Ras Pathway Components Research Articles

Novel potential mechanisms of acquired resistance to anti-EGFR monoclonal antibody (mAb) therapy detected in liquid biopsies (LBx) from patients (pts) with advanced colorectal cancer (CRC).

199 Background: While several studies have explored the evolving clonal profile of mCRC under stress of anti-EGFR using circulating tumor DNA (ctDNA), heterogeneity and small cohorts limit our understanding of acquired genomic alterations (GAs) associated with EGFR resistance. Methods: CRC LBx were profiled with FoundationOne Liquid CDx, which reports GAs in 324 cancer-related genes. Foundation Medicine’s (FMI) ctDNA tumor fraction (TF) is a composite algorithm prioritizing aneuploidy at higher levels and variant allele frequency (VAF) of canonical alterations at lower levels. Variants were considered clonal at VAF/TF ≥25%, subclonal at <25%. Among 4,546 non-MSI-H LBx, 2,776 (61%) had TF ≥1% and were used in this analysis. A subset of pts had treatment data in the US-based de-identified Flatiron Health-FM real-world clinico-genomic database (CGDB), originating from ~280 US cancer clinics (01/2011–03/2023). Results: 1,349 (49%) LBx had a clonal RAS GA, 107 (3.9%) had a clonal BRAF V600E, and 1,320 (48%) had no clonal RAS pathway driver GA. GAs in EGFR and MAP2K1 were significantly (FDR<0.0001) more common in LBx with no clonal RAS GA (162, 11%; 105, 7.4%) than with one (27, 2.0%; 24, 1.8%), consistent with prior reports of these GAs appearing in the setting of acquired resistance to anti-EGFR mAb. The 362 LBx with GAs in EGFR, MAP2K1, or subclonal RAS GAs (indicators of anti-EGFR mAb exposure based on a validated exposure signature) were compared to the 1349 LBx with a clonal RAS mutation. Post anti-EGFR LBx were significantly enriched in BRAF, TP53, FGFR3, PTPN11, ERRFI1, and MLH1 GAs (FDR<0.05) and all but TP53 had median VAF/TF <25%. The majority of clonal BRAF GAs were V600E (14/19, 74%) while the majority of subclonal BRAF GAs were Class 2 (36/61, 59%). Other subclonal GAs in receptor tyrosine kinases and RAS pathway components detected in these LBx included: NF1 (26), ROS1 (9), ALK (7), MET (7), RAF1 (7), FGFR2 (6), HRAS (6), MAP2K2 (6), and RET (4). 18/362 (4.4%) of the LBx had ≥2 of these resistance GAs (up to 5 per LBx). In CGDB, 14/25 (56%) LBx collected at progression had potential resistance GAs (median 2, maximum 10). LBx collected at progression (N = 25) had higher prevalence of GA in EGFR (28% vs. 2.3%, p=0.0003), KRAS (28% vs. 0%, p<0.0001), NRAS (20% vs. 0%, p=0.0002) and MAP2K1 (20% vs. 2.3%, p=0.008) than LBx collected before therapy start (N = 87). Conclusions: This study reports on the largest set of LBx from pts likely exposed to anti-EGFR published to date. LBx can provide a more comprehensive and quantitative picture of acquired resistance to anti-EGFR in CRC than tissue biopsy, including some potential novel mechanisms identified in this study. Further investigation into whether detection of resistance can inform clinical decisions about timing of anti-EGFR mAb use and rechallenge is warranted.

Combined Proteomic and Genetic Interaction Mapping Reveals New RAS Effector Pathways and Susceptibilities.

