Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes

Abstract

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.

In perspective: An update on telomere targeting in cancer

“…The presence of the ALT phenotype did depend on the cancer subtype, however, with a greater prevalence being observed in sarcomas (25%‐60%) and 5% to 15% in carcinomas . Conversely, the ALT phenotype was largely absent in urothelial carcinomas, gastric carcinomas, and adenocarcinomas of most major cancer types, accentuating the idea that ALT is more common in mesenchymal and neuroepithelial derived tumor types . Overall, it is believed that 5% to 15% of cancer cells lack telomerase activity and are maintained by ALT .…”

“…Treating cancer cells that express telomerase with inhibitors can potentially elicit the phenotype of ALT which is telomerase‐independent . Cells are capable of adapting to the telomerase inhibitors, leading to upregulation of more telomerase (common) or the activation of alternative lengthening of telomeres (much less common) in a telomerase‐independent fashion . In a comprehensive analysis of a large panel of primary tumors representing multiple different cancer subtypes, only 3.74% presented with a positive ALT phenotype .…”

“…Differences that distinguish ALT‐dependent cells from telomerase‐positive cancer cells include longer telomere overhangs and preferentially elongated lagging strands depicted in the S phase during replication, meaning that the length of telomeres in ALT‐positive cancer cells are heterogeneous and vary greatly in length . Recently, telomerase‐positive H1299 and SW39 telomerase positive cell lines have been modulated using the CRISPR/Cas9 technique to knockout h TERC in an attempt to activate the ALT pathway . It was shown that upon the depletion of h TERC , a very small percentage of cells were able to survive, and those that did all displayed elongated telomeres of varying lengths and the presence of ECTR (extra chromosomal telomere repeats) as determined by the C‐circle assay .…”

“…Recently, telomerase‐positive H1299 and SW39 telomerase positive cell lines have been modulated using the CRISPR/Cas9 technique to knockout h TERC in an attempt to activate the ALT pathway . It was shown that upon the depletion of h TERC , a very small percentage of cells were able to survive, and those that did all displayed elongated telomeres of varying lengths and the presence of ECTR (extra chromosomal telomere repeats) as determined by the C‐circle assay . It is speculated that those methods by which ALT is acquired in cells are affected by the degree of the depletion of ATRX or DAXX and h TERC .…”

Direct Quantitative Monitoring of Homology-Directed DNA Repair of Damaged Telomeres

“…For a review of these methods, see Jackson and Cook (2008a, 2008b, 2008c). Recent studies have utilized cell microscopy, flow cytometry, and fiber labeling of thymidine analogs to detect break-induced synthesis after replication stress (Bhowmick et al, 2016; Costantino et al, 2014; Minocherhomji et al, 2015; Sotiriou et al, 2016) and at telomeres (Dilley et al, 2016; Min, Wright, & Shay, 2017; Roumelioti et al, 2016). …”