RAF Mutations Research Articles

AML Specific Targeted Drugs Identified By Drug Sensitivity and Resistance Testing: Comparison of Ex Vivo Patient Cells with in Vitro Cell Lines

IntroductionMany drug discovery efforts and pharmacogenomic studies are based on testing established cancer cell lines for their sensitivity to a given drug or a panel of drugs. This approach has been criticized due to high selectivity and fast proliferation rate of cancer cell lines. To explore new therapeutic avenues for acute myeloid leukemia (AML) and to compare experimental model systems, we applied high-throughput Drug Sensitivity and Resistance Testing (DSRT) platform with 305 approved and investigational drugs for 28 established AML cell lines and compared their drug responses with our earlier study of 28 ex vivo AML patient samples (Pemovska et al., 2013). We then correlated drug sensitivities with genomic and molecular profiles of the samples.MethodsDSRT was carried out with 305 clinical, emerging and experimental drugs and small molecule chemical inhibitors. The drugs were tested at five different concentrations over a 10,000-fold concentration range. Cell viability was measured after 72 hours using Cell Titre Glow assay. IC50 values were calculated with Dotmatics software and drug sensitivity scores (DSS, a modified area under the curve metric) were derived for each drug (Yadav et al., 2014). Nimblegen’s SeqCap EZ Designs Comprehensive Cancer Design kit was used to identify mutations from 578 oncogenes in cell lines.ResultsThe 28 established AML cell lines were in general more sensitive to the drugs as compared to the 28 ex vivo patient samples, with some important exceptions. Sensitivity towards many targeted drugs was observed in both AML cell lines and in patient samples. These included inhibitors of MEK (e.g. trametinib in 56% of cell lines and 36% of ex vivo samples), mTOR (e.g. temsirolimus in 42% and 32%) and FLT3 (quizartinib in 28% and 18%). Overall, drug responses between cell lines and ex vivo patient cells in AML showed an overall correlation coefficient of r=0.81. BCL2 inhibitors (venetoclax and navitoclax) showed more sensitivity in ex vivo patient cells than in AML cancer cell lines, whereas responses to anti-mitotic agents (docetaxel, camptothecin, vincristine) showed stronger responses in cell lines (Figure). Only 7% of AML cell lines exhibited responses to a broad-spectrum tyrosine kinase inhibitor dasatinib, in contrast to 36% patient samples.AML cell lines that carried FLT3 mutations showed high sensitivity to FLT3 inhibitors. Similarly, cell lines harbouring mutations in RAS or RAF were strongly sensitive to MEK inhibitors. MEK and FLT3 inhibitor responses were mutually exclusive, indicating alternative pathway dependencies in cell lines. However, these pharmacogenomics correlations were not as clearly seen in the clinical samples.SummaryThese data revealed a few important differences as well as many similarities between established AML cell lines and primary AML patient samples in terms of their response to a panel of cancer drugs. The hope is that patient-derived primary cells in ex vivo testing predict clinical response better as compared to the established cancer cell lines, which indeed seem to overestimate the likelihood of responses to many drugs. On the other hand, cancer cell line studies may also underestimate the potential of dasatinib and BCL2 inhibitors as emerging AML therapeutics.

Characterization of the Mutational Landscape of Multiple Myeloma Using Comprehensive Genomic Profiling

