During CML progression, the loss of genetic stability is manifested not only by a general increase in point mutations, DNA breakages, oxidative DNA damage, or disturbances of the epigenome, but also by changes in telomeric sequences (Boultwood et al. 1999; de Oliveira et al. 2022). The analysis of telomere length in hematopoietic (HSCs) and LSCs from the same patient shows that the average length of telomere in LSCs is much shorter, and this shortening correlates with the leukemic clone size (Bouillon et al. 2018). Moreover, according to the Hasford score, high-risk patients at diagnosis, reveal significantly greater telomere shortening rate compared to low-risk score patients, while intermediate-risk score patients exhibit an intermediate telomere shortening rate (Drummond et al. 2004). The obtained results confirm that telomeres were significantly shorter in CML-BP cells compared to cells from patients in CML-CP (Drummond et al. 2004). Thus, telomere shortening can be considered as a novel prognosis marker complementary to already established markers (Wang et al. 2014). The correlation analysis of the telomere length and the level of BCR::ABL1 expression suggests that BCR::ABL1 may induce dynamic changes in telomere length. Moreover, telomere shortening may contribute to senescence-associated inflammation and in turn disease progression in CML (Braig et al. 2014). CML cells might present nonrandom individual telomere length changes, such as shortening with different shorting rates and lengthening of telomeres located at some specific chromosome ends (Samassekou et al. 2009). It has been shown that the dynamics of individual telomere lengths might lead to telomere position effects, and in consequence inappropriate gene expression at subtelomeric regions (Koering et al. 2002). The analysis of the relationship between age of CML patients and mean length of telomeres has shown that telomere length slightly decreased with age. However, generally telomere length in CML-BP cells was still shorter than in CML-CP cells, so the age-related changes in telomere length during CML  do not seem to be the major factor responsible for telomere shortening.

In cancer cells, the lengthening of telomere sequences takes place through the reactivation of telomerase enzyme activity or alternatively through the process of recombination (Okamoto and Seimiya 2019). The decreasing effectiveness of these mechanisms leads to excessive shortening of telomeres, resulting in chromosome instability, which often leads to cellular heterogeneity related to defective mechanisms of apoptosis (Murnane et al. 2012). Furthermore, chromosome changes can promote the production of many factors that work mainly locally, for instance the cytokines or growth factors, which may lead to inflammation or tumor growth (Andriani et al. 2016). Thus, understanding the mechanisms that regulate the length of telomeres may shed light on the processes of cell selection and adaptation that occur during the development of cancer, including CML. For these purposes, it would be interesting to find out the molecular factors that control the activity of telomerase. Telomerase, as a ribonucleoprotein enzyme complex, is composed of a subunit of reverse transcriptase (TERT) and an RNA component (TERC). The activity of human telomerase is controlled on three levels, namely, on the level of transcription, the assembly of subunits into an active enzyme, as well as of direct interaction of telomerase with proteins from the telomere complex.

Our comprehensive analysis of the activity of telomerase in CML CD34+ cells does not confirm earlier observations, which pointed to changes in the activity of this enzyme depending on the phase of disease in leukocyte cells of patients with CML (Ohyashiki et al. 1997). However, it should be noted that in the aforementioned work, the authors analyzed unfractionated cells from 33 CML-CP patients and 21 CML-BP patients. The cells at CML-BP exhibited a significant increase in telomerase activity (TA) (p = 0.016) and, at the same time, a statistically significant decrease in telomere length from 6.13 ± 1.68 kb in CML-CP to 4.53 ± 0.72 kb in CML-BP at p = 0.0005). The authors did not correlate their results with the level of expression or activity of BCR::ABL1 kinase. Drummond et al. arrived at similar conclusions, showing a lack of overexpression of TERT and lowered levels of TERC expression in CD34 + cells of CML-CP patients as compared with healthy subjects (Drummond et al. 2005). They postulated that the observed increase in TA in peripheral blood cells of patients with CML may be related to a heightened proportion of cells released from bone marrow into the periphery, rather than a true increase in intracellular telomerase activity.

Nevertheless, the association of telomerase upregulation with CML progression has been reported (Keller et al. 2009). Based on these results, telomere length, at least in the context of intact cell cycle checkpoints, could represent a valuable prognostic and/or predictive biomarker for disease progression, response to TKIs, and potentially for maintenance of response upon cessation of TKI treatment.

