Novel Integration of Bulk-seq and TRAP-seq to Explore Plx3397 Resistance Mechanisms in glioblastoma

AbstractBackground:Despite progress in various treatment modalities, glioma therapy remains challenging, which has poor outcomes and high recurrence rates. Preclinical studies have demonstrated that targeting tumor-associated macrophages and microglia (TAMs) through colony stimulating factor 1 receptor (CSF1R) inhibition is of significant importance for the treatment of gliomas. However, clinical trial results indicate that glioma patients have developed resistance to Plx3397(a CSF1R inhibitor), but the mechanisms underlying this resistance are not yet clear. In this study, we utilize G422TN-GBM to investigate the mechanisms of resistance to Plx3397 in order to identify appropriate combination therapy strategies.Methods: Single-cell datas from human glioblastoma (GBM) were used to analyze the role of CSF1 signaling in glioma. In vivo, we explored resistance mechanisms to Plx3397 in G422TN tumor cells using bulk sequencing and viral-based translating ribosome affinity purification (TRAP) profiling. Transmission electron microscopy was used to observe the mitochondrial morphology of tumor cells in situ. In vitro experiments assessed potential resistance mechanisms by measuring the expression of DNA damage-related genes, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), lactate production and ATP levels. Lastly, the effectiveness of combination therapies was evaluated in the G422TN-GBM model.Results: Bulk sequencing indicated that Plx3397 might reduce DNA damage repair signaling pathways. TRAP sequencing suggests that Plx3397 might affect tumor metabolic reprogramming to promote drug resistance. Transmission electron microscopy observed that the degree of mitochondrial destruction in tumor cells was greater after treatment with Plx3397. In vitro studies have confirmed that Plx3397 decreases TMZ-induced DNA damage repair gene expression. Piperlongumine (PL)and vorinostat (SAHA)restored oxidative phosphorylation inhibited by Plx3397 and decreased glycolysis. We showed that combining Plx3397 with TMZ and PL/vorinostat had a positive therapeutic effect on G422TN-GBM.Conclusions: We have identified that the combination of Plx3397/TMZ/ PL or SAHA exhibits superior therapeutic efficacy in treating gliomas, providing valuable guidance for clinical treatment selection. Our study reveals that integrating Bulk-seq and TRAP-seq is a potent approach to uncover drug resistance targets.