Characterization of DNA damage sensing mechanism by SLFN11 and its regulation by post-translational modifications

SLFN11 was initially identified as a strong retrovirus inhibitor and, only recently, emerged as an important determinant of cancer cell sensitivity to DNA damaging agents (DDAs). Strikingly, in nearly 50% of cancer cell lines SLFN11 is transcriptionally silenced, conferring chemoresistance. SLFN11 anti-viral activity was shown to depend on its nuclease domain targeting tRNAs and, thus, inhibiting viral protein synthesis.
Additionally, recent structural data revealed that SLFN11 also possesses single-stranded DNA (ssDNA) binding and a helicase/ATPase domain, which appears inactive in vitro. Other studies have reported several functions for SLFN11, such as recruitment to replication forks, replication blockage and chromatin opening.
However, several discrepancies between these reports remain unsolved, such as which factors are bona-fide SLFN11 interactors and what is the role of SLFN11 ATPase activity in vivo. Notably, the Boulton lab recently identified SLFN11 in genetic interactions screens for DNA damage response genes (manuscripts in preparation) and found that different cell lines respond differently to DDAs, regardless of SLFN11 expression. Therefore, the mechanisms by which SLFN11 responds to DNA damage and induces chemosensitivity remains to be elucidated.
First, I will investigate the substrate(s) responsible for SLFN11 recruitment to chromatin using C-trap optical tweezers. In parallel, I will treat cells with different classes of DDAs to determine which conditions lead to SLFN11 chromatin recruitment and/or tRNase/ATPase activation. Lastly, I will identify post-translational modifications and interactors that regulate SLFN11 chromatin recruitment and activity via immunoprecipitation coupled with mass spectrometry.
Overall, this work will contribute to a deeper understanding of the molecular mechanism of a crucial determinant of chemosensitivity and, thus, has the potential to impact therapeutic strategies aimed at overcoming chemoresistance.