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PLOS ONE  2014 

Identification of a Novel Function of CX-4945 as a Splicing Regulator

DOI: 10.1371/journal.pone.0094978

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Alternative splicing is a nearly ubiquitous versatile process that controls gene expression and creates numerous protein isoforms with different functions from a single gene. The significance of alternative splicing has been confirmed by the increasing number of human diseases that are caused by misregulation of splicing events. Very few compounds, however, have been reported to act as inhibitors of alternative splicing, and their potential clinical use needs to be evaluated. Here, we report that CX-4945, a previously well-characterized inhibitor of casein kinase 2 (CK2) and a molecule currently in clinical trials (Phase II) for cancer treatment, regulates splicing in mammalian cells in a CK2-independent manner. Transcriptome-wide analysis using exon array also showed a widespread alteration in alternative splicing of numerous genes. We found that CX-4945 potently inhibits the Cdc2-like kinases (Clks) in vitro and in turn, leads to suppression of the phosphorylation of serine/arginine-rich (SR) proteins in mammalian cells. Surprisingly, the overall efficacy of CX-4945 on Clks (IC50 = 3–90 nM) was stronger than that of TG-003, the strongest inhibitor reported to date. Of the Clks, Clk2 was most strongly inhibited by CX-4945 in an ATP-competitive manner. Our research revealed an unexpected activity of the drug candidate CX-4945 as a potent splicing modulator and also suggested a potential application for therapy of diseases caused by abnormal splicing.


