Non-Random Telomere Lengthening at Specific Chromosome Ends as a Novel Clonal Event in Chronic Myeloid Leukemia

Abstract It is widely accepted that chromosomal telomere dysfunction caused either by telomere shortening or lesions in the capping machinery is an important factor in carcinogenesis. However, in our recent study using quantitative FISH (Q-FISH), telomere restriction fragment (TRF) analysis and fiber FISH techniques on the measurement of telomere length in 32 cases of chronic myeloid leukemia (CML), we found that telomere lengthening at some specific chromosome ends is apparently a non-random event showing a clonal nature. Methods: TRF: genomic DNA was digested by frequently cutting restriction enzymes. After gel electrophoresis and Southern blotting, the blotted DNA was hybridized to a digoxigenin (DIG)-labeled probe specific for telomeric repeats. A DIG-specific antibody covalently coupled to alkaline phosphate followed by the chemiluminescence detection was used to detect telomere signals. The quantitative measurements of mean TRF length can be reached by scanning the signals on the film and analyzing them with the computer software. Q-FISH: chromosomes on cytogenetic slides were hybridized with a peptide nucleic acid (PNA) telomere probe (Panagene, Korea). The leukemia cells can be traced by the particular chromosome rearrangement presented in the metaphase cells, e.g. t(9;22). The signal intensity, which is proportional to telomere length, of each individual telomere was automatically measured with the software of the imaging system (ISIS 2 MetaSystems, Belmont, MA). The relative telomere length was calculated using the ratio of individual signal intensity to the standard deviation. fiber FISH: fiber FISH was performed with cohybridization of a specific subtelomere probe (with a known size and close to the telomere site of interest) and the telomere probe to the DNA/chromatin fibers preparation on the same slide. Results: The telomere lengthening at short arm of a X chromosome (Xp) was the most frequent event (seen in about 50% of CML cases we studied) in leukemia cells that can be identified by the chromosome 9 and 22 translocation [t(9;22)(q34;q11.2)]. The longest telomere length at Xp end for the CML cases reached 200 kb, which is about 20-fold longer than in normal cells. The other telomeres involved in the non-random lengthening include 18p (7/32), Yp (4/32), 4q (4/32), 5p (3/32), 7q (3/32), and 15p (3/32). Conclusion: While the relationship between the sequence organization for telomere shortening and their function is relatively clear, it has not been well stated if telomere lengthening at specific chromosome ends could be involved in cell proliferation, in maintenance of the genome stability and in evolution of the cancer. Our findings have shown the first evidence that the telomere lengthening at some specific chromosome ends is such a salient clonal event. Further investigation on picking up specific individual telomere lengthening in leukemia cells would greatly aid studies of chromosomal stability, telomerase activity, proliferative capacity and the evaluation of clinical status and thus one can use telomere length as an indicator to follow-up the cancer progression, to evaluate the treatment efficacy and to predict the prognosis in cancer.

Download Full-text

Role of Shelterin Complex and Alternative Telomere Lengthening in Genomic Instability and Disease Progression in Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.1880.1880 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1880-1880

Author(s):

Tomasz Stoklosa ◽

Anna Deregowska ◽

Katarzyna Pruszczyk ◽

Iwona Solarska ◽

Marcin M Machnicki ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Telomere Length ◽

