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MYC inhibits miR-150 expression in CML
Despite the high efficacy of TKIs, 20-30% of CML patients develop resistance during the chronic phase (CP). The frequency of TKI resistance significantly increases as the disease transforms from the CP to fatal blast crisis, which is initially a BCR-ABL1-dependent process;2 how- ever, an established network further transforms the condi- tion to BCR-ABL1 independence, resulting in a switch to a more aggressive acute leukemia-like disease.3 Although TKI treatment can successfully ablate the tumor cell pop- ulation, it does not permanently cure CML because quies- cent CML stem cells (LSCs) are often insensitive to TKIs.4,5 CML LSCs survive and are able to re-initiate the disease after the discontinuation of TKI treatment in some patients.6
The dysregulated epigenetic mechanisms previously described in CML involve microRNAs. We and others have shown that miR-150 levels are significantly reduced in CML.7-10 miR-150 is an inhibitor of the oncogenic tran- scription factor MYB, which regulates hematopoiesis at the early progenitor levels,11 while its inappropriate levels during later stages block cell differentiation.12,13 In a mouse model of CML blast crisis, c-MYB was shown to be required for BCR-ABL1-dependent leukemogenesis.14 We previously showed that miR-150 and MYB levels are inversely related, and these levels reciprocally respond to TKI treatment.10 CML in blast crisis shares certain features of acute leukemia. MYB is an upstream factor of acute myeloid leukemia (AML) aggressiveness that positively regulates miR-155. miR-155 inhibits the tumor suppressor and pro-differentiation factor PU.1.15,16 MYB expression is directly activated by the oncogenic transcription factor MYC in murine virus-induced myeloid leukemia tumor cells.17 MYC and its partner MAX directly bind the BCR promoter and up-regulate BCR-ABL1 expression.18
The functional connections among miR-150, MYC and BCR-ABL1 and the mechanism of the MYB/miR-155/PU.1 network, which is involved in acute leukemogenesis and affects its aggressiveness, led us to evaluate their relation- ship in CML and TKI resistance.
Methods
Patients’ samples
Chronic myeloid leukemia patients were diagnosed and treated at the Institute of Hematology and Blood Transfusion in Prague (UHKT), Czech Republic, and the Marlene and Stuart Greenebaum Comprehensive Cancer Center at the University of Maryland, USA. The bone marrow (BM) samples (n=46) from the CML patients in CP (n=41) and peripheral blood mononuclear cells (PBMCs) samples (n=10) from healthy volunteers were obtained with written informed consent according to the princi- ples of the Declaration of Helsinki and approval by the UHKT Ethics Committee. The samples were collected at time of diagno- sis (n=28) and at time of TKI resistance (n=18). The therapeutic response was scored according to the European LeukemiaNet rec- ommendations.19 The response to first-line treatment was assessed after 12 months of therapy (Online Supplementary Table S1). Patients' BM samples were used for FACS sorting, and subse- quently to evaluate gene expression (Figure 1A-F and Online Supplementary Figure S1A and B) and the correlations among them (Online Supplementary Figure S1C).
Additional BM samples (n=6) from CML patients in CP (n=3) or blast crisis (n=3), and the BM samples from healthy donors (n=3), were obtained and handled according to the Ethics Committee of
the University of Maryland, USA, and used for miRNA sequenc- ing (Figure 2).
The PBMC samples from CML patients in CP at the time of diagnosis (n=3) and additional PBMC samples of healthy donors (n=3) were handled according to the Ethics Committee of the UHKT. Samples were separated into CD34+ and CD34– cells, and used in the study of MYC binding to MIR150 regulatory regions by chromatin immunoprecipitation.
Leukemic cell lines
The BCR-ABL1-positive CML cell lines K562, MEG-01 and KCL-22 and the BCR-ABL1-negative AML cell lines HL-60 and KG-1 were obtained from a publicly accessible biological resource center (Leibniz Institute - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH/DSMZ, Braunschweig, Germany). The cell lines were handled and culti- vated in appropriate medium according to the recommendations of the supplier. The K562R and KCL-22R cell lines resistant to ima- tinib were established by gradually exposing naive parental cells to increasing concentrations of imatinib in the medium (See Online Supplementary Appendix for details). The leukemic cell lines were used to measure gene expression and for functional experiments.
Statistical analysis
Statistical analyses were performed using Student t-test in MS Excel (Microsoft Corporation, Redmond, WA, USA); *P<0.05, **P<0.01 and ***P<0.001. All graphs were generated using MAT- LAB version R2015b and GraphPad (GraphPad, La Jolla, CA, USA). Correlation coefficients calculated by non-parametric Spearman tests were used to determine the positive and negative correlations. Cloud-based MeV4 software (Multiple Experiment Viewer; http://mev.tm4.org/)20 was used to visualize the correlation data. The combinatory effect of miR-150 overexpression and ima- tinib treatment on MYB expression was assessed using the Highest Single Agent approach (CIB-HSA).21
Details regarding patients and primary cell samples, cell sorting and separation, cell lines, transient transfections, siRNAs design, nucleic acid isolation, RT-qPCR assays, protein isolation and immunoblotting, miRNA RNA sequencing, cell cycle and viability analyses are described in detail in the Online Supplementary Appendix.
Results
A putative BCR-ABL1/MYC/miR-150/MYB/miR-155 regulatory pathway is activated in chronic myeloid leukemia
To outline relationships among the studied molecules, gene expression levels were evaluated in primary cells sorted according to CD34 expression (see Online Supplementary Appendix) from the BM of CML patients at the time of diagnosis (n=28) and at the time of resistance to TKIs (n=18) (Figure 1). We observed significantly lower miR-150 levels and increased MYC expression in CD34+ CML cells (P<0.0001 for miR-150 and MYC) and CD34– CML cells (P=0.0001 for miR-150; P=0.0013 for MYC) at the time of diagnosis compared with those in the CD34+ and CD34– cells of healthy donors (n=10), respectively. MYB expression was significantly down-regulated in CD34+ CML cells (P<0.0001) and significantly up-regulat- ed in CD34– CML cells (P<0.0001). miR-155 levels were significantly up-regulated in the CD34+ CML cells at the time of diagnosis (P=0.0002), while the expression of the pro-differentiation transcription factor PU.1, which is the
haematologica | 2018; 103(12)
2017