All cancer cells need to maintain telomere length for immortality. While most cancer cells reactivate telomerase, a reverse transcriptase, to elongates telomere from an RNA template, about 10-15% of cancer cells are telomerase-negative and adopt a homologous-recombination based alternative lengthening of telomeres (ALT) pathway. ALT cells exhibit many abnormalities in nuclear organization, including the formation of nuclear bodies called APBs for ALT telomere-associated promyelocytic leukemia nuclear bodies, clustering of telomeres within APBs, and the formation of RNA foci on telomeres with a long non-coding RNA called telomere repeat-containing RNA (TERRA). These unique features are used as biomarkers for ALT diagnosis and can be attractive therapeutic targets because of reduced side effects on healthy cells that do not share these features. However, how these features contribute to telomere maintenance and ALT cancer cell growth remain elusive, due to the lack of conceptual model as well as experimental tools to monitor and control their assembly and function in live cells. Based on our observation that APBs exhibit liquid behavior and long non- coding RNAs can phase separate with RNA-binding proteins, we propose a liquid-liquid phase separation model for the assmembly and function of these ALT specific features. We hypothesize TERRA phase separates with its interacting proteins to nucleate APB liquid droplets. The liquid nature of APBs droplets (also called condensates) would promote coalescence of APBs to drive telomere clustering. Meanwhile, condensation of APB droplets can concentrate DNA repair factors, providing opportunities for telomeres to use one another as repair templates to elongate within APBs. To test our hypothesis, we developed a state-of-the- art optogenetic approach to control APB assembly. We demonstrate that liquid phase separation underlies APB assembly and coalescence of APB droplets indeed drives telomere clustering. Building on our ability to control telomere clustering and APB assembly and by collaborating with experts in super resolution microscopy, nuclear mechanics, chromosome organization and ALT cancer, we will investigate how DNA repair factors are recruited to and organized in APB condensates for ALT telomere DNA synthesis (Aim 1) and how telomere clustering leads to unique genome organization and gene expression in ALT cells (Aim 2). We will then extend our optogenetic tools to control RNA and dissect TERRA contributions in ALT (Aim 3). Results obtained by manipulating cultured ALT cells will be confirmed by characterizing ALT tissue or creating de novo ALT phonotypes in primary human cells. Our results will provide mechanistic understanding on how protein and/or RNA phase separation contributes to ALT cancer, which will offer the potential to develop strategies specifically targeting these unique phase separation processes, rather than the existing molecules that shared by heathy cells, for ALT cancer treatment. Such therapies are also beneficial for treating telomerase-positive cancer as these cancer cells can escape telomerase inhibition and adopt ALT for telomere maintenance.
Telomerase-negative cancer cells that use an alternative lengthening of telomeres (ALT) pathway for telomere maintenance have many unique nuclear features including the relocation of nuclear bodies to telomeres, telomere clustering, and formation of unique RNA foci on telomeres. These unique features are used for ALT diagnosis but how they contribute to ALT cancer is unknown. By combining expertise in nuclear organization and ALT cancer, we will test a liquid-liquid phase separation model for the formation and function of these unique features, providing potential to target these features for ALT cancer therapy.
