Abstract B38: Ras clipping by bacterial toxin RRSP reduces viability and proliferation of Ras-dependent cancer cell lines in 2D and 3D in vitro models

Abstract

Abstract Ras GTPases are critical molecular hubs regulating cell proliferation and survival and are among the most frequently mutated oncogenes in human cancers. In recent years, significant efforts have been made to develop Ras-targeting anticancer strategies. Although promising preclinical results were reported for approaches directed against specific KRas mutant variants (e.g., G12C), more effective tools for inhibiting the spectrum of common KRas mutants and/or aberrantly activated wild-type Ras oncoproteins are required to bring Ras-blocking strategies from the bench to the bedside. We discovered the Ras-Rap1-Specific Protease (RRSP) from bacterium Vibrio vulnificus. RRSP site-specifically cleaves the major Ras isoforms (H, N and K) and the most common mutated Ras oncoproteins (G12V, G13D and Q61R) within the Switch I region. In order to bring RRSP into cancer cells, the Melnyk’s group fused RRSP with diphtheria toxin binding subunit B (DTB). DTB is a novel tool for delivery of cargo proteins into the cytosol of target cells via binding to its receptor hHB-EGF (human heparin-binding epidermal growth factor-like growth factor), which is expressed on most human cell types. Successful RRSP intracellular delivery was assessed via Western blot as indicated by Ras cleavage and pERK1/2 dephosphorylation. In MDA-MB-436 (triple-negative breast cancer, KRAS WT) cells with hyperactive Ras signaling, RRSP-DTB efficiently cleaved Ras at concentrations as low as 1 pM. As a consequence of Ras cleavage, we observed pERK1/2 dephosphorylation and decrease in cell viability as shown by crystal violet assay. Similar results were observed in MDA-MB-231 (triple-negative breast cancer, KRAS G13D) cells. A catalytically dead mutant of RRSP carrying an amino acid substitution in its active site (H451A RRSP-DTB) did not show Ras clipping nor decrease in pERK1/2 expression. We also found that RRSP-DTB reduced viability and proliferation of HCT-116 (colorectal, KRAS G13D) cells in 2-dimensional monolayers and 3-dimensional (3D) spheroids in a time- and dose-dependent manner. Notably, 3D spheroids from HCT-116 cells showed a significant reduction in size following RRSP-DTB but not H451A RRSP-DTB treatment. Also, in vivo experiments are currently ongoing to corroborate our in vitro findings. All together our results revealed that, by physically clipping Ras, bacterial toxin RRSP strongly reduces viability and proliferation of Ras-dependent human cancer cell lines bearing either wild-type or mutant KRAS. This work supports further development of RRSP as a potential anticancer therapeutic for a wide variety of Ras-driven tumors. Citation Format: Vania Vidimar, Minyoung Park, Roman A. Melnyk, Karla J.F. Satchell. Ras clipping by bacterial toxin RRSP reduces viability and proliferation of Ras-dependent cancer cell lines in 2D and 3D in vitro models [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B38.