WRN Loss Induces Switching of Telomerase-Independent Mechanisms of Telomere Elongation

Abstract

Telomere maintenance can occur in the presence of telomerase or in its absence, termed alternative lengthening of telomeres (ALT). ALT adds telomere repeats using recombination-based processes and DNA repair proteins that function in homologous recombination. Our previous work reported that the RecQ-like BLM helicase is required for ALT and that it unwinds telomeric substrates in vitro. WRN is also a RecQ-like helicase that shares many biochemical functions with BLM. WRN interacts with BLM, unwinds telomeric substrates, and co-localizes to ALT-associated PML bodies (APBs), suggesting that it may also be required for ALT processes. Using long-term siRNA knockdown of WRN in three ALT cell lines, we show that some, but not all, cell lines require WRN for telomere maintenance. VA-13 cells require WRN to prevent telomere loss and for the formation of APBs; Saos-2 cells do not. A third ALT cell line, U-2 OS, requires WRN for APB formation, however WRN loss results in p53-mediated apoptosis. In the absence of WRN and p53, U-2 OS cells undergo telomere loss for an intermediate number of population doublings (50–70), at which point they maintain telomere length even with the continued loss of WRN. WRN and the tumor suppressor BRCA1 co-localize to APBs in VA-13 and U-2 OS, but not in Saos-2 cells. WRN loss in U-2 OS is associated with a loss of BRCA1 from APBs. While the loss of WRN significantly increases telomere sister chromatid exchanges (T-SCE) in these three ALT cell lines, loss of both BRCA1 and WRN does not significantly alter T-SCE. This work demonstrates that ALT cell lines use different telomerase-independent maintenance mechanisms that variably require the WRN helicase and that some cells can switch from one mechanism to another that permits telomere elongation in the absence of WRN. Our data suggest that BRCA1 localization may define these mechanisms.

Alternative mechanisms of telomere lengthening: Permissive mutations, DNA repair proteins and tumorigenic progression

“…Long-term WRN knockdown in WI-38 VA-13/2RA and U-2 OS cells significantly reduces telomere length and inhibits APB formation. In Saos-2 cells, depletion of WRN does not inhibit cell growth, telomere lengthening or APB formation [63]. The results from Saos-2 are consistent with the observation of an ALT phenotype in an immortalized cell line from a person with Werner’s syndrome ( WRN −/− ) [64].…”

“…WRN is a helicase that, like BLM, functions in numerous aspects of DNA repair and replication. In contrast, RNA interference-mediated depletion of WRN inhibits ALT in some, but not all, immortalized human ALT cell lines [63]. Long-term WRN knockdown in WI-38 VA-13/2RA and U-2 OS cells significantly reduces telomere length and inhibits APB formation.…”

Werner syndrome: Clinical features, pathogenesis and potential therapeutic interventions

“…Tumors derived from the G5 m Terc ^−/− Wrn ^−/− MEF showed the characteristics of ALT cells (Laud et al, 2005). Elevated SCEs, mainly localized to telomeres, were also observed in in human WRN deficient cells (Gocha et al, 2014; Hagelstrom et al, 2010). For example, the widely used AG11395 human WS cell line is ALT-positive (Fasching et al, 2005).…”

Telomere profiles and tumor-associated macrophages with different immune signatures affect prognosis in glioblastoma

“…3,4 It is possible that these tumors do not activate a telomere maintenance mechanism similar to low-grade astrocytomas, 16,17 or that their telomere maintenance mechanism remains undescribed. [18][19][20] Another possibility is that non-defined telomere maintenance mechanism tumors are comprised of telomerase and alternative lengthening of telomere-positive tumors, but complications in assaying telomerase activity and measuring the hallmarks of the alternative lengthening of telomeres mechanism lead to falsenegative results. Many glioblastomas contain a considerable proportion of non-neoplastic cells such as macrophages/microglia (tumor-associated macrophages) that can comprise over 70% of the tumor.…”