Oncogenic KRAS provides a uniquely powerful and variable oncogenic contribution among RAS family members in the colonic epithelium

Activating mutations of the RAS family of small GTPases are among the most common genetic events in human tumorigenesis. Constitutive activation of the three canonical family members, KRAS, NRAS, and HRAS segregate strongly by tissue type. Of these, KRAS mutations predominate in human tumors, including those arising from the colon and lung. We sought to compare the oncogenic contributions of different RAS isoforms in a comparable genetic setting and to explore downstream molecular changes that may explain the apparent differential oncogenic effects of the various RAS family members. We utilized colorectal cancer cell lines characterized by oncogenic KRAS in parallel with isogenically derived lines in which the mutant allele has been disrupted. We additionally attempted to reconstitute the isogenic derivatives with oncogenic forms of other RAS family members and analyze them in parallel. Pairwise analysis of HCT 116 and DLD-1 cell lines as well as their isogenic derivatives reveals distinct K-RAS(G13D) signatures despite the genetic similarities of these cell lines. In DLD-1, for example, oncogenic K-RAS enhances the motility of these cells by downregulation of Rap1 activity, yet is not associated with increased ERK1/2 phosphorylation. In HCT 116, however, ERK1/2 phosphorylation is elevated relative to the isogenic derivative, but Rap1 activity is unchanged. K-RAS is uniquely oncogenic in the colonic epithelium, though the molecular aspects of its oncogenic contribution are not necessarily conserved across cell lines. We therefore conclude that the oncogenic contribution of K-RAS is a function of its multifaceted functionality and is highly context-dependent.     

Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines

Constitutive activation of the phosphatidylinositol-3-OH kinase (PI3K) and RAS signaling pathways are important events in tumor formation. This is illustrated by the frequent genetic alteration of several key players from these pathways in a wide variety of human cancers. Here, we report a detailed sequence analysis of the PTEN, PIK3CA, KRAS, HRAS, NRAS, and BRAF genes in a collection of 40 human breast cancer cell lines. We identified a surprisingly large proportion of cell lines with mutations in the PI3K or RAS pathways (54% and 25%, respectively), with mutants for each of the six genes. The PIK3CA, KRAS, and BRAF mutation spectra of the breast cancer cell lines were similar to those of colorectal cancers. Unlike in colorectal cancers, however, mutational activation of the PI3K pathway was mutually exclusive with mutational activation of the RAS pathway in all but 1 of 30 mutant breast cancer cell lines (P = 0.001). These results suggest that there is a fine distinction between the signaling activators and downstream effectors of the oncogenic PI3K and RAS pathways in breast epithelium and those in other tissues.     

Absolute Quantification of Endogenous Ras Isoform Abundance

Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic protein standards together with selected reaction monitoring for diagnostic peptides is sensitive, robust and suitable for application to sub-milligram quantities of lysates. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ≥260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS≥HRAS. A subset of oncogenic KRAS mutants exhibit increased total cellular Ras abundance and altered the ratio of mutant versus wild type KRAS protein. These data and methodology are significant because Ras protein copy number is required to parameterise models of signalling networks and informs interpretation of isoform-specific Ras functional data.     

Oncogenic RAS induces accelerated transition through G2/M and promotes defects in the G2 DNA damage and mitotic spindle checkpoints

