Abstract LB-204: SON controls the oncogenic alternative splicing program in glioblastoma by regulating PTBP1/2 switch and RBFOX2 activity

Alternative RNA splicing is a critical regulatory step of gene expression that generates various spliced isoforms and controls RNA stability. While dysregulation of alternative splicing has been implicated in pathogenesis of several cancers, molecular mechanisms of aberrant RNA splicing in brain tumors remain largely uninvestigated. Here, we identified SON, a nuclear speckle protein containing both RNA and DNA binding domains, as a key regulator of the oncogenic alternative splicing program in glioblastoma. Analyses of brain tumor patient samples as well as publicly available databases demonstrated that SON is significantly upregulated in high grade gliomas and correlated with poor prognosis of the patients. SON depletion in glioblastoma cells resulted in inhibition of cell growth and colony formation, suggesting that SON play a pro-survival and oncogenic role in glioblastoma. Interestingly, we found that the SON level in glioblastoma patients has a strong positive correlation with the expression level of PTBP1, a well-known oncogenic RNA splicing factor, and furthermore exhibits an inverse correlation with PTBP2, a neuronal-specific PTBP1 paralog. RNA splicing analysis together with RNA-IP reveals that SON directly interacts with PTBP1 pre-mRNA, and SON knockdown leads to downregulation of PTBP1 through intron retention in PTBP1 pre-mRNA, resulting in inhibition of oncogenic RNA splicing of PTBP1 target genes. On the other hand, SON knockdown strongly induces PTBP2 cassette exon (exon 10) inclusion, resulting in generation of PTBP2 mRNA that does not bear a pre-mature stop codon and subsequent upregulation of PTBP2. To further identify the molecular mechanism of PTBP2 exon 10 inclusion upon SON knockdown, we searched for binding motifs of RNA splicing factors in the PTBP2 pre-mRNA and identified RBFOX2 binding motifs in intron sequences. Interestingly, RNA-IP experiments revealed that both SON and RBFOX2 are enriched in introns flanking the cassette exon and SON knockdown significantly increased RBFOX2-binding to PTBP2 RNA and subsequent cassette exon inclusion. Conversely, RBFOX2 knockdown facilitated SON-binding to PTBP2 RNA and induced skipping of the cassette exon. These results demonstrate that SON competes with RBFOX2 for RNA binding at PTBP2 introns to regulate exon exclusion, affecting PTBP2 expression. Finally, we verified that SON knockdown in human patient-derived glioblastoma stem cells (GSCs) indeed induces downregulation of PTBP1 and its downstream oncogenic splicing program while enhancing RBFOX2-mediated cassette exon inclusion that are found in healthy brains. Taken together, our study defines SON as a novel master splicing regulator that initiates “the oncogenic alternative splicing program” in glioblastoma by promoting PTBP1-mediated oncogenic splicing while suppressing non-oncogenic splicing mediated by RBFOX2 through its competitive binding to target RNAs. Therefore, our findings strongly implicate SON as a potential therapeutic target in malignant brain tumors. Citation Format: Jung-Hyun Kim, Joshua K. Stone, Jianfeng Li, Alexander Richard, Lana Vukadin, G. Yancey Gillespie, Robert W. Sobol, Steve Lim, Eun-Young Erin Ahn. SON controls the oncogenic alternative splicing program in glioblastoma by regulating PTBP1/2 switch and RBFOX2 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-204.