Genomic Instability ◽

Myeloid Leukemia ◽

Somatic Mutations ◽

Telomere Maintenance ◽

Telomere Shortening ◽

Shelterin Complex ◽

Telomere Lengthening

Abstract Genomic instability has many sources, among others, shortening of telomeres, nucleoprotein complexes located at the ends of chromosomes. Tumor cells have aberrant mechanisms of telomere maintenance: their telomeres are shortened, no longer preventing chromosome end-to-end fusion and recombination, but frequently not short enough to lead to cell senescence. Both telomerase and shelterin complexes are involved in telomere homeostasis. Reduction in the telomere length is considered as one of the features of chronic myeloid leukemia (CML) similar to other human malignancies and telomere shortening is correlated with disease progression from the chronic phase (CML-CP) to the blastic phase (CML-BP)1. However, recent report shows that shorter telomeres can actually be detected in patients who discontinued imatinib and are in treatment-free remission as compared to those who relapsed2. Therefore, there is no agreement on the telomere length dynamics in CML evolution. Moreover, the precise role of telomere-associated proteins, including shelterin complex in BCR-ABL1-mediated genomic instability in CML progression and resistance to TKIs, is not fully elucidated. Initially, we confirmed that the telomere shortening was positively correlated with CML progression (CML-BP in comparison to CML-CP). However, in CD34+ samples from CML-CP TKI-resistant patients in comparison to CML-CP patients, an increase in telomere length was observed. This suggests that shortening of telomeres in CML progression may have a biphasic scenario. This can be explained by alternative telomere lengthening (ALT) mechanisms, since no significant changes in the expression of subunits of the telomerase complex and its enzymatic activity were observed at different phases of the disease; enzymatic activity of telomerase was measured immunoenzymatically, while length of telomeres was determined by Southern blotting. Then we decided to analyze possible involvement of shelterin complex and of ALT mechanisms in CML progression. Importantly, expression of the three members of the shelterin complex, Protection Of Telomeres 1 (POT1), Repressor Activator Protein 1 (RAP1) and Tankyrase 1 (TNKS1) was significantly upregulated in CML-BP (10 samples) as compared to CML-CP (15 samples) and was also positively correlated with BCR-ABL1 expression. Moreover, as determined by TKI treatment of CD34+ CML-BP primary cells, expression of POT1 was BCR-ABL1-dependent. No significant changes were observed in the expression of other members of the shelterin complex, namely TINT1-PTOP-PIP1 (TPP1), TRF1 interactor 2 (TIN2) and Tankyrase 2 (TNKS2). Also telomere repeat-binding factor 1 and 2 (TRF1 and TRF2), which are responsible for anchoring shelterin complex to the double stranded telomeric repeats remain stable in the course of the disease. Expression of subunits of telomerase and shelterin complexes was examined by RT-qPCR and Western blotting. This was confirmed in K562 and K562 imatinib-resistant cell line model. Somatic mutations in POT1 have been recently described in human tumors including chronic lymphocytic leukemia (CLL). In CLL, mutations in POT1 affect telomere stability and are associated with shorter survival in patients receiving chemotherapy as a frontline treatment. We have screened our NGS data from targeted sequencing in a cohort of patients who progressed to CML-BP (paired CP and BP samples, n=10 and BP samples, n=9) but we did not detect any somatic mutations in POT1. This is in accordance with our data on POT1 upregulated expression and suggests that dysregulation of shelterin complex during progression of CML differs significantly from CLL. In conclusion, we present the first comprehensive analysis of the expression of all members of the shelterin complex in the course of CML. We postulate that abnormal expression of selected members such as POT1, RAP1 and TNKS1 may be responsible for the aberrant telomere maintenance mechanisms in CML cells and may play an important role in genomic instability associated with CML progression. References: 1. Brummendorf TH, et al. Blood 2000; 95:1883-1890. 2. Caocci et al. Journal of Hematology & Oncology 2016; 9:63; Disclosures Seferynska: Novartis: Consultancy, Honoraria.

Download Full-text

Prognostic implications of differences in telomere length between normal and malignant cells from patients with chronic myeloid leukemia measured by flow cytometry

Blood ◽

10.1182/blood.v95.6.1883 ◽

2000 ◽

Vol 95 (6) ◽

pp. 1883-1890 ◽

Cited By ~ 140

Author(s):

Tim H. Brümmendorf ◽

Tessa L. Holyoake ◽

Nathalie Rufer ◽

Michael J. Barnett ◽

Michael Schulzer ◽

...

