Oncogenic BRAF Signaling Research Articles

Abstract 78: A strategy for effective targeting of oncogenic BRAF signaling with increased therapeutic index using conformation-selective RAF and MEK inhibitors

Abstract BRAF kinase, a critical component of the RAS/MAPK signaling pathway, requires dimerization for its catalytic activity, whereas the oncoprotein BRAF(V600E) can be catalytically active as a monomer. Current clinical RAF inhibitors selectively bind and inhibit monomeric over dimeric BRAF providing a high therapeutic index and are now standard practice in combination with MEK inhibitors in the treatment of patients harboring BRAF(V600E) tumors. However adaptive resistance due to RAF dimerization limits their effectiveness. Recently, RAF inhibitors that equipotently inhibit both monomeric and dimeric RAF have been developed, but they are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues. Here we identify and characterize a third class of RAF inhibitors that preferentially binds and inhibits dimeric over monomeric BRAF(V600E). Using long-timescale molecular dynamics (MD) simulations, we observed distinct dynamic features of RAF inhibitors bound to monomeric and dimeric BRAF, thus providing a structural explanation for the selectivity of these drugs. Biochemical analysis using RAF dimer selective inhibitors revealed that the two forms of dimeric and monomeric BRAF(V600E) differ in their conformation in their active site and their strength of interaction with MEK. Further, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index, we assessed the triple combination of RAF monomer- plus RAF dimer selective inhibitor plus a MEK inhibitor that potently disrupts the BRAF-MEK complex. The triple combination potently suppressed tumor growth in multiple colorectal and melanoma BRAF(V600E) cell line- and in vivo models that were resistant to the current clinical RAF and MEK inhibitor combination. Strikingly, while the double combination treatment with the RAF dimer-selective inhibitor and MEK inhibitor was associated with a significant gradual weight loss in vivo, the triple combination showed no weight loss or other apparent toxicities, indicating a higher therapeutic index. Finally, off-label use of the triple combination in a patient with a stage IV BRAF(V600E) colorectal cancer that progressed on standard therapies achieved durable tumor control with minimal toxicities. Thus, a rationally designed combinatorial approach of conformation-selective RAF and MEK inhibitors may be a highly effective and well tolerated therapeutic strategy for patients with BRAF(V600E) tumors. Citation Format: Christos Adamopoulos, Tamer A. Ahmed, Maxwell R. Tucker, Peter M. Ung, Min Xiao, Zoi Karoulia, Xuewei Wu, Stuart A. Aaronson, Celina Ang, Vito Rebecca, Avner Schlessinger, Meenhard Herlyn, Qi Wang, David E. Shaw, Poulikos I. Poulikakos. A strategy for effective targeting of oncogenic BRAF signaling with increased therapeutic index using conformation-selective RAF and MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 78.

Exploiting Allosteric Properties of RAF and MEK Inhibitors to Target Therapy-Resistant Tumors Driven by Oncogenic BRAF Signaling.

Current clinical RAF inhibitors (RAFi) inhibit monomeric BRAF (mBRAF) but are less potent against dimeric BRAF (dBRAF). RAFi equipotent for mBRAF and dBRAF have been developed but are predicted to have lower therapeutic index. Here we identify a third class of RAFi that selectively inhibits dBRAF over mBRAF. Molecular dynamic simulations reveal restriction of the movement of the BRAF αC-helix as the basis of inhibitor selectivity. Combination of inhibitors based on their conformation selectivity (mBRAF- plus dBRAF-selective plus the most potent BRAF-MEK disruptor MEK inhibitor) promoted suppression of tumor growth in BRAFV600E therapy-resistant models. Strikingly, the triple combination showed no toxicities, whereas dBRAF-selective plus MEK inhibitor treatment caused weight loss in mice. Finally, the triple combination achieved durable response and improved clinical well-being in a patient with stage IV colorectal cancer. Thus, exploiting allosteric properties of RAF and MEK inhibitors enables the design of effective and well-tolerated therapies for BRAFV600E tumors. SIGNIFICANCE: This work identifies a new class of RAFi that are selective for dBRAF over mBRAF and determines the basis of their selectivity. A rationally designed combination of RAF and MEK inhibitors based on their conformation selectivity achieved increased efficacy and a high therapeutic index when used to target BRAFV600E tumors.See related commentary by Zhang and Bollag, p. 1620.This article is highlighted in the In This Issue feature, p. 1601.