Introduction:Multiple myeloma (MM) is a neoplastic disease of the bone marrow characterized by a malignant transformation of plasma cells. Many patients relapse after initial treatment and require additional therapies. Impaired cell cycle regulation and DNA repair mechanisms as well as exposure to genotoxic drugs leads to accumulation of genomic alterations with progressive disease. Pressure from antineoplastic agents, including novel agents, eventually leads to the selection of resistant clones. Assessing acquired somatic mutations in MM patients can identify key genomic drivers and guide the development of a rational, individualized therapy plan for each patient with advanced disease. Here we report on the mutational landscape of cancer-associated genes in 214 patients who underwent comprehensive genomic profiling.Methods:Review of this data was approved by the UAMS institutional review board. DNA and RNA were extracted from CD138+ selected cells from bone marrow aspirates. Adaptor ligated sequencing libraries from extracted nucleic acids were captured by solution hybridization using bait sets targeting 405 cancer-related and 265 frequently rearranged genes (FoundationOne Heme®; Foundation Medicine ). For samples with low cell yield only the DNA portion was performed. All samples were sequenced in a CLIA-certified, CAP-accredited laboratory to an average depth >500x.ResultsWe identified 147 clinically relevant alterations with an average of 3 alterations per patient ranging from 1 to 8. The most frequently altered genes were KRAS (29% of cases), NRAS (23%), TP53 (19%), RB1 (10%), BRAF (8%), TRAF3(8%), CDKN2C (7%), DNMT3A (5%), NF1, FAF1 and TET2 (4% each). While RAS, RAF, RB1 and TP53 mutations are also found in previously untreated patients, albeit in lower frequencies, mutations of DNTM3A and TET2 are rarely reported in the early phase of the disease, arguing for the accumulation of genomic alterations over time. We found concomitant alterations in KRAS and BRAF in 5, KRAS and NRAS in 3, and NRAS and BRAF in 2 patients. The vast majority of RAS alterations occurred at hotspots resulting in activating alterations at codons 12, 13 or 61 with mutant allele frequencies ranging from 0.01 to 0.92 with an average of 0.30. In the 17 patients with BRAF alterations the hotspot mutation V600E was found in 7 with mutant allele frequencies ranging from 0.01 to 0.48 with an average of 0.32. Overall the MAPK pathway was affected in 128 of 214 patients. 61 patients had alterations of genes associated with DNA damage repair. Among the 10 patients with DNMT3A alterations 2 also had alterations of TET2 suggesting significant epigenetic deregulation in a subset of patients. Data on subclonal structure and correlation of mutation status with paired gene expression profiles will be presented as well, as will be selected responses of patients treated on the basis of these results.ConclusionSubjecting CD138 selected bone marrow cells to comprehensive genomic profiling allows for the identification of clinically relevant alterations, which deregulate critical pathways in multiple myeloma. Small molecule inhibitors that target key genes in these affected pathways (MEK, BRAF) have recently been approved for therapy in other cancers or are being actively developed (PI3K, AKT, PARP). This comprehensive genomic characterization allows rational development of individualized clinical strategies using molecular targets for MM patients who are refractory to standard of care therapies. DisclosuresWalker:Onyx Pharmaceuticals: Consultancy, Honoraria. van Rhee:Senesco: PI Other. Zangari:Norvartis: Membership on an entity’s Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment. Morgan:Celgene Corp: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Janssen: Membership on an entity’s Board of Directors or advisory committees; Myeloma UK: Membership on an entity’s Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity’s Board of Directors or advisory committees; The Binding Site: Membership on an entity’s Board of Directors or advisory committees; MMRF: Membership on an entity’s Board of Directors or advisory committees. Barlogie:Celgene: Consultancy, Patents & Royalties, Research Funding; Millenium: Consultancy, Patents & Royalties, Research Funding.

Abstract IA06: Discovery of a first-in-class ERK 1,2 inhibitor

Abstract The RAS /RAF /MEK / ERK signal transduction pathway is frequently activated in several human cancers, due to a gain of function mutations in KRAS, NRAS, or BRAF. Because of the high frequency of activating mutations, this pathway has become a focus for targeted therapy. A wide spectrum of MEK and RAF inhibitors have been developed to inhibit components of the pathway. Vemurafenib and dabrafenib, selective BRAF inhibitors, and trametinib, an allosteric MEK inhibitor, have shown clinical efficacy in melanoma patients. However, the majority of responses to RAF inhibitors are transient and acquired resistance is often associated with pathway reactivation involving the downstream extracellular-signal-regulated kinases 1 and 2 (ERK1/2). Pathway blockade at ERK, the last signaling node prior to MAPK transcriptional programming, may not only be efficacious in MAPK-activated tumors but might also have utility in RAF or MEK inhibitor resistant settings. This abstract describes the identification and characterization of SCH772984, a potent and selective ATP competitive inhibitor of ERK1/2 which displays behaviors of both type I and type II kinase inhibitors. SCH772984 has nanomolar cellular potency on tumor cells with mutations in RAF, NRAS, or KRAS and induces tumor regressions in xenograft models at tolerated doses. SCH772984 effectively inhibited MAPK signaling and cell proliferation in RAF or MEK inhibitor resistant models as well as in the context of RAF/MEK combination resistance. Together these data support the clinical development of ERK inhibitors, not only in patients with MAPK activated tumors, but also in patients who have developed acquired resistance to RAF or MEK inhibitors or resistance to the recently described combination of these agents. Strategies to overcome acquired resistance to pathway inhibitors will be also described. Citation Format: Ahmed A. Samatar. Discovery of a first-in-class ERK 1,2 inhibitor. [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 IA06. doi: 10.1158/1557-3125.RASONC14-IA06

Abstract A41: Oncogenic RAF mutants that signal as functional dimers are resistant to current RAF inhibitors but sensitive to a novel inhibitor of RAF dimer kinase activity

