Systematic CRISPR Analysis of EBV Lymphoma Resistance Pathways to Natural Killer Cell Lysis - Abstract Epstein-Barr virus (EBV) causes lymphomas at greatly increased frequency in people living with HIV/AIDS, including diffuse large B-cell lymphoma, primary CNS lymphoma, Hodgkin lymphoma, Burkitt lymphoma, and post-transplant lymphomas. Consequently, most AIDS-related lymphomas are EBV-infected. Yet, EBV transformation sensitizes infected B-cells to attack by natural killer (NK) cells, which exert key roles in control of EBV-driven B-cell malignancies in immunocompetent hosts. Much remains unknown about how NK recognize latently EBV-infected B lymphoblastoid cells and how EBV+ B cells evolve resistance to NK control. To gain insights, we performed the first human genome-wide CRISPR screen of EBV-transformed B-cell escape from NK surveillance using a lymphoblastoid cell line (LCL), a key model for EBV-driven immunoblastic lymphomas including the AIDS defining cancer primary CNS lymphoma. Our screen identified factors known to be important for EBV+ B-cell recognition by NK, including CD48, which is highly EBV-induced. Unexpectedly, multiple CRISPR screen hits centered on the Human Silencing Hub (HUSH) complex, an epigenetic repressor that induces heterochromatin formation to silence target gene expression. HUSH is implicated in silencing of HIV genes, but has not been studied in the contexts of EBV infection, B-cell immunobiology or NK surveillance. Protocadherin gamma (PCDHG) family members, which are adhesion molecules typically expressed only by neurons, were highly enriched amongst LCL genes de-repressed by HUSH KO. Furthermore, PCDHG are necessary for NK resistance driven by HUSH KO and sufficient for inhibition of LCL lysis by NK. We therefore hypothesize that PCDHG expression represents a ‘don’t kill me’ signal to protect neurons from NK attack, but which is subverted to support EBV+ lymphomagenesis. Our central hypothesis is that NK surveillance of EBV transformed B-cells requires B-cell intrinsic HUSH complex activity, in the absence of which gamma- protocadherin are de-repressed and interact with a novel NK inhibitory receptor to prevent NK lysis. Our specific aims are: (1) Characterize key proto-cadherin gamma properties in EBV transformed B-cell evasion of NK attack, including in tumor samples from HIV+ individuals. (2) Identify the Proto-cadherin Gamma NK Counter-Receptor and Characterize its NK Inhibitory Roles. Collectively, these studies promise to identify a novel NK cell inhibitory pathway subverted by EBV+ tumors. Our studies may lay the foundation for novel therapeutic approaches that could re-sensitize tumors that subvert this pathway to NK attack.
