Replication stress and the alternative lengthening of telomeres pathway
In an effort to achieve replicative immortality, human cancer cells must avoid the constant telomere attrition that accompanies DNA replication. Cancer cells accomplish this by employing mechanisms to lengthen their telomeres. Approximately 10 percent of all cancers utilize the Alternative Lengthening of Telomeres (ALT) pathway to maintain telomere length. Although ALT is known to rely on homologous recombination between two telomeric sequences, the exact mechanism and regulators of the ALT pathway remain elusive. As common fragile sites, telomeres pose a challenge to the replication machinery. This replication challenge is exacerbated in ALT cells due to defects in nucleosome assembly, suggesting the importance of managing replication stress at telomeres in these cells. ATR (ataxia telangiectasia and Rad3-related) is an important kinase in the response to replication stress. The work in this thesis demonstrates that ATR is also a key mediator of ALT activity. Due to the highly recombinogenic state of ALT telomeres, these cells depend on ATR activity. In fact, we illustrate that small molecule inhibition and siRNA mediated loss of ATR disrupts ALT activity and promotes cell death specifically in ALT positive cancer cells. Although we establish ATR as a critical regulator and effective therapeutic target in ALT cancers, the exact mechanism of ATR in this pathway remains elusive. Recently, the chromatin remodeling enzyme SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A-like protein 1) was identified as one of the most abundant proteins bound to sites of replication stress. We demonstrate by combined immunofluorescence-FISH and chromatin immunoprecipitation that SMARCAL1 associates with ALT telomeres to resolve replication stress and maintain telomere stability. Specifically, we illustrate that siRNA mediated loss of SMARCAL1 in ALT cancer cells results in persistently stalled replication forks that collapse into DNA double strand breaks, which promotes the formation of chromosome fusions. Ultimately, we illustrate that loss of SMARCAL1 in ALT cancer cells promotes genomic instability through telomere dysfunction. Although great strides have been made in defining the ALT mechanism, the drivers of this pathway remain elusive. These studies highlight the importance of replication stress in both activation and maintenance of the ALT pathway. Our data demonstrate chronic replication stress as a key feature at ALT telomeres. Importantly, we were able to exploit this feature to identify a novel therapeutic avenue for ALT positive cancers.