Abstract Dysregulation of ERK signaling is a common event in malignancy. Receptor activation of RAS leads to dimerization and activation of RAF kinase, which in turn activates ERK. Physiologic activation of the pathway is limited by ERK dependent feedback inhibition of RAS activation and other components of the pathway. Oncogenic mutations and translocations of RAF family members have been identified in many human tumors and serve as drivers of tumor growth. We now show that these mutants are active despite feedback inhibition of RAS and that this is associated with dysregulated ERK output. All translocations and almost all of the RAF mutants tested so far form RAS-independent constitutive dimers, the activity of which is dependent on their dimerization. Mutations at one site, V600E and V600K allow RAF to function as a monomer in cells with low RAS activity and as a RAS-dependent dimer in cells with adequate RAS activity. In tumors expressing any of these mutants, RAF/MEK/ERK activity is elevated despite feedback inhibition of RAS. Currently available RAF inhibitors bind to one protomer of the RAF dimer and cause negative cooperativity of binding to the second site. This is sometimes associated with transactivation of the unbound protomer. These inhibitors cannot be used to inhibit ERK signaling in tumors in which ERK is driven by RAF-dimers (those with mutant RAS, NF1 loss, atypical RAF mutations, RAF translocations.) In contrast, in tumors with V600E BRAF mutations, ERK causes strong feedback inhibition of RAS and V600E BRAF exists as a monomer that is inhibited by these drugs. Acquired resistance to RAF inhibitors usually results from a molecular lesion that causes RAS-dependent or independent dimerization of V600E (NRAS mutation, V600E BRAF amplification, V600E aberrantly spliced isoform.) Current RAF inhibitors inhibit V600E BRAF dimers at concentrations 10-100 fold greater than those required to inhibit the corresponding monomer in cells. We take this as a provisional measure of the degree of negative cooperativity of binding to the second site. We have now identified inhibitors that inhibit V600E dimers at concentrations close (1-3 fold higher) than those required to inhibit the monomer. They do not cause dissociation of dimers and their inhibition is abolished by gatekeeper mutations. Phenotypically, therefore, binding of these compounds to dimers is associated with only minimal negative cooperativity. These drugs inhibit ERK signaling and the proliferation of tumor cells with atypical BRAF mutations and translocations as well as V600E melanoma models with acquired resistance to Vemurafenib. However, they are much less active in cancer cells with KRAS mutation or receptor-dependent activation of RAF. Selective inhibitors of subsets of RAF dimers may allow effective ERK inhibition of large subset of tumors without inhibiting signaling in normal cells. Citation Format: Zhan Yao, Torres N Merna, Wei Min, Hanrahan Aphrothiti, Abdel-Wahab Omar, Solit David, Luo Lusong, Poulikakos Poulikos, Rosen Neal. Oncogenic RAF mutants that signal as functional dimers are resistant to current RAF inhibitors but sensitive to a novel inhibitor of RAF dimer kinase activity. [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 A41. doi: 10.1158/1557-3125.RASONC14-A41

