BRAF-mutant Cells Research Articles

Low-concentration vemurafenib induces the proliferation and invasion of human HaCaT keratinocytes through mitogen-activated protein kinase pathway activation.

Cutaneous squamous cell carcinomas and keratoacanthomas commonly occur in patients treated with BRAF inhibitors. We investigated the effect of the BRAF inhibitor vemurafenib on normal immortalized human HaCaT keratinocytes to explore the mechanism of hyperproliferative cutaneous neoplasia associated with the use of BRAF inhibitors. Vemurafenib induced an increase in viable cell number in BRAF wild-type cell lines (SK-MEL-2 and HaCaT) but not in BRAF mutant cell lines (SK-MEL-24 and G361). In HaCaT keratinocytes, a low concentration (2μmol/L) of vemurafenib increased cell proliferation and activated mitogen-activated protein kinase kinase/extracellular signal-regulated kinase in a CRAF-dependent manner. Invasiveness of HaCaT cells in a Matrigel assay significantly increased upon cultivation of cells with 2μmol/L vemurafenib for 24h. Gelatin zymography, reverse transcription polymerase chain reaction and western blot results revealed that 2μmol/L vemurafenib treatment increased matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities in HaCaT cells. These results offer additional insight into the complex mechanism of paradoxical mitogen-activated protein kinase signaling involved in hyperproliferative cutaneous neoplasias that arise after BRAF inhibition and suggest a possible role for MMP in tumor progression and invasion.

Aloe-emodin exerts a potent anticancer and immunomodulatory activity on BRAF-mutated human melanoma cells

Aim of this study was to extend the knowledge on the antineoplastic effect of aloe-emodin (AE), a natural hydroxyanthraquinone compound, both in metastatic human melanoma cell lines and in primary stem-like cells (melanospheres). Treatment with AE caused reduction of cell proliferation and induction of SK-MEL-28 and A375 cells differentiation, characterized by a marked increase of transamidating activity of transglutaminase whose expression remained unmodified. In vitro antimetastatic property of AE was evaluated by adhesion and Boyden chamber invasion assays. The effect of AE on melanoma cytokines/chemokines production was determined by a multiplex assay: interestingly AE showed an immunomodulatory activity through GM-CSF and IFN-γ production. We report also that AE significantly reduced the proliferation, stemness and invasive potential of melanospheres. Moreover, AE treatment significantly enhanced dabrafenib (a BRAF inhibitor) antiproliferative activity in BRAF mutant cell lines. Our results confirm that AE possesses remarkable antineoplastic properties against melanoma cells, indicating this anthraquinone as a promising agent for differentiation therapy of cancer, or as adjuvant in chemotherapy and targeted therapy. Further, its mechanisms of action support a potential efficacy of AE treatment to counteract resistance of BRAF-mutated melanoma cells to target therapy.

Phase I trial of the CDK 4/6 inhibitor, P1446A-05 (voruciclib) in combination with the BRAF inhibitor (BRAFi), vemurafenib in advanced, BRAF-mutant melanoma.

9076 Background: P1446A-05 is a potent, selective CDK 4/6 inhibitor with activity in multiple BRAF-mutant and wild type cell lines. The BRAFi have been transformative in the therapy of BRAF-mutant ...

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Global gene expression profiling analysis reveals reduction of stemness after B-RAF inhibition in colorectal cancer cell lines.

Cancer cell differentiation is an important field of discussion in the light of cancer stem cells. In a recent study by Herr et al. (2015) “B-RAF inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells” we described how inhibition of mutant BRAF in colorectal cancer cell lines induces cell re-differentiation that is correlated with the loss of tumor growth in vitro and in vivo. We used Illumina HumanHT-12 v4 Expression BeadChip to characterize the gain of differentiation of PLX4720-treated 3D cultures of HT29 and Colo-205 cells. Here, we describe the experimental design and statistical analysis that were performed on the data set leading to the above hypothesis. The data are publicly available at the Gene Expression Omnibus (GEO) database under the accession number GSE50791.

BRAF-induced tumorigenesis is IKKα-dependent but NF-κB-independent.

