Abstract B046: Dimerization of SLFN12 by the PDE3A-SLFN12 complex is required for velcrin-induced toxicity in cancer cells

Abstract Our group discovered a class of compounds, called velcrins, that kill cancer cells expressing elevated levels of PDE3A and SLFN12. Velcrins bind PDE3A and induce PDE3A-SLFN12 complex formation, resulting in an upregulation of the RNase activity of SLFN12. We recently identified the physiological substrate of SLFN12 as the four isodecoders of tRNA-Leu-TAA. Velcrin treatment of biomarker-positive cancer cell lines activates the SLFN12 RNase, resulting in degradation of tRNA-Leu-TAA, ribosome stalling at cognate TTA codons in mRNA, and global inhibition of protein synthesis, culminating in cell cycle arrest and apoptosis. We hypothesized that the primary function of PDE3A in the PDE3A-SLFN12 heterotetramer is the dimerization of SLFN12. Based on our cryo-EM structure of the PDE3A-SLFN12 complex, we identified SLFN12 I131 as a critical residue in the SLFN12 homodimer interface. We showed that a SLFN12 I131Q mutation disrupts SLFN12 dimerization and prevents tRNA-Leu-TAA cleavage in vitro. We have also shown that wild-type SLFN12 can dimerize independently of PDE3A or PDE3A-velcrin interactions. In PDE3A-depleted cells, the overexpression of wild-type SLFN12 results in cell death, but expression of SLFN12 I131Q does not lead to cellular toxicity. To further show the importance of SLFN12 dimerization for its catalytic activity, we prepared cell lines expressing truncated SLFN12 linked to FKBP-based dimerization domain. These chemical dimerization experiments demonstrated that the forced dimerization leads to depletion of tRNA-Leu-TAA, translation inhibition and apoptotic cell death, mimicking the mechanism of velcrin-induced apoptosis. Citation Format: Kristyna Kotynkova, Sawyer Andersen, Daniel Denney, Colin Garvie, Xiaoyun Wu, Sooncheol Lee, Joseph McGaunn, Matthew Meyerson, Heidi Greulich. Dimerization of SLFN12 by the PDE3A-SLFN12 complex is required for velcrin-induced toxicity in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B046.