(A) A schematic representation of bifunctional PROTAC ARV-825.
(B) ARV-825 leads to fast and efficient degradation of BRD4. (top panel) Namalwa and CA-46 cells were treated overnight with increasing doses of ARV-825, lysates were analyzed for BRD4 levels by immunoblot with actin serving as loading control; (bottom panel) Namalwa and Ramos cells were treated with ARV-825 (0.1 µM) for indicated time points, lysates were collected and subjected to immunoblot analysis with antibodies for BRD4 and actin.
(C) Confirmation of cereblon-based mechanism in driving BRD4 degradation upon ARV-825 treatment. Namalwa (left panel) and Ramos (right panel) cells were treated overnight with various concentrations of ARV-825 or pomalidomide (10 µM), or combination of ARV-825 and pomalidomide; lysates were analyzed by immunoblot for BRD4 and actin.
(D) Confirmation of proteasome-based mechanism in driving BRD4 degradation upon ARV-825 treatment. Namalwa cells were treated overnight with ARV-825 (+/0.01 µM and ++/0.1 µM) alone, MG132 (5 µM) or carfilzomib (5 µM) alone, or combination of ARV-825 with MG132 or with carfizomib; lysates were collected and analyzed by immunoblot for BRD4 and actin.