In advanced prostate cancer (PC), in particular after acquisition of resistance to androgen receptor (AR) signaling inhibitors (ARSI), upregulation of AR splice variants compromises endocrine therapy efficiency. Androgen receptor splice variant-7 (ARV7) is clinically the most relevant and has a distinct 3' untranslated region (3'UTR) compared to the AR full-length variant, suggesting a unique post-transcriptional regulation. Here, we set out to evaluate the applicability of the ARV7 3'UTR as a therapy target. A common single nucleotide polymorphism, rs5918762, was found to affect the splicing rate and thus the expression of ARV7 in cellular models and patient specimens. Serine/arginine-rich splicing factor 9 (SRSF9) was found to bind to and increase the inclusion of the cryptic exon 3 of ARV7 during the splicing process in the alternative C allele of rs5918762. The dual specificity protein kinase CLK2 interferes with the activity of SRSF9 by regulating its expression. Inhibition of the Cdc2-like kinase (CLK) family by the small molecules cirtuvivint or lorecivivint results in the decreased expression of ARV7. Both inhibitors show potent anti-proliferative effects in enzalutamide-treated or -naive PC models. Thus, targeting aberrant alternative splicing at the 3'UTR of ARV7 by disturbing the CLK2/SRSF9 axis might be a valuable therapeutic approach in late stage, ARSI-resistant PC.

Department of Experimental Biology Faculty of Science Palacký University Olomouc Czech Republic

Division of Experimental Urology Department of Urology Medical University of Innsbruck Austria

Institute of Cell Biology Biocenter Medical University of Innsbruck Austria

Institute of Pathology Neuropathology and Molecular Pathology Medical University of Innsbruck Austria

Institute of Pathology University Hospital Bonn Germany

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Dai C, Dehm SM, Sharifi N. Targeting the androgen signaling axis in prostate cancer. J Clin Oncol. 2023;41:4267–4278. PubMed PMC

Scher HI, Lu D, Schreiber NA, Louw J, Graf RP, Vargas HA, et al. Association of AR‐V7 on circulating tumor cells as a treatment‐specific biomarker with outcomes and survival in castration‐resistant prostate cancer. JAMA Oncol. 2016;2:1441–1449. PubMed PMC

Mayr C. Regulation by 3’‐untranslated regions. Annu Rev Genet. 2017;51:171–194. PubMed

Skeeles LE, Fleming JL, Mahler KL, Toland AE. The impact of 3′UTR variants on differential expression of candidate cancer susceptibility genes. PLoS One. 2013;8:e58609. PubMed PMC

Pamuła‐Piłat J, Tęcza K, Kalinowska‐Herok M, Grzybowska E. Genetic 3′UTR variations and clinical factors significantly contribute to survival prediction and clinical response in breast cancer patients. Sci Rep. 2020;10:5736. PubMed PMC

Bonnal SC, López‐Oreja I, Valcárcel J. Roles and mechanisms of alternative splicing in cancer – implications for care. Nat Rev Clin Oncol. 2020;17:457–474. PubMed

Liu LL, Xie N, Sun S, Plymate S, Mostaghel E, Dong X. Mechanisms of the androgen receptor splicing in prostate cancer cells. Oncogene. 2014;33:3140–3150. PubMed PMC

Jiménez‐Vacas JM, Herrero‐Aguayo V, Montero‐Hidalgo AJ, Gómez‐Gómez E, Fuentes‐Fayos AC, León‐González AJ, et al. Dysregulation of the splicing machinery is directly associated to aggressiveness of prostate cancer. EBioMedicine. 2020;51:102547. PubMed PMC

Sanjana NE, Shalem O, Zhang F. Improved vectors and genome‐wide libraries for CRISPR screening. Nat Methods. 2014;11:783–784. PubMed PMC

Furlan T, Kirchmair A, Sampson N, Puhr M, Gruber M, Trajanoski Z, et al. MYC‐mediated ribosomal gene expression sensitizes enzalutamide‐resistant prostate cancer cells to EP300/CREBBP inhibitors. Am J Pathol. 2021;191:1094–1107. PubMed

