Triple-negative breast cancer (TNBC) is a major cause of death among breast cancer patients that results from intrinsic and acquired resistance to systemic chemotherapies. To identify novel targets for effective treatment of TNBC through combination strategies with MEK inhibitor (AS703026), we used a novel method of combining high-throughput two- and three-dimensional (2D and 3D) RNAi screening. TNBC cells were transfected with a kinome siRNA library comprising siRNA targeting 790 kinases under both 2D and 3D culture conditions with or without AS703026. Molecule activity predictor analysis revealed the PI3K pathway as the major target pathway in our RNAi combination studies in TNBC. We found that PI3K inhibitor SAR245409 (also called XL765) combined with AS703026 synergistically inhibited proliferation compared with either drug alone (P < 0.001). Reduced in vitro colony formation (P < 0.001) and migration and invasion ability were also observed with the combination treatment (P<0.01). Our data suggest that SAR245409 combined with AS703026 may be effective in patients with TNBC. We conclude that a novel powerful high-throughput RNAi assays were able to identify anti-cancer drugs as single or combinational agents. Integrated and multi-system RNAi screening methods can complement difference between in vitro and in vivo culture conditions, and enriches targets that are close to the in vivo condition.

Activating mutations of KRas is the most common proto-oncogenic event in human cancer but there remains no effective therapy for patients harboring mutated KRas (mut-KRas). Despite intense efforts, tight nucleotide binding, few defined pockets, and redundant localization signals have impeded the development of compounds that bind or inhibit KRas. We have identified connector enhancer of kinase suppressor of Ras 1 (Cnk1) as a critical mediator for growth driven by mut-KRas in human cancer cells. Cnk1 co-localizes with mutant KRas at the membrane and deletion of Cnk1 abrogates KRas activation and the activation of the Ras effectors Ral and Rho. Cnk1 deletion caused cells with mutant KRas to accumulate at the G1 checkpoint similar to selective deletion of mutant KRas itself. Following a screen and initial structural optimization a small molecule probe compound PHT-7.3 was identified and shown to bind selectively to the pleckstrin homology PH domain of Cnk1 preventing Cnk1 and mut-KRas co-localization. PHT-7.3 inhibited mut-KRas but not wt-KRas non small cell lung cancer (nsclc) cell growth, and selectively blocks mut-KRas downstream signaling in cells. PHT-7.3 exhibited cytostatic antitumor activity in the mut-KRas(G12S) A549 and mut-KRas(G12V) H441 nsclc xenografts, but not in the wt-KRas H1975 nsclc xenograft. Mut-KRas downstream signaling was inhibited by PHT-7.3 in the xenografts with downregulation of activated Rho and Ral signaling. PHT-7.3 showed further increased antitumor activity in A549 xenografts in combination with erlotinib or trametinib. Thus, the work identifies the PH domain of Cnk1 as a druggable target whose inhibition selectively blocks mutant-KRas activation, and PHT-7.3 as a lead agent in the development of therapies for KRas tumors. Citation Format: Roisin Delaney, Marco Maruggi, Martin Indarte, Robert Lemos, Geoff Grandjean, Lynn Kirkpatrick, Garth Powis. Selective inhibition of mutant KRAS cell and tumor growth by PHT-7.3, an inhibitor of the KRas signaling nanocluster protein Cnk1 [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 3015. doi:10.1158/1538-7445.AM2017-3015