[1]  Black DL (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72: 291–336.
[2]  Chabot B (1996) Directing alternative splicing: cast and scenarios. Trends Genet 12: 472–478. doi: 10.1016/0168-9525(96)10037-8
[3]  Blencowe BJ (2006) Alternative splicing: new insights from global analyses. Cell 126: 37–47. doi: 10.1016/j.cell.2006.06.023
[4]  Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 40: 1413–1415. doi: 10.1038/ng.259
[5]  Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, et al. (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456: 470–476. doi: 10.1038/nature07509
[6]  Lukong KE, Chang KW, Khandjian EW, Richard S (2008) RNA-binding proteins in human genetic disease. Trends Genet 24: 416–425. doi: 10.1016/j.tig.2008.05.004
[7]  Martinez-Contreras R, Cloutier P, Shkreta L, Fisette JF, Revil T, et al. (2007) hnRNP proteins and splicing control. Adv Exp Med Biol 623: 123–147. doi: 10.1007/978-0-387-77374-2_8
[8]  Faustino NA, Cooper TA (2003) Pre-mRNA splicing and human disease. Genes Dev 17: 419–437. doi: 10.1101/gad.1048803
[9]  Ng B, Yang F, Huston DP, Yan Y, Yang Y, et al. (2004) Increased noncanonical splicing of autoantigen transcripts provides the structural basis for expression of untolerized epitopes. J Allergy Clin Immunol 114: 1463–1470. doi: 10.1016/j.jaci.2004.09.006
[10]  Matlin AJ, Clark F, Smith CW (2005) Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 6: 386–398. doi: 10.1038/nrm1645
[11]  Sanford JR, Longman D, Caceres JF (2003) Multiple roles of the SR protein family in splicing regulation. Prog Mol Subcell Biol 31: 33–58. doi: 10.1007/978-3-662-09728-1_2
[12]  Valcarcel J, Green MR (1996) The SR protein family: pleiotropic functions in pre-mRNA splicing. Trends Biochem Sci 21: 296–301. doi: 10.1016/0968-0004(96)10039-6
[13]  Fu XD (1995) The superfamily of arginine/serine-rich splicing factors. RNA 1: 663–680.
[14]  Hertel KJ, Graveley BR (2005) RS domains contact the pre-mRNA throughout spliceosome assembly. Trends Biochem Sci 30: 115–118. doi: 10.1016/j.tibs.2005.01.002
[15]  Xiao SH, Manley JL (1997) Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing. Genes Dev 11: 334–344. doi: 10.1101/gad.11.3.334
[16]  Caceres JF, Screaton GR, Krainer AR (1998) A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm. Genes Dev 12: 55–66. doi: 10.1101/gad.12.1.55
[17]  Misteli T, Caceres JF, Clement JQ, Krainer AR, Wilkinson MF, et al. (1998) Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo. J Cell Biol 143: 297–307. doi: 10.1083/jcb.143.2.297
[18]  Colwill K, Pawson T, Andrews B, Prasad J, Manley JL, et al. (1996) The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO J 15: 265–275.
[19]  Nayler O, Stamm S, Ullrich A (1997) Characterization and comparison of four serine- and arginine-rich (SR) protein kinases. Biochem J 326 (Pt 3): 693–700.
[20]  Younis I, Berg M, Kaida D, Dittmar K, Wang C, et al. (2010) Rapid-response splicing reporter screens identify differential regulators of constitutive and alternative splicing. Mol Cell Biol 30: 1718–1728. doi: 10.1128/mcb.01301-09
[21]  Gui JF, Tronchere H, Chandler SD, Fu XD (1994) Purification and characterization of a kinase specific for the serine- and arginine-rich pre-mRNA splicing factors. Proc Natl Acad Sci U S A 91: 10824–10828. doi: 10.1073/pnas.91.23.10824
[22]  Kuroyanagi N, Onogi H, Wakabayashi T, Hagiwara M (1998) Novel SR-protein-specific kinase, SRPK2, disassembles nuclear speckles. Biochem Biophys Res Commun 242: 357–364. doi: 10.1006/bbrc.1997.7913
[23]  Kaida D, Motoyoshi H, Tashiro E, Nojima T, Hagiwara M, et al. (2007) Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA. Nat Chem Biol 3: 576–583. doi: 10.1038/nchembio.2007.18
[24]  Kotake Y, Sagane K, Owa T, Mimori-Kiyosue Y, Shimizu H, et al. (2007) Splicing factor SF3b as a target of the antitumor natural product pladienolide. Nat Chem Biol 3: 570–575. doi: 10.1038/nchembio.2007.16
[25]  Levinson N, Hinman R, Patil A, Stephenson CR, Werner S, et al. (2006) Use of transcriptional synergy to augment sensitivity of a splicing reporter assay. RNA 12: 925–930. doi: 10.1261/rna.8306
[26]  Muraki M, Ohkawara B, Hosoya T, Onogi H, Koizumi J, et al. (2004) Manipulation of alternative splicing by a newly developed inhibitor of Clks. J Biol Chem 279: 24246–24254. doi: 10.1074/jbc.m314298200
[27]  Pilch B, Allemand E, Facompre M, Bailly C, Riou JF, et al. (2001) Specific inhibition of serine- and arginine-rich splicing factors phosphorylation, spliceosome assembly, and splicing by the antitumor drug NB-506. Cancer Res 61: 6876–6884.
[28]  Soret J, Bakkour N, Maire S, Durand S, Zekri L, et al. (2005) Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors. Proc Natl Acad Sci U S A 102: 8764–8769. doi: 10.1073/pnas.0409829102
