Modulating TERRA G-quadruplexes with ligands: impact on telomeric DNA:RNA hybrids and ALT mechanisms

Telomeres are transcribed into the long non-coding RNA TERRA, which is essential for telomere protection and maintenance. In cancer cells, telomere lengthening occurs via telomerase reactivation or the alternative lengthening of telomeres (ALT). TERRA is highly overexpressed in ALT cells and directly influences this process. However, due to the lack of efficient tools to investigate TERRA biology, its role in cancer progression and its potential as a therapeutic target remains unclear. Both telomeric DNA and TERRA form noncanonical structures called G-quadruplexes (GQs) on their G-rich strands, which can be the targets of GQ ligands. Using a ligand-based virtual screening of FDA-approved drugs, we identified novel TERRA GQ ligands capable of stabilizing TERRA binding to chromatin. This interaction increased telomeric DNA:RNA hybrids, induced telomeric defects, and elevated ALT-associated PML bodies formation in both telomerase- and ALT-positive cancer cells in an RNAseH1 dependent manner. These ligands also partly increased C-circle levels. In vitro, these ligands recognized and stabilized DNA:RNA GQ hybrids, revealing a novel mechanism of TERRA binding to telomeric DNA, which may contribute to replication stress, sister-telomere disjunction impairment, and enhanced ALT activity, offering new insights into TERRA's multifaceted role in telomere dynamics and its implications for cancer biology.