Genome-wide CRISPR screens reveal synthetic lethality of RNASEH2 deficiency and ATR inhibition

Abstract

Ataxia telangiectasia mutated and RAD3 related (ATR) protein kinase plays critical roles in ensuring DNA replication, DNA repair, and cell cycle control in response to replication stress, making ATR inhibition a promising therapeutic strategy for cancer treatment. To identify genes whose loss makes tumor cells hypersensitive to ATR inhibition, we performed CRISPR/Cas9-based whole-genome screens in 3 independent cell lines treated with a highly selective ATR inhibitor, AZD6738. These screens uncovered a comprehensive genome-wide profile of ATR inhibitor sensitivity. From the candidate genes, we demonstrated that RNASEH2 deficiency is synthetic lethal with ATR inhibition both in vitro and in vivo . RNASEH2-deficient cells exhibited elevated levels of DNA damage and, when treated with AZD6738, underwent apoptosis (short-time treated) or senescence (long-time treated). Notably, RNASEH2 deficiency is frequently found in prostate adenocarcinoma; we found decreased RNASEH2B protein levels in prostate adenocarcinoma patient derived xenograft (PDX) samples. Our findings suggest that ATR inhibition may be beneficial for cancer patients with reduced levels of RNASEH2 and that RNASEH2 merits further exploration as a potential biomarker for ATR inhibitor-based therapy.

Molecular and physiological consequences of faulty eukaryotic ribonucleotide excision repair

“…This is supported by the notion that PARP1 can sense unligated Okazaki fragments due to the presence of DNA nicks between them, and signal for their repair (Hanzlikova et al, 2018). The loss of RNase H2 activity further renders cells hypersensitive to ATR inhibition, which is likely linked to replication stress when rNMPs are encountered by the replisome (Wang et al, 2018;Hustedt et al, 2019).…”

“…This is particularly important given the recent findings with regard to RNase H2 status and sensitivity to PARP or ATR inhibitors, as it may have important consequences with respect to therapeutic approaches in patients (Wang et al, 2018;Zimmermann et al, 2018;Hustedt et al, 2019). This is particularly important given the recent findings with regard to RNase H2 status and sensitivity to PARP or ATR inhibitors, as it may have important consequences with respect to therapeutic approaches in patients (Wang et al, 2018;Zimmermann et al, 2018;Hustedt et al, 2019).…”

“…A detailed analysis of existing human cancer genomes could reveal other types of cancer that coincide with a loss of RNase H2 function. This is particularly important given the recent findings with regard to RNase H2 status and sensitivity to PARP or ATR inhibitors, as it may have important consequences with respect to therapeutic approaches in patients (Wang et al, 2018;Zimmermann et al, 2018;Hustedt et al, 2019). Therefore, it seems that the status of the p53 and/or pRb checkpoints may be critical in the fate of RER-defective cells.…”

“…Mouse xenograft studies confirmed that tumors arising from implanted RER-deficient CLL cells are similarly sensitized. The loss of RNase H2 activity further renders cells hypersensitive to ATR inhibition, which is likely linked to replication stress when rNMPs are encountered by the replisome (Wang et al, 2018;Hustedt et al, 2019). However, substrates for PARP1 could also arise in a TOP1-independent manner through spontaneous hydrolysis of DNA at unrepaired rNMPs.…”

Identification of MCM8IP, an interactor of MCM8-9 and RPA1 that promotes homologous recombination and DNA synthesis in response to DNA damage

“…Our work identifies the importance of the MCM8IP-MCM8-9 complex in mediating cellular resistance to cisplatin and olaparib. Interestingly, MCM8IP and MCM8-9 have recently been identified in genetic screens for mediators of ATR inhibitor and temozolomide resistance 66,67 . Collectively, these studies implicate a broad role for the MCM8IP-MCM8-9 complex in promoting chemoresistance to clinically relevant chemotherapeutic agents.…”