Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults and is thought to be driven by a subset of self-renewing Glioblastoma stem cells (GSCs) that persist after therapy and seed treatment refractory recurrent tumors. GBM tumors display a high degree of intra- and intertumoral heterogeneity that is a prominent barrier to targeted treatment strategies. This heterogeneity extends to GSCs that exists on a gradient between proneural/developmental to mesenchymal/injury-response subtypes. New drug targets for each subtype are needed to effectively target these heterogenous cancers. Previous genome-wide CRISPR fitness screens performed by our group revealed differences in genetic dependencies between subtypes. To identify conserved and subtype-specific genetic dependencies across a large and heterogeneous panel of GSCs we leveraged our genome-wide screening data to design a targeted gRNA library (GBM5K) and performed fitness screens in a total of 30 patient-derived GSC cultures. Our focused CRISPR screens identified the most conserved subtype-specific vulnerabilities in GSCs and reinforce our understanding of the functional gradient between the developmental and injury-response states. Developmental-specific fitness genes are enriched for master transcriptional regulators of neurodevelopmental fates, whereas Injury-response-specific fitness genes are enriched for members of B1-Integrin mediated signaling pathways. These context-specific vulnerabilities give rise to differential sensitivity to inhibitors of B1-Integrin and FAK signaling. Interestingly, our screens revealed convergence of upstream signaling pathways on differential Cyclin D (CCND1 vs CCND2) dependencies between subtypes. Our data provides biological insight and novel mechanistic understanding of heterogeneity in GBM and points to opportunities for precision targeting of defined GBM subtypes.
