AbstractApproximately 45% of colorectal cancers harbor mutations in the KRAS gene, resulting in constitutive activation of RAS signaling through effector proteins, primarily RAF and PI3K. Members of the RAS family are GTPases that function as a molecular switch, cycling between inactive (GDP-bound) and active (GTP-bound) states in cells to regulate proliferation and survival. Constitutive signaling from mutant RAS drives tumorigenesis, in part, by deregulation of the cell cycle, resulting in increased proliferation (mitosis) and decreased apoptosis of tumor cells. From an extensive medicinal chemistry/screening campaign, we identified a novel compound class that potently and selectively inhibits the growth of tumor cell lines harboring constitutively activated RAS by a mechanism involving the disruption of RAS nucleotide loading, blockage of effector activation, and consequent inhibition of signaling. A prototype of the class, MCI-062, inhibits the growth of KRAS-driven (HCT-116) colon tumor cells with low nanomolar IC50 values, while RAS-independent (HT-29) colon tumor cells harboring the BRAF V600E mutation, are essentially insensitive. A strong correlation was measured among a larger panel of tumor cell lines between potency to inhibit tumor cell growth and levels of activated RAS. The growth inhibitory effects of MCI-062 were sustained and irreversible as demonstrated by colony formation and apoptosis assays. MCI-062 treatment of HCT-116 colon tumor cells reduced levels of activated RAS and RAS-mediated signaling as measured by GST-RBD pulldown assays and phospho-specific immunoblotting. Within the same concentration range, MCI-062 induced mitotic arrest as measured by cell cycle analysis of DNA content and phospho-histone H3B immunofluorescence. Further analysis revealed that MCI-062 interfered with localization of the mitosis-inducing protein, PLK1 to kinetochores and decreased nuclear localization of its substrate, Cdc25C, a downstream target of RAS-RAF signaling involved in both mitotic entry and exit checkpoints. In vivo testing of MCI-062 in a syngeneic mouse model of KRAS-driven colon cancer (CT-26) demonstrated that MCI-062 engages its molecular target, depleting GTP-RAS and suppressing activation of the MAPK signal transduction pathway, and inhibiting tumor growth. MCI-062 also suppresses PD-L1 expression and activates anti-tumor immunity, which may contribute to its antitumor activity and suggests potential benefits of combining with immunotherapy. From these studies, we have identified a novel class of RAS inhibitors that potently and selectively inhibits RAS-driven tumor growth by disrupting downstream signaling, leading to cell cycle arrest and apoptosis. These findings support further development of MCI-062 for treatment of RAS-driven colorectal and other cancers. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638.Citation Format: Adam B. Keeton, Antonio Ward, Xi Chen, Jacob Valiyaveettil, Bing Zhu, Veronica Ramirez-Alcantara, Yulia Maxuitenko, Kristy Berry, Tyler E. Mattox, Michael R. Boyd, Gary A. Piazza. A novel RAS inhibitor, MCI-062, inhibits colon tumor growth in vivo and activates antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2707.