KRAS mutations contribute to cell proliferation and survival in numerous cancers, including colorectal cancers (CRC). One pathway through which mutant KRAS acts is an inflammatory pathway that involves the kinase IKK and activates the transcription factor NF-κB. BRAF, a kinase that is downstream of KRAS, is mutated in a subset of CRC and is predictive of poor prognosis and therapeutic resistance. We found that, in contrast to mutant KRAS, mutant BRAF (BRAF(V600E)) did not trigger NF-κB activation but instead triggered the phosphorylation of a proteolytic fragment of IKKα (p45-IKKα) in CRC cells. BRAF(V600E) CRC cells had a high abundance of phosphorylated p45-IKKα, which was decreased by a RAF inhibitor. However, the abundance and DNA binding of NF-κB in these cells were unaffected by the RAF inhibitor, and expression of BRAF(V600E) in human embryonic kidney-293T cells did not activate an NF-κB reporter. Moreover, BRAF-induced transformation of NIH-3T3 cells and BRAF-dependent transcription required phosphorylation of p45-IKKα. The kinase TAK1, which was associated with the endosomal compartment, phosphorylated p45-IKKα. Inhibition of endosomal vacuolar adenosine triphosphatase (V-ATPase) with chloroquine or bafilomycin A1 blocked p45-IKKα phosphorylation and induced apoptosis in BRAF-mutant CRC cells independent of autophagy. Treating mice with V-ATPase inhibitors reduced the growth and metastasis of BRAF(V600E) xenograft tumors in the cecum of mice.

Effects of RAL signal transduction in KRAS- and BRAF-mutated cells and prognostic potential of the RAL signature in colorectal cancer

Our understanding of oncogenic signaling pathways has strongly fostered current concepts for targeted therapies in metastatic colorectal cancer. The RALA pathway is novel candidate due to its independent role in controlling expression of genes downstream of RAS.We compared RALA GTPase activities in three colorectal cancer cell lines by GTPase pull-down assay and analyzed the transcriptional and phenotypic effects of transient RALA silencing. Knocking-down RALA expression strongly diminished the active GTP-bound form of the protein. Proliferation of KRAS mutated cell lines was significantly reduced, while BRAF mutated cells were mostly unaffected. By microarray analysis we identified common genes showing altered expression upon RALA silencing in all cell lines. None of these genes were affected when the RAF/MAPK or PI3K pathways were blocked.To investigate the potential clinical relevance of the RALA pathway and its associated transcriptome, we performed a meta-analysis interrogating progression-free survival of colorectal cancer patients of five independent data sets using Cox regression. In each dataset, the RALA-responsive signature correlated with worse outcome.In summary, we uncovered the impact of the RAL signal transduction on genetic program and growth control in KRAS- and BRAF-mutated colorectal cells and demonstrated prognostic potential of the pathway-responsive gene signature in cancer patients.

BRAF V600E mutations: a series of case reports in patients with non–small cell lung cancer

We present a series of five patients with BRAF-mutated non-small cell lung cancer (NSCLC) from the Moffitt Cancer Center and a brief literature review. Information utilized included outside medical records, imaging studies, pathology reports in which simultaneous mutation testing was performed, and clinic visit notes. In addition, we conducted a literature search of background information using the following search terms: "BRAF mutations", "non-small cell lung cancer", and "driver mutations". Several retrospective studies on BRAF mutations in patients with NSCLC found that the majority of these mutations occur in adenocarcinomas and are V600E mutations. From our patients and literature search, we found that BRAF-V600E mutations occur predominantly in female smokers with adenocarcinomas.

Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAF(V600E) melanoma.

