BRAFV600E Mutant Cell Lines Research Articles

Polyamine and EIF5A hypusination downstream of c-Myc confers targeted therapy resistance in BRAF mutant melanoma

BackgroundBRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms.MethodsCRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts.ResultsWe elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings.ConclusionsOur findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors.

NF-κB signaling activation and roles in thyroid cancers: implication of MAP3K14/NIK

Among follicular-derived thyroid cancers (TC), those with aggressive behavior and resistance to current treatments display poor prognosis. NF-κB signaling pathways are involved in tumor progression of various cancers. Here, we finely characterize the NF-κB pathways and their involvement in TC. By using immunoblot and gel shift assays, we demonstrated that both classical and alternative NF-κB pathways are activated in ten TC-derived cell lines, leading to activated RelA/p50 and RelB/p50 NF-κB dimers. By analyzing the RNAseq data of the large papillary thyroid carcinoma (PTC) cohort from The Cancer Genome Atlas (TCGA) project, we identified a tumor progression-related NF-κB signature in BRAFV600E mutated-PTCs. That corroborated with the role of RelA and RelB in cell migration and invasion processes that we demonstrated specifically in BRAFV600E mutated-cell lines, together with their role in the control of expression of genes implicated in invasiveness (MMP1, PLAU, LCN2 and LGALS3). We also identified NF-κB-inducing kinase (NIK) as a novel actor of the constitutive activation of the NF-κB pathways in TC-derived cell lines. Finally, its implication in invasiveness and its overexpression in PTC samples make NIK a potential therapeutic target for advanced TC treatment.

BRAFV600E Mutation Enhances Estrogen-Induced Metastatic Potential of Thyroid Cancer by Regulating the Expression of Estrogen Receptors.

Cross-talk between mitogen-activated protein kinase and estrogen has been reported; however, the role of BRAFV600E in the estrogen responsiveness of thyroid cancer is unknown. We elucidated the effect of BRAFV600E on the estrogen-induced increase in metastatic potential in thyroid cancer. Using a pair of cell lines, human thyroid cell lines which harbor wild type BRAF gene (Nthy/WT) and Nthy/BRAFV600E (Nthy/V600E), the expression of estrogen receptors (ERs) and estrogen-induced metastatic phenotypes were evaluated. Susceptibility to ERα- and ERβ-selective agents was evaluated to confirm differential ER expression. ESR expression was analyzed according to BRAFV600E status and age (≤50 years vs. >50 years) using The Cancer Genome Atlas (TCGA) data. Estradiol increased the ERα/ERβ expression ratio in Nthy/V600E, whereas the decreased ERα/ERβ expression ratio was found in Nthy/WT. BRAFV600E-mutated cell lines showed a higher E2-induced increase in metastatic potential, including migration, invasion, and anchorage-independent growth compared with Nthy/WT. An ERα antagonist significantly inhibited migration in Nthy/V600E cells, whereas an ERβ agonist was more effective in Nthy/WT. In the BRAFV600E group, ESR1/ESR2 ratio was significantly higher in younger age group (≤50 years) compared with older age group (>50 years) by TCGA data analysis. Our data show that BRAFV600E mutation plays a crucial role in the estrogen responsiveness of thyroid cancer by regulating ER expression. Therefore, BRAFV600E might be used as a biomarker when deciding future hormone therapies based on estrogen signaling in thyroid cancer patients.

STEM-15. THERAPY-INDUCED CHANGES BY BRAF AND MEK INHIBITORS IN BRAF V600E-MUTATED GLIOMA MODELS PROVIDE POTENTIAL NOVEL THERAPEUTIC OPPORTUNITIES