Schmidt O, Weyer Y, Baumann V, Widerin MA, Eising S, Angelova M, et al. Endosome and Golgi‐associated degradation (EGAD) of membrane proteins regulates sphingolipid metabolism. EMBO J. 2019;38:e101433. PubMed PMC

Brinkman EK, van Steensel B. Rapid quantitative evaluation of CRISPR genome editing by TIDE and TIDER. Methods Mol Biol. 2019;1961:29–44. PubMed

Tolkach Y, Kremer A, Lotz G, Schmid M, Mayr T, Förster S, et al. Androgen receptor splice variants contribute to the upregulation of DNA repair in prostate cancer. Cancers (Basel). 2022;14:4441. PubMed PMC

Watson MJ, Thoreen CC. Measuring mRNA decay with roadblock‐qPCR. Curr Protoc. 2022;2:e344. PubMed PMC

Gaildrat P, Killian A, Martins A, Tournier I, Frébourg T, Tosi M. Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants. Methods Mol Biol. 2010;653:249–257. PubMed

Kamiyama D, Sekine S, Barsi‐Rhyne B, Hu J, Chen B, Gilbert LA, et al. Versatile protein tagging in cells with split fluorescent protein. Nat Commun. 2016;7:11046. PubMed PMC

Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L, Johnson LA, et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature. 2010;468:968–972. PubMed PMC

Girstun A, Ishikawa T, Staron K. Effects of SRSF1 on subnuclear localization of topoisomerase I. J Cell Biochem. 2019;120:11794–11808. PubMed

Chang C‐T, Tsai C‐N, Tang CY, Chen C‐H, Lian J‐H, Hu C‐Y, et al. Mixed sequence reader: a program for analyzing DNA sequences with heterozygous base calling. ScientificWorldJournal. 2012;2012:365104. PubMed PMC

Cancer Genome Atlas Research Network . The molecular taxonomy of primary prostate cancer. Cell. 2015;163:1011–1025. PubMed PMC

Tang Z, Li C, Kang B, Gao G, Li C, Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017;45:W98–W102. PubMed PMC

Zivanovic A, Miller JT, Munro SA, Knutson TP, Li Y, Passow CN, et al. Co‐evolution of AR gene copy number and structural complexity in endocrine therapy resistant prostate cancer. NAR Cancer. 2023;5:zcad045. PubMed PMC

Henzler C, Li Y, Yang R, McBride T, Ho Y, Sprenger C, et al. Truncation and constitutive activation of the androgen receptor by diverse genomic rearrangements in prostate cancer. Nat Commun. 2016;7:13668. PubMed PMC

Li Y, Alsagabi M, Fan D, Bova GS, Tewfik AH, Dehm SM. Intragenic rearrangement and altered RNA splicing of the androgen receptor in a cell‐based model of prostate cancer progression. Cancer Res. 2011;71:2108–2117. PubMed PMC

Dadaev T, Saunders EJ, Newcombe PJ, Anokian E, Leongamornlert DA, Brook MN, et al. Fine‐mapping of prostate cancer susceptibility loci in a large meta‐analysis identifies candidate causal variants. Nat Commun. 2018;9:2256. PubMed PMC

Kote‐Jarai Z, Olama AAA, Giles GG, Severi G, Schleutker J, Weischer M, et al. Seven novel prostate cancer susceptibility loci identified by a multi‐stage genome‐wide association study. Nat Genet. 2011;43:785–791. PubMed PMC

Lorenz R, Bernhart SH, Höner Zu Siederdissen C, Tafer H, Flamm C, Stadler PF, et al. ViennaRNA package 2.0. Algorithms Mol Biol. 2011;6:26. PubMed PMC

Robinson D, Van Allen EM, Wu Y‐M, Schultz N, Lonigro RJ, Mosquera J‐M, et al. Integrative clinical genomics of advanced prostate cancer. Cell. 2015;161:1215–1228. PubMed PMC

Abida W, Cyrta J, Heller G, Prandi D, Armenia J, Coleman I, et al. Genomic correlates of clinical outcome in advanced prostate cancer. Proc Natl Acad Sci USA. 2019;116:11428–11436. PubMed PMC

