of drugs that induced synergistic cytotoxicity. One robust and clinically relevant pair identified was the BRAF inhibitor PLX4720 and the EGFR/HER2 inhibitor lapatinib. This drug combination was synergistic in half of the BRAF mutant cell lines tested, including a line that was completely resistant to PLX4720. The combination was also effective in the two NRAS mutant lines tested. Xenograft experiments testing the combination of PLX4720 and lapatinib revealed a significant reduction in tumor volume compared to either single agent alone in both PLX4720 sensitive and resistant BRAF mutant tumors, providing preclinical data supporting the efficacy of this drug combination. Synergy of the PLX4720-lapatinib combination did not correlate with EGFR/HER2 expression, activity state, or mutational status. This further indicates that sensitivity of melanoma to drug combinations is not determined by the targeted oncogenic drivers, but by the existence of unique secondary, compensatory survival responses. We hypothesize that mutationally activated BRAF is wired into the constitutive signaling network differently in each of these cells, presumably a consequence of the diverse secondary mutations that characterize melanomas. Consistent with this, whole exome-sequencing data revealed a diversity of somatic variation among the synergistic and non-synergistic melanomas, while gene expression analysis demonstrated both distinct, line-specific basal transcriptional profiles and the lack of common gene expression signatures in melanomas synergistically inhibited by treatment with PLX4720-lapatinib. Collectively, these data point out the extraordinary robustness of cancer cell signaling networks and the challenges of individualizing therapies. This study has uncovered novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially depending on unexplored components of the cell genotype and cell signaling network. Citation Format: Devin Roller, Brian Capaldo, Aaron J. Mackey, Mark Conaway, Michael J. Weber, Daniel G. Gioeli. Small molecule biological screens and global analytical approaches reveal the complexity of cancer cell signaling networks and the challenge of personalized medicine. [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 3400. doi:10.1158/1538-7445.AM2013-3400

MicroScale: miniaturizing cell arrays for macrogenetic screens

MicroScale: miniaturizing cell arrays for macrogenetic screens

Synthesis and evaluation of apoptosis induction of thienopyrimidine compounds on KRAS and BRAF mutated colorectal cancer cell lines

Monoclonal antibodies (MoAb) and tyrosine kinase inhibitors (TKI) targeting the EGFR (Epidermal Growth Factor Receptor) pathways are currently used in colorectal cancer treatment. Despite the improvement of median overall survival, resistance is observed notably due to KRAS and BRAF gene mutations. We synthesized four series of thienopyrimidines whose scaffold is structurally close to TKI used in clinical practice. We evaluated apoptosis induced by these compounds using flow cytometry on KRAS and BRAF mutated cell lines. Our results confirm that the mutated cell lines (HCT116 and HT29) are more resistant to apoptosis than the non-mutated cell line (Hela). Interestingly, among the 13 compounds tested, three of them (5b, 6b and 6d) and gefitinib exhibited a noteworthy pro-apoptotic effect, especially on mutated cell lines with an IC50 value between 70 and 110μM. These three compounds seem particularly attractive for the development of novel treatments for colorectal cancer patients harboring EGFR pathway mutations.

1145 - Sensitive Detection of BRAF Mutations Using Mutant-Enriched PCR and Reverse-Hybridization Teststrips

ABSTRACT Background BRAF plays a key role in growth factor receptor induced signalling pathways. Somatic BRAF mutations are involved in oncogenesis and are found in various types of tumors, including colorectal, thyroid and skin cancer. Activating BRAF mutations confer resistance to anti-EGFR monoclonal antibody therapy. Moreover mutated BRAF is a prominent target of the drug vemurafenib. Materials and methods We have developed a reverse-hybridization StripAssay detecting nine BRAF mutations: c.1799T > C (V600A), c.1799_1800TG > AT (V600D), c.1799T > A (V600E), c.1799_1800TG > AA (V600E), c.1799T > G (V600G), c.1798_1799GT > AA (V600K), c.1798G > A (V600M), c.1798_1799GT > AG (V600R) and c.1801A > G (K601E). The test is based on mutant-enrichted PCR in the presence of a BRAF wild-type suppressor, followed by hybridization of PCR products to teststrips presenting a parallel array of allele-specific oligonucleotide probes. Bound sequences are visualized using streptavidin-alkaline phosphatase conjungate and colour substrates. The hybridization and detection steps can be carried out fully automated using commercially available instrumentation. Results Plasmid clones served as reference DNA templates to control for specificity. StripAssay performance was evaluated on genomic DNA obtained from cultured cell lines and formalin-fixed paraffine-embedded (FFPE) tissue. Using normal DNA spiked with serial dilutions of DNA from the BRAF-mutant tumor cell lines HT29 or IGR-1, the mutations c.1799T > A (V600E) and c.1798_1799GT > AA (V600K) were shown to be detectable at a level of 1%. Conclusions The simultaneous detection of nine different mutations with high sensitivity will make the StripAssay a very useful tool for the assessment of the BRAF mutation status of cancer patients. Disclosure All authors have declared no conflicts of interest.

