MEK1 Blockade Sensitizes Multiple Myeloma Tumor Cells to Arsenic Trioxide (ATO)-Induced Apoptosis: A Functional and Molecular Study.

Abstract

Preliminary data from clinical studies indicate that ATO has clinical activity as a single agent in human multiple myeloma (MM), and combination therapies are being investigated. We recently reported that PD184352 (PD) ( Pfizer), a highly selective inhibitor of MEK1, strikingly enhances ATO-mediated apoptosis in NB4, K562 cell lines and in Acute Myelogenous Leukemia via multiple intrinsic apoptotic pathways activation. The aim of this study was to investigate whether the combined treatment with PD and ATO has cytotoxic effects on MM cells. We first analyzed the pharmacologic interactions between PD (2 μM) and ATO (1–2 μM) using a fixed-ratio experimental design on 7 human myeloma cell lines (HMCL) with varying p53 status (RPMI 8226, U266, XG-1, XG-6, JJN3, HS-SULTAN, NCI-H929) and found that the combined treatment resulted in the synergistic (Combination Index <1.0) induction of apoptosis in NCI-H929, XG-1, XG-6, RPMI 8226, SULTAN and JJN3 HMCL. Conversely, the combination of PD plus ATO had a slight antagonistic effect on apoptosis induction in U266 HMCL (Combination Index >1). Moreover, PD plus ATO-induced cytotoxicity on HMCL was maintained also in presence of IL-6 (20 ng/ml) or in a co-culture system with bone marrow stromal cells. Similarly to HMCL we found that the treatment with PD significantly enhanced the apoptosis of fresh purified MM cells induced by ATO (P< .01) in 8 out of 11 patients of MM analyzed. Conversely, PD treatment partially attenuated (n=2) or did not affect (n=1) the ATO cytotoxicity in normal bone marrow B cells. To investigate the molecular mechanisms by which PD plus ATO induced MM cell apoptosis first we compared the effect on caspase activation in myeloma cells expressing wild type (wt) p53 or mutated p53. Using caspase blocking peptides, specific siRNA against caspase-8 or caspase-9 and Western immunoblotting we demonstrated the involvement of primarily caspase-8 and -3 in PD plus ATO-induced apoptosis in myeloma cells with mutated p53 and primarily caspase-9 and -3 in dual treated cells expressing wt p53. In addition, PD plus ATO induced a p53-dependent up regulation of Puma, Bax and Bak in HMCL with wt p53, and p53-specific siRNA significantly (P< .01) reduced the induction of apoptosis in dual treated cells. The caspase-8-mediated proteolytic activation of BID, a key protein involved in the cross-talk between the intrinsic and extrinsic apoptotic pathways, closely correlated with the caspase-9 activation and loss of mitochondrial membrane potential observed in dual treated HMCL with mutated p53. Finally, in the responsive HMCL both with wt or mutated p53, the combined treatment increased the level of the pro-apoptotic Bim (PD-mediated) and decreased its neutralizing anti-apoptotic protein Mcl-1 (ATO-mediated) causing an imbalance between these proteins that positively influenced the pro-apoptotic efficacy of the combination. In conclusion, our data indicate that the disruption of MEK pathway potentiates the apoptotic effect of ATO in MM cells through the activation of both extrinsic and intrinsic (caspase-8-BID-mediated) pathways in HMCL with mutated p53 or through the primarily activation of intrinsic pathway in HMCL with wt p53, with the positive contribution of Bim pathway. These findings suggest that a strategy combining ATO with disruption of MEK pathway could represent an effective therapeutic strategy for the treatment of MM.