PI3Ka E545 mutations have already been observed in clinical samples of strong tumors as well as the E545A mutation has been shown to constitutively activate the PI3K pathway. These information recommend that also the PI3Ka E545G muta tion that we identified in cell line KCL 22 may be responsible for the constitutive activity from the PI3K AKT1 pathway conferring TKI resistance to the cells. Deep sequencing may possibly enable to elucidate no matter whether acti vating mutations in oncogenes apart from BCR ABL1 or PIK3CA, or loss of tumor suppressor genes trigger the PI3K in cell lines NALM 1, SD 1, SUP B15 and MHH TALL1, thus causing TKI resistance. Conclusion In this study an unexpectedly high variety of Ph ALL and CML cell lines tested imatinib resistant. The unresponsiveness of the cell lines was not attributa ble to recognized causes as BCR ABL1 mutations or activa tion of SRC kinases.
Even though the BCR ABL1 triggered The PI3K subunit p85b plus the Casitas B Cell lymphoma gene belong to those seven genes identified as core elements for coordinating the oncogenic functions of BCR ABL1. Phosphory lation of CBL recruits the p85 subunit of PI3K major to activation selleck of PI3K AKT1 mTOR pathway. Quan titative RT PCR did not reveal key variations in the expression of CBL and p85 involving imatinib sensitive and resistant cell lines. Besides, we didn’t detect alterations in exons 7 9 of CBL, described not shown. Class I PI3Ks are heterodimeric proteins consisting of a catalytic in addition to a regulatory adaptor subunit.
To locate out which precise PI3K may be involved in imatinib resistant activation of AKT1 mTOR, we applied inhibitors with differing specificities for the JAK2 STAT5 and ERK1 2 pathways had been inhibited by imatinib, the resistant cell lines stand out by the consti tutive activation in the PI3K AKT1 mTOR pathway. The mTOR inhibitor rapamycin inhibited cell development, but did not induce apoptosis selleck chemical and didn’t sensitize resis tant cells to imatinib. As an alternative, inhibition of AKT1 induced apoptosis in TKI resistant cell lines. Cell line KCL 22 carries a heterozygous mutation within the helical domain of PIK3CA, a web-site critical for activation from the gene. These final results suggest that activating mutations within the PI3K itself or in PI3K stimulating oncogenes might be the molecular cause for TKI resistance. Approaches Human cell lines The cell lines applied within this study were taken in the stock with the cell bank or have been offered by originators. Detailed references and cultivation protocols have been described previously. Inhibitors Imatinib and nilotinib had been generously supplied by Novartis. Ten mM stock solutions had been ready in H2O or DMSO. Dasatinib was obtained from LC Laboratories. The SRC inhibitor SU 6656 was obtained from Cayman Chemical. Rapamycin was purchased from Cell Signalling.