Enhancing radiosensitization in EphB4 receptor-expressing Head and Neck Squamous Cell Carcinomas

Abstract

Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas.

Inhibition of EphB4–Ephrin-B2 Signaling Reprograms the Tumor Immune Microenvironment in Head and Neck Cancers

“…The soluble EphB4 extracellular domain fused to human serum albumin (sEphB4-HSA; ref. 16) was used to inhibit EphB4ephrin-B2 interaction in a PDX immunocompromised mouse model. The sEphB4-HSA protein was provided by Vasgene Therapeutics, Inc. For immunocompetent mouse models, a plasmid encoding the 15 amino acids long TNYL-RAW peptide fused with the Fc portion of human IgG 1 (TNYL-RAW-Fc, an EphB4 antag-onist) was used to block EphB4-ephrin-B2 signaling (25).…”

“…The HNSCC PDX tumors CUHN013 and CUHN004 (F8-F16 generation) were obtained from Dr. Antonio Jimeno's lab (University of Colorado, Anschutz Medical Campus, Aurora, CO). Tumor implantations were performed as described earlier (16). When tumor volumes reached approximately 50 to 150 mm 3 , mice were randomized into four groups (i) PBS, (ii) sEphB4-HSA, (iii) PBSþRT, and (iv) sEphB4-HSAþRT.…”

“…When tumor volumes reached approximately 50 to 150 mm 3 , mice were randomized into four groups (i) PBS, (ii) sEphB4-HSA, (iii) PBSþRT, and (iv) sEphB4-HSAþRT. Mice were either injected with PBS or with a 20 mg/kg dose of sEphB4-HSA (three times/week) and/or subjected to RT (5 Gy/fraction  4 fractions) as described earlier (16).…”

“…We previously reported that blockade of EphB4-ephrin-B2 signaling delays tumor growth and enhances radiosensitization in HNSCC PDX models (16). To determine the role of EphB4ephrin-B2 interaction in supporting tumor growth in immunocompetent models, we used two different orthotopic models of HNSCC: Ly2 (BALB/c) and Moc2 (C57BL/6).…”

“…It has been shown to be safe in multiple clinical trials (NCT01642342, NCT02717156, NCT02767921) with minimal to no toxicity (15), likely due to low levels of expression in normal tissue. In our laboratory, we have previously shown that EphB4-ephrin-B2 inhibition in different patient-derived xenograft (PDX) HNSCC models enhances tumor response to RT (16) as well as to combined EGFR inhibition and RT (17). Although direct evidence that implicates EphB4-ephrin-B2 interaction in the cancer-related immune response is lacking, multiple reports have documented that Eph/ ephrin gene family members modulate immune cell processes in inflammatory models, such as arteriosclerosis and wound healing (18)(19)(20).…”