Combining immunotherapy and BRAF targeted therapy may result in improved antitumor activity with the high response rates of targeted therapy and the durability of responses with immunotherapy. However, the first clinical trial testing the combination of the BRAF inhibitor vemurafenib and the CTLA4 antibody ipilimumab was terminated early because of substantial liver toxicities. MEK [MAPK (mitogen-activated protein kinase) kinase] inhibitors can potentiate the MAPK inhibition in BRAF mutant cells while potentially alleviating the unwanted paradoxical MAPK activation in BRAF wild-type cells that lead to side effects when using BRAF inhibitors alone. However, there is the concern of MEK inhibitors being detrimental to T cell functionality. Using a mouse model of syngeneic BRAF(V600E)-driven melanoma, SM1, we tested whether addition of the MEK inhibitor trametinib would enhance the antitumor activity of combined immunotherapy with the BRAF inhibitor dabrafenib. Combination of dabrafenib and trametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression, increased T cell infiltration into tumors, and improved in vivo cytotoxicity. Single-agent dabrafenib increased tumor-associated macrophages and T regulatory cells (Tregs) in tumors, which decreased with the addition of trametinib. The triple combination therapy resulted in increased melanosomal antigen and major histocompatibility complex (MHC) expression and global immune-related gene up-regulation. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen-specific ACT, we tested the combination of dabrafenib, trametinib, and anti-PD1 therapy in SM1 tumors, and observed superior antitumor effect. Our findings support the testing of triple combination therapy of BRAF and MEK inhibitors with immunotherapy in patients with BRAF(V600E) mutant metastatic melanoma.

MTORC1 upregulation via ERK-dependent gene expression change confers intrinsic resistance to MEK inhibitors in oncogenic KRas-mutant cancer cells.

Cancer cells harboring oncogenic BRaf mutants, but not oncogenic KRas mutants, are sensitive to MEK inhibitors (MEKi). The mechanism underlying the intrinsic resistance to MEKi in KRas-mutant cells is under intensive investigation. Here, we pursued this mechanism by live imaging of extracellular signal-regulated kinases (ERK) and mammalian target of rapamycin complex 1 (mTORC1) activities in oncogenic KRas or BRaf-mutant cancer cells. We established eight cancer cell lines expressing Förster resonance energy transfer (FRET) biosensors for ERK activity and S6K activity, which was used as a surrogate marker for mTORC1 activity. Under increasing concentrations of MEKi, ERK activity correlated linearly with the cell growth rate in BRaf-mutant cancer cells, but not KRas-mutant cancer cells. The administration of PI3K inhibitors resulted in a linear correlation between ERK activity and cell growth rate in KRas-mutant cancer cells. Intriguingly, mTORC1 activity was correlated linearly with the cell growth rate in both BRaf-mutant cancer cells and KRas-mutant cancer cells. These observations suggested that mTORC1 activity had a pivotal role in cell growth and that the mTORC1 activity was maintained primarily by the ERK pathway in BRaf-mutant cancer cells and by both the ERK and PI3K pathways in KRas-mutant cancer cells. FRET imaging revealed that MEKi inhibited mTORC1 activity with slow kinetics, implying transcriptional control of mTORC1 activity by ERK. In agreement with this observation, MEKi induced the expression of negative regulators of mTORC1, including TSC1, TSC2 and Deptor, which occurred more significantly in BRaf-mutant cells than in KRas-mutant cells. These findings suggested that the suppression of mTORC1 activity and induction of negative regulators of mTORC1 in cancer cells treated for at least 1 day could be used as surrogate markers for the MEKi sensitivity of cancer cells.

Abstract A23: eIF4F is a key and targetable convergence nexus of multiple mechanisms of resistance to anti-BRAF and anti-MEK cancer therapies

Abstract The V600E mutation in BRAF is the most frequent oncogenic mutation in human cancers, and can be targeted by specific anti-BRAF agents. However, several mechanisms of resistance have been identified, leading to reactivation of the initially blocked MAP-kinase pathway (known as ERK-dependent mechanisms) or reactivation of alternative ones, like the PI3K-AKT-mTOR pathway, or inhibition of the apoptotic cascade (ERK-independent). These pathways converge to regulate the formation of the eIF4F translation initiation complex that binds to the 7-methylguanosine cap at the 5′ end of mRNAs, thereby modulating mRNA translation of specific mRNAs. We addressed the potential role of the eIF4F eukaryotic translation initiation complex in resistance or sensitivity to anti-BRAF and anti-MEK agents in several BRAF mutant cells, xenografts and tumors. Our data demonstrate that the formation of the eIF4F complex is associated with most resistance mechanisms to these targeted therapies, independently of their capacity to reactivate or not the MAP-kinase pathway. Formation of this complex can be explored in tumor samples using a new in situ technology. Inhibitors of the eIF4F complex synergize with anti-BRAF agents and can thus reverse resistance. Citation Format: Lise Boussemart, Helene Malka-Mahieu, Isabelle Girault, Delphine Allard, Oskar Hemmingson, Nyam Kamsu-Kom, Sandrine Agoussi, Alexander Eggermont, Laurent Desaubry, Caroline Robert, Stephan Vagner. eIF4F is a key and targetable convergence nexus of multiple mechanisms of resistance to anti-BRAF and anti-MEK cancer therapies. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A23.

