Abstract 867: Velcrin-induced cleavage of tRNA-Leu-TAA by SLFN12 RNase causes cancer cell death

Abstract Velcrins are small molecules that induce apoptosis of cancer cells expressing high levels of phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) by inducing PDE3A-SLFN12 complex formation. Recently, we found that SLFN12 is an RNase and PDE3A binding dramatically activates SLFN12 RNase activity, resulting in velcrin-mediated apoptosis. However, the mechanism of cancer cell death by the PDE3A-SLFN12 complex is poorly understood. In this study, we found that SLFN12 specifically recognizes tRNA-Leu-TAA and induces its cleavage in velcrin-sensitive cell lines. According to tRNA-sequencing, tRNA-Leu-TAA isodecoders decreases by 2-fold to 5-fold in Hela cells treated with DNMDP compared to DMSO control, while other tRNAs are not differentially expressed. Down-regulation of tRNA-Leu-TAA by velcrin treatment is observed only in velcrin-sensitive cell lines but not insensitive cell lines. Furthermore, we tested whether SLFN12 digests tRNA-Leu-TAA in vitro. Purified wild-type SLFN12 cleaves tRNA-Leu-TAA but not heat-denatured or catalytically inactive SLFN12. Even though tRNA-Leu-TAA is weakly digested by low concentrations of SLFN12, co-incubation with purified PDE3A increases SLFN12 RNase activity, which is even further upregulated by velcrin treatment. To verify specificity for tRNA-Leu-TAA, we assessed activity of SLFN12 against a panel of in vitro transcribed tRNAs. We found that purified SLFN12 is significantly more efficient at degrading tRNA-Leu-TAA when compared with tRNA-Leu-TAG or tRNAs for other amino acids. Digestion of tRNA-leu-TAA by SLFN12 could inhibit mRNA translation due to the delivery of leucine amino acid to elongating ribosomes. To test this hypothesis, we designed the mutant tRNA-Leu-[CAG:TAA] in which the anticodon of tRNA-Leu-CAG was replaced by TAA anticodon. Ectopic expression of wild-type tRNA-Leu-TAA or tRNA-Leu-CAG has no effect on viability of DNMDP-treated cells, whereas expression of tRNA-Leu-[CAG:TAA] genes partially rescues the cells from DNMDP-induced cytotoxicity. These data confirm that impaired leucine delivery is responsible at least in part for the observed effects of DNMDP treatment. Citation Format: Sooncheol Lee, Stephanie Hoyt, Xiaoyun Wu, Colin Garvie, Joseph McGaunn, Andrew D. Cherniack, Matthew Meyerson, Heidi Greulich. Velcrin-induced cleavage of tRNA-Leu-TAA by SLFN12 RNase causes cancer cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 867.