Activating mutations in RAS GTPases drive many cancers, but limited understanding of less-studied RAS interactors, and of the specific roles of different RAS interactor paralogs, continues to limit target discovery. We developed a multistage discovery and screening process to systematically identify genes conferring RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry, we generated a protein-protein interaction map of RAS interactors and pathway components containing hundreds of interactions. From this network, we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for KRAS-dependent genetic interactions (GI). This approach identified new RAS effectors, including the adhesion controller RADIL and the endocytosis regulator RIN1, and >250 synthetic lethal GIs, including a potent KRAS-dependent interaction between RAP1GDS1 and RHOA. Many GIs link specific paralogs within and between gene families. These findings illustrate the power of multiomic approaches to uncover synthetic lethal combinations specific for hitherto untreatable cancer genotypes. SIGNIFICANCE: We establish a deep network of protein-protein and genetic interactions in the RAS pathway. Many interactions validated here demonstrate important specificities and redundancies among paralogous RAS regulators and effectors. By comparing synthetic lethal interactions across KRAS-dependent and KRAS-independent cell lines, we identify several new combination therapy targets for RAS-driven cancers.This article is highlighted in the In This Issue feature, p. 1775.

RAS pathway influences the number of melanocytic nevi in cardiofaciocutaneous and Costello syndromes

To the Editor: The number of melanocytic nevi is one of the strongest risk factors for melanoma,1 yet the reasons for the interpersonal variability in this number are largely unknown. Both nevi and melanoma show somatic mutations in the RAS pathway components, most commonly in BRAF.2 Germline mutations in these same genes cause a group of developmental syndromes termed RASopathies,3 some of which characteristically display melanocytic nevi.4 However, the effects of germline RAS pathway mutations on the number of nevi are poorly understood.

ERK1/2 inhibitors: New weapons to inhibit the RAS-regulated RAF-MEK1/2-ERK1/2 pathway.

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is de-regulated in a variety of cancers due to mutations in receptor tyrosine kinases (RTKs), negative regulators of RAS (such as NF1) and core pathway components themselves (RAS, BRAF, CRAF, MEK1 or MEK2). This has driven the development of a variety of pharmaceutical agents to inhibit RAF-MEK1/2-ERK1/2 signalling in cancer and both RAF and MEK inhibitors are now approved and used in the clinic. There is now much interest in targeting at the level of ERK1/2 for a variety of reasons. First, since the pathway is linear from RAF-to-MEK-to-ERK then ERK1/2 are validated as targets per se. Second, innate resistance to RAF or MEK inhibitors involves relief of negative feedback and pathway re-activation with all signalling going through ERK1/2, validating the use of ERK inhibitors with RAF or MEK inhibitors as an up-front combination. Third, long-term acquired resistance to RAF or MEK inhibitors involves a variety of mechanisms (KRAS or BRAF amplification, MEK mutation, etc.) which re-instate ERK activity, validating the use of ERK inhibitors to forestall acquired resistance to RAF or MEK inhibitors. The first potent highly selective ERK1/2 inhibitors have now been developed and are entering clinical trials. They have one of three discrete mechanisms of action - catalytic, "dual mechanism" or covalent - which could have profound consequences for how cells respond and adapt. In this review we describe the validation of ERK1/2 as anti-cancer drug targets, consider the mechanism of action of new ERK1/2 inhibitors and how this may impact on their efficacy, anticipate factors that will determine how tumour cells respond and adapt to ERK1/2 inhibitors and consider ERK1/2 inhibitor drug combinations.

Divergent effects of intrinsically active MEK variants on developmental Ras signaling.

Germline mutations in Ras pathway components are associated with a large class of human developmental abnormalities, known as RASopathies, that are characterized by a range of structural and functional phenotypes, including cardiac defects and neurocognitive delays. Although it is generally believed that RASopathies are caused by altered levels of pathway activation, the signaling changes in developing tissues remain largely unknown. We used assays with spatiotemporal resolution in Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish) to quantify signaling changes caused by mutations in MAP2K1 (encoding MEK), a core component of the Ras pathway that is mutated in both RASopathies and cancers in humans. Surprisingly, we discovered that intrinsically active MEK variants can both increase and reduce the levels of pathway activation in vivo. The sign of the effect depends on cellular context, implying that some of the emerging phenotypes in RASopathies may be caused by increased, as well as attenuated, levels of Ras signaling.

FGFR1 and NTRK3 actionable alterations in "Wild-Type" gastrointestinal stromal tumors.

