ABERRANT MECHANISMS OF TELOMERE MAINTENANCE IN CHRONIC MYELOID LEUKEMIA - THE POTENTIAL ROLE OF POT1
(Abstract release date: 05/19/16)
EHA Library. Deregowska A. 06/09/16; 132632; E1083
Ms. Anna Deregowska
Contributions
Contributions
Abstract
Abstract: E1083
Type: Eposter Presentation
Background
As in most cancers, a reduction in the telomere length is one of the features of chronic myeloid leukaemia (CML) and telomere shortening is correlated with disease progression from the chronic phase (CML-CP) to the blastic phase (CML-BP). Targeted therapy in CML with tyrosine kinase inhibitors (TKIs), such as imatinib, gives long-lasting remission in the majority of patients. However, significant proportion of patients may develop resistance and progress to advanced phases with limited therapeutic options. Majority of studies are focused on monitoring the telomere length during the progression of the disease. However the precise role of telomere-associated proteins, including shelterin complex in BCR/ABL1-mediated genomic instability in CML progression and resistance to TKIs have not been fully elucidated.
Aims
The aim of our study was to determine the role of the members of the shelterin complex in aberrant telomere maintenance mechanisms in CML progression with special focus on POT1.
Methods
We employed human BCR-ABL1-positive cells (K562), and BCR-ABL1-negative cells (HL60), as well as CD34+ primary cells isolated from peripheral blood leukocytes of CML patients at various stages of the disease (chronic phase, chronic phase TKI-resistant and blastic phase). Blood samples were taken after informed consent. The components of telomere complex were studied at the mRNA level (RT-qPCR) and protein level (Western blotting). The activity of telomerase was measured using ELISA. The mean TRF length was measured using the TeloTAGGG telomere length assay and individual telomere length using primed in-situ labelling technique. Global DNA damage was assessed using comet assay. ROS and RNS were analysed using fluorogenic probes.
Results
Initially, we confirmed with Southern blotting that the telomere shortening was positively correlated with CML progression (CML-CP in comparison to CML-BP). However, in samples from CD34+ CML-CP TKI-resistant patients in comparison to CML-CP patients, an increase in telomere length was observed. Dynamic changes in telomere length were neither associated with expression of TERT/TERC nor with enzymatic activity of telomerase in the course of the disease. Therefore, we decided to examine the potential role of shelterin complex in telomere maintenance in disease progression looking at the expression pattern of selected members of telomeric complex. We found that the expression of POT1 was significantly upregulated in CML-BP as compared to CML-CP (Fig.1). Moreover, expression of POT1 was positively correlated with BCR-ABL1 expression. To verify the hypothesis that telomeres are elongated in TKI-resistant cells, imatinib-resistant K562 cells were used. We showed that telomeres in the resistant cells were significantly longer which was associated with differential expression of shelterin complex members, including POT1. These findings suggest that actual telomere length in disease progression may change in biphasic mode and may include initial telomere lengthening (possibly by alternative telomere lengthening mechanisms) in TKI-resistant cells. We also found that the changes in telomere length in TKI-resistant cells were accompanied by the genomic changes at chromosome level and oxidative/nitrosative stress
Conclusion
In conclusion, we postulate that abnormal expression of members of the shelterin complex, such as POT1, may be responsible for aberrant telomere maintenance mechanisms in CML cells and may play role in genomic instability associated with CML progression as well as the clonal selection and resistance to TKIs.
Session topic: E-poster
Keyword(s): CML blast crisis, Genomic instability, Telomere length, Tyrosine kinase inhibitor
Type: Eposter Presentation
Background
As in most cancers, a reduction in the telomere length is one of the features of chronic myeloid leukaemia (CML) and telomere shortening is correlated with disease progression from the chronic phase (CML-CP) to the blastic phase (CML-BP). Targeted therapy in CML with tyrosine kinase inhibitors (TKIs), such as imatinib, gives long-lasting remission in the majority of patients. However, significant proportion of patients may develop resistance and progress to advanced phases with limited therapeutic options. Majority of studies are focused on monitoring the telomere length during the progression of the disease. However the precise role of telomere-associated proteins, including shelterin complex in BCR/ABL1-mediated genomic instability in CML progression and resistance to TKIs have not been fully elucidated.
Aims
The aim of our study was to determine the role of the members of the shelterin complex in aberrant telomere maintenance mechanisms in CML progression with special focus on POT1.