Activating mutations of RAS are prevalent in thyroid follicular neoplasms, which commonly have chromosomal losses and gains. In thyroid cells, acute expression of HRAS(V12) increases the frequency of chromosomal abnormalities within one or two cell cycles, suggesting that RAS oncoproteins may interfere with cell cycle checkpoints required for maintenance of a stable genome. To explore this, PCCL3 thyroid cells with conditional expression of HRAS(V12) or HRAS(V12) effector mutants were presynchronized at the G(1)/S boundary, followed by activation of expression of RAS mutants and release from the cell cycle block. Expression of HRAS(V12) accelerated the G(2)/M phase by approximately 4 h and promoted bypass of the G(2) DNA damage and mitotic spindle checkpoints. Accelerated passage through G(2)/M and bypass of the G(2) DNA damage checkpoint, but not bypass of the mitotic spindle checkpoint, required activation of mitogen-activated protein kinase (MAPK). However, selective activation of the MAPK pathway was not sufficient to disrupt the G(2) DNA damage checkpoint, because cells arrested appropriately in G(2) despite conditional expression of HRAS(V12,S35) or BRAF(V600E). By contrast to the MAPK requirement for radiation-induced G(2) arrest, RAS-induced bypass of the mitotic spindle checkpoint was not prevented by pretreatment with MEK inhibitors. These data support a direct role for the MAPK pathway in control of G(2) progression and regulation of the G(2) DNA damage checkpoint. We propose that oncogenic RAS activation may predispose cells to genomic instability through both MAPK-dependent and independent pathways that affect critical checkpoints in G(2)/M.     

Reduced HRASG12V-Driven Tumorigenesis of Cell Lines Expressing KRASC118S

In many different human cancers, one of the HRAS, NRAS, or KRAS genes in the RAS family of small GTPases acquires an oncogenic mutation that renders the encoded protein constitutively GTP-bound and thereby active, which is well established to promote tumorigenesis. In addition to oncogenic mutations, accumulating evidence suggests that the wild-type isoforms may also be activated and contribute to oncogenic RAS-driven tumorigenesis. In this regard, redox-dependent reactions with cysteine 118 (C118) have been found to promote activation of wild-type HRAS and NRAS. We sought to determine if this residue is also important for the activation of wild-type KRAS and promotion of tumorigenesis. Thus, we mutated C118 to serine (C118S) in wild-type KRAS to block redox-dependent reactions at this site. We now report that this mutation reduced the level of GTP-bound KRAS and impaired RAS signaling stimulated by the growth factor EGF. With regards to tumorigenesis, we also report that oncogenic HRAS-transformed human cells in which endogenous KRAS was knocked down and replaced with KRAS^C118S exhibited reduced xenograft tumor growth, as did oncogenic HRAS-transformed Kras^C118S/C118S murine cells in which the C118S mutation was knocked into the endogenous Kras gene. Taken together, these data suggest a role for redox-dependent activation of wild-type KRAS through C118 in oncogenic HRAS-driven tumorigenesis.     

Differential Oncogenic Ras Signaling and Senescence in Tumor Cells

Several studies have shown that forced expression of oncogenic H-ras can induce a senescence-like permanent growth arrest in normal cells. Here we report that expression of oncogenic H-ras in human osteosarcoma U2OS cells also resulted in a senescence-like flat and enlarged cell morphology and permanent growth arrest. In contrast to normal human fibroblasts, U2OS cells were arrested independently of the p16 and ARF tumor suppressors. Treatment with a MEK inhibitor or a p38MAPK inhibitor interrupted oncogenic H-ras-induced growth arrest in U2OS cells, suggesting that activation of MAPK pathways is important. To further determine whether this process is unique to oncogenic H-ras signaling, we examined the effect of oncogenic K-ras on normal cells and human osteosarcoma cells. Similar to oncogenic H-ras, oncogenic K-ras also induced senescence in normal fibroblasts, while transforming immortalized mouse fibroblasts. However, in contrast to oncogenic H-ras, oncogenic K-ras failed to induce a permanent growth arrest in osteosarcoma U2OS cells. Additionally, cells transduced with oncogenic K-ras exhibited distinguishable cellular changes compared to those transduced with oncogenic H-ras. In summary, we report for the first time that oncogenic H-ras signaling can trigger a senescence-like growth arrest in tumor cells, independent of the p16 and ARF tumor suppressors. This result suggests that tumor cells may harbor a senescence-like program that can be activated by ras signaling. Moreover, our study uncovered a cell type-dependent differential response to oncogenic K-ras, as compared to oncogenic H-ras.     

RAS and RHO family GTPase mutations in cancer: twin sons of different mothers?