Abstract Combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib (BRAFi+MEKi) show rapid and sustained responses in patients with BRAF V600E-mutated low-grade glioma, but tumor rebound after treatment discontinuation is frequent. Moreover, a lack of response is common in patient with high-grade glioma raising the need for further research into BRAFi+MEKi effects on tumors. We showed previously that BRAF V600E-mutated glioma cells positive for CD133 (Prominin-1), a marker of brain tumor stem cells, show decreased sensitivity to BRAFi, indicative of their role in promoting therapy resistance. BRAF V600E-mutated murine and patient-derived glioma cell lines (STN-10049, SU-aGBM5) were generated and together with established BRAF V600E-mutated cell lines (DBTRG, AM38) were analyzed for changes in gene expression in response to 48 hrs treatment with BRAFi dabrafenib and MEKi trametinib. Cells were analyzed by RNA-seq and gene enrichment analyses while cell culture supernatant was analyzed for cytokine production using an ELISA. Syngeneic, orthotopic BRAF V600E-mutated tumor allograft-bearing mice were treated with BRAFi+MEKi, with therapeutic antibodies against immune checkpoint molecules (anti-PD-L1 and anti-CTLA-4) and with combination of all four agents, and tumors were analyzed by mass cytometry and immunofluorescence for stem and T cell markers. BRAFi+MEKi treatment induced an interferon gamma (IFNg) response gene signature in BRAF V600E-mutated glioma cells and increased HLA gene expression. The frequency of tumor-infiltrating CD4+ CD8+ T cells in syngeneic BRAF V600E-mutated tumor allografts increased with BRAFi+MEKi treatment. Combining BRAFi+MEKi with anti-PD-L1 and anti-CTLA-4 treatment decreased CD133+ cells more effectively than either therapy alone, and resulted in a T cell-dependent survival benefit of mice with orthotopic BRAF V600E-mutated high-grade glioma. Combination of BRAFi+MEKi with immune checkpoint inhibition should be further explored as a viable option to prevent tumor rebound and therapy resistance in patients with BRAF V600E-mutated glioma.

BRAFV600E Overrides NOTCH Signaling in Thyroid Cancer.

Background: Several mechanisms likely cooperate with the mitogen-activated protein (MAP)-kinase pathway to promote cancer progression in the thyroid. One putative pathway is NOTCH signaling, which is implicated in several other malignancies. In thyroid cancer, data regarding the role of the NOTCH pathway are insufficient and even contradictory. Methods: A BRAFV600E-driven papillary thyroid carcinoma (PTC) mouse model was subjected to NOTCH pathway genetic alterations, and the tumor burden was followed by ultrasound. Further analyses were performed on PTC cell lines or noncancerous cells transfected with NOTCHIC or BRAFV600E, which were then subjected to pharmacological treatment with MAP-kinase or NOTCH pathway inhibitors. Results: The presence of the BRAFV600E mutation coupled with overexpression of the NOTCH intracellular domain led to significantly bigger thyroid tumors in mice, to a more aggressive carcinoma, and decreased overall survival. Although more cystic, the tumors did not progress into anaplastic thyroid carcinomas. On the contrary, the deletion of RBP-jκ (a major cofactor involved in NOTCH signaling) did not alter the phenotype in mice. BRAFV600E-mutated PTC cell lines were resistant to pharmacological inhibition of the NOTCH pathway. Inhibition of MEK1/2 uncovered a predominant effect on Hes1/Hey1 transcription compared with NOTCH inhibition in BRAFV600E-mutated cell lines. Finally, γ-secretase activity and γ-secretase subunit transcription levels were dependent on ERK activation. Our findings suggest that MAP-kinase activity overrides the NOTCH pathway in the context of thyroid cancer. Conclusions: The interaction between the BRAF and NOTCH pathways demonstrates that the BRAFV600E mutation might bypass NOTCH and exert a strong positive effect on NOTCH downstream targets in thyroid carcinoma.

Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment

Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear. We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification. HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC). Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation. Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification. Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX. These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells. Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF. The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.

Abstract 5087: Human melanoma exosomes induce metabolic reprogramming in human adult dermal fibroblasts