Oncogene addiction: pathways of therapeutic response, resistance, and road maps toward a cure.

A key goal of cancer therapeutics is to selectively target the genetic lesions that initiate and maintain cancer cell proliferation and survival. While most cancers harbor multiple oncogenic mutations, a wealth of preclinical and clinical data supports that many cancers are sensitive to inhibition of single oncogenes, a concept referred to as 'oncogene addiction'. Herein, we describe the clinical evidence supporting oncogene addiction and discuss common mechanistic themes emerging from the response and acquired resistance to oncogene-targeted therapies. Finally, we suggest several opportunities toward exploiting oncogene addiction to achieve curative cancer therapies.

Structural investigation of B-Raf paradox breaker and inducer inhibitors.

The V600E missense mutation in B-Raf kinase leads to an anomalous regulation of the MAPK pathway, uncontrolled cell proliferation, and initiation of tumorigenesis. While the ATP-competitive B-Raf inhibitors block the MAPK pathway in B-Raf mutant cells, they induce conformational changes to wild-type B-Raf kinase domain leading to heterodimerization with C-Raf causing a paradoxical hyperactivation of MAPK pathway. A new class of inhibitors (paradox breakers) has been developed that inhibit B-Raf(V600E) activity without agonistically affecting the MAPK pathway in wild-type B-Raf cells. In this study, we explore the structural, conformational, and cellular effects on the B-Raf kinase domain upon binding of paradox breakers and inducers. Our results indicate that a subtle structural difference between paradox inducers and breakers leads to significant conformational differences when complexed with B-Raf. This study provides a novel insight into the activation of B-Raf by ATP-competitive inhibitors and can aid in the design of more potent and selective inhibitors without agonistic function.

The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies.

Resistance to RAF- and MEK-targeted therapy is a major clinical challenge. RAF and MEK inhibitors are initially but only transiently effective in some but not all patients with BRAF gene mutation and are largely ineffective in those with RAS gene mutation because of resistance. Through a genetic screen in BRAF-mutant tumor cells, we show that the Hippo pathway effector YAP (encoded by YAP1) acts as a parallel survival input to promote resistance to RAF and MEK inhibitor therapy. Combined YAP and RAF or MEK inhibition was synthetically lethal not only in several BRAF-mutant tumor types but also in RAS-mutant tumors. Increased YAP in tumors harboring BRAF V600E was a biomarker of worse initial response to RAF and MEK inhibition in patients, establishing the clinical relevance of our findings. Our data identify YAP as a new mechanism of resistance to RAF- and MEK-targeted therapy. The findings unveil the synthetic lethality of combined suppression of YAP and RAF or MEK as a promising strategy to enhance treatment response and patient survival.

Interleukin-1 loop model for pathogenesis of Langerhans cell histiocytosis.