BackgroundAbout 10–15% of adult, and most pediatric, gastrointestinal stromal tumors (GIST) lack mutations in KIT, PDGFRA, SDHx, or RAS pathway components (KRAS, BRAF, NF1). The identification of additional mutated genes in this rare subset of tumors can have important clinical benefit to identify altered biological pathways and select targeted therapies.MethodsWe performed comprehensive genomic profiling (CGP) for coding regions in more than 300 cancer-related genes of 186 GISTs to assess for their somatic alterations.ResultsWe identified 24 GIST lacking alterations in the canonical KIT/PDGFRA/RAS pathways, including 12 without SDHx alterations. These 24 patients were mostly adults (96%). The tumors had a 46% rate of nodal metastases. These 24 GIST were more commonly mutated at 7 genes: ARID1B, ATR, FGFR1, LTK, SUFU, PARK2 and ZNF217. Two tumors harbored FGFR1 gene fusions (FGFR1–HOOK3, FGFR1–TACC1) and one harbored an ETV6–NTRK3 fusion that responded to TRK inhibition. In an independent sample set, we identified 5 GIST cases lacking alterations in the KIT/PDGFRA/SDHx/RAS pathways, including two additional cases with FGFR1–TACC1 and ETV6–NTRK3 fusions.ConclusionsUsing patient demographics, tumor characteristics, and CGP, we show that GIST lacking alterations in canonical genes occur in younger patients, frequently metastasize to lymph nodes, and most contain deleterious genomic alterations, including gene fusions involving FGFR1 and NTRK3. If confirmed in larger series, routine testing for these translocations may be indicated for this subset of GIST. Moreover, these findings can be used to guide personalized treatments for patients with GIST. Trial registration NCT 02576431. Registered October 12, 2015Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1075-6) contains supplementary material, which is available to authorized users.

Inhibition of Ras/Raf/MEK/ERK Pathway Signaling by a Stress-Induced Phospho-Regulatory Circuit

Ras pathway signaling plays a critical role in cell growth control and is often upregulated in human cancer. The Raf kinases selectively interact with GTP-bound Ras and are important effectors of Ras signaling, functioning as the initiating kinases in theERK cascade. Here, we identify a route for the phospho-inhibition of Ras/Raf/MEK/ERK pathway signaling that is mediated by the stress-activated JNK cascade. We find that key Ras pathway components, the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in response to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as rigosertib and paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and may functionas a signaling checkpoint to suppress the Ras pathway during conditions of cellular stress.

Abstract 2002: Assembling test points for quantitative proteomics to elucidate RAS signaling

Abstract RAS mutation is a critical player in cancer development, particularly in lung, colon, and pancreas. However, mutant RAS phenotypes have not produced viable therapeutic options. Therefore, additional reagents and techniques are required to study the functionality of RAS pathway components in model systems and patient specimens. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has been applied to study cancer signaling by quantification of protein expression, mutation status, and post-translational modification in a variety of sample types. LC-MRM with immunoaffinity enrichment (ImmunoMRM) enables multiplexed analysis of target peptides with little background interference. Here, we describe the selection of 100 targets for assay development and provide insight into the utility of the immunoMRM platform as part of the NCI's RAS Initiative. Provided with biologically relevant proteins, several approaches were used for peptide selection. Complementary contributions were obtained from analysis of individual proteins, targeted LC-MRM screening, phosphoproteomics, and global discovery proteomics datasets (both in-house and publically available). For phosphorylation and mutation sites, the ability to quantify those molecular endpoints is directly limited by the primary amino acid sequence produced by trypsin digestion. Peptides selected for assay development quantify biological endpoints indicating the expression levels of wild-type and mutant proteins and signaling of RAS pathway components. Literature reviews show the prospective value of each assay in their specific biological context. Assays are developed for RAS signaling proteins in three categories: upstream proteins, RAS isoforms, and downstream effectors. As an example of upstream proteins, EGFR expression and tyrosine phosphorylation will be quantified, which enables comparison of ImmunoMRM to prior techniques. Extensive characterization is planned for RAS isoforms, particularly to monitor the expression of wild-type and mutant proteins. Measurements focused on downstream proteins include Erk and pErk to examine the activation of that MAPK pathway as well as disparate processes, like epithelial-to-mesenchymal transition (N-Cad/E-Cad). A comprehensive circuit diagram is prepared to illustrate the assembled test points for discussion with the cancer biology community. Development of these immunoMRM assays enables evaluation of multiple RAS signaling events and downstream outcomes in one sample. The emerging paradigm of peptide enrichment for protein biomarker measurement provides a novel platform for the study of critical processes in cancer. Although currently early in development, the implementation of this platform can ultimately demonstrate the utility of ImmunoMRM reagent development and be a first step for understanding RAS biology in human tumors and developing novel treatment options for patients. Citation Format: Eric Kuhn, Jeffrey R. Whiteaker, Melissa Martinez, Matthew Holderfield, Jacob Kennedy, Jacob Jaffe, Ping Yan, ChenWei Lin, Gordon Whiteley, Steven A. Carr, Amanda G. Paulovich, John M. Koomen. Assembling test points for quantitative proteomics to elucidate RAS signaling. [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 2002. doi:10.1158/1538-7445.AM2015-2002

