a) Schematic showing the genomic position of the EGFR-AS1 lncRNA gene relative to exon 20 of the EGFR gene, and the position of the SNP encoding EGFR p.Gln787Gln. Arrows correspond to the positions of the siRNAs designed to knock down EGFR-AS1. TK, tyrosine kinase; TM, transmembrane domain. ( b, c) Graphs showing levels of the EGFR-AS1 lncRNA (measured by real-time RT–PCR) in untreated lines ( b) and the trend in expression over time after treatment with actinomycin D in cells with the A/A (NCC-HN19 and NCC-HN64) and G/G (NCC-HN1 and NCC-HN43) genotypes and in the isogenic NCC-HN1 clones with correct editing (G/A AAV: CL16, CL63 and CL19) and controls (G/G AAV: CL12, CL76 and CL77) ( c). ( d, e) Graphs of IC 50 values for gefitinib in cell lines with knockdown of EGFR-AS1 by two different siRNAs (siAS1.1 and siAS1.2) as compared to nontargeting controls (NT) ( d) and EGFR-AS1 overexpression (AS1 OE) as compared to vector controls (Ctrl) ( e). ( f) Tumor growth in PDXs HN124 and HN159 (with high expression of EGFR-AS1) after treatment with EGFR-AS1-targeting LNA as compared to treatment with nontargeting control ( P = 0.0012 and P = 0.0059, respectively, determined by two-way ANOVA with Bonferroni correction). Red arrows indicate the start of weekly LNA treatment (i.e., when tumor volume was 0.5 cm 3); for HN124, mice were also treated with gefitinib 1 week after the start of LNA treatment. In b– f, each data point corresponds to a cell culture replicate, with the midline (where applicable) representing the average of these; error bars indicate 1 s.d. In b and c, levels were derived on the basis of triplicate technical replicates (RT–PCR reactions per sample) from one cell culture experiment per cell line condition; in d and e, levels were derived on the basis of triplicate technical replicates from two and three separate cell culture experiments per cell line condition, respectively. Asterisks denote significance as determined by Student's t-test (two-tailed). * P < 0.05, ** P < 0.01, *** P < 0.001. Detailed statistics are provided in the Supplementary Data.