Paz I, Kosti I, Ares M, Cline M, Mandel‐Gutfreund Y. RBPmap: a web server for mapping binding sites of RNA‐binding proteins. Nucleic Acids Res. 2014;42:W361–W367. PubMed PMC

Piva F, Giulietti M, Burini AB, Principato G. SpliceAid 2: a database of human splicing factors expression data and RNA target motifs. Hum Mutat. 2012;33:81–85. PubMed

Cook KB, Kazan H, Zuberi K, Morris Q, Hughes TR. RBPDB: a database of RNA‐binding specificities. Nucleic Acids Res. 2011;39:D301–D308. PubMed PMC

Shepard PJ, Hertel KJ. The SR protein family. Genome Biol. 2009;10:242. PubMed PMC

Song M, Pang L, Zhang M, Qu Y, Laster KV, Dong Z. Cdc2‐like kinases: structure, biological function, and therapeutic targets for diseases. Signal Transduct Target Ther. 2023;8:1–25. PubMed PMC

Jin H‐J, Kim J, Yu J. Androgen receptor genomic regulation. Transl Androl Urol. 2013;2:158–177. PubMed PMC

Venkadakrishnan VB, Ben‐Salem S, Heemers HV. AR‐dependent phosphorylation and phospho‐proteome targets in prostate cancer. Endocr Relat Cancer. 2020;27:R193–R210. PubMed PMC

Guo Z, Yang X, Sun F, Jiang R, Linn DE, Chen H, et al. A novel androgen receptor splice variant is up‐regulated during prostate cancer progression and promotes androgen depletion‐resistant growth. Cancer Res. 2009;69:2305–2313. PubMed PMC

Tam BY, Chiu K, Chung H, Bossard C, Nguyen JD, Creger E, et al. The CLK inhibitor SM08502 induces anti‐tumor activity and reduces Wnt pathway gene expression in gastrointestinal cancer models. Cancer Lett. 2020;473:186–197. PubMed

Deshmukh V, Hu H, Barroga C, Bossard C, Kc S, Dellamary L, et al. A small‐molecule inhibitor of the Wnt pathway (SM04690) as a potential disease modifying agent for the treatment of osteoarthritis of the knee. Osteoarthr Cartil. 2018;26:18–27. PubMed

Lindberg MF, Deau E, Arfwedson J, George N, George P, Alfonso P, et al. Comparative efficacy and selectivity of pharmacological inhibitors of DYRK and CLK protein kinases. J Med Chem. 2023;66:4106–4130. PubMed

Navone NM, Olive M, Ozen M, Davis R, Troncoso P, Tu SM, et al. Establishment of two human prostate cancer cell lines derived from a single bone metastasis. Clin Cancer Res. 1997;3:2493–2500. PubMed

Cato L, de Tribolet‐Hardy J, Lee I, Rottenberg JT, Coleman I, Melchers D, et al. ARv7 represses tumor‐suppressor genes in castration‐resistant prostate cancer. Cancer Cell. 2019;35:401–413.e6. PubMed PMC

Di Veroli GY, Fornari C, Wang D, Mollard S, Bramhall JL, Richards FM, et al. Combenefit: an interactive platform for the analysis and visualization of drug combinations. Bioinformatics. 2016;32:2866–2868. PubMed PMC

Sharp A, Coleman I, Yuan W, Sprenger C, Dolling D, Rodrigues DN, et al. Androgen receptor splice variant‐7 expression emerges with castration resistance in prostate cancer. J Clin Invest. 2019;129:192–208. PubMed PMC

Kim J, Park RY, Kee Y, Jeong S, Ohn T. Splicing factor SRSF3 represses translation of p21cip1/waf1 mRNA. Cell Death Dis. 2022;13:933. PubMed PMC

Schwich OD, Blümel N, Keller M, Wegener M, Setty ST, Brunstein ME, et al. SRSF3 and SRSF7 modulate 3′UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels. Genome Biol. 2021;22:82. PubMed PMC

