Lizotte P.H.RUEY-LONG HONGLuster T.A.Cavanaugh M.E.Taus L.J.Wang S.Dhaneshwar A.Mayman N.Yang A.Kulkarni M.Badalucco L.Fitzpatrick E.HSIANG-FONG KAOKuraguchi M.Bittinger M.Kirschmeier P.T.Gray N.S.Barbie D.A.Janne P.A.2021-03-092021-03-0920182326-6066https://www.scopus.com/inward/record.uri?eid=2-s2.0-85057886812&doi=10.1158%2f2326-6066.CIR-18-0193&partnerID=40&md5=d9935f4f36f10f7225d3e26b680341f5https://scholars.lib.ntu.edu.tw/handle/123456789/551227We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8? T cells specifically recognizing the model antigen in an H-2b–restricted manner. The assay was screened with a small-molecule library to identify compounds that inhibit or enhance T cell–mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNg-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8? cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting EGFR that sensitized tumor cells to T cell–mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small-molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted. ?2018 American Association for Cancer Research.[SDGs]SDG3afatinib; epidermal growth factor receptor kinase inhibitor; erlotinib; major histocompatibility antigen class 1; pembrolizumab; programmed death 1 receptor; afatinib; antineoplastic agent; epidermal growth factor receptor; firefly luciferase; monoclonal antibody; PDCD1 protein, human; pembrolizumab; programmed death 1 receptor; protein kinase inhibitor; animal cell; animal experiment; animal model; antigen specificity; Article; cancer patient; cancer research; cancer screening; CD8+ T lymphocyte; cell killing; clustered regularly interspaced short palindromic repeat; computer assisted tomography; controlled study; CRISPR-CAS9 system; cytolysis; cytotoxic T lymphocyte; drug efficacy; drug potentiation; enhancer region; enzyme linked immunosorbent assay; flow cytometry; genome-wide association study; head and neck cancer; high throughput screening; high throughput sequencing; hypopharynx squamous cell carcinoma; ID8 cell line; immunomodulation; in vivo study; larynx squamous cell carcinoma; medical record; molecular library; monotherapy; mouse; mouth squamous cell carcinoma; nonhuman; nuclear magnetic resonance imaging; oncology; open reading frame; oropharynx squamous cell carcinoma; retrospective study; target cell; treatment duration; tumor cell; animal; C57BL mouse; coculture; CRISPR Cas system; cytotoxic T lymphocyte; drug effect; drug screening; genetics; head and neck tumor; high throughput screening; human; immunology; procedures; tumor cell line; Afatinib; Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Coculture Techniques; CRISPR-Cas Systems; Drug Screening Assays, Antitumor; ErbB Receptors; Head and Neck Neoplasms; High-Throughput Screening Assays; Humans; Luciferases, Firefly; Mice, Inbred C57BL; Programmed Cell Death 1 Receptor; Protein Kinase Inhibitors; Squamous Cell Carcinoma of Head and Neck; T-Lymphocytes, Cytotoxicjournal article10.1158/2326-6066.CIR-18-0193302420212-s2.0-85057886812