Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction

Abstract

Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds.

Fig 1. Identification and characterization of RPA:RAD52 inhibitors.

(A) Chemical structures of the top eleven FluorIA hits. The strongest inhibitors are boxed (FDA-approved in solid-line, Chembridge dashed-line). (B) Comparison of hits for the inhibition of the RPA:RAD52 interaction at a single concentration (62.5 μM). The FluorIA inhibition data for the full concentration range for each hit was included in S1 Fig.

Fig 2. BRCA1-deficient HCC1937 breast cancer cell line survival assay.

BRCA1-deficient HCC1937 cells corrected with wild-type BRCA1 gene (BRCA1+; closed triangle), and with an empty vector (BRCA1-; closed circle) were examined. For each experiment (Figs 2–4), cell lines were treated with nine concentrations of (A) quinacrine, (B) mitoxantrone, and (C) doxorubicin in 96-well culture plates at 5x10³ cell/well density. Each treatment point was made in sextuplicate. Treated cells were incubated for 72 hours at 37°C before assessing cell viability using PrestoBlue. Data were normalized to vehicle control. The experiment was repeated three times. Error bars indicate the standard deviation. Significantly different data are indicated with an asterisk. EC₅₀ values, estimated from the graphs, are given.

Fig 3. BRCA1-deficient UWB1.289 ovarian cancer cell line survival assay.

BRCA1-deficient UWB1.289 cells corrected with wild-type BRCA1 gene (BRCA1+; closed triangle) and parental BRCA1-deficient UWB1.289 (BRCA1-; closed circle). Two PARPi-resistant cell lines derived from UWB1.289, UWB1.289.SYr12 (closed diamond) and UWB1.SYr13 (X), were also examined. Cell lines were treated with (A) quinacrine, (B) mitoxantrone, and (C) doxorubicin as described in Fig 2.

Fig 4. BRCA2-deficient PE01 ovarian cancer cell line survival assay.

BRCA2-deficient PE01(BRCA2-; closed circle) and BRCA2-revertant PE01 C4-2 (BRCA2+; closed triangle) were examined. Cell lines were treated with (A) quinacrine, (B) mitoxantrone, and (C) doxorubicin as described in Fig 2.

Fig 5. Mitoxantrone induces apoptosis preferentially in the BRCA1-deficient UWB1.289.

UWB1.289 cells with or without restoration of the BRCA1 expression were treated with 0.2 μM of mitoxantrone (MX) for three days. Olaparib (Ola) at 1 μM was used as control for the induction of apoptosis. Near EC₅₀ concentrations were selected for each compound. After the treatment with each compound, cell lysates were prepared and cleaved PARP was detected with (A) anti-cleaved PARP antibody (Cell Signaling Technology #9541) and (B) anti-PARP antibody (Cell Signaling Technology #9542). Tubulin detected with anti-beta-tubulin antibody (Cell Signaling Technology #15115) was used as an internal control. (C) Ratios of cleaved PARP to tubulin were determined with ImageJ and graphed. Three independent experiments were performed and the mean values are shown. The bars indicate standard deviations. Statistical significance of differences was determined with Student t-test (* p<0.05 and ** p<0.01).

Fig 6. The effect of mitoxantrone treatment on RAD52 levels and DNA damage.

(A, C, and E) RAD52 and γH2AX (B, D, and F) levels were probed in the three parental (black) and (BRCA1 or BRCA2 proficient, grey) cell lines indicated was assessed with Simple Western using lysate post 72 h treatment with mitoxantrone at doses near their EC₅₀ values (see Figs 2–4). Data and ANOVA statistical analyses are described in the material and methods section. Significance statistics are shown with single, double, or triple asterisks representing P<0.05, P<0.005, or P<0.0005, respectively.

Fig 7. RAD52 inhibitor mitoxantrone selectively suppresses SSA.

(A) The effect of mitoxantrone (MX) on SSA and HR were studied with cell-based DSB repair assays. U2OS-SA and U2OS-DR cells were pre-treated with mitoxantrone for two hours before transfection of the SceI-expression plasmid. Then cells were grown in the presence of mitoxantrone for three days. DSB repair activities were determined by (B) FACS analysis. FACS data are provided in S3 Fig. (C) Inhibition of the expression of I-SceI (SceI) by mitoxantrone. U2OS-SA cells were pre-treated with indicated concentrations of mitoxantrone for two hours, before I-SceI-expression vector transfection. After 72 hours incubation, the cells were harvested, and lysates were prepared. Lysate from each treatment was analyzed by 12% SDS-PAGE and the expression levels of I-SceI was determined by the western blot using anti-HA antibody. The p70 of RPA was used as a loading control. Ratios of I-SceI to p70 was obtained with ImageJ and are shown at the bottom of the image. C: control experiment without the addition of mitoxantrone. (D, E) Analysis of DSB repair activiies by western blots. Graphs showed relative DSB repair activities with mitoxantrone to control experiments without mitoxantrone. At least three independent experiments were performed. Error bars represent standard deviations. The differences between the SSA inhibition and the HR suppression by mitoxantrone were statistically significant (p<0.05 at 4 nM, and p<0.05 at 3 nM). Western blot data are provided in S4 Fig.

Fig 8. Effect of RAD52 inhibitor mitoxantrone on radiation-induced GFP-RAD52 and GFP-RAD51 foci formation in the HR-proficient ovarian cancer cells.

PE01 C4-2 cells constitutively expressing (A) GFP-RAD52 and (B) GFP-RAD51 were treated with mitoxantrone (MX, 3 nM) alone, radiation alone (IR, 15 Gy, RAD Source RS2000) or both. Cells without mitoxantrone and radiation (labeled C) were controls. The average percent of cells with foci (more than 5 foci per cell) are graphed from three independent experiments. More than 30 cells per experiment were analyzed. Error bars represent standard deviation. GFP-RAD52 and GFP-RAD51 were stably expressed individually in PE01 C4-2 (S5 Fig).

Fig 9. Mitoxantrone disrupts the RPA:RAD52 PPI in cells.

(A) RAD52 was co-immunoprecipitated with RPA in the fractions 5, 8, and 10 in the control experiment. The elution positions of the molecular weight standards, thyroglobulin (669 kDa), apoferritin (443 kDa), beta-amylase (200 kDa), and bovine serum albumin (66 kDa), are indicated below the western blot. The data indicate that RAD52 and RPA were in complexes with sizes of ~400 kDa and ~200 kDa in the control. (B) The treatment of the cells with 3 nM mitoxantrone resulted in the loss of the co-immunoprecipitation of RAD52 with RPA in the large complexex. These data suggest that mitoxantrone disrupted the interaction of RAD52 and RPA in cells.