KRAS-mutant Cancer Cell Lines Research Articles

Abstract A44: SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS-mutant tumors treated with MEK inhibitors

Abstract FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTKs) are also feedback activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using a SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS mutant cancer cells and involves several RTKs including EGFR, FGFR, and MET. We further demonstrate this pathway feedback activation is mediated through mutant KRAS. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS mutant cancer cell proliferation in vitro and in vivo. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic. Citation Format: Hengyu Lu, Chen Liu, Roberto Velazquez, Hongyun Wang, Lukas M. Dunkl, Malika Kazic-Legueux, Anne Haberkorn, Eric Billy, Eusebio Manchado, Saskia M. Brachmann, Susan Moody, Jeffrey A. Engelman, Peter S. Hammerman, Giordano Caponigro, Morvarid Mohseni, Huai-Xiang Hao. SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS-mutant tumors treated with MEK inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A44.

Abstract 954: SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS mutant tumors treated with MEK inhibitors

Abstract Introduction: FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with MEK inhibitor (MEKi) such as trametinib in several KRAS mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTKs) are also feedback activated in KRAS mutant cell lines after MEKi treatment. Experimental procedures: We profiled a large panel (n>32) of KRAS mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using a SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We then performed in vitro and in vivo combination efficacy studies and pathway analysis in various KRAS mutant cancer models. Results: We find that RTK-driven feedback activation widely exists in KRAS mutant cancer cells and involves several RTKs including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS in KRAS G12C or G12D models. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting MAPK pathway and KRAS mutant cancer cell proliferation in vitro and in vivo. Conclusions: Our findings suggest that MAPK inhibition in KRAS mutant cancer provokes feedback re-activation of the pathway that often involves RTK activity and SHP2 inhibition may enhance the efficacy of MEKi in KRAS mutant tumors. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS mutant cancers in the clinic. Citation Format: Hengyu Lu, Chen Liu, Roberto Velazquez, Hongyun Wang, Lukas M. Dunkl, Malika Kazic-Legueux, Anne Haberkorn, Eric Billy, Eusebio Manchado, Saskia M. Brachmann, Susan Moody, Jeffrey A. Engelman, Peter S. Hammerman, Giordano Caponigro, Morvarid Mohseni, Huaixiang Hao. SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS mutant tumors treated with MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 954.

SHP2 Inhibition Overcomes RTK-Mediated Pathway Reactivation in KRAS-Mutant Tumors Treated with MEK Inhibitors.

FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with the MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTK) are also feedback-activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using an SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS-mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs, including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D, and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS-mutant cancer cell proliferation in vitro and in vivo These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic.

Abstract 3973: Dabrafenib suppresses the growth of BRAF-WT cancers through inhibition of novel targets Nek9 and Cdk16

Abstract Although the BRAF inhibitors dabrafenib and vemurafenib have both proven successful against BRAF-mutant melanoma, there seem to be differences in their mechanisms of action. Here, we show that dabrafenib is more effective at inhibiting the growth of NRAS-mutant and KRAS-mutant cancer cell lines than vemurafenib. Using mass spectrometry-based chemical proteomics we identified NEK9 and CDK16 as unique targets of dabrafenib. Both NEK9 and CDK16 were highly expressed in specimens of advanced melanoma, with high expression of both proteins correlating with a worse overall survival. A role for NEK9 in the growth of NRAS and KRAS-mutant cell lines was suggested by siRNA studies in which silencing was associated with decreased proliferation, cell cycle arrest associated with increased p21 expression, inhibition of phospho-CHK1, decreased CDK4 expression and the initiation of a senescence response. Inhibition of CDK4 but not CHK1 recapitulated the effects of NEK9 silencing, indicating this to be the likely mechanism of growth inhibition. We next turned our attention to CDK16 and found that its knockdown inhibited the phosphorylation of the Rb protein at S780 and increased expression of p27. Both of these effects were phenocopied in NRAS and KRAS-mutant cancer cells by dabrafenib but not vemurafenib. Combined silencing of NEK9 and CDK16 were associated with enhanced inhibition of melanoma cell proliferation and a greater induction of cell cycle arrest. In summary, we have identified dabrafenib as a potent inhibitor of NEK9 and CDK16, and suggest that inhibition of these kinases may have activity against cancers that do not harbor BRAF mutations. Citation Format: Manali S. Phadke, Lily Rix, Inna Smalley, Annamarie Bryant, Harshani Lawrence, Braydon Schaible, Ann Chen, Uwe Rix, Keiran Smalley. Dabrafenib suppresses the growth of BRAF-WT cancers through inhibition of novel targets Nek9 and Cdk16 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3973.

