Contributions
Abstract: S483
Type: Oral Presentation
Presentation during EHA22: On Saturday, June 24, 2017 from 16:30 - 16:45
Location: Room N101
Background
The expression of miRNAs is regulated at transcriptional and posttranscriptional levels. Dysregulation of miRNAs could directly induce or be a consequence of oncogenic pathways. Chronic myeloid leukemia (CML) is characterized by reduced miR-150 levels leading to an insufficient repression of its target, oncogene MYB. CML treatment with imatinib normalizes miR-150 levels. Thus miR-150 is crucial for CML biology, however, little is known about its upstream transcriptional regulation. MiR-150 is an inhibitor of oncogene MYB, which is required for BCR-ABL1-dependent leukemogenesis in CML blast crisis. Our recent work brought an evidence of mutual BCR-ABL1/MYC/miR-150/MYB regulatory links in CML, sustained in CML resistant cells. We found low levels of miR-150 to be a hallmark of CML and impaired signaling pathway MYB/BCR-ABL1/MYC/miR-150/miR-155/PU.1 leading to a progressive cell differentiation block.
Aims
To delineate potential mechanisms of the miR-150 transcription regulation via the oncogenic transcription factor MYC in CML.
Methods
Primary bone marrow cells from CML (N=28), CML cell lines K562 and KCL-22 and imatinib resistant (K562R, KCL-22R). Expression analysis: RT-PCR. Protein levels: WB. ChIP: chromatin from the cell lines. SiRNA inhibition: AMAXA electroporation. DNA methylation analysis: Methylated DNA immunoprecipitation.
Results
We observed that unlike MLL-AML diagnosis (Jiang et al. 2012), CML is not characterized by a block of miR-150 maturation and that miR-150 levels negatively correlated with MYC mRNA levels in CML HSPCs (p˂0.001). Role of MYC in CML was further strengthen by imatinib induced MYC downregulation and restored miR-150 levels in K562 and KCL22. Imatinib resistance in K562R and KCL-22R was characterized by further miR-150 downregulation. To assess the MYC role on regulating miR-150 levels we tested the MYC binding sites upstream the miR-150 gene. We detected MYC binding to the upstream CpG of the MIR150 gene in K562 and KCL-22. We also found a depletion of MYC from the miR-150 locus after the imatinib treatment. We suggested potentially synergistic route for imatinib-induced BCR-ABL1 inhibition. This could be processed not only directly but also through an inhibition of a mutual positive regulatory loop between MYC and BCR-ABL1 (Xie et al. 2002). We also noticed MIR150-neighboring gene FCGRT (which is adjacent to the studied miR-150 CpG) to become activated by imatinib. We observed MYC levels dependent regulation of both genes, but FCGRT is activated by MYC. This different MYC regulatory role may be facilitated by the detected transcription factor CTCF binding to an insulator site between miR-150 promoter and the CpG. An activation of the insulator via CTCF binding changes an interaction between enhancers and promoters (Bell et al. 2000). CTCF was previously described to be an inhibitor of MYC transcription and we show CTCF transcription to be induced by imatinib. CTCF binding to DNA is prevented by DNA methylation. We did not detect DNA methylation within MIR150 upstream region.
Conclusion
Session topic: 7. Chronic myeloid leukemia - Biology
Keyword(s): Transcriptional regulation, c-Myc, Chronic myeloid leukemia, Chromatin structure
Abstract: S483
Type: Oral Presentation
Presentation during EHA22: On Saturday, June 24, 2017 from 16:30 - 16:45
Location: Room N101
Background
The expression of miRNAs is regulated at transcriptional and posttranscriptional levels. Dysregulation of miRNAs could directly induce or be a consequence of oncogenic pathways. Chronic myeloid leukemia (CML) is characterized by reduced miR-150 levels leading to an insufficient repression of its target, oncogene MYB. CML treatment with imatinib normalizes miR-150 levels. Thus miR-150 is crucial for CML biology, however, little is known about its upstream transcriptional regulation. MiR-150 is an inhibitor of oncogene MYB, which is required for BCR-ABL1-dependent leukemogenesis in CML blast crisis. Our recent work brought an evidence of mutual BCR-ABL1/MYC/miR-150/MYB regulatory links in CML, sustained in CML resistant cells. We found low levels of miR-150 to be a hallmark of CML and impaired signaling pathway MYB/BCR-ABL1/MYC/miR-150/miR-155/PU.1 leading to a progressive cell differentiation block.
Aims
To delineate potential mechanisms of the miR-150 transcription regulation via the oncogenic transcription factor MYC in CML.
Methods
Primary bone marrow cells from CML (N=28), CML cell lines K562 and KCL-22 and imatinib resistant (K562R, KCL-22R). Expression analysis: RT-PCR. Protein levels: WB. ChIP: chromatin from the cell lines. SiRNA inhibition: AMAXA electroporation. DNA methylation analysis: Methylated DNA immunoprecipitation.
Results
We observed that unlike MLL-AML diagnosis (Jiang et al. 2012), CML is not characterized by a block of miR-150 maturation and that miR-150 levels negatively correlated with MYC mRNA levels in CML HSPCs (p˂0.001). Role of MYC in CML was further strengthen by imatinib induced MYC downregulation and restored miR-150 levels in K562 and KCL22. Imatinib resistance in K562R and KCL-22R was characterized by further miR-150 downregulation. To assess the MYC role on regulating miR-150 levels we tested the MYC binding sites upstream the miR-150 gene. We detected MYC binding to the upstream CpG of the MIR150 gene in K562 and KCL-22. We also found a depletion of MYC from the miR-150 locus after the imatinib treatment. We suggested potentially synergistic route for imatinib-induced BCR-ABL1 inhibition. This could be processed not only directly but also through an inhibition of a mutual positive regulatory loop between MYC and BCR-ABL1 (Xie et al. 2002). We also noticed MIR150-neighboring gene FCGRT (which is adjacent to the studied miR-150 CpG) to become activated by imatinib. We observed MYC levels dependent regulation of both genes, but FCGRT is activated by MYC. This different MYC regulatory role may be facilitated by the detected transcription factor CTCF binding to an insulator site between miR-150 promoter and the CpG. An activation of the insulator via CTCF binding changes an interaction between enhancers and promoters (Bell et al. 2000). CTCF was previously described to be an inhibitor of MYC transcription and we show CTCF transcription to be induced by imatinib. CTCF binding to DNA is prevented by DNA methylation. We did not detect DNA methylation within MIR150 upstream region.
Conclusion
Session topic: 7. Chronic myeloid leukemia - Biology
Keyword(s): Transcriptional regulation, c-Myc, Chronic myeloid leukemia, Chromatin structure