# ZEB1 Promotes Alternate Lengthening of Telomeres at Multiple Levels

## TL;DR

ZEB1 promotes the ALT pathway in cancer cells by regulating key proteins involved in telomere maintenance.

## Contribution

ZEB1 is shown to regulate the ALT pathway at multiple levels, including transcription and splicing.

## Key findings

- ZEB1 regulates multiple ALT-associated genes like PML, RMI2, and POLD4.
- ZEB1-deficient cells show reduced telomere length and ALT hallmarks like C-circles and APBs.
- ESRP1 repression by ZEB1 is linked to the production of PML isoform IV, a key APB component.

## Abstract

About 10–15% of cancers–often difficult to treat, pediatric tumors–maintain their telomeres through the Alternate Lengthening of Telomeres (ALT) pathway, the mechanistic details of which are still being elucidated. A significant fraction of these is characteristically mesenchymal–more stem cell-like–in nature, and previous studies have shown a positive correlation between factors that can induce the mesenchymal state and ALT. In this study, we investigated how a pro-mesenchymal gene regulatory protein, called ZEB1, affects ALT mechanistically. We found that it regulates several key factors involved in the initiation, execution and resolution of this pathway, in particular the levels of a protein that forms the major cellular compartment in which ALT is carried out. As ALT is utilized exclusively in cancer cells, identifying potential molecular targets in this pathway offers the possibility of developing efficacious therapies.

Background/Objectives: Approximately 10–15% of cancers rely on Alternate Lengthening of Telomeres (ALT), which is a telomerase-independent, BIR (Break-Induced Replication)-based pathway for maintaining long-term replicative potential. Methods: As ALT is over-represented in mesenchymal-type tumors, we investigated, via RNA-seq, the extent to which the EMT-promoting factor ZEB1 regulates this pathway. Results: The ALT-associated genes targeted by ZEB1, including PML, RMI2, POLD4, RPA3 (induced), SLX4, and WRN (repressed), in the aggregate, suggest that it regulates ALT at multiple steps in that pathway. ZEB1-deficient cells showed a significant reduction in telomere length as well as in two hallmarks of ALT, C-circle levels and the size and number of ALT-associated PML Bodies (APBs), which are the telomere-aggregating compartments in which BIR occurs. As one of the most highly regulated genetic targets of ZEB1 was the pro-epithelial alternative splicing factor ESRP1, we investigated whether the repression of this factor was required to generate the PML splice variant isoform IV, which is the major structural component of APBs. We found an inverse relationship between the expression of this protein and levels of PML isoform IV mRNA. Conclusions: These findings suggest a novel role for ZEB1 in promoting ALT both transcriptionally and post-transcriptionally at multiple levels.