Tomas Goncalves

Telomeres are the protective ends of chromosomes. After every cell division, telomeres shorten due to the end-replication problem. This continues until they reach a critically shortened state where they elicit DNA damage checkpoint activation, stopping further divisions. Therefore, for cancer cells to divide indefinitely, they must maintain the ends of their chromosomes through a telomere maintenance mechanism to prevent replicative senescence or apoptosis. 

In about 90% of cancers, the enzyme telomerase, which replenishes telomeric DNA, is reactivated, permitting indefinite cell divisions. In the absence of telomerase activity, about 10-15% of tumours can gain a telomerase independent, alternative pathway to maintain their telomeres, termed the alternative lengthening of telomeres (ALT), which is dependent on homologous recombination. The ALT pathway is more common in certain cancers, mainly those of a mesenchymal origin, including some brain cancers and certain sarcomas, particularly osteosarcoma. ALT activity appears to be unique to cancer cells. However, despite this novel feature of telomerase-independent telomere maintenance, effective drugs that selectively kill ALT-positive cancer cells have yet to be discovered. As many as yet untreatable childhood cancers extend their telomeres via ALT, a greater understanding of this pathway may provide much needed treatments for such cancers.

Recent studies have provided important clues as to the molecular mechanisms behind the ALT pathway. It has been shown that ALT is strongly associated with dysfunction of ATRX (alpha-thalassemia/mental retardation syndrome X-linked), a chromatin remodelling protein that, together with the death domain associated protein DAXX, inserts histone variant H3.3 into telomeric and pericentromeric heterochromatin. Interestingly, ectopic expression of ATRX in the ALT-positive, ATRX-negative U-2 OS cell line reduced the presence of ALT hallmarks such as C-circles and ALT-associated PML bodies (APBs), suggesting that ATRX functions as a suppressor of the ALT mechanism and loss of ATRX is a necessary step for ALT to occur. However, knocking down ATRX in telomerase-positive cells fails to promote the ALT phenotype, suggesting that ATRX loss alone is insufficient to promote ALT and other as yet unidentified alterations must occur in cancer cells for ALT to occur.

ATRX is thought to play an important role in DNA replication and repair. Previous studies have shown that knockout of ATRX causes replication defects, including a significantly higher frequency of stalled replication forks, particularly during replication stress.

Further studies are required to better understand where ATRX functions in relation to the replication fork at telomeres. My project involves identifying the proteins that are present at telomeric replication forks using novel proteomic techniques. This would help answer a number of unresolved questions regarding the molecular mechanisms behind the ALT pathway, such as whether ATRX is always travelling with the fork at telomeres or if it only appears upon particular damage. It could also help us understand what recruits ATRX to the replication fork at telomeres.  To do this, I will utilise commercially available cell lines along with a number of molecular biology and proteomics techniques, including co-immunoprecipitations, microscopy and iPOND as well as numerous ALT-specific assays.