Abstract PD1-14: Induction of epigenetic BRCAness in BRCA1 wild-type triple negative breast cancer: BET inhibition as a therapeutic strategy
Authors: Gerratana L , Puglisi F , Damante G , Mio C 
Published: February 2018

Background: Pharmacological induced BRCAness and synthetic lethality could represent a potential therapeutic strategy in triple negative breast cancer (TNBC). Bromodomain and Extra-Terminal proteins (BET) such as BRD2 and BRD4, are involved in controlling RNA polymerase II pause/release transition mediating transcriptional activation and gene bookmarking. BRD4 is found to be associated to enhancer and promoter regions to foster gene expression. Furthermore, BRD4 overexpression is described to be associated with worse outcome and increased metastasizing potential. Aim of this study was to explore novel therapeutic strategies through pharmacological induced epigenetic BRCAness in BRCA1 wild-type TNBC cells by means of BET inhibition.

Methods: The experimental models were based on the BT-549, MDA-MB-157 and MDA-MB-231 TNBC BRCA1 wild type cells lines. Two different pan-BET inhibitors were explored (i.e. JQ1 and I-BET762). BRCA1 and RAD51 levels were evaluated by both qPCR and western blotting as a marker of BRCAness phenotype onset. Chromatin immunoprecipitation (ChIP) and RNA interference (RNAi)-mediated BRD4 silencing were performed on TNBC cells. Synthetic lethality induced by JQ1 (0.5 µM) and the effects of co-treatment with the PARP inhibitor PJ34 (5-15 µM) or cisplatin (2-10 µM) were evaluated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay.

Results: A dose-dependent reduction in BRCA1 and RAD51 protein levels was observed after treatment with both JQ1 and I-BET762 in all the cell lines tested. To verify the direct relationship between BRD4 and BRCA1/RAD51 expression, a dual approach was performed. First, BRCA1 and RAD51 protein levels were evaluated after RNAi-mediated BRD4 knockdown, verifying that the BRCAness phenotype (exemplified by BRCA1 and RAD51 protein downregulation) was due to the absence of BRD4 protein. Furthermore, a ChIP assay was performed to confirm the direct regulation of BRCA1 and RAD51 promoters by BRD4. To test the gain in sensitivity of BRCAness-displaying TNBC cells towards platinum salts, the effects of cisplatin alone or in combination with BET inhibitors were tested. JQ1 and cisplatin combinations induced a strong reduction in TNBC cell viability in all the three cell lines tested. Moreover, the pharmacological combination between JQ1 and PJ34 displayed a significant cell viability decrease in TNBC wild type cells, corroborating the synthetic lethality mechanism.

Conclusions: A decrease in BRCA1 and RAD51 levels was observed after BET inhibition by means of the JQ1 drug, exploiting synergism with PARP inhibition and use of platinum salts. Moreover, the present results demonstrate the direct interaction between BRD4, BRCA1 and RAD51, supporting the therapeutic rationale behind this novel strategy.

Citation Format: Gerratana L, Puglisi F, Damante G, Mio C. Induction of epigenetic BRCAness in BRCA1 wild-type triple negative breast cancer: BET inhibition as a therapeutic strategy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-14.