Abstract 1845: BRAF-mediated NAMPT overexpression induces a melanoma cell dedifferentiation program leading to metabolic reprogramming and intrinsic resistant to BRAF inhibitors

Abstract INTRODUCTION: In order to support continued proliferation and growth, tumor cells must metabolically adapt to balance their bioenergetic and biosynthetic needs. In metastatic melanoma (MM) oncogenic BRAFV600E signaling is a critical regulator of this process. Consistently, treatment with BRAF/MEK inhibitors (BRAFi/MEKi) leads to a block of glycolysis, promoting energy stress mediated-apoptosis. However, resistance to therapy develops rapidly, with paradoxical activation of the MAPK/ERK signaling pathway and the acquisition of novel metabolic phenotypes. Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme in the biosynthesis of NAD, an essential cofactor in redox reactions and a substrate of key cellular enzymes, including sirtuins. This enzyme may also be secreted in the extracellular space (eNAMPT), where it helps establish a cyto-protective microenvironment, favoring immunosuppression and resistance to therapy. Consequently, NAMPT inhibitors are being tested as cancer therapeutics. METHODS: NAMPT expression and activity was studied using biochemical, enzymatic, immunohistochemical and ELISA assays. In silico analysis using TCGA database, RNAseq and functional/metabolic analyses were performed. Stable or inducible NAMPT overexpression was established using lentiviral vector. Xenografts of melanoma were set up using immunocompromised mice treated with NAMPT inhibitor (NAMPTi) alone or in combination with BRAFi. RESULTS: We found that the BRAF oncogenic signature converges transcriptionally on the overexpression of NAMPT, leading to a marked increase in NAD levels, which in turn support a metabolic switch toward glycolysis or OXPHOS, both strategies exploited by BRAF-mutated melanomas to adapt to chronic exposure to BRAFi. NAMPT up-regulation in BRAFi resistant cells was confirmed in serial biopsies from melanoma patients. Furthermore, NAMPT could be dosed in patient plasma where it correlated with disease burden, response to therapy and overall survival. Treatment of melanoma cells with NAMPTi depleted NAD, inducing mitochondrial stress, cell cycle arrest and apoptosis in vitro, while they were highly effective in controlling the disease in melanoma xenografts. Exogenous overexpression of NAMPT in BRAF-mutated melanoma cell lines leads to a net increase of NAD and ATP, rendering cells intrinsically resistant to BRAFi and supporting a proliferative/invasive phenotype in vitro and in vivo. Mechanistically, NAMPT overexpressing cells switch to a dedifferetiation/epigenetic program leading to a downregulation of microphthalmia-associated transcription factor (MITF). CONCLUSION: In conclusion, this work links oncogenic BRAF signaling to metabolic reprogramming through NAD biosynthesis and identifies NAMPT as an actionable target for melanoma patients with BRAF mutations. Citation Format: Valentina Audrito, Antonella Managò, Federica Gaudino, Ilaria Manfredonia, Vincenzo Gianluca Messana, Nicoletta Vitale, Lorenzo Brandimarte, Enrico Moiso, Roberto Piva, Nadia Raffaelli, Mario Mandalà, Silvia Deaglio. BRAF-mediated NAMPT overexpression induces a melanoma cell dedifferentiation program leading to metabolic reprogramming and intrinsic resistant to BRAF inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1845.

