Abstract 2671: Targeting the KRAS signaling naocluster protein CNKSR1 provides antitumor activity against mutant KRAS xenografts
Authors: Triana-Balzer Gallen , Indarte Martin , Scott Mike , Powis Garth , Kirkpatrick D. Lynn 
Published: May 2015

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. Its effects are so powerful that it overrides the activity of many molecularly targeted signaling drugs being developed for cancer today such that they cannot be used in patients with mut-KRAS. We have identified genes that when inhibited block the growth of mut-KRAS cancer cells without affecting wild type-KRAS (wt-KRAS) cell growth using a global siRNA screen. A top hit was CNKSR1 (connector enhancer of kinase suppressor of Ras 1), a protein associated with KRAS in the RAS membrane signaling nanocluster. siRNA knockdown of CNKSR1 shows selective inhibition of the growth of mut-KRAS versus wilt type non-small cell lung cancer (NSCLC) cell lines. CNKSR1 is a multidomain protein with a pleckstrin homology (PH)-domain, a 100 to 120 amino acid, highly conserved but low sequence identity 3D superfold. This domain is found in a number of signaling proteins, and is responsible for binding to membrane phosphatidylinositol-3-phosphates (PIP2/3). Over expression of the PH-domain of CNKSR1 in H1373 mut-KRAS NSCLC cells inhibits cell growth suggesting a dominant negative activity, and that the PH-domain of CNKSR1 is necessary for the effects on mut-KRAS activity. PHusis is developing PH-domain inhibitors of signaling proteins through use of its in silico computational modeling platform PhuDock, and has used this approach to identify inhibitors that bind to the PH-domain of CNKSR1. Through rounds of compound refinement we have been optimizing the activity of leads using surface plasmon resonance spectroscopy (SPR), in vitro cell line screening and in vivo xenograft evaluation. Micromolar binders have been improved to nanomolar inhibitors (SPR) that also demonstrate in vitro cytotoxicity. The initial lead PHT-782 (Kd1.8 μM) inhibited mut-KRAS signaling and growth of mut-KRAS isogenic NSCLC cell lines (IC50 ∼30 μM) as effectively as siRNA knockdown of KRAS. Importantly, PHT-782 did not block the growth of wt-KRAS cell lines at concentrations up to 100 μM. In mice PHT-782 exhibited oral bioavailability and had moderate antitumor activity against a mut-KRAS xenograft model without observable toxicity. Refinement has provided PHT-7571 and PHT-7327 with improved PH-domain binding. Additionally, PHT-7327 displays more potent in vivo antitumor activity, both as a single agent in mut-KRAS xenografts and in combination with chemotherapy or EGRF inhibitors. Thus, our studies have shown that the PH-domain of CNKSR1 can be targeted by small molecule inhibitors, selectively blocking mut-KRAS signaling and cell growth, thus creating a novel therapeutic potential for patients with oncogenic KRAS for which there is currently no effective therapy.

Citation Format: Gallen Triana-Balzer, Martin Indarte, Mike Scott, Garth Powis, D. Lynn Kirkpatrick. Targeting the KRAS signaling naocluster protein CNKSR1 provides antitumor activity against mutant KRAS xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2671. doi:10.1158/1538-7445.AM2014-2671