MEK Inhibition Is a Promising Therapeutic Strategy for MLL-Rearranged Infant ALL Patients Carrying RAS Mutations

Background: Acute Lymphoblastic Leukemia (ALL) in infants is characterized by a high incidence (~80%) of chromosomal rearrangements of the Mixed Lineage Leukemia (MLL) gene, fusing the N-terminal portion of MLL to the C-terminal region of one of its translocation partner genes. MLL-rearranged infant ALL patients are challenged by a very poor prognosis (i.e. 30-40% 5-year EFS), hence the need for better risk stratification and improved therapeutic solutions is evident. We recently screened a relatively large cohort (n=109) infant ALL patients (all enrolled in INTERFANT treatment protocols) for the presence of KRAS and NRAS mutations and found that the incidence of such mutations ranges between 14-24%, depending on the type of MLL translocation. Moreover, these mutations were found to represent independent predictors of exceedingly poor prognosis; patients carrying RAS mutations essentially stand no chance in surviving their malignancy, as all RAS mutation-positive MLL-rearranged infant ALL patients deceased within 3 years from diagnosis.Aims: Here we aimed to identify a therapeutic strategy to improve the prognosis of MLL-rearranged infant ALL patients carrying RAS mutations.Methods: For this, 8 small molecule inhibitors against different RAS-pathway components were selected and initially tested for anti-leukemic activity against the MLL-rearranged ALL cell lines SEM and RS4;11 (RASwt) and KOPN8 (RASmut) using MTS cell viability assays. Next, primary MLL-rearranged infant ALL samples (n=20) all carrying MLL translocation t(4;11) (giving rise to the MLL-AF4 fusion protein) either with (n=6) or without (n=14) RAS mutations were exposed to these inhibitors in MTT cytotoxicity assays. In addition, we assessed the RAS activity in RAS mutated and wild-type MLL-rearranged infant ALL cells, and performed immunoblotting analysis of downstream MEK and ERK both in the absence and presence of the MEK inhibitors.Results: We found that the MEK inhibitors MEK162, Selumetinib and Trametinib effectively reduced the viability of KOPN8 cells (RASmut), whereas SEM and RS4;11 cells (RASwt) largely remained unaffected. In line with this, MLL-AF4+ infant ALL patient samples carrying RAS mutations were significantly more sensitive to these MEK inhibitors when compared with patients carrying wild-type RAS genes: LC50 values for MEK162 were 0.04 vs. 26.9 µM (p<0.01), for Selumetinib 0.04 and 23.7 µM (p<0.01), and for Trametinib 0.01 vs. 26.5 µM (p<0.01), respectively. Furthermore, the presence of RAS mutations in primary MLL-rearranged infant ALL samples was associated with significantly increased RAS activity, as determined by immunoprecipitation of GTP-bound RAS. Remarkably, however, enhanced RAS activation did not manifest itself in the form of increased phosphorylation of the downstream ERK protein, while a slight increase of MEK phosphorylation was observed. Yet, MEK inhibitor exposure in both KOPN8 and SEM cells resulted in nearly complete abrogation of ERK phosphorylation, without affecting total ERK protein levels, suggesting that the loss of ERK activation plays an important role in the observed anti-leukemic effects. Furthermore, MEK162 and Selumetinib seemed to induce accumulation of phosphorylated MEK, while MEK phosphorylation was hardly affected by Trametinib. Interestingly, a subgroup of patient samples (n=5) with wild-type RAS also showed sensitivity towards MEK inhibition, similar to the primary cells with RAS mutations. However, this observation could not be explained by increased RAS activation, nor by the phosphorylation levels of either MEK or ERK.Conclusions: Our data show that MEK inhibition represents a promising therapeutic approach for MLL-rearranged infant ALL patients carrying additional RAS mutations. Furthermore, the mechanism of action provoked by these MEK inhibitors seems to involve abrogation of ERK phosphorylation, but the initial level of ERK phosphorylation did not correlate with MEK inhibitor sensitivity, and has no predictive value. Currently we are in the process of testing the efficacy of the above mentioned MEK inhibitors in vivo using a xenograft mouse model, while further elucidation of the molecular mechanisms underlying the anti-leukemic effects of these inhibitors in MLL-rearranged ALL cells is in progress. DisclosuresNo relevant conflicts of interest to declare.

