RAS Mutant Cancers Research Articles

Abstract 3725: B-Raf inhibition-induced paradoxical activation of MEK/ERK signaling enhances DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells

Abstract B-Raf inhibitors have been used for the treatment of some B-Raf-mutated cancers such as melanoma and thyroid cancer. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but lead to a paradoxical activation of MEK/ERK signaling in Ras-mutant cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. Given the critical role of MEK/ERK signaling in the positive regulation of DR5 expression as we have previously demonstrated, this study focuses on determining the impact of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Ras-mutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-treatment of Ras-mutated cancer cells with PLX4032 sensitizes them to TRAIL-induced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells. Our results may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition. (This study was supported by Emory Winship Cancer Institute Robbins Scholar awards to YTO and to JD and Halpern Research Scholar award to SYS. SYS is a Georgia Research Alliance Distinguished Cancer Scientist and Halpern Research Scholar) Citation Format: You-Take Oh, Jiusheng Deng, Ping Yue, Shi-Yong Sun. B-Raf inhibition-induced paradoxical activation of MEK/ERK signaling enhances DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3725.

Abstract 282: Pan-RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4 and 6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1

Abstract KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, including melanoma, lung, colorectal, and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor currently in phase I clinical trial, has been shown to inhibit cell proliferation of RAS- or BRAF-mutant tumor cells in vitro and xenograft tumor growth in vivo. An unbiased screen for compounds that synergize with LY3009120 in RAS/BRAF mutant cancers identified inhibitors of CDK4 among the top hits. In this study, we found that combined inhibition of RAF and CDK4 and 6 by LY3009120 and abemaciclib cooperatively reduced viability of tumor cells with KRAS, NRAS or BRAF mutation in vitro. In animal models, the LY3009120 and abemaciclib combination exhibited synergistic regression of tumor growth in multiple xenograft models with KRAS, NRAS, or BRAF mutation. Molecular mechanistic analysis revealed that pan-RAF inhibitor treatment suppressed the cyclin D1 upregulation which was mediated through CDK4 and CDK6 inhibition by abemaciclib, and the combination treatment cooperatively demonstrated more complete inhibition of RB phosphorylation. These results were further verified by CDK4 and CDK6 siRNA knockdown and another CDK4 and CDK6 selective inhibitor palbociclib. Importantly, the more complete phospho-RB inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combinational treatment led to more significant cell cycle G0/G1 arrest and apoptosis of tumor cells. These preclinical findings suggest that the combinational inhibition of RAF and CDK4 and CDK6 signaling by LY3009120 and abemaciclib is synergistic and should be further studied compared to single agents in the treatment of cancer in patients with KRAS, NRAS or BRAF mutations. Citation Format: Shih-Hsun Chen, Youyan Zhang, Robert D. Van Horn, Tinggui Yin, Lysiane Huber, Teresa F. Burke, Xueqian Gong, Wenjuan Wu, Shripad Bhagwat, Sean Buchanan, Richard P. Beckmann, Ramon V. Tiu, Sheng-Bin Peng. Pan-RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4 and 6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 282.

Paradoxical activation of MEK/ERK signaling induced by B-Raf inhibition enhances DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells.

B-Raf inhibitors have been used for the treatment of some B-Raf–mutated cancers. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but paradoxically activates MEK/ERK in Ras-mutated cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. This study focused on determining the effects of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Ras-mutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-exposure of Ras-mutated cancer cells to PLX4032 sensitizes them to TRAIL-induced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively, our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells and hence may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition.

(18)F-FDG PET/CT imaging in rectal cancer: relationship with the RAS mutational status.

