Benefits Kinase ORF expression screen. To recognize kinases whose expression can mediate resistance to PI3K inhibitors, we performed open reading frame expression screens in breast cancer cell lines within the presence of BEZ235 or BKM120. Both of these compounds are at present in clinical development. This ORF library is composed of 597 kinases and kinase related genes in lentiviral expression vectors containing a blasticidin resistance marker for efficient transduction and stable overexpression in target cells. We chose to perform a focused screen with kinases, as they represent a set of readily druggable targets, facilitating validation and potentially clinical translation. We screened MCF7 and BT474 cells, as they represent the 2 genotypes of breast cancer cells previ ously established as exhibiting sensitivity to PI3K inhibition, MCF7 and BT474.
The criteria made use of to pick kinases that support proliferation following PI3K mTOR blockade within the ORF screen have been elevated cell numbers within the presence of BEZ235 or BKM120 by a minimum of three SD above the mean and corresponding increases within the ratio of cell number in treated versus untreated wells to eliminate kinases that simply stimulate common proliferation. We performed validation experi ments selleckchem C59 wnt inhibitor on the ORFs with all the strongest phenotypes in the MCF7 screens for resistance against BEZ235 and BKM120 and had been in a position to confirm PI3K inhibitor resistance phenotype for many of those candidates using 2 independent assays for viability. Unsurprisingly, vali dated candidates integrated the receptor tyrosine kinases ERBB2 and IGF1R, each of that are known to be upstream of PI3K dependent signaling and PI3K independent signaling too as AKT1 and AKT3, crucial effectors in the PI3K pathway.
In the remaining candidates, we have been specifically keen on RPS6KA2 and RPS6KA6, selleck as these 2 genes pro vided robust resistance against PI3K inhibition. RSKs mediate resistance to PI3K inhibition. Because RSK3 and RSK4 overexpressing cells exhibited a profound decrease in PI3K inhibitor sensitivity, we sought to figure out regardless of whether other RSK family members exhibited similar properties. In contrast to RSK3 and RSK4, expression of RSK1 and RSK2 only slightly decreased the sensitivity to PI3K inhibition, although the very related mito gen and stress activated protein kinases exhibited no activity, and this was irrespective of expression levels. We for that reason chose to focus on RSK3 and RSK4 for subsequent analyses. To decide whether or not the resistance phenotypes of RSK more than expressing cell lines extended to other PI3K pathway inhibitors, we determined the sensitivity of these cells to other inhibitors cur rently in early stage clinical testing, like GDC 0941, a pan PI3K inhibitor, and MK 2206, an allosteric pan AKT inhibitor.