Telomeric DNA breaks in human induced pluripotent stem cells trigger ATR-mediated arrest and telomerase-independent telomere length maintenance

Although mechanisms of telomere protection are well-defined in differentiated cells, it is poorly understood how stem cells sense and respond to telomere dysfunction. Recent efforts have characterized the DNA damage response (DDR) following progressive telomere erosion in human pluripotent cells, yet the broader impact of telomeric double-strand breaks (DSBs) in these cells is poorly characterized. Here, we report on DNA damage signaling, cell cycle, and transcriptome-level changes in human induced pluripotent stem cells (iPSCs) in response to telomere-internal DSBs. We engineered a novel human iPSC line with a targeted doxycycline-inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres. Using this model, we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DDR in iPSCs, in contrast to an established ATM-dependent response to telomeric FokI breaks in differentiated cells. ATR activation leads to a potent cell cycle arrest in G2, which we show is p53-independent and can be rescued by treatment with an ATR inhibitor. Telomere lengths are remarkably well-maintained in the face of persistent TRF1-FokI induction. Using CRISPR-Cas9 to cripple the catalytic domain of telomerase, we show that telomerase is largely dispensable for survival and telomere length maintenance following telomeric breaks, which instead appear to be repaired by a mechanism bearing hallmarks of lengthening mediated by homologous recombination, so-called alternative lengthening of telomeres (ALT). Our findings suggest a previously unappreciated role for ALT in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric damage.