Abstract

The RAS and RHO family comprise two major branches of the RAS superfamily of small GTPases. These proteins function as regulated molecular switches and control cytoplasmic signaling networks that regulate a diversity of cellular processes, including cell proliferation and cell migration. In the early 1980s, mutationally activated RAS genes encoding KRAS, HRAS and NRAS were discovered in human cancer and now comprise the most frequently mutated oncogene family in cancer. Only recently, exome sequencing studies identified cancer-associated alterations in two RHO family GTPases, RAC1 and RHOA. RAS and RHO proteins share significant identity in their amino acid sequences, protein structure and biochemistry. Cancer-associated RAS mutant proteins harbor missense mutations that are found primarily at one of three mutational hotspots (G12, G13 and Q61) and have been identified as gain-of-function oncogenic alterations. Although these residues are conserved in RHO family proteins, the gain-of-function mutations found in RAC1 are found primarily at a distinct hotspot. Unexpectedly, the cancer-associated mutations found with RHOA are located at different hotspots than those found with RAS. Furthermore, since the RHOA mutations suggested a loss-of-function phenotype, it has been unclear whether RHOA functions as an oncogene or tumor suppressor in cancer development. Finally, whereas RAS mutations are found in a broad spectrum of cancer types, RHOA and RAC1 mutations occur in a highly restricted range of cancer types. In this review, we focus on RHOA missense mutations found in cancer and their role in driving tumorigenesis, with comparisons to cancer-associated mutations in RAC1 and RAS GTPases.     

Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation

ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS^G12C/G13D or BRAF^V600E. Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.     

Anti-oncogenic role of the endoplasmic reticulum differentially activated by mutations in the MAPK pathway

Dysfunction of the endoplasmic reticulum (ER) has been reported in a variety of human pathologies, including cancer. However, the contribution of the ER to the early stages of normal cell transformation is largely unknown. Using primary human melanocytes and biopsies of human naevi (moles), we show that the extent of ER stress induced by cellular oncogenes may define the mechanism of activation of premature senescence. Specifically, we found that oncogenic forms of HRAS (HRAS(G12V)) but not its downstream target BRAF (BRAF(V600E)), engaged a rapid cell-cycle arrest that was associated with massive vacuolization and expansion of the ER. However, neither p53, p16(INK4a) nor classical senescence markers--such as foci of heterochromatin or DNA damage--were able to account for the specific response of melanocytes to HRAS(G12V). Instead, HRAS(G12V)-driven senescence was mediated by the ER-associated unfolded protein response (UPR). The impact of HRAS on the UPR was selective, as it was poorly induced by activated NRAS (more frequently mutated in melanoma than HRAS). These results argue against premature senescence as a converging mechanism of response to activating oncogenes and support a direct role of the ER as a gatekeeper of tumour control.     

Organization of Farnesylated,Carboxymethylated KRAS4B on Membranes

Mutations of the Ras proteins HRAS, KRAS4A, KRAS4B, and NRAS are associated with a high percentage of all human cancers. The proteins are composed of highly homologous N-terminal catalytic or globular domains, plus C-terminal hypervariable regions (HVRs). Post-translational modifications of all RAS HVRs helps target RAS proteins to cellular membrane locations where they perform their signaling functions. For the predominant KRAS4 isoform, KRAS4B, post-translational farnesylation and carboxymethylation, along with a patch of HVR basic residues help foster membrane binding. Recent investigations implicate membrane-bound RAS dimers, oligomers, and nanoclusters as landing pads for effector proteins that relay RAS signals. The details of these RAS signaling platforms have not been elucidated completely, in part due to the difficulties in preparing modified proteins. We have employed properly farnesylated and carboxymethylated KRAS4B in lipid monolayer incubations to examine how the proteins assemble on membranes. Our results reveal novel insights into to how KRAS4B may organize on membranes.     

