Abstract Prostate cancer (CaP) is a leading cause of cancer deaths in American men. There is an urgent need for novel treatments that are mechanistically different and target biological processes that drive CaP progression. We previously uncovered a critical role for the poorly characterized mitotic citron kinase (CIT) as a driver of CaP growth and treatment resistance, irrespective of CaP stage or lineage. CIT did not impact proliferation of benign cells, indicating a large therapeutic window to interfere with CIT action. CIT’s effects on CaP cells relied entirely on its kinase activity, but selective CIT inhibitors that prevent CaP growth are not available. We hypothesized that understanding how CIT kinase’s substrates impact CaP cell behavior will result in novel therapeutic strategies. The majority of the CIT substrates that we previously identified function in alternative splicing. Here, we focus on Thyroid Hormone Receptor-Associated Protein 3 (THRAP3), an RNA-binding protein implicated in mRNA splicing and stability. In vitro kinase assays revealed the CIT-dependent phosphosites in THRAP3. The impact of mutating these sites was determined in cell proliferation assays, cellular distribution assays and THRAP3 RNA binding studies. CLIP-Seq assays defined CIT-dependent RNA-binding for THRAP3 in CaP cells. Motif and pathway analyses were done on CLIP-Seq data, which were validated in RIP and qRT-PCR studies. Clinical relevance of CLIP-Seq data was verified in patients’ CaP transcriptomics datasets. The effect of overexpressing CIT- and THRAP3- (in) dependent transcripts was defined in in vitro cell proliferation, xenograft, patient-derived organoid (PDO) and PDX-derived organoid (PDXO) studies. Antisense oligos (ASOs) that prevent THRAP3 RNA-binding and resemble those that are FDA-approved for genetic diseases were developed. THRAP3 silencing and overexpression diminished and increased, respectively, CaP cell proliferation, mimicking CIT’s effects. Moreover, THRAP3 overexpression rescued the inhibitory effects of CIT depletion on CaP cell proliferation. CIT phosphorylated THRAP3 at S243, a phosphomark enriched in clinical CaP phosphoproteomics data. S243A mutation decreased CaP cell proliferation and THRAP3’s binding to pre-mRNA targets. We identified 311 CIT-dependent THRAP3-bound genes that were preferentially involved in cell proliferation. THRAP3 RNA-binding was enriched in genes that undergo alternative splicing during CaP progression. CIT-dependent THRAP3-bound transcripts stimulated the growth of CaP cell lines, xenografts, PDOs and PDXOs whereas CIT-independent THRAP3-bound transcripts from the same gene did not. An ASO that blocks THRAP3’s recruitment to a CIT-dependent RNA-binding site prevented the emergence of CIT- and THRAP3-dependent transcripts and inhibited the growth of cell lines and PDXOs representing different CaP stages and lineages, but not of benign cells. These findings define a novel mechanism by which CIT controls CaP growth by rewiring alternative splicing and present new therapeutic opportunities to treat aggressive CaP. Citation Format: Chitra Rawat, Ujjwal R. Dahiya, Sarah Ng, Nidhi Singh, Qiang Hu, Dingxiao Zhang, Shaun R. Stauffer, Amina Zoubeidi, Eva Corey, Jesse McKenney, Christopher Weight, Samuel C. Haywood, Song Liu, Tao Liu, Hannelore V. Heemers. Citron kinase-driven alternative splicing controls prostate cancer growth abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (2Suppl): Abstract nr B029.
Rawat et al. (Tue,) studied this question.