Selective killing of tumor cells requires the identification of drug targets critical to pathways that drive or support cancer progression. Protein kinases are an important class of intracellular enzymes involved in the regulation of biochemical pathways, deregulation of these kinases has been strongly implicated in cancer progression. To identify possible oncogenic kinases to which tumor cells might be selectively addicted, we screened the ON108 series of ATP-mimetic small molecule inhibitors in various triple negative breast cancer (TNBC) and normal cell lines. This approach led us to the identification of a novel kinase inhibitor, ON108600. We first examined the in vitro and in vivo effects of ON108600. ON108600 was found to be a potent inhibitor of Casein Kinase 2 (CK2) and the Dual-Specificity-Tyrosine (Y)-Phosphorylation-Regulated-Kinase (DYRK) family of serine-threonine kinases, both of which have been implicated in cancer progression. ON108600 showed broad-spectrum anti-proliferative and cytotoxic activity in multiple TNBC cell lines whilst having little or no effect on normal cells. Treatment of cancer cells with ON108600 resulted in inhibition of downstream signaling mediated by substrates of CK2. Further, ON108600 selectively arrested cancer cells in the mitotic phase of the cell cycle and activated the caspase-signaling cascade. We next performed x-ray crystallographic studies of ON108600-CK2 to determine the structural basis of ON108600-CK2 interaction. The co-crystal structure of ON108600-CK2 revealed that ON108600 binds in the active site pocket of CK2α wherein it mimics the binding of ATP and GTP in the CK2 active site. Notably, ON108600 mimics not only the shape and electrostatics of ATP/GTP, but also their hydration patterns in the CK2 active site pocket. Structural studies further revealed that ON108600 induces a conformational change in the β4-β5 loop of the catalytic subunit, which is known to interact with the β-regulatory subunit of CK2 and is critical for substrate recognition and activation. Lastly, we examined the efficacy of ON108600 in Triple Negative Breast Cancer (TNBC) and its ability to target and eliminate chemo-resistant Tumor-Initiating Stem Cells (TI-SCs) in TNBC. Clonogenic survival and sphere forming ability of purified CD44high CD24-/low TI-SCs from MDAMB-231 and Hs578t cells was potently inhibited by ON108600 treatment. We also observed that paclitaxel-resistant MDAMB-231 cells had increased levels of the CD44high CD24-/low stem cell- like population that correlated with increased expression of kinases CK2α2, DYRK1A and DYRK1B and these cells were sensitive to ON108600 treatment. Significantly, ON108600 showed robust antitumor efficacy as a single agent in a highly aggressive orthotopic TNBC xenograft model showing ~60% tumor growth inhibition. Immunohistochemical analysis of ON108600 treated tumors showed that a significant percentage of cells were apoptotic, indicating that activation of caspase mediated apoptosis contributes to the mechanism of action of ON108600 in vivo. Taken together, our results demonstrate that ON108600 is a novel and potent inhibitor of the CK2α1, CK2α2, DYRK1A and DYRK1B kinases. ON108600 binds in the active site pocket of CK2α and mimics ATP-GTP binding. ON108600 inhibits CK2-mediated signaling; arrests cancer cells in mitosis and induces apoptotic cell death via activation of caspases. Importantly, ON108600 is able to effectively kill the CD44high CD24-/low breast-cancer stem cell like population from TNBC cells. Finally, taxol-resistant MDAMB-231 TNBC cells express high levels of CD44, CK2α2, DYRK1a and DYRK1b and are sensitive to ON108600 treatment. Our study represents the first attempt to associate protein kinase CK2, DYRK1A and DYRK1B with TNBC and TI-SCs in TNBC and identifies a novel kinase inhibitor, ON108600 which effectively kills TI-SCs and taxol-resistant cells in TNBC. / Molecular Biology and Genetics
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/3375 |
| Date | January 2014 |
| Creators | Padgaonkar, Amol |
| Contributors | Reddy, E. Premkumar, Graña-Amat, Xavier, Shore, Scott K., Liebermann, Dan A., 1949-, Testa, Joseph R. |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 115 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/3357, Theses and Dissertations |
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