SUMMARY, Project 1 Treatment resistance remains a challenging problem for high-risk head and neck cancer (HNSCC) patients. Current trials with immune checkpoint inhibitors have yielded sub-optimal response rates. This could be due to TME factors that substantially modulate the immune landscape’s composition, phenotype, and function. Preferential recruitment of unique immunosuppressive cell subtypes to the tumor bed may play an important role in contributing to the promotion of tumor growth and progression. The tumor endothelium can act as a selective barrier that regulates entry, stability, and activation status of immune cells, but the mechanistic underpinnings remain poorly understood. The EphB4 receptor tyrosine kinase and its ligand EFNB2 define novel molecular targets. Although they have been studied in cell migration and angiogenesis in early embryonic development, little has been published on their role in modulating the cancer immune microenvironment. Our preliminary data show that HNSCCs are enriched in regulatory T-cells (Tregs) and tumor associated macrophages (TAMs), both of which contribute to resistance after RT. EFNB2 is upregulated on tumor vasculature after RT whereas EphB4 is expressed on Tregs and TAMs, and among all immune cells, Tregs and TAMs are most sensitive to either pharmacologic inhibition with TNYL-RAW or genetic knockdown of endothelial EFNB2 in the context of RT. We therefore hypothesize that RT upregulates EFNB2 on tumor vascular endothelial cells, which acts preferentially to recruit EphB4 expressing Tregs and TAMs. Blockade of EphB4-EFNB2 signaling at the tumor endothelial barrier will hinder Tregs’ and TAMs’ ability to infiltrate and promote cancer cell survival or suppress Teff function. In Aim1, we will analyze the mechanistic outcome of the interaction between immune cell EphB4 and endothelial EFNB2 on immune trans-endothelial trafficking, survival, polarization, and differentiation of immune cells as well as vascular normalization of endothelial cells in response to RT. We will use pharmacological inhibitors as well as GEMMs with EFNB2 deletion on endothelial cells or EphB4 deletion on Tregs or TAMs. Aim 2 will study the cellular and molecular mechanisms triggered by the interaction between endothelial EFNB2 and EphB4-expressing immune cells on immune cell processes in response to RT using endothelial-immune cells co-culture assays. Targeted inhibition of STAT3, AKT, and Erk pathway will be done with pharmacological inhibitors based on our preliminary data. Aim 3 will use tissue from a human clinical trial with sEphB4-HSA (an inhibitor currently in Phase I and II trials) before and after drug alone or drug and chemoradiation alone. This will serve to identify the molecular mediators that drive the anti-tumor immune response to this therapy. These studies will also collectively elucidate the molecular and cellular parameters of EphB4-EFNB2 inhibitors and will allow for the identification of potential predicti...