Identifying small cell lung cancer resistance mechanisms to cytotoxic T cells in vitro using whole genome CRISPR screening.

Abstract

e20075 Background: Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine neoplasm associated with high tumor mutation burden (TMB), a paucity of compelling therapeutic options, and very poor survival. In comparison to other malignancies with high TMB, SCLC patients tend to experience lower response rates and shorter response durations with immune checkpoint blockade (ICB) targeting the PD-1 and/or CTLA-4 axes, and chemotherapy with ICB in first line does not exhibit the dramatic synergy seen in non-small cell lung cancer. To identify additional immunosuppressive mechanisms that could serve as novel biomarkers and therapeutic targets in SCLC, we adapted a recently described method to screen for genes that alter the sensitivity of cancer cells to killing by cytotoxic CD8+ T cells. Methods: We introduced Cas9 nuclease and a genome-wide gene inactivation library of ~80,000 single guide RNA (sgRNA) templates into a cell line derived from a genetically engineered murine model of SCLC (mSCLC). In parallel, we isolated and stimulated CD8+ T cells from the OT-I transgenic mouse, which express a rearranged T cell receptor with very high avidity for the ovalbumin (OVA) SIINFEKL peptide presented on H2-K(b) class I MHC. mSCLC cells were loaded with OVA peptide or vehicle and co-cultured with T cells for 24 hours. We collected surviving cells, isolated DNA, and used next-generation DNA sequencing to tabulate the relative representation of sgRNAs in each experimental condition. From these data, we inferred which gene knockouts were protective from, or sensitizing to, killing by CD8+ T cells. Results: We observed expected mediators, including resistance via knockout of antigen presentation machinery and sensitization via knockout of pro-survival pathways ( e.g. NF-kB), and unexpected hits spanning diverse cellular functions. Conclusions: Genome-wide screening for mediators of in vitro killing by CD8+ T cells in a murine model of SCLC is feasible and reveals that both expected and novel pathways may contribute to resistance to immunotherapy in SCLC. Future studies are directed at validating these hits in vitro and in vivo, incorporating new complex gene perturbation libraries, and scaling to additional cell lines.