KRAS(G12V) enhances proliferation and initiates myelomonocytic differentiation in human stem/progenitor cells via intrinsic and extrinsic pathways

In human hematopoietic malignancies, RAS mutations are frequently observed. Yet, little is known about signal transduction pathways that mediate KRAS-induced phenotypes in human CD34(+) stem/progenitor cells. When cultured on bone marrow stroma, we observed that KRAS(G12V)-transduced cord blood (CB) CD34(+) cells displayed a strong proliferative advantage over control cells, which coincided with increased early cobblestone (CAFC) formation and induction of myelomonocytic differentiation. However, the KRAS(G12V)-induced proliferative advantage was transient. By week three no progenitors remained in KRAS(G12V)-transduced cultures and cells were all terminally differentiated into monocytes/macrophages. In line with these results, LTC-IC frequencies were strongly reduced. Both the ERK and p38 MAPK pathways, but not JNK, were activated by KRAS(G12V) and we observed that proliferation and CAFC formation were mediated via ERK, while differentiation was predominantly mediated via p38. Interestingly, we observed that KRAS(G12V)-induced proliferation and CAFC formation, but not differentiation, were largely mediated via secreted factors, since these phenotypes could be recapitulated by treating non-transduced cells with conditioned medium harvested from KRAS(G12V)-transduced cultures. Multiplex cytokine arrays and genome-wide gene expression profiling were performed to gain further insight into the mechanisms by which oncogenic KRAS(G12V) can contribute to the process of leukemic transformation. Thus, angiopoietin-like 6 (ANGPTL6) was identified as an important factor in the KRAS(G12V) secretome that enhanced proliferation of human CB CD34(+) cells.     

A Real-World Study in Advanced Non–Small Cell Lung Cancer with KRAS Mutations

BACKGROUND: KRAS gene mutations are well known as a key driver of advanced non–small cell lung cancer (NSCLC). The impact of KRAS-mutant subtypes on the survival benefit from salvage chemotherapy is controversial. Here, we present a real-world study in patients across China with advanced NSCLC with KRAS mutations using a website-based patient self-report system. METHODS: We identified a total of 75 patients diagnosed with KRAS-mutant (determined by molecular sequencing) advanced NSCLC between 2014/5/9 and 2019/5/30. KRAS mutation subtypes were divided into G12C and non-G12C groups for statistical analysis. The clinicopathological characteristics and treatment survival benefit in all patients with a KRAS mutation were evaluated. Programmed death-ligand 1 (PD-L1) expression data were collected from 30 patients in the same cohort. RESULTS: In this study, 23 patients with stage IIIB NSCLC and 52 patients with stage IV NSCLC were enrolled with 58 men and 17 women; the median age was 60 years (39–84). All patients received regular chemotherapy/radiotherapy/targeted therapy/immune therapy as per the disease condition. Four main KRAS mutation subtypes were detected: G12C (33%), G12V (19%), G12A (12%), and G12D (12%). Three predominant KRAS comutations were detected: TP53-KRAS (31%), EGFR-KRAS (11%), and STK11-KRAS (8%). Compared with the KRAS non-G12C mutation subtype, patients with the KRAS G12C mutation had potentially longer progression-free survival (PFS) after first-line chemotherapy (4.7 vs. 2.5 months, p < 0.05). Pemetrexed-based chemotherapy appeared to be superior to taxanes- and gemcitabine-based chemotherapies in all patients (PFS: 5.0 vs. 1.5 and 2.3 months, respectively, p > 0.05). Cox regression analysis showed that the KRAS G12C mutation and pemetrexed-based first-line chemotherapy were positive influencers for PFS after first-line (hazard ratios = 0.31 and 0.55, respectively, P < 0.05), but not second-line chemotherapies. CONCLUSION: The KRAS G12C mutation could be a predictive biomarker for better survival benefit from first-line chemotherapy in patients with advanced NSCLC and KRAS mutations. The first-line chemotherapy regimen could possibly influence the outcome in patients with KRAS mutations. Larger and prospective clinical trials are warranted to confirm our conclusions.     

