Abstract 4272: Cirtuvivint inhibits Ewing sarcoma (ES) growth by suppressing EWS-FLI-1 expression and inducing alternate EWS-FLI1 splice variants

Abstract Background: The transcription factor EWS-FLI1 is responsible for malignant transformation and ES cell survival. The breakpoints observed typically occur within introns of EWSR1 or FLI1, and expression of EWS-FLI1 requires the splicing machinery to generate an in-frame transcript. Cirtuvivint (SM08502, Biosplice Therapeutics Inc) has been shown to disrupt spliceosome activity by inhibiting Cdc2-like (CLK) and dual-specificity tyrosine phosphorylation-regulated (DYRK) kinases that regulate pre-mRNA splicing. In view of this, we elected to test its effect on ES cell growth, EWS-FLI-1 expression, and whether it would generate alternate splice variants that could suppress ES cell growth. Results: Treatment of ES cells (A673, CHP100, and MHH ES1) with increasing concentrations of cirtuvivint, as well as its analog SM09419, significantly reduced cell viability (LD50 ∼250 nM) both in vitro and in vivo, This treatment suppressed EWS-FLI-1 expression and generated multiple alternative EWS-FLI-1 variants within 4-6 hours, marked by the loss of whole exons. Mechanistic studies showed reduced β-catenin and c-myc expression, however, siRNA experiments targeting these genes did not affect cell growth. To investigate the role of these alternative EWS-FLI1 splice variants, transcripts generated by cirtuvivint treatment were cloned, sequenced, and inserted into eukaryotic expression vectors, including a Tet-inducible system and vectors for cell-free protein expression. A luciferase reporter vector containing the EWSR1-FLI1 binding site (GGAAx20) was constructed to assess the impact of wild-type and variant EWSR1-FLI1 on downstream gene expression. Results showed that the expression of specific alternative EWSR1-FLI1 variants in ES cells significantly reduced cell viability, whereas others had no effect. Moreover, cirtuvivint-treated cells transfected with the luciferase reporter exhibited decreased luciferase expression, indicating a loss of gene expression regulated by the oncogenic fusion protein. Comprehensive transcriptome analysis (RNAseq) 4-6 hours post-treatment exhibited significant changes in gene expression, including those associated with alternate splicing, offering new insights on the molecular shifts induced by splicing modulation in these cells. Conclusion. These findings suggest that cirtuvivint, as a novel CLK/DYRK inhibitor, offers a new therapeutic strategy for targeting EWS-FLI-1 in ES. The induction of specific alternative splice variants has the ability to inhibit cell growth with the associated suppression of gene expression. A clinical trial in recurrent or refractory ES is planned. Citation Format: Sergei Vatolin, Gary K. Schwartz. Cirtuvivint inhibits Ewing sarcoma (ES) growth by suppressing EWS-FLI-1 expression and inducing alternate EWS-FLI1 splice variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4272.