Defining key signaling nodes and therapeutic biomarkers in NF1-mutant cancers.

NF1 encodes a RAS GTPase-activating protein. Accordingly, aberrant RAS activation underlies the pathogenesis of NF1-mutant cancers. Nevertheless, it is unclear which RAS pathway components represent optimal therapeutic targets. Here, we identify mTORC1 as the key PI3K effector in NF1-mutant nervous system malignancies and conversely show that mTORC2 and AKT are dispensable. However, we find that tumor regression requires sustained inhibition of both mTORC1 and MEK. Transcriptional profiling studies were therefore used to establish a signature of effective mTORC1-MEK inhibition in vivo. We unexpectedly found that the glucose transporter GLUT1 was potently suppressed, but only when both pathways were inhibited. Moreover, unlike VHL- and LKB1-mutant cancers, reduction of (18)F-FDG uptake required the suppression of both mTORC1 and MEK. Together, these studies identify optimal and suboptimal therapeutic targets in NF1-mutant malignancies and define a noninvasive means of measuring combined mTORC1-MEK inhibition in vivo, which can be readily incorporated into clinical trials. This work demonstrates that mTORC1 and MEK are key therapeutic targets in NF1-mutant cancers and establishes a noninvasive biomarker of effective, combined target inhibition that can be evaluated in clinical trials.

Integrated Genetic Profiling Of JAK2 Wildtype Chronic-Phase Myeloproliferative Neoplasms

