Abstract 879: SLFN12-mediated depletion of tRNA-Leu-TAA by velcrin treatment induces ribosome pausing at Leu-TTA codons and represses global translation initiation

Abstract

Abstract Velcrin compounds kill cancer cells expressing high levels of PDE3A and SLFN12 by inducing complex formation between these two proteins, but the mechanism of cancer cell killing by the PDE3A-SLFN12 complex is not fully understood. We previously determined that SLFN12 is an RNase, that PDE3A binding upregulates SLFN12 RNase activity, and that SLFN12 RNase activity is required for velcrin response. Here, we report that the depletion of tRNA-Leu-TAA by velcrin treatment induces ribosome pausing at Leu-TTA codons and global inhibition of protein synthesis. We performed ribosome profiling in HeLa cells treated with DMSO or DNMDP and found that ribosome footprints containing Leu-TTA codons at the A-site of the ribosome were significantly more abundant in DNMDP-treated cells than in cells treated with DMSO, indicating a pausing or stalling of ribosomes at Leu-TTA upon velcrin treatment. Although the overall number of ribosome pausing sites across the transcriptome was doubled by DNMDP treatment, ribosome pausing at Leu-TTA was increased &gt 1000-fold, also resulting in an increased number of genes with TTA pause sites. Even though Leu-TTA is a rare codon, over 70% of all transcripts contain at least one TTA codon, implying that ribosome pausing at TTA codons could directly impact many genes. Genes with TTA pause sites most enriched upon velcrin treatment prominently include those involved in control of mRNA translation and protein homeostasis. These results imply that, in addition to direct effects on mRNA translation due to ribosome pausing, indirect effects could occur due to decreased availability of particular components of the translation machinery, resulting in a widespread impact on protein synthesis. To test our hypothesis, we measured the effects of velcrin treatment on nascent protein abundance. Treatment of HeLa cells with DNMDP resulted in downregulation of newly synthesized proteins, whereas no significant changes in total protein content were observed. Downregulation of protein translation was observed only in velcrin-sensitive cell lines, indicating that inhibition of protein translation could be the mechanism of action of cancer cell killing by PDE3A-SLFN12 complex formation. Citation Format: Stephanie Hoyt, Sooncheol Lee, Joseph McGaunn, Andrew Cherniack, Matthew Meyerson, Heidi Greulich. SLFN12-mediated depletion of tRNA-Leu-TAA by velcrin treatment induces ribosome pausing at Leu-TTA codons and represses global translation initiation [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 879.