Human BRCA2 Knockout Cell Line-DLD-1

Poly ADP-ribose polymerase 1 (PARPi) inhibitors are highly efficient in killing cells deficient in homologous recombination (HR). As a result, PARPi have been used successfully in the clinic to treat BRCA2-mutant tumors. However, positive responses to PARPi are not universal, even in patients with HR defects. Here, researchers present results from a genome-wide CRISPR knockout and activation screen that reveals genetic determinants of PARPi response in wild-type or BRCA2 knockout cells. Surprisingly, the report reports that depletion of the ubiquitin ligase HUWE1 or the histone acetyltransferase KAT5 (the top hits from the screen) strongly reverses PARPi sensitivity caused by BRCA2 deficiency. The researchers identify distinct mechanisms of resistance, where HUWE1 loss increases RAD51 levels to partially restore HR, while KAT5 depletion rewires double-strand break repair by promoting 53BP1 binding to double-strand breaks. This work provides a comprehensive set of putative biomarkers that advance the understanding of PARPi responses and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.

Few studies have reported proteins that cause PARPi resistance when overexpressed. Interestingly, however, the best gene in the researchers' screen, ABCB1, had previously been identified as a mechanism of PARPi resistance. This gene encodes the protein MDR-1 (multidrug resistance protein 1), a drug efflux pump whose overexpression has been associated with acquired resistance to olaparib in ovarian cancer cell lines. To validate these results, the researchers generated HeLa BRCA2KO cells with transcriptional activation of the ABCB1 gene (Figure 1a). They found that the BRCA2 knockout, ABCB1-overexpressing cell line was as resistant to PARPi as the BRCA2-proficient parental HeLa line (Figure 1b). Previously, PARPi treatment has been shown to induce apoptosis in BRCA2-deficient cells. Consistent with this, the study found that olaparib treatment led to a more than sixfold increase in cells positive for Annexin V, a marker of apoptosis, in BRCA2 knockout cells. However, overexpression of ABCB1 restored olaparib-induced apoptosis to control levels (Figure 1c). Overall, these findings validated the results of the CRISPR activation screen.

Figure 1. Overexpression of ABCB1, the top hit from the CRISPR activation screen, causes PARPi resistance in BRCA2 knockout cells. (Clements K E, et al., 2020)