Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2019/09/25/mol.119.117804.DC1 1521-0111/96/6/862–870$35.00 https://doi.org/10.1124/mol.119.117804 MOLECULAR PHARMACOLOGY Mol Pharmacol 96:862–870, December 2019 Copyright ª 2019 by The American Society for Pharmacology and Experimental Therapeutics Functional RNAi Screens Define Distinct Protein Kinase Vulnerabilities in EGFR-Dependent HNSCC Cell Lines s Trista K. Hinz,1 Emily K. Kleczko,1 Katherine R. Singleton, Jacob Calhoun, Lindsay A. Marek, Jihye Kim, Aik Choon Tan, and Lynn E. Heasley Departments of Craniofacial Biology (T.K.H., E.K.K., K.R.S., J.C., L.A.M., L.E.H.) and Medicine (J.K., A.C.T.), University of Colorado Anschutz Medical Campus, Aurora, Colorado Received July 9, 2019; accepted September 14, 2019 Downloaded from ABSTRACT The inhibitory epidermal growth factor receptor (EGFR) antibody, signal-regulated kinase (ERK) mitogen-activated protein kinase cetuximab, is an approved therapy for head and neck squamous pathway, consistent with ERK reactivation and/or incomplete cell carcinoma (HNSCC). Despite tumor response observed in ERK pathway inhibition in response to EGFR inhibitor mono- some HNSCC patients, cetuximab alone or combined with radio- therapy. As validation, distinct mitogen-activated protein kinase or chemotherapy fails to yield long-term control or cures. We kinase (MEK) inhibitors yielded synergistic growth inhibition molpharm.aspetjournals.org hypothesize that a flexible receptor tyrosine kinase coactivation when combined with the EGFR inhibitors, gefitinib and AZD8931. signaling network supports HNSCC survival in the setting of The findings identify ERBB3 and MTOR as important phar- EGFR blockade, and that drugs disrupting this network will macological vulnerabilities in HNSCC and support combin- provide superior tumor control when combined with EGFR ing MEK and EGFR inhibitors to enhance clinical efficacy in inhibitors. In this work, we submitted EGFR-dependent HNSCC HNSCC. cell lines to RNA interference-based functional genomics screens to identify, in an unbiased fashion, essential protein kinases for SIGNIFICANCE STATEMENT growth and survival as well as synthetic lethal targets for Many cancers are driven by nonmutated receptor tyrosine kinase combined inhibition with EGFR antagonists. Mechanistic target coactivation networks that defy full inhibition with single targeted of rapamycin kinase (MTOR) and erythroblastosis oncogene B drugs. This study identifies erythroblastosis oncogene B (ERBB) at ASPET Journals on September 24, 2021 (ERBB)3 were identified as high-ranking essential kinase hits in 3 as an essential protein kinase in epidermal growth factor the HNSCC cell lines. MTOR dependency was confirmed by receptor–dependent head and neck squamous cell cancer (HNSCC) distinct short hairpin RNAs (shRNAs) and high sensitivity of the cell lines and a synthetic lethal interaction with the extracellular cell lines to AZD8055, whereas ERBB3 dependency was signal-regulated kinase mitogen-activated protein kinase path- validated by shRNA-mediated silencing. Furthermore, a syn- way that provides a rationale for combining pan-ERBB and thetic lethal kinome shRNA screen with a pan-ERBB inhibitor, mitogen-activated protein kinase inhibitors as a therapeutic AZD8931, identified multiple components of the extracellular approach in subsets of HNSCC. Introduction Atlas project has failed to provide abundant targets for effective precision medicine strategies. Although epidermal Comprehensive identification of diverse, but often growth factor receptor (EGFR) is not subject to frequent therapeutically-tractable oncogenes in cancers such as lung somatic mutations in HNSCC, genomic amplification and adenocarcinoma and melanoma, permits implementation of high expression provided a rationale for clinical testing of precision medicine, which matches oncogenic vulnerabil- the anti-EGFR antibody, cetuximab, and ultimately led to ities with specific drugs, yielding greatly enhanced patient Food and Drug Administration approval in combination benefit (Politi and Herbst, 2015; Bivona and Doebele, 2016). with radio- or chemotherapy (Bonner et al., 2006, 2010; By contrast, the mutational landscape of head and neck Vermorken et al., 2007, 2008). EGFR-specific tyrosine squamous cell cancer (HNSCC) defined by The Cancer Genome kinase inhibitors (TKIs) have not exhibited clear efficacy, although pan-erythroblastosis oncogene B (ERBB)–active The studies were supported by a VA grant (Merit Award 1BX001994-01) to LEH and the UC Cancer Center Support Grant (P30 CA046934). TKIs induce responses in HNSCC equal to cetuximab (Seiwert 1T.K.H. and E.K.K. contributed equally to this work. et al., 2014). Still, the therapeutic