Impact of Combined mTOR and MEK Inhibition in Uveal Melanoma Is Driven by Tumor Genotype

Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.

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 4170: Mutation and chemosensitivity profiling of 18 human melanoma cell lines

Abstract Malignant melanomas account for a substantial proportion of cancers worldwide and are largely resistant to conventional therapy. The heterogeneity and the invasiveness of melanomas present notable challenges for anti-cancer treatment. Recently, several mutations in RAF, RAS, and PTEN were identified that allow for prognosis of cancer development and progression. Therefore, inhibitors of the RAF-MEK-ERK pathway form attractive candidates for molecular-based targeted treatment of melanoma cancer. Here, we present the molecular characterization and chemosensitivity profiling of 18 human melanoma cell lines. Eight of these were established by Oncotest from human tumor xenografts originally derived from primary patient material. The molecular profiling included mutational analysis for BRAF, PIK3CA, KRAS, NRAS, TP53, and PTEN by sequencing. The chemosensitivity profiles were determined in vitro using a fluorescence-based cytotoxicity assay and included commonly used chemotherapeutic agents for treatment of melanoma, targeted agents (sorafenib and bortezomib) and various BRAF (PLX-4032, PLX-4720, GDC-0879) and MEK (CI-1040, PD0325901) inhibitors. The clinically relevant BRAF V600E substitution was the most prevalent mutation and was found in more than 50% of the melanoma cell lines. The chemosensitivity profiling revealed diverse patterns of selectivity and potency with mean IC50 values between 0.4 nM (vinblastine) and 8.3 µM (carboplatin). Each of the BRAF inhibitors tested was clearly more active in melanoma cell lines carrying a BRAF mutation. On average, IC50 values of BRAF inhibitors were more than 20-fold lower for the BRAF V600E mutated cell lines compared to the wild type cell lines. However, the BRAF mutated cell line MEXF 462NL was resistant towards BRAF inhibitors and it is warranted to compare its gene expression profile and mutational status with BRAF mutated cell lines responding to BRAF inhibitors. By using an ex vivo clonogenic assay, similar activity patterns for BRAF inhibitors were found, with mutated BRAF tumor xenografts showing markedly higher sensitivity. Potential synergistic activity of BRAF inhibitors with other inhibitors of the RAF-MEK-ERK pathway or with standard chemotherapeutic agents will be presented. The mutational status of selected cancer related genes, as well as responsiveness towards many cytotoxic and targeted agents was analyzed for 18 melanoma cell lines. This extensive genotypic and phenotypic characterization makes this melanoma cell line panel a valuable tool for in vitro profiling of novel compounds, in particular inhibitors of the RAF-MEK-ERK pathway. 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 4170. doi:1538-7445.AM2012-4170

Abstract 4709: Combined mitogen-activated protein kinase pathway inhibition with short-term romidepsin treatment induces proapoptotic Bim and cell death in BRAF mutant cancers

