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K. Srtutova et al.
the time of diagnosis (Figure 7). The proposed scheme combines the data obtained from expression analyses, the data obtained from functional experiments and chromatin immunoprecipitation assays performed in cell cultures and primary cells to present a simplified model of the reg- ulatory interactions in CML hematopoiesis.
Based on our data and that from the literature, we pro- pose a model illustrating relatively lower levels of MYC and higher levels of miR-150 in normal CD34+ and CD34– cells (Figure 7A and B) compared with those observed in corresponding leukemic cells. As the binding of MYC to the regulatory regions of MIR150 is weak in normal CD34+ cells (i.e. slightly above background) (Figure 5), the model assumes no inhibition of miR-150 expression by MYC in CD34+ cells to ensure the physiological levels of miR-150 required for the early stages of hematopoiesis. MYC transcription has previously been shown to be repressed by PU.1.24 MYB levels are high in the normal CD34+ cells, suggesting that MYB is required during the early stages of hematopoiesis;11,12 however, MYB levels are subsequently sharply reduced by abundant miR-150 and, supposedly, transcriptional repression by PU.125 in CD34– cells (Figure 7B). The miR-155 levels were lower in healthy CD34+ cells (compared with those in the corre- sponding leukemic subpopulation), allowing for the prop- er expression of its targets MYB and PU.1 in healthy hematopoietic stem and progenitor cells. Indeed, PU.1 expression in the normal cells was further increased in the more mature subpopulation, which is consistent with its requirement for proper myeloid lineage development and differentiation.26 In summary, the model proposes the existence of few 'core' interactions between the studied molecules ensuring the strict regulation of MYC and MYB expression during normal hematopoietic cell differentia- tion.
In contrast, the suggested model of interactions among the studied molecules in CD34+ and the more differentiat- ed CD34– CML cells (Figure 7C and D) is characterized by the establishment of several aberrant connections caused by BCR-ABL1 activity, resulting in the consistently increased expression of MYC and MYC occupancy in the
MIR150 regulatory regions in CML. As a consequence, the expression levels of miR-150 and PU.1 were lower than those observed in the corresponding cell populations from healthy controls. Due to the repressed expression of miR- 150, we would expect MYB overexpression in CML CD34+ cells compared with the expression in normal CD34+ cells, which was not demonstrated. Decreased lev- els of MYB may be explained by the increased levels of miR-155 targeting MYB in leukemic CD34+ cells.27 Moreover, miR-155 overexpression in CML CD34+ cells suggests the presence of inhibitory effect on the expres- sion of its target PU.1.15
The significantly higher expression of MYC in concert with the BCR-ABL1 activity resulted in downregulation of miR-150 and, consequently, in higher MYB expression levels in CD34– cells compared with the levels in cells from healthy donors.
No marked differences in miR-155 levels were observed between the leukemic and healthy cells in the CD34– sub- population, and the role of miR-155 is uncertain in these more differentiated cells. The trend for increased PU.1 lev- els and decreased MYB and BCR-ABL1 levels was found in leukemic CD34– compared with CD34+ cells, correspon- ding to the fact that the myeloid lineage development and differentiation in CML-CP is not fully arrested (Online Supplementary Figure S1A and B).
Discussion
This study provides new evidence to show that BCR- ABL1 establishes the MYC/miR-150/MYB/miR-155/PU.1 leukemic pathway in CML pathogenesis. First, we deter- mined the expression levels of these molecules in sorted cell populations from CML-CP patients. Consistent with previous reports7,8 and our prior data,10 we observed that miR-150 levels in primary CML samples were lower than those in the corresponding cells of healthy donors.
Next, we investigated the mechanism of miR-150 sup- pression in CML. Previously, the repression of miR-150 expression by MYC had been demonstrated in an immor-
AB
Figure 5. miR-150 regulation by MYC in chronic myeloid leukemia (CML) primary cells. MYC occupancy at putative regulatory loci of the MIR150 gene in separated (A) CD34+ and (B) CD34– cell subpopulations isolated from healthy donors [control (Ctrl) n=3] and CML-chronic phase (CP) patients at the time of diagnosis (CML; n=3), respectively. Separated cells from 2 healthy donors were pooled due to very low yields. The columns represent the fold change (FC) in the % of DNA input obtained in the leukemic or healthy cells compared with the non-specific IgG precipitation and equalized to 1 (dashed line). Unpaired two-tailed Student t-test was used to determine P-values. *P<0.05, **P<0.01. Error bars represent standard deviations.
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haematologica | 2018; 103(12)