The Regulation of Replication Stress Response at ALT telomeres by Timeless‐1

Abstract

All cancer cells need to maintain their telomeres in order to continue proliferating. In 85–90% of all human cancers, maintenance and extension of these telomeres is accomplished by telomerase, which adds a short repetitive sequence to the ends of chromosomes. 10–15% of cancers adopt another method, called the Alternative Lengthening of Telomeres (ALT) pathway. ALT pathway uses homologous recombination to maintain the telomere length of ALT cancers and is vital for the continued growth of ALT cancers. Previously, our lab reported that depletion of FANCM, a key member of the Fanconi Anemia family of genes, induced replication stress response primarily at the ALT telomeres (Pan, PNAS 2017). The objectives of our study were: (1) Elucidate the biological function of human Timeless (Tim1) protein, a very important DNA repair protein, in DNA replication stress response at ALT telomeres. (2) Determine whether depletion of Tim1 affects the viability of ALT cells. To accomplish these goals, we first used siRNA transfection to deplete either Tim1, or FANCM, or both in ALT cells. These cells were then co‐stained with antibodies recognizing TRF2 (a telomere marker) and either BLM (a DNA repair marker) or pRPA (a DNA damage checkpoint marker). The percentage of cells with TRF2 & BLM as well as TRF2 & pRPA foci were quantified. In addition, we also monitored the amount of micronuclei formed in those cells. Formation of micronuclei often represent the unrepaired DNA damages. Finally, cell viability assays of the siRNA transfected cells were done. Cells were allowed to grow for 10 days, fixed and stained with crystal violet. Our results show that: siRNA depletion of FANCM and Tim1 in ALT cells had synergistic/additive foci formation for TRF2 & BLM, and TRF2 & pRPA. siRNA depletion of FANCM and Tim1 in ALT cells had no significant effect on micronuclei formation alone, but increased formation when combined. siRNA depletion of FANCM and Tim1 in ALT cells appears to decrease cell viability to the greatest extent when co‐depleted, as opposed to either one depleted alone. Our findings suggest: (1) ALT cancer cells utilize both FANCM and Tim1 to resolve the replication stress at ALT telomeres; (2) co‐depletion of FANCM and Tim1 is synthetically lethal in ALT cancers. (3) Targeting both FANCM and Tim1 is a potential efficacious strategy in treating ALT cancers.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.