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. 2019 Dec;96(6):862-870.
doi: 10.1124/mol.119.117804. Epub 2019 Sep 25.

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

Trista K Hinz  1 , Emily K Kleczko  1 , Katherine R Singleton  1 , Jacob Calhoun  1 , Lindsay A Marek  1 , Jihye Kim  1 , Aik Choon Tan  2 , Lynn E Heasley  3
Affiliations

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

Trista K Hinz et al. Mol Pharmacol. 2019 Dec.

Erratum in

Abstract

The inhibitory epidermal growth factor receptor (EGFR) antibody, cetuximab, is an approved therapy for head and neck squamous cell carcinoma (HNSCC). Despite tumor response observed in some HNSCC patients, cetuximab alone or combined with radio- or chemotherapy fails to yield long-term control or cures. We hypothesize that a flexible receptor tyrosine kinase coactivation signaling network supports HNSCC survival in the setting of EGFR blockade, and that drugs disrupting this network will provide superior tumor control when combined with EGFR inhibitors. In this work, we submitted EGFR-dependent HNSCC cell lines to RNA interference-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 antagonists. Mechanistic _target of rapamycin kinase (MTOR) and erythroblastosis oncogene B (ERBB)3 were identified as high-ranking essential kinase hits in the HNSCC cell lines. MTOR dependency was confirmed by distinct short hairpin RNAs (shRNAs) and high sensitivity of the cell lines to AZD8055, whereas ERBB3 dependency was validated by shRNA-mediated silencing. Furthermore, a synthetic lethal kinome shRNA screen with a pan-ERBB inhibitor, AZD8931, identified multiple components of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, consistent with ERK reactivation and/or incomplete ERK pathway inhibition in response to EGFR inhibitor monotherapy. As validation, distinct mitogen-activated protein kinase kinase (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 clinical efficacy in HNSCC. SIGNIFICANCE STATEMENT: Many cancers are driven by nonmutated receptor tyrosine kinase coactivation networks that defy full inhibition with single _targeted drugs. This study identifies erythroblastosis oncogene B (ERBB)3 as an essential protein kinase in epidermal growth factor receptor-dependent head and neck squamous cell cancer (HNSCC) cell lines and a synthetic lethal interaction with the extracellular signal-regulated kinase mitogen-activated protein kinase pathway that provides a rationale for combining pan-ERBB and mitogen-activated protein kinase inhibitors as a therapeutic approach in subsets of HNSCC.

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Figures

Fig. 1.
Fig. 1.
Essential protein kinases identified by the functional shRNA screen. (A) The average ranks of 521 protein kinases from the essential shRNA screen performed in UMSCC25, UMSCC8, JHU011, HN6, HN12, and Cal27 cells are graphed along with the 10 murine control genes indicated with green symbols. The ranks for the protein kinases and murine control genes in the individual cell lines are presented in Supplemental Table 1. EGFR, ERBB2, MET, and FGFR1 were not identified as highly essential as single protein kinases and are shown with yellow symbols. Based on the published literature (see text in Results), the top-ranked essential kinases were assembled into interaction networks centered on MTOR (B) and ERBB3 (C) in which the average ranks of the indicated protein kinases among the six HNSCC cell lines are denoted by the colors shown in the color scale bar.
Fig. 2.
Fig. 2.
Validation of MTOR as an essential protein kinase in HNSCC cell lines. UMSCC25, UMSCC8, and Cal27 cells in six-well plates were transduced with a control shRNA _targeting GFP and two MTOR shRNAs (TRCN0000363722 and TRCN0000332888) not present in the kinome shRNA library. The cells were submitted to selection in puromycin (1 μg/ml) for 5 to 10 days and stained with crystal violet. Representative images of stained wells are shown and were quantified by measuring the crystal violet absorbance of the fixed cells at 590 nM. The data are the means and S.D. of duplicate wells from two independent experiments normalized to the GFP shRNA controls. Cell extracts were prepared from replicate wells and submitted to immunoblot analysis for MTOR. The filters were stripped and reprobed for the α-subunit of NaK-ATPase (Na/K) as a loading control. The data demonstrate efficient inhibition of MTOR protein levels with the MTOR shRNAs relative to the GFP control shRNA.
Fig. 3.
Fig. 3.
Validation of ERBB3 as an essential protein kinase in HNSCC cells. UMSCC8 (A, B), UMSCC25 (C), and HN6 cells (D) were transduced with lentiviruses encoding a control GFP shRNA or two ERBB3-_targeted shRNAs (TRCN199364 and TRCN40108). After ∼10 days of culture under puromycin selection, the cells were submitted to RNA purification and reverse-transcription PCR (A) or the wells were fixed and stained with crystal violet and quantified by measuring the absorbance of the fixed cells at 590 nm (B–D). The data are the means and S.D. of duplicate wells from two independent experiments normalized to their GFP shRNA controls.
Fig. 4.
Fig. 4.
Identification of protein kinases that exhibit synthetic lethality in combination with AZD8931. (A) Average ranks of 521 protein kinases from the synthetic lethal shRNA screen performed in UMSCC25, UMSCC8, JHU011, HN12, and Cal27 cells are graphed along with the 10 murine control genes indicated with green symbols. The ranks for the protein kinases and murine control genes in the individual cell lines are presented in Supplemental Table 2. (B) ERK MAPK pathway components from the synthetic lethal screen are assembled into a putative pathway, in which the strength of the hit is indicated by the color bar.
Fig. 5.
Fig. 5.
Synergistic growth inhibition of UMSCC25 cells with combined AZD8931 and MEK inhibitor, selumetinib. (A) UMSCC25 cells were seeded in 96-well plates at 100 cells/well and incubated in triplicate with the indicated combinations of AZD8931 and selumetinib for 7 to 10 days. Cell number was estimated by measuring DNA content with CyQUANT reagent, and the mean fluorescence values are plotted. (B) The resulting data in (A) were submitted to the CalcuSyn program (Biosoft) to calculate combination indices (CI) for inferring synergy as shown by the color scale. The data shown are representative of another independent experiment.
Fig. 6.
Fig. 6.
Effect of combined AZD8931 and trametinib treatment on phospho-ERK and phospho-EGFR levels in UMSCC25 cells. UMSCC25 cells were treated with AZD8931 (0–100 nM) in the presence or absence of 3 nM trametinib, and cell extracts were prepared after 1, 6, 24, and 72 hours of treatment. The extracts were submitted to SDS-PAGE and immunoblotted for phospho-EGFR (mixture of anti-Y1068 and Y1148) as well as phospho-ERK and phospho-Ser473-AKT. The filters were stripped and reprobed for total EGFR, ERK, AKT, and Na/K-ATPase α-subunit levels as loading controls. The relative levels of p-EGFR/EGFR and p-ERK/ERK following densitometry of the autoradiographic films are shown, and the data are representative of another independent experiment.
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