Methods
We employed human BCR-ABL1-positive cells (K562), and BCR-ABL1-negative cells (HL60), as well as CD34+ primary cells isolated from peripheral blood leukocytes of CML patients at various stages of the disease (chronic phase, chronic phase TKI-resistant and blastic phase). Blood samples were taken after informed consent. The components of telomere complex were studied at the mRNA level (RT-qPCR) and protein level (Western blotting). The activity of telomerase was measured using ELISA. The mean TRF length was measured using the TeloTAGGG telomere length assay and individual telomere length using primed in-situ labelling technique. Global DNA damage was assessed using comet assay. ROS and RNS were analysed using fluorogenic probes.
Results
Initially, we confirmed with Southern blotting that the telomere shortening was positively correlated with CML progression (CML-CP in comparison to CML-BP). However, in samples from CD34+ CML-CP TKI-resistant patients in comparison to CML-CP patients, an increase in telomere length was observed. Dynamic changes in telomere length were neither associated with expression of TERT/TERC nor with enzymatic activity of telomerase in the course of the disease. Therefore, we decided to examine the potential role of shelterin complex in telomere maintenance in disease progression looking at the expression pattern of selected members of telomeric complex. We found that the expression of POT1 was significantly upregulated in CML-BP as compared to CML-CP (Fig.1). Moreover, expression of POT1 was positively correlated with BCR-ABL1 expression. To verify the hypothesis that telomeres are elongated in TKI-resistant cells, imatinib-resistant K562 cells were used. We showed that telomeres in the resistant cells were significantly longer which was associated with differential expression of shelterin complex members, including POT1. These findings suggest that actual telomere length in disease progression may change in biphasic mode and may include initial telomere lengthening (possibly by alternative telomere lengthening mechanisms) in TKI-resistant cells. We also found that the changes in telomere length in TKI-resistant cells were accompanied by the genomic changes at chromosome level and oxidative/nitrosative stress
Conclusion
In conclusion, we postulate that abnormal expression of members of the shelterin complex, such as POT1, may be responsible for aberrant telomere maintenance mechanisms in CML cells and may play role in genomic instability associated with CML progression as well as the clonal selection and resistance to TKIs.
Session topic: E-poster
Keyword(s): CML blast crisis, Genomic instability, Telomere length, Tyrosine kinase inhibitor
Abstract: E1083
Type: Eposter Presentation
Background
As in most cancers, a reduction in the telomere length is one of the features of chronic myeloid leukaemia (CML) and telomere shortening is correlated with disease progression from the chronic phase (CML-CP) to the blastic phase (CML-BP). Targeted therapy in CML with tyrosine kinase inhibitors (TKIs), such as imatinib, gives long-lasting remission in the majority of patients. However, significant proportion of patients may develop resistance and progress to advanced phases with limited therapeutic options. Majority of studies are focused on monitoring the telomere length during the progression of the disease. However the precise role of telomere-associated proteins, including shelterin complex in BCR/ABL1-mediated genomic instability in CML progression and resistance to TKIs have not been fully elucidated.
Aims
The aim of our study was to determine the role of the members of the shelterin complex in aberrant telomere maintenance mechanisms in CML progression with special focus on POT1.
Methods
We employed human BCR-ABL1-positive cells (K562), and BCR-ABL1-negative cells (HL60), as well as CD34+ primary cells isolated from peripheral blood leukocytes of CML patients at various stages of the disease (chronic phase, chronic phase TKI-resistant and blastic phase). Blood samples were taken after informed consent. The components of telomere complex were studied at the mRNA level (RT-qPCR) and protein level (Western blotting). The activity of telomerase was measured using ELISA. The mean TRF length was measured using the TeloTAGGG telomere length assay and individual telomere length using primed in-situ labelling technique. Global DNA damage was assessed using comet assay. ROS and RNS were analysed using fluorogenic probes.
