Abstract
OBJECTIVE: Developing a high-throughput, in vivo, loss-of-function screening platform for GBM immune-escape mechanisms and identifying novel immuno-therapy targets. BACKGROUND: While recent success of cancer immunotherapy asserts the importance of immune-mediated tumor eradication, patients respond heterogeneously. Expanding the clinical utility of immunotherapy requires a rational method for identifying new combination therapies and resistance mechanisms. These mechanisms can be discovered using high-throughput functional genetic screens and we have adapted a novel loss-of-function in vivo screen to identify mechanisms of immune-evasion in the CNS. DESIGN/METHODS: B16-melanoma or CT2A- and GL261-astrocytoma cells bearing Cas9-endonuclease were engineered to express a library of barcoded guide RNAs. These cells form tumors when implanted in the flank (B16) or intracranially (CT2A, GL261) in immunocompetent animals. Tumor-bearing animals were subjected to immunotherapy with vaccination or PD-1 checkpoint blockade. Barcode-relative representation was measured by next-generation sequencing at the time of tumor implantation and tumor harvest post-immunotherapy. RESULTS: Here we report an in vivo, pooled, loss-of-function genetic screen using Cas9/CRISPR genome editing in mouse transplantable tumors treated with vaccination and PD-1 checkpoint blockade. Screening 2,400 genes expressed by melanoma cells for those that synergize with or cause resistance to checkpoint blockade recovered known immune-evasion molecules PD-L1 and CD47. Novel immunotherapy targets validated individually, identifying essential pathways of immune-evasion. When recapitulating this approach in the CNS, 500-1000 genes can be functionally screened under immune-pressure. We are currently screening a GBM-specific library based on differential in vitro gene expression in the tumor cytokine-environment in an in vivo pooled loss-of-function genetic screen using graded immune-pressure to identify novel immunotherapy targets in GBM. CONCLUSIONS: Our screening assay provides the first high-throughput method for systematically identifying resistance mechanisms and new candidate targets for immunotherapy in CNS tumors and can screen multiple immunotherapy combinations, serving as an important tool for the identification of next-generation combination immunotherapies.