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
Authors: Piya Sujan , McQueen Teresa , Mak Duncan , Konopleva Marina , Zweidler-McKay Patrick , Lu Jing , Qian Yimin , Andreeff Michael , Borthakur Gautam 
Published: October 2019

Abstract
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.
Figure 1. OCI-AML3 cells treated for 24 hours, re-suspended without drug, stained for Annexin V at post 48 hours. Primary AML cells treated with ARV-825 or JQ1 and cell viability tested with Cell Titer-Glo 2.0® PROMEGA. (A) OCI-AML3 treated for 24 hrs and stained with CXCR4-APC antibody. (B) OCI-AML3 cells treated for 24 hrs and percentage of cells migrating at 4 hrs to the lower chamber containing recombinant SDF-1 was counted using Vi-Cell (Trypan blue staining assay). Figure 1. OCI-AML3 cells treated for 24 hours, re-suspended without drug, stained for Annexin V at post 48 hours. / Primary AML cells treated with ARV-825 or JQ1 and cell viability tested with Cell Titer-Glo 2.0® PROMEGA. / (A) OCI-AML3 treated for 24 hrs and stained with CXCR4-APC antibody. (B) OCI-AML3 cells treated for 24 hrs and percentage of cells migrating at 4 hrs to the lower chamber containing recombinant SDF-1 was counted using Vi-Cell (Trypan blue staining assay).

Disclosures
Konopleva: 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.