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Amplification of MDM2 on supernumerary chromosomes is a common mechanism of P53 inactivation across tumors. Here, we investigated the impact of MDM2 overexpression on chromatin, gene expression, and cellular phenotypes in liposarcoma. Three independent regulatory circuits predominate in aggressive, dedifferentiated tumors. RUNX and AP-1 family transcription factors bind mesenchymal gene enhancers. P53 and MDM2 co-occupy enhancers and promoters associated with P53 signaling. When highly expressed, MDM2 also binds thousands of P53-independent growth and stress response genes, whose promoters engage in multi-way topological interactions. Overexpressed MDM2 concentrates within nuclear foci that co-localize with PML and YY1 and could also contribute to P53-independent phenotypes associated with supraphysiologic MDM2. Importantly, we observe striking cell-to-cell variability in MDM2 copy number and expression in tumors and models. Whereas liposarcoma cells are generally sensitive to MDM2 inhibitors and their combination with pro-apoptotic drugs, MDM2-high cells tolerate them and may underlie the poor clinical efficacy of these agents., Competing Interests: B.E.B. declares outside interests in Fulcrum Therapeutics, Arsenal Biosciences, HiFiBio, Cell Signaling Technologies, Design Pharmaceuticals, and Chroma Medicine. G.D.D. reports leadership as co-founder of IDRX; stocks/options/shares in IDRX, Blueprint Medicines, G1 Therapeutics, Caris Life Sciences, Erasca Pharmaceuticals, RELAY Therapeutics, Bessor Pharmaceuticals, CellCarta, IKENA Oncology, and Kojin Therapeutics; paid consulting fees from Bayer, Pfizer, Novartis, Roche/Genentech, GSK, PharmaMar, Daiichi Sankyo, EMD-Serono/Merck KGaA, Mirati, WCG/Arsenal Capital, G1 Therapeutics, Caris Life Sciences, RELAY Therapeutics, CellCarta, IKENA Oncology, Kojin Therapeutics, RAIN Therapeutics, Jazz Pharmaceuticals, Aadi Biosciences, and IDRX; royalties, patents, or licenses from Novartis to Dana-Farber Cancer Institute for “use patent” of imatinib in GIST; and non-financial interests in AACR Science Policy and Government Affairs Committee and Alexandria Real Estate Equities summit conference series., (© 2023 The Author(s).)

A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance., Competing Interests: Declaration of interests J.K.N., J.C., G.C.P., L.A.G., and J.S.W. have filed patent applications related to CRISPRoff, CRISPRon, and CRISPRi/a screening. J.M.R. consults for Maze Therapeutics. L.A.G., J.S.W., H.Y.C., and B.E.B. consult for and hold equity in Chroma Medicine. J.S.W. declares outside interest in KSQ Therapeutics, Maze Therapeutics, Amgen, and Tessera Therapeutics. M.K. serves on the Scientific Advisory Boards of Engine Biosciences, Casma Therapeutics, and Cajal Neuroscience. B.E.B. declares outside interests in Fulcrum Therapeutics, Arsenal Biosciences, HiFiBio, and Cell Signaling Technologies. H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, and is an advisor for 10x Genomics, Arsenal Biosciences, and Spring Discovery., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic., Competing Interests: Declaration of Interests N.H. is an equity holder of BioNTech and a consultant for Related Sciences. M.J.A. declares outside interest in Excelsior Genomics. B.E.B. declares outside interests in Fulcrum Therapeutics, 1CellBio, HiFiBio, Arsenal Biosciences, Cell Signaling Technologies, BioMillenia, and Nohla Therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