BackgroundThe Myeloproliferative Neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF) are clonal hematopoietic disorders. JAK2V617F mutations are observed in approximately 90-95% of PV cases, but only 40-50% of ET and PMF cases. Although JAK2 exon 12 and LNK mutations are observed in the majority of JAK2V617F-negative PV patients, candidate gene and exome sequencing studies to date have not identified activating oncogenes in the majority of JAK2V617F-negative ET/PMF patients. Thus, further genetic investigations are needed to define the mutational architecture of these JAK2 wildtype MPNs in order to gain insight into the biology of these diseases, the clinical implications of genetic events that do occur, and the elucidation of potential therapeutic targets. AimsTo characterize the spectrum of genetic alterations in JAK2 wildtype chronic-phase myeloproliferative neoplasms. MethodsWe identified 32 patients with a confirmed diagnosis of an MPN (per 2008 WHO criteria), including MF, PV and ET, who were negative for JAK2V617F using a CLIA-certified allele specific assay for the JAK2 disease allele. Genomic DNA and total RNA was isolated from formalin fixed paraffin embedded (FFPE) tissue, blood and bone marrow aspirates. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. ResultsHigh coverage sequencing allowed us to identify JAK2V617F mutations in two patients (allele burden 3-5%) that were below the limit of detection of the CLIA assay. The most common mutations observed in JAK2V617F-negative MPN were in ASXL1 (22% of patients) and in TET2 (9%). Taken together, mutations in known epigenetic modifiers (ASXL1, TET2, DNMT3A, EZH2, MLL) were observed in 43% of samples, including a MLL-PTD mutation in one patient with PMF. We identified mutations in spliceosome components (SRSF2, U2AF1), in a subset of patients, consistent with previous reports, and in each case mutations in spliceosome components were mutually exclusive.We identified mutations in the JAK-STAT pathway (MPL, TYK2) and the RAS pathway components (KRAS, NF1) in 9% of this patient cohort, suggesting that there are alternate disease alleles that activate signaling in JAK2V617F-negative MPN. RNA-sequencing identified a ETV6-ABL1 fusion in one patient, and we identified amplification of PIK3CA in one patient in our cohort; these data suggest fusion genes and amplifications activate signaling in a subset of patients with JAK2V617F-negative MPN. We also identified novel mutations in MPN patients which have not been reported to date, including mutations in DNA repair genes (ATM and BRCA) in 25% of cases and mutations in the Notch signaling pathway (NOTCH1-4) in 31% of cases. The functional implications of these novel mutations remain to be elucidated.In univariate analysis, ASXL1 mutations were found to associate with impaired overall survival (Figure 1, p=0.049). These findings are consistent with data demonstrating an impaired survival in patients with MDS and PMF, and suggest that ASXL1 mutations represent an important biomarker for adverse outcome in JAK2V617F-negative MPN. [Display omitted] ConclusionsThese data demonstrate that the mutational spectra of JAK2V617F-negative MPN includes genes implicated in epigenetic regulation, novel mutations which activate gene signaling, and fusion genes/copy number alterations which provide a novel mechanism of oncogenic activation not previously reported in MPN. ASXL1 mutations occur frequently in JAK2 wildtype Philadelphia-Chromosome negative MPNs, and are associated with impaired overall survival. Collectively, these findings support the importance of ASXL1 mutations in predicting outcome in JAK2V617F-negative MPN, demonstrate that mutations in signaling effectors and in epigenetic regulators are common in MPN, and illustrate the genetic heterogeneity of JAK2V617F-negative MPN. Disclosures:Rampal:Foundation Medicine: Consultancy. Levine:Foundation Medicine, Inc: Consultancy.

High Incidence of RAS Signalling Pathway Mutations in MLL–rearranged Acute Myeloid Leukemia

