Functional RNAi Screens Define Distinct Protein Kinase Vulnerabilities in EGFR-Dependent HNSCC Cell Lines

Cetuximab, an epidermal growth factor receptor (EGFR) inhibitory antibody, is an approved therapy for head and neck squamous cell carcinoma (HNSCC). Despite the EGFR dependency of HNSCC, cetuximab alone or combined with radio- or chemotherapy fails to yield long-term control or cures. Herein, we submitted EGFR-dependent HNSCC cell lines to RNAi-based functional genomics screens to identify, in an unbiased fashion, essential protein kinases for growth and survival as well as synthetic lethal targets for combined inhibition with EGFR inhibitors. MTOR and ERBB3 were identified as the highest ranking essential kinase hits. MTOR dependency was confirmed by distinct shRNAs and high sensitivity of the cell lines to AZD8055 while ERBB3 dependency was validated by shRNA-mediated silencing. Further, a synthetic lethal kinome shRNA screen with a pan-ERBB inhibitor, AZD8931, identified multiple components of the ERK MAPK pathway, consistent with ERK reactivation and/or incomplete ERK pathway inhibition in response to EGFR inhibitor monotherapy. As validation, distinct MEK inhibitors yielded synergistic growth inhibition when combined with the EGFR inhibitors, gefitinib and AZD8931. The findings identify ERBB3 and MTOR as important pharmacological vulnerabilities in HNSCC and support combining MEK and EGFR inhibitors to enhance efficacy of agents such as cetuximab. Overall design: Five EGFR-dependent HNSCC cell lines treated 72 hours with DMSO or a pan-ERBB inhibitor, AZD8931 to identify synthetic lethal targets (a kinome-targeting shRNA library). Also, genomic DNA (gDNA) in the five cell lines and HN6 cell line was isolated from replicate plates (n=4, exception in JHU011 and UMSCC25) after 48 hrs and 14 days of culture post lentiviral transduction.