Telomere dysfunction and fusion during the progression of chronic lymphocytic leukemia: evidence for a telomere crisis

Abstract

We performed single-molecule telomere length and telomere fusion analysis in patients at different stages of chronic lymphocytic leukemia (CLL). Our work identified the shortest telomeres ever recorded in primary human tissue, reinforcing the concept that there is significant cell division in CLL. Furthermore, we provide direct evidence that critical telomere shortening, dysfunction, and fusion contribute to disease progression. The frequency of short telomeres and fusion events increased with advanced disease, but importantly these were also found in a subset of early-stage patient samples, indicating that these events can precede disease progression. Sequence analysis of fusion events isolated from persons with the shortest telomeres revealed limited numbers of repeats at the breakpoint, subtelomeric deletion, and microhomology. Array-comparative genome hybridization analysis of persons displaying evidence of telomere dysfunction revealed large-scale genomic rearrangements that were concentrated in the telomeric regions; this was not observed in samples with longer telomeres. The telomere dynamics observed in CLL B cells were indistinguishable from that observed in cells undergoing crisis in culture after abrogation of the p53 pathway. Taken together, our data support the concept that telomere erosion and subsequent telomere fusion are critical in the progression of CLL and that this paradigm may extend to other malignancies. (Blood. 2010;116(11): 1899-1907) IntroductionNonreciprocal translocations (NRTs) are considered to be key mutational events that can drive many types of malignancy. 1 The underlying mechanisms that result in these types of events can include, among others, deficiencies in double-strand break repair, 2 mitotic checkpoints, 3,4 and telomere dysfunction. 5 Telomeres play a key role in upholding genomic integrity; in the context of DNA damage checkpoint defects, cells in culture undergo crisis and have extensive telomere erosion, chromosomal fusion, and genomic rearrangements. 6,7 NRTs, as well as localized gene amplification, 8 can arise as a consequence of cycles of anaphase-bridging, breakage, and fusion initiated by the formation of dicentric chromosomes after telomere fusion. 9 This paradigm is exemplified in vivo by telomerase knockout mice, where short telomeres appear to drive the formation of tumors containing NRTs. 5 However, evidence for this phenomenon in humans is circumstantial. Numerous malignancies, including breast, prostate, colorectal, and chronic lymphocytic leukemia (CLL), [10][11][12][13][14][15] have been documented to exhibit shorter telomeres compared with normal tissues. These data are consistent with the expected levels of cell division during the progression to malignancy but do not indicate that telomeres become short enough to lose their end-capping function. Telomere fusion, as well as other chromosomal defects, can lead to the formation of anaphase bridges; in situ data show an increase in anaphase bridges, often interpreted as a surrogate marker for telomere f...

Extreme telomere erosion in ATM-mutated and 11q-deleted CLL patients is independent of disease stage

“…These data indicate that there were no differential rates of erosion at specific chromosome ends and thus it is unlikely that chromosome-specific telomere dynamics drive the loss of specific chromosome ends. These data, together with our previous analysis of specific telomeres with single telomere length analysis and the correlation of single telomere length analysis data with genome wide telomere length analysis, 5 indicate that a single telomere is sufficient to represent the genome wide telomere length distributions.…”

“…Together, these data Table 1 Demographics and clinical characteristics of the CLL patient cohort separated by ATM mutation status Letters to the Editor provide a clear demonstration that CLL B-cells exhibiting ATM mutations display extreme telomere shortening, with telomeres in the length range at which telomere fusion has been detected in both in vitro cell culture and ex vivo. 5 Within the ATM-mutated and wild-type cohorts there was no difference between the mean telomere lengths determined at all the three chromosome ends (wild type P ¼ 0.98, mutant P ¼ 0.14, ANOVA, Figure 2a). These data indicate that there were no differential rates of erosion at specific chromosome ends and thus it is unlikely that chromosome-specific telomere dynamics drive the loss of specific chromosome ends.…”

“…We have previously described short telomeres in Binet stage C CLL patients, the majority of which were prone to telomere fusion events. 5 Comparison of this data set with the ATM-mutated cohort at the XpYp and 17p telomeres revealed that ATM mutation confers more homogeneous and extensive telomere erosion, a difference of 0.56 kb (2.58 vs 2.03 kb, P ¼ 0.038, Figures 2a and b). Together, these data Table 1 Demographics and clinical characteristics of the CLL patient cohort separated by ATM mutation status Letters to the Editor provide a clear demonstration that CLL B-cells exhibiting ATM mutations display extreme telomere shortening, with telomeres in the length range at which telomere fusion has been detected in both in vitro cell culture and ex vivo.…”

“…It is clear from these in vitro studies that there is a subtle distinction between the length of telomere that elicits a cell cycle checkpoint response, triggering p53-dependent replicative senescence or apoptosis and the shorter telomeres that are capable of fusion in cells undergoing 'crisis'. 5 Based on these observations, we hypothesised that in order for CLL cells to achieve substantial telomere erosion and dysfunction they must be compromising in their ability to…”

“…6 The telomere dynamics described in CLL are indistinguishable from those observed experimentally in fibroblast cells undergoing a telomere driven 'crisis' in culture following the abrogation of the p53 pathway. 5 In this situation, the loss of cell cycle checkpoints in response to DNA doublestrand breaks (DSBs) facilitates the ongoing cell division beyond the point that would normally trigger telomere-dependent replicative senescence. It is clear from these in vitro studies that there is a subtle distinction between the length of telomere that elicits a cell cycle checkpoint response, triggering p53-dependent replicative senescence or apoptosis and the shorter telomeres that are capable of fusion in cells undergoing 'crisis'.…”