Abstract B18: Clostridium perfringens lethal toxin specifically targets RAS and disrupts RAS signaling pathway

Clostridium perfringens lethal toxin (TpeL) belongs to the family of large clostridial glucosylating cytotoxins. Clostridial toxins are glucosyltransferases that modify and deactivate small GTPases of the RHO and RAS subfamily. TpeL mono-glucosylates in the switch I domain (Thr35) of RAC and RAS small GTPases. RAS family proteins function as key regulators of cell proliferation, differentiation, survival and gene expression. Moreover, mutations in RAS proteins are highly prevalent in human cancers. Considering the specificity of TpeL for RAS, we decided to investigate the biochemical interaction between TpeL and RAS, and potentially develop TpeL as a tool to disrupt the RAS signaling pathway in vivo. First, we used RAS-dependent MEFs expressing different KRAS mutations and BRAF V600E to assess the specificity of TpeL in vivo. We expected that proliferation should be inhibited in MEF cells expressing RAS isoforms, but MEF cells expressing BRAF V600E should not be affected. We found that TpeL treatment did not affect the viability of the BRAF V600E cells and only induced toxicity in RAS expressing cells. Consistent with this result, TpeL treatment inhibited MAPK signal transduction in the RAS expressing cells, but not in the BRAF V600E cells. Surprisingly, both cell viability as well as pERK levels remained unaffected in the KRAS Q61R MEF cells, suggesting that KRAS Q61R is resistant to TpeL. Furthermore, TpeL treatment of cancer cell lines shows significant differences in potency, with KRAS G12C cell lines being more sensitive to the toxin. Our cellular data suggest a correlation between TpeL sensitivity and intrinsic hydrolysis rate of the KRAS alleles. Biochemical assays corroborate our hypothesis that TpeL preferentially glucosylates KRAS GDP loaded in vitro. Moreover, TpeL glucosylation inhibits GEF-mediated nucleotide exchange, effectively blocking KRAS on the GDP inactive state. To further follow up the correlation between KRAS GDP levels and TpeL sensitivity, we used a combination treatment of TpeL and EGFR/FGFR inhibitors. Combination screen shows a synergistic effect; we observed increased TpeL sensitivity in combination with the inhibitors. By elucidating the mechanism of substrate binding and substrate specificity, we have gained insight into the TpeL-RAS interaction and we are working on developing the toxin as a therapeutic tool. Citation Format: Maria Abreu-Blanco, Ming Yi, Jean-Paul Denson, Matthew Holderfield. Clostridium perfringens lethal toxin specifically targets RAS and disrupts RAS signaling pathway [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 B18.