Genomic heterogeneity and instability in colorectal cancer: spectral karyotyping, glutathione transferase-Ml and ras

Genomic instability in cancer is frequently described as being either chromosomal instability or microsatellite instability, although when events within chromosomes are monitored, extensive intrachromosomal instability is also found. Spectral karyotyping was used to visualize how extensively genomic instability gives rise to intratumor genomic heterogeneity in sporadic colorectal carcinomas. Two factors were then examined which might relate to intrachromosomal instability in colorectal cancers: the presence of the glutathione transferase-Ml gene to detoxify potential carcinogens, and the presence of activated ras which has been associated with chromosomal instability when first expressed. Intrachromosomal genomic instability was previously determined by inter-(simple sequence repeat) PCR (inter-SSR PCR) and by fractional allelic loss rate for 348 markers. GSTM1 status was determined for each of 49 tumors through use of specific PCR, and 28 of the tumors showed the GSTM1 null genotype. A significant association was found between GSTMl-null status and elevated inter-(simple sequence repeat) PCR instability. In contrast, no association was found with fractional allelic loss rate. The first exons of the K-ras and H-ras oncogenes were sequenced in 72 colorectal cancers; 19 of the tumors had a mutation in codon 12 of the K-ras gene (24.5%), but no H-ras mutations were found. A weak correlation (p=0.10) was observed between mutant K-ras and inter-(simple sequence repeat) PCR genomic instability, and no association existed with fractional allelic loss rate.     

Oncogenic RAS-induced downregulation of ATG12 is required for survival of malignant intestinal epithelial cells

Activating mutations of RAS GTPase contribute to the progression of many cancers, including colorectal carcinoma. So far, attempts to develop treatments of mutant RAS-carrying cancers have been unsuccessful due to insufficient understanding of the salient mechanisms of RAS signaling. We found that RAS downregulates the protein ATG12 in colon cancer cells. ATG12 is a mediator of autophagy, a process of degradation and reutilization of cellular components. In addition, ATG12 can kill cells via autophagy-independent mechanisms. We established that RAS reduces ATG12 levels in cancer cells by accelerating its proteasomal degradation. We further observed that RAS-dependent ATG12 loss in these cells is mediated by protein kinases MAP2K/MEK and MAPK1/ERK2-MAPK3/ERK1, known effectors of RAS. We also demonstrated that the reversal of the effect of RAS on ATG12 achieved by the expression of exogenous ATG12 in cancer cells triggers both apoptotic and nonapoptotic signals and efficiently kills the cells. ATG12 is known to promote autophagy by forming covalent complexes with other autophagy mediators, such as ATG5. We found that the ability of ATG12 to kill oncogenic RAS-carrying malignant cells does not require covalent binding of ATG12 to other proteins. In summary, we have identified a novel mechanism by which oncogenic RAS promotes survival of malignant intestinal epithelial cells. This mechanism is driven by RAS-dependent loss of ATG12 in these cells.     

The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae

Sequencing of the neurofibromatosis gene (NF1) revealed a striking similarity among NF1, yeast IRA proteins, and mammalian GAP (GTPase-activating protein). Using both genetic and biochemical assays, we demonstrate that this homology domain of the NF1 protein interacts with ras proteins. First, expression of this NF1 domain suppressed the heat shock-sensitive phenotype of yeast ira1 and ira2 mutants. Second, this NF1 domain, after purification as a glutathione S-transferase (GST) fusion protein, strongly stimulated the GTPase activity of yeast RAS2 and human H-ras proteins. The GST-NF1 protein, however, did not stimulate the GTPase activity of oncogenic mutant ras proteins, H-rasVal-12 and yeast RAS2Val-19 mutants, or a yeast RAS2 effector mutant. These results establish that this NF1 domain has ras GAP activity similar to that found with IRA2 protein and mammalian GAP, and therefore may also regulate ras function in vivo.     

