January 1, 1970 (Vol. , No. )
Jessica Hunt Senior Scientist Horizon Discovery
The epidermal growth factor receptor (EGFR) is highly expressed or mutated in cancers such as non-small cell lung cancer and plays a critical role in the development of cancer, causing increased proliferation, decreased apoptosis, enhanced motility and angiogenesis. Patients with EGFR-mutant tumors have increased sensitivity to EGFR inhibitors such as the tyrosine kinase inhibitors Gefitinib and Erlotinib.
Horizon Discovery has used its proprietary rAAV gene engineering technology (GENESIS™) to generate a suite of MCF10A cell lines focused on EGFR mutations. This includes one of the most common mutations, ΔE746-A750, clinically relevant double mutations such as PI3Kα & EGFR, and mutations conferring drug resistance such as the T790M mutation. All lines engineered are genetically identical to MCF10A parental cells except for their EGFR status.1 Historically, cell lines such as PC-9 have been used to model EGFR overexpression;2 however these cell lines are often compared to cell lines that differ not only in EGFR status but also differ with respect to other genetic mutations and tissue type background. The X-MAN™ cell system allows the role of EGFR to be studied without the influence of other factors.
The EGFR isogenic system offers an attractive method for studying sensitivity to inhibitors in a clean and patient-relevant system. The permanent and specific nature of the gene-targeting technology overcomes limitations of RNA interference strategies such as transient gene disruption and off-target effects. In addition, the isogenic system can be used throughout the drug discovery process, to identify and validate novel targets both in vitro and in vivo.
Cells were seeded into 96-well plates in media containing 1% charcoal stripped FBS and 0.2 ng/mL EGF, and allowed to adhere overnight. Cells were then treated with compounds for 96 h. Cell viability was quantified using alamar blue.
Results & Discussion
Erlotinib and Gefitinib, clinically relevant EGFR inhibitors, were investigated in MCF10A EGFR isogenic cells using a proliferation assay. EGFR ΔE746-A750 cells were sensitive to EGFR inhibition, showing a selectivity over parental cells that was most pronounced following Gefitinib treatment. In contrast, introduction of the T790M mutation-conferred drug resistance.
The PI3Kα & EGFR double mutant cell line harbors the H1047R point mutation within exon 20 of the kinase domain of PI3Kα. This cell line was used to study the effect of PI3Kα mutations on EGFR inhibitor selectivity. The PI3Kα & EGFR double mutant cell line displayed attenuated sensitivity to Gefitinib compared to the single mutant EGFR cell line, indicating the need for rational therapeutic strategies dependant on tumor genetic background.
EGFR isogenic cells are a powerful and relevant tool for drug discovery and development, enabling single and double gene mutations to be studied without the need for large cell panels. The EGFR isogenic cell panel clearly demonstrates the significance of genetic background in the sensitivity of EGFR mutant cells to EGFR inhibitors, exemplifying the need for personalized medicine. Such approaches to drug screening aim to facilitate the discovery of effective anticancer drugs that exploit vulnerabilities unique to cancer cells by virtue of the mutations they have accrued during tumor progression.
1. Di Nicolantonio et al., PNAS 2008 (105) p20864
2. Mukohara et al., J Natl Cancer Inst. 2005 (16) p1169