Degradation Of BRD4 Research Articles

BRD4 Proteolysis Targeting Chimera (PROTAC) Leads to Sustained Degradation of BRD4 with Broad Activity Against Acute Leukemias and Overcomes Stroma Mediated Resistance By Modulating Surface Expression of CXCR4

Background: Hematological neoplasms are characterized by aberrant epigenetic events that modify the chromatin regulatory machinery to enhance oncogene expression (Zuber et al. Nature 2011). BRD4, a member of the bromodomain and extra terminal domain (BET) family, is critical in the assembly of a 'super enhancer complex' that drives expression of oncogenes MYC, CCND1, SOX2 and NF-kB and anti-apoptotic proteins e.g. Bcl-2 and BCL-XL (Klapproth et al. Br J Haematol 2010; Wang et al. Cancer Res 2014; Zou et al. Oncogene 2014). Small molecule BRD4 inhibitors lead to rapid accumulation of BRD4 that may partially account for their moderate suppression of MYC (Lu et al. Chem Biol 2015).ARV-825 is hetero-bifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon, leading to fast, efficient, and prolonged degradation of BRD4 and sustained down-regulation of MYC (Lu et al. Chem Biol 2015).Aim: We investigated the activity of ARV-825 against leukemia cell lines representing acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) including gamma secretase inhibitor (GSI) resistant T-ALL and against primary AML blasts and compared it to that of the BRD4 inhibitor, JQ1. As stroma renders leukemia resistant, we tested if and through what mechanism ARV-825 can overcome stroma mediated drug resistance.Results: The IC50s for all tested cell lines at 72 hours were in very low nanomolar range; AML-OCIAML3-8.3 nM, HL60-2.4nM; ALL-KOPT-K1/SUPTI-1.3nM (GSI resistant T-ALL), LOUCY-2.3nM (early T-cell phenotype ALL), MOLT4-3.12nM and HPB-ALL-4.6nM (T- ALL) while the same for JQ1 was 102.6 nM, 5 nM, 1.5nM/8.8 nM, 4.5nM, 9.4 and 8.4 nM respectively. Consistent with sustained degradation of BRD4, the apoptotic effect of ARV-825 is sustained longer than that of JQ1 after the removal of compounds following a 24-hr treatment at IC50 (Annexin V positive 70% vs. 4% at 48 hrs post-washout respectively) (Fig. 1). Importantly, IC50 values for primary AML samples for ARV-825 were approximately 6-100 times lower than those for JQ1 (Fig. 2).ARV-825 led to sustained decreases of BRD4 and MYC proteins with corresponding increase in cleaved PARP, cleaved Caspase-3 and γH2AX in MOLT4 and OCIAML3 cells. While Bcl-2 level remained suppressed with ARV-825, it recovered at 48 hrs of treatment with JQ1.We co-cultured OCI-AML3 cells with normal bone marrow derived mesenchymal stromal cells (MSCs) to mimic the bone marrow microenvironment. While MSC protected OCI-AML3 cells from cytarabine (25 % reduction in cell death), there was no stromal protection for ARV-825. To gain mechanistic insight, we used a Mass cytometry (CyTOF) based approach to analyze the changes in apoptotic, cell adhesion and signaling proteins (panel of 24 antibodies) in OCI-AML3 cells after 12 and 24-hour treatment with ARV-825. MYC and surface CXCR4 were the two most down-regulated proteins. The reduction of surface CXCR4 was confirmed by conventional flow cytometry (Fig. 3A) and the functional relevance was confirmed in migration assays against the CXCR4 ligand SDF-1 (29.2% Control vs. 9.03% ARV-825) (Fig. 3B).Conclusion: ARV-825, a BRD4 PROTAC, has substantial anti-leukemia activity across a range of acute leukemias and importantly can overcome stroma-mediated drug resistance. ARV-825 promises to be an exciting molecule in the quest towards efficient BRD4 inhibition. [Display omitted] DisclosuresKonopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Lu:Arvinas, LLC: Employment, Equity Ownership. Qian:Arvinas, LLC: Employment, Equity Ownership.

Proteolytic Targeting Chimeric Molecules (PROTACs) Specific for Bromodomain-Containing Protein (BRD) 4 Are Active Against Pre-Clinical Models of Multiple Myeloma

