Abstract 1238: A novel Ras inhibitor potently and selectively suppresses lung tumor cell growth by blocking Ras-Raf binding

Abstract

Abstract Mutations in ras genes that result in constitutive activation of Ras proteins are key drivers of oncogenesis, but no effective drugs have been developed that target these aberrant gene products. Screening using a phenotypic assay designed to select for Ras inhibitors and iterative chemical synthesis, identified a preclinical drug development candidate with attractive drug-like properties designated as DC070-547. Here we evaluated the sensitivity of non-small cell lung cancer (NSCLC) cell lines to DC070-547. The compound potently suppresses the growth of human A549, H460, HOP62, H1299, H1975 lung tumor cells having high levels of activated Ras with low nanomolar IC50 values. By contrast, normal human airway epithelial cells (NHAECs) and H322 lung tumor cells with low levels of activated Ras displayed IC50 values in the mid micromolar range. The Ras selectivity of DC070-547 was confirmed by ectopic expression of mutant H-Ras-G12V (H-Rasm) in H322 cells using a retroviral construct, in which the IC50 value of DC070-547 for growth inhibition was reduced approximately 2,500 fold compared with vector control cells. To study the mechanism of action, Ras activation status was measured by Ras-Raf pull-down assays using GTP-bound Ras from cell lysates with GST-Raf1-RBD/GSH sepharose and followed by western blotting with Ras antibodies. Direct binding to Ras was apparent by the ability of DC070-547 to inhibit Ras-Raf binding at concentrations that inhibit Ras-dependent tumor cell growth. Pull-down assays also showed a dose-dependent inhibition of Ras-GTP levels in intact H-Rasm-H322 cells treated with DC070-547 at the same concentration range. Western blot analysis of Ras-immunoprecipitated proteins revealed that DC070-547 also attenuated high levels of EGFR phosphorylation at Y1068 in H-Rasm-H322 cells but not in control cells. These results demonstrate novel Ras inhibitory activities of DC070-547 in NSCLC cell lines. DC070-547 and its pipeline analogs are being evaluated for antitumor efficacy in preclinical mouse models of lung cancers. Citation Format: Bing Zhu, Xi Chen, Veronica Ramirez-Alcantara, Kevin Lee, Jacob Valiyaveettil, Joshua Canzoneri, Sara Sigler, Kristy Berry, Ashley Lindsey, Adam Keeton, Michael R. Boyd, Gary A. Piazza. A novel Ras inhibitor potently and selectively suppresses lung tumor cell growth by blocking Ras-Raf binding. [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 1238.