Abstract 4367: The small GTPase ARF6 is necessary for melanomagenesis

Abstract ARF6 is a member of the adenosine diphosphate (ADP)-ribosylation factor (ARF) family of small GTPases and is part of the RAS superfamily. We have shown that pharmacologic inhibition of ARF6 can inhibit proliferation of uveal melanoma and invasion/metastasis of cutaneous melanoma. Most cutaneous melanomas are defined by mutually-exclusive driver mutations in BRAF, NRAS, or NF1. These subtypes share similar mechanisms of oncogenesis because they align along a continuum of RAS signaling. Mutations in the PI3K pathway occur in a fraction of all melanoma subtypes, indicating a selective advantage for PI3K/AKT activation beyond BRAF or RAS activation. We and others have reported ARF6-dependent ERK activation occurring upon either extracellular ligand stimulation or mutant oncoprotein signaling. Recently, ARF6 has also been shown to be upstream of PI3K signaling. Based on these findings, we hypothesized that ARF6 is critical for both the RAF and PI3K arms of the RAS pathway and that loss of ARF6 will impede tumor growth driven by these oncogenes. We tested this hypothesis using human melanoma cell lines and with an inducible, genetically engineered mouse model (GEMM) of melanoma. Our data reveal that pharmacologic inhibition of ARF6 can reduce proliferation of BRAF, NRAS and NF1-mutated melanoma cells. Furthermore, loss of Arf6 causes a reduction in mean tumor size and improves survival in BRAFV600E/CDKN2ANULL/PTENNULL/AKTE17K and BRAFV600E/CDKN2ANULL/PTENNULL mice. These results indicate that ARF6 is necessary for melanomagenesis and for oncogenic BRAF and/or PI3K signaling. Our data suggest that ARF6 may be an actionable node in the RAS pathway and that therapeutic targeting of ARF6 has the potential to improve current treatment methods for progressive disease. Citation Format: Lehi Acosta, Aaron Rogers, Jingfu Peng, Alan Mueller, Zongzhong Tong, Donghan Shin, Jae Hyuk Yoo, Dean Y. Li, Shannon J. Odelberg, Sheri L. Holmen, Allie H. Grossmann. The small GTPase ARF6 is necessary for melanomagenesis [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 4367.

Oncogenic BRAF mutation induces DNA methylation changes in a murine model for human serrated colorectal neoplasia

ABSTRACTColorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.

Nuclear effectors of oncogenic BRAF signaling and their pharmacological inhibition

Nuclear effectors of oncogenic BRAF signaling and their pharmacological inhibition

An Integrated Model of RAF Inhibitor Action Predicts Inhibitor Activity against Oncogenic BRAF Signaling

The complex biochemical effects of RAF inhibitors account for both the effectiveness and mechanisms ofresistance to these drugs, but a unified mechanistic model has been lacking. Here we show that RAF inhibitors exert their effects via two distinct allosteric mechanisms. Drug resistance due to dimerization is determined by the position of the αC helix stabilized by inhibitor, whereas inhibitor-induced RAF primingand dimerization are the result of inhibitor-induced formation of the RAF/RAS-GTP complex. The biochemical effect ofRAF inhibitor in cells is the combined outcome of the two mechanisms. Therapeutic strategies including αC-helix-IN inhibitors are more effective in multiple mutant BRAF-driven tumor models, including colorectal and thyroid BRAF(V600E) cancers, in which first-generation RAF inhibitors have been ineffective.

Cooperative Activity of BRAF F595L and Mutant HRAS in Histiocytic Sarcoma Provides New Insights into Oncogenic BRAF Signaling

Activating BRAF mutations, in particular V600E/K, drive many cancers, including a substantial proportion of systemic histiocytic disorders, and mutant BRAF-selective inhibitors are promising therapeutics for these diseases. Activating BRAF alleles are considered mutually exclusive with mutations in RAS family members, whereas inactivating BRAF mutations in the D(594)F(595)G(596) motif can coexist with oncogenic RAS and cooperate via transactivation of wildtype RAF proteins and paradoxical MEK/ERK activation. Due to the increasing use of global approaches to tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We used several in vitro experimental systems, including Braf-deficient murine embryonic fibroblasts expressing a regulatable HRAS oncogene, to determine the biochemical properties and cellular effects of a largely uncharacterized mutation, F595L, in the DFG motif of the BRAF activation segment that was identified by clinical exome sequencing in a patient with histiocytic sarcoma and multiorgan involvement and also occurs as somatic alteration in colorectal adenoma or carcinoma, non-small cell lung cancer, cholangiocarcinoma, urothelial cancer, melanoma, and neuroblastoma and as germline mutation in cardio-facio-cutaneous syndrome. In addition, we investigated the interaction between BRAF F595L and a concomitant HRAS Q61R allele, which was present in the same tumor cell clone and occurs as acquired alteration in multiple tumor types and as inherited variant in Costello syndrome. Unlike previously described DFG motif mutants, BRAF F595L is a gain-of-function variant with intermediate activity towards MEK that, in sharp contrast to BRAF V600E, requires an intact dimer interface for downstream signaling. Furthermore, BRAF F595L does not act paradoxically, but nevertheless cooperates with mutant HRAS to induce maximal activity of the MEK-ERK signaling pathway. Of immediate clinical relevance, BRAF F595L shows divergent responses to the mutant BRAF-selective inhibitors vemurafenib and dabrafenib, whereas signaling driven by BRAF F595L with and without mutant HRAS is efficiently blocked by the pan-RAF inhibitors sorafenib and AZ628 and the MEK inhibitor trametinib. Consistent with this, sorafenib treatment led to abrogation of aberrant MEK/ERK signaling in the index case with histiocytic sarcoma driven by BRAF F595L and HRAS Q61R. Mutation data from patients and cell lines, representing 18 different tumor entities, show that BRAF F595L as well as other BRAF mutants with intermediate signaling activity coincide with mutant RAS in at least 40% and 23% of cases, respectively. These data define a distinct class of activating BRAF mutations that cooperate with oncogenic RAS in a non-paradoxical fashion, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway, and underscore the value of comprehensive genomic profiling for uncovering the vulnerabilities of individual tumors. DisclosuresOff Label Use: Administration of sorafenib in a patient with histiocytic sarcoma.