Treating metastatic colorectal cancer with anti-EGFR monoclonal antibodies is recommended only for patients whose tumour does not harbour mutations of KRAS or NRAS. The aim of this study was to investigate the biology of rectal cancers and specifically to evaluate the relationship between fluorine-18 fludeoxyglucose ((18)F-FDG) positron emission tomography (PET) intensity and heterogeneity parameters and their mutational status. 151 patients with newly diagnosed rectal cancer were included in this retrospective study. All patients underwent a baseline (18)F-FDG PET/CT within a median time interval of 27 days of tumour tissue sampling, which was performed before any treatment. Standardized uptake values (SUVs), volume-based parameters and texture analysis were studied. We retrospectively performed KRAS genotyping on codons 12, 13, 61, 117 and 146, NRAS genotyping on codons 12, 13 and 61 and BRAF on codon 600. Associations between PET/CT parameters and the mutational status were assessed using univariate and multivariate analysis. 83 (55%) patients had an RAS mutation: 74 KRAS and 9 NRAS, while 68 patients had no mutation (wild-type tumours). No patient had BRAF mutation. First-order features based on intensity histogram analysis were significantly associated with RAS mutations: maximum SUV (SUVmax) (p-value = 0.002), mean SUV (p-value = 0.006), skewness (p-value = 0.049), SUV standard deviation (p-value = 0.001) and SUV coefficient of variation (SUVcov) (p-value = 0.001). Both SUVcov and SUVmax showed an area under the curve of 0.65 with sensitivity of 56% and 69%, respectively, and specificity of 64% and 52%, respectively. None of the volume-based (metabolic tumour volume and total lesion glycolysis), nor local or regional textural features were associated with the presence of RAS mutations. Although rectal cancers with KRAS or NRAS mutations display a significantly higher glucose metabolism than wild-type cancers, the accuracy of the currently proposed quantitative metrics extracted from (18)F-FDG PET/CT is not sufficiently high for playing a meaningful clinical role. RAS-mutated rectal cancers have a significantly higher glucose metabolism. However, the accuracy of (18)F-FDG PET/CT quantitative metrics is not as such as the technique could play a clinical role.

The renewed battle against RAS-mutant cancers.

The RAS genes encode for members of a large superfamily of guanosine-5'-triphosphate (GTP)-binding proteins that control diverse intracellular signaling pathways to promote cell proliferation. Somatic mutations in the RAS oncogenes are the most common activating lesions found in human cancers. These mutations invariably result in the gain-of-function of RAS by impairing GTP hydrolysis and are frequently associated with poor responses to standard cancer therapies. In this review, we summarize key findings of past and present landmark studies that have deepened our understanding of the RAS biology in the context of oncogenesis. We also discuss how emerging areas of research could further bolster a renewed global effort to target the largely undruggable oncogenic RAS and/or its activated downstream effector signaling cascades to achieve better treatment outcomes for RAS-mutant cancer patients.

Direct Inhibitors of Ras-Effector Protein Interactions.

Activating Ras mutations are associated with ~30% of all human cancers, which often respond poorly to standard therapies. The four Ras isoforms are therefore highly attractive targets for anticancer drug discovery. However, Ras proteins function through protein-protein interactions and their surfaces lack any major pockets for small molecules to bind; as a result they have been declared "undruggable" for the past 30 years. Several breakthroughs during the past few years may finally remove Ras from the list of undruggable proteins. This mini-review discusses the current approaches to developing inhibitors especially cyclic peptides that physically block the interaction between Ras and its downstream effector proteins, which is potentially the most effective approach for treating Ras mutant cancers.

RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies.

Activating mutations of oncogenic RAS genes are frequently detected in human cancers. The studies in genetically engineered mouse models (GEMMs) reveal that Kras-activating mutations predispose mice to early onset tumors in the lung, pancreas, and gastrointestinal tract. Nevertheless, most of these tumors do not have metastatic phenotypes. Metastasis occurs when tumors acquire additional genetic changes in other cancer driver genes. Studies on clinical specimens also demonstrated that KRAS mutations are present in premalignant tissues and that most of KRAS mutant human cancers have co-mutations in other cancer driver genes, including TP53, STK11, CDKN2A, and KMT2C in lung cancer; APC, TP53, and PIK3CA in colon cancer; and TP53, CDKN2A, SMAD4, and MED12 in pancreatic cancer. Extensive efforts have been devoted to develop therapeutic agents that target enzymes involved in RAS posttranslational modifications, that inhibit downstream effectors of RAS signaling pathways, and that kill RAS mutant cancer cells through synthetic lethality. Recent clinical studies have revealed that sorafenib, a pan-RAF and VEGFR inhibitor, has impressive benefits for KRAS mutant lung cancer patients. Combination therapy of MEK inhibitors with either docetaxel, AKT inhibitors, or PI3K inhibitors also led to improved clinical responses in some KRAS mutant cancer patients. This review discusses knowledge gained from GEMMs, human cancer cells, and patient-related studies on RAS-mediated tumorigenesis and anti-RAS therapy. Emerging evidence demonstrates that RAS mutant cancers are heterogeneous because of the presence of different mutant alleles and/or co-mutations in other cancer driver genes. Effective subclassifications of RAS mutant cancers may be necessary to improve patients' outcomes through personalized precision medicine.

Long-Term ERK Inhibition in KRAS-Mutant Pancreatic Cancer Is Associated with MYC Degradation and Senescence-like Growth Suppression

Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.

Inhibitors of Ras-SOS Interactions.