Abstract The “seed and soil” hypothesis of cancer metastasis has led us to examine the role of tumor-derived exosomes on reprogramming stromal cells composing the tumor microenvironment. We used human adult dermal fibroblasts (HADF) in vitro as a model of the premetastatic niche. Specifically, we determined the role of human melanoma-derived exosomes (HMEX) on governing metabolic reprogramming of healthy HADF. HMEX from six melanoma cell lines (3 BRAF V600E mutant cell lines (526-mel, 888-mel and Hs 294T) and 3 BRAF wild-type cell lines (1300-mel, 2183-Her4 and HMCB)) were purified by size-exclusion chromatography. Using the fluorescent tag CellVue (Maroon) we demonstrated via ImageStream that HMEXs are rapidly taken up by HADF. Following 18 hours of exposure to HMEX, HADF increased aerobic glycolysis and reduced oxidative phosphorylation (OXPHOS) in vitro, as measured by Agilent Seahorse XFe96. We have also developed an immuno-biochip that captures exosomes via anti-CD63 antibodies and simultaneously detects exosomal microRNAs by cationic lipoplex nanoparticles (tCLN) containing molecular beacons. With this immuno-biochip, we demonstrated the presence of microRNAs miR-155 and miR-210 in HMEX, both of which work in concert to promote glycolysis and inhibit OXPHOS. The result has been consistent across all HMEXs from the six different melanoma cell lines. Our findings reveal a correlation between metabolic function and the presence of metabolically active microRNAs in HMEX that control aerobic glycolysis and OXPHOS of normal fibroblasts. Melanoma exosomes are potentially key players in promoting tumor progression by modulating normal stromal cell metabolism and creating an environment that is conducive to metastasis. Oxygen Consumption Rate (OCR) pmol/minHADF onlyHADF + HMEX 17.5 μgHADF + HMEX 25 μgHADF + HMEX 50μgHADF + HMEX100μgBasal OCR52.6753.5850.9638.2031.56 Citation Format: Shin La Shu, Cheryl L. Allen, Yunchen Yang, Orla Maguire, Hans Minderman, Arindam Sen, Michael J. Ciesielski, Katherine A. Collins, Peter J. Bush, Martin Morgan, Yun Wu, Richard B. Bankert, Marc S. Ernstoff. Human melanoma exosomes induce metabolic reprogramming in human adult dermal fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5087.

DUSP5 and DUSP6, two ERK specific phosphatases, are markers of a higher MAPK signaling activation in BRAF mutated thyroid cancers.

BackgroundMolecular alterations of the MAPK pathway are frequently observed in papillary thyroid carcinomas (PTCs). It leads to a constitutive activation of the signalling pathway through an increase in MEK and ERK phosphorylation. ERK is negatively feedback-regulated by Dual Specificity Phosphatases (DUSPs), especially two ERK-specific DUSPs, DUSP5 (nuclear) and DUSP6 (cytosolic). These negative MAPK regulators may play a role in thyroid carcinogenesis.MethodsMAPK pathway activation was analyzed in 11 human thyroid cancer cell lines. Both phosphatases were studied in three PCCL3 rat thyroid cell lines that express doxycycline inducible PTC oncogenes (RET/PTC3, H-RASV12 or BRAFV600E). Expression levels of DUSP5 and DUSP6 were quantified in 39 human PTCs. The functional role of DUSP5 and DUSP6 was investigated through their silencing in two human BRAFV600E carcinoma cell lines.ResultsBRAFV600E human thyroid cancer cell lines expressed higher phospho-MEK levels but not higher phospho-ERK levels. DUSP5 and DUSP6 are specifically induced by the MEK-ERK pathway in the three PTC oncogenes inducible thyroid cell lines. This negative feedback loop explains the tight regulation of p-ERK levels. DUSP5 and DUSP6 mRNA are overexpressed in human PTCs, especially in BRAFV600E mutated PTCs. DUSP5 and/or DUSP6 siRNA inactivation did not affect proliferation in two BRAFV600E mutated cell lines, which may be explained by a compensatory increase in other phosphatases. In the light of this, we observed a marked DUSP6 upregulation upon DUSP5 inactivation. Despite this, DUSP5 and DUSP6 positively control cell migration and invasion.ConclusionsOur results are in favor of a stronger activation of the MAPK pathway in BRAFV600E PTCs. DUSP5 and DUSP6 have pro-tumorigenic properties in two BRAFV600E PTC cell line models.

Abstract 2086: Dominant role of receptor tyrosine kinase in the activation of MAPK signaling in BRAF non-V600 mutant cancers

