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.
RAD50 and RAD51 Define Two Pathways That Collaborate to Maintain Telomeres in the Absence of Telomerase
