P04.74 Preclinical evaluation of combinations targeting the DNA damage response in 2D and 3D models of glioblastoma stem cells

Background
Despite surgical resection followed by DNA-damaging adjuvant therapies, glioblastoma remain incurable. Increasing evidence demonstrates that aberrations within the DNA damage response (DDR) of cancer stem cells contribute to treatment resistance. We have previously shown that the Fanconi Anaemia (FA) pathway, a key DDR process, remains inactive in normal brain but is re-activated in glioblastoma, making it an appealing foundational target for cancer-specific combination therapies. Since intratumoural heterogeneity in glioblastoma and inherent capacity for functional redundancy within DDR networks are established concepts - we aimed to determine whether combined and hypothesis-driven targeting of the FA pathway along with interconnected DDR processes could form a basis for effective multimodal therapies.

Material and Methods
Bioinformatic analysis of mRNA expression data (REMBRANDT database) was used to confirm the relevance of FA pathway activity in glioma. Subsequently, immunofluorescence and cell viability assays were used to validate and establish the therapeutic potential of novel FA pathway inhibitors (nFAPi) and inhibition of related DDR targets in established cell models. Finally, combinations targeting the DDR were optimised using immunoblotting, and assessed using clonogenic survival in 2D and novel 3D patient-derived glioblastoma stem cell models.

Results
High expression of downstream FA pathway genes is strongly associated with poor survival (-17.1% 5-year OS, n=329, Log-rank, P
Conclusion
Simultaneously targeting the FA pathway and interconnected DDR processes in glioblastoma represents a promising therapeutic strategy. Early mechanistic studies suggest this approach augments DNA damage and enhances IR-induced cell cycle arrest in G2/M, however further preclinical evaluation is ongoing.