Quantitative Proteomic Characterization of CX-4945, a Clinical Stage Inhibitor of Protein Kinase CK2

Protein phosphorylation is controlled by protein kinases, and represents a critical signaling mechanism involved in the regulation of fundamental biological processes. Furthermore, the aberrant regulation of kinase activity is implicated in diseases such as cancer and has resulted in efforts to target kinases therapeutically. Protein kinase CK2, although frequently considered constitutively active, has emerged as a clinical target on the basis of its altered expression in different types of human cancers and its regulatory participation in multiple biological processes. In fact, CX-4945, a small molecule ATP-competitive inhibitor of CK2 has advanced to clinical trial and has been widely used to interrogate CK2-dependent signaling events in cells. Despite its widespread applications, an understanding of the mechanism of action of CX-4945 on cells remains limited. In this thesis, comparison of proteomic sample preparation strategies led to the development of a phosphoproteomic workflow that enabled the enrichment of phosphopeptides conforming to the CK2 consensus sequence. Using the optimized workflow, phosphoproteomic profiling was conducted in HeLa cells treated with CX-4945. Several phosphorylation sites conforming to the recognition motif for CK2 phosphorylation displayed significantly decreased phosphorylation in response to CX-4945. Kinase substrate enrichment analysis also revealed a broad impact of CX-4945 on several kinases other than CK2. Profiling of the kinome utilizing multiplexed inhibitor beads also revealed changes in the activity of other kinases including activation of the ERK MAPK pathway and inhibition of the PI3K/Akt/mTOR pathway. Studies with Inhibitor VIII, an unrelated CK2 inhibitor, also resulted in activation of the ERK MAPK pathway suggesting that CK2 has a role in regulating this pathway. By comparison, the PI3K/Akt/mTOR pathway was not affected by Inhibitor VIII indicating that the effects of CX-4945 on that pathway are independent of CK2. Overall, this investigation provides valuable insight into the regulation of the phosphoproteome and the kinome in response to CX-4945 in HeLa cells. Dynamic markers of CK2 activity in cells were identified and putative CK2-independent effects of CX-4945 were revealed. Collectively, these studies illustrate the utility of global proteomic approaches to elucidate the cellular effects of clinical-stage kinase inhibitors

Molecular pathways: Emergence of protein kinase CK2 (CSNK2) as a potential target to inhibit survival and DNA damage response and repair pathways in cancer cells

©2016 AACR. Protein kinase CK2 (designated CSNK2) is a constitutively active protein kinase with a vast repertoire of putative substrates that has been implicated in several human cancers, including cancer of the breast, lung, colon, and prostate, as well as hematologic malignancies. On the basis of these observations, CSNK2 has emerged as a candidate for targeted therapy, with two CSNK2 inhibitors in ongoing clinical trials. CX-4945 is a bioavailable small-molecule ATP-competitive inhibitor targeting its active site, and CIGB-300 is a cell-permeable cyclic peptide that prevents phosphorylation of the E7 protein of HPV16 by CSNK2. In preclinical models, either of these inhibitors exhibit antitumor efficacy. Furthermore, in combinations with chemotherapeutics such as cisplatin or gemcitabine, either CX-4945 or CIGB-300 promote synergistic induction of apoptosis. While CSNK2 is a regulatory participant in many processes related to cancer, its potential to modulate caspase action may be particularly pertinent to its emergence as a therapeutic target. Because the substrate recognition motifs for CSNK2 and caspases are remarkably similar, CSNK2 can block the cleavage of many caspase substrates through the phosphorylation of sites adjacent to cleavage sites. Phosphoproteomic strategies have also revealed previously underappreciated roles for CSNK2 in the phosphorylation of several key constituents of DNA damage and DNA repair pathways. Going forward, applications of proteomic strategies to interrogate responses to CSNK2 inhibitors are expected to reveal signatures for CSNK2 inhibition and molecular insights to guide new strategies to interfere with its potential to inhibit caspase action or enhance the susceptibility of cancer cells to DNA damage

Protein kinase CK2: Intricate relationships within regulatory cellular networks

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. Protein kinase CK2 is a small family of protein kinases that has been implicated in an expanding array of biological processes. While it is widely accepted that CK2 is a regulatory participant in a multitude of fundamental cellular processes, CK2 is often considered to be a constitutively active enzyme which raises questions about how it can be a regulatory participant in intricately controlled cellular processes. To resolve this apparent paradox, we have performed a systematic analysis of the published literature using text mining as well as mining of proteomic databases together with computational assembly of networks that involve CK2. These analyses reinforce the notion that CK2 is involved in a broad variety of biological processes and also reveal an extensive interplay between CK2 phosphorylation and other post-translational modifications. The interplay between CK2 and other post-translational modifications suggests that CK2 does have intricate roles in orchestrating cellular events. In this respect, phosphorylation of specific substrates by CK2 could be regulated by other post-translational modifications and CK2 could also have roles in modulating other post-translational modifications. Collectively, these observations suggest that the actions of CK2 are precisely coordinated with other constituents of regulatory cellular networks