Abstract IA14: Understanding the complex biology of KRAS mutant cancers using genetic screens

Abstract Apart from identifying synthetic lethal genes, genetic screens have the potential to reveal much about the basic biology of cell lines. We utilize two techniques, one analytical and one experimental, to demonstrate how genetic screens can also reveal which pathways are on in cell lines and what are the phenotypic consequences of pathway perturbation. First, we apply a clustering analysis of genome-scale CRISPR screening data to map out active pathways in cancer cell lines and to discover novel members of signaling transduction pathways and protein complexes. Secondly, we performed a combinatorial siRNA screen that knocks out multiple genes at once and measures five functional parameters to comprehensively describe the effects of pathway inhibition. Application of these two techniques to KRAS mutant cancer cell lines reveals a trove of biological information that highlights the vast heterogeneity of this disease and subtype-specific opportunities for therapeutic intervention. Citation Format: Tina L. Understanding the complex biology of KRAS mutant cancers using genetic screens [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA14.

A Quadruple‐Action Platinum(IV) Prodrug with Anticancer Activity Against KRAS Mutated Cancer Cell Lines

AbstractWe developed a novel PtIV prodrug that simultaneously releases four different bioactive moieties inside the cancer cell. Its cytotoxicity against monolayer cultures (2D) and spheroid (3D) cancer cells is significantly better than cisplatin. It is 200–450‐fold more potent than cisplatin against KRAS mutated pancreatic and colon cancers and is 40‐fold more selective towards KRAS mutated cells compared to non‐cancerous. This is important since RAS proteins play a role in regulating cell differentiation, proliferation, and survival and KRAS is mutated in 90 % of pancreatic adenocarcinomas, 45 % of colorectal cancers, and 35 % of lung adenocarcinomas. The selectivity index, determined by dividing the IC50 value in non‐cancerous cells by that of a cancerous cell line, is two‐fold better than cisplatin, attesting to preferential cytotoxicity towards neoplastic cells.

A Quadruple-Action Platinum(IV) Prodrug with Anticancer Activity Against KRAS Mutated Cancer Cell Lines.

We developed a novel PtIV prodrug that simultaneously releases four different bioactive moieties inside the cancer cell. Its cytotoxicity against monolayer cultures (2D) and spheroid (3D) cancer cells is significantly better than cisplatin. It is 200-450-fold more potent than cisplatin against KRAS mutated pancreatic and colon cancers and is 40-fold more selective towards KRAS mutated cells compared to non-cancerous. This is important since RAS proteins play a role in regulating cell differentiation, proliferation, and survival and KRAS is mutated in 90 % of pancreatic adenocarcinomas, 45 % of colorectal cancers, and 35 % of lung adenocarcinomas. The selectivity index, determined by dividing the IC50 value in non-cancerous cells by that of a cancerous cell line, is two-fold better than cisplatin, attesting to preferential cytotoxicity towards neoplastic cells.

Abstract 5161: Inhibition of TBK1/IKBKE impacts tumor growth of KRAS-mutant colorectal cancers