Introduction: KRAS, the predominant form of mutated RAS (mut-KRAS), is found in ∼25% of patient tumors across many cancer types and plays a critical role in driving tumor growth and resistance to therapy. We identified CNKSR1 (connector enhancer of kinase suppressor of Ras 1) to be critical for mut-KRAS but not wild type (wt)-KRAS signaling and cell proliferation. Its product CNK1 is a multi-domain organizer protein found as part of the Ras membrane signaling nanocluster where it binds to mut-KRas although not to wt-KRas, and is necessary for mut-KRas cell growth and signaling. We have exploited the pleckstrin homology (PH)-domain of CNK1 as a target for drug discovery to inhibit mut-KRas. To understand the role of CNK1 as a regulator of KRas cell growth, evaluation of siKRAS and siCNK1 on 2D and 3D growth were evaluated. We also studied the effects of KRAS cell growth conditions on response to selective inhibitors the PH-domain of CNK1 identified using a computational modeling approach. Results: Using a panel of twelve NSCLC lines and siRNA knockdown of KRAS we found that the reported growth addiction of some cancer cell lines for mut-KRAS, and the resistance of others, is most likely an artifact of 2D culture. Mut-KRAS NSCLC cell lines showing addiction in 2D growth did not show addiction in 3D anchorage independent growth in agarose, where all cell lines were more sensitive. Wt-KRAS cell lines were largely unaffected by siKRAS knockdown. Additionally, the CNK1 inhibitor PHT-7390 IC50s for 2D growth inhibition of a panel of 8 mut-KRAS NSCLC lines ranged from 0.60 to 100 μM (ave 18.7 μM) yet was found to be considerably more potent in 3D culture with IC50s from 0.03 to 2.99 μM (ave 0.69 μM). The most pronounced difference was seen in H2009 and Calu1 mut-KRAS cells where PHT-7390 IC50s in 2D were &gt;100 μM, while 0.093 and 0.35 μM in 3D. Wt-KRas NSCLC growth inhibition (3 lines) was largely unaffected by PHT-7390 under either condition with average IC50s of 68 and 69 μM in 2D and 3D conditions, respectively. Target engagement in 2D has shown these CNK1 inhibitors block mut-KRAS signaling but not that of wt-KRAS. Conclusion: Variable enhanced growth dependence on mut-KRAS (“addiction”) is seen in 2D but not 3D. Potent and specific inhibition of mut-KRAS cell line growth by CNK1 PH-domain inhibitors is considerably more robust in 3D culture suggesting a novel approach to inhibit mut-KRAS effect on cancer growth. Citation Format: D. Lynn Kirkpatrick, Roisin Delaney, Geoff Grandjean, Assael Madrigal, Martin Indarte, Mike Scott, Garth Powis. ‘KRAS addiction’ an artifact of 2D culture? Inhibitors of the mut-KRAS NSCLC 3D growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C131.

KRAS is the predominant form of mutated RAS (mut-KRAS) and is found in 25% of patient tumors across many cancer types. Mut-KRAS is well known to play a critical role in driving tumor growth and resistance to therapy, and its effects are so powerful that it overrides the activity of many of the new molecularly targeted signaling drugs being developed for cancer today such that they cannot be used in patients with mut-KRAS. However, despite extensive effort, there is no effective treatment for mut-KRAS. The effects of mut-KRAS are mediated through multiple downstream signaling pathways which have been independently associated with tumorigenesis, including RAF1, RALGDS, and PI3K. Current efforts to treat mut-KRAS tumors employ concurrent treatment with inhibitors of the RAF and PI3K pathways but this does not address the potential contribution of other pathways for which there are currently no inhibitors. Using a global siRNA screen we searched for genes that when inhibited would block the growth of mut-KRAS cancer cells without affecting wild type-KRAS (wt-KRAS) cell growth using an isogenic MiaPaCa-2 pancreatic cell line with and without oncogenic KRAS, and validated these hits in a similar isogenic HCT-116 colon cell line. From the screen we identified CNKSR1 (connector enhancer of kinase suppressor of Ras 1) as a top hit. CNKSR1 is found associated with KRAS in the RAS membrane associated signaling nanocluster that KRAS has to be associated with to provide growth signals. Knockdown of CNKSR1 with siRNA inhibited the growth of a panel of 10 mut-KRAS non small cell lung cancer (NSCLC) cell lines but not of 4 NSCLC cell lines with wt-KRAS. CNKSR1 is a multidomain protein that has a potentially druggable plekstrin homology (PH) domain responsible for binding to membrane phosphatidylinositols-3-phosphates. In order to demonstrate whether the PH domain of CNKSR1 is necessary for mut-KRAS activity we over expressed the PH domain in H1373 mut-KRAS NSCLC cells and found that it acted as a dominant negative and inhibited cell growth. We suggest that the PH domain fragment competes with the full length CNKSR1 in the cell. We also showed that knockdown of CNKSR1 inhibited KRAS dependent phosphorylation of RAF1 in multiple non small cell lung cancer cell lines. Together our results suggest that the CNKSR1 protein, acting through it's PH domain, is necessary for cell growth and down stream signaling by the KRAS oncogene. The PH domain can be targeted by inhibitors thus potentially providing agents that will selectively block mut-KRAS signaling and cell growth, creating a therapeutic potential for patients with oncogenic KRAS for which there is currently no effective therapy. 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 A197.