Abstract LB-060: A phosphoproteomic comparison of BRAF(V600E) and MKK1/2 inhibition in melanoma

Abstract Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4,722 proteins. This included 1,317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. While the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nM), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation. Citation Format: Scott Stuart, Natalie Ahn, Stephane Houel, William Old. A phosphoproteomic comparison of BRAF(V600E) and MKK1/2 inhibition in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-060. doi:10.1158/1538-7445.AM2015-LB-060

A Phosphoproteomic Comparison of B-RAFV600E and MKK1/2 Inhibitors in Melanoma Cells*

Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4784 proteins. This included 1317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. Although the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nm), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation.

Biophysical Characterization and Expression Analysis of Kv1.3 Potassium Channel in Primary Human Leukemic B Cells

Background/Aims: Pharmacological inhibition of the potassium channel Kv1.3 has been shown to selectively kill B cells from patients with chronic lymphocytic leukemia (B-CLL). Here we aimed to biophysically characterize and compare Kv1.3 channel activity in B cells isolated either from healthy subjects or patients and investigated the mechanism accounting for the increased protein expression in B-CLL cells. Methods: Kv1.3 activity was measured by patch clamp, while expression of the channel protein was assessed by Western blot and FACS analysis. B-CLL cells were co-cultured with mesenchymal stromal cells (MSC) and Kv1.3 inhibitor-induced apoptosis was assessed. Results: We demonstrate that Kv1.3 is highly expressed and is more active at resting membrane potential in human B-CLL cells than in healthy cells. Channel expression in pathologic cells decreased by the B-RAF kinase inhibitor PLX-4720, while it increased with Doxazosin, an α1-adrenoceptor antagonist. Kv1.3 inhibitors induced death in B-CLL cells also when co-cultured with MSC. Conclusion: Our results contribute to the characterization of B-CLL cells, as it shows that upregulation of Kv1.3 in pathologic B lymphocytes is linked to the oncogenic B-RAF signalling. We also conclude that Kv1.3 inhibitors represent a valuable tool to induce apoptosis of B-CLL cells even in the presence of MSC.

B-Raf inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells.

BRAF mutations are associated with aggressive, less-differentiated and therapy-resistant colorectal carcinoma. However, the underlying mechanisms for these correlations remain unknown. To understand how oncogenic B-Raf contributes to carcinogenesis, in particular to aspects other than cellular proliferation and survival, we generated three isogenic human colorectal carcinoma cell line models in which we can dynamically modulate the expression of the B-Raf(V600E) oncoprotein. Doxycyclin-inducible knockdown of endogenous B-Raf(V600E) decreases cellular motility and invasion in conventional and three-dimensional (3D) culture, whereas it promotes cell-cell contacts and induces various hallmarks of differentiated epithelia. Importantly, all these effects are recapitulated by B-Raf (PLX4720, vemurafenib, and dabrafenib) or MEK inhibitors (trametinib). Surprisingly, loss of B-Raf(V600E) in HT29 xenografts does not only stall tumor growth, but also induces glandular structures with marked expression of CDX2, a tumor-suppressor and master transcription factor of intestinal differentiation. By performing the first transcriptome profiles of PLX4720-treated 3D cultures of HT29 and Colo-205 cells, we identify several upregulated genes linked to epithelial differentiation and effector functions, such as claudin-1, a Cdx-2 target gene encoding a critical tight junction component. Thereby, we provide a mechanism for the clinically observed correlation between mutant BRAF and the loss of Cdx-2 and claudin-1. PLX4720 also suppressed several metastasis-associated transcripts that have not been implicated as targets, effectors or potential biomarkers of oncogenic B-Raf signaling so far. Together, we identify a novel facet of clinically applied B-Raf or MEK inhibitors by showing that they promote cellular adhesion and differentiation of colorectal carcinoma cells.

