Abstract 4822: Targeting RAS and downstream signaling in high-grade serous ovarian carcinoma with novel RAS inhibitors
Authors: Mattox Tyler E. , Norton Tiffany S. , Keeton Adam B. , Ward Antonio B. , Maxuitenko Yulia Y. , Berry Kristy L. , Zhu Bing , Musiyenko Alla , Gavin Elaine , Ramirez-Alcantara Veronica , Chen Xi , Valiyaveettil Jacob , Scalici Jennifer , Rocconi Rodney P. , Piazza Gary A. , Silva Luciana Madeira da 
Published: August 2019

Abstract
High grade serous ovarian carcinoma (HGSOC), which accounts for 70-80% of ovarian cancer deaths, is characterized by TP53 mutations and about half of these tumors present defects in homologous recombination DNA repair pathway genes. PARP inhibitors have now become FDA-approved for the treatment of recurrent and BRCA-associated ovarian cancer. Despite providing a major advance in ovarian cancer treatment in recent years, resistance to PARP inhibitors is often encountered and new treatment strategies must be devised. Oncogenic RAS and constitutively active RAS signaling regulate multiple downstream cascades to increase cell proliferation, survival, and malignant transformation. Despite the absence of RAS mutations, upregulation in the RAS pathway is prevalent in HGSOC, suggesting that these represent key, yet under-explored, inhibition targets for the treatment of ovarian cancer. Further bolstering the importance of RAS pathway inhibition are reports on cytotoxic synergistic effects for the combination of MEK and PARP inhibitors in ovarian cancer. The development of inhibitors directly targeting RAS has been hindered by the lack of suitable surfaces on the protein for small molecule binding and its high affinity for GTP binding. We developed a novel series of indene derivatives that showed highly selective growth inhibitory activity in tumor cells harboring constitutively active RAS vs. cells with low levels of active RAS. Chemical optimization led to a series of compounds that potently and selectively inhibit RAS-dependent tumor cell growth by blocking GTP binding to RAS. Here we examined the effects of RAS inhibition by two novel compounds, MCI-059 and MCI-062, in a panel of HGSOC cell lines with variable baseline levels of active RAS. We observed potent growth inhibitory activity for these novel inhibitors in most of the 15 ovarian cancer cell lines tested (IC50s ~25nM and 7nM for MCI-059 and MCI-062, respectively), with the lesser sensitivity in OV-90 cells correlating with its lowest basal levels of active RAS measured by a pull down assay using GST-RAF1-RBD/ glutathione agarose beads. Time-course experiments revealed that MCI-062 induces apoptosis beginning at 18-24 hours. Furthermore, both MCI-059 and MCI-062 inhibited: i) RAS-RAF1-RBD binding in GTPγS loaded recombinant human KRAS; ii) RAS-RAF1-RBD binding in OVCAR8 and SKOV3ip cells under normal culture growth conditions or under serum starvation followed by EGF-stimulation; iii) EGF-stimulated activation of the RAF-MEK-ERK and PI3K-AKT cascades in OVCAR-8 and SKOV3ip cells. We also confirmed that MCI-062 decreases several components of the RAS downstream signaling pathway via RPPA analysis. In summary, our results demonstrate that MCI-059 and MCI-062 inhibit HGSOC cell growth by blocking RAS-effector interactions, and support further evaluation of our novel RAS inhibitors for the treatment of ovarian cancer.
Citation Format: Tyler E. Mattox, Tiffany S. Norton, Adam B. Keeton, Antonio B. Ward, Yulia Y. Maxuitenko, Kristy L. Berry, Bing Zhu, Alla Musiyenko, Elaine Gavin, Veronica Ramirez-Alcantara, Xi Chen, Jacob Valiyaveettil, Jennifer Scalici, Rodney P. Rocconi, Gary A. Piazza, Luciana Madeira da Silva. Targeting RAS and downstream signaling in high-grade serous ovarian carcinoma with novel RAS inhibitors [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 4822.