nce Celecoxib of inhibitor. These results were compared to clinical resistance mutants by sequencing tumors from melanoma patients who had relapsed upon treatment with AZD6244. These , which is analogous to two secondary mutations that were discovered in the random mutagenesis screen. The Pro124Leu MEK1 mutant provided a modest increase in AZD6244 GI50 when expressed in parental A375 melanoma cells. A drug resistance study has also been performed with the phosphatidylinositol 3 kinase p110, which is a lipid kinase that generates phosphatidylinositol 3,4,5 trisphosphate from phosphatidylinositol 4,5 bisphosphate. p110 is the most frequently mutated gene in human cancer, with the activating mutation His1047Arg in the kinase domain being the most common.
For this reason, a number of ATP competitive small molecule inhibitors of p110have been developed and are undergoing clinical trials for the treatment of cancer. To facilitate the identification of p110 resistance mutations in vitro, Shokat and co workers developed a PI3K inhibitor screen in the yeast S. cerevisiae. Over expression of membranelocalized p110 inhibits the growth BI6727 of S. cerevisiae, most likely because these yeast lack the ability to degrade any PIP3 that is generated. However, small molecule inhibitors of PI3K can rescue growth. Through the use of replica plating and robotic pinning this screen allows the rapid assessment of a large number of mutants under various conditions.
A library of high copy plasmids containing mutants of p110 CAAX, which were generated by site directed saturation mutagenesis, was transformed into the drug permeable yeast strain YRP1. The library of p110 CAAX variants was then screened on glucose and galactose media to determine which mutants retain catalytic activity. Active mutants that were growth inhibited on galactose in the presence of high p110 inhibitor concentrations, for example PI 103, were selected and sequenced. In contrast to protein kinases, the gatekeeper residue of p110 was found to be intolerant to mutation and, therefore, not a likely site of resistance. However, another residue that lines the ATP binding pocket, Ile800, was found to confer resistance without compromising kinase activity. The identified resistance mutations did not affect all of the p110 inhibitors uniformly, one drug resistant mutant, Ile800Leu, sensitized p110 to dual PI3K/mTOR inhibitor BEZ 235 and multi targeted kinase inhibitor PW 12.
The functional relevance of these resistance mutations was validated with in vitro activity assays and in the non tumorigenic mammary epithelial cell line MCF10A. Conclusions The emergence of drug resistance to targeted cancer therapies is an ongoing clinical problem. While resistance to small molecule kinase inhibitors can be caused by the amplification of the oncogenic kinase gene being targeted or the re wiring of signaling cascades, the emergence of mutations in the catalytic domain that hinder drug binding is a common mechanism. However, the range of mutations that are available to a kinase to confer drug resistance are limited due to the necessity of these enzymes maintaining their cellular functions. Several general themes emerge by comparing drug resistance mutations in BCR ABL, EGFR, MEK1, p110 and the Aurora kinases. First, point mutations that generate resistance to small mo