Wang X, Lu X, Wang P, Chen Q, Xiong L, Tang M, et al. SRSF9 promotes colorectal cancer progression via stabilizing DSN1 mRNA in an m6A‐related manner. J Transl Med. 2022;20:198. PubMed PMC

Raffetseder U, Frye B, Rauen T, Jürchott K, Royer H‐D, Jansen PL, et al. Splicing factor SRp30c interaction with Y‐box protein‐1 confers nuclear YB‐1 shuttling and alternative splice site selection. J Biol Chem. 2003;278:18241–18248. PubMed

Simard MJ, Chabot B. SRp30c is a repressor of 3′ splice site utilization. Mol Cell Biol. 2002;22:4001–4010. PubMed PMC

Haltenhof T, Kotte A, De Bortoli F, Schiefer S, Meinke S, Emmerichs A‐K, et al. A conserved kinase‐based body‐temperature sensor globally controls alternative splicing and gene expression. Mol Cell. 2020;78:57–69.e4. PubMed

Yoshida T, Kim JH, Carver K, Su Y, Weremowicz S, Mulvey L, et al. CLK2 is an oncogenic kinase and splicing regulator in breast cancer. Cancer Res. 2015;75:1516–1526. PubMed

Heidenreich A, Pfister D, Merseburger A, Bartsch G, German Working Group on Castration‐Resistant Prostate Cancer (GWG‐CRPC) . Castration‐resistant prostate cancer: where we stand in 2013 and what urologists should know. Eur Urol. 2013;64:260–265. PubMed

Zhang D, Zhao S, Li X, Kirk JS, Tang DG. Prostate luminal progenitor cells in development and cancer. Trends Cancer. 2018;4:769–783. PubMed PMC

Pritsker M, Doniger TT, Kramer LC, Westcot SE, Lemischka IR. Diversification of stem cell molecular repertoire by alternative splicing. Proc Natl Acad Sci USA. 2005;102:14290–14295. PubMed PMC

Tian L, Jabbari JS, Thijssen R, Gouil Q, Amarasinghe SL, Voogd O, et al. Comprehensive characterization of single‐cell full‐length isoforms in human and mouse with long‐read sequencing. Genome Biol. 2021;22:310. PubMed PMC

Fu Y, Huang B, Shi Z, Han J, Wang Y, Huangfu J, et al. SRSF1 and SRSF9 RNA binding proteins promote Wnt signalling‐mediated tumorigenesis by enhancing β‐catenin biosynthesis. EMBO Mol Med. 2013;5:737–750. PubMed PMC

Zhang G, Liu B, Shang H, Wu G, Wu D, Wang L, et al. High expression of serine and arginine‐rich splicing factor 9 (SRSF9) is associated with hepatocellular carcinoma progression and a poor prognosis. BMC Med Genomics. 2022;15:180. PubMed PMC

Murillo‐Garzón V, Kypta R. WNT signalling in prostate cancer. Nat Rev Urol. 2017;14:683–696. PubMed

Haynes C, Iakoucheva LM. Serine/arginine‐rich splicing factors belong to a class of intrinsically disordered proteins. Nucleic Acids Res. 2006;34:305–312. PubMed PMC

Havens MA, Hastings ML. Splice‐switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Res. 2016;44:6549–6563. PubMed PMC

Luna Velez MV, Verhaegh GW, Smit F, Sedelaar JPM, Schalken JA. Suppression of prostate tumor cell survival by antisense oligonucleotide‐mediated inhibition of AR‐V7 mRNA synthesis. Oncogene. 2019;38:3696–3709. PubMed PMC

Qiu X, Boufaied N, Hallal T, Feit A, de Polo A, Luoma AM, et al. MYC drives aggressive prostate cancer by disrupting transcriptional pause release at androgen receptor targets. Nat Commun. 2022;13:2559. PubMed PMC

Sabha M, Siaton BC, Hochberg MC. Lorecivivint, an intra‐articular potential disease‐modifying osteoarthritis drug. Expert Opin Investig Drugs. 2020;29:1339–1346. PubMed