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K. Srtutova et al.
vation by MYC and the post-transcriptional inhibition of miR-150 maturation by MYC-driven Lin28 described using in vitro and ex vivo AML models with MLL rearrangements.29 However, our data did not reveal the repression of an miR- 150 maturation blockade following the BCR-ABL1 inhibi- tion in CML cells. The specific regulatory mechanisms of miR-150 expression were identified in CML, as we identi- fied MYC-binding loci neighboring the MIR150 TSS in K562 and KCL-22 cells, while there was no MYC binding in AML HL-60 cells. Recently, MYC was reported to exert opposite effects on the expression of a specific gene depending on the local epigenetic pattern;30 this would be interesting to investigate for the MIR150 gene given the presence of a CpG island near the -11.7 kb locus.
Together our data suggest that BCR-ABL1 inhibits miR- 150 expression in CML cells via the transcriptional activa- tion of MYC and its simultaneous recruitment to a specific -11.7 and -0.35 kb loci of the MIR150 gene, where MYC binds and acts as a direct repressor of miR-150 transcrip- tion (Figure 7). This conclusion is reliable since MYC has recently been found to act with p53 as a key CML regula- tor to maintain CML LSC survival.31
Furthermore, our data indicated that low miR-150 levels drive MYB expression in CD34– CML cells and hinder cell differentiation,9 which is consistent with other reports32,33 and our finding regarding low PU.1 expression in the pri- mary CML–CP CD34+ populations and CD34– CML blast cell lines. Our data showed a significant downregulation of PU.1 following imatinib treatment in K562 cells (a model of erythroid lineage), while the opposite effect on PU.1 expression was observed following imatinib treat- ment in KCL-22 cells (a model of myeloid lineage), likely reflecting the distinct nature of these cell lines. We hypothesize that BCR-ABL1 inhibition generally relieves blocked cell differentiation by manipulating PU.1 levels because either low or high levels of PU.1 are required for the terminal differentiation of K562 (erythroblasts) and KCL-22 (myeloblasts) cells, respectively. Conversely, both PU.1 upregulation in the K562R imatinib-resistant cells and PU.1 downregulation in the KCL-22R imatinib-resis- tant cells, together with reduced miR-150 levels, may impose a differentiation block, similar to that described in a murine erythroleukemia (MEL) cell model.34
In addition, we found that miR-155 levels in the CD34+ CML-CP cells from patients at diagnosis and with TKI
resistance were higher than those in healthy CD34+ cells. miR-155 was previously identified to be an oncogenic miRNA that is up-regulated in a variety of malignancies.35- 37 Notably, the sustained miR-155 overexpression in the CML was associated with the induction of myeloid disor- der in mice.38 However, the inhibition of BCR-ABL1 activ- ity by imatinib increased miR-155 levels in the KCL-22 and K562 cells, which is consistent with a previous report showing miR-155 upregulation in leukocytes from CML patients following prolonged imatinib treatment.39 The functionally distinct, dose-dependent effects of miR-155 expression have been recently described in AML, high- lighting the importance of the cell context and fine regula- tion in assessing its role in leukemogenesis.40
In summary, we propose a leukemic network model including a novel mechanism of BCR-ABL1-dependent recruitment of MYC oncoprotein to bind and inhibit MIR150 gene expression in CML cells. Compared with healthy cells, the CD34– leukemic cells showed that down- regulation of miR-150 expression by MYC resulted in sig- nificantly higher MYB levels. miR-150 expression is reduced at the time of TKI resistance and further dimin- ished in resistant CML cell lines, emphasizing the increased aggressiveness of the disease as well as the links between TKI resistance and disrupted cell differentiation. The key connecting nodes of the described leukemic net- work established by aberrant activity of BCR-ABL1 may serve as potential druggable targets, as was recently shown for the transcription factor MYC31 to overcome the resistance of CML LSCs.
Funding
This study was supported by the Ministry of Education Youth and Sports grant project MSMT LH15104, Charles University grant GAUK 178215, GACR 18-18407S, Czech Ministry of Health project no. 00023736 for the conceptual development of a research institute, Charles University Research Centers grant UNCE/MED/016 and program Progres Q26 and NCI-NIH grant CA163800.
Acknowledgments
We thank Prof. Jianjun Chen, Section of Hematology/ Oncology, Department of Medicine, University of Chicago, Chicago, IL, USA for providing the primer sequences for pri-miR-150 and pre-miR-150 transcript quantification.
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