Background. Disrupting cancer cell dependency on the replication stress (RS) response by ATR inhibition (ATRi) is a promising strategy to improve outcomes for children with high-risk neuroblastoma. However, combination therapies are needed to overcome resistance. Epigenetic regulation is essential for DNA replication and repair, making it an attractive target for combination treatment with ATRi.
Aims. Exploit the interplay between the DNA Damage Response (DDR) and epigenetic regulation as a personalised High-Risk Neuroblastoma treatment.
Methods. Whole genome and RNA-sequencing data from patients enrolled onto the Australian ZERO Childhood Cancer program (n=157) were analysed to define DDR and epigenetic landscapes of neuroblastoma. Gene Set Enrichment Analysis (GSEA) was performed to identify pathway regulation, and unsupervised clustering defined transcriptional subgroups. ATR inhibitor Cerelasertib and epigenetic targeted drugs were tested alone using ZERO neuroblastoma samples ex vivo (n=16) or in combination using classical neuroblastoma cell lines (n=11) (MYCN-amplified: NGP, SK-N-BE-(2C), KCNR, IMR-32, SJNB6, KELLY; non-MYCN: SH-SY-5Y, CH-LA-90, GI-M-EN, SK-N-FI, SHEP) and patient-derived cell lines (n=3) to assess effects on cell viability. Bliss synergy scores were calculated. Dose-response curves established half-maximal inhibitory (IC50) and -lethal (LC50) concentrations.
Results. DDR and epigenetic gene alterations were reported in 17.9% (27/157) and 23.5% (37/157) neuroblastoma tumours, respectively, alterations co-occurred in only 3.8% (6/157). Transcriptomic profiling identified three subgroups with low, intermediate, or high enrichment of DNA replication, DDR, and epigenetic pathways. Significant association with samples at relapse (24/37, 65%; p=0.0089), MYCN-amplification (15/37, 40.5%; p=1.6×10⁻⁵), and chromosome 11q loss (17/37, 45.9%; p=0.049) was observed exclusively in the high-enrichment cluster. Cerelasertib combined with BET/BRD4, LSD1 or p300 inhibition, induced strongest synergism and potent cell killing in classical and patient-derived cell lines, independent of MYCN-amplification. Combined ATR and BET/BRD4 inhibition enhanced sub-G1 and G2/M accumulation beyond either agent alone.
Conclusion. Distinct transcriptional expression profiles highlight targetable DDR or epigenetic pathways in relapsed and/or High-Risk Neuroblastoma subtypes. Combined ATR and BRD4 or p300 inhibition is strongly synergistic, with high efficacy in classical and patient-derived Neuroblastoma models in vitro, supporting further mechanistic studies and in vivo validation.