Activating Ras mutations are found in about 30 % of human cancers. Ras activation is regulated by guanine nucleotide exchange factors, such as the son of sevenless (SOS), which form protein-protein interactions (PPIs) with Ras and catalyze the exchange of GDP by GTP. This is the rate-limiting step in Ras activation. However, Ras surfaces lack any evident suitable pockets where a molecule might bind tightly, rendering Ras proteins still 'undruggable' for over 30 years. Among the alternative approaches is the design of inhibitors that target the Ras-SOS PPI interface, a strategy that is gaining increasing recognition for treating Ras mutant cancers. Herein we focus on data that has accumulated over the past few years pertaining to the design of small-molecule modulators or peptide mimetics aimed at the interface of the Ras-SOS PPI. We emphasize, however, that even if such Ras-SOS therapeutics are potent, drug resistance may emerge. To counteract this development, we propose "pathway drug cocktails", that is, drug combinations aimed at parallel (or compensatory) pathways. A repertoire of classified cancer, cell/tissue, and pathway/protein combinations would be beneficial toward this goal.

NF2 Loss Promotes Oncogenic RAS-Induced Thyroid Cancers via YAP-Dependent Transactivation of RAS Proteins and Sensitizes Them to MEK Inhibition.

Ch22q LOH is preferentially associated with RAS mutations in papillary and in poorly differentiated thyroid cancer (PDTC). The 22q tumor suppressor NF2, encoding merlin, is implicated in this interaction because of its frequent loss of function in human thyroid cancer cell lines. Nf2 deletion or Hras mutation is insufficient for transformation, whereas their combined disruption leads to murine PDTC with increased MAPK signaling. Merlin loss induces RAS signaling in part through inactivation of Hippo, which activates a YAP-TEAD transcriptional program. We find that the three RAS genes are themselves YAP-TEAD1 transcriptional targets, providing a novel mechanism of promotion of RAS-induced tumorigenesis. Moreover, pharmacologic disruption of YAP-TEAD with verteporfin blocks RAS transcription and signaling and inhibits cell growth. The increased MAPK output generated by NF2 loss in RAS-mutant cancers may inform therapeutic strategies, as it generates greater dependency on the MAPK pathway for viability. Intensification of mutant RAS signaling through copy-number imbalances is commonly associated with transformation. We show that NF2/merlin inactivation augments mutant RAS signaling by promoting YAP/TEAD-driven transcription of oncogenic and wild-type RAS, resulting in greater MAPK output and increased sensitivity to MEK inhibitors.

Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

Abstract 669: BGB-283 effectively enhances MEK inhibitor induced tumor suppression in RAS mutant cancers

Abstract Mutation in K-RAS, which drives constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway, is one of the most frequent genetic alterations in human cancers. Pharmacological targeting RAS mutants directly is extremely challenging and targeted inhibition of K-RAS downstream effector MEK has shown limited clinical activity in suppressing the progression of K-RAS mutant tumors. The reduced sensitivity of K-RAS-mutated cancer cells to MEK inhibitors (MEKi) is associated with feedback phosphorylation of MEK and rebound of phosphorylated ERK levels after prolonged treatment. A synthetic lethal screen provided the rational of targeting both RAF1 and MEK in K-RAS mutated cancers. In this study, we investigated whether combining BGB-283, a potent inhibitor to RAF dimers, and a series of MEKi could achieve better therapeutic effect of the latter. We confirmed that selumetinib (AZD6244) induced strong MEK phosphorylation in K-RAS mutated cancer cells. Either knockdown of B-RAF/C-RAF or addition of BGB-283 in the presence of selumetinib potently inhibited RAF-dependent MEK phosphorylation and led to sustained inhibition of ERK activity. Furthermore, BGB-283 and selumetinib showed synergistic effect in inhibiting proliferation of several non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines harboring RAS mutations. In vivo, combined dosing of BGB-283 and AZD6244 also exhibited enhanced antitumor activity in K-RAS mutant NSCLC and CRC xenograft models compared to monotherapies. Pharmacodynamic (PD) analysis revealed improved inhibition of ERK phosphorylation in dually treated tumors. Together, these findings suggested that combining RAF dimer inhibitor BGB-283 and MEKi could be a promising strategy in treating RAS mutated cancers. Citation Format: Xi Yuan, Zhiyu Tang, Rong Du, Shing-Hu Cheung, Jing Wei, Yuan Zhao, Yunguang Du, Rui Hao, Xiaoxia Hu, Wenfeng Gong, Yong Liu, Yajuan Gao, Min Wei, Changyou Zhou, Lai Wang, Lusong Luo. BGB-283 effectively enhances MEK inhibitor induced tumor suppression in RAS mutant cancers. [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 669. doi:10.1158/1538-7445.AM2015-669

Melanoma and non-melanoma skin cancers in hairy cell leukaemia: a Surveillance, Epidemiology and End Results population analysis and the 30-year experience at Memorial Sloan Kettering Cancer Center.