Abstract PR09: Genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes

Abstract Mouse models have proven their utility in establishing causal roles and addressing genetic interactions of candidate alterations in tumorigenesis. Previously, our cross-species comparison of human and mouse melanoma genomes led to the identification of NEDD9 (Neural precursor cell Expressed, Developmentally Down-regulated 9) as a novel melanoma metastasis gene that is up-regulated in 35~50% of metastatic melanomas. We have shown that NEDD9 enhances proliferation and drives invasion in vitro and lung seeding in vivo of melanoma cells. In this study, to explore the biological roles of NEDD9 in melanoma tumorigenesis, we have generated tet-inducible NEDD9 mouse model that carries a doxycycline-responsive, melanocyte-targeted NEDD9. Two independent transgenic founder lines (line A and D) were obtained and genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes have been tested. Melanoma displays frequent activation of NRAS and BRAF and inactivation of CDKN2A (INK4A/ARF) and PTEN. First, we asked whether NEDD9 cooperates with RAS/RAF/MAPK pathway activation by crossing Nedd9 alleles onto the non-metastatic Tyr-RAS* Ink4a/Arf-/- melanoma model to establish a colony of compound mutant mice designated as “iNEDD9” (e.g. Tyr-HRAS*;Tyr-rtTA/Tet-NEDD9;Ink4a/Arf-/-). These mice developed spontaneous cutaneous nodular type melanomas and ocular melanomas indistinguishable from the ones developed in Tyr-RAS* Ink4a/Arf-/- model. In these mice, NEDD9 expression in melanocytes via doxycycline administration led to decreased tumor latency (Median latency: 88 (NEDD9 ON) vs. 120 (NEDD9 OFF) days (p=0.041, log-rank test)) and increased tumor initiation without signs of metastasis. Median melanoma-free survival of iNEDD9 mice was 111 (line A, p=0.009, log-rank test) and 114.5 (line D, p=0.032) days, lower than 166 days observed in control mice without NEDD9. This data supports the cooperation of NEDD9 with RAS/RAF/MAPK pathway activation in melanoma tumorigenesis. Secondly, we addressed whether NEDD9 can synergize with loss of PTEN and INK4a/ARF in vitro and in vivo. We observed that NEDD9 cooperated with PTEN loss in Ink4a/Arf-/- melanocytes without activating RAS or RAF mutation and enhanced proliferation, anchorage independent growth, and invasion in vitro and tumor growth in nude mice. NEDD9 expression correlated with increased phosphorylation of SFKs (Src family kinases), AKT2, and STAT3. To analyze the biological consequence of NEDD9 expression in vivo, we generated a mouse model based on loss of PTEN and CDKN2A (INK4A/ARF) with or without NEDD9 up-regulation designated as “NIP” (e.g. Tyr-CreERT2;Tyr-rtTA/Tet-NEDD9;cInk4a/ArfL/L;cPtenL/L). These mice developed cutaneous and ocular melanomas following tamoxifen treatment, which are under characterization. This study will generate a body of knowledge for the in vivo roles of NEDD9 in melanoma tumorigenesis and invaluable melanoma models driven by genetic lesions observed in human melanoma patients. Since most of the current mouse melanoma models are driven by activating mutations in NRAS or BRAF genes, our model may provide a novel opportunity to understand melanomas without these mutations. This abstract is also presented as Poster B04. Citation Format: Hyeran Sung, Jong Woo Lee, Gretchen Alicea, Ji-Hyun Lee, Jane Messina, Scott Granter, Lynda Chin, Minjung Kim. Genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR09.

Abstract 740: Phenotypic profiling and selectivity optimization of ERK inhibitors using a high-throughput imaged-based cell cycle assay

Abstract The extracellular-signal-regulated kinases (ERK-1 and ERK-2) represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated due to oncogenic mutations in RAF or RAS or due to upstream oncogenic signaling, such as through receptor tyrosine kinase (RTK) signaling. We have discovered and developed potent and selective small molecule inhibitors of both ERK-1 and 2. Optimization of kinase selectivity was a critical goal in order for ATP-competitive ERK inhibitors to maintain parity with the safety profile of highly selective allosteric MEK inhibitors. As a complement to enzymatic kinase panel profiling, we employed an imaged-based cell cycle assay, based on the expected on-target phenotype of cytostatic G1 arrest in B-Raf-mutant cell lines. We were thus able to simultaneously measure on-target and off-target activities. Inhibition of Cdk2, one of the primary off-targets, was associated with a distinct G2 cell cycle arrest, which enabled us to quantify the functional selectivity margin between ERK and Cdk2 in a single assay, and select ERK inhibitors with acceptable selectivity margins for further development. Note: This abstract was not presented at the meeting. Citation Format: Grace Ka-Yan Chan, Tracy Kleinheinz, Matthew Martinson, Hok-Sum Cheung, John G. Moffat. Phenotypic profiling and selectivity optimization of ERK inhibitors using a high-throughput imaged-based cell cycle assay. [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 740. doi:10.1158/1538-7445.AM2014-740

Abstract 3440: Cell-based screening identify gene expression signature correlated with sensitivity to an MEK1/2 inhibitor

Abstract The mitogen-activated extracellular signal-regulated kinase (MEK) family is one of the key kinase regulators involved in tumor oncogenic growth and progression. Multiple MEK inhibitors are currently under clinical investigation for cancer treatment and more agents targeting MEK are under preclinical development. The mechanisms underlying resistance to MEK inhibitor are unclear. Here we examined the anti-proliferation activity of a new small molecule MEK inhibitor in a panel of cell lines harboring either ras or raf mutation. We found 23 lines that are sensitive to the inhibitor and 24 lines are resistant. We compared gene expression by Affymetrix U219 arrays and gene copy number by Affymetrix SNP6.0 arrays in these two cell line groups. With a p-value cutoff set at0.01, we identified 549 genes differentially expressed and 328 genes demonstrated different copy numbers between the two groups. 23 genes were both detected by gene expression profiling and gene copy number data. These genes include MEK family members MAP2K3 and MAP2K7, as well as other important molecule markers in cancer biology, including PIK3R2 and BRCA2. The results suggested a potential genomic signature that could be predictive of response to the MEK inhibitor in vitro. Further investigation and validation in patient-derived xenograft (PDX) models will undoubtfully provide valuable information for selecting patients that are likely to receive clinical benefit. Citation Format: Jing Zhang. Cell-based screening identify gene expression signature correlated with sensitivity to an MEK1/2 inhibitor. [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 3440. doi:10.1158/1538-7445.AM2014-3440

