Contributions
Type: Oral Presentation + travel grant
Presentation during EHA20: From 13.06.2015 11:45 to 13.06.2015 12:00
Location: Room Stolz 1
Background
Chronic Myeloid Leukemia(CML) is caused by the BCR/ABL fusion gene. Both the presence and the levels of BCR/ABL expression seems critical for CML progression from Chronic phase(CP) to Blast crisis(BC). At the molecular level BC is a heterogeneous disease. Regardless of the additionally secondary changes, one common feature during the evolution from CP to BC is a marked increase in BCR/ABL expression. After the oncogenic translocation, the BCR/ABL gene is under the transcriptional control of BCR promoter but the molecular mechanisms involved in the regulation of oncogene expression are mostly unknown.
Aims
The aim of this study is the identification of molecular mechanisms responsible for BCR promoter regulation and BCR/ABL expression.
Methods
To identify the transcription factors directly acting on BCR promoter, we studied in-silico a region of 1443bp upstream of the human BCR gene coding sequence(Gene ID:613). Chromatin Immunoprecipitation was used to confirm in-silico data. The role of transcription factors on BCR and BCR/ABL expression have been determined through transfection of the K562 cell line with selected expression vectors and through specific silencing in K562, LAMA-84 and KCL-22 cells. mRNA and protein expression levels of BCR and BCR-ABL were detected by quantitative PCR(RT-qPCR) and western blot. BCR reporter activity was also analyzed by reporter luciferase assay in the 293T cell line. The role of the identified transcription factors in CML progression was investigated with in-vitro experiments.
Results
These data demonstrate that MYC-MAX heterocomplex binds to the BCR promoter at four different binding sites(PBS1-4), leading to up-regulation of BCR and BCR/ABL at both transcriptional and protein levels in the K562 cells. We found that both MYC and MYC-MAX overexpression significantly upregulate BCR/ABL compared to control cells, as assessed by RT-qPCR (MYC_BCR/ABL fold induction: 2.27±0.48,p=0.01; MYC-MAX_BCR/ABL fold induction: 2.55±0.34,p=0.002) and western blot. Accordingly, silencing of MYC(shMYC) expression in various BCR/ABL+ cell lines causes significant down-regulation of BCR and BCR/ABL compared to the negative controls(BCR fold change: KCL-22shMYC 0.49 ± 0.07, p=0.013; LAMA-84shMYC 0.07 ± 0.03, p=0.0003. BCR/ABL fold change: KCL-22shMYC 0.58 ± 0.02, p=0.0007; LAMA-84shMYC 0.38 ± 0.006, p<0.0001). In addition, MYC silencing in these cell lines leads to decreased proliferation and induction of cell death as assessed by cell cycle analysis and PARP-1 cleavage. In order to assess the effect of MYC-MAX on BCR promoter, we tested the activity of the luciferase reporter assay in presence of MYC expression and in a lentiviral MYC silencing model, showing that MYC silencing significantly decreases BCR promoter activity in all of the constructs analysed(p<0.0001). Notably, deletion of PBS1 and PBS2 and/or deletion of PBS3 and PBS4, dramatically decreases the promoter strength, therefore confirming the critical role of these region in controlling BCR promoter activity. Interestingly, the deletion of PBS3 and PBS4 induces a greater down-modulation of luciferase activity compared to PBS1 and PBS2 deletion (BCRpromoter: 6.11± 0.11; PBS3-4deleted: 1.86±0.14; PBS1-2deleted: 3.92±0.07).
Summary
This is the first description of a new pathway which places BCR and BCR/ABL under the transcriptional control of the MYC-MAX heterodimer. Since MYC is frequently over-expressed in BC, this phenomenon could play a critical role in BCR/ABL up-regulation and blast aggressiveness acquired during CML evolution.