Abstract BRAF mutations are found in ~8% of all cancers. In addition to the commonest mutation in kinase domain at the V600, a wide range of other missense mutations (non-V600) have been reported. Non-V600 mutant BRAF protein was also shown to enhance MAPK signaling, suggesting the effectiveness of MEK inhibitors. However, the existence of feedback mechanism in MAPK singling and the roles of receptor tyrosine kinases (RTKs) in these cells are not fully understood. To interrogate this, BRAF non-V600 cell lines with intermediate/impaired kinase activity were treated with MEK inhibitor. RTKs involved in the upregulation of P-MEK following MEK inhibition in BRAF non-V600E mutant cell lines were determined by phosphor-RTK arrays. The effect of combinatorial inhibition of RTKs and MEK in BRAF non-V600 cancer was assessed in vitro and in vivo. By analyzing drug screen data, we first found cells with BRAF non-V600E mutation were less sensitive to MEK inhibition compared with BRAF V600E mutant cell lines and almost comparable to RAS/RAF wild-type cell lines. MEK inhibition did not induce P-MEK suppression and it resulted in significant upregulation of p-MEK compared to BRAF V600E mutant cell lines. Phospho-RTK arrays in the presence or absence of trametinib displayed basal phosphorylation of RTKs including EGFR, MET, and IGF1R, although the phosphorylation level was not consistently changed following MEK inhibition. Among these RTKs, treatment with the EGFR inhibitor erlotinib led to more complete suppression of P-ERK upon trametinib treatment. Furthermore, we found that erlotinib monotherapy downregulated P-ERK in BRAF non-V600 mutant cell lines in contrast that inhibition of RTKs had no effect on MAPK signaling in BRAF V600 mutant cells. While erlotinib treatment led to MAPK inhibition both in intermediate active and impaired kinase active BRAF mutant cells, p-MEK and p-ERK were more strongly suppressed in impaired type BRAF non-V600 mutant cells. The combination of EGFR inhibitor and MEK inhibitor suppressed cell growth and achieved tumor shrinkage in mice xenograft. These results suggest that RTKs, especially EGFR, are dominantly involved in the regulation of MAPK signaling in BRAF non-V600 cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Hiromichi Ebi, Hiroshi Kotani, Hidenori Kitai, Yuta Adachi, Seiji Yano. Dominant role of receptor tyrosine kinase in the activation of MAPK signaling in BRAF non-V600 mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2086. doi:10.1158/1538-7445.AM2017-2086

Targeting of super-enhancers and mutant BRAF can suppress growth of BRAF-mutant colon cancer cells via repression of MAPK signaling pathway

Bromodomain and extra-terminal (BET) inhibitors suppress super-enhancers and show cytotoxicity against multiple types of tumors. However, early clinical trials with BET inhibitors showed severe hematopoietic toxicities, highlighting the need for sensitive tumors and rational combination strategies to enhance their therapeutic potential. Here, we identified colon cancer-specific super-enhancers that were associated with multiple oncogenic pathways, including the mitogen-activated protein kinase (MAPK) signaling pathway. Among the 14 colon cancer cell lines tested, their sensitivity to JQ1, a BET inhibitor, was not correlated with c-MYC expression. Three of four BRAFV600E-mutant cell lines were sensitive. Addition of JQ1 to vemurafenib, a specific mutant BRAF inhibitor, suppressed cell growth by arresting cell cycle progression and inducing apoptosis in the BRAFV600E-mutant cells. Mechanistically, the feedback activation of MAPK signaling pathway by vemurafenib was repressed by JQ1. Further, the addition of JQ1 to a BRAF inhibitor enhanced the in vivo anti-tumor effect. Thus, this study indicates the therapeutic potential of targeting of super-enhancers and mutant BRAF in patients with BRAFV600E-mutant colorectal cancer.

Study of negative feedback between wild-type BRAF or RAFV600E and Mps1 in melanoma

To study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma. Melanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression. In melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector. Based on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.

Abstract 5475: Interrogation of NCI-60 patterns of growth inhibition in conjunction with investigational oncology agents kinase profiling for the elucidation of mechanistic targets

