Alternative Lengthening of Telomeres (ALT) in Human Cancers

Kaitlin Raseley

doi:10.17918/00010762

In humans, telomeres are nucleoprotein complexes made up of tandem 5′TTAGGG3′ DNA repeats and associated proteins located at the ends of all 46 chromosomes. Telomere repeat tract loss beyond a certain threshold changes the telomere structure ("uncapping"), causing telomere dysfunction and leading to senescence or apoptosis. Cancer cells utilize one of the two Telomere Maintenance Mechanisms (TMM) to maintain their telomeres during uncontrolled proliferation; telomerase or alternative lengthening of telomeres (ALT).  ALT is thought to occur in about 10–20% of all tumors and relies on many DNA damage response (DDR) proteins, including those involved in break-induced repair (BIR) in the homology-dependent repair (HDR) pathway.   Single Molecule Telomere Assay Optical Mapping (SMTA-OM) assay is the only telomere measurement technology capable of quantifying individual telomeres from single molecules genome-wide to analyze the telomeric features in ALT+ cancer cells. SMTA-OM has also identified unique telomeric features of ALT+ cancer, like fusions with internal telomere-like sequence (ITS+), fusions with loss of internal telomere-like sequence (ITS-), telomere-free ends (TFEs), superlong telomeres, and elevated telomere length heterogeneity. Here, we validate the advantages of SMTA-OM for telomere length and structure analysis, which is especially helpful in ALT+ cancers with high-frequency recombination. Next, we identified SMTA-OM readouts are potential biomarkers for ALT+ cancer. The telomere mean lengths between ALT+ and TEL+ cell lines are not substantially different, but differences are significant between the frequencies of telomeric structural features observed in ALT+ and TEL+ cells using SMTA-OM. Additionally, we used the C-circle assay to verify the reliability of SMTA-OM readouts for determining ALT activity since it is considered the gold standard for detecting ALT activity. Recently, C-circles have been detected in TEL+ cells with unusually long telomeres. Although it is not entirely clear why these TEL+ cells form C-circles, it is hypothesized they assist in the inhibition of "telomere trimming" to maintain telomere length homeostasis of extremely long telomeres. This highlights the potential for a false positive when trying to detect ALT activity with the C-circle assay. Since SMTA-OM utilizes three parameters for the determination of ALT activity, it provides a more accurate diagnosis than the C-circle assay alone. Finally, we applied the SMTA-OM to further construe the underlying mechanisms of ALT pathways. Our results show that targeting telomeric sequence with a catalytically inactive Cas9 (dCas9) physically prevents the progression of DNA replication machinery. Blocked replication machinery led to an increase in DNA damage and an increase in unique structural changes in the telomere and adjacent subtelomere, likely resulting from the upregulation of the ALT pathways. We hypothesize the damage to telomeres activates the break-induced replication pathway. These studies also provide substantial insight into the breakage-fusion-bridge cycle and how telomeric structural changes and molecular fusions are formed. It is essential to continue investigating TMM in ALT+ cancer cells, especially at the single-molecule level, which is possible with SMTA-OM. Despite recent advances in this area of research, there are still critical unanswered questions and a need for a reliable diagnostic method for ALT cancer.

Alternative Lengthening of Telomeres (ALT) in Human Cancers

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Alternative Lengthening of Telomeres (ALT) in Human Cancers

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