Few studies have examined melanoma and non-melanoma skin cancer (NMSC) incidence rates after a diagnosis of hairy cell leukaemia (HCL). We assessed 267 HCL patients treated at Memorial Sloan Kettering Cancer Center (MSKCC) and Surveillance, Epidemiology and End Results (SEER) data for melanoma and NMSC incidence rates after HCL. Incidence data from MSKCC patients demonstrated a 10-year combined melanoma and NMSC skin cancer rate of 11·3%, melanoma 4·4% and NMSC 6·9%. Molecular analysis of skin cancers from MSKCC patients revealed activating RAS mutations in 3/9 patients, including one patient with melanoma. Of 4750 SEER patients with HCL, 55 (1·2%) had a subsequent diagnosis of melanoma. Standardized incidence ratios (SIRs) did not show that melanoma was more common in HCL patients versus the general population (SIR 1·3, 95% CI 0·78-2·03). Analysis of SEER HCL patients diagnosed before and after 1990 (approximately before and after purine analogue therapy was introduced) showed no evidence of an increased incidence after 1990. A better understanding of any potential association between HCL and skin cancer is highly relevant given ongoing trials using BRAF inhibitors, such as vemurafenib, for relapsed HCL, as RAS-mutant skin cancers could be paradoxically activated in these patients.

Discovery of 1-(3,3-dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-6-yl)phenyl)urea (LY3009120) as a pan-RAF inhibitor with minimal paradoxical activation and activity against BRAF or RAS mutant tumor cells.

The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.

The Hippo effector YAP regulates the response of cancer cells to MAPK pathway inhibitors

RAF- and MEK-targeted therapies are approved for patients with BRAFV600E melanoma and under investigation in a several other tumor types, but resistance remains a major challenge. We uncovered yes-associated protein 1 (YAP1) as a mechanism of resistance to RAF-MEK inhibition in BRAF- and RAS-mutant cancers, providing a rationale for co-targeting YAP and RAF-MEK to enhance patient outcomes.

The RAS-RAL axis in cancer: evidence for mutation-specific selectivity in non-small cell lung cancer.

Activating RAS mutations are common in human tumors. These mutations are often markers for resistance to therapy and subsequent poor prognosis. So far, targeting the RAF-MEK-ERK and PI3K-AKT signaling pathways downstream of RAS is the only promising approach in the treatment of cancer patients harboring RAS mutations. RAL GTPase, another downstream effector of RAS, is also considered as a therapeutic option for the treatment of RAS-mutant cancers. The RAL GTPase family comprises RALA and RALB, which can have either divergent or similar functions in different tumor models. Recent studies on non-small cell lung cancer (NSCLC) have showed that different RAS mutations selectively activate specific effector pathways. This observation requires broader validation in other tumor tissue types, but if true, will provide a new approach to the treatment of RAS-mutant cancer patients by targeting specific downstream RAS effectors according to the type of RAS mutation. It also suggests that RAL GTPase inhibition will be an important treatment strategy for tumors harboring RAS glycine to cysteine (G12C) or glycien to valine (G12V) mutations, which are commonly found in NSCLC and pancreatic cancer.

Molecular pathways: targeting RAC-p21-activated serine-threonine kinase signaling in RAS-driven cancers.

Cancers driven by oncogenic Ras proteins encompass some of the most deadly human cancer types, and there is a pressing need to develop therapies for these diseases. Although recent studies suggest that mutant Ras proteins may yet be druggable, the most promising and advanced efforts involve inhibitors of Ras effector signaling. Most efforts to target Ras signaling have been aimed at the ERK mitogen-activated protein kinase and the phosphoinositide 3-kinase signaling networks. However, to date, no inhibitors of these Ras effector pathways have been effective against RAS-mutant cancers. This ineffectiveness is due, in part, to the involvement of additional effectors in Ras-dependent cancer growth, such as the Rac small GTPase and the p21-activated serine-threonine kinases (PAK). PAK proteins are involved in many survival, cell motility, and proliferative pathways in the cell and may present a viable new target in Ras-driven cancers. In this review, we address the role and therapeutic potential of Rac and group I PAK proteins in driving mutant Ras cancers.

Molecular pathways: the basis for rational combination using MEK inhibitors in KRAS-mutant cancers.

