Enhanced Anti-Tumor Activity with Exposure to BCL-2 and BRD4 Inhibitors in Mantle Cell Lymphoma

Introduction Mantle Cell Lymphoma (MCL) is the third most common B Cell lymphoma and is currently an incurable malignancy with a poor long-term survival. MCL affects 1 in 200,000 people per year and has a median survival rate of 3 to 4 years - indicating the need for novel therapies. MCL has been characterized with a high frequency of Bcl-2 overexpression, shifting cells to an anti-apoptotic state. Venetoclax, a Bcl-2 inhibitor, is an approved treatment for MCL. Venetoclax binds to Bcl-2, blocking the binding of Bax or Bak, allowing Bax and Bak to bind to the outer mitochondrial membrane and permeabilize the membrane. PLX51107, a BRD4 inhibitor, is currently in phase I of clinical trials in Acute Myelogenous Leukemia (AML) patients. BRD4 is involved in the preinitiation complex for the transcription of miRNA, which targets mRNA encoding for BIM protein, a member of the Bcl-2 family that serves as a pro-apoptotic initiator. Our lab aims to elucidate if a synergistic therapeutic effect exists between Venetoclax and PLX51107.

Methods Using a panel of MCL cell lines (HBL-2, Granta, Z-138, Mino, Jeko-1, and Rec-1) we conducted pre-clinical targeting of Bcl-2 and BRD4 with Venetoclax and PLX51107 respectively. Cells were exposed to Venetoclax and PLX51107 as a single agent (0 - 4,000nM and 0 - 10,000nM respectively) over 24, 48, and 72 hrs. IC₅₀ concentrations were calculated for each cell line. Using appropriate doses seen in single agent exposure, double agent exposure was performed at 72 hrs where coefficients of synergy were assessed using CalcuSyn. Cellular viability was determined using Cell Titer-Glo (Promega) and was read using a BioTek Synergy HTX multimode reader. Induction of apoptosis was measured via conventional flow cytometry using Annexin V and Propidium Iodide (Annexin V/PI). Bcl-2 family member protein expression was determined via western blot. Cell viability with double agent exposure was assessed in combination with caspase inhibition using a pan caspase inhibitor, QVD-OPh (Thermo Fisher Scientific). In vivo experiments are ongoing.

Results Concurrent PLX51107 and Venetoclax exposure in vitro induced apoptotic cell death in a dose and time dependent manner. Synergy was observed in vitro via viability assays and confirmed via Annexin V/ PI staining across cell lines tested. The greatest synergistic effect was observed in cell lines Jeko-1 and Rec-1exhibiting strong synergy; and the least synergistic effect was observed in the Z-138 cell line. The combination of PLX51107 and Venetoclax shifted cells to a pro-apoptotic state, with a decrease in anti-apoptotic proteins (Bcl x/L and Mcl-1), and an increase in pro-apoptotic proteins (Bak, Bax and BIM). Downregulation of p21 and c-MYC following Venetoclax and PLX51107 exposure was also observed driving the cells to a more apoptotic state.

Conclusion Elucidating the proliferative and survival pathways within MCL offers insight into novel therapeutic targets, and subsequently heightened therapeutic success. Our data suggests that Venetoclax in combination with PLX51107 exhibits a synergistic effect across a broad range of MCL cell lines. Further elucidation of the mechanism by which PLX51107 and Venetoclax establish synergy is necessary for therapeutic advancement within the field of MCL.

No relevant conflicts of interest to declare.