Inhibition of the Alternative Lengthening of Telomeres (ALT) Pathway

Inhibition of the Alternative Lengthening of Telomeres (ALT) Pathway

Download the Summer 2015 issue of Advances PDF

Can the ALT pathway be disrupted to treat cancer?

The telomere plays a key role in the continuous duplication of proliferating cells, and its erosion eventually leads to a cell’s senescence. Cancer cells overcome this replicative senescence in one of two ways: through activating telomerase, an enzyme that extends telomeres, or using another process called the alternative lengthening of telomeres (ALT) pathway.

In contrast to the RNA-directed DNA synthesis by telomerase, ALT relies on recombination and replication of telomere DNA to extend telomeres. But precisely how the ALT pathway is activated in cancer cells and how it works mechanistically remain largely unclear.

Roughly 5 percent of human cancers, and possibly more, utilize ALT; the process is prevalent in up to 60 percent of osteosarcomas and 40 percent to 60 percent of glioblastomas. Currently, no cancer therapies specifically disrupt ALT pathways.1 Lee Zou, PhD, and his team at the Massachusetts General Hospital Cancer Center investigated the molecules and genes that drive the ALT pathway and how new treatments might disrupt this process.

Terra Controls Single-Stranded DNA Binding Protein RPA

In a paper published in Science in January 2015,2 Dr. Zou and his team mapped a model of critical steps along the ALT pathway. The processes that maintain telomeres employ replication protein A (RPA), a single-stranded DNA binding protein. Dr. Zou and his team had previously investigated the role for RPA at telomeres3 and found that it associated transiently with telomeres during S phase of DNA replication. This process is facilitated by a type of RNA called telomeric repeat-containing RNA (TERRA). Levels of TERRA fluctuate during the cell cycle as RPA binds and detaches from telomeres.

Some of the cell lines under investigation, however, did not show this fluctuation in TERRA throughout the cell cycle. Dr. Zou and his team postulated that such cancer cell lines were those without active telomerase, relying instead on the ALT pathway, which lengthens telomeres through recombination with telomeric DNA sequences from the same or other chromosomes.

Dr. Zou and his team then looked to other known factors of ALT pathways that might intersect with TERRA fluctuation. Previously published research had shown that cancer cell lines using ALT commonly carry mutations in the ATRX gene, but researchers did not have a working model of its mechanisms. Knockdown of ATRX protein in ATRX-expressing cancer cells disrupted TERRA’s fluctuation, recapitulating the situation in ALT-positive cancer cells. The study predicted that if TERRA levels did not change, RPA binding must be dysregulated as well. And indeed the team found that in cells that use the ALT pathway, RPA binds persistently to telomeres, not detaching after replication.

Inhibiting ATR Disrupts ALT

This insight into the role of RPA proved to be key in mapping the ALT pathway. RPA functions as a DNA repair protein that promotes DNA recombination, and the ALT pathway is known to be a process that is dependent on recombination. Dr. Zou’s team discovered that loss of ATRX leads to dysregulation of TERRA, which in turn causes RPA to bind persistently to telomeres. Then the persistent binding of RPA promotes recombination and the activation of the ALT pathway, allowing the cancer to continue to proliferate.

A further investigation, however, revealed that the ATRX mutation alone is not enough to activate this ALT pathway, and a fuller understanding of this mechanism is needed. Dr. Zou’s team postulates that ALT is established via a multistep process that includes loss of ATRX, as well as additional genetic or epigenetic changes.

The model presented a novel avenue for treatment. Previous research by Dr. Zou and his colleagues had shown that RPA-bound single-stranded DNA activates the kinase ATR, which is known to be a master regulator of DNA repair and recombination. The implication of RPA in ALT suggests that this pathway may also be regulated by ATR and susceptible to ATR inhibitors.

Dr. Zou and his team tested the ATR inhibitors VE-821 and AZ20. These selectively eliminated ALT-positive osteosarcoma and glioblastoma cancer cells, leading to the rapid death of these cells. (The expectation was that cell death would happen more gradually.) While the mechanism is not yet fully understood, Dr. Zou speculates that cancer cells relying on the ALT pathway have rewired their DNA repair pathways to maintain telomeres. Thus, eliminating ALT would have a dramatic effect on cellular survival.

With further research and clinical trials, ATR inhibitors such as VE-821 and AZ20 could offer a promising treatment for those tumors dependent on the ALT pathway.

References:

(1) Shay, Jerry W., Roger R. Reddel and Woodring E. Wright, “Cancer and Telomeres—An ALTernative to Telomerase,” Science, vol. 336, no. 1687 (June 15, 2012): 1388–90.

(2) Flynn, Rachel L, Kelli E. Cox, Maya Jeitany, Hiroaki Wakimoto, et al., “Alternative Lengthening of Telomeres Renders Cancer Cells Hypersensitive to ATR Inhibitors,” Science, vol. 347, no. 6219 (January 16, 2015): 273–277.

(3) Flynn, Rachel L., Richard C. Centore, Roderick J. O’Sullivan, Rekha Rai, et al., “TERRA and hnRNPA1 Orchestrate an RPA-to-POT1 Switch on Telomeric Single-Stranded DNA,” Nature, vol. 471, no. 7339 (March 24, 2011): 532–6.