Abstract Mutations in RAS occur in an estimated one third of all human cancers. While direct targeting of RAS remains challenging, targeting downstream RAS effector molecules has been shown to impact the oncogenic function of RAS and elicit anti-tumor activity. TANK Binding Kinase 1 (TBK1) and I-Kappa B Kinase Epsilon (IKBKE) serine/threonine kinases are non-canonical IKK family members. TBK1 is directly downstream of the RAS effector Ral Guanine Nucleotide Dissociation Stimulator (RALGDS) and may be required for RAS to elicit its full oncogenic potential. To explore this possibility, we identified a potent and selective TBK1/IKBKE inhibitor to determine whether inhibition of TBK1 impacts the growth of RAS-mutant cancer models. KRAS-mutant cancer cell lines showed preferential sensitivity to TBK1/IKBKE inhibition over KRAS wild-type cell lines. The TBK1/IKBKE inhibitor was then evaluated in vivo with two KRAS-mutant colorectal cancer patient-derived xenograft (PDX) models, one of which harbors co-occurring PIK3CA and KRAS mutations. The TBK1/IKBKE inhibitor caused moderate tumor growth inhibition in comparison to vehicle in both PDX models. Since RAS exerts its oncogenic effects via additional effectors, such as RAF, and that inhibition of MEK1/2 resulted in compensatory activation of TBK1 in a KRAS-mutant cancer cell line, we tested if inhibition of TBK1/IKBKE in combination with the MEK inhibitor, trametinib, would result in superior efficacy in PDX models. The combination of a TBK1/IKBKE inhibitor and trametinib resulted in greater than 90% tumor growth inhibition within both of these KRAS mutant PDX models compared to either single agent alone. We also tested TBK1/IKBKE inhibition in a KRAS wild-type colorectal PDX model containing a BRAF mutation. In this model, TBK1/IKBKE did not inhibit tumor growth and, although there was significant tumor growth inhibition with trametinib alone, there was no further enhancement of efficacy with the addition of a TBK1/IKBKE inhibitor. Together, these data suggest that a TBK1/IKBKE inhibitor in combination with a MEK inhibitor may impact KRAS-mutant cancers. Citation Format: Susanna Stinson, Jianhua He, Zhiheng Jia, Katie S. Walker, Zhi-Hua Cui, Joshua A. Kaplan, Chandrasekar Venkataramani, Yujin Wang, Fatima Guevara, Timothy Nelson, Ewa Sicinska, Kimmie Ng, Charles S. Fuchs, David Dornan. Inhibition of TBK1/IKBKE impacts tumor growth of KRAS-mutant colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5161. doi:10.1158/1538-7445.AM2017-5161

KRAS oncogene repression in colon cancer cell lines by G-quadruplex binding indolo[3,2-c]quinolines.

KRAS is one of the most frequently mutated oncogenes in human cancer, yet remaining undruggable. To explore a new therapeutic strategy, a library of 5-methyl-indolo[3,2-c]quinoline derivatives (IQc) with a range of alkyldiamine side chains was designed to target DNA and RNA G-quadruplexes (G4) in the promoter and 5′-UTR mRNA of the KRAS gene. Biophysical experiments showed that di-substituted IQc compounds are potent and selective KRAS G4 stabilizers. They preferentially inhibit the proliferation of KRAS mutant cancer cell lines (0.22 < IC50 < 4.80 μM), down-regulate KRAS promoter activity in a luciferase reporter assay, and reduce both KRAS mRNA and p21KRAS steady-state levels in mutant KRAS colon cancer cell lines. Additionally, IQcs induce cancer cell death by apoptosis, explained in part by their capacity to repress KRAS expression. Overall, the results suggest that targeting mutant KRAS at the gene level with G4 binding small molecules is a promising anticancer strategy.

Abstract A20: ARHGEF2 inhibition impairs cell growth and viability in KRAS-dependent cancer cells

Abstract Sporadic tumours carrying activating mutations of the RAS family of GTPases are characterized by a progressive and multiple step tumorigenesis. Among the three isoforms, K-RAS is the most commonly mutated (Prior et al., 2012). ARHGEF2 is a microtubule-associated guanine nucleotide exchange factor (GEF) for the Rho family of small GTPases whose expression is up-regulated (more than 30 times) upon Ras neoplastic transformation in Rat fibroblasts (Zuber et al., 2000). A truncated version of the protein can transform NIH 3T3 cells (Whitehead et al., 1995) and induce tumour formation in nude mice (Brecht et al., 2005). We recently identified ARHGEF2 as a transcriptional target of the RAS/MAPK pathway and its expression contributes to cell survival and growth in RAS-transformed cells (Cullis et al., in revision). We aimed to determine if ARHGEF2 is required for tumour cell viability in different KRAS mutant cancer cell lines. We used an RNAi approach using two different ARHGEF2 and KRAS shRNAs to monitor cell growth and viability in six different cancer cell lines harbouring a KRAS mutation: four pancreatic adenocarcinomas (PL45, Panc 02.03, Panc 04.03 and KP4), one endometrial adenocarcinoma (HEC-1-A) and one colorectal adenocarcinoma (LS 174T). We found that ARHGEF2 is required for cell growth and survival in half of the cell lines analyzed. We established an ARHGEF2 and KRAS dependency index for those cell lines and further analysis allowed us to determine a positive correlation (correlation coefficient of 0.64, p value &amp;lt;0.03) between ARHGEF2 and KRAS dependency. In conclusion, our findings suggest that: i/ ARHGEF2 plays a role in the tumorigenesis and survival of KRAS dependent cell lines, but further in vitro and in vivo studies are required; ii/ ARHGEF2 inhibition could be a potential therapeutic target in tumours harbouring KRAS mutations.

