Accelerated Degradation of cFLIP Mediated By Inhibition of Methyltransferases Sensitizes B-Cell Lymphoma Cells to TRAIL-Induced Apoptosis

Introduction

Non-Hodgkin lymphomas (NHLs) are characterized by specific abnormalities that alter cell cycle regulation, DNA damage response, and apoptotic signaling. Cancer cells can be particularly sensitive to cell death induced by tumor necrosis factor a–related apoptosis-inducing ligand (TRAIL). However, many cancer cells show blocked TRAIL signaling because up-regulated expression of anti-apoptotic factors such as cFLIP. Thus, by further understanding the underpinnings of apoptosis resistance, we will be in a better position to develop strategies that improve TRAIL-induced killing of lymphoma cells.

Methods/Results

NHL cell lines, including mantle cell lymphoma (MCL; Mino, JeKo-1, JVM-2 and Z-138), Burkitt lymphoma (BL; Bjab, Ramos, Daudi) and diffuse large B-cell lymphoma (DLBCL; SU-DHL-4, SU-DHL -6, SU-DHL-9) were treated with 3-Deazaneplanocin A (DZNep); 0.2-5µM) for 24 hours followed by incubation with TRAIL (10-20ng/ml, 6-16hours). Cell death, DNA fragmentation, and mitochondrial membrane potential (Δψm) were determined by calcein staining, subG1 analysis, and TMRM staining, respectively. Neither DZNep alone nor in combination with TRAIL showed a significant induction of necrosis as determined by LDH-release levels, but DZNep alone showed strong anti-proliferative properties at higher concentrations. Activation of the caspase signaling cascade (caspase-8, -3, and PARP cleavage) was analyzed by Western blotting. TRAIL-induced signaling was significantly increased and caspase-8 processing enhanced in DZNep pretreated cells indicating a regulation at the TRAIL/DISC assembly. Although we observed a reduced expression of DR5 in total cell lysates of DZNep treated cells, the surface receptor levels were not altered. Interestingly, down regulation of the well-known caspase inhibitor, cFLIP, correlated with the DZNep-induced increased TRAIL cell killing in all sensitive NHL cells. The DZNep-treatment did not block NF-kB signaling related to cFLIP expression but profoundly affected cFLIP mRNA and protein stability. Elevated levels of cFLIP targeting miRNA’s were observed and were attributed to some extend with cFLIP regulation.

Conclusions: In this study, we investigated the impact of a pan-methyltransferase inhibitor (3-deazaneplanocin A, or DZNep) on TRAIL-induced apoptosis in aggressive NHLs: mantle cell, Burkitt, and diffuse large B-cell. We characterized TRAIL apoptosis regulation and caspase activation in several NHL cell lines pre-treated with DZNep. We found that DZNep increased cancer cell sensitivity to TRAIL signaling by promoting caspase-8 processing through accelerated cFLIP degradation. Blockage of methyltransferase activity induced by DZNep profoundly affected cFLIP mRNA and protein stability, at least in part through increased levels of cFLIP-targeting microRNAs (miR-512-3p and miR-346). However, additional microRNAs and cFLIP-regulating mechanisms appear to be involved in DZNep-mediated enhanced response to extrinsic apoptotic stimuli. The capacity of DZNep to target cFLIP expression on multiple levels underscores its potential in TRAIL-based therapies for NHLs.