Exploring Genomic Essentialities of Minimal Residual Disease in Adult Glioblastoma by Integrating CRISPR cas9 and Single Cell Technologies

Introduction: Glioblastoma (GBM) is a common and remarkably resilient brain tumour. Its ability to evade treatment remains one of the enigmas in neuro-oncology. The answer likely lies in understanding the biology of minimal residual disease (MRD), the state that tumour cells retreat into and tolerate pressures of treatment. We investigate gene essentialities of MRD with CRISPR-Cas9 screens, integrated with single cell gene expression analysis (scRNA-Seq) in humanized xenograft GBM models of MRD. We identify key biological programs, genomic essentialities and plausible targets for treatment of resistant GBM states.

Methods: We carried out CRISPR Cas-9 screen on two primary, patient-derived treatment-naïve cell lines with adaptation of standard of care (SOC) treatments temozolomide and radiation. For in-vivo scRNA-seq analysis of MRD we generated humanized xenograft model in nude mice subjected to the adopted SOC regime. CRISPR Cas-9 outputs were analyzed using the BAGEL algorithm, genes were selected based on sum Z scores < -2, FDR < 0.5 and adjusted p< 0.005. Gene annotation was carried using MSigDB and single cell data was analyzed with Seurat package.

Results: CRISPR Cas-9 screen identified several known essentialities of MRD for example DNA repair, ubiquitinoylation and cellular response to stress. Upregulated genes of MRD in PDX models included marker of therapy resistance (ID2) or oncogenesis (EGFR). Gene list annotation overlapped with several mechanisms in the CRISPR Cas-9 screen highlighting axonal guidance pathways and oxidative phosphorylation. There were mechanisms exclusive to the in-vivo model such as ferroptosis, iron dependent cell death which is an emerging target in tumor biology.

Conclusion : Our results define and validate the biological program of MRD in GBM by integrating CRISPR Cas-9 screen and scRNA-seq. Vulnerabilities in MRD include axonal guidance pathways and oxidative phosphorylation which are both targetable mechanisms. Our analysis also identifies ferroptosis as a likely contextual essentiality occurring in the tumor microenvironment.