Further analyses showed that also dysregulation of drug transporters was improb able unlike imatinib, nilotinib is neither imported via hOCT 1, nor exported via ABCB1. All five imati nib resistant cell lines were nilotinib resistant. Therefore, it appeared unlikely that imatinib resistance was caused by deregulated transport proteins. Finally, the finding that both imatinib and nilotinib induced dephosphorylation of signal transducer and activator of transcription 5 in the TKI resistant cell line SUP B15 as shown in Figure 2 further excludes resis tance being due to low intracellular drug levels. Both drugs were transported into the cells which responded by dephosphorylating STAT5 while retaining viability. SRC kinases SRC kinases had been described to play an important role in BCR ABL1 positive ALL.

Interest ingly, 4/5 imatinib resistant Ph cell lines were from patients with pre B ALL, T ALL, or CML in B cell blast crisis. Among lymphoid Ph cell lines 5/7 were imatinib resistant, including TOM 1, a pre B cell line classed semiresistant displaying normal IC50 values in the thymidine uptake assay while remaining relatively unresponsive to higher concentrations. There fore, we applied dasatinib to elucidate whether activity of SRC kinases was important for the growth of imatinib resistant cells. Dasatinib is a dual BCR ABL1 and SRC kinase inhibitor, as evidenced by its ability to inhibit phosphorylation of SRC and STAT5 in TKI responsive JURL MK2 cells. However, two of three imatinib resistant cell lines tested were resistant to dasatinib in the proliferation assay.

Furthermore, TKI resistant SUP B15 cells did not express an active, phosphorylated SRC kinase and dasatinib did not affect RSP6 Brefeldin_A phosphorylation in this cell line. These results are not consistent with the notion that SRC kinases are the cause of imati nib resistance in these cell lines. Imatinib induces dephosphorylation of ERK1/2 and of STAT5 in TKI resistant cell lines BCR ABL1 positive cells are characterized by stimulation of the Janus kinase 2 /STAT5, extracellular signal regulated kinase 1/2 and phosphoinositide 3 kinase/v Akt murine thymoma viral oncogene homolog 1/mammalian target of rapamycin pathways. To determine the activity of these signalling cascades, we assessed the phosphoryla tion status of STAT5, ERK1/2 and of the mTOR complex 1 substrate ribosomal S6 protein.

In TKI sensitive cells, imatinib induced dephosphory lation of all three proteins. In TKI resistant cell lines, treatment with TKI reduced phosphorylation of STAT5 and of ERK1/2 but did not comparably affect phosphorylation of RPS6. This observation allowed three con clusions cells that survive in the presence of imatinib are not necessarily completely unresponsive to the drug. activation of ERK1/2 and the JAK/STAT5 pathway is not obligatory for short term proliferation of Ph posi tive cell lines.