responses are invariably https://doi.org/10.1124/mol.119.117804. s This article has supplemental material available at molpharm. incomplete and eventually followed by tumor progression and aspetjournals.org. treatment failure. Because receptor tyrosine kinases (RTKs) ABBREVIATIONS: BINGS!, Bioinformatics for Next Generation Sequencing; EGFR, epidermal growth factor receptor; ERBB, erythroblastosis oncogene B; ERK, extracellular signal-regulated kinase; FGFR, fibroblast growth factor receptor; gDNA, genomic DNA; HNSCC, head and neck squamous cell cancer; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; MLK1, mixed-lineage protein kinase 1; MTOR, mechanistic target of rapamycin kinase; p, phosphorylated; PCR, polymerase chain reaction; RNAi, RNA interference; RTK, receptor tyrosine kinase; shRNA, short hairpin RNA; TKI, tyrosine kinase inhibitor. 862 Protein Kinase Vulnerabilities in EGFR-Dependent HNSCC 863 such as EGFR rarely signal in isolation, even when oncogeni- and 3.0 mg kinome library. The virus-containing media from the cally mutated, a deeper understanding of the functional EGFR 293T cells was then filtered through a 0.45-mm filter after adding network in HNSCC is warranted. 1 mg/ml polybrene and either used immediately or stored at 4°C until Distinct from mutated RTKs that function as dominant ready for use. oncogenes in lung adenocarcinoma, we and others have suggested that networks of nonmutated RTKs and down- Functional Genomics Screens stream effector pathways serve as oncogene inputs in many Essential Kinome Screen. Target cell lines (UMSCC8, UMSCC25, cancers, including HNSCC (Xu and Huang, 2010; Singleton HN6, HN12, Cal27, JHU011) were plated at 5  106 cells per 15-cm plate, et al., 2013; Tan et al., 2017). Indeed, Singleton et al. (2013) two plates per cell line, in 20 ml growth medium. The following day, m defined a coactivation network comprised of EGFR, ERBB2, cells were pretreated with 1 g/ml polybrene for 30 minutes. The fibroblast growth factor receptor (FGFR) family members, and media containing the packaged lentiviral kinome shRNA library supplemented with 50 shRNAs (5 per gene) targeting 10 murine hepatocyte growth factor receptor (MET) in HNSCC cell lines genes (b-actin, Actb; aldolase A fructose-bisphosphate, Aldoa; Rho and demonstrated maximal growth inhibition with a triple GDP dissociation inhibitor a, Arhgdia; copine I, Cpne1; glyceraldehyde- TKI cocktail. Considering the toxicity often associated when 3-phosphate dehydrogenase, Gapdh; heterogeneous nuclear even two drugs are combined, therapeutic approaches in- ribonucleoprotein D, Hnrnpd; lactate dehydrogenase A, Ldha; non- volving such complex combinations of TKIs seem unlikely to POU domain–containing octamer-binding protein, Nono; phospho- move forward through clinical trials. Instead, an unbiased glycerate kinase 1, Pgk1; ribosomal protein L11, Rpl11) was added Downloaded from screen for novel and druggable vulnerabilities might identify to each plate of target cells at a multiplicity of infection of ∼0.2, incubated safer and more effective combination therapies. In this regard, for 16 hours, and then removed and replaced with fresh growth medium functional genomics techniques provide a path forward for (lacking puromycin). Forty-eight hours postinfection, each cell line was  6 dissecting the complexity of oncogene signaling in cancer cells trypsinized and plated at a density of at least 2 10 cells per 15-cm plate in quadruplicate. Additionally, at the same time point, four replicates of and identifying and prioritizing therapeutic vulnerabilities 4  106 cells each from each cell line were pelleted, and the genomic DNA molpharm.aspetjournals.org (Singleton et al., 2015, 2017; Tsherniak et al., 2017). (gDNA) was isolated using the Quick g-DNA MiniPrep kit (Zymo With the goal of defining targetable vulnerabilities in Research, Irvine, CA). DNA was eluted with 100 ml elution buffer and HNSCC and developing TKI-based combinations, we per- stored at 220°C. On day 14 postinfection, 4  106 cells per replicate from formed functional genomics-based RNA interference (RNAi) each cell line were pelleted, and the gDNA was isolated. screens for both essential protein kinases and kinases whose Synthetic Lethal Screen. Target HNSCC cell lines (UMSCC8, inhibition in combination with an ERBB-active TKI induces UMSCC25, HN12, Cal27, JHU011) were plated at 3  106 cells per 15- synergistic growth inhibition. We found that short hairpin cm plate in 20 ml growth media with two plates per cell line. Cells were RNAs (shRNAs) targeting mechanistic target of rapamycin maintained at cell densities that are at least 10 times the number of kinase (MTOR) and ERBB3 were significantly lost over time shRNAs in