Summary BRD4, a bromodomain and extraterminal domain (BET) family member, is an attractive target in multiple pathological settings, particularly cancer. While BRD4 inhibitors have shown some promise in MYC-driven malignancies such as Burkitt’s Lymphoma (BL), we show that BRD4 inhibitors lead to robust BRD4 protein accumulation, which may account for their limited suppression of MYC expression, modest anti-proliferative activity and lack of apoptotic induction. To address these limitations, we designed ARV-825, a heterobifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon leading to fast, efficient, and prolonged degradation of BRD4 in all BL cell lines tested. Consequently, ARV-825 more effectively suppresses c-MYC levels and downstream signaling than small molecule BRD4 inhibitors resulting in more effective cell proliferation inhibition and apoptosis induction in BL. Our findings provide strong evidence that cereblon-based PROTACs provide a better and more efficient strategy in targeting BRD4 than traditional small molecule inhibitors.

Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extraterminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC.BET | BRD4 | protein degradation | prostate | PROTAC

Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach.

The androgen receptor is a major driver of prostate cancer and inhibition of its transcriptional activity using competitive antagonists, such as enzalutamide remains a frontline therapy for prostate cancer management. However, the majority of patients eventually develop drug resistance. We propose that targeting the androgen receptor for degradation via Proteolysis Targeting Chimeras (PROTACs) will be a better therapeutic strategy for targeting androgen receptor signaling in prostate cancer cells. Here we perform a head-to-head comparison between a currently approved androgen receptor antagonist enzalutamide, and its PROTAC derivative, ARCC-4, across different cellular models of prostate cancer drug resistance. ARCC-4 is a low-nanomolar androgen receptor degrader able to degrade about 95% of cellular androgen receptors. ARCC-4 inhibits prostate tumor cell proliferation, degrades clinically relevant androgen receptor point mutants and unlike enzalutamide, retains antiproliferative effect in a high androgen environment. Thus, ARCC-4 exemplifies how protein degradation can address the drug resistance hurdles of enzalutamide.

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that recruit an E3 ligase to a target protein to facilitate ubiquitination and subsequent degradation of that protein. While the field of targeted degraders is still relatively young, the potential for this modality to become a differentiated and therapeutic reality is strong, such that both academic and pharmaceutical institutions are now entering this interesting area of research. In this article, we describe a broadly applicable process for identifying degrader hits based on the serine/threonine kinase TANK-binding kinase 1 (TBK1) and have generalized the key structural elements associated with degradation activities. Compound 3i is a potent hit (TBK1 DC = 12 nM, D = 96%) with excellent selectivity against a related kinase IKKε, which was further used as a chemical tool to assess TBK1 as a target in mutant K-Ras cancer cells.