Abstract 3706: Oncogenic RAF mutants that signal as functional dimers are resistant to current RAF inhibitors but sensitive to a novel inhibitor of RAF dimer kinase activity

Abstract Dysregulation of ERK signaling is a common event in malignancy. Receptor activation of RAS leads to dimerization and activation of RAF kinase, which in turn activates ERK. Physiologic activation of the pathway is limited by ERK dependent feedback inhibition of RAS activation and other components of the pathway. Oncogenic mutations and translocations of RAF family members have been identified in many human tumors and serve as drivers of tumor growth. We now show that these mutants are active despite feedback inhibition of RAS and that this is associated with dysregulated ERK output. All translocations and almost all of the RAF mutants tested so far form RAS-independent constitutive dimers, the activity of which is dependent on their dimerization. Mutations at one site, V600E and V600K allow RAF to function as a monomer in cells with low RAS activity and as a RAS-dependent dimer in cells with adequate RAS activity. In tumors expressing any of these mutants, RAF/MEK/ERK activity is elevated despite feedback inhibition of RAS. Currently available RAF inhibitors bind to one protomer of the RAF dimer and cause negative cooperativity of binding to the second site. This is sometimes associated with transactivation of the unbound protomer. These inhibitors cannot be used to inhibit ERK signaling in tumors in which ERK is driven by RAF-dimers (those with mutant RAS, NF1 loss, atypical RAF mutations, RAF translocations.) In contrast, in tumors with V600E BRAF mutations, ERK causes strong feedback inhibition of RAS and V600E BRAF exists as a monomer that is inhibited by these drugs. Acquired resistance to RAF inhibitors usually results from a molecular lesion that causes RAS-dependent or independent dimerization of V600E (NRAS mutation, V600E BRAF amplification, V600E aberrantly spliced isoform.) Current RAF inhibitors inhibit V600E BRAF dimers at concentrations 10-100 fold greater than those required to inhibit the corresponding monomer in cells. We take this as a provisional measure of the degree of negative cooperativity of binding to the second site. We have now identified inhibitors that inhibit V600E dimers at concentrations close (1-3 fold higher) than those required to inhibit the monomer. They do not cause dissociation of dimers and their inhibition is abolished by gatekeeper mutations. Phenotypically, therefore, binding of these compounds to dimers is associated with only minimal negative cooperativity. These drugs inhibit ERK signaling and the proliferation of tumor cells with atypical BRAF mutations and translocations as well as V600E melanoma models with acquired resistance to Vemurafenib. However, they are much less active in cancer cells with KRAS mutation or receptor-dependent activation of RAF. Selective inhibitors of subsets of RAF dimers may allow effective ERK inhibition of large subset of tumors without inhibiting signaling in normal cells. Citation Format: Zhan Yao, Merna Torres, Min Wei, Aphrothiti Hanrahan, Omar Abdel-Wahab, David Solit, Lusong Luo, Poulikos Poulikakos, Neal Rosen. Oncogenic RAF mutants that signal as functional dimers are resistant to current RAF inhibitors but sensitive to a novel inhibitor of RAF dimer kinase activity. [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 3706. doi:10.1158/1538-7445.AM2014-3706

O4.06 * ANALYSIS OF RAF PROTEINS IN MALIGNANT GLIOMAS

OBJECTIVE: Low grade astrocytoma WHO grade II (LGA) display an increased expression of the platelet-derived growth factor receptor. The epidermal growth factor receptor is overexpressed in 60% of glioblastoma multiforme (GBM) and amplified in 40%. Both of these tyrosine kinase receptors signal to the mitogen activated Ras-RAF-MEK-ERK signaling pathway. RAF proteins are well known oncogenes, comprised by a family of three members: A-RAF, B-RAF and C-RAF. Especially B-RAF has been defined to be mutationally activated in a high percentage of melanomas, ovarial, thyroidal and colon carcinomas. C-RAF is discussed to be involved in lung cancer development. However, the role of Raf proteins during development of astrocytic tumors has only rarely been addressed in the literature. METHODS: The mutational status of A-RAF and B-RAF was assessed by sequencing in 66 and 44 human GBM, respectively. A panel of three normal brain samples, 15 LGA and 15 GBM was analyzed for gene amplification by dot blot hybridization, for mRNA expression by semiquantitative RT-PCR and for protein expression by Western blotting. The results were correlated with patients' prognosis. Finally, we performed functional assays using transient overexpression and siRNA mediated knock-down to address a putative function of A-RAF in glioma cell proliferation and migration. RESULTS: RAF mutations are rare events in GBM. No mutations were found in the A-RAF gene and only 2% of the tumors contained activating B-RAF mutations. A-RAF gene amplification could be detected more often. However, all three Raf proteins were overexpressed in astrocytic tumors. Both, A-RAF and C-RAF expression was negatively correlated with the patients' survival. In contrast, B-RAF expression had a positive effect. Neither A-RAF, nor C-RAF had any influence on proliferation or migration of GBM cells in functional assays. There are hints for an involvement of A-RAF in the regulation of the tumor cells metabolism and of C-RAF in angiogenesis. CONCLUSION: Raf proteins are clearly overexpressed in GBM and play a role during progression of these tumors. Therefore, the RAF-isoforms are possible candidates for small molecule therapies in conjunction with targeting parallel signaling cascades. However, initially specific functions of RAF during tumorigenesis of human gliomas have to be elucidated.

