Genome-wide CRISPR screen to identfy immune evasion genes in pancreatic cancer

Here we systematically dissected PDA intrinsic mechanisms of immune evasion by in vitro and in vivo CRISPR screening. Beyond the conserved set of genes regulating anti-cancer immunity we identified Rnf31 and Vps4b as essential factors required for escaping CD8+ T cell-killing. While the absence of Rnf31 induced sensitivity to T cell killing through TNF-mediated apoptosis in murine cancer cells and human PDA organoids, loss of Vps4b abrogated functional autophagy, resulting in accumulation of CD8+ T cell-derived granzyme B and subsequent tumor cell lysis. Overall design: Genome-wide CRISPR screen in pancreatic cancer cells using the Brie sgRNA library. Cancer cells were co-cultured with CD8 T cells and resistors/sensitizer (genes) were identified after T cell selection. Candidates Rnf31 and Vps4b were chosen for further analysis and RNA-Seq was performed on WT-KPC cells or KPC cells with Rnf31/Vps4b knockout with and without T cell co-culture. The same as done for Stat1-KO KPC cells as a resistor to T cell killing.