AbstractAbstract ▪539▪This icon denotes a clinically relevant abstract Background:Chromosomal translocations of the MLL gene on chromosome 11q23 are associated with a unique subset of acute lymphoblastic or acute myeloid leukemias (AML). In adults, MLL rearrangements are detected in 3% of de novo AML and in 10% of therapy-related AML (t-AML) cases and are associated with poor prognosis. In addition to disease defining mutations recent high-throughput sequencing studies had shown that almost all myeloid malignancies accumulate a large number of cooperating gene mutations. Aim:Determination of somatic mutations occurring in cases harboring MLL rearrangements and investigation of the prognostic impact of molecular and additional chromosomal aberrations. Patients and Methods:We investigated a cohort of 110 adult AML (80 de novo, 30 t-AML) cases harboring an 11q23 translocation. The cohort was composed of 66 females and 44 males; median age: 55.8 years. MLL translocation partners were as follows: MLLT3 (n=46), MLLT4 (n=15), ELL (n=15); MLLT10 (n=8), others (n=26). Chromosome banding analysis data was available in all cases and survival data in 78 cases (median overall survival (OS) was 10.1 months). Patients were screened for mutations in ASXL1 (n=98), CBL (n=62), CEBPA (n=61), FLT3-ITD (n=103), FLT3-TKD (n=95), IDH1 (n=96), IDH2 (n=84), KRAS (n=107), NPM1 (n=101), NRAS (n=106), PTPN11 (n=99), RUNX1 (n=110), and TP53 (n=110) using amplicon deep-sequencing (454 Roche Life Sciences, Branford, CT), direct Sanger sequencing or melting curve analysis. Results:Overall, mutations were detected in 59/110 (53.6%) cases. We discovered that 42/110 (38.2%) MLL-translocated AML cases harbored mutations within the RAS signalling pathway (KRAS mut: 23/107; 21.5%; NRAS mut: 22/106; 20.8%; PTPN11 mut: 3/99, 3.0%) or alterations in the RAS regulating FLT3 gene (FLT3-ITD: 4/103, 3.9%, and FLT3-TKD: 10/95, 10.5%). Additional mutations were detected in the tumor suppressor gene TP53 (8/110; 7.3%), ASXL1 (6/98; 6.1%), RUNX1 (4/110; 3.6%), and IDH1 (1/96). No mutation was detected in IDH2, CBL, CEBPA, and NPM1. Most cases showed only one mutation (n=39, 66.1%), whereas 17 cases (28.8%) showed two and 3 cases (5.1%) three mutations in different genes. No difference of mutation distribution was seen between de novo and t-AML. In this cohort, no associations amongst gene mutations were observed, however, FLT3-ITD was associated with MLL-ELL (3/14 vs 1/89, P=0.008) and PTPN11 mutations with MLLT10-MLL (2/8 vs 1/91, P=0.017) alterations. In addition, KRAS mut and NRAS mut correlated with high WBC count (KRAS mut: 103.0±79 vs 59.2±67 x109/L, P=0.016; NRAS mut: 94.7±57 vs 60.4±72 x109/L, P=0.080). Further, we were interested in the prognostic impact of single gene mutations. NRAS mut and TP53 mut showed both a non-significant inferior impact on OS, i.e. OS after 2 years: 19.1% vs 46.4%, P=0.62; 0% vs 41.3%, P=0.114. Further, TP53 mutations were correlated with shorter event-free survival (EFS) (EFS after 2 years: 0% vs 20.0%, P=0.029). No associations with prognosis were observed for the remaining genes and translocation partners.In contrast, age was associated with OS and EFS (<60 years, n=59 vs ≥60 years, n=51: OS after 2 years: 51.4% vs 26.3%, P=0.003, EFS after 2 years: 28.0% vs 7.7%, P=0.004). Within the cohort of cases ≥60 years, TP53 mutations (n=5) were associated with worse EFS and OS in comparison to TP53 wild-type cases (n=45) (EFS after 2 years: 8.4% vs 0%, P= 0.006; OS after 2 years: 28.5% vs 0%, P=0.045). Of note, no correlations between mutation frequency and age were observed. We next focused on whether the number of mutations showed any impact on survival. This analysis revealed that cases with more than one mutation (n=20) showed shorter EFS (EFS after 2 years: 10.0% vs 27.3%, P=0.020).Finally, we concentrated on AML with t(9;11)(p22;q23)/MLLT3-MLL, recognized as a distinct WHO-entity. We neither detected an association of MLLT3-MLL (n=46) with OS (P=0.445) or EFS (P=0.644) in comparison to the remaining translocation partners nor a distinct gene mutation profile. However, NRAS mutations correlated with shorter OS and EFS in cases with MLLT3-MLL (after 2 years OS: 17.8% vs 48.3%, P=0.045; after 2 years EFS: 17.8% vs 35.2%, P=0.056). Conclusions:In patients with MLL-translocations a high number of secondary alterations (53.6%), predominantly in RAS pathway components (38.2%), were detected. This may have implication on novel therapeutic options in this unfavorable AML subset. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

H-ras resides on clathrin-independent ARF6 vesicles that harbor little RAF-1, but not on clathrin-dependent endosomes

Internalization of H-Ras from the cell surface onto endomembranes through vesicular endocytic pathways may play a significant role(s) in regulating the outcome of Ras signaling. However, the identity of Ras-associated subcellular vesicles and the means by which Ras localize to these internal sites remain elusive. In this study, we show that H-Ras is absent from endosomes initially derived from a clathrin-dependent endocytic pathway. Instead, both oncogenic H-Ras-61L and wild type H-Ras (basal or EGF-stimulated) bind Arf6-associated clathrin-independent endosomes and vesicles of the endosomal-recycling center (ERC). K-Ras4B-12V can also be internalized via Arf6 endosomes, and the C-terminal tails of both H-Ras and K-Ras4B are sufficient to mediate localization of GFP chimeras to Arf6-associated vesicles. Interestingly, little Raf-1 was found on these Arf6-associated endosomes even when active H-Ras was present. Instead, endogenous Raf-1 distributed primarily on EEA1-containing vesicles, suggesting that this H-Ras effector, although accessible for H-Ras interaction on the plasma membrane, appears to separate from its regulator during early stages of endocytosis. The discrete and dynamic distribution of Ras pathway components with spatio-temporal complexity may contribute to the specificity of Ras:effector interaction.

