Abstract 5076: IHC and flow cytometry quantifies BRD4 levels in surrogate tissues after ex-vivo and in-vivo dosing with a BRD4 degrading PROTAC
Authors: Gough Sheryl M. , Raina Kanak , Gordon Debbie , Willard Ryan , Altieri Martha , Shen Angela , Qian Yimin , Neklesa Taavi , Coleman Kevin , Taylor Ian 
Published: July 2017

The Bromodomain and Extra-Terminal (BET) protein BRD4 has been identified as a key transcriptional regulator of various oncogenes, most notably MYC, in numerous human malignancies. Efforts have been extensive over recent years to inhibit the gene regulatory action of BRD4 and BET inhibitors have entered clinical trials. In a new approach to cancer therapeutics, we have developed a proteolysis-targeting chimera (PROTAC) molecule, ARCC-29, which induces degradation of BET (BRD4/3/2) proteins via the proteasome. ARCC-29 degrades BRD4 in several human malignant cell lines and in in-vivo rodent xenograft tumors. In mice xenograft models of prostate (22Rv1), DLBCL (SU-DHL-6) and ovarian (A2780) tumors, this molecule demonstrates a strong PK/PD/efficacy relationship. Obtaining tumor tissue from patients often requires invasive procedures and may be unavailable. We have developed two assays using surrogate tissues to evaluate PROTAC mechanism of action which could be applied in the clinical setting. We have developed a flow cytometry protocol to quantitatively assess intra-cellular BRD4 levels in peripheral blood mononuclear cells (PBMCs). Frozen or fresh human PBMCs were cultured for six hours ex-vivo with ARCC-29, fixed and permeabilized for intra-cellular BRD4 staining, and BRD4 levels quantified in gated lymphocytes. ARCC-29 reduced BRD4 levels to 13, 10, 16, and 9 percent of vehicle in four fresh PBMC samples, and to 10, 9, 11, 6, 8 and 35 percent in six frozen PBMC samples. Treatment of PBMCs with the BRD4 inhibitor OTX-015, showed similar or increased levels of BRD4 compared to vehicle control. Inactive stereoisomers of ARCC-29 also showed BRD4 levels increased or similar to vehicle control. Duplicates of selected experiments were analyzed by western blot, corroborating the depletion of BRD4. The second assay uses immunohistochemistry (IHC) to evaluate BRD4 degradation in skin biopsies of Sprague Dawley and nude male rats dosed with 2mg/kg intravenous (i.v.) BRD4 PROTAC ARCC-29. IHC analysis of rat skin eight hours after in-vivo dosing with ARCC-29 demonstrated a dose-dependent reduction in BRD4 protein. Rats dosed once a week with ARCC-29 [2 mg/kg] showed a 60-90% reduction in BRD4 levels compared to vehicle, while 0.3 mg/kg twice per week showed a 10-30% decrease in BRD4 levels. These experiments demonstrate that BRD4 degradation can be evaluated in surrogate tissue. Flow cytometry using PBMCs and skin IHC provide potential alternative methods for confirming the PROTAC mechanism-of-action in the clinic using pre- and post-treatment patient samples. DISCLOSURES All authors are employed by Arvinas, LLC.

Citation Format: Sheryl M. Gough, Kanak Raina, Debbie Gordon, Ryan Willard, Martha Altieri, Angela Shen, Yimin Qian, Taavi Neklesa, Kevin Coleman, Ian Taylor. IHC and flow cytometry quantifies BRD4 levels in surrogate tissues after ex-vivo and in-vivo dosing with a BRD4 degrading PROTAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5076. doi:10.1158/1538-7445.AM2017-5076