Abstract C131: ‘KRAS addiction’ an artifact of 2D culture? Inhibitors of the mut-KRAS NSCLC 3D growth

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 u003e100 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.