Abstract B23: DC070-547, a novel Ras inhibitor potently and selectivity inhibits colon tumor growth in vitro and in vivo

Abstract

Abstract Introduction: Activating mutations in Ras oncogenes play a critical role in the development of colorectal cancer and are an indicator of poor prognosis. Constitutive activation of Ras proteins is also associated with resistance to chemotherapy and radiation in which treatment options are limited. Despite extensive efforts, no drugs have been successfully developed that target these aberrant gene products, in part because of the high affinity of Ras binding to GTP, which is essential for Ras activation. Using a phenotypic screening assay designed to select for Ras inhibitors and follow-up chemical optimization, a novel Ras inhibitor, DC070-547 was identified, which potently and selectively inhibits the growth of colon tumor cells with activated Ras in vitro and in vivo. Methods: Tumor cell growth inhibitory activity of DC070-547 was measured in a panel of human colon tumor cell lines using the CellTiter Glo assay following 72 h of treatment. Ras activation levels were measured by precipitating GTP-bound Ras from human colon tumor cell lysates with GST-Raf1-RBD/GSH Sepharose followed by western blotting using anti-Ras antibody. Isogenic cell lines were established by transfecting Ras wild-type HT-29 colon tumor cells with mutant H-Ras. Disruption of Ras-Raf binding was determined by pre-incubating cell lysates or intact cells in the presence of DC070-547 for 30 or 45 min followed by Ras pull-down with GST-Raf beads and western blotting using anti-Ras or an anti-GST antibody as a loading control. Cell cycle arrest and apoptosis were measured by DNA content and annexin V levels, respectively. Antitumor activity was determined in a mouse xenograft model subcutaneously implanted with mutant K-Ras HCT-116 colon tumor cells. Mice were treated with DC070-547 administered i.p. for 14 days at a dose of 2.5 mg/kg bid in a co-solvent formulation. Results: DC070-547 potently inhibits the growth of mutant K-Ras HCT-116 and other colon tumor cell lines having constitutively activated Ras with IC50 values as low as 2 nM and selectivity indices of approximately 100-fold for HT-29 and other colon tumor cells lacking activated Ras. Among a panel of six colon tumor cell lines, a strong correlation was measured between IC50 values for growth inhibition and the level of Ras activation, suggesting that activated Ras is the primary target. Isogenic cell line pairs involving transfecting Ras wild-type cells with mutant H-Ras confirmed that sensitivity to DC070-547 required activated Ras. DC070-547 also blocked Ras-Raf binding in cell lysates and intact cells at concentrations that inhibit the growth of tumor cells with activated Ras. The mechanism of growth inhibition by DC070-547 appears to involve mitotic arrest and apoptosis. Moreover, cells obtained from normal colon mucosa were essentially refractory to treatment with DC070-547. In a mouse xenograft model involving mutant K-Ras human HCT-116 colon tumors, DC070-547 was well tolerated and caused a sustained inhibition of tumor growth in which there was complete tumor regression in 3 of 7 mice. Conclusions: DC070-547 potently and selectively inhibits the growth of colon tumor cells with constitutively activated Ras by disrupting Ras-effector interactions and represents a first-in-class drug development candidate for the treatment of Ras-driven colorectal cancer. Citation Format: Veronica Ramirez-Alcantara, Adam B. Keeton, Bing Zhu, Kevin J. Lee, Joshua Canzoneri, Ashley S. Lindsey, Luciana Madeira da Silva Barnes, Kristy Berry, Jacob Valiyaveettil, Xi Chen, Michael R. Boyd, Gary Piazza. DC070-547, a novel Ras inhibitor potently and selectivity inhibits colon tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B23.