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. 2015 Apr 9;520(7546):239-42.
doi: 10.1038/nature14122. Epub 2015 Jan 28.

EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors

Christine M Fillmore  1 , Chunxiao Xu  2 , Pooja T Desai  3 , Joanne M Berry  3 , Samuel P Rowbotham  1 , Yi-Jang Lin  4 , Haikuo Zhang  2 , Victor E Marquez  5 , Peter S Hammerman  6 , Kwok-Kin Wong  2 , Carla F Kim  1
Affiliations

EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors

Christine M Fillmore et al. Nature. .

Erratum in

Abstract

Non-small-cell lung cancer is the leading cause of cancer-related death worldwide. Chemotherapies such as the topoisomerase II (TopoII) inhibitor etoposide effectively reduce disease in a minority of patients with this cancer; therefore, alternative drug targets, including epigenetic enzymes, are under consideration for therapeutic intervention. A promising potential epigenetic target is the methyltransferase EZH2, which in the context of the polycomb repressive complex 2 (PRC2) is well known to tri-methylate histone H3 at lysine 27 (H3K27me3) and elicit gene silencing. Here we demonstrate that EZH2 inhibition has differential effects on the TopoII inhibitor response of non-small-cell lung cancers in vitro and in vivo. EGFR and BRG1 mutations are genetic biomarkers that predict enhanced sensitivity to TopoII inhibitor in response to EZH2 inhibition. BRG1 loss-of-function mutant tumours respond to EZH2 inhibition with increased S phase, anaphase bridging, apoptosis and TopoII inhibitor sensitivity. Conversely, EGFR and BRG1 wild-type tumours upregulate BRG1 in response to EZH2 inhibition and ultimately become more resistant to TopoII inhibitor. EGFR gain-of-function mutant tumours are also sensitive to dual EZH2 inhibition and TopoII inhibitor, because of genetic antagonism between EGFR and BRG1. These findings suggest an opportunity for precision medicine in the genetically complex disease of non-small-cell lung cancer.

