Exploring the Immunosuppressive Role of Human Endogenous Retrovirus K in the Glioblastoma Microenvironment - PROJECT SUMMARY Glioblastoma (GBM) is the most lethal primary brain tumor with a five-year survival rate of less than 5%. The GBM tumor microenvironment (TME) is highly immunosuppressive, conferring resistance to immunotherapies that have proven groundbreaking in treating other malignancies. Thus, the need to identify and target drivers of immunosuppression in the GBM TME remains critically important. Notably, our lab recently uncovered robust overexpression of Human Endogenous Retrovirus K (HERV-K) in GBM that is associated with poor patient outcomes. HERVs are remnants of ancient germ-line retroviral infections that comprise ~8% of the human genome. They are generally transcriptionally silenced, but dysregulated states like cancer can lead to increased expression of canonical HERV genes and proteins, including env (envelope protein). This project proposes HERV-K Env as a viable candidate to target therapeutically to improve GBM patients’ response to immunotherapy due to robust evidence that retroviral Env proteins have immunosuppressive properties and contain a highly conserved immunosuppressive domain (ISD). Recent results, including our preliminary data, show that HERV-K Env inhibits proliferation of T cells, modulates cytokine secretion, and increases expression of immune checkpoint receptors. Further, immune deconvolution of bulk RNA sequencing and spatial transcriptomics show that high HERV-K Env expression is associated with low immune cell infiltration in GBM and decreased survival of GBM patients. However, the mechanism by which HERV-K Env suppresses T cell function remains elusive, and HERV-K Env’s exact role in the immunosuppressive GBM TME remains unclear. This proposal will test the central hypothesis that HERV-K Env’s ISD mediates binding to critical surface receptors, including CD98hc (roles in amino acid transport and integrin signaling) on T cells, altering their downstream functions and leading to T cell dysfunction and to greater immunosuppression in the GBM TME. Specific Aim 1 will identify critical mediators of HERV-K Env’s immunosuppressive effect on T cells via the use of co-immunoprecipitation and mass spectrometry to identify the binding partners of Herv-K Env on the T cell surface and to elucidate the ISD’s role; further, it will explore alterations in downstream pathways of these receptors via methods including Western blot, qPCR, flow cytometry, and ELISA. Specific Aim 2 will characterize HERV-K Env-mediated immunosuppression in the GBM TME by utilizing GBM-T cell co- cultures, HERV-K Env-overexpressing orthotopic mouse models paired with single cell RNA sequencing and multiplex flow cytometry, and spatial transcriptomics analysis of patient specimens. Altogether, this study’s results will explain critical immunosuppressive mechanisms of a retroviral protein, HERV-K Env, in GBM and elucidate its role in GBM’s highly immunosuppressive TME. It possesses robust translational potential by providing a candidate to target therapeutically and by identifying key pathways in Env’s immunosuppressive effect in GBM.
