Establishment and characterization of an erlotinib-resistant clone derived from the EGFR^L858R NSCLC cell line H3255

Abstract

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PURPOSE: A subset of non-small cell lung cancer (NSCLC) patients that have somatic EGFR mutations have a dramatic initial response to EGFR tyrosine kinase inhibitors (Erlotinib or Gefitinib), but eventually relapse and die of advanced cancer. In some patients, a second EGFR^T790M mutation has been identified. To gain insights into the mechanism of acquired resistance to erlotinib, we have generated and characterized an erlotinib-resistant clone from the parental NSCLC cell line H3255, which contains a heterozygous EGFR^L858R/EGFR^WT genotype. EXPERIMENTAL DESIGN and RESULTS: The H3255 cell line was derived from a female non-smoker with adenocarcinoma (a generous gift from Dr. Pasi Jänne) and was very sensitive to erlotinib (IC₅₀ = 0.029 ± 0.019 μM). We generated 3 erlotinib-resistant clones by exposing the H3255 cells to a step-wise escalating concentration of erlotinib (0.01-1.0 μM) over an average period of 3 months. By DNA profiling, clone R#2 shared the closest short tandem repeat (STR) allele pattern with parental H3255 cells. Chromatographic sequencing tracing revealed lack of the EGFR^L858R allele in R#2 cells and the presence of almost exclusive EGFR^WT allele in H3255 cells maintained in culture for > 3 months. No EGFR^T790M mutation was found in R#2 or H3255 cells. Compared with the parental H3255 cells, R#2 cells were 200-fold more resistant to erlotinib (IC₅₀: 9.9±2.1 versus 0.05±0.01 μM; p<0.01) and at least 100-fold more resistant to cetuximab (IC₅₀: >10 versus 0.10±0.06 μg/mL; p<0.05) by the MTT assay. Parental H3255 and R#2 cells had similar cytotoxicity to several chemotherapy agents: cisplatin, paclitaxel, irinotecan, and VP-16. Compared with the parental H3255 cells, R#2 cells were more resistant to the PI3-K inhibitors: 1.9-fold more resistant to LY-294002 (IC₅₀: 9.2 versus 4.9 μM; p=0.04) and 3.5-fold more resistant to Wortaminnin (IC₅₀: 18.5 versus 5.3 μM; p=0.03). No differences in cytotoxicity to other signal transduction pathway inhibitors (such as the p42-44 MAPK inhibitors, FTI inhibitors or ERK inhibitors) were observed in R#2 and H3255 cells (p>0.01 for all comparisons). CONCLUSIONS: Continuous exposure to erlotinib might lead to the preferable expansion of pre-existing EGFR^WT cells in NSCLC tumors, which ultimately leads to resistance to erlotinib. This might be an alternative mechanism of acquired resistance to erlotinib in addition to the emerging of a second EGFR^T790M mutation in NSCLC patients. Supported by NIH grants CA 91784 and CA 84119.