Elimination of B-RAF in Oncogenic C-RAF-expressing Alveolar Epithelial Type II Cells Reduces MAPK Signal Intensity and Lung Tumor Growth

Tumors are often greatly dependent on signaling cascades promoting cell growth or survival and may become hypersensitive to inactivation of key components within these signaling pathways. Ras and RAF mutations found in human cancer confer constitutive activity to these signaling molecules thereby converting them into an oncogenic state. RAF dimerization is required for normal Ras-dependent RAF activation and is required for the oncogenic potential of mutant RAFs. Here we describe a new mouse model for lung tumor development to investigate the role of B-RAF in oncogenic C-RAF-mediated adenoma initiation and growth. Conditional elimination of B-RAF in C-RAF BxB-expressing embryonic alveolar epithelial type II cells did not block adenoma formation. However, loss of B-RAF led to significantly reduced tumor growth. The diminished tumor growth upon B-RAF inactivation was due to reduced cell proliferation in absence of senescence and increased apoptosis. Furthermore, B-RAF elimination inhibited C-RAF BxB-mediated activation of the mitogenic cascade. In line with these data, mutation of Ser-621 in C-RAF BxB abrogated in vitro the dimerization with B-RAF and blocked the ability to activate the MAPK cascade. Taken together these data indicate that B-RAF is an important factor in oncogenic C-RAF-mediated tumorigenesis.

Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond.

The identification of mutationally activated BRAF in many cancers altered our conception of the part played by the RAF family of protein kinases in oncogenesis. In this Review, we describe the development of BRAF inhibitors and the results that have emerged from their analysis in both the laboratory and the clinic. We discuss the spectrum of RAF mutations in human cancer and the complex interplay between the tissue of origin and the response to RAF inhibition. Finally, we enumerate mechanisms of resistance to BRAF inhibition that have been characterized and postulate how strategies of RAF pathway inhibition may be extended in scope to benefit not only the thousands of patients who are diagnosed annually with BRAF-mutated metastatic melanoma but also the larger patient population with malignancies harbouring mutationally activated RAF genes that are ineffectively treated with the current generation of BRAF kinase inhibitors.

“RAF” neighborhood: Protein–protein interaction in the Raf/Mek/Erk pathway

The Raf/Mek/Erk signaling pathway, activated downstream of Ras primarily to promote proliferation, represents the best studied of the evolutionary conserved MAPK cascades. The investigation of the pathway has continued unabated since its discovery roughly 30 years ago. In the last decade, however, the identification of unexpected in vivo functions of pathway components, as well as the discovery of Raf mutations in human cancer, the ensuing quest for inhibitors, and the efforts to understand their mechanism of action, have boosted interest tremendously. From this large body of work, protein–protein interaction has emerged as a recurrent, crucial theme. This review focuses on the role of protein complexes in the regulation of the Raf/Mek/Erk pathway and in its cross-talk with other signaling cascades. Mapping these interactions and finding a way of exploiting them for therapeutic purposes is one of the challenges of future molecule-targeted therapy.

RAF1 mutations in childhood-onset dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ~9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.

