Mechanisms of Telomere Protection and Deprotection in Human Cells

Telomeres, the nucleo-protein complexes at the ends of linear chromosomes, have critical roles in genome stability, cancer, and aging. Early work by B. McClintock and H.J. Muller demonstrated that eukaryotic chromosome ends contain specialized structures that prevent recognition and processing by the DNA repair machinery. The importance of these structures is illustrated by studies showing that loss of chromosome end protection results in massive genome instability and cell death. Although Muller and McClintock's initial observations were made several decades ago, little progress has been made in understanding the molecular markers that distinguish naturally occurring chromosome ends from de novo DNA double strand breaks, especially in humans. Using a novel system to specifically target proteins of interest to human telomeres, we have uncovered a role for hRAP1 in protecting telomeres from non-homologous end joining (NHEJ). We find that telomeric DNA containing hRAP1, but not TRF2, is protected from NHEJ in vitro. Furthermore, we show that telomeres containing TRF2 but not hRAP1 can be fused by NHEJ in vivo, and we also demonstrate that targeting hRAP1 to telomeres in vivo, even when TRF2 is not detected, is sufficient to protect telomeres from NHEJ. These results identify hRAP1 as a critical mediator of telomere protection and genome stability in humans. Related to this work, we have also identified a new type of telomere dysfunction associated with semi-conservative replication stress at human telomeres. This new type of telomere dysfunction is telomerase and NHEJ-independent and may require the RecQ helicase WRN for its formation, suggesting that it is related to telomere entanglements observed upon induction of replication stress in fission yeast. The finding that this type of dysfunction is conserved from yeast to man is a testament to the underappreciated role of semi-conservative DNA synthesis in maintaining telomere structure and function

Human RAP1 inhibits non-homologous end joining at telomeres

Telomeres, the nucleoprotein structures at the ends of linear chromosomes, promote genome stability by distinguishing chromosome termini from DNA double-strand breaks (DSBs). Cells possess two principal pathways for DSB repair: homologous recombination and non-homologous end joining (NHEJ). Several studies have implicated TRF2 in the protection of telomeres from NHEJ, but the underlying mechanism remains poorly understood. Here, we show that TRF2 inhibits NHEJ, in part, by recruiting human RAP1 to telomeres. Heterologous targeting of hRAP1 to telomeric DNA was sufficient to bypass the need for TRF2 in protecting telomeric DNA from NHEJ in vitro. On expanding these studies in cells, we find that recruitment of hRAP1 to telomeres prevents chromosome fusions caused by the loss of TRF2/hRAP1 from chromosome ends despite activation of a DNA damage response. These results provide the first evidence that hRAP1 inhibits NHEJ at mammalian telomeres and identify hRAP1 as a mediator of genome stability

Short H2A histone variants are expressed in cancer

International audienceShort H2A (sH2A) histone variants are primarily expressed in the testes of placental mammals. Their incorporation into chromatin is associated with nucleosome destabilization and modulation of alternate splicing. Here, we show that sH2As innately possess features similar to recurrent oncohistone mutations associated with nucleosome instability. Through analyses of existing cancer genomics datasets, we find aberrant sH2A upregulation in a broad array of cancers, which manifest splicing patterns consistent with global nucleosome destabilization. We posit that short H2As are a class of "ready-made" oncohistones, whose inappropriate expression contributes to chromatin dysfunction in cancer

Automated in situ chromatin profiling efficiently resolves cell types and gene regulatory programs

Abstract Background Our understanding of eukaryotic gene regulation is limited by the complexity of protein–DNA interactions that comprise the chromatin landscape and by inefficient methods for characterizing these interactions. We recently introduced CUT&RUN, an antibody-targeted nuclease cleavage method that profiles DNA-binding proteins, histones and chromatin-modifying proteins in situ with exceptional sensitivity and resolution. Results Here, we describe an automated CUT&RUN platform and apply it to characterize the chromatin landscapes of human cells. We find that automated CUT&RUN profiles of histone modifications crisply demarcate active and repressed chromatin regions, and we develop a continuous metric to identify cell-type-specific promoter and enhancer activities. We test the ability of automated CUT&RUN to profile frozen tumor samples and find that our method readily distinguishes two pediatric glioma xenografts by their subtype-specific gene expression programs. Conclusions The easy, cost-effective workflow makes automated CUT&RUN an attractive tool for high-throughput characterization of cell types and patient samples

Optimal therapeutic targeting by HDAC inhibition in biopsy-derived treatment-naïve diffuse midline glioma models

BACKGROUND Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine gliomas (DIPGs), have a dismal prognosis with less than 2% surviving 5-years post-diagnosis. The majority of DIPGs and all DMGs harbor mutations altering the epigenetic regulatory histone tail (H3 K27M). Investigations addressing DMG epigenetics have identified few promising drugs, including the HDAC inhibitor (HDACi) panobinostat. Here, we use clinically-relevant DMG models to identify and validate other effective HDACi and their biomarkers of response. METHODS HDACi were tested across biopsy-derived treatment-naïve in vitro and in vivo DMG models with biologically-relevant radiation-resistance. RNA sequencing was performed to define and compare drug efficacy, and to map predictive biomarkers of response. RESULTS Quisinostat and romidepsin showed efficacy with a low nanomolar IC50 values (~50 and ~5 nM, respectively). Comparative transcriptome analyses across quisinostat, romidepsin, and panobinostat showed a greater degree of shared biological effects between quisinostat and panobinostat, and less overlap with romidepsin. However, some transcriptional changes were consistent across all three drugs at similar biologically effective doses, such as overexpression of TNNT1 and downregulation of COL20A1, identifying these as potential vulnerabilities or on-target biomarkers in DMG. Quisinostat and romidepsin significantly (p <0.0001) inhibited in vivo tumor growth. CONCLUSIONS Our data highlights the utility of treatment-naïve biopsy-derived models; establishes quisinostat and romidepsin as effective in vivo; illuminates potential mechanisms and/or biomarkers of DMG cell lethality due to HDAC inhibition; and emphasizes the need for brain-tumor-penetrant versions of potentially efficacious agents

CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy