in the absence of telomerase , telomere shortening limits the replicative lifespan of human cells thus providing a stringent tumor suppressive mechanism . however if dna damage checkpoints are compromised , cells can continue to divide until the telomeres lose their end - capping function and are subjected to dna double - strand break repair activity that results in telomere - telomere fusion events .
the resulting dicentric chromosomes can initiate cycles of anaphase - bridging , breakage , and fusion that generate the large - scale genomic rearrangements common in human cancer .
telomere - driven mutation is therefore a mechanism that generates variation in tumor cell populations , upon which clonal selection can operate and facilitate progression .
we have developed high - resolution , single - molecule technologies to study telomere length and fusion in detail ; these methods have provided a level of clarity that was hitherto impossible to achieve in human cells . in colorectal cancer
we showed that telomere erosion , dysfunction , and fusion not only precede the adenoma / carcinoma transition , but may also be pre - existent in the normal cells in which the initial mutation occurs . in chronic lymphocytic leukemia ( cll )
we observed extreme telomere erosion and fusion , consistent with cll b - cells undergoing a telomere - driven crisis .
importantly , this was also detected in a subset of patients with early - stage disease prior to clinical progression .
our recent large - scale analysis of telomere length and fusion in early - stage cll has allowed definition of the telomere length threshold below which telomere fusion is detected and revealed the prognostic value of stratifying patients according to this parameter .
patients with telomeres below the telomere fusion threshold had a significantly shorter overall survival that was even more prognostic in early - stage disease patients ( p < 00001 , hr = 19.3 ) , and telomere dysfunction was the dominant variable in a multivariate analysis . based on these observations
we hypothesize that short dysfunctional telomeres provide a mutator mechanism capable of driving genomic instability and disease progression .
it is therefore important to understand the molecular basis of telomere fusion and how this drives mutation and clonal evolution .
we have also investigated the process of fusion between short dysfunctional telomeres in human cells by the direct isolation and characterization of the dna sequence of telomere fusion events .
irrespective of the tissue or cell model analyzed , we observed a consistent mutational profile with deletion into the subtelomeric dna and microhomology at the fusion junction ; this mutational profile is consistent with alternative non - homologous end - joining ( a - nhej ) processes .
a - nhej involves the coordinated interaction of multiple proteins with nucleating , scaffolding , and resection activity , as well as those ultimately executing dna ligation .
we therefore examined the contribution of dna ligase iii ( lig3)-dependent a - nhej and dna ligase iv ( lig4)-dependent classical - nhej ( c - nhej ) pathways in mediating fusion between short dysfunctional telomeres . using a dominant negative telomerase ( dn - htert ) we induced telomere erosion , fusion , and the onset of a telomere crisis in hct116 cells in which the lig3 or lig4 genes had been inactivated using recombinant adeno - associated virus - mediated gene targeting . both wild - type and lig4 clones displayed large - scale genomic rearrangements and telomere fusions , but readily escaped crisis following the re - establishment of telomerase activity .
fusions were also detected in lig3 cells but strikingly no clones escaped crisis ; after 2 to 3 months no cells remained in these cultures .
all lig3 clones escaped crisis following complementation with a wild - type lig3 cdna , but none escaped following complementation with cdnas containing either a deletion in the lig3 brca1 c - terminal domain or a a874d point mutation , both of which are required for the interaction of lig3 with x - ray repair cross - complementing protein 1 ( xrcc1 ) .
these data demonstrate an absolute requirement for lig3 in mediating the escape from a telomere - driven crisis . in order to gain some insight into the underlying mechanisms by which lig3 facilitated the escape from crisis
, we undertook a detailed molecular characterization of telomere fusion events mediated by lig3 or lig4 .
sister chromatid telomere fusion events were detected in both lig3 and lig4cells , however there was a marked reduction in interchromosomal events in lig4 cells .
sequencing of interchromosomal fusions from lig3 cells revealed a higher incidence of breakpoints within telomere repeats and a reduction in microhomology at the fusion junction .
our data demonstrated the involvement of both lig3 and lig4 in the fusion of short dysfunctional telomeres , but also indicated that fusions involving lig3 provide a selective advantage to cells undergoing a telomere - driven crisis that facilitates clonal evolution and escape from crisis .
the mechanism by which lig3 facilitates the escape from crisis is not clear ; our hypothesis is that interchromosomal fusions , which predominate in cells that can not escape crisis , are more mutagenic and detrimental to the cells in which they occur ; whereas fusion between sister chromatids  resulting in localized amplification and deletion events  may confer a selective advantage .
we consider that the relative balance between these events dictates the ability of cells to escape crisis and that this is modulated by the activities of a - nhej and c - nhej at short dysfunctional telomeres ( fig .
1 ) . further work should elucidate the contributions of other components of the a - nhej pathway in telomere fusion and the escape from crisis , in addition to examining the mutational impact that these pathways have in the context of the evolving cancer genome . 

figure 1.schematic illustrating our hypothesis for how lig3 may affect the ability of cells to escape a telomere - driven crisis .
cells with short dysfunctional telomeres undergo crisis , during which telomere fusion results in the creation of dicentric chromosomes and the initiation of anaphase - bridging , breakage , and fusion cycles ; the ensuing genomic instability leads to large - scale genomic rearrangements .
clonal evolution results in cells that escape crisis ; these cells exhibit fewer interchromosomal fusion events compared to fusions between sister chromatids , whereas interchromosomal fusions predominate in cells that can not escape crisis .
we consider that this is governed by the relative activities of ligase iii - dependent alternative non - homologous end - joining ( a - nhej ) compared to ligase iv - dependent classic non - homologous end - joining ( c - nhej ) pathways . 
 
schematic illustrating our hypothesis for how lig3 may affect the ability of cells to escape a telomere - driven crisis .
cells with short dysfunctional telomeres undergo crisis , during which telomere fusion results in the creation of dicentric chromosomes and the initiation of anaphase - bridging , breakage , and fusion cycles ; the ensuing genomic instability leads to large - scale genomic rearrangements .
clonal evolution results in cells that escape crisis ; these cells exhibit fewer interchromosomal fusion events compared to fusions between sister chromatids , whereas interchromosomal fusions predominate in cells that can not escape crisis .
we consider that this is governed by the relative activities of ligase iii - dependent alternative non - homologous end - joining ( a - nhej ) compared to ligase iv - dependent classic non - homologous end - joining ( c - nhej ) pathways .
telomere fusion during crisis is ultimately a cellular survival mechanism that provides short - term relief from telomere erosion , but importantly also facilitates genome instability that allows for the generation of the genomic rearrangements that can facilitate progression .
our work identifies the a - nhej pathway as essential for the ability of cells to clonally evolve and escape a telomere crisis .
this indicates that intervention(s ) in this pathway may sensitize cells with short dysfunctional telomeres and thus provide a potential therapeutic target in the subsets of tumors in which short dysfunctional telomeres confer such a poor prognosis .
moreover , given that telomere dysfunction occurs early in tumorigenesis this may open up the possibility of therapeutic intervention prior to clinical progression .
this work was supported by funding to the baird laboratory from cancer research uk , leukemia and lymphoma research and the national institute for social care and health research ; and to the hendrickson laboratory from the united states national institutes of health ( gm088351 ) and the national cancer institute ( ca15446 ) .
 telomere dysfunction and fusion play key roles in driving genomic instability and clonal evolution in many tumor types . 
 we have recently described a role for dna ligase iii ( lig3 ) in facilitating the escape of cells from crisis induced by telomere dysfunction . 
 our data indicate that lig3-mediated telomere fusion is important in facilitating clonal evolution . 
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