SUMMARYIn the developing retina, multipotent neural progenitors undergo unidirectional differentiation in a precise spatiotemporal order. Here we profile the epigenetic and transcriptional changes that occur during retinogenesis in mice and humans. Although some progenitor genes and cell cycle genes were epigenetically silenced during retinogenesis, the most dramatic change was derepression of cell type–specific differentiation programs. We identified developmental stage–specific super-enhancers and showed that most epigenetic changes are conserved in humans and mice. To determine how the epigenome changes during tumorigenesis and reprogramming, we performed integrated epigenetic analysis of murine and human retinoblastomas and induced pluripotent stem cells (iPSCs) derived from murine rod photoreceptors. The retinoblastoma epigenome mapped to the developmental stage when retinal progenitors switch from neurogenic to a terminal patterns of cell division. The epigenome of retinoblastomas was more similar to that of normal retina than was that of retina-derived iPSCs, and we identified retina-specific epigenetic memory.
Ewing sarcoma (EWS) is a tumor of the bone and soft-tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using 3 different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.
Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G/M pathway is the most effective approach in vivo for high-risk RMS.
Summary Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known if the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated iPSCs murine rod photoreceptors (r-iPSCs) and scored their ability to make retina using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs were more efficient at producing differentiated retina than embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had a reduction in amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod specific CTCF insulator protein binding sites may promote retinogenes in r-iPSCs. Taken together, our data suggest that the source of stem cells are important for producing retinal neurons in 3D organ cultures.
Highlights d There are dynamic changes in chromatin compartments and looping during retinogenesis d Euchromatin and heterochromatin localization can be predicted by machine learning d Vsx2 has a cell-type-and stage-specific core regulatory circuit super-enhancer d Vsx2 core regulatory circuit super-enhancer deletion eliminates bipolar neurons
Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumorsuppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.
DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.Oxidative DNA damage, which occurs frequently in all cells, is linked to aging and human disease, such as cancer and various degenerative disorders (6,13,45,81,83). Reactive oxygen species (ROS) are a by-product of normal cellular metabolic processes that can cause oxidative damage to DNA, lipids, and proteins (79). Unrepaired oxidative DNA lesions can result in mutations and lead to arrest of both DNA replication and transcription (34). In order to combat such continuous insults to the genome, cells have evolved DNA repair and DNA damage tolerance pathways (2).Base excision repair (BER) is the primary process by which oxidative DNA damage is repaired (74,90). BER is initiated by the recognition and excision of a base lesion by an N-glycosylase, resulting in an apurinic/apyrimidinic (AP) site (47, 48). The resulting AP site is processed by an AP endonuclease or an AP lyase, which cleaves the sugar-phosphate DNA backbone on the 5Ј side or 3Ј side of the AP site, respectively (5). Subsequent processing involving DNA repair polymerases replaces the excised nucleotides, and DNA ligase completes the repair process (8).Very little is known about how eukaryotic cells regulate events that initiate BER in response to oxidative stress. Deleterious oxidative DNA damage can occur in both nuclear and mitochondrial genomes, adding a level of complexity to this cellular response. In this case, the intracellular localization of BER proteins would be regulated dynamically in response to the introduction of either nuclear or mitochondrial DNA damage. Controlled protein localization has been implicate...
Highlights d ATRX IFFs are redistributed genome wide and are enriched at active promoters d The neuronal silencing transcription factor REST is an ATRX IFF target gene d REST and EZH2 silence neuronal gene programs in ATRX IFF NB d REST loss or EZH2 inhibition induces neuronal gene expression programs and NB cell death
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