The Preliminary Study Of ARNO Mediated BCR-ABL-ARF6 Signaling Pathway And The Promotion Of Chronic Myeloid Leukemia Cell Drug Resistance

Chronic myeloid leukemia(CML) is a clonal hematologic malignancy which originates from pluripotent hematopoietic stem cell, accounting for 20% of adult leukemia. About 95% of CML patients were caused by BCR-ABL fusion gene, which results from the 9th and 22 nd chromosome translocation, encoding a 210 k Da constitutively activated tyrosine kinase which further induces the occurrence of CML. For CML therapies, imatinib, targeting the BCR-ABL kinase, has achieved good therapeutic effect, which can prolong the survival time of CML patients and has replaced bone marrow transplantation to be the preferred treatment options. However, imatinib can not cure CML, because its long-term application easily leads to gene mutation of BCR-ABL and enhances drug resistance. More importantly, Imatinib can not eliminate leukemia stem cells(LSCs) and will results in the relapse of disease, so to search the other drug targets except BCR-ABL kinase and to completely remove LSCs will be a key point to cure CML. However, imatinib does not cure CML. Long term administration of imatinib easily lead to BCR-ABL gene mutation and the emergence of drug resistance, which drives CML in chronic phase into the accelerated phase, or to acute myeloid leukemia(AML) even more difficult to control. Therefore, it is of great significance to clarify the mechanisms of drug resistance in CML and to find new drug targets for the future diagnosis and treatment of CML.ADP-ribosylation factors(ARFs), comprising of a conserved family of proteins of the Ras superfamily, are highly conserved GTP binding proteins and widely expressed in eukaryotic cells. Our previous experiments showed that reduced expression of ARF6 protein by impression of Arf6 gene had a significant impact on the proliferation and drug resistance of K562 cells. Therefore, we speculated that ARF6 plays a key role in BCR-ABL induced CML. ARF nucleotide-binding-site opener(ARNO) is the most frequently reported GEF protein which can catalysis ARF6 protein. We detected the required expression amount of GEFs protein for ARF6 activation in K562 cell by RT-PCR. The expression of ARNO was significantly higher than that of other 3 GEFs proteins(Cytohesin 1, Grp1 and Cytohesin 4), suggesting that it might be ARNO that plays the catalytic effect in CML. According to the results of preliminary experiments and previous literatures worldwide, we have speculated and summarized the upstream and downstream signal transduction pathways of ARF6. BCR-ABL promotes ARF6 activation by ARNO phosphorylation, therefore regulates the expression of downstream target gene(c-myc, cyclin B1, p21, MDR1 and TERT, etc.) and promotes the proliferation and drug resistance of CML cells. In order to test this hypothesis, four eukaryotic expression vectors bearing the mutant ARNO(Y6F, Y326 F, Y381 F, E156K) were constructed and transfected into 293 T cells. The molecular mechanisms of ARF6 activation promoted through BCR-ABL mediated ARNO phosphorylation was explored by CO-IP and western blot. Moreover, K562 cell was also treated with Secin H3(ARNO inhibitor) to detect the effect of ARNO on drug resistance of K562 cell.Method: ARNO-mutant expression vectors were constructed using point mutation kit and co-transfected 293 T cells with BCR-ABL expression vector. 2. The phosphorylation level of ARNO and the expression level of ARF6-GTP were detected by CO-IP and western blot. 3. After treated with Secin H3, K562 cells were detected by flow cytometry to determine the effect of ARNO on the drug resistance phenomenon.Results: 1. Results of western blot and CO-IP showed that, among the three ARNO-mutant vectors, Y326 F presented low phosphorylation level of ARNO and low Arf6-GTP expression level, which was similar to that of the negative control E156 K. 2. Flow cytometry showed that Secin H3 treatment significantly increased the apoptosis rate of K562 cells, suggesting a decreased drug resistance to imatinib.Conclusion: BCR-ABL can promote the phosphorylation of the 326 th tyrosine of ARNO and further stimulate the activation of ARF6; ARNO plays a significant role in the process of drug resistance of K562 cells.