Sensitivity to poly(ADP-ribose) polymerase (PARP) inhibition identifies ubiquitin-specific peptidase 11 (USP11) as a regulator of DNA double-strand break repair

Abstract

DNA damage repair and checkpoint responses prevent genome instability and provide a barrier to the development of cancer. Inherited mutations in DNA damage response (DDR) genes such as those that encode the homologous recombination (HR) proteins BRCA1 and BRCA2 cause cancer predisposition syndromes. PARP inhibitors are an exciting new class of targeted therapy for treating patients with HR repair-defective tumors. In this study, we use an RNAi screen to identify genes that when silenced cause synthetic lethality with the PARP inhibitor AZD2281. This screen identified the deubiquitylating enzyme USP11 as a participant in HR repair of DNA double-strand breaks. Silencing USP11 with siRNA leads to spontaneous DDR activation in otherwise undamaged cells and hypersensitivity to PARP inhibition, ionizing radiation, and other genotoxic stress agents. Moreover, we demonstrate that HR repair is defective in USP11-silenced cells. Finally, the recruitment of a subset of double-strand break repair proteins including RAD51 and 53BP1 to repair foci is misregulated in the absence of USP11 catalytic activity. Thus, our synthetic lethal approach identified USP11 as a component of the HR double-strand break repair pathway.

FIGURE 1.

USP11 silencing causes AZD2281 hypersensitivity and spontaneous DDR activation. A, diagram of AZD2281 synthetic lethal screen. U2OS cells were split 24 h after transfection with siRNA and treated with AZD2281 for 96 h prior to measurement of cell survival. B, graph of all siRNAs screened. C, survival of USP11-silenced cells compared with non-targeting or BRCA1 siRNA controls after PARP inhibition. D, sensitivity index was calculated as previously described (42). Genes scored as >0.1 were considered as positive for hypersensitivity to AZD2281. E and F, non-targeting and USP11 siRNA were transfected into U2OS cells and γH2AX foci were counted 72 h after transfection (Mock, mock transfection with no siRNA). Graph represents percentage of cells with >5 foci, and error bars are S.D. (n = 3). G, immunoblot analysis of protein knockdown 72 h after transfection of control and USP11 siRNA.

FIGURE 2.

USP11 silencing impairs HR repair at double-strand breaks. A, RAD51 foci were scored by immunofluorescent imaging at the indicated times after treatment with IR (*, p = 0.001; **, p = 0.038). B, mock, non-targeting and USP11-silenced cells were plated in 60-mm dishes and treated with the indicated doses of IR. Colonies of >50 cells were scored 7–10 days after irradiation. Error bars represent S.D. (n = 3). C–E, non-targeting, BRCA1- and USP11-silenced U2OS cells were treated with C, bleomycin (1 μg/ml), D, cisplatin (5 μm), or E, mitomycin C (0.2 μg/ml) for 96 h followed by measurement of viability (*, p < 0.05). Error bars are S.D. (n = 4). F, non-targeting, BRCA1- and USP11-silenced HEK293 cells containing an integrated HR repair substrate were transfected with I-SceI to induce a double-strand break in the reporter gene. The percentage of GFP+ cells was scored by flow cytometry. (Error bars are S.D., n = 3, *, p = 0.046). G, USP11 and BRCA1 protein levels in the cells used in F were measured by immunoblotting.

FIGURE 3.

USP11 regulates 53BP1 foci after IR. A–C, mock, non-targeting and USP11-silenced U2OS cells were treated with IR and allowed to recover for the indicated time prior to fixation and staining with antibodies to A, γH2AX; B, BRCA1; or C, 53BP1 (*, p = 0.0006; **, p = 0.074). The percentage of cells with >5 foci were scored at each time point. Error bars are S.D. (n = 3).

FIGURE 4.

USP11 is a chromatin-associated protein, and its DUB activity is required for its activity in double-strand break repair. A, whole cell extracts (WCE), soluble fractions (S2 and S3) and chromatin fractions (P3) from cells treated with IR and allowed to recover for the indicated times were immunoblotted for USP11, ORC2, and GAPDH. B–E, HA-tagged wild-type (WT), and catalytic inactive (C318S) USP11 cDNAs that are insensitive to the USP11 siRNAs were stably expressed in U2OS cells. B, expression levels of HA-tagged proteins after non-targeting and USP11 siRNA transfections were determined by immunoblotting. C, parental-, WT-, and C318S USP11-expressing cells were transfected with the indicated siRNAs and scored for γH2AX foci in undamaged cells. D and E, parental-, WT-, and C318S-expressing cells were transfected with the indicated siRNAs, irradiated with 1 or 4 Gy of IR, allowed to recover for 15 h, then scored for RAD51 and 53BP1 foci by immunofluorescent imaging. Error bars in C–E are S.D. (n = 3).