We propose Langerhans cell histiocytosis (LCH) is an inflammatory process that is prolonged by mutations. We hypothesize that Merkel cell polyomavirus (MCPyV) infection triggers an interleukin-1 (IL-1) activation loop that underlies the pathogenesis of LCH. Langerhans cells (LCs) are antigen presenting cells in the skin. When LCs encounter exogenous antigens, they migrate from the epidermis into draining lymphoid tissues to initiate T-cell activity. It has been proposed that LC migration-related factors, including E-cadherin, matrix metalloproteinase, and Notch ligand induce LCH activity. We found that the tyrosine phosphatase SHP-1, which binds IL-1 receptor-associated kinase 1, is expressed at a significantly higher level in LCH affecting multiple organ systems (MS-LCH) than in LCH affecting a single organ system (SS-LCH). IL-1 stimulates T helper 17 cells and their signature cytokine IL-17 had been a matter of controversy. We detected higher levels of IL-17A receptor expression in MS-LCH than in SS-LCH and proposed an IL-17 endocrine model that could settle the controversy. IL-1 is the first cytokine secreted in response to sensitizers and promotes LC migration from sentinel tissues. Myeloid differentiation primary response 88 (MyD88), downstream of the IL-1 receptor, has functions in both RAS signaling and inflammation, leading to human cell transformation. In 2010, an activating mutation in the B-rapidly accelerated fibrosarcoma gene (BRAF) V600E was found in LCH. This BRAF mutation induces phosphorylation of the extracellular signal-regulated kinase (ERK) that may play an important role with MyD88 in LCH pathogenesis. However, phosphorylated ERK (pERK) is rapidly dephosphorylated by dual specificity phosphatase 6 (DUSP6), and limited proliferation is predicted in BRAF mutant cells. MyD88 binds pERK via its D-domain, thereby preventing pERK–DUSP6 interaction and maintaining ERK in an active, phosphorylated state. We detected MCPyV-DNA in the peripheral blood cells of two out of three patients with LCH in high-risk organs but not in those of patients with LCH in non–high-risk organs (0/12; P = .029). MCPyV infection can trigger precursor LCH cells with BRAF mutation to produce IL-1; the IL-1 loop is amplified in all LCH subclasses. Our model indicates both BRAF mutation and IL-1 loop regulation as potential therapeutic targets.

Preexisting MEK1P124 mutations diminish response to BRAF inhibitors in metastatic melanoma patients.

MEK1 mutations in melanoma can confer resistance to BRAF inhibitors, although preexisting MEK1(P124) mutations do not preclude clinical responses. We sought to determine whether recurrent, preexisting MEK1(P124) mutations affected clinical outcome in BRAF inhibitor-treated patients with melanoma. Data from four published datasets were analyzed to determine whether preexisting MEK1(P124) mutations affect radiologic response or progression-free survival (PFS) in patients with BRAF(V600)-mutant metastatic melanoma treated with vemurafenib or dabrafenib. The effects of MEK1(P124) mutations on MAPK pathway activity and response to BRAF inhibition were also investigated in a series of cell models. In a pooled analysis of 123 patients, the presence of a pretreatment MEK1(P124) mutation (N = 12, 10%) was associated with a poorer RECIST response (33% vs. 72% in MEK1(P124Q/S) vs. MEK1(P124) wild-type, P = 0.018), and a shorter PFS (median 3.1 vs. 4.8 months, P = 0.004). Furthermore, MEK1(P124Q/S) mutations were shown to have independent kinase activity and introduction of these mutations into a BRAF-mutant melanoma cell line diminished inhibition of ERK phosphorylation by dabrafenib and enhanced clonogenic survival in the presence of dabrafenib compared with cells ectopically expressing wild-type MEK1. Consistent with these data, two BRAF-mutant cell lines with endogenous MEK1(P124) mutations showed intermediate sensitivity to dabrafenib, but were highly sensitive to downstream inhibition of MEK or ERK. Taken together, our data indicate that preexisting MEK1(P124) mutations are associated with a reduced response to BRAF inhibitor therapy and identify a subset of patients with BRAF-mutant melanoma likely to benefit from combination therapies involving MEK or ERK inhibitors.

Sema6A and Mical1 control cell growth and survival of BRAFV600E human melanoma cells.

We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAFV600E vs NRASQ61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAFV600E and NRASQ61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAFV600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRASQ61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAFV600E melanoma.

