Patients with Chronic Myeloid Leukemia Show Different Modulation of MYB-Dependent Oncogenic Pathway in the Course of Hematopoietic Differentiation upon Sensitivity to the TKI Treatment

Objectives: We hypothesize that BCR-ABL activity together with low levels of miR-150 activates putative oncogenic signaling pathway MYB/miR-155/PU.1 in CML leading to a progressive blockade of cell differentiation and possibly also of resistance to TKIs.

Material and Method: Bone marrow (BM) cells of CML-CP patients at the time of diagnosis (n=10), and of treated patients either sensitive (n=8) or resistant (n=11) to TKIs, were FACS sorted for CD34 and CD38 into subpopulations representing the stages of cell differentiation. CD34+ and CD34- leukocytes from peripheral blood of healthy donors (n=10) were isolated by MACS and used as control. Levels of miR-150, BCR-ABL, MYB, miR-155 and PU.1 were determined by qRT-PCR. Expression of surface differentiation markers on BM cells was determined using flow cytometry at the time of diagnosis (n=10) and upon TKIs resistance (n=5). The effect of miR-150 overexpression and BCR-ABL inhibition was studied in CML-BC cell line K562 using nucleofection of the synthetic miR-150 and siRNA BCR-ABL (and by cell cultivation with TKI imatinib). mRNA and protein expression of BCR-ABL, MYB and miR-150 levels were determined by qRT-PCR and immunoblotting.

Results: In the groups of healthy donors and de novo diagnosed CML patients we observed significant positive or negative correlations of expression levels between studied molecules among the sorted cell subpopulations, which outlined the activity of the putative pathway BCR-ABL/miR-150/MYB/miR-155/PU.1 in CML-CP. Interestingly, in the group of patients resistant to TKI, we found that correlation between MYB and miR-155 was not observed suggesting this pathway is deregulated upon TKI resistance. Conversely, we found gain of negative correlation between expression of MYB and PU.1 in the group of patients responding to TKI suggesting that MYB and PU.1 oppose each other upon normal cell differentiation. We also observed significant differences of the surface marker phenotype profiles between BM cells of de novo diagnosed CML patients and patients resistant to TKIs treatment. While relatively differentiated (granulocyte) CD34- CD38- CD11b+ CD14- population presents about 7 % of BM cells for the group of patients at diagnosis, it comprises only 0.3 % of BM cells for resistant group (P= 0.0027) indicating that TKI resistance associates with significant blockade of myeloid differentiation.

We next utilized K562 cells showing that levels of miR-150 significantly increased after BCR-ABL inhibition by imatinib (P=0.0017) and also upon BCR-ABL knockdown with siRNA (P=0.002). This suggests that miR-150 is regulated by BCR-ABL. Similarly, MYB mRNA and protein levels were markedly decreased upon miR-150 overexpression and this was enhanced by inhibition of BCR-ABL activity with imatinib.

Conclusions: Dysregulation of BCR-ABL/miR-150/MYB/miR-155/PU.1 pathway was observed in CML stages and stage-specific cell populations in relation to TKI-sensitivity and disease progression. Our model consists of the BCR-ABL upregulating MYB both through miR-150 inhibition and by other downstream mechanisms by additive effect, thus the cooperation of miR-150 and imatinib through inhibiting MYB may represent an important barrier to CML progression. Even more our results show that regulation of miR-155 and PU.1 may play role in resistance to TKI treatment.

Supported by GAUK 178215 and by project 00023736 from the Czech Ministry of Health