Keyword(s):

Flow Cytometry ◽

Chronic Myeloid Leukemia ◽

T Lymphocytes ◽

Telomere Length ◽

Myeloid Leukemia ◽

Philadelphia Chromosome ◽

Surrogate Marker ◽

Chronic Phase ◽

Telomere Shortening ◽

Normal Individuals

Abstract Chronic myeloid leukemia (CML) is a clonal, multilineage myeloproliferative disorder characterized by the Philadelphia chromosome (Ph) and a marked expansion of myeloid cells. Previous studies have indicated that the telomere length in blood cells may indicate their replicative history. However, the large variation in telomere length between individuals complicates the use of this parameter in CML and other hematologic disorders. To circumvent this problem, we compared the telomere length in peripheral blood or bone marrow cells with purified normal (Ph−) T lymphocytes from the same CML patient using fluorescence in situ hybridization and flow cytometry. Overall telomere fluorescence was significantly reduced in Ph+ cells from patients with CML compared to blood leukocytes from normal individuals (P < 0.001) or normal (Ph−) T lymphocytes from the same individuals (n = 51, P < 0.001). Cells from patients in accelerated phase or blast phase (AP/BP) showed significantly shorter average telomere length than cells from patients in chronic phase (CP,P = 0.02) or cytogenetic remission (CR,P = 0.03). Patients in CP who subsequently developed BP within 2 years had significantly shorter telomeres than those who did not develop BP for at least 2 years (P < 0.05). Accelerated replication-dependent telomere shortening in Ph+ versus Ph− leukocytes supports previous evidence that Ph+ stem cells cycle more actively than their counterparts in normal individuals. Our data further suggest that telomere shortening may serve as a surrogate marker of disease progression in patients with CP CML, supporting a mechanistic link between CML stem cell turnover, genetic instability, and malignant evolution in this disease. (Blood. 2000;95:1883-1890)

Download Full-text

Characterization of Bcr-Abl Positive Leukemic Cells under Long-Term In Vitro Treatment with Telomerase Inhibitor BIBR1532.

Blood ◽

10.1182/blood.v108.11.2185.2185 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2185-2185

Author(s):

Ute Brassat ◽

Stefan Balabanov ◽

Ulrike Hartmann ◽

Daniel Rössler ◽

Kerstin Borgmann ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Chromosome 9 ◽

Leukemia Cell Line ◽

Telomere Shortening ◽

Telomerase Inhibition ◽

Telomerase Inhibitor

Abstract In normal somatic cells telomeres shorten with each cell division because of the end-replication problem. The ribonucleoprotein enzyme telomerase is able to prevent replicative telomere shortening and to maintain or elongate telomere length. In 90 % of tumour cells the enzyme telomerase is found to be upregulated. Chronic myeloid leukemia is a disorder characterized by a reciprocal translocation between Chromosome 9 and 22, leading to the so called Philadelphia chromosome harbouring the BCR-ABL translocation. BCR-ABL positive leukemic stem cells are characterized by increased turnover leading to accelerated telomere shortening as opposed to their normal counterparts. It is unclear to date whether accelerated telomere shortening in Bcr-Abl-positive cells is linked to genetic instability eventually leading to the acquisition of secondary clonal events that might propagate acceleration of the disease to blast crisis. Therefore we aimed to characterize Bcr-Abl positive chronic myeloid leukemia cell line K562 with or without inhibition of telomerase activity under long-term culture conditions. K652 cells were expanded for 400 populations doublings (PD) with or without treatment with the small molecule telomerase inhibitor BIBR1532 in vitro. While telomeres in untreated control cells remained relatively constant, telomeres in BIBR1532 treated cells underwent replicative shortening from 10 kb to 3 kb (as measured by flow FISH), reflecting a rate of 22 base pairs (bp) lost per PD. No difference in growth kinetics were observed until that stage. We next characterized treated K562 with short telomeres (K562-S) in contrast to control cells with long telomeres (K562-L) for the expression of telomere and telomerase-binding proteins. No difference in mRNA expression for any of the candidate proteins were observed by RT-PCR. Comparative analysis of global protein expression was performed by 2D gel electrophoresis. Taken together, 23 protein spots were found to be differentially expressed between treated and untreated cells, fifteen of which were already identified by mass spectometry. Additionally, we analysed the cells for the acquisition of additional cytogenetic abnormalities by M-FISH. Interestingly, in this ongoing study, we consistently found acquisition of genetic material on chromosome 7 in treated as compared to untreated cells. To study radiation sensitivity under BIBR1532 treatment, K562 cells were exposed to increasing doses of irradiation. Interestingly, despite of a dose-dependent increase in the fraction of apoptotic cells in the pre-treated as opposed to control cells, no accumulation in the number of double strand breaks or lethal aberrations were detected. Interestingly, telomere shortening after telomerase inhibition translated to increased sensitivity to Imatinib (IC50 0.6 μM vs. IC50 1.2 μM). Taken together, telomerase inhibition represent a attractive new therapeutic strategy in Bcr-Abl positive leukemias. However, careful evaluation of side effects need to be studied on the proteomics and cytogenetic level.

