Telomerase-Mediated Healing of Double-Strand Breaks in Human Cells

PROJECT SUMMARY/ABSTRACT Telomeres?which define and protect the ends of humans? linear chromosomes?serve as a natural check on carcinogenesis.

Genome stability requires cells to differentiate telomeres from perilous DNA double-strand breaks (DSBs) to block inappropriate DSB repair and DNA damage response (DDR) signaling, which humans accomplish with the shelterin complex.

Telomerase maintains telomere length in the gonads and some stem cells, but telomeres in somatic cells shorten with each cell division due to developmental silencing of telomerase.

Unfettered cell division in early neoplasms eventually leads to a few telomeres becoming critically short and activating persistent DDR signaling, which causes cells with functional p53 and Rb pathways to undergo senescence or apoptosis.

Cells defective in these pathways continue to divide until multiple telomeres become de-protected and then enter telomere crisis, defined by poor cell viability due to intolerable genomic instability, as chromosomes repeatedly fuse at their ends and break. Clinical tumors emerge from crisis with rearranged, aneuploid genomes and a telomere maintenance mechanism.

To escape from telomere crisis, I predict that malignant cells must reconstitute their telomeres and that telomerase may accomplish this by directly repairing non-telomeric chromosome ends with neotelomeres.

The objective of the proposed project is to identify and mechanistically characterize telomerase- mediated DSB repair in human cells.

In vitro, telomerase can add TTAGGG repeats to a non-telomeric breakpoint sequence derived from a patient with ?-thalassemia due to a terminal chromosomal truncation.

Using this sequence, I have designed a PCR-based reporter assay to detect neotelomere formation in cells at an inducible DSB and have gathered evidence that suggests that telomere healing occurs in human cells in a telomerase-dependent manner.

I will improve this assay with TaqMan probes on a qPCR platform to rigorously quantify telomere healing events and will perform further experiments to demonstrate that telomerase is responsible for TTAGGG repeat addition.

Because telomerase-mediated repair threatens to convert DSBs into terminal chromosome deletions, I hypothesize that human cells have evolved mechanisms to block telomerase activity at DSBs.

I will implement a genetic approach with my telomere healing assay to identify the physiologic repressors of this aberrant mode of DSB repair.

Ultimately, I aim to unveil a new role for telomerase in enabling incipient cancers to traverse the bottleneck of telomere crisis.

This leap in our understanding of genomic instability in early tumorigenesis may lead to unexpected ways to detect and prevent cancer in patients.

With the aid of this award and the stimulating environment of the Tri-Institutional MD/PhD Program, I will grow scientifically, medically, and professionally in ways that will enable me to advance toward my long-term career goal of leading a cancer-centric lab while providing patient care as a transformative physician-scientist.