Abstract Solid tumor trials with histone deacetylase inhibitors (HDIs) have been largely disappointing. We recently characterized a romidepsin-resistant T-cell lymphoma cell line that was found to have activation of the mitogen-activated protein kinase (MAPK) pathway, leading to subsequent phosphorylation and degradation of the proapoptotic protein Bim, suggesting that activation of this pathway may also confer resistance to romidepsin and other HDIs. The V600E BRAF mutation has been found in approximately 60% of melanomas and approximately 15% of colon cancers and leads to constitutive activation of the MAPK pathway. We thus hypothesized that combined treatment with romidepsin and BRAF or mitogen-activated protein kinase kinase (MEK) inhibitors may be effective in cancers that harbor the V600E BRAF mutation. To more closely simulate clinical administration of romidepsin, 11 V600E BRAF mutant cell lines (8 melanomas and 3 colorectal cancers) were exposed to 25 ng/ml romidepsin for 6 h after which romidepsin was removed, cells were incubated in fresh medium for an additional 42 h and subsequently apoptosis was measured by Annexin V and propidium iodide staining. Of the 11 cell lines, 10 exhibited significantly higher annexin staining after short-term romidepsin treatment (control 7.1% ± 3.8% vs. treated 34.7% ± 17.8%, p<0.001). Cells were subsequently treated with medium containing 25 ng/ml romidepsin with 250 nM of the MEK inhibitors AZD6244 (selumetinib) and PD0325901 or 1 µM of the mutant BRAF inhibitor PLX4032 (vemurafenib) for 6 h, after which the cells were incubated an additional 42 h in medium containing the inhibitors alone. Combined treatment with romidepsin and AZD6244 resulted in a significantly higher percent of annexin positive cells (p<0.05) in 9 of the 11 BRAF mutant cell lines examined. Immunoblot analysis demonstrated inhibition of the MAPK pathway at the concentrations used and that apoptosis was associated with increased expression of Bim, as well as increased poly (ADP-ribose) polymerase cleavage. Our results suggest that combined treatment with romidpesin and BRAF or MEK inhibitors may be useful in the treatment of cancers that express mutant BRAF. 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 4709. doi:1538-7445.AM2012-4709

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 4570: Functional analysis of gene expression profiling of primary and metastatic melanoma: Possible implications from BRAF mutant and wild-type cell lines

Abstract Metastatic melanoma is largely refractory to existing therapies and has a very poor prognosis. Despite the recent development of new therapies, the median survival rate is only 6-9 months and 5-year survival is less than 5%. Better understanding of melanoma biology, specifically the gene expression profile between primary and metastatic melanoma, may provide useful information for developing new therapeutic approaches to this disease. The gene expression profile from 89 primary and metastatic melanoma tissues was investigated by cDNA microarray. 8261 differentially expressed genes were identified between primary and metastatic melanoma at a statistical level p <0.01 and false discovery rate (FDR) of 5%. Panther Pathway Classification showed that the 8261 differentially expressed genes were enriched with genes which were involved in cytoskeletal regulation by Rho GTPase, inflammation mediated by chemokine and cytokine signaling pathways, Notch signalling pathways, p53 and angiogenesis pathways. The primary melanoma microarrays were analyzed according to the thickness of tumour. Group A: the thickness of a tumour less than 2.0 mm, 20 microarrays. Group B: the thickness of a tumour was equal or more than 2.0 mm, 11 microarrays. 981 genes were identified as differentially expressed between Group A and Group B at statistical level p<0.01 and FDR=5%. 657 out of 981 differentially expressed genes were shared with the 8261 differentially expressed genes. We believed these 657 genes were related to malignant potential of melanoma; they were enriched with genes which were involved in apoptosis, cell adhesion, cell cycle regulation and immune system processing. 218 out of the 657 genes overlapped with a published gene list (308 genes) which have been implicated in melanoma metastases. We further evaluated the expression of the transcription factor gene: ETV1. Ets variant 1 (ETV1) gene expression was significantly increased in metastatic melanoma and in primary melanoma with tumour thickness equal or more than 2.0 mm. We investigated the ETV1 expression in human melanoma cell lines. ETV1 mRNA level in 7 BRAF inhibitor sensitive cell lines was significantly lower than that in 6 BRAF inhibitor resistant cell lines (0.230±0.057 vs 0.927±0.6, p<0.0001). The ETV1 expression in BRAF-induced resistant melanoma cell lines was significantly (p<0.05) higher than in their parental cell lines. While a variety of genes are differentially expressed in primary and metastatic melanoma, the transcription factor–ETV1 gene may be an important target for study and as a therapeutic target as its expression may be associated with BRAF inhibitor resistance in melanoma cells. 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 4570. doi:1538-7445.AM2012-4570

Antitumor Activity of BRAF Inhibitor Vemurafenib in Preclinical Models of BRAF-Mutant Colorectal Cancer

The protein kinase BRAF is a key component of the RAS-RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAF(V600E) in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAF(V600)-expressing cell lines and inhibiting tumor growth in BRAF(V600E) bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAF(V600E)-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAF(V600E) mutation.

