118P Aberrant splicing in Ewing sarcoma: Preclinical evaluation of SM08502 in combination with vincristine and biomarkers of response

Background: Ewing sarcoma (ES) is an aggressive pediatric tumor driven by the EWS-FLI1 fusion oncoprotein, which not only alters transcription but also reprograms pre-mRNA splicing to sustain tumor survival.This highlights spliceosome regulation as a promising therapeutic vulnerability.Cirtuvivint (SM08502), a dual CLK/DYRK inhibitor, disrupts phosphorylation of SR splicing factors and remodels oncogenic splicing programs.Vincristine (VCR), a standard agent in ES treatment, induces microtubule destabilization, mitotic arrest, and apoptosis.We hypothesized that simultaneous perturbation of aberrant splicing and mitotic checkpoints may produce enhanced antitumor responses.Methods: ES cell lines were treated with cirtuvivint, VCR, or the combination.Viability and apoptosis were assessed by multicolor flow cytometry.Protein expression of EWS-FLI1 and splicing regulators was analyzed by Western blotting.Drug interactions were evaluated by combination index analysis.Results: Cirtuvivint reduced phosphorylation of spliceosomal SR proteins and downregulated EWS-FLI1 expression.Flow cytometry showed increased apoptosis with cirtuvivint, further enhanced when combined with VCR.The combination significantly decreased viable cells compared with either monotherapy.Western blotting confirmed modulation of splicing regulators and increased markers of mitotic stress under combinatorial treatment, supporting mechanistic synergy.Conclusions: CLK/DYRK inhibition disrupts oncogenic splicing programs and sensitizes ES cells to VCR-induced mitotic arrest.The cirtuvivint-VCR combination demonstrates synergistic cytotoxicity and represents a promising strategy to exploit splicing vulnerabilities in ES.Furthermore, these findings could extend to other fusion driven and splicing dysregulated pediatric cancers, establishing a broader paradigm for targeting oncogenic splicing programs and improving outcomes in children and adolescents.This project will deliver mechanistic, preclinical, and translational evidence to support the clinical testing of splicing modulators, aiming to improve outcomes for children and adolescents with Ewing sarcoma.