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Figures

Figure 1
Figure 1. EZH2i sensitizes BRG1 or EGFR mutants to TopoIIi
a, Director’s Challenge samples were hierarchically clustered into two risk groups using the EZH2 co-expression signature (Table S1). The Kaplan-Meier curves for only Stage 1 (n=94) or only moderately differentiated tumors (n=142) to 6 years post diagnosis are shown. b, Western Blot for EZH2 and H3K27me3 on indicated transduced lines, total Histone H3 is shown as loading control. CR indicates a coding region targeting hairpin. c, Fold change +/− s.e.m. in etoposide IC50 between transduced lines, n=3 biological replicates for HCC15, A549, PC9, H23 and Sw1573, n=4 biological replicates for HCC15 and H460, rescues n=3 biological replicates, * P<0.04, ** P<0.01. d, Western Blot for EZH2 and H3K27me3 on lines treated with indicated drugs. e, Fold change +/− s.e.m. in etoposide IC50 between vehicle treated and drug treated lines, n=3 biological replicates for all except n=4 biological replicates for H157 + DZNep, * P<0.04, ** P<0.01. f, Average Chou-Talalay combination index (CI) values +/− s.e.m. (also see SI Table 3) for fractions affected equivalent to IC25 through IC75; n=3 biological replicates.
Figure 2
Figure 2. In vitro sensitivities to EZH2i + TopoIIi predict in vivo responses
Either the H157 (a) or H23 (b) cell line was injected into the flanks of Nude mice and tumors were allowed to form. On day 12, mice were randomly segregated into cohorts that received either placebo, DZNep, etoposide or dual therapy for 2 weeks, and tumor size +/− s.e.m. were graphed, n for tumors/mice in each arm indicated on graphs, * P=0.002, ** P=0.0005 dual vs. etoposide. c, The PC9 cell line was injected into the flanks of Nude mice and tumors were allowed to grow to 70mm3. Mice were then treated with etoposide, GSK126, dual therapy or gefitinib (as a positive control) for 2 weeks, and tumor size +/− s.e.m. were graphed, n indicated on legend, mice with one tumor each, P<0.008 for dual vs etoposide or GSK126 alone. d, Representative MR images of mice of indicated genotypes on combination etoposide+DZNep treatment at 0 and 4 weeks post treatment initiation. H indicates heart area. e, Waterfall plot depicting tumor growth +/− s.e.m. of EGFRT790M;L858R tumors after 2 weeks and 4 weeks of treatment with vehicle (blue), etoposide (green), DZNep (red) and etoposide+DZNep (purple). The y-axis indicates % tumor growth vs. day 0. Each bar represents an individual mouse. Statistical analyses were performed on the 4 week log2 transformed data, P=0.008 dual vs. DZNep and P=0.004 dual vs. etoposide. f, Waterfall plot depicting tumor growth +/− s.e.m. of KrasG12D/+; p53Δ/Δ tumors after 2 weeks and 4 weeks of treatment with vehicle (blue) and etoposide+DZNep (purple).
Figure 3
Figure 3. Dual EZH2i and TopoIIi differentially affects cell cycle, apoptosis and anaphase bridging
a, RT-qPCR for BRG1 levels +/− s.e.m. in indicated cell lines in response to 4 days of 1μM DZNep or 10μM GSK126, n=6 biological replicates for DZNep, n=3 biological replicates for GSK126, * P<0.05, ** P<0.008. b, Percentage of anaphase structures with bridges +/− s.e.m. in vehicle-, DZNep- treated or GSK126-treated cell lines, n≥3 biological replicates and indicated in Methods, * P<0.04, ** P<0.02. c, Annexin V+/7AAD− cells +/− s.e.m. quantified by flow cytometry on cell lines cultured with indicated treatments for 3 days, n=4 biological replicates, * P<0.03, ** P<0.001 for Etop vs Dual treated or DZNep vs Dual treatment for PC9. d, 7-AAD cell cycle flow cytometry was performed on cell lines with or without 5μM etoposide or 1μM DZNep for 4 days. The average % S phase +/− s.e.m. of each culture is plotted, n=3 biological replicates for HCC15, A549, PC9 and H23, n=4 biological replicates for H157 and Sw1573, * P<0.05 ** P<0.009.
Figure 4
Figure 4. BRG1 and EGFR are genetically antagonistic and control the sensitized phenotype
a, EGFR and BRG1 expression in tumors from the Director’s Challenge were plotted and correlation was assessed, n=36. b, RT-qPCR for expression of BRG1 and EGFR +/− s.e.m. in indicated transduced HCC15 and H460 cell lines, * P<0.04, **P<0.0001, n=5 biological replicates for H460, n=4 biological replicates for HCC15, oe indicates over-expression. c, The average etoposide IC50 +/− s.e.m. in the indicated vehicle- and DZNep-treated cells, n=3 biological replicates, * P<0.04, ** P<0.0001. d, Average percentage of anaphase structures with bridges +/− s.e.m. in indicated vehicle- or DZNep-treated cell lines, n=3 biological replicates, * P<0.04, ** P<0.02. e, Depiction of EGFR regulatory element including H3K27ac and BRG1 binding peaks and from ENCODE database. f, Immunofluorescence for EGFR and BRG1 on HCC15 shGFP (control) and BRG1 over-expressing cell lines, scale bar = 30μm. g, Chromatin immunoprecipitation for GFP (control), BRG1 or the FLAG-tag on the exogenously expressed BRG1 in the HCC15 shGFP (control) and BRG1 expressing cell lines. qPCR was performed with primers for indicated genomic sites and enrichment over GFP +/− s.e.m. was plotted, n=4 biological replicates, One-way ANOVA * P=0.02. h, In response to EZH2i: WT cells show increased BAF complex function and subsequent decrease in etoposide sensitivity; BRG1 mutant cells have increased etoposide sensitivity; and EGFR mutant cells act like BRG1 mutant cells due to genetic antagonism of EGFR by BRG1.
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