Novel strategies for the targeted elimination of glioblastoma stem-like cells

• Applicant: Professor Dr. Donat Kögel, since 5/2023
• Subject Area: Molecular and Cellular Neurology and Neuropathology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 449700301

Glioblastoma is the most common primary brain cancer and is associated with a dismal prognosis and very short survival, despite aggressive multi-modal therapy. It is hypothesized that GBMs contain a population of glioma stem-like cells (GSC) that differ from the majority of tumor cells, as they are highly resistant to conventional therapy and can therefore cause therapy failure and recurrence. These GSCs express marker proteins of normal stem cells are slow-cycling and have a high differentiation potential. The proposed project is based on findings from our group showing that a differentiation therapy combined with potent cell death induction using arsenic trioxide (ATO) and (-)-gossypol (Gos) can deplete GSCs in vitro and ex vivo. Accordingly, we observed a reduction in genetic and functional stem cell features accompanied by proteomic changes indicating decreased migratory potential and DNA repair, and increased radiosensitivity. One major task of the proposed project is the implementation of a novel multi-FLuOrescence-REporter System (multiFLORES) in order to analyze genetic dependencies regulating the stemness-related proteins OLIG2, SOX2 and SOX9 in GSC. I will employ this assay to globally analyze genes regulating stemness using an unbiased, genome-wide CRISPR/Cas9 gene perturbation screen. Importantly, this model system will be designed as a toolbox that on a long term perspective can be adapted to other cancer and tissue types. After target selection of hits from this screen, findings will be validated using phenotypical assays and the role of novel candidates for the GSC phenotype will be analyzed in detail in vitro, ex vivo and in vivo. In parallel to this part, I will investigate candidate proteins with high potential based on our existing data and expressional analysis in primary GBM specimens (human protein atlas, TCGA) in a hypothesis-driven approach. In order to better understand the molecular events triggered by combined ATO+Gos treatment and how these proteins relate to GSC biology, the following target proteins will be scrutinized: Chordin-like 1 (CHRDL1) implicated in maintaining stemness and BRCA1-associated ATM activator 1 (BRAT1) that might play an important role in GBM radiation resistance. These proteins will be analyzed using established GSC lines and primary cultures using stem-cell specific (limiting dilution assays), radiation specific (induction of DNA damage) and tumor-biologically relevant (cell death induction) in vitro assays. The results obtained in vitro will be complemented and validated using ex vivo and state-of-the-art in vivo orthotopic transplantation experiments. Using this tandem approach of unbiased genome-wide CRISPR/Cas9 screening in parallel to a knowledge-based candidate approach will likely allow identifying novel key signaling events in GSCs as potential targets of therapy.

• DFG Programme: Research Grants
• Ehemaliger Antragsteller: Dr. Benedikt Linder, until 4/2023