Search

Summary Introduction: We investigated the second-generation tyrosine kinase inhibitors, dasatinib and nilotinib, for their potential to overcome resistance in the imatinib-resistant K562 cell line, and evaluated whether rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, and bortezomib, a proteasome inhibitor, could increase imatinib sensitivity in resistant cell lines. Methods: Cell lines, including K562 (chronic myeloid leukaemia in blast crisis) and K562r (imatinib-resistant K562), were exposed to dasatinib, nilotinib, rapamycin, bortezomib, and rapamycin plus imatinib. Cell proliferation was measured by 3-[4, 5-dimethylthia-zol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and mTOR signaling pathways were assessed by Western blotting. Results: Dasatinib, nilotinib, and bortezomib inhibited proliferation of K562 and K562r cell lines at nm concentrations. Resistance of K562r due to duplication of autophosphorylation of wild-type Bcr-Abl was not overcome by dasatinib and nilotinib, but was sensitive to bortezomib. Rapamycin partially inhibited proliferation of K562 and K562r cell lines. Rapamycin plus imatinib did not have a more inhibitory effect on the proliferation of K562 and K562r cell lines. Conclusion: K562r due to duplication of autophosphorylation of wild-type Bcr-Abl induced by imatinib was still partially resistant to dasatinib and nilotinib, but this was overcome by incremental dosing. Rapamycin did not enhance imatinib sensitivity. The blockade of the ubiquitin-proteasome pathway could be effective in overcoming resistance in the K562r imatinib-resistant cell line.

Transcription factor stem cell leukemia (SCL), also known as the T-cell acute lymphocytic leukemia 1 (TAL1), plays a key role in the regulation of hematopoiesis, but the molecular mechanisms are not well understood. The aim of the present study is to elucidate the effects of the epidermal growth factor receptor (EGFR) signal pathways underlying the biologic activity of SCL/TAL1 on normal hematopoietic development. Lentiviral vectors with up or down-regulation of SCL/TAL1 were transfected into umbilical cord blood CD34 stem cells. EGFR signaling pathways (including MEK/ERK and Akt/mTOR) and surface hematopoietic markers were analyzed in the process of hematopoietic differentiation. The data revealed that up or down-regulation of SCL/TAL1 gene was accompanied positively by the expressions of p-MEK and p-ERK1/2 protein, but the changes of Akt/mTOR were unobvious. MEK/ERK inhibitor U0126 and SCL/TAL1 down-regulation showed similar inhibitory effects on erythroid, myeloid, and megakaryoid differentiation. However, Akt/mTOR pathway altered insignificantly. MEK/ERK inhibitor U0126 could not affect the expression of SCL/TAL1 mRNA or protein. Taken together, these findings fully illustrated that SCL/TAL1 is located in the up-stream of MEK/ERK pathway and partially regulates hematopoiesis by modulating the phosphorylation level of the key proteins in MEK/ERK pathway.

In this study, we established an imatinib resistant Ph+ acute lymphoblastic leukemia (ALL) cell line SUP-B15/RI in vitro and studied the mechanism of imatinib resistance. Our results showed that the BCR-ABL1 fusion gene and the mdr1 gene were 6.1 times and 1.7 times, respectively, as high as that of parental SUP-B15 cell line. We found no mutation in the Abl kinase domain of SUP-B15/RI. Furthermore, the detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κB, JNK and STAT showed the up-regulation of phosphorylation of AKT, mTOR, P70S6K, and RAF, ERK, and MEK, down-regulation of PTEN and 4EBP-1, and no change in other cell signaling pathways in SUP-B15/RI. However, dasatinib and nilotinib showed partial resistance. Interestingly, bortezomib had no resistance. Imatinib combination with rapamycin had synergistic effect on overcoming the resistance. Altogether, over-expression of BCR-ABL1 and mdr1 gene were involved in the resistance mechanisms, and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line may be correlated with them. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinase, dasatinib, and nilotinib, not bortezomib. The combination of imatinib with rapamycin can partially overcome the resistance and blockade of the ubiquitin-proteasome can be also a promising pathway to overcome imatinib resistance.

Abstract 2135 Objective To establish an imatinib resistant Bcr-Abl positive acute lymphoblastic leukemia (ALL) cell line in vitro and to study imatibin resistance in Ph+ ALL. The reversal of the imatinib resistance by rapamycin, the second generation tyrosine kinase inhibitor and proteasome inhibitor was studied. Methods Ph(+) ALL SUP-B15 cell line was cultured in gradually increasing concentrations of imatinib to generate the imatinib resistant cell line at 6 μM imatinib. The cytotoxic effect of imatinib and other drugs was analyzed by MTT assay. RT-PCR, flow cytometry, Western blot analyses of proteins, DNA sequence analysis of ABL kinase domain were used to clarify the possible mechanisms of the imatinib resistance in the SUP-B15/RI cell line. Results We established the imatinib resistant Ph+ ALL cell line. The fusion bcr-abl gene was 6.1 times as high as that of the parental sensitive cell, and the mdr1 gene also increased 1.7 times in SUP-B15/RI cell line by the RT-PCR detection. However, the expression of hoct1 Abcl–2 and topoIIα gene were no difference between two cell lines by the RT-PCR detection. A K362S point mutation in the Abl kinase domain of SUP-B15/RI was found. The detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κBA JNK and STAT showed the expression of PTEN and 4EBP-1 was down-regulated, AKT, mTOR and P70S6K was up-regulated and the expression of other cell signaling pathways in SUP-B15/RI was similar to its parental sensitive cell line. Dasatinib, nilotinib, and bortezomib could inhibit proliferation of SUP-B15/RI cells at nM concentration. SUP-B15/RI cell line also showed partial resistance to dasatinib and nilotinib, but not bortezomib. The combination of imatinib with rapamycin had synergistic effect to the resistance cell line. Conclusion In vitro, we establish imatinib resistant Ph + ALL cell line. Overexpression of bcr-abl and mdr1 gene, K362S point mutation in ABL kinase domain and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line were involved in the resistance mechanisms. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinaeses dasatinib and nilotinib,not bortezomib in vitro. However, the combination of imatinib with rapamycin can partially overcome the resistance. Blockade of the ubiquitin-proteasome could be a promising pathway to overcome resistance to imatinib. Disclosures: No relevant conflicts of interest to declare.