Abstract PL03-01: Discovery of candidate biomarkers of anticancer drug sensitivity by high-throughput cell line screening.

Abstract PL03-01: Discovery of candidate biomarkers of anticancer drug sensitivity by high-throughput cell line screening.

Abstract C70: Integrative analysis of pharmacologic, genomic, and transcriptomic assays in cancer

Abstract We describe an integrative analysis approach leveraging the publicly available Cancer Cell Line Encyclopedia to uncover regulatory pathways in cancer. Six different lines of evidence are considered in identifying candidate factors associated with disease: 1) gene mutation status, 2) copy number variations, 3) RNAi growth inhibitions, 4) pharmacological growth inhibitions, 5) baseline gene expression data, and 6) physical protein-protein interactions. As each of these evidence sources measure a different aspect of cellular regulation, they may be considered in combination to uncover regulatory mechanisms associated with tumorigenesis. For example, gene mutation status or copy number variation data may be combined with baseline gene expressions to identify genes that are differentially expressed in cell lines with aberrant or deactivated versions of specific tumor suppressors and oncogenes. To assess our approach, we applied it to identification of genes involved in the well-known RAS-RAF regulatory signaling pathway in both tissuespecific and nonspecific contexts. The RAS-RAF signaling pathway plays a key role in cellular differentiation and proliferation and is frequently associated with tumorigenesis. Specifically, we identified differentially expressed genes in BRAF mutant vs. wild-type cell lines, genes associated with high and low RAF copy number alteration cell lines, genes correlating in expression to growth inhibition by RAF-targeting compounds, and genes that either rescue or sensitize cell lines to BRAF knockdown by RNA interference. We further considered these genes within the overall context of physical interactions between their protein products. Highlighting the significant, research potential of our approach, our RAF-focused analysis identified both well-known and novel genes, supported by different lines of evidence, that mediate RAS-RAF signaling across multiple tissues. Positive control genes include members of the classical pathway, such as MAPK1, MAP2K1, and PI3K. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C70.

Mechanism of antitumor activity of E7080, a selective VEGFR and FGFR tyrosine kinase inhibitor (TKI), in combination with selective mutant BRAF inhibition.

8567 Background: E7080 is a TKI targeting VEGFR1-3, FGFR1-4, KIT, RET and PDGFR. Anti-tumor activity has been observed in melanoma patients in phase I. The objective of this study was to examine the effect of E7080 alone and in combination with PLX-4032 on tumor response, gene expression and tumor microvasculature in BRAFmu melanoma. Methods: BRAF mutant (mu) cell lines were created by transfection of BRAF V600E constructs into BRAF wild type (wt) cell lines. Anti-tumor activity of E7080 alone or in combination with the selective BRAF inhibitor PLX-4032 was examined in xenografted models bearing mock and BRAFmu transfectants. Results: The ratio of ANGPT1/2 expression was significantly higher in BRAFmu/ PTENwt compared to BRAFwt/PTENwt and BRAFmu/PTENmu among 14 melanoma cell lines. ANGPT1 expression was up-regulated in BRAFmu by 330%, and ANGPT2 in PTENmu by 290% compared to wt. PLX-4032 decreased ANGPT1 expression and pericyte coverage of tumor vessels in BRAFmu/PTENwt A375 melanoma. Similarly, overexpression of BRAF V600E in BRAFwt SK-MEL-2 melanoma resulted in increased ANGPT1 expression along with increased pericyte coverage. E7080 at 3, 10, 30 to 100 mg/kg showed significant anti-tumor activity against mock transfectants, but only at 100 mg/kg against BRAF V600E transfectants. Combination of E7080 at 10 mg/kg with PLX-4032 at 100 mg/kg showed synergistic anti-tumor activity against A375 melanoma and caused tumor regression, not observed by either single agent E7080 or PLX-4032. E7080 alone increased pericyte coverage of tumor vessels, but in combination with PLX-4032 resulted in marked decreased in pericyte coverage and microvessel density. Conclusions: The BRAFmu/ PTENwt melanomas studied demonstrate high pericyte coverage and resistance to E7080. Combination of E7080 with PLX-4032 achieves synergistic antitumor effect with enhanced reduction of pericyte coverage. These data provide a mechanistic basis for clinical study of BRAF inhibitors in combination with VEGFR and FGFR targeted agents.