NEAT1 is one of the most studied lncRNAs, in part because its silencing in mice causes defects in mammary gland development and corpus luteum formation and protects them from skin cancer development. Moreover, depleting NEAT1 in established cancer cell lines reduces growth and sensitizes cells to DNA damaging agents. However, NEAT1 produces two isoforms and because the short isoform, NEAT1_1 , completely overlaps the 5' part of the long NEAT1_2 isoform; the respective contributions of each of the isoforms to these phenotypes has remained unclear. Whereas NEAT1_1 is highly expressed in most tissues, NEAT1_2 is the central architectural component of paraspeckles, which are nuclear bodies that assemble in specific tissues and cells exposed to various forms of stress. Using dual RNA-FISH to detect both NEAT1_1 outside of the paraspeckles and NEAT1_2/NEAT1 inside this nuclear body, we report herein that NEAT1_1 levels are dynamically regulated during the cell cycle and targeted for degradation by the nuclear RNA exosome. Unexpectedly, however, cancer cells engineered to lack NEAT1_1 , but not NEAT1_2 , do not exhibit cell cycle defects. Moreover, Neat1_1 -specific knockout mice do not exhibit the phenotypes observed in Neat1 -deficient mice. We propose that NEAT1 functions are mainly, if not exclusively, attributable to NEAT1_2 and, by extension, to paraspeckles., (© 2019 Adriaens et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The past decades have provided remarkable insights into how the eukaryotic cell nucleus and the genome within it are organized. The combined use of imaging, biochemistry and molecular biology approaches has revealed several basic principles of nuclear architecture and function, including the existence of chromatin domains of various sizes, the presence of a large number of non-membranous intranuclear bodies, non-random positioning of genes and chromosomes in 3D space, and a prominent role of the nuclear lamina in organizing genomes. Despite this tremendous progress in elucidating the biological properties of the cell nucleus, many questions remain. Here, we highlight some of the key open areas of investigation in the field of nuclear organization and genome architecture with a particular focus on the mechanisms and principles of higher-order genome organization, the emerging role of liquid phase separation in cellular organization, and the functional role of the nuclear lamina in physiological processes.

Many patients with advanced cancers achieve dramatic responses to a panoply of therapeutics yet retain minimal residual disease (MRD), which ultimately results in relapse. To gain insights into the biology of MRD, we applied single-cell RNA sequencing to malignant cells isolated from BRAF mutant patient-derived xenograft melanoma cohorts exposed to concurrent RAF/MEK-inhibition. We identified distinct drug-tolerant transcriptional states, varying combinations of which co-occurred within MRDs from PDXs and biopsies of patients on treatment. One of these exhibited a neural crest stem cell (NCSC) transcriptional program largely driven by the nuclear receptor RXRG. An RXR antagonist mitigated accumulation of NCSCs in MRD and delayed the development of resistance. These data identify NCSCs as key drivers of resistance and illustrate the therapeutic potential of MRD-directed therapy. They also highlight how gene regulatory network architecture reprogramming may be therapeutically exploited to limit cellular heterogeneity, a key driver of disease progression and therapy resistance., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In a search for mediators of the p53 tumor suppressor pathway, which induces pleiotropic and often antagonistic cellular responses, we identified the long noncoding RNA (lncRNA) NEAT1. NEAT1 is an essential architectural component of paraspeckle nuclear bodies, whose pathophysiological relevance remains unclear. Activation of p53, pharmacologically or by oncogene-induced replication stress, stimulated the formation of paraspeckles in mouse and human cells. Silencing Neat1 expression in mice, which prevents paraspeckle formation, sensitized preneoplastic cells to DNA-damage-induced cell death and impaired skin tumorigenesis. We provide mechanistic evidence that NEAT1 promotes ATR signaling in response to replication stress and is thereby engaged in a negative feedback loop that attenuates oncogene-dependent activation of p53. NEAT1 targeting in established human cancer cell lines induced synthetic lethality with genotoxic chemotherapeutics, including PARP inhibitors, and nongenotoxic activation of p53. This study establishes a key genetic link between NEAT1 paraspeckles, p53 biology and tumorigenesis and identifies NEAT1 as a promising target to enhance sensitivity of cancer cells to both chemotherapy and p53 reactivation therapy.