Abstract As oncology treatment moves toward personalized targeted therapeutic agents, the NCI-60 human tumor cell line panel is an ideal community-wide tool to further understanding of the disease and molecular targets of new agents. The panel includes cell lines from nine tumor types, and is extremely well characterized at the molecular level, enabling interrogation of patterns of growth inhibition by a set of targeted investigational oncology agents looking for characteristics of the cell lines that determine sensitivity. We have used a number of online tools to enable data analysis, including COMPARE (http://dtp.nci.nih.gov/compare/), which provided the identification of compounds and/or genes that have highly correlated response patterns for any selected ‘seed’ compound. These data enable comparisons between drug sensitivity profiles that lead to the elucidation of common mechanistic targets or pathways, associations with potential response biomarkers, the confirmation of mechanism of action or identification of novel mechanisms, and the uncovering of unexpected “off-target” activities. For example, using the allosteric Akt inhibitor MK-2206 as the seed compound, response patterns for the ATP-competitive Akt inhibitors PF-4173640 (0.84), GDC-0068 (0.80), AZD-5363 (0.83), GSK-690693 (0.67), and CCT-128930 (0.69) are highly correlated. In addition, examination of the response profile for vandetinib produced a set of highly correlative agents including the corresponding EGFR inhibitors sapatinib (0.81) and AEE-788 (0.83), as well as the recently FDA-approved BTK inhibitor, ibrutinib (0.72) and the SRC inhibitor, AZD-0530 (0.74). Not surprisingly, kinase profiling of these 5 agents (0.5 uM) showed >90% inhibition of EGFR in all cases. In a third example, the BRAF V600E mutated cell lines were found to be sensitive to the bcr-abl inhibitors bafetinib and rebastinib, similarly to vemurafenib. This association suggested BRAF inhibitory activity for the former agents, which was confirmed through kinase profiling. Moreover, the NCI-60 response pattern for the androgen receptor modulator AZD-3514 has a high correlation with the BET bromodomain inhibitors JQ-1 (0.77), I-Bet-151 (0.80), and I-Bet-762 (0.78), suggesting a commonality of target/pathway for these compounds. Further correlations, associations and hypotheses generated from interrogating the compound response patterns, gene expression profiles, mutations and other characteristics will be presented. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Joel Morris, Mark Kunkel, Eric Polley, Susan Holbeck, Anne Monks, David Evans, Annamaria Rapisarda, Jerry Collins, Beverly Teicher. Interrogation of NCI-60 patterns of growth inhibition in conjunction with investigational oncology agents kinase profiling for the elucidation of mechanistic targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2014-5475

Genotype-dependent cooperation of ionizing radiation with BRAF inhibition in BRAF V600E-mutated carcinomas

A substantial proportion of solid tumors carry the BRAF V600E mutation, which causes activation of the MEK/MAPK pathway and is a poor prognostic indicator. Patients with locally advanced human cancers are often treated with external beam radiation therapy. Given the association of Raf overactivation with radioresistance, we hypothesized that, in BRAF V600E-mutated carcinomas, there would be combinatorial activity between radiation and PLX4720, a specific BRAF V600E-inhibitor. Two BRAF V600E-mutated cancer cell lines and one BRAF-V600E wildtype (WT) cancer cell line were obtained. We performed cell viability assays and clonogenic assays using combinations of radiation and PLX4720. We assessed MEK and MAPK phosphorylation at different PLX4720 concentrations with western blotting, and cell cycle progression was evaluated by flow cytometry. Our results show combinatorial, additive activity between radiation and PLX4720 in BRAF V600E-mutated cell lines, but not in the BRAF WT line. In BRAF V600E-mutated cells, there was a PLX4720 concentration-dependent decrease in MEK and MAPK phosphorylation. In cells with BRAF V600E mutations, PLX4720 caused cell cycle arrest at G1, and, when combined with radiation, caused a combined G1 and G2 cell cycle arrest; this pattern of cell cycle effects was not seen in the BRAF WT cell line. These data suggest additive, combinatorial activity between radiation and PLX4720 in cancers carrying BRAF V600E mutations. Our data has potential for translation into the multimodality treatment of BRAF V600E-mutated cancers.

Abstract 2437: MEK162 (ARRY 438162), a MEK1/2 inhibitor, has activity in melanoma cells independent of BRAF and NRAS mutation status.