GeLC-MRM quantitation of mutant KRAS oncoprotein in complex biological samples

Tumor-derived mutant KRAS (v-Ki-ras-2 Kirsten rat sarcoma viral oncogene) oncoprotein is a critical driver of cancer phenotypes and a potential biomarker for many epithelial cancers. Targeted mass spectrometry analysis by multiple reaction monitoring (MRM) enables selective detection and quantitation of wild-type and mutant KRAS proteins in complex biological samples. A recently described immunoprecipitation approach (Proc. Nat. Acad. Sci.2011, 108, 2444-2449) can be used to enrich KRAS for MRM analysis, but requires large protein inputs (2-4 mg). Here, we describe sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based enrichment of KRAS in a low molecular weight (20-25 kDa) protein fraction prior to MRM analysis (GeLC-MRM). This approach reduces background proteome complexity, thus, allowing mutant KRAS to be reliably quantified in low protein inputs (5-50 μg). GeLC-MRM detected KRAS mutant variants (G12D, G13D, G12V, G12S) in a panel of cancer cell lines. GeLC-MRM analysis of wild-type and mutant was linear with respect to protein input and showed low variability across process replicates (CV = 14%). Concomitant analysis of a peptide from the highly similar HRAS and NRAS proteins enabled correction of KRAS-targeted measurements for contributions from these other proteins. KRAS peptides were also quantified in fluid from benign pancreatic cysts and pancreatic cancers at concentrations from 0.08 to 1.1 fmol/μg protein. GeLC-MRM provides a robust, sensitive approach to quantitation of mutant proteins in complex biological samples.     

Design,synthesis and biological evaluation of new Myo-inositol derivatives as potential RAS inhibitors

Ras is a small family of GTPases that control numerous cellular functions like cell proliferation, growth, survival, gene expression, and is closely engaged in cancer pathogenesis. The ras-targeted methodology entails a holy grail in oncology. Nevertheless, there are no specific molecules reported targeting the same, although it is a known oncogene for more than three decades. In this study, we have designed and synthesized new phosphate derivatives of Myo-inositol to inhibit the oncogenic KRAS pathway in breast cancer cells, which has been validated by cellular and theoretical studies. The synthesized compound 1b (C2-O-phosphate derivative of Myo-inositol 1,3,5-orthobenzoate) inhibited the downstream signaling pathway of oncogenic KRAS, RAF/MEK/ERK. Furthermore, we also found that this compound induced necrosis/apoptosis and causes cell cycle arrest. This class of molecules may work as a potential inhibitor of breast cancer caused by a mutation in KRAS and its downstream proteins. Though the efficacy of the molecules is in the micromolar scale, they have not been explored previously for RAS inhibition. Impressive preliminary results are presented in this article which could be further explored for its detailed biological studies to get better candidates as RAS inhibitors.     

Ras Effector Mutant Expression Suggest a Negative Regulator Inhibits Lung Tumor Formation

Lung cancer is currently the most deadly malignancy in industrialized countries and accounts for 18% of all cancer-related deaths worldwide. Over 70% of patients with non-small cell lung cancer (NSCLC) are diagnosed at a late stage, with a 5-year survival below 10%. KRAS and the EGFR are frequently mutated in NSCLC and while targeted therapies for patients with EGFR mutations exist, oncogenic KRAS is thus far not druggable. KRAS activates multiple signalling pathways, including the PI3K/Akt pathway, the Raf-Mek-Erk pathway and the RalGDS/Ral pathway. Lung-specific expression of BrafV600E, the most prevalent BRAF mutation found in human tumors, results in Raf-Mek-Erk pathway activation and in the formation of benign adenomas that undergo widespread senescence in a Cre-activated Braf mouse model (Braf^CA). However, oncogenic KRAS expression in mice induces adenocarcinomas, suggesting additional KRAS-activated pathways cooperate with sustained RAF-MEK-ERK signalling to bypass the oncogene-induced senescence proliferation arrest.To determine which KRAS effectors were responsible for tumor progression, we created four effector domain mutants (S35, G37, E38 and C40) in G12V-activated KRAS and expressed these alone or with BrafV600E in mouse lungs… The S35 and E38 mutants bind to Raf proteins but not PI3K or RalGDS; the G37 mutant binds to RalGDS and not Raf or PI3K and the C40 mutant is specific to PI3K. We designed lentiviral vectors to code for Cre recombinase along with KRAS mutants (V12, V12/S35, V12/G37, V12/E38 or V12/C40) or EGFP as a negative control.. These lentiviruses were used to infect Braf^CA and wild-type mice. Surprisingly there was a significant decrease in tumor number and penetrance with each KRAS effector domain mutant relative to controls, suggesting that KRAS directly activates effectors with tumor suppressive functions.