Rongwei Zhao1, Meng Xu1, Xiaoyang Yu1, Anne R. Wondisford2, Rachel M. Lackner3, Jayme Salsman4, Graham Dellaire4, David M. Chenoweth3, Roderick J. O'Sullivan2, Xiaolan Zhao5 and Huaiying Zhang1 1Department of Biology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA; 2Department of Pharmacology and Chemical Biology, University of Pittsburgh Medical Center Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA; 3Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19014, USA; 4Department of Pathology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada; 5Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA Corresponding author: huaiyinzandrew.cmu.edu Abstract

The alternative lengthening of telomeres (ALT) pathway maintains telomere length in a significant fraction of cancers that are associated with poor clinical outcomes. A better understanding of ALT mechanisms is therefore necessary for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins contributes to the formation of ALT telomere-associated PML bodies (APBs), in which telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, it is still unknown whether—and if so, how—SUMO supports ALT beyond APB formation. Here, we show that SUMO condensates that contain DNA repair proteins enable telomere maintenance in the absence of APBs. In PML knockout ALT cell lines that lack APBs, we found that SUMOylation is required for manifesting ALT features independent of PML and APBs. Chemically induced telomere targeting of SUMO produces condensate formation and ALT features in PML-null cells. This effect requires both SUMOylation and interactions between SUMO and SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are associated with the accumulation of DNA repair proteins, including Rad52, Rad51AP1, RPA, and BLM, at telomeres. Furthermore, Rad52 can undergo phase separation, enrich SUMO at telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that SUMO condensate formation promotes collaboration among DNA repair factors to support ALT telomere maintenance without PML. Given the promising effects of SUMOylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

Received February 29, 2024. Accepted July 8, 2024.