Abstract A021: CLK inhibition induces mis-splicing of SOX8 in alveolar rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is a malignant tumor originating from skeletal muscle precursor cells and is the most common soft tissue sarcoma in children. RMS is classified histologically as embryonic (ERMS) or alveolar (ARMS). ARMS is generally more aggressive and has poorer outcomes. ARMS is mainly driven by oncogenic fusion proteins Pax3::Foxo1 (P3F1) or Pax7::Foxo1 (P7F1). SOX8, a transcription factor essential for embryonic development, is highly expressed in embryonic skeletal muscle but decreases as development advances. SOX8 plays a vital role in the survival of ARMS by maintaining muscle cells in a less differentiated state. P3F1 enhances SOX8 expression via super-enhancers. Our preliminary data indicate that, in the presence of T025, a Cdc2-like kinase (CLK) inhibitor, SOX8 undergoes mRNA mis-splicing, leading to the loss of normal SOX8 protein function. Methods: ARMS cell lines (RH30/RH4) were plated in six-well plates and allowed to adhere for 24 hours before treatment with different concentrations of T025 (0, 12, 37, 111, 333, and 1000 nm) for 48 hours. After treatment, cells were harvested and analyzed for changes in protein and RNA. Western blotting measured levels of SOX8, CLK2, MYOD1, and FOXO1 proteins. Total RNA was converted to cDNA to identify alternative splicing events in SOX8 mRNA. Results: Western blot analysis of both RH30 and RH4 shows a dose-dependent decrease in the expression of the SOX8 protein as T025 concentrations increase. Additionally, a reduction in the expression of MYOD1 and FOXO1 proteins was observed with higher doses. CLK2 protein levels were also suppressed with increasing T025 doses, confirming its activity. While both cell lines exhibited a dose-dependent response, RH4 appeared to be more significantly affected. Subsequent PCR analysis of RH30 cells treated with T025 revealed the presence of a mis-spliced isoform containing intronic sequences in exon 2, consistent with our RNA-seq data. Conclusion: Our findings reveal that CLK2 inhibition via T025 causes mis-splicing in the mRNA of the SOX8 in alveolar rhabdomyosarcoma. This results in reduced SOX8 protein levels and downregulation of other key proteins involved in rhabdomyosarcoma survival, impairing tumor proliferation and survival. This discovery further supports the idea that CLK inhibition is a promising target for future studies to disrupt mRNA splicing in ARMS. Citation Format: Vincent A. Terta, Qiong Wu, Jun Yang. CLK inhibition induces mis-splicing of SOX8 in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr A021.