Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

Cancer Therapy Resistance Cluster and Bern Center for Precision Medicine Department for Biomedical Research University of Bern 3088 Bern Switzerland

Department of Molecular Genetics Erasmus MC Cancer Institute Erasmus University Medical Center 3015GD Rotterdam The Netherlands

Division of Molecular Carcinogenesis The Netherlands Cancer Institute 1066CX Amsterdam The Netherlands

Division of Molecular Pathology The Netherlands Cancer Institute 1066CX Amsterdam The Netherlands

Institute of Animal Pathology Vetsuisse Faculty University of Bern 3012 Bern Switzerland

Laboratory of Genome Dynamics Institute of Molecular Genetics of the Czech Academy of Sciences 142 20 Prague 4 Czech Republic

Oncode Institute Amsterdam The Netherlands

The Netherlands Cancer Institute Robotics and Screening Center The Netherlands Cancer Institute 1066CX Amsterdam The Netherlands

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