Potentiation of UCN-01 lethality by MEK1/2 inhibitors in human multiple myeloma cells involves modulation of Bim phosphorylation and degradation

Abstract

3210

In previous studies, our group reported that exposure of human myeloma, leukemia, and solid tumor cells to the PKC and Chk1 inhibitor UCN-01 induced the marked activation of MEK1/2/ERK1/2. Furthermore, prevention of MEK1/2/ERK1/2 activation (e.g., by pharmacologic MEK1/2 inhibitors) dramatically increased apoptosis in UCN-01-treated cells, suggesting that UCN-01-mediated MEK1/2/ERK1/2 activation represents a cytoprotective response. However, the downstream target(s) of MEK1/2/ERK1/2 signaling in this setting remains unclear. Among numerous possibilities, recent attention has focused on post-translational regulation of Bim, an “activator” BH3-only protein, by ERK1/2. Consequently, modulation of Bim phosphorylation and its functional role in cell death induced by UCN-01 ± MEK1/2 inhibitors (e.g., PD184352) was examined. Exposure (8-24h) of U266 cells to UCN-01 dramatically induced Bim phosphorylation and degradation, associated with ERK1/2 activation. These events were largely blocked by co-administration of PD184352, resulting in accumulation of unphosphorylated Bim and a marked increase in caspase-3 activation and PARP degradation. Identical events occurred in RPMI8226 cells. Enforced activation of ERK1/2 by stable transfection with constitutively active (CA)-MEK1 increased Bim phosphorylation/degradation, which was further enhanced by UCN-01 exposure. Ectopic expression of CA-MEK1 diminished the inhibitory effects of PD98059 but not PD184352 on UCN-01-mediated ERK1/2 activation and Bim phosphorylation, as well as caspase-3 cleavage. Knockdown of Bim by transient or stable transfection with a construct encoding shRNA directed against Bim diminished the lethality of the UCN-01/MEK1/2 inhibitor regimen without modifying ERK1/2 inactivation. Co-immunoprecipitation analysis revealed that exposure to UCN-01 markedly decreased binding of Bim to Bcl-2 and Bcl-xL. These events were reversed by co-administration of PD184352, resulting in conformational change of Bax/Bak and Bax translocation. Ectopic expression of Bcl-xL sequestered Bim and blunted the lethality of the UCN-01/MEK1/2 inhibitor regimen, without modulating Bim phosphorylation. Similar results were observed in cells overexpressing Bcl-2. Lastly, treatment with PD184352 either alone or in combination with UCN-01 diminished IL-6- or IGF-1-induced ERK1/2 activation and Bim phosphorylation, potentially accounting for failure of these survival factors to protect myeloma cells from the lethality of this combinational regimen. These studies indicate that Bim represents a critical downstream target of MEK1/2/ERK1/2 in cells exposed to UCN-01 and that Bim activation contributes to synergism between UCN-01 and MEK1/2 inhibitors.