Characterization of RAS Alterations in Myeloma: Why Direct Targeting of RAS May be the Most Appropriate Therapeutic Approach

Recent studies have shown that the RAS pathway is a major therapeutic target with which to develop personalized treatment strategies for myeloma patients. In order to translate this knowledge into patient benefit, we have taken a systematic approach by characterizing the mutational profile of the RAS pathway and examining its role in disease progression and patient outcome. Finally we investigated a targeted treatment approach using small molecules to inhibit the RAS MAPK signalling cascade at different levels.In order to gain a deeper understanding of RAS pathway alterations in myeloma, next-generation sequencing data was analyzed from 463 patients entered into the MRC Myeloma XI trial. Using published papers and online databases, a panel of 72 genes were identified as RAS MAPK family genes and were grouped into 5 categories: RAS GTPases, guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), downstream pathway or RAS processing genes. Thirty seven of these genes were mutated in 50% of patients, with novel mutations described for genes in each of these categories. The mutation frequency of KRAS (22.5%), NRAS (19.7%) and BRAF (7.8%) was in-line with other reports and KRAS was found mutated at more codons compared to NRAS. Additionally, recurrent mutations at the same codon were identified only in KRAS, NRAS and BRAF, attesting to their importance in this disease. A quarter of patients (n=59) had more than one RAS MAPK mutation. When more than one mutation was present, the majority were in two RAS GTPases (n=19) (most commonly KRAS and NRAS (n=8) or both in KRAS (n=6)) or in a RAS GTPase and a downstream pathway gene (n=12). One patient had mutations in KRAS, NRAS and BRAF but at sub-clonal levels, indicating they may be present in different cells.We also assessed the role of this pathway in myeloma by examining gene expression profiling data from plasma cells at various myeloma disease stages (GSE2113, GSE5900 and GSE6477). This analysis highlighted roles for KRAS, RAF1, MAPK1, MAP2K1, NF1, RASGRP1 and FNTA in disease progression from MGUS to PCL. Additionally, survival analysis suggested important roles for MAPK1 in determining outcome in newly diagnosed patients in the HOVON65/GMMG-HD4 (GSE19784) trial and FNTA, KRAS, MAP2K1 and RAF1 in determining outcome in relapsed patients in the APEX (GSE9782) trial. These results suggest the highlighted genes are important in mediating clinical outcome and could be therapeutic targets.Given the biological importance of this pathway, we have explored its therapeutic potential by using inhibitors that target the pathway at different levels. Inhibitors included those targeting RAS (n=2), RAF1 (n=2), RAF1/BRAF (n=2), MEK (n=2) and ERK (n=1). The effect of these inhibitors was explored in a panel of 8 myeloma cell lines chosen based on their differing RAS mutational status. Using Western blot analysis, all cell lines showed evidence of activation of the RAS MAPK pathway. No consistent pattern of expression was observed in terms of the levels of activated pathway proteins in KRAS mutant cells compared to NRAS or non-mutated cells. In contrast NRAS and BRAF mutant cells contained elevated levels of activated MEK compared to the other cell lines and were the most sensitive to a MEK inhibitor. Interestingly all cell lines, regardless of mutational status, were similarly sensitive to a novel KRAS inhibitor which prevents KRAS reaching its correct localization. It was shown to be effective at inducing apoptosis, even in the presence of the bone marrow stromal cells. Importantly, protein analysis demonstrated these effects were target-driven with Western blot analysis indicating inhibition of RAS signalling within two to six hours of treatment. Thus, the direct targeting of RAS early in the signalling cascade may be effective against myeloma, irrespective of the RAS mutation status. DisclosuresWalker:Onyx Pharmaceuticals: Consultancy, Honoraria.