Abstract 3495: Targeted therapies in melanoma – in vitro effects of single and combined drug treatment

Abstract Malignant melanoma is one of the most rapidly increasing malignancies in Caucasian populations. So far, curative treatment is achieved only by surgical resection at an early stage, while the prognosis in cases with disseminated melanoma is poor, and we lack predictive tools to identify the few patients who respond to existing chemotherapy. Promising clinical trials of targeted therapies are ongoing, however, it is already clear that drug resistance will be a clinical reality also for these new therapies. Thus, novel predictive markers are urgently needed to identify responders to different types of therapy in melanoma. The aim of the study is to unravel molecular mechanisms and pathways underlying resistance to new therapies in melanoma. We are studying the effects of a set of MAPK and PI3K pathways inhibitors in melanoma cell lines that are wildtype or have different mutations/deletions in genes affecting these pathways (BRAF, NRAS, PTEN etc.). Cells are exposed to single drugs as well as combinations of inhibitors and/or chemotherapeutic agents. Our results have shown that there is a wide range of sensitivity to the BRAF inhibitor PLX4720 among BRAF mutated cell lines, with IC50 values varying from 0.3 µM to 30 µM. We are investigating the underlying mechanisms causing this variation in sensitivity to the drug. Using gene expression profiling of a clinical material, we have identified several candidate genes that may predict response to DNA alkylating agents dacarbazine and temozolomide. The expression of some of these genes can be affected by PLX4720, and their potential relevance for response to different treatments is currently tested in vitro. 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 3495. doi:10.1158/1538-7445.AM2011-3495

Abstract 5365: Molecular and pharmacological (EGFRi, MEKi) characterisation of a colorectal cancer (CRC) cell line panel to evaluate cellular phenotype and efficacy of targeted therapies in CRC

Abstract A broad range of targeted agents are in early development for treatment of solid tumours. It is important that patients receive treatments which are tailored to work optimally based on their individual tumour biology. Retrospective analysis of clinical data for the EGFR tyrosine kinase inhibitor, Iressa, in lung cancer demonstrated cell line panels can provide a platform to direct targeted therapies towards specific patient subpopulations. In order to evaluate targeted agents in colorectal cancer we have characterized a panel of 49 colorectal tumour cell lines derived from Dukes stage A-D of CRC for commonly occurring mutations (KRas, BRAF, PI3Ka, PTEN), microsatellite instability, gene copy number alterations (Agilent 244K ArrayCGH), mRNA expression (Affymetrics HG_U133_plus_2) and miRNA expression (TLDA – 177 miRNAs). These data have been used to characterize the differentiation status of the cell lines and to link to compound activity. We have probed the anti-proliferative activity of compounds from several growth factor pathways, EGFR, RAS/MEK and PI3K to evaluate pathway dependence and linkage to molecular data. The greatest activity of the EGFR TKI inhibitor was in Ras, Raf, PTEN, PI3K wild type (quadruple negative (QN)) CRC lines, in agreement with clinical data for EGFR antibodies. However, of the 9 QN lines profiled, only 4 were hypersensitive (GI50 <0.5uM), the hypersensitive lines had an epithelial phenotype (E-cadherin mRNA, miR200c & B-Cat protein positive; vimentin mRNA negative). In contrast the QN lines that did not respond to EGFR inhibition had either a mesenchymal (E-cadherin mRNA, miR200c, B-Catenin protein negative, vimentin positive) or an intermediate phenotype (E-cadherin mRNA, miR200c & B-Cat protein low; vimentin negative). KRAS and BRAF mutant cell lines were relatively resistant to EGFRi compared to QN cell lines (p = 0.009 and 0.06 respectively), with the exception of two lines with G13D KRAS mutations which had GI50s of 0.3 and 1.8uM. Interestingly, the epithelial QN lines were co-sensitive to EGFR, MEK and PI3K inhibitors. In contrast the mesenchymal QN lines were relatively insensitive to the kinase inhibitors used. The QN lines with an intermediate phenotype did not respond to EGFR or MEKi but did respond to PI3K inhibition. In conclusion, the data suggests that both mutation status and cellular differentiation status contribute to the response to targeted agents. Through profiling the anti-proliferative activity of inhibitors from across a range of pathways co-dependencies of kinase targets were revealed as well as anti-correlations which indicate potential combination strategies. 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 5365. doi:10.1158/1538-7445.AM2011-5365