PDGF/VEGF signaling controls cell size in Drosophila

Pvr and its ligands, Pvf 2 and 3, which are upstream of Ras and PI3kinase, are identified from a genome-wide screen in Drosophila cells, as regulators of cell growth.

The influence of Ras pathway signaling on tumor radiosensitivity.

Radiotherapy is used to treat approximately 60% of solid tumors in the US. The relative radiosensitivity of these tumors can have a significant impact upon local control. One factor that has been shown to contribute to the increased survival of tumor cells is the activation of signaling pathways in which oncogene products play a central role. The Ras oncoprotein family, comprised of H-, K-, and N-Ras are frequently activated by mutation in certain tumors such as pancreatic and non-small cell lung cancers and are activated by receptor tyrosine kinase activity in an even wider range of tumor types. The role of ras mutation and more recently Ras signaling has been an area of intense study in both radiobiology and tumor biology in general. In this review, we focus on findings from our lab and others that led to the current hypotheses relating to the role of Ras signaling in tumor radiation survival and the strategies used to block Ras activation. We will also point out new means of studying the contribution of Ras and Ras pathway components that could contribute to defining new targets for inhibition in the context of radiation therapy.

Early occurrence of RASSF1A hypermethylation and its mutual exclusion with BRAF mutation in thyroid tumorigenesis.

Follicular epithelial cell-derived thyroid tumors are common neoplasms comprised mainly of benign thyroid adenomas, follicular thyroid cancers, and papillary thyroid cancers (PTCs). Hypermethylation of the tumor suppressor gene RASSF1A and activating mutation of BRAF gene have been reported recently in thyroid cancers. To additionally investigate the roles of these two epigenetic/genetic alterations in thyroid tumor progression, we examined their occurrences and relationship in both benign and malignant thyroid neoplasms. With real-time quantitative methylation-specific PCR, we found that 4 of 9 (44%) benign adenomas, 9 of 12 (75%) follicular thyroid cancers tumors, and 6 of 30 (20%) of PTC tumors harbored promoter methylation in > or = 25% of RASSF1A alleles. Additional quantitative analysis revealed RASSF1A methylation only in BRAF mutation-negative PTCs. A similar inverse correlation of RASSF1A methylation with BRAF mutation was seen in thyroid tumor cell lines. Our results, therefore, suggest that epigenetic inactivation of RASSF1A through aberrant methylation is an early step in thyroid tumorigenesis. Like the previously reported mutually exclusive relationship between BRAF mutation and other Ras pathway components such as RET/PTC rearrangement, a mutually exclusive relationship also exists between BRAF mutation and RASSF1A methylation in thyroid tumorigenesis.

Ras Pathway Specificity Is Determined by the Integration of Multiple Signal-Activated and Tissue-Restricted Transcription Factors

Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. We demonstrate that Wingless (Wg) and Decapentaplegic (Dpp) confer competence for receptor tyrosine kinase–mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated (dTCF, Mad, and Pointed—functioning in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted (Twist and Tinman) transcription factors on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development.

Characterization of rho GTPase family homologues in Drosophila melanogaster: overexpressing Rho1 in retinal cells causes a late developmental defect.

The rho family of GTPases has been implicated in regulating changes in cell morphology in response to extracellular signals. We have cloned three widely expressed members of this family from Drosophila melanogaster; a rho homologue (Rho1) and two rac homologues (Rac1 and Rac2). Flies harbouring a Rho1 transgene that is specifically expressed in the eye exhibit a dramatic dose dependent disruption of normal eye development. Flies bearing at least two copies of the transgene display a severe rough eye phenotype characterized by missing secondary and tertiary pigment cells, a substantial reduction in the number of photoreceptor cells and a grossly abnormal morphology of the rhabdomeres. Cell fate determination in the imaginal disc occurs normally and abnormalities become manifest late in pupariation, coincident with the phase when the cells undergo major morphological changes. This phenotype is modified by mutations at several other loci that have been implicated in signal transduction, but not by mutations in ras pathway components.