Abstract IA09: Copper is required for oncogenic BRAF signaling and tumorigenesis

Abstract The BRAF serine/threonine kinase is mutated, typically at V600, to induce an active oncogenic state in a large fraction of melanoma, thyroid, hairy cell leukemia, and to a lesser extent, a wide spectrum of other cancers. BRAFV600E activates the kinases MEK1 and MEK2, which stimulate the MAPK pathway to promote cancer. ATP inhibitors of oncogenic BRAF and MEK1/2 provide a survival advantage in metastatic melanoma, an effect enhanced by co-administering two such inhibitors. Thus, combining multiple approaches to inhibit MAPK signaling is a promising strategy to treat BRAF mutation-positive cancers. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of its substrates ERK1 and ERK2 through a Cu-MEK1 interaction. We now show here that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced MAPK signaling and oncogenic BRAFV600E-mediated tumorigenesis. Loss of tumor growth was rescued by expressing a MEK1/5 chimera that phosphorylates ERK1/2 independent of Cu or an activated ERK2. Importantly, Cu chelators used in the treatment of Wilson disease reduced tumor growth of not only BRAFV600E-transformed cells, but also cells resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat BRAFV600E mutation-positive cancers. Citation Format: Donita Brady, Matt Crowe, Michelle Turski, Aaron Hobbs, Xiaojie Yao, Apirat Chaikuad, Stefan Knapp, Kunhong Xiao, Sharon Campbell, Dennis Thiele, Chris Counter. Copper is required for oncogenic BRAF signaling and tumorigenesis. [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 IA09. doi: 10.1158/1557-3125.RASONC14-IA09

Abstract 5245: A network of microRNAs contolled by oncogenic B-Raf signaling in melanoma cells

Abstract Constitutive activation of the B-Raf/MKK/ERK signaling pathway a result of B-Raf activating mutations is a hallmark of over two-thirds of melanomas. The most prevalent mutation, B-RafV600E, promotes cancer cell behavior through mechanisms which are still incompletely defined. We utilized microRNA (miRNA) microarray profiling to identify a network of more than 20 miRNAs deregulated by B-Raf/MKK/ERK in melanoma cells, the majority of which modulate expression of key cancer regulatory genes and functions. Importantly, miRNAs within the network converge on protein regulation and cancer phenotypes, suggesting that these miRNAs might function combinatorially. Additionally, by altering functional levels of these miRNAs in cells with miRNA mimics and inhibitors we demonstrate that miRNAs augment effects on protein repression and cell invasion when co-expressed, and we also observe gene-specific latency and interference effects between miRNAs. Thus, B-Raf/MKK/ERK controls key aspects of cancer cell behavior and gene expression by modulating a network of miRNAs with cross-regulatory functions. The findings highlight the potential for complex interactions between coordinately regulated miRNAs within a gene expression network. Citation Format: Kasey L. Couts, Emily M. Anderson, Maren M. Gross, Kevin Sullivan, Natalie G. Ahn. A network of microRNAs contolled by oncogenic B-Raf signaling in melanoma cells. [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 5245. doi:10.1158/1538-7445.AM2014-5245

