CRISPR screens identify EXO1 as a therapeutic target for cancers with Fanconi anemia pathway and BRCA1-A complex deficiencies [PM22329]

EXO1 is a conserved multi-functional exonuclease that performs roles in mismatch repair, homologous recombination, nucleotide excision repair, translesion DNA synthesis, resection of stalled replication forks and processing of Okazaki fragments. However, EXO1 loss is well tolerated, suggesting the existence of compensatory mechanisms that could be exploited therapeutically in DNA damage repair (DDR) deficient cancers. Using CRISPR screening, we find that EXO1 loss is synthetic lethal with many somatically inactivated DDR genes identified as frequent vulnerabilities in cancers, including Fanconi anemia (FA) pathway and BRCA1-A complex genes. We also identify the spliceosome factor and tumour suppressor ZRSR2 as synthetic lethal with loss of EXO1 and show that ZRSR2-deficient cells are attenuated for FA-pathway activation, exhibiting cisplatin sensitivity and radial chromosome formation, which we attribute to discreet splicing defects that compromise FA pathway genes. Finally, we show that vulnerabilities caused by mutations in FA or ZRSR2 genes depend on the catalytic activity of EXO1 and can be further sensitised in combination with either PARP inhibitors or ionising radiation. Altogether, we establish the rationale for development of EXO1 inhibitors for treatment across a broad spectrum of cancers with distinct DDR vulnerabilities, which are not effectively targeted with current therapeutic approaches. Overall design: To investigate drivers of synthetic lethal interactions between EXO1 and FANCG or EXO1 and ZRSR2, we performed genome-wide CRISPR rescue screens. eHAP iCas9 EXO1 KO cells, that were previously transduced with Brunello library in triplicate for the dropout screen and frozen, were recovered and transduced with lenti-sgRNA-Hygro with sgRNAs for either FANCG or ZRSR2 or non-targeting control (at MOI of 1 based on predetermined titer). Transduced cells were selected with hygromycin and Cas9 expression was induced with doxycycline. Cells were passaged every 2-3 days (keeping 40 x 10^6 cells representation) and samples were collected 10 days after Cas9 expression induction. Genomic DNA was extracted with the PureLink Genomic DNA Mini Kit (Invitrogen; 16 columns per sample). 200ug gDNA was used for PCRs with Ex Taq polymerase (TaKaRa) to prepare sgRNA libraries following Broad Institute's protocol with P5 mix of oligos and barcoded P7 oligos. Libraries (sgRNA amplicons) were gel purified with QIAquick Gel Extraction Kit (QIAGEN), followed by MinElute PCR Purification kit (QIAGEN) for buffer exchange, and analysed with Agilent 2100 Bioanalyzer.