Abstract 3562: Loss of the PTEN and RB1 tumor suppressors attenuates RAF-dependence in melanomas

Abstract Oncogenic BRAF mutations are found in ∼8% of human tumors, with the highest frequency observed in melanoma (40-70%). BRAF mutations are common in nevi suggesting that BRAF mutation alone is insufficient for tumorigenesis. To identify concurrent mutations that condition BRAF/MEK dependence in V600E BRAF melanoma, we genotyped a panel of 149 melanoma cell lines. Seventy-eight cell lines (52%) harbored BRAF mutations with 72 of which are V600E mutants. Proteomic profiling of 41 out of the 72 V600E BRAF mutant cell lines demonstrated significant variability in the expression of PTEN and pAKT. Nine cell lines harboring V600E BRAF mutations express no PTEN protein and high pAKT by immunoblotting. All of the nine cell lines harbored small (1-2) base-pair insertions or deletions, missense mutations or focal deletions encompassing the PTEN locus. We have previously reported that mutations of BRAF are associated with enhanced and selective sensitivity to MEK inhibition when compared to either receptor tyrosine kinase-driven cells or cells harboring a RAS mutation. To determine the MAPK pathway dependence of V600E BRAF mutant cell lines as a function of PTEN expression, we used PD0325901, a selective allosteric inhibitor of MEK1/2. All V600E BRAF mutant, PTEN expressing cell lines were sensitive to PD0325901. All but two of the V600E BRAF mutant, PTEN null cells were also dependent upon MEK for proliferation. These data suggest that PTEN loss is insufficient to confer resistance to MAPK-pathway inhibition. Examination of the two models which displayed complete resistance to MEK inhibition (IC50 > 3,000 nM) revealed the presence of concurrent loss of PTEN and RB1. Inactivation of Rb functions by expressing human papillomavirus E7 protein in three BRAF mutant, PTEN-null models led to failure of the cells to arrest in G1 and complete resistance to MEK-inhibition. Moreover, RB1 alterations were mutually exclusive with loss of p16INK4A, suggesting that whereas p16INK4A and RB1 may have overlapping roles in preventing tumor formation, tumors with functional loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. V600EBRAF/RB-null/PTEN-null models were also resistant to the selective RAF inhibitor PLX4720. Consistent with these in vitro results, V600EBRAF/RB-null/PTEN-null xenografts were resistant to RAF and MEK inhibition whereas V600EBRAF/WTRB/PTEN-null cells exhibited an intermediate phenotype. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the MAP kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor 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 3562. doi:10.1158/1538-7445.AM2011-3562

Basal and Treatment-Induced Activation of AKT Mediates Resistance to Cell Death by AZD6244 (ARRY-142886) in Braf-Mutant Human Cutaneous Melanoma Cells

The majority of melanomas show constitutive activation of the RAS-RAF-MAP/ERK kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway. AZD6244 is a selective MEK1/2 inhibitor that markedly reduces tumor P-MAPK levels, but it produces few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the phosphoinositide 3-kinase (PI3K)-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines showed AZD6244-induced activation of AKT. In contrast, sensitive cell lines showed AZD6244 treatment-induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or insulin-like growth factor I receptor blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells and the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients.

Abstract 69: Functional metabolomic profiling of human melanocyte and melanoma cells during hypoxic adaptation

