In Vivo Screening of Immunotherapy Resistant Head and Neck Squamous Cell Carcinoma Identifies B7-H3 Inhibition as a Potential Radiosensitizer

Abstract

<h3>Purpose/Objective(s)</h3> Patients with locally advanced head and neck squamous cell carcinoma (HNSCC) have poor outcomes following conventionally fractionated radiation (conRT) alone or in combination with chemotherapy. Unfortunately, the addition of anti-PD-1/L1 therapy to conRT has not been found to improve patient outcome. To identify other, more active conRT and immunotherapy combinations, we performed an <i>in vivo</i> shRNA screen of immune-related and other "druggable" genes in an immunotherapy and comparative conRT resistant murine HNSCC model (MOC2) to identify potential radiosensitizers. <h3>Materials/Methods</h3> The <i>in vivo</i> screen utilized shRNA libraries focused on immune-related and other "druggable" targets (n=242) in murine immunoevasive HNSCC tumor cells (MOC2), which were implanted into C57BL/6 flanks and treated with conRT (2Gy x 5) or sham. For validation, the same MOC2 tumor model was treated with murine isotype IgG or anti-B7H3 antibody with conRT. Flow cytometry was performed on single tumor cells approximately 10 days following the last conRT treatment using a murine specific adaptive immunotherapy panel. Data from the HNSCC Cancer Genome Atlas (TCGA) cohort was used for expression of B7-H3 in HPV-negative (-) and HPV-positive (+) HNSCC tumors. A subset of these patients with HPV (-) tumors (n=94) treated uniformly with surgery and post-operative radiation were analyzed for locoregional recurrence (LRR). Statistical analysis was performed in a scientific 2-D graphing and statistics software. <h3>Results</h3> Our <i>in vivo</i> shRNA screen identified several immune-related genes associated with conRT sensitization, including the under-studied CD276 (gene encoding immune checkpoint molecule B7-H3) (RSA log p-value -2.8). Notably, PD-L1 was predicted to have a relatively poor response in combination with conRT (RSA log p-value -0.38). Although ongoing at the time of writing, the validation study showed that conRT + anti-B7-H3 treatment led to a median survival of at least 28.3 days, significantly greater than RT + IgG of 22.3 days (p=3.5e⁻³), IgG (p=4.4e⁻⁶) or anti-B7-H3 alone (p=7e⁻⁶) (both 15.5 days). On initial flow cytometry, RT + anti-B7H3 had a potential increase in NK cells (1.55) compared to no RT + anti-B7H3 (0.22) or no RT + IgG (1.00). B7-H3 was expressed at a significantly higher level in HPV (-) versus HPV (+) HNSCC tumors from the TCGA (p=1.37e-13). In a subset of HPV (-) tumors from the HNSCC TCGA cohort treated with surgery and conRT, B7-H3 expression was significantly associated with increased LRR (p=0.035). <h3>Conclusion</h3> Our immunocompetent <i>in vivo</i> screen identified B7H3 inhibition as a potential novel radiosensitizer in HNSCC. In an immunocompetent syngeneic model, conRT + anti-B7H3 treatment demonstrated improved survival. As B7-H3 is targetable clinically, it provides an exciting and rational target for combinational therapy with RT.