Determine the mechanism of ALT-mediated telomere elongation

Division Of Basic Sciences - Nci

Investigators

Abstract

This project aims to define the mechanisms underlying the alternative lengthening of telomeres (ALT) pathway. This year, we developed and applied high-throughput screening platforms to identify regulators of ALT and performed structural analysis of ALT telomeres. Development of ALT Discovery Platforms: We established two complementary screening tools to interrogate ALT regulation at scale. The first, based on high-content imaging of native telomeric DNA FISH, enables arrayed-format screening for genes that promote or suppress ALT phenotypes. This platform was used to identify multiple regulators of ALT activity and was published in Cell Reports (PMC11844024). The second platform adapts this method for optical pooled screening and was published in Methods (PMID 40324704). Both tools are now publicly available and being used by collaborating labs. Structural Features of ALT Telomeres: Using an adapted END-seq protocol, we mapped terminal telomeric DNA features in ALT-positive cancer cells. This analysis, published as a reviewed preprint in eLife, reveals conserved and unique characteristics of ALT telomere architecture, providing new insight into the structural consequences of recombination-based maintenance. Together, these advances create a foundation for understanding how ALT is regulated and provide tools for future discovery and therapeutic targeting. Publications Cell Reports, Jan 2025: "Identification of ALT regulators using native FISH screening" (PMCID: PMC11844024) Describes the development of an arrayed high-content imaging screen to identify genetic modulators of ALT. Demonstrates practical utility in pinpointing both suppressors and enhancers of ALT activity. Methods, May 2025 (online): "Optical pooled ALT screening workflow" (PMID: 40324704) Presents a pooled screening approach compatible with native telomeric FISH readouts. Expands the scalability of ALT discovery efforts and enables genome-wide interrogation with reduced resource requirements. eLife Reviewed Preprint, July 2025: "Terminal telomere architecture in ALT-positive cells" Uses END-seq to define telomere end structure in ALT-positive cells. Identifies features that distinguish ALT from telomerase-based maintenance and provides a reference framework for future studies.