Abstract Background. Activation of the Ras/Raf/MEK/MAP kinase pathway is implicated in uncontrolled cell proliferation and tumor growth. Mutated, oncogenic forms of Ras are found in colon, pancreatic, and lung cancers; BRAF mutations have been identified in more than 60% of malignant melanomas and from 40-60% of papillary thyroid cancers. MEK, a dual specific kinase, is a key player in this pathway; it is downstream of both Ras and Raf and activates ERK1/2 through phosphorylation of key tyrosine and threonine residues. MEK162 (ARRY 438162) is a novel small molecule ATP-uncompetitive inhibitor of the kinases MEK1 and MEK2. MEK162 showed promising data in an ongoing Phase 2 Clinical Trial in patients with BRAF and NRAS mutated advanced melanoma. This is the first targeted therapy to show activity in patients with NRAS mutated melanoma. Methods and Results. The melanoma cell line panel was the most sensitive after investigating the growth inhibitory effect of MEK162 on 328 cancer cell lines from diverse histologies including melanoma, head and neck, colon, pancreas, lung, ovarian, liver, kidney, breast and endometrial. When a cutoff of IC50 <500nM or >70% Inhibition at 1uM after 6 days of culture was used 83% out of 47 melanoma cell lines were sensitive to the treatment with the MEK inhibitor. Sensitivity to MEK162 was independent of BRAFV600E andNRASQ61mutation status in this cell line panel. Cell cycle arrest and apoptosis was assessed upon exposure to MEK162 using flow cytometry. MEK162 led to a G1 arrest and marked increase in apoptotic cells in the majority of the sensitive melanoma cell lines regardless of their origin and oncogenic driver mutations. Western blots were used to characterize the changes induced by exposure to MEK162 in the MAPK and PI3K/mTOR pathways. MEK1/2 inhibition resulted in a decrease in pERK in all the cell lines tested regardless of their mutational status and the in vitro sensitivity. We observed an increase in pMEK more prominently in NRASQ61L mutant and wild type for NRAS and BRAF mutations cell lines than in BRAFV600Emutant cell lines. We found pAKT and pS6 decreased in the NRAS and BRAF mutant cell lines after treatment, suggesting that the inhibition of the mTOR pathway by MEK162 may be crucial for the sensitivity to the drug. Conclusion. These data provide evidence for supporting the use of MEK162 in the treatment of patients with melanoma. Citation Format: Erika M. Von Euw, Hong-Mei Rong, Neil O'Brien, Dylan Conklin, Veerauo Konkankit, Ke-Wei Gong, Angela Zubel, Ronald Linnartz, Richard Finn, Bartosz Chmielowski, Dennis Slamon. MEK162 (ARRY 438162), a MEK1/2 inhibitor, has activity in melanoma cells independent of BRAF and NRAS mutation status. [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 2437. doi:10.1158/1538-7445.AM2013-2437

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

Antitumor effects of the investigational selective MEK inhibitor TAK733 against cutaneous and uveal melanoma cell lines

BackgroundTAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2.MethodsThe growth inhibitory effects of TAK733 were assessed in a panel of 27 cutaneous and five uveal melanoma cell lines genotyped for driver oncogenic mutations. Flow cytometry, Western blots and metabolic tracer uptake assays were used to characterize the changes induced by exposure to TAK733.ResultsFourteen cutaneous melanoma cell lines with different driver mutations were sensitive to the antiproliferative effects of TAK733, with a higher proportion of BRAFV600E mutant cell lines being highly sensitive with IC50s below 1 nM. The five uveal melanoma cell lines had GNAQ or GNA11 mutations and were either moderately or highly sensitive to TAK733. The tested cell lines wild type for NRAS, BRAF, GNAQ and GNA11 driver mutations were moderately to highly resistant to TAK733. TAK733 led to a decrease in pERK and G1 arrest in most of these melanoma cell lines regardless of their origin, driver oncogenic mutations and in vitro sensitivity to TAK733. MEK inhibition resulted in increase in pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines. Uptake of the metabolic tracers FDG and FLT was inhibited by TAK733 in a manner that closely paralleled the in vitro sensitivity assays.ConclusionsThe MEK inhibitor TAK733 has antitumor properties in melanoma cell lines with different oncogenic mutations and these effects could be detectable by differential metabolic tracer uptake.

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 3584: Effects of the investigational selective MEK inhibitor TAK-733 against cutaneous and uveal melanoma cell lines

