EGFR and Therapeutic Resistance

Patient Responses to Gefitinib

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In this issue we are highlighting the Epidermal Growth Factor Receptor (EGFR) and its role in non-small cell lung cancer. The EGFR family consists of four members (EGFR, ERBB2, ERBB3 and ERBB4) that are each integral membrane proteins capable of forming homo- or hetero-oligomers within the family. Ligand binding leads to a conformational change in the receptor and activation of the intracellular tyrosine kinase domain. The resulting intracellular signaling cascade(s) promote a variety of biological processes including cellular proliferation, differentiation and survival.

Improved Mutant Responses

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Although EGFR is present in normal cells, its expression on a substantial percentage of NSCLCs compelled the development of small-molecule inhibitors capable of blocking kinase function, of which Gefitinib was the first to be evaluated clinically. While the initial phase II studies were promising (and actually led to accelerated FDA approval), an ensuing phase III trial showed no efficacy relative to placebo and the license for the drug was revoked worldwide (with the exception of Asia). Fortunately, thanks to the perseverance of a dedicated group of researchers, this was not the end of the story. What we now know is that a small subset of NSCLC patients actually showed favorable responses to Gefitinib, but only if their cancer expressed a mutant form of the receptor. There are actually two different mutations that are typically observed – a single amino acid substitution at position 858 of the protein (Leucine replaced by Arginine) and a deletion of five amino acids spanning positions 746 through 750. Either of these mutations dramatically improves Gefitinib efficacy and patient outcome.

Double Mutant Responses

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Unfortunately for afflicted patients, their responses do not persist and their cancer typically returns. This is due to the development of secondary mutations that cause Gefitinib-resistance. In the case of the EGFR, this seecondary mutation occurs in a region of the enzyme known as the “gatekeeper” located at position 790, where a relatively small threonine is replaced by a much bulkier methionine. With the discovery of this bulky substitution, it was assumed that the lack of Gefitinib efficacy could be explained – the larger amino acid was simply interfering with Gefitinib binding to the kinase. However, this hypothesis was at odds with biochemical data that suggested that Gefitinib binding to these “double mutant” receptors was relatively unaffected. The story became further complicated when X-ray crystallography confirmed that Gefitinib could in fact bind to these double mutant receptors.

Biochemical Versus Cellular Responses

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The apparent solution to this paradox was recently revealed by Yun and colleagues, who discovered that the “gatekeeper” mutation dramatically alters the affinity of ATP for the kinase domain. This altered affinity in turn impacts the ability of Gefitinib to bind and hence the observed reduction in affinity. This also explains why this affect was not observed in biochemical assays; these are usually performed at the enzymes Km for ATP. This concentration is typically much lower than found within the cell, where the much higher ATP concentration in turn makes it much more difficult for Gefitinib to bind to the EGFR kinase domain. This of course is one of the benefits of using ACDs cell-based assay services – our kinases are already in their native environment and more accurately capture the biological influences of the intracellular environment.