Prostate cancer ETS rearrangements switch a cell migration gene expression program from RAS/ERK to PI3K/AKT regulation

BackgroundThe RAS/ERK and PI3K/AKT pathways induce oncogenic gene expression programs and are commonly activated together in cancer cells. Often, RAS/ERK signaling is activated by mutation of the RAS or RAF oncogenes, and PI3K/AKT is activated by loss of the tumor suppressor PTEN. In prostate cancer, PTEN deletions are common, but, unlike other carcinomas, RAS and RAF mutations are rare. We have previously shown that over-expression of “oncogenic” ETS transcription factors, which occurs in about one-half of prostate tumors due to chromosome rearrangement, can bypass the need for RAS/ERK signaling in the activation of a cell migration gene expression program. In this study we test the role of RAS/ERK and PI3K/AKT signaling in the function of oncogenic ETS proteins.ResultsWe find that oncogenic ETS expression negatively correlates with RAS and RAF mutations in prostate tumors. Furthermore, the oncogenic ETS transcription factors only increased cell migration in the absence of RAS/ERK activation. In contrast to RAS/ERK, it has been reported that oncogenic ETS expression positively correlates with PI3K/AKT activation. We identified a mechanistic explanation for this finding by showing that oncogenic ETS proteins required AKT signaling to activate a cell migration gene expression program through ETS/AP-1 binding sequences. Levels of pAKT correlated with the ability of oncogenic ETS proteins to increase cell migration, but this process did not require mTORC1.ConclusionsOur findings indicate that oncogenic ETS rearrangements cause a cell migration gene expression program to switch from RAS/ERK control to PI3K/AKT control and provide a possible explanation for the high frequency of PTEN, but not RAS/RAF mutations in prostate cancer.

Cardiovascular disease in Noonan syndrome

BackgroundNoonan syndrome (NS), a relatively common autosomal dominant disorder with an incidence of 1 in 1000 to 2500 live births, is the most common syndromic cause of congenital heart disease...

Evaluation of reverse phase protein array (RPPA)-based pathway-activation profiling in 84 non-small cell lung cancer (NSCLC) cell lines as platform for cancer proteomics and biomarker discovery

The reverse phase protein array (RPPA) approach was employed for a quantitative analysis of 71 cancer-relevant proteins and phosphoproteins in 84 non-small cell lung cancer (NSCLC) cell lines and by monitoring the activation state of selected receptor tyrosine kinases, PI3K/AKT and MEK/ERK1/2 signaling, cell cycle control, apoptosis, and DNA damage. Additional information on NSCLC cell lines such as that of transcriptomic data, genomic aberrations, and drug sensitivity was analyzed in the context of proteomic data using supervised and non-supervised approaches for data analysis. First, the unsupervised analysis of proteomic data indicated that proteins clustering closely together reflect well-known signaling modules, e.g. PI3K/AKT- and RAS/RAF/ERK-signaling, cell cycle regulation, and apoptosis. However, mutations of EGFR, ERBB2, RAF, RAS, TP53, and PI3K were found dispersed across different signaling pathway clusters. Merely cell lines with an amplification of EGFR and/or ERBB2 clustered closely together on the proteomic, but not on the transcriptomic level. Secondly, supervised data analysis revealed that sensitivity towards anti-EGFR drugs generally correlated better with high level EGFR phosphorylation than with EGFR abundance itself. High level phosphorylation of RB and high abundance of AURKA were identified as candidates that can potentially predict sensitivity towards the aurora kinase inhibitor VX680. Examples shown demonstrate that the RPPA approach presents a useful platform for targeted proteomics with high potential for biomarker discovery. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Human relevance of NRAS/BRAF mouse melanoma models

Melanoma is a major problem for many individuals worldwide. Although no effective treatment is available, promising new strategies are being developed. A better understanding of the inner workings of the disease would undoubtedly lead to improved treatments. Mouse melanoma models have been used to elucidate many key regulatory pathways involved in melanoma initiation and progression, and models with mutations in the oncogenes RAF and RAS have been particularly informative. Here, we summarize and evaluate the human relevance of various RAF and RAS mouse melanoma models and their contribution to our understanding of melanoma.

PAF-Mediated MAPK Signaling Hyperactivation via LAMTOR3 Induces Pancreatic Tumorigenesis

Deregulation of mitogen-activated protein kinase (MAPK) signaling leads to development of pancreatic cancer. Although Ras-mutation-driven pancreatic tumorigenesis is well understood, the underlying mechanism of Ras-independent MAPK hyperactivation remains elusive. Here, we have identified a distinct function of PCNA-associated factor (PAF) in modulating MAPK signaling. PAF is overexpressed in pancreatic cancer and required for pancreatic cancer cell proliferation. In mouse models, PAF expression induced pancreatic intraepithelial neoplasia with expression of pancreatic cancer stem cell markers. PAF-induced ductal epithelial cell hyperproliferation was accompanied by extracellular signal-regulated kinase (ERK) phosphorylation independently of Ras or Raf mutations. Intriguingly, PAF transcriptionally activated the expression of late endosomal/lysosomal adaptor, MAPK and mTOR activator 3 (LAMTOR3), which hyperphosphorylates MEK and ERK and is necessary for pancreatic cancer cell proliferation. Our results reveal an unsuspected mechanism of mitogenic signaling activation via LAMTOR3 and suggest that PAF-induced MAPK hyperactivation contributes to pancreatic tumorigenesis.

