Abstract 4710: BRD4 degradation by PROTACs represents a more effective therapeutic strategy than BRD4 inhibitors in ovarian cancer
Authors: Raina Kanak , Lu Jing , Qian Yimin , Altieri Martha , Dong Hanqing , Wang Jing , Chen Xin , Crew Andrew , Coleman Kevin , Crews Craig , Winkler James 
Published: July 2016

BRD4, a member of the bromodomain and extraterminal domain (BET) family of chromatin readers, localizes to super-enhancer regions of DNA, and turns on the expression of a number of oncogenes. Therefore, it has attracted considerable interest as a potential drug target in a variety of cancers, and several inhibitors that block its binding to acetylated histones have been discovered. The BRD4 genomic region is amplified in approximately 17% of ovarian serous adenocarcinomas, and a recent shRNA screen identified it as a bona fide target in high-grade serous ovarian carcinoma (HGSOC). BRD4 inhibitors were shown in the same study to be efficacious in cell culture and PDX models of HGSOC. However, the inhibitor paradigm of drug discovery has several limitations, for example, the requirement of high target occupancy for activity, and the inability to block potentially critical scaffolding functions of the drug target. To circumvent these limitations, we have designed small molecule BRD4 degraders based on our PROTAC (PROteolysis TArgeting Chimera) platform, and assessed their activity across a panel of ovarian carcinoma cell lines.

BRD4 PROTACs, containing a BRD4 ligand connected to a VHL E3 ubiquitin ligase binding moiety, successfully promote proteasome-mediated degradation of BRD4 at sub- to low-nM concentrations in a number of ovarian cancer cell lines. Furthermore, this BRD4 degradation results in a very potent anti-proliferative effect in the cells. All the cell lines tested showed modest anti-proliferative effects when treated with BRD4 inhibitors (ED50 ∼ 0.5 μM-1μM). In contrast, when treated with BRD4 PROTACs the cell lines separated into three profoundly different groups (most sensitive, moderately sensitive, least sensitive), with a difference of several orders of magnitude (ED50 ∼ 0.03nM-500nM). Thus BRD4 PROTACs are up to 10,000-fold more potent than BRD4 inhibitors in the most sensitive cell lines. Moreover, the ED50 curves closely track with BRD4 degradation, and result in a much more profound apoptotic response than BRD4 inhibition. In addition, we have also shown that the anti-proliferative effect of BRD4 PROTACs is decoupled from their effect on the levels of cMYC. Finally, we have carried out deep RNA sequencing in five cell lines of variable sensitivity to BRD4 PROTACs in order to thoroughly define the molecular subtype of the malignancies represented and have identified gene signatures that correlate with efficacy of BRD4 PROTACs and BRD4 inhibitors in these cells.

In summary, PROTACs designed to target BRD4 for proteasomal degradation are highly potent in ovarian cancer cells, and elicit a powerful pro-apoptotic response in the same setting. The cells can be grouped by their level of sensitivity to PROTAC-mediated BRD4 degradation, and carry gene expression biomarkers which we hope will enable a patient selection strategy as BRD4 PROTACs are advanced into the clinic.

Citation Format: Kanak Raina, Jing Lu, Yimin Qian, Martha Altieri, Hanqing Dong, Jing Wang, Xin Chen, Andrew Crew, Kevin Coleman, Craig Crews, James Winkler. BRD4 degradation by PROTACs represents a more effective therapeutic strategy than BRD4 inhibitors in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4710.