[29]  Stoilov P, Lin CH, Damoiseaux R, Nikolic J, Black DL (2008) A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators. Proc Natl Acad Sci U S A 105: 11218–11223. doi: 10.1073/pnas.0801661105
[30]  Sumanasekera C, Watt DS, Stamm S (2008) Substances that can change alternative splice-site selection. Biochem Soc Trans 36: 483–490. doi: 10.1042/bst0360483
[31]  Siddiqui-Jain A, Drygin D, Streiner N, Chua P, Pierre F, et al. (2010) CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy. Cancer Res 70: 10288–10298. doi: 10.1158/0008-5472.can-10-1893
[32]  Venkatachalam CM, Jiang X, Oldfield T, Waldman M (2003) LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites. J Mol Graph Model 21: 289–307. doi: 10.1016/s1093-3263(02)00164-x
[33]  Litchfield DW (2003) Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochem J 369: 1–15. doi: 10.1042/bj20021469
[34]  Guerra B, Issinger OG (2008) Protein kinase CK2 in human diseases. Curr Med Chem 15: 1870–1886. doi: 10.2174/092986708785132933
[35]  Tawfic S, Yu S, Wang H, Faust R, Davis A, et al. (2001) Protein kinase CK2 signal in neoplasia. Histol Histopathol 16: 573–582.
[36]  Ahmad KA, Wang G, Unger G, Slaton J, Ahmed K (2008) Protein kinase CK2—a key suppressor of apoptosis. Adv Enzyme Regul 48: 179–187. doi: 10.1016/j.advenzreg.2008.04.002
[37]  Trembley JH, Wang G, Unger G, Slaton J, Ahmed K (2009) Protein kinase CK2 in health and disease: CK2: a key player in cancer biology. Cell Mol Life Sci 66: 1858–1867. doi: 10.1007/s00018-009-9154-y
[38]  Dalma-Weiszhausz DD, Warrington J, Tanimoto EY, Miyada CG (2006) The affymetrix GeneChip platform: an overview. Methods Enzymol 410: 3–28. doi: 10.1016/s0076-6879(06)10001-4
[39]  Takehara T, Liu X, Fujimoto J, Friedman SL, Takahashi H (2001) Expression and role of Bcl-xL in human hepatocellular carcinomas. Hepatology 34: 55–61. doi: 10.1053/jhep.2001.25387
[40]  Bardella C, Costa B, Maggiora P, Patane S, Olivero M, et al. (2004) Truncated RON tyrosine kinase drives tumor cell progression and abrogates cell-cell adhesion through E-cadherin transcriptional repression. Cancer Res 64: 5154–5161. doi: 10.1158/0008-5472.can-04-0600
[41]  Duncan PI, Stojdl DF, Marius RM, Bell JC (1997) In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase. Mol Cell Biol 17: 5996–6001.
[42]  Sarno S, Reddy H, Meggio F, Ruzzene M, Davies SP, et al. (2001) Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 (‘casein kinase-2′). FEBS Lett 496: 44–48. doi: 10.1016/s0014-5793(01)02404-8
[43]  Pagano MA, Poletto G, Di Maira G, Cozza G, Ruzzene M, et al. (2007) Tetrabromocinnamic acid (TBCA) and related compounds represent a new class of specific protein kinase CK2 inhibitors. Chembiochem 8: 129–139. doi: 10.1002/cbic.200600293
[44]  Di Maira G, Salvi M, Arrigoni G, Marin O, Sarno S, et al. (2005) Protein kinase CK2 phosphorylates and upregulates Akt/PKB. Cell Death Differ 12: 668–677. doi: 10.1038/sj.cdd.4401604
[45]  Zahler AM, Lane WS, Stolk JA, Roth MB (1992) SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev 6: 837–847. doi: 10.1101/gad.6.5.837
[46]  Ferguson AD, Sheth PR, Basso AD, Paliwal S, Gray K, et al. (2011) Structural basis of CX-4945 binding to human protein kinase CK2. FEBS Lett 585: 104–110. doi: 10.1016/j.febslet.2010.11.019
[47]  Dredge BK, Polydorides AD, Darnell RB (2001) The splice of life: alternative splicing and neurological disease. Nat Rev Neurosci 2: 43–50. doi: 10.1038/35049061
[48]  Licatalosi DD, Darnell RB (2006) Splicing regulation in neurologic disease. Neuron 52: 93–101. doi: 10.1016/j.neuron.2006.09.017
[49]  Kim E, Goren A, Ast G (2008) Alternative splicing and disease. RNA Biol 5: 17–19. doi: 10.4161/rna.5.1.5944
[50]  Dahmus ME (1981) Purification and properties of calf thymus casein kinases I and II. J Biol Chem 256: 3319–3325.
[51]  Cabrejos ME, Allende CC, Maldonado E (2004) Effects of phosphorylation by protein kinase CK2 on the human basal components of the RNA polymerase II transcription machinery. J Cell Biochem 93: 2–10. doi: 10.1002/jcb.20209
[52]  Coombs TC, Tanega C, Shen M, Wang JL, Auld DS, et al. (2013) Small-molecule pyrimidine inhibitors of the cdc2-like (Clk) and dual specificity tyrosine phosphorylation-regulated (Dyrk) kinases: development of chemical probe ML315. Bioorg Med Chem Lett 23: 3654–3661. doi: 10.1016/j.bmcl.2013.02.096
[53]  Rodgers JT, Haas W, Gygi SP, Puigserver P (2010) Cdc2-like kinase 2 is an insulin-regulated suppressor of hepatic gluconeogenesis. Cell Metab 11: 23–34. doi: 10.1016/j.cmet.2009.11.006


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