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Biochemical crosstalk between two or more histone modifications is often observed in epigenetic enzyme regulation, but its functional significance in cells has been difficult to discern. Previous enzymatic studies revealed that Lys14 acetylation of histone H3 can inhibit Lys4 demethylation by lysine-specific demethylase 1 (LSD1). In the present study, we engineered a mutant form of LSD1, Y391K, which renders the nucleosome demethylase activity of LSD1 insensitive to Lys14 acetylation. K562 cells with the Y391K LSD1 CRISPR knockin show decreased expression of a set of genes associated with cellular adhesion and myeloid leukocyte activation. Chromatin profiling revealed that the cis-regulatory regions of these silenced genes display a higher level of H3 Lys14 acetylation, and edited K562 cells show diminished H3 mono-methyl Lys4 near these silenced genes, consistent with a role for enhanced LSD1 demethylase activity. These findings illuminate the functional consequences of disconnecting histone modification crosstalk for a key epigenetic enzyme., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Transcriptional coregulators and transcription factors (TFs) contain intrinsically disordered regions (IDRs) that are critical for their association and function in gene regulation. More recently, IDRs have been shown to promote multivalent protein-protein interactions between coregulators and TFs to drive their association into condensates. By contrast, here we demonstrate how the IDR of the corepressor LSD1 excludes TF association, acting as a dynamic conformational switch that tunes repression of active cis-regulatory elements. Hydrogen-deuterium exchange shows that the LSD1 IDR interconverts between transient open and closed conformational states, the latter of which inhibits partitioning of the protein's structured domains with TF condensates. This autoinhibitory switch controls leukemic differentiation by modulating repression of active cis-regulatory elements bound by LSD1 and master hematopoietic TFs. Together, these studies unveil alternative mechanisms by which disordered regions and their dynamic crosstalk with structured regions can shape coregulator-TF interactions to control cis-regulatory landscapes and cell fate., Competing Interests: Declaration of interests B.B.L. has received research funding from Eisai and AstraZeneca and is a shareholder and member of the scientific advisory board of Light Horse Therapeutics. P.A.C. is a cofounder of Acylin Therapeutics (advisor and equity holder) and has been a paid consultant for the following epigenetics companies: Epizyme, GSK, and Constellation (now MorphoSys). S.A.C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, Seer, and PrognomIQ., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity., Competing Interests: Declaration of interests D.E.F. has a financial interest in Soltego. S.M. and D.A.H. are cofounders of TellBio. G.M.B. has sponsored research agreements with Olink Proteomics, Teiko Bio, InterVenn Biosciences, and Palleon Pharmaceuticals; served on advisory boards for Iovance, Merck, Nektar Therapeutics, Novartis, and Ankyra Therapeutics; consults for Merck, InterVenn Biosciences, Iovance, and Ankyra Therapeutics; and holds equity in Ankyra Therapeutics. M.S.-F. received funding from Calico Life Sciences, Bristol-Myers Squibb, Istari Oncology, and consultants for Galvanize Therapeutics. R.W.J. is a member of the advisory board for/has a financial interest in Xsphera Biosciences Inc. R.W.J. has received honoraria from Incyte (invited speaker), G1 Therapeutics (advisory board), and Bioxcel Therapeutics (invited speaker). R.W.J. has ownership interest in U.S. patents US20200399573A9 and US20210363595A1. A.N.H. has received grants/research support from Amgen, Blueprint Medicines, BridgeBio, Bristol-Myers Squibb, C4 Therapeutics, Eli Lilly, Novartis, Nuvalent, Pfizer, Roche/Genentech, and Scorpion Therapeutics. A.N.H. has served as a compensated consultant for Amgen, Engine Biosciences, Nuvalent, Oncovalent, Pfizer, TigaTx, and Tolremo Therapeutics. M.L.S. is an equity holder and scientific co-founder/advisory board member of Immunitas Therapeutics. C.J.O. received funding from Scorpion Therapeutics, Gilead Sciences, and eFFECTOR Therapeutics. L.B.-P. is a founder/consultant/holds privately held equity in Scorpion Therapeutics. All relationships for investigators have been reviewed/managed by their respective institutions in accordance with their conflict-of-interest policies. M.M., B.H., R.D.W., H.E.M., C.-C.T., G.K., N.B., J.R.N., and B.R.M. are employees of Scorpion Therapeutics and some hold equity., (Copyright © 2024 Elsevier Inc. All rights reserved.)

DNA methyltransferase 3A (DNMT3A) is a de novo cytosine methyltransferase responsible for establishing proper DNA methylation during mammalian development. Loss-of-function (LOF) mutations to DNMT3A, including the hotspot mutation R882H, frequently occur in developmental growth disorders and hematological diseases, including clonal hematopoiesis and acute myeloid leukemia. Accordingly, identifying mechanisms that activate DNMT3A is of both fundamental and therapeutic interest. Here, we applied a base editor mutational scanning strategy with an improved DNA methylation reporter to systematically identify DNMT3A activating mutations in cells. By integrating an optimized cellular recruitment strategy with paired isogenic cell lines with or without the LOF hotspot R882H mutation, we identify and validate three distinct hyperactivating mutations within or interacting with the regulatory ADD domain of DNMT3A, nominating these regions as potential functional target sites for pharmacological intervention. Notably, these mutations are still activating in the context of a heterozygous R882H mutation. Altogether, we showcase the utility of base editor scanning for discovering functional regions of target proteins.