Fluorescence resonance energy transfer based quantitative analysis of feedforward and feedback loops in epidermal growth factor receptor signaling and the sensitivity to molecular targeting drugs.

The Ras-ERK and PI3K-mTOR pathways are hyperactivated in various malignant tumors. Feedforward (FF) and feedback (FB) regulations between the Ras-ERK and the PI3K-mTOR pathways have been suggested to attenuate sensitivity to drugs targeting these pathways and confer tumor resistance to therapies. However, because analyses of such regulations require measurements and perturbations with high temporal resolution, the quantitative roles played by FF and FB regulations in the intrinsic resistance to molecular targeting drugs still remain unclear. To address this issue, we quantified FF and FB regulations of the epidermal growth factor receptor (EGFR) signaling pathway by Förster/fluorescence resonance energy transfer (FRET) imaging. EGF-induced activation of EGFR, Ras, extracellular-signal-regulated kinase and S6K with or without inhibitors was measured by FRET imaging, and analyzed by semi-automatic image processing. Based on the imaging data set and kinetic parameters determined by our previous studies, we identified the roles played by a coherent FF regulation and two negative FB regulations, one of which was not recognized previously. The systems analyses revealed how these FF and FB regulations shape the temporal dynamics of extracellular-signal-regulated kinase activity upon EGF stimulation. Furthermore, the simulation model predicts the response of molecular targeting drugs applied solely or in combination with each other to BRaf- or KRas-mutated cancer cell lines, indicating the validity of a quantitative model integrating FF and FB regulations.

Abstract C142: Mortalin/HSPA9 regulates p21CIP1 expression in Raf/MEK/ERK-activated cancer cells.

Abstract Dysregulated Raf/MEK/ERK signaling is a common hallmark of tumorigenesis. However intriguingly, aberrant Raf/MEK/ERK activation triggers senescence-like growth arrest as a primary response in many cell types. This response is recognized as an innate tumor suppressive mechanism, which should be bypassed for Raf/MEK/ERK to drive carcinogenesis. Therefore, understanding of the bypass mechanisms is likely to provide an opportunity to design a novel therapeutic strategy. In this study, we report that mortalin, a member of the heat shock protein 70 (HSP70) family, has a role in bypassing the Raf/MEK/ERK-induced growth inhibition in cancer. Using tandem affinity purification and proteomic mass spectrometry, we found that mortalin is present in the MEK1/2 proteome. A specific physical interaction between mortalin and MEK was validated by co-immunoprecipitation analyses and in vitro binding assays of recombinant proteins. Immunohistochemical analysis revealed that mortalin is upregulated in human melanoma biopsies in correlation with tumor malignancy. Of note, mortalin levels were inversely correlated with p21CIP1 levels in a number of Raf/MEK/ERK-activated cancer cell lines, suggesting a role for mortalin in p21CIP1 regulation. Indeed, mortalin depletion elicited increased p21CIP1 transcription and MEK/ERK activation which were accompanied by G2/M phase cell cycle arrest and cell death in different B-Raf or K-Ras mutated cancer cell lines. Remarkably, blocking MEK/ERK activation, using the MEK1/2 inhibitor, AZD6244, dominant negative mutants of MEK or ERK, or RNA interference, abrogated p21CIP1 induction by mortalin depletion in cancer cells harboring B-RafV600E regardless of their p53 status. In contrast, mortalin overexpression suppressed B-RafV600E or ΔRaf-1:ER-induced MEK/ERK activation, p21CIP1 expression, and cell cycle arrest in cell types exhibiting normal MEK/ERK status. Other HSP70 family chaperones could not effectively replace mortalin for the observed effects on p21CIP1 regulation, suggesting a unique role for mortalin. These findings suggest that mortalin upregulation is a mechanism that underlies p21CIP1 silencing in Raf/MEK/ERK-activated cancer, identifying mortalin as a novel negative regulator of the pathway signaling. Mortalin may provide a target to reactivate the tumor suppressive signaling of Raf/MEK/ERK in cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C142. Citation Format: Pui Kei Wu, Seung-Keun Hong, Sudhakar Veeranki, Mansi Karkhanis, Dmytro Starenki, Jose A. Plaza, Jong-In Park. Mortalin/HSPA9 regulates p21CIP1 expression in Raf/MEK/ERK-activated cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C142.