Mutations in RAS oncogenes are frequently observed in human cancers, and the mutations result in activation of the RAS-RAF-MEK-ERK pathway, leading to cell proliferation and survival. The pathway is, therefore, a potent therapeutic target in the RAS-mutant cancers. MEK inhibitors can specifically block the pathway and are one of the key types of drugs for the treatment of the RAS-mutant cancers. As RAS proteins activate other downstream signaling proteins in addition to the RAS-RAF-MEK-ERK pathway, combination therapeutic approaches with MEK inhibitors are also being evaluated. Moreover, MEK inhibitors can arrest cancer cells in G1 phase and repress prosurvival Bcl2 family proteins such as MCL1 and BCL2/BCLXL, and increase expression of Bim, a proapoptotic BH3-only family protein. This mechanism may explain the efficacy of the combination of MEK inhibitors with cytotoxic agents or other targeted inhibitors. A better understanding of the pathway will help us with development of rational combinations for the treatment of the RAS-mutant cancers.

Dual targeting of RAF-MEK-ERK and PI3K-AKT-mTOR pathways in RAS-mutant cancers: Preclinical insights and institutional experience from a clinical trial of binimetinib (MEK162) plus BYL719.

e13559 Background: Current development is focused on agents targeting the RAF-MEK-ERK and PI3K-AKT-mTOR pathways. Combinations of these agents may address causes of de novo resistance. Identifying patients (pts) most likely to benefit from these combinations is important. Methods: A panel of 20 cell lines (5 BRAFM, 5 KRASM, 5 PIK3CAM and 5 BRAF/PIK3CA/KRASWT) were exposed to MEK and/or AKT inhibitors and evaluated for inhibition of pathway signaling and cell growth. Inhibition of signaling output of the RAF-MEK-ERK (p-ERK) and PI3K-AKT-mTOR (p-S6) pathways was quantified by ELISA. Growth inhibition was determined using sulforhodamine B assay. Based on the results of preclinical experiments, at our center we prioritized enrolling pts with RAS mutant tumors (including ovarian cancer and non-small cell lung cancer [NSCLC]) into an ongoing phase 1b clinical trial (CMEK162X2109) evaluating the combination of MEK inhibitor binimetinib (MEK162) and the PI3K-α inhibitor BYL719 in pts with RAS and RAF mutant tumor...

Abstract PL04-04: Synthetic lethal approaches to targeting Ras mutant cancers.

Abstract The RAS oncogenes are very frequently activated by mutation in human tumors, which very often show continued dependency on the signaling functions of RAS. In addition to controlling the RAF/MEK/ERK pathway, RAS proteins are established to be important direct regulators of type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino terminal RAS binding domain (RBD). Disruption of PI3K p110alpha activation by oncogenic KRAS has been shown to prevent tumor development in a mouse model of lung cancer. Disruption of RAS-PI3K p110alpha interaction in established lung tumors prevents tumor progression and results in long-term stabilization of lung tumors. Coordinate inhibition of the MAPK pathway at the same time promotes tumor regression and dramatically reduces tumor burden. However, as yet it is unclear whether the combined targeting of pathways that RAS controls, such as RAF/ERK and PI 3-kinase/AKT, will prove effective in the treatment of RAS mutant cancers in the clinic, or whether this approach will prove unacceptably toxic. It is therefore important that other means of targeting this critical oncogene are considered. One such approach is the development of agents that directly inhibit the function of RAS proteins, for example by blocking interaction with effectors. Another approach, which will be explored here, is the identification of unique dependencies of RAS mutant tumor cells through the use of functional genomic screens. We have used RNA interference libraries on a panel of lung cancer cell lines to determine genes that are required for the survival of cells with oncogenic KRAS mutations, but not wild type KRAS. This has led to the identification of signaling networks that are essential for RAS mutant cells, including a transcriptional programme controlled by the GATA2 transcription factor. In mouse models of lung cancer, deletion of GATA2 leads to dramatic regression of RAS induced tumors. Although GATA2 itself is likely to be undruggable, investigation of the transcriptional targets of GATA2 has revealed three key pathways that are much more tractable: the proteasome, NF-kB and Rho signaling. Combined suppression of GATA2 regulated pathways with clinically approved inhibitors of the proteasome and Rho kinase causes marked tumor clearance. Discovery of the non-oncogene addiction of KRAS mutant lung cancers to GATA2 function presents a network of druggable pathways for therapeutic exploitation. We are currently testing combinations of GATA2 functional targeting with other approaches that show potential, such as MEK plus PI 3-kinase inhibition, and immune checkpoint modulators. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL04-04. Citation Format: Julian Downward. Synthetic lethal approaches to targeting Ras mutant cancers. [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 PL04-04.