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

Although glutamic acid decarboxylase (GAD) has been implicated in IDDM, there is no direct evidence showing GAD-reactive T cells are diabetogenic in vivo. To address this issue, 3-wk-old NOD mice received two injections of purified rat brain GAD; one mouse rapidly developed diabetes 3 wk later. Splenocytes from this mouse showed a proliferative response to purified GAD, and were used to generate a CD4+ T cell line, designated 5A, that expresses TCRs encoding Vbeta2 and Vbeta12. 5A T cells exhibit a MHC restricted proliferative response to purified GAD, as well as GAD65 peptide 524-543. After antigen-specific stimulation, 5A T cells secrete IFNgamma and TNFalpha/beta, but not IL-4. They are also cytotoxic against NOD-derived hybridoma cells (expressing I-Ag7) that were transfected with rat GAD65, but not nontransfected hybridoma cells. Adoptive transfer of 5A cells into NOD/SCID mice produced insulitis in all mice. Diabetes occurred in 83% of the mice. We conclude that GAD injection in young NOD mice may, in some cases, provoke diabetes due to the activation of diabetogenic T cells reactive to GAD65 peptides. Our data provide direct evidence that GAD65 autoimmunity may be a critical event in the pathogenesis of IDDM.

Although DQA1*0301/DQB1*0302 is the human histocompatibility leukocyte antigen (HLA) class II gene most commonly associated with human type 1 diabetes, direct in vivo experimental evidence for its diabetogenic role is lacking. Therefore, we generated C57BL/6 transgenic mice that bear this molecule and do not express mouse major histocompatibility complex (MHC) class II molecules (DQ8+/mII−). They did not develop insulitis or spontaneous diabetes. However, when DQ8+/mII− mice were bred with C57BL/6 mice expressing costimulatory molecule B7-1 on β cells (which normally do not develop diabetes), 81% of the DQ8+/mII−/B7-1+ mice developed spontaneous diabetes. The diabetes was accompanied by severe insulitis composed of both T cells (CD4+ and CD8+) and B cells. T cells from the diabetic mice secreted large amounts of interferon γ, but not interleukin 4, in response to DQ8+ islets and the putative islet autoantigens, insulin and glutamic acid decarboxylase (GAD). Diabetes could also be adoptively transferred to irradiated nondiabetic DQ8+/mII−/B7-1+ mice. In striking contrast, none of the transgenic mice in which the diabetes protective allele (DQA1*0103/DQB1*0601, DQ6 for short) was substituted for mouse MHC class II molecules but remained for the expression of B7-1 on pancreatic β cells (DQ6+/mII−/B7-1+) developed diabetes. Only 7% of DQ−/mII−/B7-1+ mice developed diabetes at an older age, and none of the DQ−/mII+/B7-1+ mice or DQ8+/mII+/B7-1+ mice developed diabetes. In conclusion, substitution of HLA-DQA1*0301/DQB1*0302, but not HLA-DQA1*0103/DQB1*0601, for murine MHC class II provokes autoimmune diabetes in non–diabetes-prone rat insulin promoter (RIP).B7-1 C57BL/6 mice. Our data provide direct in vivo evidence for the diabetogenic effect of this human MHC class II molecule and a unique “humanized” animal model of spontaneous diabetes.

Insulin-dependent diabetes mellitus in humans is linked with specific HLA class II genes, e.g., HLA-DQA1*0301/ DQB1*0302 (DQ8). To investigate the roles of HLA-DQ8 molecules and glutamic acid decarboxylase (GAD) in disease development, we generated DQ8