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.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA