The majority of PIK3CA mutations occur either in the helical or in the kinase domain of the gene. Thus, we sequenced the respective regions of PIK3CA in all imatinib resistant cell lines. We did not find mutations in the kinase domain, but cell line KCL 22 carried a heterozygous point mutation in the helical domain, leading to the amino acid change PI3Ka E545G. PI3Ka E545 mutations have been observed in clinical samples of solid tumors and the E545A mutation has been shown to constitutively activate the PI3K pathway. These data suggest that also the PI3Ka E545G muta tion that we identified in cell line KCL 22 may be responsible for the constitutive activity of the PI3K/ AKT1 pathway conferring TKI resistance to the cells.

Deep sequencing might help to elucidate whether acti vating mutations in oncogenes other than 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 number of Ph ALL and CML cell lines tested imatinib resistant. The unresponsiveness of the cell lines was not attributa ble to known causes as BCR ABL1 mutations or activa tion of SRC kinases. While the BCR ABL1 triggered The PI3K subunit p85b and the Casitas B Cell lymphoma gene belong to those seven genes identified as core components for coordinating the oncogenic functions of BCR ABL1. Phosphory lation of CBL recruits the p85 subunit of PI3K leading to activation of PI3K/AKT1/mTOR pathway. Quan titative RT PCR did not reveal major differences in the expression of CBL and p85 between imatinib sensitive and resistant cell lines.

Besides, we did not detect alterations in exons 7 9 of CBL, described as transforming mutations in myeloid malignancies. Class I PI3Ks are heterodimeric proteins consisting AV-951 of a catalytic and a regulatory adaptor subunit. To find out which specific PI3K might be involved in imatinib resistant activation of AKT1/mTOR, we applied inhibitors with differing specificities for the JAK2/STAT5 and ERK1/2 pathways were inhibited by imatinib, the resistant cell lines stand out by the consti tutive activation of the PI3K/AKT1/mTOR pathway. The mTOR inhibitor rapamycin inhibited cell growth, but did not induce apoptosis and did not sensitize resis tant cells to imatinib. Instead, inhibition of AKT1 induced apoptosis in TKI resistant cell lines.

Cell line KCL 22 carries a heterozygous mutation in the helical domain of PIK3CA, a site critical for activation of the gene. These results suggest that activating mutations in the PI3K itself or in PI3K stimulating oncogenes might be the molecular cause for TKI resistance. Methods Human cell lines The cell lines applied in this study were taken from the stock of the cell bank or were provided by originators. Detailed references and cultivation protocols have been described previously. Inhibitors Imatinib and nilotinib were generously provided by Novartis.