# Phase separation-induced nuclear organization in ALT Cancer

> **NIH NIH U01** · CARNEGIE-MELLON UNIVERSITY · 2022 · $530,948

## Abstract

Abstract: 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 canc...

## Key facts

- **NIH application ID:** 10479118
- **Project number:** 5U01CA260851-03
- **Recipient organization:** CARNEGIE-MELLON UNIVERSITY
- **Principal Investigator:** Huaiying Zhang
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $530,948
- **Award type:** 5
- **Project period:** 2020-09-10 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10479118

## Citation

> US National Institutes of Health, RePORTER application 10479118, Phase separation-induced nuclear organization in ALT Cancer (5U01CA260851-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10479118. Licensed CC0.

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