Carnie, C. J. | Search bioRxiv

bioRxiv2023

The dCMP deaminase DCTD and the E3 ligase TOPORS are central mediators of decitabine cytotoxicity

The nucleoside decitabine (5-aza-dC) is used to treat several hematological cancers. Upon triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNMT1 DNA-protein crosslinks (DPCs) and DNA hypomethylation. However, 5-aza-dC treatment success varies, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC susceptibility. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation underlies most 5-aza-dC cytotoxicity in wild-type cells. Combining results from a subsequent genetic screen in DCTD-deficient cells with identification of the proximal proteome of DNMT1-DPCs, we uncover the ubiquitin/SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to DNMT1-DPCs in a SUMO-dependent manner and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism and lays the foundations for the development of predictive biomarkers for decitabine treatment.

cell biology↗

bioRxiv2023

The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a stress-responsive hub that inhibits the translation elongation factor eEF2, and consequently mRNA translation elongation, in response to hypoxia and nutrient deprivation. EEF2K is also involved in the response to DNA damage but its role in response to DNA crosslinks, as induced by cisplatin, is not known. Here we found that eEF2K is critical to mediate the cellular response to cisplatin. We uncovered that eEF2K deficient cells are more resistant to cisplatin treatment. Mechanistically, eEF2K deficiency blunts the activation of the DNA damage response associated ATM and ATR pathways, in turn preventing p53 activation and therefore compromising induction of cisplatin-induced apoptosis. We also report that loss of eEF2K delays the resolution of DNA damage triggered by cisplatin, suggesting that eEF2K contributes to DNA damage repair in response to cisplatin. In support of this, our data shows that eEF2K promotes the expression of the DNA repair protein ERCC1, critical for the repair of cisplatin-caused DNA damage. Finally, using Caenorhabditis elegans as an in vivo model, we find that deletion of efk-1, the worm eEF2K ortholog, mitigates the induction of germ cell death in response to cisplatin. Together, our data highlight that eEF2K represents an evolutionary conserved mediator of the DNA damage response to cisplatin which promotes p53 activation to induce cell death, or alternatively facilitates DNA repair, depending on the extent of DNA damage.

molecular biology↗