Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells.

The relationship between the checkpoint kinase Chk1 and the STAT3 pathway was examined in multiple myeloma (MM) cells. Gene expression profiling of U266 cells exposed to low (nM) Chk1 inhibitor (PF-477736) concentrations revealed STAT3 pathway-related gene down-regulation (e.g., BCL-XL, MCL-1, c-Myc), findings confirmed by RT-PCR. This was associated with marked inhibition of STAT3 Tyr705 (but not Ser727) phosphorylation, dimerization, nuclear localization, DNA binding, STAT3 promoter activity by ChIP assay, and down-regulation of STAT-3-dependent proteins. Similar findings were obtained in other MM cells and with alternative Chk1 inhibitors (e.g., prexasertib, CEP3891). While PF did not reduce GP130 expression or modify SOCS or PRL-3 phosphorylation, the phosphatase inhibitor pervanadate antagonized PF-mediated Tyr705 dephosphorylation. Significantly, PF attenuated Chk1-mediated STAT3 phosphorylation in in vitro assays. SPR analysis suggested Chk1/STAT3 interactions and PF reduced Chk1/STAT3 co-immunoprecipitation. Chk1 CRISPR knockout or shRNA knockdown cells also displayed STAT3 inactivation and STAT-3-dependent protein down-regulation. Constitutively active STAT3 diminished PF-mediated STAT3 inactivation and down-regulate STAT3-dependent proteins while significantly reducing PF-induced DNA damage (rH2A.X formation) and apoptosis. Exposure of cells with low basal phospho-STAT3 expression to IL-6 or human stromal cell conditioned medium activated STAT3, an event attenuated by Chk1 inhibitors. PF also inactivated STAT3 in primary human CD138+ MM cells and tumors extracted from an NSG MM xenograft model while inhibiting tumor growth. Implications: These findings identify a heretofore unrecognized link between the Chk1 and STAT3 pathways and suggest that Chk1 pathway inhibitors warrant attention as novel and potent candidate STAT3 antagonists in myeloma.