CRISPR screens identify EXO1 as a therapeutic target for cancers with Fanconi anemia pathway and BRCA1-A complex deficiencies [PM20138]

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 synthetic lethal interactions with EXO1 loss, we performed a genome-wide CRISPR-Cas9 dropout screen. Wild type and EXO1 KO cell lines were transduced with Brunello library from Addgene containing sgRNAs targeting 19114 genes, with 4 sgRNAs per gene, and with 1000 non-targeting sgRNAs. Transductions (performed in a biological triplicate on cells that were passaged separately through expansion) were achieved through transduction of 100 x 10^6 cells in 12 well plates at MOI of 0.4 for 500x representation of each sgRNA (titer required for both cell lines was determined prior to experiment). Cells with viruses were spun at 290 x g for 90 minutes at 37 degrees. The following day cells were trypsinised to transfer into 5-layer flasks and puromycin was added for selection. 40 x 10^6 cells for each replicate was passaged onwards every two days to ensure that sgRNA representation is maintained. Replicates were then treated with doxycycline (1ug/ml final concentration) for 6 days. Samples were collected at day 6 and day 16 for library preparation and sequencing (harvesting 60 x 10^6 cells per sample). 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 amplicon 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.