Functional genomic studies of cancer immune evasion using in vitro and in vivo CRISPR/Cas9 genetic screens

Recent advances in cancer immunology have shed light on the mechanisms of cancer immune evasion. An accumulation of data in recent years has identified several immune checkpoints, whose expression by cancer cells allows them to evade anti-cancer immune responses. The great potential of these findings is highlighted by the recent approval of therapeutic antibodies blocking PD1 and CTLA4, for the treatment of various cancers. Development of novel immunotherapies constitutes an extremely promising clinical prospect. However, target identification was limited in the past due to lack of high-throughput technologies. The advent of CRISPR/Cas9 genetic screens has revolutionised our ability to interrogate the cancer genome and identify its genetic vulnerabilities. With these in mind, I have carried out pooled in vitro and in vivo CRISPR/Cas9 genome-wide genetic screens, in order to identify genetic targets that sensitise cancer cells to the immune system and characterise cancer immune evasion pathways. I have verified that genome-wide gRNA library coverage in vivo is feasible in models of MC38 (mouse colon cancer) and B16F10 (mouse melanoma) cell isografts. Furthermore, two separate genome-wide in vivo screens have been completed in MC38 cells, which have enabled the identification of a number of candidate genes. Initial results indicated that disruption of antigen presentation pathways and NF-κB signalling are lethal to tumour cells in vivo. Moreover, the screen identified known cancer immunotherapy targets, such as Cd274 (PDL1) and Ptpn2, among other genes. Candidate genes were validated in vivo, in immunocompetent and immunocompromised mice, by designing targeted gRNA libraries. I am currently in the process of carrying out individual gRNA knockout experiments. Finally, I will further characterise the immune responses elicited by specific mutations and identify the main effector cells. I anticipate that these findings have the potential to advance our understanding of cancer immune evasion, while enabling the development of immunotherapies, by providing a framework for high-throughput target identification and validations.