Background:BRD4, a bromodomain and extraterminal domain (BET) family member, has an important role in modulating the expression of essential oncogenes such as c-MYC, and is emerged as a promising therapeutic target in diverse cancer types. Pharmacologic BET inhibitors in development such as JQ1 and OTX015 display preclinical anti-myeloma activity, and induce preferential loss of BRD4 bound to super-enhancers leading to transcriptional repression of c-MYC. Another approach to target this pathway is through the use of bi-functional molecules, which incorporate a small molecule BRD4 binding moiety with an E3 ubiquitin ligase recognition motif, such as ARV-825 and dBET1 (Lu et al. Chem Biol. 22:755, 2015, Winter et al. Science 348:1376, 2015). These agents induce Cereblon (CRBN)-dependent BRD4 ubiquitination and then proteasome-mediated degradation, thereby also reducing downstream c-MYC protein levels.Methods:We performed pre-clinical studies in myeloma cell lines and primary samples using ARV-825 and ARV-763, which are PROTACs that target BRD4 to either the CRBN or the Von Hippel-Lindau (VHL) E3 ligases, respectively. Downstream effects were studied using viability and apoptosis assays, cell cycle profiling, and Western blotting, among others.Results:Tetrazolium assays showed that both PROTACs were able to reduce the viability of a panel of myeloma cell lines, including MM1.S, U266, RPMI 8226, ANBL-6, KAS-6/1, and OPM-2 cells, and this occurred with greater potency than was the case for the BRD4 inhibitors JQ1 or OTX015. Median inhibitory concentrations were 5.66-91.98 nM for ARV-825, and 13.22-1522 nM for ARV-763, respectively. This reduction in viability was both time- and concentration-dependent, and was associated with a reduction of cells in the S phase, and an increase in G0/G1 cells, as well as cells with sub-G0/G1 DNA content, suggesting the onset of apoptosis. Programmed cell death was indeed found to be induced based on the appearance of an increase in Annexin V-positive cells by flow cytometry, and in cleaved caspase 8, caspase 9, caspase 3, and poly-ADP-ribose polymerase by Western blotting. The latter was associated with a specific reduction in the expression levels of both BRD4 and c-MYC that did not influence the abundance of other cellular proteins that were not BRD4 targets, and in a reduction in BRD4 and c-MYC mRNA. In contrast, JQ1 and OTX015 exposure resulted in a slight increase in BRD4 protein expression and a lesser decrease of c-MYC protein. Studies of drug combinations showed that, as expected, lenalidomide and pomalidomide were antagonistic to the effects of the CRBN-targeted ARV-825 PROTAC, but these immunomodulatory drugs showed additive or synergistic effects in combination with the VHL-targeted agent ARV-763. Also as expected, bortezomib and carfilzomib reduced the ability of both ARV-825 and ARV-763 to induce BRD4 degradation, but enhanced anti-proliferative and pro-apoptotic effects were seen in a manner that was influenced by the sequence of drug addition. In studies of drug-resistant cell lines, both PROTACs were able to overcome dexamethasone, melphalan, lenalidomide, and bortezomib resistance, but cross-resistance was seen in RPMI 8226/Dox40 cells, suggesting that these compounds are substrates for P-glycoprotein, which is over-expressed in these cells. Finally, we tested BRD4 PROTACs in primary cells isolated from patients with multiple myeloma, and observed rapid loss of viability of these plasma cells.Conclusions:Taken together, our data demonstrate that BRD4 degraders have promising activity against pre-clinical models of multiple myeloma, and support their translation to the clinic for patients with relapsed/refractory disease. Additional combination and mechanistic studies, as well as data from ongoing in vivo studies, will be presented at the meeting. DisclosuresLu:Arvinas, LLC: Employment, Equity Ownership. Qian:Arvinas, LLC: Employment, Equity Ownership. Orlowski:Acetylon: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Research Funding; Forma Therapeutics: Consultancy; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Research Funding.

Abstract LB-010: Hijacking the E3 ubiquitin ligase cereblon to create efficient BRD4 degraders

Abstract BRD4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as an attractive target in multiple pathological settings. For example, recent studies have demonstrated its importance in driving the proliferation of several cancers including NUT midline carcinoma, acute myelogenous leukemia (AML), multiple myeloma, Burkitt's Lymphoma (BL), and prostate cancer. Thus, BET bromodomain inhibitors have showed promising effects in certain preclinical settings, particularly in MYC-driven hematological malignancies, such as BL. Interestingly, we found that BRD4 inhibitors lead to a rapid and robust accumulation of BRD4 that, together with the reversible nature of binding to BRD4, may account for their moderate suppression of MYC expression and inhibition of cell proliferation. To circumvent these limitations, we designed a Proteolysis Targeting Chimera (PROTAC) compound, containing a BRD4 binding moiety and an E3 ubiquitin ligase cereblon ligand. By actively engaging the E3 ligase-proteasome degradation machinery, BRD4 PROTAC leads to fast and efficient degradation of BRD4. Consequently, it is more effective than small molecule BRD4 inhibitors in suppressing MYC levels and downstream signaling, which are associated with proliferation and survival of malignant cells. More importantly, we also demonstrated that BRD4 PROTAC is more effective in inhibiting BL cell proliferation and inducing apoptosis compared to BRD4 inhibitors. Our findings strongly demonstrate that a degrader of BRD4, in the form of a cereblon-based PROTAC, provides a better and more efficient strategy in targeting BRD4 than traditional small molecule inhibitors. Citation Format: Jing Lu, Yimin Qian, Martha Altieri, Hanqing Dong, Jing Wang, Kanak Raina, Jim Winkler, Andy Crew, Kevin Coleman, John Hines, Craig Crews. Hijacking the E3 ubiquitin ligase cereblon to create efficient BRD4 degraders. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-010. doi:10.1158/1538-7445.AM2015-LB-010

Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4.

The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra-BET selectivity limits the scope of current inhibitors as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis Targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent with selective suppression of BRD4. Our discovery opens up new opportunities to elucidate the cellular phenotypes and therapeutic implications associated with selective targeting of BRD4.

Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4

BRD4, a bromodomain and extraterminal domain (BET) family member, is an attractive target in multiplepathological 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 antiproliferative activity, and lack of apoptotic induction. To address these limitations we designed ARV-825, a hetero-bifunctional 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 inBL. Our findings provide strong evidence that cereblon-based PROTACs provide a better and more efficient strategy in targeting BRD4 than traditional small-molecule inhibitors.

BRD4 degraders produce long-lasting loss of BRD4 Ppotein and robust efficacy in Burkitt’s lymphoma cells.

8557 Background: We have created specific BRD4 degraders using PROTAC (PROteolysis TArgeting Chimera) technology. This involves creating bi-functional molecules, with one end having a ligand for BR...