Ras-dependent activation of ERK1/2 regulates the lethality of Chk1 inhibitors in multiple myeloma cells

C180 Checkpoint kinases (Chk) represent key components of the DNA damage checkpoint pathway and attractive targets for anticancer therapies. Our group has reported that exposure of human multiple myeloma (MM) and leukemia cells to the Chk1 inhibitor UCN-01 results in pronounced activation of the MEK/ERK pathway, while disruption of this event by pharmacologic MEK inhibitors or farnesyltransferase inhibitors (FTIs) leads to a dramatic increase in apoptosis. Here we attempted to define the mechanism(s) by which interruption of such pro-survival pathways lower the apoptotic threshold in response to Chk1 inhibitors in vitro and in vivo in MM cells employing both pharmacologic and genetic approaches. Exposure of MM cells (e.g., U266, RPMI8226, MM.1S and MM.1R) to either UCN-01 or a novel Chk1/Wee1 inhibitor resulted in cdc2/cdk1 Thr14/Tyr15 dephosphorylation/activation, accompanied by a marked increase in ERK1/2 phosphorylation/activation. Furthermore, treatment with both Chk1 inhibitors induced translocation of Ras to membrane fractions and marked increases in Ras activity. Co-administration of either the FTI R115777 (tipifarnib) or the MEK1/2 inhibitor PD184352 attenuated ERK1/2 activation, enhanced cdc2 activation, increased DNA damage (manifested by γH2AX expression), and strikingly induced apoptosis. Notably, stable transfection with dominant-negative Ras (S17N) or MEK1 knockdown by siRNA largely abrogated Chk1 inhibitor-mediated ERK1/2 activation, and sensitized cells to cdc2 activation, DNA damage, and cell death induced by the Chk1 inhibitors. Conversely, transfection with a constitutively active Ras (Q61L) or ectopic expression of B-Raf prevented R115777 from blocking ERK1/2 activation, promoting cdc2 dephosphorylation, or increasing γH2AX expression in cells exposed to the Chk1 inhibitors, substantially reducing apoptosis. In contrast, transfection with either Q61L Ras or B-Raf failed to prevent PD184352 from blocking Chk1 inhibitor-induced ERK1/2 activation, and such cells remained fully sensitive to cdc2 activation, DNA damage and lethality mediated by the PD184352/Chk1 inhibitor regimen. Finally, administration of UCN-01 resulted in a marked increase in expression of phospho-ERK in tumor cells in athymic nude mice inoculated with MM.1S cells, an event prevented by co-treatment with R115777. Moreover, combined treatment with R115777 and UCN-01 resulted in a significant reduction in tumor size, compared to the effects of these agents administered individually. This was associated with a dramatic inhibition of cell proliferation (reflected by reduced expression of the nuclear proliferation antigen Ki67) and a striking induction of tumor cell apoptosis (manifested by TUNEL staining). Together, these findings indicate that activation of Ras/Raf/MEK/ERK pathway plays a critical role in regulating the lethality of Chk1 inhibitors in vitro and in vivo, while interruption of this compensatory cytoprotective response dramatically potentiates the lethal actions of Chk1 inhibitors (e.g., DNA damage and “unscheduled” cdc2 activation), thereby triggering cell death. They also suggest that co-targeting the checkpoint machinery (e.g., by Chk1 inhibitors) and the pro-survival Ras/Raf/MEK/ERK pathway (by FTIs or MEK1/2 inhibitors) may represent a useful therapeutic strategy in MM and other hematological malignancies.