1 The outcomes for high-risk HPV-negative head and neck squamous cell carcinoma (HNSCC) remain poor 2 despite aggressive therapy. One possible explanation is the development of therapeutic resistance by aberrant 3 regulation of tumor angiogenesis. The receptor tyrosine kinase receptor, EphB4, and its membrane-bound 4 ligand, ephrinB2 (EFNB2), can both signal and have been shown to play pro-tumorigenic and pro-angiogenic 5 roles in numerous malignancies and in early embryonic development. Our data demonstrate strong correlation 6 between elevated gene expression of EphB4 and EFNB2 and survival outcomes in HPV negative HNSCC 7 patients. We show that EphB4 is predominantly expressed on cancer cells and EFNB2 is predominantly 8 present on endothelial cells (EC). Our data also show that targeting HNSCC tumors with a recombinant fusion 9 protein that blocks EphB4-EFNB2 interaction enhances cancer cell apoptosis, reduces 10 angiogenesis/lymphangiogenesis, and decreases tumor growth in patient-derived xenograft models. This, we 11 demonstrate, is associated with an increase in FasR, Bax, Bim, and caspase 3 cleavage apoptotic markers as 12 well as a decrease in VEGFR2/3, JAK2 and Stat3 signaling components. Based on these data, we hypothesize 13 that the interaction between EphB4 and EFNB2 at the cancer-EC junction simultaneously leads to activation of 14 survival and anti-apoptotic pathways for the cancer cell and angiogenic pathways for the EC. Therefore, we 15 propose, its inhibition will decrease tumor vascular/lymph angiogenesis and decreases cancer cell apoptosis. 16 This EphB4-EFNB2 interaction defines a novel cancer-EC “synapse,” where the focus is on the crosstalk and 17 not just on ECs, thus providing an opportunity for greater therapeutic efficacy than the currently available anti- 18 angiogenic therapies. In Aim 1, we will differentiate the compartmental effects of targeting the EphB4- 19 expressing cancer cell versus EFNB2-expressing ECs, by using an EFNB2 inducible knockout murine model 20 with selective deletion of EFNB2 in the adult ECs. Mouse HNSCC cell lines with shRNA and dominant negative 21 (DN) knockdown of EphB4 on cancer cells will be implanted and perfusion and immunofluorescence will be 22 used to assess effects on growth and angiogenesis/lymphangiogenesis. In Aims 2 and 3, in-depth interrogation 23 of signaling mechanisms underlying EphB4-EFNB2 interaction will be tested on in vivo tissue as well as 24 cancer-EC co-culture assays using shRNA and DN knockdowns for EphB4 and EFNB2. Targeted inhibition of 25 JAK2-STAT3-ERK and FasR, BAX, and PI3K will be done with genetic and pharmacologic inhibitors to assess 26 their role in vascular sprouting and cancer cell apoptosis. Proximity ligation assay will be done to evaluate 27 intracellular and intercellular interactions. Finally, tissue from the in vivo mouse model as well as from a clinical 28 trial incorporating an EphB4-EFNB2 inhibitor in HNSCC will be assessed using RNAseq,...