The analysis of expression profiles of TERC and TERT in two groups of patients showed a statistically significant increase only in the expression of TERT in the CML-BP group of patients. Thereby, the obtained results do not confirm earlier observations, which point to lowered expression of TERT along with the progression of CML (Campbell et al. 2006). Campbell et al., comparing the gene expression in the CML CD34+ cells isolated from 22 CML patients’ samples to the normal CD34+ cells, showed that expression of TERT was downregulated in over half of the samples from patients in the chronic phase, significantly downregulated in two out of three patients in the accelerated phase and in all CML CD34+ cells isolated from patients in blastic phase. The same authors also postulated that lowered transcription of TERT in the CML-BP stage is associated with the levels of C-MYC, the expression of which decreased as the disease progresses. Due to these divergences, extended research in this area is required. Nevertheless, our results show that the level of expression and number of copies of TERT cannot be considered as the main cause of changes in telomere length during progression. In this context, we checked the transcriptional activity of the DKC1 gene–nucleolar protein, which is responsible for maintaining TERC stability. The role of DKC1 in the progression and development of hematopoietic and solid tumors has been already described i.e., DKC1 dysfunction leads to diminished TERC levels, a decrease in telomerase activity, and premature telomere shortening in males (Montanaro et al. 2010; Hirvonen et al. 2019). The conducted comparison between the expression of the DKC1 gene in two groups of CML CD34+ showed a significant increase (2.1-fold) of DKC1 expression in CML-BP cells in comparison with CML-CP cells. This result is in contrast with the observed decrease in the length of telomeres in CML-BP. This may suggest that DKC1 overexpression in CML cells is not related to telomerase activity. A likely explanation for this biological phenomenon is the increase of CML cancer cells’ demand for DKC1 due to its role in post-transcriptional modification of rRNA necessary for the maintenance of an effective process of translation (Ge et al. 2010; Jack et al. 2011).

Comparative analysis of the expression profile of genes of the telomere complex showed a significant increase in the expression of two genes: TNKS1 and RAP1 in CML-BP cells. Campbell et al. 2006 previously showed that the expression of telomeric-associated proteins TEP1, TRF1, TRF2, TNKS1, and PinX1 was elevated in the majority of CML-CP and CML-AP patients and decreased during disease progression, with the exception of TEP1 (Campbell et al. 2006). However, it ought to be noted that the analysis of the expression of the genes studied had not been correlated with the expression of BCR::ABL1 in individual samples, and the analysis was performed on one reference gene (B2M), which may have an impact on the obtained results, while our results were normalized to B2M and GUSB.

Moreover, contrary to the other researchers we have shown a positive correlation between increased expression of TRF2, RAP1, TTP1, TNKS1, and TNKS2 genes and the level of expression of BCR::ABL1, and also simultaneously with a decrease in the length of telomeres. Nevertheless, one ought to remember that an increase in the expression of RAP1 observed here may not be related to changes in telomeres and could be merely another form of adaptation of CML cells to increased metabolic activity characteristic for cancer cells (Deregowska and Wnuk 2021). It is well known that RAP1 is a pleiotropic protein that is responsible for the regulation of cell metabolism, the production of conditions associated with inflammation, response to oxidative stress (Cai et al. 2017) and regulation of hematopoietic stem cell survival (Khattar et al. 2019). Therefore, due to the observed correlation between increased expression of BCR::ABL1 and levels of TRF2 expression, an alternative explanation may also be found in the following scenario: the increase of expression of BCR::ABL1 during the progression of CML leads to an increase in levels of shelterin complex proteins, including the overexpression of TRF1 and TRF2, which are known to be negative regulators of telomere length, and whose binding to telomeres is dependent on posttranslational modification of the poly-ADP-ribosylate by tankyrases 1 and 2 (van Steensel et al. 1998; Smogorzewska et al. 2000; Smogorzewska and de Lange 2004). Furthermore, overexpression of TRF1 and TRF2 may promote the nucleolytic activity of XPF on chromosome endings, leading to acceleration of telomere shortening (Muñoz et al. 2005).