Telomeric DNA breaks in human induced pluripotent stem cells trigger ATR-mediated arrest and telomerase-independent telomere damage repair.

Although mechanisms of telomere protection are well-defined in differentiated cells, it is poorly understood how stem cells sense and respond to telomere dysfunction. In particular, 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 human iPSCs with an 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, which leads to p53-independent cell cycle arrest in G2. Using CRISPR-Cas9 to cripple the catalytic domain of telomerase, we show that telomerase is largely dispensable for survival and lengthening of TRF1-FokI-cleaved telomeres, which instead are effectively repaired by robust homologous recombination (HR). In contrast to HR-based telomere maintenance in mouse embryonic stem cells, we find neither evidence that HR causes extension of telomeres beyond their initial lengths, nor an apparent role for ZSCAN4 in this process. Rather, HR-based repair of telomeric breaks is sufficient to maintain iPSC telomeres at a normal length which is compatible with sustained survival of the cells over several days of TRF1-FokI induction. Our findings suggest a previously unappreciated role for HR in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric damage.

Patient-Induced Pluripotent Stem Cell-Derived Hepatostellate Organoids Establish a Basis for Liver Pathologies in Telomeropathies.

BACKGROUND & AIMS: Dyskeratosis congenita (DC) is a telomere biology disorder caused primarily by mutations in the DKC1 gene. Patients with DC and related telomeropathies resulting from premature telomere dysfunction experience multiorgan failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. However, the mechanism responsible for telomere dysfunction-induced liver disease remains unclear. METHODS: We used isogenic human induced pluripotent stem cells (iPSCs) harboring a causal DC mutation in DKC1 or a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9)-corrected control allele to model DC liver pathologies. We differentiated these iPSCs into hepatocytes (HEPs) or hepatic stellate cells (HSCs) followed by generation of genotype-admixed hepatostellate organoids. Single-cell transcriptomics were applied to hepatostellate organoids to understand cell type-specific genotype-phenotype relationships. RESULTS: Directed differentiation of iPSCs into HEPs and stellate cells and subsequent hepatostellate organoid formation revealed a dominant phenotype in the parenchyma, with DC HEPs becoming hyperplastic and also eliciting a pathogenic hyperplastic, proinflammatory response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes in DKC1-mutant HEPs and hepatostellate organoids could be rescued via suppression of serine/threonine kinase AKT (protein kinase B) activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. CONCLUSIONS: Isogenic iPSC-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.

Potential Roles for G-Quadruplexes in Mitochondria.

Some DNA or RNA sequences rich in guanine (G) nucleotides can adopt noncanonical conformations known as G-quadruplexes (G4). In the nuclear genome, G4 motifs have been associated with genome instability and gene expression defects, but they are increasingly recognized to be regulatory structures. Recent studies have revealed that G4 structures can form in the mitochondrial genome (mtDNA) and potential G4 forming sequences are associated with the origin of mtDNA deletions. However, little is known about the regulatory role of G4 structures in mitochondria. In this short review, we will explore the potential for G4 structures to regulate mitochondrial function, based on evidence from the nucleus.

YU238259 Is a Novel Inhibitor of Homology-Dependent DNA Repair That Exhibits Synthetic Lethality and Radiosensitization in Repair-Deficient Tumors.

UNLABELLED: Radiotherapy and DNA-damaging chemotherapy are frequently utilized in the treatment of solid tumors. Innate or acquired resistance to these therapies remains a major clinical challenge in oncology. The development of small molecules that sensitize cancers to established therapies represents an attractive approach to extending survival and quality of life in patients. Here, we demonstrate that YU238259, a member of a novel class of DNA double-strand break repair inhibitors, exhibits potent synthetic lethality in the setting of DNA damage response and DNA repair defects. YU238259 specifically inhibits homology-dependent DNA repair, but not non-homologous end-joining, in cell-based GFP reporter assays. Treatment with YU238259 is not only synergistic with ionizing radiation, etoposide, and PARP inhibition, but this synergism is heightened by BRCA2 deficiency. Further, growth of BRCA2-deficient human tumor xenografts in nude mice is significantly delayed by YU238259 treatment even in the absence of concomitant DNA-damaging therapy. The cytotoxicity of these small molecules in repair-deficient cells results from an accumulation of unresolved DNA double-strand breaks. These findings suggest that YU238259 or related small molecules may have clinical benefit to patients with advanced BRCA2-negative tumors, either as a monotherapy or as an adjuvant to radiotherapy and certain chemotherapies. IMPLICATIONS: We have identified a novel series of compounds that demonstrate synthetic lethality in DNA repair-deficient cell and animal models and have strong potential for clinical translation.