Keyword(s): BCR-ABL, MYC
Session topic: Novel actors in chronic myeloid leukemia biology
Type: Oral Presentation + travel grant
Presentation during EHA20: From 13.06.2015 11:45 to 13.06.2015 12:00
Location: Room Stolz 1
Background
Chronic Myeloid Leukemia(CML) is caused by the BCR/ABL fusion gene. Both the presence and the levels of BCR/ABL expression seems critical for CML progression from Chronic phase(CP) to Blast crisis(BC). At the molecular level BC is a heterogeneous disease. Regardless of the additionally secondary changes, one common feature during the evolution from CP to BC is a marked increase in BCR/ABL expression. After the oncogenic translocation, the BCR/ABL gene is under the transcriptional control of BCR promoter but the molecular mechanisms involved in the regulation of oncogene expression are mostly unknown.
Aims
The aim of this study is the identification of molecular mechanisms responsible for BCR promoter regulation and BCR/ABL expression.
Methods
To identify the transcription factors directly acting on BCR promoter, we studied in-silico a region of 1443bp upstream of the human BCR gene coding sequence(Gene ID:613). Chromatin Immunoprecipitation was used to confirm in-silico data. The role of transcription factors on BCR and BCR/ABL expression have been determined through transfection of the K562 cell line with selected expression vectors and through specific silencing in K562, LAMA-84 and KCL-22 cells. mRNA and protein expression levels of BCR and BCR-ABL were detected by quantitative PCR(RT-qPCR) and western blot. BCR reporter activity was also analyzed by reporter luciferase assay in the 293T cell line. The role of the identified transcription factors in CML progression was investigated with in-vitro experiments.
Results
These data demonstrate that MYC-MAX heterocomplex binds to the BCR promoter at four different binding sites(PBS1-4), leading to up-regulation of BCR and BCR/ABL at both transcriptional and protein levels in the K562 cells. We found that both MYC and MYC-MAX overexpression significantly upregulate BCR/ABL compared to control cells, as assessed by RT-qPCR (MYC_BCR/ABL fold induction: 2.27±0.48,p=0.01; MYC-MAX_BCR/ABL fold induction: 2.55±0.34,p=0.002) and western blot. Accordingly, silencing of MYC(shMYC) expression in various BCR/ABL+ cell lines causes significant down-regulation of BCR and BCR/ABL compared to the negative controls(BCR fold change: KCL-22shMYC 0.49 ± 0.07, p=0.013; LAMA-84shMYC 0.07 ± 0.03, p=0.0003. BCR/ABL fold change: KCL-22shMYC 0.58 ± 0.02, p=0.0007; LAMA-84shMYC 0.38 ± 0.006, p<0.0001). In addition, MYC silencing in these cell lines leads to decreased proliferation and induction of cell death as assessed by cell cycle analysis and PARP-1 cleavage. In order to assess the effect of MYC-MAX on BCR promoter, we tested the activity of the luciferase reporter assay in presence of MYC expression and in a lentiviral MYC silencing model, showing that MYC silencing significantly decreases BCR promoter activity in all of the constructs analysed(p<0.0001). Notably, deletion of PBS1 and PBS2 and/or deletion of PBS3 and PBS4, dramatically decreases the promoter strength, therefore confirming the critical role of these region in controlling BCR promoter activity. Interestingly, the deletion of PBS3 and PBS4 induces a greater down-modulation of luciferase activity compared to PBS1 and PBS2 deletion (BCRpromoter: 6.11± 0.11; PBS3-4deleted: 1.86±0.14; PBS1-2deleted: 3.92±0.07).
Summary
This is the first description of a new pathway which places BCR and BCR/ABL under the transcriptional control of the MYC-MAX heterodimer. Since MYC is frequently over-expressed in BC, this phenomenon could play a critical role in BCR/ABL up-regulation and blast aggressiveness acquired during CML evolution.
Keyword(s): BCR-ABL, MYC
Session topic: Novel actors in chronic myeloid leukemia biology