Results
Initially, we confirmed with Southern blotting that the telomere shortening was positively correlated with CML progression (CML-CP in comparison to CML-BP). However, in samples from CD34+ CML-CP TKI-resistant patients in comparison to CML-CP patients, an increase in telomere length was observed. Dynamic changes in telomere length were neither associated with expression of TERT/TERC nor with enzymatic activity of telomerase in the course of the disease. Therefore, we decided to examine the potential role of shelterin complex in telomere maintenance in disease progression looking at the expression pattern of selected members of telomeric complex. We found that the expression of POT1 was significantly upregulated in CML-BP as compared to CML-CP (Fig.1). Moreover, expression of POT1 was positively correlated with BCR-ABL1 expression. To verify the hypothesis that telomeres are elongated in TKI-resistant cells, imatinib-resistant K562 cells were used. We showed that telomeres in the resistant cells were significantly longer which was associated with differential expression of shelterin complex members, including POT1. These findings suggest that actual telomere length in disease progression may change in biphasic mode and may include initial telomere lengthening (possibly by alternative telomere lengthening mechanisms) in TKI-resistant cells. We also found that the changes in telomere length in TKI-resistant cells were accompanied by the genomic changes at chromosome level and oxidative/nitrosative stress
Conclusion
In conclusion, we postulate that abnormal expression of members of the shelterin complex, such as POT1, may be responsible for aberrant telomere maintenance mechanisms in CML cells and may play role in genomic instability associated with CML progression as well as the clonal selection and resistance to TKIs.
Session topic: E-poster
Keyword(s): CML blast crisis, Genomic instability, Telomere length, Tyrosine kinase inhibitor
Type: Eposter Presentation
Background
As in most cancers, a reduction in the telomere length is one of the features of chronic myeloid leukaemia (CML) and telomere shortening is correlated with disease progression from the chronic phase (CML-CP) to the blastic phase (CML-BP). Targeted therapy in CML with tyrosine kinase inhibitors (TKIs), such as imatinib, gives long-lasting remission in the majority of patients. However, significant proportion of patients may develop resistance and progress to advanced phases with limited therapeutic options. Majority of studies are focused on monitoring the telomere length during the progression of the disease. However the precise role of telomere-associated proteins, including shelterin complex in BCR/ABL1-mediated genomic instability in CML progression and resistance to TKIs have not been fully elucidated.
Aims
The aim of our study was to determine the role of the members of the shelterin complex in aberrant telomere maintenance mechanisms in CML progression with special focus on POT1.
Methods
We employed human BCR-ABL1-positive cells (K562), and BCR-ABL1-negative cells (HL60), as well as CD34+ primary cells isolated from peripheral blood leukocytes of CML patients at various stages of the disease (chronic phase, chronic phase TKI-resistant and blastic phase). Blood samples were taken after informed consent. The components of telomere complex were studied at the mRNA level (RT-qPCR) and protein level (Western blotting). The activity of telomerase was measured using ELISA. The mean TRF length was measured using the TeloTAGGG telomere length assay and individual telomere length using primed in-situ labelling technique. Global DNA damage was assessed using comet assay. ROS and RNS were analysed using fluorogenic probes.
Results
Initially, we confirmed with Southern blotting that the telomere shortening was positively correlated with CML progression (CML-CP in comparison to CML-BP). However, in samples from CD34+ CML-CP TKI-resistant patients in comparison to CML-CP patients, an increase in telomere length was observed. Dynamic changes in telomere length were neither associated with expression of TERT/TERC nor with enzymatic activity of telomerase in the course of the disease. Therefore, we decided to examine the potential role of shelterin complex in telomere maintenance in disease progression looking at the expression pattern of selected members of telomeric complex. We found that the expression of POT1 was significantly upregulated in CML-BP as compared to CML-CP (Fig.1). Moreover, expression of POT1 was positively correlated with BCR-ABL1 expression. To verify the hypothesis that telomeres are elongated in TKI-resistant cells, imatinib-resistant K562 cells were used. We showed that telomeres in the resistant cells were significantly longer which was associated with differential expression of shelterin complex members, including POT1. These findings suggest that actual telomere length in disease progression may change in biphasic mode and may include initial telomere lengthening (possibly by alternative telomere lengthening mechanisms) in TKI-resistant cells. We also found that the changes in telomere length in TKI-resistant cells were accompanied by the genomic changes at chromosome level and oxidative/nitrosative stress
Conclusion
In conclusion, we postulate that abnormal expression of members of the shelterin complex, such as POT1, may be responsible for aberrant telomere maintenance mechanisms in CML cells and may play role in genomic instability associated with CML progression as well as the clonal selection and resistance to TKIs.
Session topic: E-poster
Keyword(s): CML blast crisis, Genomic instability, Telomere length, Tyrosine kinase inhibitor
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