DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Pola/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

Highlights d VCP and USP7 control the SUMO/ubiquitin landscape during DNA replication d VCP is recruited by FAF1 to chromatin-associated SUMO/ ubiquitylated proteins d FAF1 recognizes both SUMO and ubiquitin on target proteins d The control of DNA replication by VCP FAF1 and USP7 is evolutionarily conserved

Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53-independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.

Diffuse large B cell lymphoma (DLBCL) is the most common aggressive B cell lymphoma and accounts for nearly 40% of cases of B cell non-Hodgkin lymphoma. DLBCL is generally treated with R-CHOP chemotherapy, but many patients do not respond or relapse after treatment. Here, we analyzed the therapeutic potential of the tumor suppressor microRNA-28 (miR-28) for DLBCL, alone and in combination with the Brutons tyrosine kinase inhibitor ibrutinib. Combination therapy with miR-28 plus ibrutinib potentiated the anti-tumor effects of monotherapy with either agent by inducing a specific transcriptional cell-cycle arrest program that impairs DNA replication. Moreover, we found that downregulation of the miR-28-plus-ibrutinib gene signature correlates with better survival of ABC-DLBCL patients. These results provide evidence for the effectiveness of a new miRNA-based ibrutinib combination therapy for DLBCL and unveil the miR-28-plus-ibrutinib gene signature as a new predictor of outcome in ABC-DLBCL patients.