Abstract 941: Discovery of novel VEGFR2 and Raf-1 kinase inhibitorsuppressing MAPK signaling pathway in K-Ras mutant cancer cell-lines.

Abstract Introduction: Signal transduction in the Ras-Raf-MEK-ERK (MAPK) pathway plays a key role in cell survival, growth and proliferation. The pathway is controlled by extracellular signals through receptor tyrosine kinases (RTK) and is activated by oncogenic mutations in many types of cancer. The B-Raf mutants are found in various cancers, such as melanoma, colorectal, ovarian, and prostate cancer. The Ras mutants are also found in various carcinomas including colon cancer, pancreatic cancer, non-small-cell lung cancer, and liver cancer. The mutated K-Ras gene sends continuous signals to the colorectal cancer cells, telling them to grow independent of the way ERBITUX blocks the EGFR growth signals. Especially, over 90% of detected mutations in B-Raf are a V600E which leads to constitutive kinase activity 500-fold greater than B-Raf wild type and correlates with increased malignancy. Vascular endothelial growth factor (VEGF) and its receptors have been implicated in the angiogenesis that is essential for growth and metastasis of solid tumors. Since formation of solid tumors are angiogenesis dependent, several strategies have been developed to inhibit VEGF signal transduction as part of anticancer therapy. The first VEGFR2 and Raf-1 inhibitor to enter clinical development was the multikinase inhibitor SORAFENIB, which is now approved for the treatment of patients with advanced renal cell carcinoma. More potent dual inhibitors against VEGFR2 and Raf-1 may be beneficial for patients suffering from various tumors. In this study, some of rational-designed compounds were synthesized and carried out the in vitro and in vivo studies to find more efficient VEGFR2 and Raf-1 inhibitors. Methods: About two hundreds of compounds were designed and synthesized. in vitro assays and in vivo studies were performed to characterize the potential of VEGFR2 and Raf-1 inhibitors for solid tumors. Results: Compound UI-162 showed strong kinase inhibition against VEGFR2 (12 nM), B-Raf V600E mutant (0.2 nM), B-Raf wildtype (2.0 nM) and Raf-1(0.6 nM). The compound showed good inhibitory effect in against malignant cells expressing K-Ras or B-Raf V600E mutant type of cell-line (SK-MEL-2 GI50: 133 nM / HCT-116 GI50: 229 nM / MDA-MB231 GI50: 278 nM / A375P GI50: 51 nM / HT29 GI50: 40 nM / COLO 205 GI50: 12 nM, respectively). In mechanism studies, the phosphorylation of MEK and ERK were suppressed by the UI-162 compound in HCT-116 cell-line. In addition, paradoxical effects were not detected compared with VEMURAFENIB. The compound had also a good anti-proliferative activity against HUVEC and showed a good oral bioavailability. Conclusions: The results of in vitro and in vivo studies suggests that dual inhibition of VEGFR2 and Raf-1 may provide strong antitumor efficacy and that UI-162 is a promising candidate for the treatment of various human cancers harboring the K-Ras and B-Raf V600E mutation. Citation Format: Seung Yong Kim, Younho Lee, Hyungtae Bang, Kihwan Eum, Sungpyo Hong, Ky-Youb Nam, Nam Song Choi, Ju Hee Kang, Hara Kang, Kil Won Kim, Michael Lee, Soon Kil Ahn. Discovery of novel VEGFR2 and Raf-1 kinase inhibitorsuppressing MAPK signaling pathway in K-Ras mutant cancer cell-lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 941. doi:10.1158/1538-7445.AM2013-941

Abstract C152: Mechanisms of KRAS dependency in pancreatic ductal adenocarcinoma.