Download Full-text

Sensitivity to Imatinib In BCR-ABL1-Positive Chronic Myeloid Leukemia Cells Depends on the Presence of Normal ABL1.

Blood ◽

10.1182/blood.v116.21.3405.3405 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3405-3405

Author(s):

Yashodhara Dasgupta ◽

Anna Virgili ◽

Mateusz Koptyra ◽

Elisabeth P. Nacheva ◽

Tomasz Skorski

Keyword(s):

Cell Cycle ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Myeloid Leukemia ◽

Chromosome 9 ◽

Leukemia Cells ◽

Wild Type ◽

Tki Resistance ◽

Tki Treatment

Abstract Abstract 3405 Background: BCR-ABL1 encoding for oncogenic tyrosine kinase results from t(9;22)(q34;q11) reciprocal translocation or variants generating the Philadelphia chromosome (Ph), which initiates chronic myeloid leukemia in chronic phase (CML-CP). The second (wild-type) ABL1 and BCR alleles in CML-CP cells remain intact on the non-rearranged homologues of chromosome 9 and 22, respectively. Accordingly, CML-CP cells at early stages express both forms of the ABL1 kinase, oncogenic BCR-ABL1 and normal ABL1. ABL1 tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib revolutionized the treatment of CML-CP, but they do not eradicate leukemia. Patients who do achieve complete cytogenetic remission (CCyR) may eventually stop responding and acquire resistance to TKIs, which may lead to disease relapse and malignant progression. Mutations in the sequence encoding BCR-ABL1 kinase have been detected in approximately 50% of CML-CP patients resistant to TKIs, but other factors contributing to this phenomenon are poorly characterized. Results: Here, we identified a novel mechanism of TKI resistance: loss of the remaining normal ABL1 allele resulting from cryptic deletion in the 9q34 region in the normal chromosome 9 [del(9)(q34)]. Using bacterial artificial chromosome probes (BACs) for dual color/dual probe fluorescent in situ hybridization (D-FISH), and oligonucleotide array comparative genomic hybridization (aCGH) we show that genomic deletion of ABL1 allele in non-translocated chromosome 9 acquired during TKI therapy in CML-CP patients was associated with resistance to imatinib and dasatinib. del(9)(q34) was detected in approximately 10% of the patients who initially failed to achieve CCyR within 12 months of TKI treatment. Moreover, BCR-ABL1-positive Abl1-/- murine leukemia cells were refractory to imatinib in comparison to BCR-ABL1–positive Abl1+/+ counterparts as indicated by persistent BCR-ABL1 –mediated tyrosine phosphorylation, lack of BCR-ABL1 protein degradation, increased cell survival and clonogenic activity. Expression of exogenous ABL1 kinase in BCR-ABL1–positive Abl1-/- cells restored their sensitivity to imatinib. These results provide direct evidence that Abl1 plays a crucial role in regulation of the sensitivity of BCR-ABL1-positive leukemia cells to imatinib. ABL1 is regarded as a cell cycle regulatory and pro-apoptotic protein, thus antagonistic to BCR-ABL1. The cell cycle inhibitory activity is independent of ABL1 kinase, whereas the pro-apoptotic function is dependent on its kinase activity. However, acquired resistance to TKIs caused by a loss of the wild-type ABL1 