Abstract B37: Prenylation inhibition induces cell death in NRAS mutant melanoma cells

Abstract While targeted therapeutic approaches are now available for the treatment of BRAF mutant melanomas, non-BRAF mutant melanoma still lacks this modality. In order to explore the potential of prenylation inhibition we investigated the response of NRAS or BRAF mutant and double wild-type melanoma to zoledronic acid treatment. Effect of zoledronic acid on proliferation, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were characterized in vitro. In vivo subcutaneous xenograft and spleen to liver colonization models were used to evaluate the influence of zoledronic acid treatment on primary tumor growth and metastatic potential of melanoma cells. Zoledronic acid significantly decreased proliferation in NRAS mutant cells as compared to BRAF mutant and double wild-type cells, in vitro. Similarly, TUNEL assay revealed apoptosis induction in an oncogenic mutation specific manner. In line with this finding, activation of ribosomal protein S6 was decreased. Unexpected activation of the downstream effector Erk1/2 and increase in migration was found in BRAF mutant cells. Zoledronic acid failed to show significant synergism with cytotoxic cisplatin or DTIC treatment in vitro. In vivo zoledronic acid did not inhibit the subcutaneous growth of melanoma cells irrespective of oncogenic mutations. In contrast, the lower dose of zoledronic acid resulted in significantly higher liver metastasis in double wild-type cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target melanoma cells. Citation Format: Tamas Garay, Istvan Kenessey, Viktoria Laszlo, Jozsef Tovari, Walter Berger, Laszlo Kovacs, Jozsef Timar, Balazs Hegedus. Prenylation inhibition induces cell death in NRAS mutant melanoma cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B37. doi: 10.1158/1557-3125.RASONC14-B37

Abstract PR06: Impact of RAL signal transduction on genetic program and growth control in KRAS- and BRAF-mutated colorectal cells and prognostic potential of pathway-responsive genes in cancer patients

Abstract The EGFR/RAS signaling system plays an imperative role in tumor pathogenesis and therapy response. Downstream effects of the EGFR/RAS signaling axis are mediated by three major pathway branches: the RAF/MAPK, PI3K and RALA pathways. While the RAF/MAPK and PI3K pathways have been extensively characterized in mechanistic, preclinical and clinical studies, the RAL pathway is less well understood. Here we compared RALGTPase activities in three colorectal cancer cell lines by active GTPase pull-down assay and analyzed the effects of transient silencing of RALA expression by siRNA. RALA activity was highest in SW480 [KRAS codon 12 mutation] and HCT116 cells [KRAS codon 13 mutation] and also detectable in HT29 colorectal cancer cells [KRAS wild-type, BRAF V600E mutation]. Silencing of RALA expression strongly diminished the active GTP-bound form of the protein in all cell lines. The proliferation of the two KRAS mutated cell lines was significantly reduced. BRAF mutated cells showed a slight increase in cell death only. Next we interrogated microarrays using RNA prepared from the cell lines treated with siRNA and controls. We identified 550 common genes the expression of which was up-regulated or down-regulated after RALA knock-down (“RALA pathway-responsive genes”). None of them were affected when the RAF/MAPK or PI3K pathways were blocked. To investigate the potential clinical relevance of transcriptional targets regulated by the RALA pathway, we performed a meta-analysis utilizing expression profiles of 1,424 colorectal cancers documented in 8 independent publicly available data sets comprising information on patient survival. Of 19 RALA pathway-responsive genes correlated with progression-free survival, IQGAP1 (IQ-motif containing GTPase-activating protein 1), TOP1 (topoisomerase 1), LGALS1 (lectin galactoside-binding soluble 1), FILIP1L (filamin A-interacting protein 1-like) and TCF4 (transcription factor 4) were identified as the most important RALA pathway targets capable of predicting survival. In conclusion, the RALA pathway impinges on the transcription of a distinct subset of target genes in colorectal cancer cells independent of the KRAS and BRAF mutational status. RALA pathway-responsive genes were unaffected by RAF/MAPK and PI3K signaling. These findings support the concept of a pathway-sensitive modular organization of the transcriptome. In view of the correlation of RAL pathway-responsive genes and patient survival, further investigations of the diagnostic impact in prospective trials and exploitation of therapeutic approaches are warranted. This abstract is also presented as Poster B32. Citation Format: Balazs Gyorffy, Iwona Stelniec-Klotz, Christian Sigler, Attila Szijarto, Yu Qian, Reinhold Schäfer. Impact of RAL signal transduction on genetic program and growth control in KRAS- and BRAF-mutated colorectal cells and prognostic potential of pathway-responsive genes in cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR06. doi: 10.1158/1557-3125.RASONC14-PR06