Abstract 4427: Copper is required for oncogenic BRAF signaling and tumorigenesis

Abstract The BRAF serine/threonine kinase is mutated, typically at V600, to induce an active oncogenic state in a large fraction of melanoma, thyroid cancers, hairy cell leukemia, and to a lesser extent, a wide spectrum of other cancers. BRAFV600E in turn activates the kinases MEK1 and MEK2, which stimulates the MAPK pathway to promote cancer. Excitingly, ATP inhibitors of oncogenic BRAF and MEK provide a survival advantage in metastatic melanoma, an effect enhanced by combining the two inhibitors. Thus, combining multiple approaches to inhibit MAPK signaling holds great promise for the treatment of BRAF mutation-positive cancers, especially in terms of overcoming resistance. In this regard, we previously found that copper (Cu) influx enhanced MEK1 phosphorylation of its substrates ERK1/2 through a Cu-MEK1 interaction. We show here that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced MAPK signaling and oncogenic BRAFV600E-mediated tumorigenesis, which was rescued by expressing activated MEK5 engineered to phosphorylate ERK1/2 or activated ERK2. Importantly, Cu chelators used in the treatment of Wilson disease reduced tumor growth of not only BRAFV600E-transformed cells but also cells resistant to a BRAF inhibitor. Taken together, these results suggest that Cu-chelation therapy could be repurposed for the treatment of BRAFV600E mutation-positive cancers. Citation Format: Donita C. Brady, Matthew S. Crowe, Michelle L. Turski, G Aaron Hobbs, Apirat Chaikuad, Stefan Knapp, Sharon L. Campbell, Dennis J. Thiele, Christopher M. Counter. Copper is required for oncogenic BRAF signaling and tumorigenesis. [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 4427. doi:10.1158/1538-7445.AM2014-4427

Signalling: Inhibiting oncogenic BRAF signalling by copper depletion.

Brady et al. demonstrate that BRAF-driven MEK signalling can be inhibited using copper chelators.

Copper is required for oncogenic BRAF signalling and tumorigenesis

The BRAF kinase is mutated, typically V600E, to induce an active oncogenic state in a large fraction of melanoma, thyroid, hairy cell leukemia, and to a lesser extent, a wide spectrum of other cancers1,2. BRAFV600E phosphorylates and activates the kinases MEK1 and MEK2, which in turn phosphorylate and activate the kinases ERK1 and ERK2, stimulating the MAPK pathway to promote cancer3. Targeting MEK1/2 is proving to be an important therapeutic strategy, as a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma4, which is increased when co-administered with a BRAFV600E inhibitor5. In this regard, we previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction6. We now show that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced BRAFV600E-driven signaling and tumorigenesis. Conversely, a MEK1-MEK5 chimera that phosphorylates ERK1/2 independent of Cu or an active ERK2 restored tumor growth to cells lacking Ctr1. Importantly, Cu chelators used in the treatment of Wilson disease7 reduced tumor growth of both BRAFV600E-transformed cells and cells resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat BRAFV600E mutation-positive cancers.

Is Mcl‐1L the new anti‐apoptotic effector of B‐RAFV600E in melanoma?

A recent report has unveiled a novel mechanism by which oncogenic BRAF signalling might trigger apoptotic resistance in melanoma by selectively affecting the expression of Bcl-2 family member Mcl-1L (Exp Dermatol 2013: 22: 767). Correlation of Mcl-1 splice variants and B-RAF mutational status was determined in a panel of melanoma cell lines. In vivo validation of this mechanism, which is supported by recent literature, might provide novel therapeutic approaches such as the use of targeted Mcl-1 inhibitors to improve outcome in melanoma.

Copper is required for oncogenic BRAF signalling and tumorigenesis

Copper is required for oncogenic BRAF signalling and tumorigenesis

Oncogenic BRAF signalling increases Mcl‐1 expression in cutaneous metastatic melanoma

The Bcl-2 family member Mcl-1 is essential for melanoma survival; however, the influence of oncogenic BRAF signalling remains elusive. In this study, Mcl-1 splice variant expression was determined in a panel of melanoma cell lines in relation to BRAF mutational status. Mcl-1L mRNA expression was increased in melanoma cells compared with primary melanocytes with significantly increased mRNA and protein expression observed in BRAF(V600E) mutant melanoma cells. Although no change in Mcl-1S mRNA was observed, Mcl-1S protein expression also increased in BRAF mutant melanoma cells. Additionally, while over-expression of mutant BRAF(V600E) increased both Mcl-1L and Mcl-1S expression, inhibition of hyperactive BRAF signalling resulted in decreased Mcl-1L expression. These studies suggest that the regulation of Mcl-1 expression by BRAF signalling is increased by oncogenic activation of BRAF, revealing a mechanism of apoptotic resistance which may be overcome by the use of more specifically targeted Mcl-1 inhibitors.