Abstract Background. There are several MEK inhibitors currently under clinical investigation, including, TAK-733, a novel non-ATP-competitive allosteric selective inhibitor of MEK. We have analyzed the sensitivity of human melanoma cell lines of different origins to TAK-733. Methods. The half maximal inhibitory concentration (IC50) of TAK-733 was assessed in a panel of 27 skin melanoma cell lines and 5 ocular melanoma cell lines genotyped for driver and secondary oncogenic mutations. Flow cytometry was used to analyze effects on cell cycle progression, and Western blots to analyze the downstream signaling changes upon exposure to TAK-733. Results. Among 12 BRAFV600E mutated cell lines tested, 6 were sensitive to TAK-733 with IC50s less than 10 nM, including 4 highly sensitive cell lines with IC50s below 1 nM. In 5 cell lines with BRAFV600E mutation the IC50 was higher than 100 nM and were considered highly resistant. Among 10 NRASQ61L mutant melanoma cell lines, 6 were sensitive with IC50s below 10 nM. Among 5 melanoma cell lines with wild type sequences for NRAS, BRAF, GNAQ and GNA11 genes only 1 was sensitive to TAK-733. All 5 of the uveal melanoma cell lines were highly sensitive to TAK-733 IC50 regardless of having a GNAQ or GNA11 mutations. MEK inhibition resulted in increase of pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines. In all cases TAK-733 induced marked dose-dependent inhibition of pERK, regardless of the driver oncogenic mutation or the sensitivity or resistance to this agent in cell viability assays. Cell lines resistant to TAK-733 showed no inhibition of pAKT or pS6K, while there was a general trend towards inhibition of these two phosphorylated molecules in sensitive cell lines. Conclusions. TAK-733 is a potent MEK inhibitor with high antitumor activity against human melanoma cell lines with BRAF, NRAS, GNAQ and GNA11 driver mutations. 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 3584. doi:10.1158/1538-7445.AM2011-3584

Abstract 5769: A novel, high-throughput approach to discovering drug-resistance genes identifies CRAF, CrkL, and TPL2/COT as drivers of resistance to BRAFV600E inhibition

Abstract Activating mutations in the BRAF kinase are found in 50-70% of melanomas and have nominated BRAF, along with downstream components of the MAP kinase pathway, as therapeutic targets. Until recently, MAPK pathway inhibitors have shown poor efficacy in clinical trials, leading many to doubt the therapeutic potential of BRAF/MAPK inhibition. However, the recent clinical success of the BRAFV600E-specific kinase inhibitor PLX4032 has validated MAPK pathway inhibition as a viable treatment strategy. Despite initial success, most targeted therapeutics will eventually succumb to acquired resistance. It is therefore imperative that we identify drivers of targeted drug resistance in a rapid, systematic manner that identifies parallel drug targets, ultimately facilitating an effective long-term treatment strategy. Accordingly, we report here a novel, high-throughput approach to systematically identify kinases capable of driving resistance to clinically efficacious, BRAFV600E-specific kinase inhibitors. We screened an open reading frame (ORF) expression library consisting of ∼600 kinases and kinase-related ORFs for their ability to drive resistance to PLX4720 in BRAFV600E-mutant cell lines. Parallel analytical approaches and subsequent validation identified RAF1 (CRAF), CRKL (CrkL) and MAP3K8 (TPL2/COT) as the strongest candidate resistance genes. CRAF and TPL2/COT are both MAP3 kinases that directly phosphorylate and activate MEK, whereas CrkL is an adapter protein that is amplified and phosphorylated in melanoma. CRAF, CrkL and TPL2/COT expressing cells proliferate and show constitutive MAPK pathway activation at concentrations of PLX4720 that are 10-fold over IG50. While CRAF-expressing cells show a dose-dependent increase in MAPK activation when treated with PLX4720, MAPK pathway activation is weakly dampened in CrkL and TPL2/COT-expressing cells upon PLX4720 treatment, suggesting that CrkL and TPL2/COT drive resistance largely, but not exclusively, through MAPK pathway activation. Accordingly, CRAF, CrkL and TPL2/COT segregate into two categories when queried for their ability to drive resistance to a panel of MAPK pathway inhibitors: CRAF mediates resistance only to BRAFV600E-specific inhibitors, while CrkL and TPL2/COT drive resistance to BRAFV600E-specific inhibitors and modify sensitivity to allosteric inhibitors of MEK kinases. Combinatorial treatment with BRAFV600E inhibitors and MEK inhibitors effectively inhibits the proliferation of cells expressing CRAF, CrkL and TPL2/COT, suggesting that combinatorial strategies may address acquired resistance in the clinic. These data highlight the utility and broad applicability of rapid, ORF-based methods of uncovering drivers of targeted resistance and identify CRAF, CrkL and TPL2/COT as putative resistance alleles to BRAFV600E-specific inhibition. 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 5769.