Abstract
Survival and proliferation of Multiple Myeloma plasma cells (MMPCs) depend on the activation of signaling pathways through the interaction with the surrounding bone marrow microenvironment. CK2 is a ubiquitous cellular serine-threonine kinase, whose involvement in oncogenic transformation, apoptosis and cell cycle progression has recently become matter of intense research. Due to its connection with signaling molecules pivotal for plasma cell (PCs) survival, such as those implicated in the TNF-α/NF-κB, IGF1/PI3K/AKT and Wnt/β-catenin pathways, CK2 is likely to play a central role in MM biology. We investigated CK2 function in MMPCs survival and cell cycle progression, in the modulation of the sensitivity to chemotherapeutics and in the regulation of the I-κB/NF-κB dependent pathway.
We first analysed the CK2 protein levels and specific kinase activity in MMPCs. Different cell lines and highly purified CD138+ PCs from 5 patients were used. We observed higher protein levels of the CK2 catalytic subunit αin the neoplastic MMPCs than in controls (resting peripheral blood and splenic B lymphocytes). Moreover, also the total CK2-dependent kinase activity was found significantly increased in MMPCs. We also assessed the levels and pattern of total protein phosphorylation by radioactive phosphate incorporation assay. We found that MMPCs share a similar pattern of phoshorylated proteins. The degree of phosphorylation of some of these proteins was significantly reduced in the presence of specific CK2 inhibitors. Next, using a panel of highly specific CK2 inhibitors, we studied the effects of hampering CK2 function in MMPCs. A dose-dependent cytotoxic effect was observed after the treatment with such compounds that was associated with the activation of both the extrinsic and intrinsic caspase-dependent pathways, the release from mitochondria of cytochrome c and smac/diablo and cell cycle arrest in G2-M. A possible role for CK2 inhibition in sensitising MMPCs to melphalan-induced apoptosis was also investigated. Indeed, CK2 blockade lowered the threshold of sensitivity of MMPCs to the cytotoxic effect of melphalan. We then looked at the consequences of CK2 blockade on the NF-κB dependent signaling cascade. Basal and TNF-α-dependent I-κB-αdegradation, as well as NF-κB transcriptional activity upon TNF-αstimulation, were partially impaired by CK2 blockade in MMPCs. Finally, we detected association between the endogenous αcatalytic subunit of CK2 and the NF-κB p50/p105 member by confocal microscopy and co-immunoprecipitation.
Altogether, our data suggest a pivotal role for CK2 in controlling survival, proliferation and sensitivity to chemotherapeutics of MMPCs and implicate this kinase in the regulation of the NF-κB pathway in MM through the modulation of I-κB protein levels and NF-κB transcriptional activity. This latter effect is possibly exerted through physical association of CK2 with NF-κB transcription factors. Our findings also suggest that CK2 inhibition could be exploited as a novel therapeutic approach for MM.
Tyr198 is the Essential Autophosphorylation Site for STK16 Localization and Kinase Activity