Abstract Currently, pancreatic ductal adenocarcinoma (PDAC) has a dismal &amp;lt;5% survival rate two years after initial diagnosis. Despite significant advances in development of signal transduction targeted therapies for some cancers, erlotinib, an EGFR inhibitor, in combination with gemcitabine, is the only targeted drug approved for treatment of PDAC. The frequency of KRAS oncogene mutations in PDAC is greater than 90%, thus inhibition of K-Ras4B protein function may have a significant impact on PDAC tumorigenesis. In support of this hypothesis, several recent studies demonstrated that KRAS mutant cancer cell lines display a strong dependence or addiction to mutant K-Ras for survival. Surprisingly, K-Ras addiction was not attributed to either the Raf or phosphoinositide 3-kinase (PI3K) effector pathways, which are two of the best-characterized Ras signaling effectors. These results suggest that other pathways are key for mutant KRAS dependency. Elucidating the effector mechanism(s) necessary for KRAS addiction may implicate signaling pathways, which harbor novel therapeutic targets for future anti-K-Ras drug development. To address this question, we first verified that shRNA silencing of mutant KRAS in a panel of pancreatic cell lines led to a strong reduction in anchorage-independent growth. Notably and in contrast to published studies, this was not due to decreased cell survival or increased anoikis. Using KRAS mutant PDAC cell lines, we purpose to use effector domain mutants of oncogenic K-Ras4B that are differentially impaired in effector interaction and constitutively activated effectors to define the key effector(s) involved in KRAS dependency. These studies will help to define key signaling activities of mutant KRAS that are critical for oncogenesis in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C152.

Abstract 956: Receptor tyrosine kinases, not KRAS, activate PI3K in KRAS mutant colorectal cancers

Abstract Introduction: Therapies inhibiting receptor tyrosine kinases (RTKs) are effective when they lead to simultaneous downregulation of phosphoinositide 3-kinase (PI3K)-AKT and mitogen-activated protein kinase (MEK)-ERK signaling. Although cetuximab has demonstrated activity in KRAS wildtype cancers, it remains unknown if RTK inhibition alone can suppress one or both of these pathways in colorectal cancers, especially in cancers with KRAS mutations. Experimental Procedures and Results: Colorectal cancer cells were treated with gefitinib (1 μM) or Cetuximab (10 μg/mL) to compare the consequences of EGFR inhibition on downstream signaling events between KRAS wt (n=6) and mt (n=8) cell lines. In KRAS wt cancers, either cetuximab or gefitinib led to loss of ERK phosphorylation, but had only a modest effect on PI3K signaling. However, in most KRAS mutant cancers, EGFR inhibition minimally impacted ERK signaling and had neglible effects on PI3K signaling. To determine if KRAS constitutively activates ERK and PI3K signaling in KRAS mutant colorectal cancers, we utilized shRNA in eight KRAS mutant cancer cell lines and abruptly turned off KRAS expression in an vivo transgenic model. In all models, the phosphorylation of ERK was markedly decreased by KRAS knockdown, but this was not associated with downregulation of PI3K signaling. To determine how PI3K was activated in KRAS mutant cancers, we immunoprecipitated the p85 regulatory subunit and identified that IRS proteins co-precipitated with PI3K in KRAS mutant cancer. These complexes were disrupted by an IGF-IR inhibitor, NVP-AEW541, but not by gefitinib, and accordingly, NVP-AEW541 led to loss of AKT phosphorylation. Of the eight KRAS mutant cell lines, we found p85/IRS-1 or p85/IRS-2 complexes in 7 cell lines. We also observed that a monoclonal anti-IGF-IR antibody R1507 (18 mg/kg twice a week) downregulated PI3K signaling in SW837 xenografts in vivo. Biochemical assessment of patient specimens with KRAS mutations also suggested that PI3K was regulated primarily by IGF-IR. Importantly, by combining RTK inhibitors with MEK inhibitors in KRAS mutant cancers, both the PI3K and ERK pathways were concomitantly downregulated leading to marked growth suppression and robust apoptosis. Furthermore, combining R1507 (18 mg/kg twice a week) with a MEK inhibitor (AZD6244 25 mg/kg twice daily) induced marked tumor regressions of a KRAS mutant colorectal cancer in vivo. Conclusions: In this study, we provide evidence that KRAS does not drive PI3K signaling in KRAS mutant cancers, and demonstrate that specific RTK inhibition potently suppresses PI3K signaling in vitro and in vivo. These findings provide a framework for utilizing RTK inhibitors in the treatment of KRAS mutant colorectal cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 956. doi:10.1158/1538-7445.AM2011-956