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Targeting radioresistance and replication fork stability in prostate cancer

The bromodomain and extraterminal (BET) family of chromatin reader proteins bind to acetylated histones and regulate gene expression. The development of BET inhibitors (BETi) has expanded our knowledge of BET protein function beyond transcriptional regulation and has ushered several prostate cancer (PCa) clinical trials. However, BETi as a single agent is not associated with antitumor activity in patients with castration-resistant prostate cancer (CRPC). We hypothesized novel combinatorial strategies are likely to enhance the efficacy of BETi. By using PCa patient-derived explants and xenograft models, we show that BETi treatment enhanced the efficacy of radiation therapy (RT) and overcame radioresistance. Mechanistically, BETi potentiated the activity of RT by blocking DNA repair. We also report a synergistic relationship between BETi and topoisomerase I (TOP1) inhibitors (TOP1i). We show that the BETi OTX015 synergized with the new class of synthetic noncamptothecin TOP1i, LMP400 (indotecan), to block tumor growth in aggressive CRPC xenograft models. Mechanistically, BETi potentiated the antitumor activity of TOP1i by disrupting replication fork stability. Longitudinal analysis of patient tumors indicated that TOP1 transcript abundance increased as patients progressed from hormone-sensitive prostate cancer to CRPC. TOP1 was highly expressed in metastatic CRPC, and its expression correlated with the expression of BET family genes. These studies open new avenues for the rational combinatorial treatment of aggressive PCa.

. The role of BET proteins and the effect of BETi treatment on HR DNA repair pathway in PCa cell lines.
(A) HR DNA repair pathway activity was analyzed using HR QPCR assay in PCa cell lines LNCaP, VCaP, 22Rv1 and DU145 upon BETi treatment. (B) HR DNA repair pathway activity was analyzed using HR QPCR assay in PCa cell lines LNCaP, VCaP, 22Rv1 and DU145 upon various siRNA treatments. (C) siRNA knockdown validation. Unpaired two-tailed Student's t-test was applied for all panels (***P < 0.001; **P < 0.01; *P < 0.5; Error bars, SD of three technical replicates for all panels). (A) Representative images of RAD51 nuclear foci in mock-irradiated or gammairradiated U2OS cells. Cells were treated with JQ1 (1 µM or 10 µM) for 24 hrs prior to IR treatment. Cells were immunostained with an anti-RAD51 antibody (red) and the nuclei were stained with DAPI (blue). Cells were analyzed 4 hours post IR treatment. Scale bar = 20 µm. (B) Quantification of RAD51 foci in U2OS cells treated with JQ1 for 24 hrs prior to IR. Cells were analyzed 4 hrs post IR-treatment. (C) Quantification of RAD51 foci in U2OS cells that were simultaneously co-treated with JQ1 and IR. Cells were analyzed 4 hrs post JQ1+IR treatment. (D) Quantification of RAD51 foci in U2OS cells that were treated by dBET1 24 hrs prior to IR. Cells were analyzed 4 hrs post IRtreatment.  (A) Overexpression of RAD51 in U2OS cells was verified by QPCR. ***P < 0.001, Student's t-test. (B) Representative images for RAD51 foci analysis in U2OS cells upon RAD51 over-expression with or without irradiation (IR 6 Gy). pcDNA3.1 was used as a mock control. Cells were analyzed 4 hrs post IR treatment. (C) Homologous Recombination (HR) activity was analyzed upon RAD51 over-expression in LNCaP, VCaP, 22Rv1 and DU145 PCa cells. ***P < 0.001, **P < 0.01, Student's t-test. (D) Over-expression of RAD51 in the four cell lines was verified by QPCR. ***P < 0.001, **P < 0.01, Student's t-test. (A-E) Relative transcript expression of the indicated DNA repair genes upon JQ1 treatment was analyzed by QPCR in LNCaP, VCaP, 22Rv1 and DU145 PCa cells. ***P < 0.001, **P < 0.01, *P < 0.05, Student's t-test (top panel). Integrated genome view of the indicated genes in VCaP cells. Each row represents RNA Pol II Chip-Seq data with the indicated treatment. The effect of JQ1 + CPT co-treatment in the four PCa cell lines was tested. CI > 1 indicates antagonism; CI = 1 indicates additive effect; CI < 1 indicates synergism. Figure S8. DNA fiber assay with dBET1 and LMP400 in LNCaP cells.

Figure S9. Expression of TOP1 protein in various cancers.
TOP1 protein expression was analyzed in breast cancer, ovarian cancer, lung adenocarcinoma and colon cancer. Box plots were adapted from UALCAN (Two-tailed Student's t-test; ***P < 0.0001).      Tables   Table S1. Related to Figure 1A-B. Baseline characteristics of prostate cancer patients whose primary prostate cancer explants were analyzed.

DNA fiber assay
The DNA fiber assays were performed in LNCaP and 22Rv1 cell lines using published protocols (

Cell proliferation assay
Cell proliferation assays were performed using the IncuCyte® S3 Live-Cell Analysis System (Sartorius/ Essen BioScience). Data analysis was conducted using IncuCyte S3 2018B software (Sartorius/ Essen BioScience) together with Graphpad Prism version 8 for windows. Combination Index (CI) was calculated using CompuSyn software (4).

HR & NHEJ flow cytometry assay and HR QPCR assay
HR DNA repair activity was evaluated by transfection of a plasmid harboring an I-SceI endonuclease in engineered HEK293 cells expressing a GFP reporter, DR-GFP, followed by flow cytometry analysis as described elsewhere (5).
NHEJ DNA repair activity was evaluated using a flow cytometry protocol as described elsewhere (1).
QRT-PCR was carried out on QuantStudio 6 Flex Real-Time PCR System (#44856901, ThermoFisher/Applied Biosystems™) to verify the knock-down efficiency and/or relative gene expression.

Irradiation of cultured cells
IR was administered to cells using a Shepherd Mark I-68 Cesium-137 irradiator (JL Shepherd). Cells were maintained in a 37 o C incubator with 5% CO2 concentration before and immediately after IR treatment.

Chromatin fractionation assay, Immunoblot analysis and Immunoprecipitation (IP)
Cells were collected 48 hr post transfection of plasmids or 16 hr of BETi treatment and lysed with RIPA buffer supplemented with protease inhibitor cocktail (#11873580001, cOmplete, EDTA free, Roche). Chromatin fractionation was performed as previously described (1). Briefly, cells were harvested after 16 hr of JQ1 (1 uM) treatment and washed with PBS. Cells were suspended for 5 min on ice in the fractionation buffer I (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 5 mM Sodium Butyrate) supplemented with 0.2% Nonidet P-40 and protease inhibitors (cOmplete, EDTA free, Roche). Following centrifugation at 1000 g for 5 min, the cytoplasmic supernatant was discarded, then the nuclear pellets were further extracted for 40 min on ice with 150 ul of fractionation buffer II (same as fractionation buffer I, except with 0.5% Nonidet P-40). The pellets containing chromatin were obtained by centrifugation for 15 min at maximum speed at 4°C, and then resuspended in fractionation buffer II.