High-risk neuroblastoma is a severe paediatric malignancy of the sympathetic nervous system, characterised by frequent drug resistance and low survival rates. Notably, MYCN-amplified high-risk neuroblastoma has been reported to be sensitive to disruptions in RNA splicing. Indisulam, a potent degrader of RNA Binding Motif 39 (RBM39), disrupts RNA splicing and affects multiple cellular pathways. We hypothesise that by interfering with alternative splicing, indisulam can render cancer cells more susceptible to additional perturbations, particularly in neuroblastoma.

Drug combination screens were conducted using indisulam and a library of 147 FDA-approved drugs across various cancer types. Cell proliferation was assessed using the Sulforhodamine B (SRB) assay, while apoptosis and cell cycle analysis were performed via flow cytometry. Immunofluorescence (IF) was used to detect R-loops, and γ-H2AX staining was employed to measure DNA damage. PCR was utilised to evaluate splicing events. Additionally, the ADP-Glo kinase assay system was used to confirm kinase inhibition. The in vivo efficacy of the combination treatment was validated in the Th-MYCN/ALK^F1174L allograft neuroblastoma mouse model.

The combination of indisulam and alectinib consistently emerged as one of the top synergistic hits in two independent drug synergy screens. Furthermore, co-treatment with indisulam and alectinib synergistically inhibited cell growth, induced apoptosis, and caused cell cycle arrest in a panel of neuroblastoma cell lines. Mechanistic studies revealed that alectinib decreased the kinase activity of Serine/Arginine Protein Kinase 1 (SRPK1), a key regulator of RNA splicing. This co-treatment exacerbated RNA splicing defects in genes involved in DNA damage response and R-loop resolution, leading to R-loop accumulation and increased DNA damage. In the Th-MYCN/ALK^F1174L neuroblastoma mouse model, combination therapy induced complete tumour regression and significantly improved survival rates compared with monotherapies.

Our findings highlight that targeting complementary RNA splicing components SRPK1 and RBM39 offers a promising strategy for enhancing therapeutic efficacy and improving outcomes in high-risk neuroblastoma. This approach represents a potential advancement in the treatment of this challenging paediatric cancer.