The Histone Deacetylase Inhibitor SAHA Induces Caspase-Dependent Apoptosis in Chronic Lymphocytic Leukemia Cells.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation of CD5+ B cells with significant resistance to apoptosis and therefore prolonged survival. CLL remains an incurable disease requiring innovative new approaches to improve overall patient outcome.

OBJECTIVE: Histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (SAHA) have shown antitumoral activity at micromolar concentrations in a variety of human cancers in vitro and in vivo. These different studies suggested extrinsic, intrinsic and caspase-independent apoptosis as relevant death pathway depending upon cell types.

RESULTS: In this study, we examined the effects of SAHA on CLL cells in vitro. SAHA induced apoptosis in a dose-dependent manner in all (n=25) CLL samples tested, including previously untreated and chemo-resistant CLL patients. The level of apoptosis, as measured by annexin binding to exposed PS residues, increased after 48 hours of SAHA treatment and was significant in the treated cells at concentrations of 10 and 20 μM (respectively 45±5 and 52±4% of apoptotic cells versus 29.5±4 for untreated cells, p<0.0001). The pre-treatment of cells with the pan-caspase inhibitor Z-VAD before SAHA-treatment had no effect on PS externalization but inhibited DNA degradation demonstrating that caspases are critical for inducing DNA fragmentation. Using specific caspase inhibitors (DEVD, VEID and IETD) we demonstrated the participation of caspases-3, -6 and -8 in cell apoptosis. In addition, inhibition of the initiator caspase in the intrinsic/mitochondrial pathway, caspase-9, did not influence cell apoptosis. Thus, extrinsic pathway seems activated during SAHA-induced apoptosis. We have next investigated the expression of FAS and TRAIL-R1 (DR4) by CLL cells after SAHA treatment. Only a small proportion of CLL cells displayed detectable expression of CD95 (12.5±2% CD19+CD95+). 24h treatment with SAHA resulted in increase in FAS expression compared to control (33±5.6%, p<0.02). However, a FAS-blocking antibody (ZB4) did not inhibit SAHA-induced apoptosis arguing against a role of FAS/FAS-L signaling pathway in the induction of apoptosis by SAHA. The expression of TRAIL-R1 was very low and not upregulated by SAHA treatment. To explore the mechanism by which SAHA triggers the extrinsic pathway in CLL cells, the effects of SAHA on the level of various apoptosis-regulatory proteins (FLIP, FADD…) are now evaluated.

CONCLUSIONS: SAHA induces apoptosis in CLL cells via the extrinsic pathway involving caspase-8 activation. Since the majority of cytotoxic agents operate via the intrinsic pathway and since defects in the mitochondrial pathway exist in chemoresistant CLL patients, it is important to identify agents that exert their cytotoxic effect via the extrinsic pathway. Moreover, the combination of SAHA with conventional drugs could be of therapeutic effect in CLL.