Abstract Adaptation to hypoxia is an important part of the cellular progression from normal melanocyte to malignant melanoma. This adaptation can be comprised of short-term remodeling of the cellular metabolic network and/or the permanent, gene-driven development of a new metabolic program. In either case, low oxygen conditions require a unique metabolic program for continued survival and proliferation. However, beyond a general shift from respiration to glycolysis, the details of this permissive metabolic program in melanoma are generally unknown. In order to better understand the changes to both the metabolome and metabolic flux in this system, we applied functional metabolomic profiling to seven human melanoma cell lines and to two human melanocyte cell lines under both hypoxic and normoxic conditions. The melanoma panel consists of four cell lines driven by B-raf mutations (WM35, UACC903, WM793 and LU1205), two containing N-ras mutations (MEL501 and WM1346) and one p53 mutant (MeWo). These cells, along with the two melanocyte lines, were grown in media containing a 1:1 mixture of natural and [U-13C]-labeled glucose for 24 hours in either 1% oxygen or atmospheric conditions. We then employed GCMS, NMR and biochemical experiments to map flux through five metabolic hubs and quantify the abundance of 28 key metabolites for each cell line and condition. These maps of central carbon metabolism encompass glycolysis, the pentose phosphate pathway, fatty acid biosynthesis, the glutamate-glutamine-proline network, and glycine/serine de novo synthesis, along with a number of other important metabolites. Analyzing these data with unbiased hierarchical clustering and Ingenuity Pathway Analysis, we discovered a metabolite-derived signature for hypoxic adaptation in melanocytes. Furthermore, we found that certain hypoxic responses are unique to either the B-raf or N-ras of the cell line subset. Interestingly, the hypoxic response of the mutant p53 cell line is more like that of the B-raf mutant cell lines than the B-raf and N-ras sets are to each other. The information gained from this functional metabolomic profiling advances our ability to identify hypoxia-adapted melanomas and develop metabolism-based therapeutic interventions. This work was supported by P01 CA128814-01 from the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 69.

Abstract 1971: Concurrent loss of PTEN and RB is sufficient to confer BRAF independence in melanomas harboring V600E BRAF mutations

Abstract BRAF mutations are found in ∼8% of human tumors, with the highest frequency observed in melanoma (40-70%). Supporting its classification as an oncogene, V600E BRAF stimulates ERK signaling, induces proliferation and is capable in model systems of promoting transformation. BRAF mutations are, however, common in nevi suggesting that BRAF mutation alone is insufficient for tumorigenesis. With the goal of identifying concurrent mutations that condition BRAF/MEK dependence in V600E BRAF melanoma, we performed MALDI-TOF mass spectrometry genotyping on a panel of 149 melanoma cell lines. Seventy-nine (53%) harbored BRAF mutations. Proteomic profiling of the class of V600E BRAF mutant cell lines demonstrated significant variability in the expression of PTEN and pAKT. Nine cells lines harboring V600E BRAF mutations were found to express no PTEN protein and high pAKT by immunoblotting. The majority of these cell lines had small (1-2) base-pair insertions or deletions leading to a frame shift and early truncation. Array CGH confirmed focal homozygous deletion of the PTEN gene in three of the models. These deletions were small and in one case detected by the Agilent 1M but not the 244K platform. We have previously reported that mutations of BRAF are associated with enhanced and selective sensitivity to MEK inhibition when compared to either receptor tyrosine kinase-driven cells or cells harboring a RAS mutation. To determine the MAPK pathway dependence of V600E BRAF mutant cell lines as a function of PTEN expression, we used PD0325901, a selective allosteric inhibitor of MEK1/2. All V600E BRAF mutant, PTEN expressing cell lines were sensitive to PD0325901. All but two of the V600E BRAF mutant, PTEN null cells were also dependent upon MEK for proliferation with IC50 values ranging from 1.1 to >500nM. These data suggest that PTEN loss is insufficient to confer resistance to MAPK-pathway inhibition. Further characterization of the two models which displayed complete resistance to MEK inhibition revealed that both harbored concurrent loss of PTEN and RB. These models were also resistant to the selective RAF inhibitor PLX4720. To determine whether loss of RB function was sufficient to confer RAF/MEK-independence in a BRAF mutant, PTEN-null context, we stably infected viral E7 into three MEK-dependent, BRAF mutant, PTEN null models. Infection of E7 led to resistance to MEK-inhibition in all three models. These data suggest that no single mutational event is likely associated with de novo resistance to MEK-inhibition in V600E BRAF mutant melanoma. Loss of PTEN expression and activation of AKT may however be associated with diminished responsiveness to RAF/MEK-inhibition. Further, tumors with concurrent PTEN and RB loss may represent a subset of BRAF mutant patients who derive no clinical benefit from inhibitors of the MAPK pathway such as PLX4032. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1971.