kinase does not appear to depend directly on the lack of ABL1-induced cell cycle arrest and/or apoptosis. In contrast to BCR-ABL1 –positive Abl1+/+ leukemia cells, imatinib exerted only modest effect on BCR-ABL1 kinase-dependent tyrosine phosphorylation and did not downregulate BCR-ABL1 protein in Abl1-/- leukemia cells, suggesting the role of Abl1 in cellular uptake of the drug and/or BCR-ABL1 degradation. Expression of imatinib cellular importer Oct-1 and cellular exporters Abcb1 and Abcg2 does not appear to favor the resistance to TKI in BCR-ABL1-positive Abl1-/- cells, but the impact of ABL1 on intracellular metabolism of imatinib cannot be excluded. In addition, expression of chaperone protein Hsp90, which protects BCR-ABL1 from proteasomal degradation, is not affected by Abl1. However, >3-fold downregulation of cathepsin B may be responsible for lack of degradation of BCR-ABL1 protein in imatinib-treated Abl1-/- cells. Conclusions: Altogether, it can be postulated that loss of expression of ABL1 kinase plays an important role in TKI resistance in CML. It can be achieved by interstitial deletion in chromosome 9 [del(9)(q34)] causing loss of normal ABL1 allele, which could be eventually combined with epigenetic silencing of the alternative ABL1 promoter retained in the CpG island of the BCR-ABL1 gene in t(9;22)(q34:q11). Detection of the del(9)(q34) is beyond the resolution of conventional karyotyping currently used to monitor TKI treatment response. In contrast, D-FISH using commercially available probes can identify such loss in both quiescent and dividing cells. In summary, downregulation of ABL1 caused by del(9)(q34) may serve as an important prognostic factor and have a significant impact on CML treatment. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Faculty Opinions recommendation of Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.12652957.13903055 ◽

2011 ◽

Author(s):

Catriona Jamieson ◽

Anil Sadarangani

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemia Cells ◽

Quantitative Phosphoproteomics

Download Full-text

THE CASE VARIANT TRANSLOCATION T(3;9;22)(P24;Q34;Q11) IN CHRONIC MYELOID LEUKEMIA

Problems in oncology ◽

10.37469/0507-3758-2018-64-6-810-814 ◽

2018 ◽

Vol 64 (6) ◽

pp. 810-814

Author(s):

Kodirzhon Boboev ◽

Yuliana Assesorova ◽

Kh. Karimov ◽

B. Allanazarova

Keyword(s):

Adverse Effect ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Genetic Locus ◽

Philadelphia Chromosome ◽

Chromosome 9 ◽

Banding Technique ◽

Variant Translocation

This paper presents a case of chronic myeloid leukemia with an earlier unknown variant translocation t (3; 9; 22) (p24; q34; q11) detected by cytogenetic research using the GTG-banding technique. Despite the absence of the classical Philadelphia chromosome, the presence of chromosome 9 and 22 derivatives, as well as the BCR-ABL fusion gene, allow this translocation to be considered pathogenetic for CML. A good response of the patient to the treatment with glivec is that there is no adverse effect on the pathogenesis of the disease of an additional genetic locus (3p24) involved in complex restructuring.

Download Full-text