Management of rasopathies

Noonan syndrome (NS) and NS-related disorders (Cardio-Facio-Cutaneous (CFC) syndrome, Costello syndrome, LEOPARD (Lentigines, ECG conduction abnormalities, Ocular hypertelorism, Pulmonic stenosis, Abnormal genitalia, Retardation of growth and sensory neural Deafness) syndrome) share common clinical features characterized by unique facial features, postnatal growth failure, psychomotor retardation, ectodermal abnormalities, congenital heart diseases, chest & skeletal deformity and delayed puberty. During last decade, strident progress has been made in molecular understanding of NS. The functional alterations of the Ras-mitogen-activated protein kinase (MAPK) pathway are caused by the mutation in more than 10 genes (PTPN11, SOS1, RAF1, SHOC2, BRAF, KRAS, NRAS, HRAS, MEK1, MEK2). Thus, NS and NS-related disorders are called RASopathies as a disease group. PTPN11 (40-50%), SOS1 (10%–20%), and RAF1 (3%-17%) mutations are common in NS patients. Noonan syndrome and its related disorders are not rare as a whole. Since their disease natural course and management are different, it is important to recognize RASopathies and differentiate them primarily based on typical clinical feature. By utilizing DNA testing, the confirmatory diagnosis can be made. Multi-systemic involvement in RASopathies requires multidisciplinary evaluation and regular monitoring for each special clinical issue. It involves whole spectra of clinical issues of cardiovascular, growth & endocrine, neuro-cognitive, developmental, skeletal & orthopedic, opthalmo-otolaryngological, GI-nutritional, dental, hemato-oncological and ectodermal systems. For instances, surgical intervention is required for congenital heart defects and cryptoorchidism. Before the surgery, bleeding diathesis should be excluded. Also the risk for malignant hyperthermia has to be considered in choosing anesthetics. Special education might be required in 10-40% of NS patients. However, NS patients carrying the mutation in the SOS1 gene and N380D or N380S mutation in the PTPN11 gene tend to show normal cognitive function. Many NS infants have feeding difficulties with poor suck and prolonged feeding time and may require tube feeding in 24% of NS infants. Most NS patients show normal levels of IGF-1 and IGF-BP3, indicating growth hormone (GH) deficiency is not culpable for postnatal growth failure. However, some studies have demonstrated subnormal overnight mean growth hormone concentration, suggestive of impaired GH secretion. The rhGH therapy in NS has been reported to be effective to improve both the height velocity and the final adult height. In this review, the constellation of overlapping clinical features of RASopathies will be described based on genotype as well as their differential diagnostic points and management.

Expanding the SHOC2 mutation associated phenotype of noonan syndrome with loose anagen hair: Structural brain anomalies and myelofibrosis

Noonan syndrome is a heterogenous rasopathy typically presenting with short stature, characteristic facial features, cardiac abnormalities including pulmonic valve stenosis, ASD and hypertrophic cardiomyopathy (HCM), cryptorchidism, ectodermal abnormalities, and learning differences. The phenotype is variable, and limited genotype phenotype correlation exists with SOS1 mutations often associated with normal cognition and stature, RAF1 mutations entailing a high HCM risk, and certain PTPN11 mutations predisposing to juvenile myelomonocytic leukemia. The recently identified SHOC2 mutation (p.Ser2Gly) causes Noonan syndrome with loose anagen hair. We report five patients with this mutation. All had skin hyperpigmentation, sparse light colored hair, increased fine wrinkles, ligamentous laxity, developmental delay, and 4/4 had a structural cardiac anomaly. Hypotonia and macrocephaly occurred in 4/5 (80%); 3/5 (60%) had polyhydramnios, increased birth weight or required use of a feeding tube. Distinctive brain abnormalities included relative megalencephaly and enlarged subarachnoid spaces suggestive of benign external hydrocephalus, and a relatively small posterior fossa as indicated by a vertical tentorium. The combination of a large brain with a small posterior fossa likely resulted in the high rate of cerebellar tonsillar ectopia (3/4; 75%). Periventricular nodular heterotopia was seen in one patient with a thick and dysplastic corpus callosum. We report on the first hematologic neoplasm, myelofibrosis, in a 2-year-old patient with SHOC2 mutation. Myelofibrosis is exceedingly rare in children and young adults. The absence of a somatic JAK2 mutation, seen in the majority of patients with myelofibrosis, is noteworthy as it suggests that germline or somatic SHOC2 mutations are causally involved in myelofibrosis.