STEM-14. Single-cell CRISPR profiling unmasks targetable chemoradiation-induced plasticity in glioblastoma

Glioblastoma is the most lethal primary brain tumor in adults with over 75% of all patients succumbing to the disease within two years after diagnosis. Extensive research of the past decades has shed light on the potential causes of this dismal prognosis including the roles of glioma stem-like cells, inter-patient and intratumor heterogeneity and the tumor microenvironment in this tumor’s strong resistance to standard-of-care treatment (surgery and chemoradiotherapy). Yet, standard-of-care has remained the same for over 20 years and nearly all clinical trials have resulted in failure to substantially improve patient outcome. In this study, we put cellular plasticity forward as one of the main drivers of treatment resistance as it allows the glioma stem-like cells to survive and adapt to imposed stress conditions. Based on advanced single-cell RNA sequencing upon chemoradiation in a large selection of patient-derived glioma stem-like cells (PDGSCLs), we identified several treatment-induced metaprograms and shifts in gene regulatory networks that are potential drivers of plasticity. To investigate their functional role in these plastic cell state shifts, we used pooled CRISPR screening with single-cell transcriptome resolution (CROPseq) for a selection of 250 candidate targets in three heterogeneous PDGSCLs treated with (chemo)radiation. Several potential targets were identified to modulate chemoradiation-induced plasticity as their knockout locked cells in their current state, even after treatment. Response to chemoradiation was further investigated in vitro and in silico after (pharmacological) inhibition of these targets. Together, our findings indicate new vulnerabilities of glioma stem-like cells, thereby proposing promising new targets to combat acquired treatment resistance in glioblastoma. This study is funded by FWO (11B2325N) and the Foundation against Cancer (F/2022/2026).