The BET inhibitors JQ1, AZD5153, and I-BET151 co-opt ubiquitin proteasome system components for altering expression of the BRD4 interactome in a human B cell line.

Chemoproteomic analysis of the BET inhibitors, JQ1, AZD5153, and I-BET151, identified an extremely large signature of ubiquitin modified proteins associating in vitro with a recombinant BRD4 N-terminal protein fragment. The identified proteins included those with known functions in BRD4 complexes for transcriptional and epigenetic control (acetylated histones, the MED complex, BAF complex, RNA pol II transcription complexes, and chromatin-associated complexes). The BRD4 interactome in response to BET inhibitors is suggested to be orchestrated by compound-specific differential actions of up to 16 E3 ligases, 4 deubiquitinase enzymes, and 51 accessory proteins of the ubiquitin proteasome system (UPS). The UPS response of BET inhibition also involves proteins necessary for Myc enhancer binding and Myc response gene expression. A large cohort of UPS substrates commonly responsive to JQ1 and AZD5153 treatments suggests the existence of distinct mechanisms, one involving compound-activated UPS proteins, and another via their direct actions on BRD4. The findings raise the intriguing possibility that UPS triggers promoting proteostasis changes to the BRD4 interactome may be mechanistically coupled with BRD4 function in a proximity-dependent, chromatin-associated manner. Consequently, BET inhibitors and their downstream effects present highly complex environments which may lead to polypharmacology, the phenotypic outcomes or overall clinical benefits of which are hard to assess. However, many new targets and small molecule combinations suggested in this study may afford a path forward for narrowly and more selectively targeting Myc in the clinic with potentially cleaner profiles compared with BET inhibitors or BRD4 as target.