Drug addiction, a phenomenon where cancer cells paradoxically depend on continuous drug treatment for survival, has uncovered cell signaling mechanisms and cancer codependencies. Here we discover mutations that confer drug addiction to inhibitors of the transcriptional repressor polycomb repressive complex 2 (PRC2) in diffuse large B-cell lymphoma. Drug addiction is mediated by hypermorphic mutations in the CXC domain of the catalytic subunit EZH2, which maintain H3K27me3 levels even in the presence of PRC2 inhibitors. Discontinuation of inhibitor treatment leads to overspreading of H3K27me3, surpassing a repressive methylation ceiling compatible with lymphoma cell survival. Exploiting this vulnerability, we show that inhibition of SETD2 similarly induces the spread of H3K27me3 and blocks lymphoma growth. Collectively, our findings demonstrate that constraints on chromatin landscapes can yield biphasic dependencies in epigenetic signaling in cancer cells. More broadly, we highlight how approaches to identify drug addiction mutations can be leveraged to discover cancer vulnerabilities., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

A fundamental challenge in biology is understanding the molecular details of protein function. How mutations alter protein activity, regulation, and response to drugs is of critical importance to human health. Recent years have seen the emergence of pooled base editor screens for in situ mutational scanning: the interrogation of protein sequence-function relationships by directly perturbing endogenous proteins in live cells. These studies have revealed the effects of disease-associated mutations, discovered novel drug resistance mechanisms, and generated biochemical insights into protein function. Here, we discuss how this "base editor scanning" approach has been applied to diverse biological questions, compare it with alternative techniques, and describe the emerging challenges that must be addressed to maximize its utility. Given its broad applicability toward profiling mutations across the proteome, base editor scanning promises to revolutionize the investigation of proteins in their native contexts., Competing Interests: Declaration of interests B.B.L. holds sponsored research projects with Eisai and AstraZeneca, is a scientific consultant for Imago BioSciences and Exo Therapeutics, and is a shareholder and member of the scientific advisory board of Light Horse Therapeutics., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H 2 O 2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H 2 O 2 . Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H 2 O 2 accumulation and mediate resistance to platinum-based agents in ovarian cancers., Competing Interests: Declaration of interests A.P.P., C.M.S., M.P.S., and S.A.B. are employees of Cell Signaling Technology, Inc. L.B.-P. is a founder, consultant, and holds privately held equity in Scorpion Therapeutics., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite the fundamental importance of allosteric sites, their identification remains highly challenging. Here, we perform CRISPR scanning on the essential maintenance methylation machinery-DNMT1 and its partner UHRF1-with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. In contrast to non-covalent DNMT1 inhibition, activity-based selection implicates numerous regions outside the catalytic domain in DNMT1 function. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function, exhibiting increased DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD-PBR interface. Collectively, our study highlights the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, and more broadly, introduces new analytical tools that further refine the CRISPR scanning framework., Competing Interests: KN, SH, HK, NL, PG, DT, EG, CL No competing interests declared, BL holds sponsored research projects with Eisai and AstraZeneca, is a scientific consultant for Imago BioSciences and Exo Therapeutics, and is a shareholder and member of the scientific advisory board of Light Horse Therapeutics, (© 2023, Ngan et al.)

DNA methylation is critical for regulating gene expression, necessitating its accurate placement by enzymes such as the DNA methyltransferase DNMT3A. Dysregulation of this process is known to cause aberrant development and oncogenesis, yet how DNMT3A is regulated holistically by its three domains remains challenging to study. Here, we integrate base editing with a DNA methylation reporter to perform in situ mutational scanning of DNMT3A in cells. We identify mutations throughout the protein that perturb function, including ones at an interdomain interface that block allosteric activation. Unexpectedly, we also find mutations in the PWWP domain, a histone reader, that modulate enzyme activity despite preserving histone recognition and protein stability. These effects arise from altered PWWP domain DNA affinity, which we show is a noncanonical function required for full activity in cells. Our findings highlight mechanisms of interdomain crosstalk and demonstrate a generalizable strategy to probe sequence-activity relationships of nonessential chromatin regulators., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Chromatin regulators are frequently mutated in human cancer and are attractive drug targets. They include diverse proteins that share functional domains and assemble into related multi-subunit complexes. To investigate functional relationships among these regulators, here we apply combinatorial CRISPR knockouts (KOs) to test over 35,000 gene-gene pairings in leukemia cells, using a library of over 300,000 constructs. Top pairs that demonstrate either compensatory non-lethal interactions or synergistic lethality enrich for paralogs and targets that occupy the same protein complex. The screen highlights protein complex dependencies not apparent in single KO screens, for example MCM histone exchange, the nucleosome remodeling and deacetylase (NuRD) complex, and HBO1 (KAT7) complex. We explore two approaches to NuRD complex inactivation. Paralog and non-paralog combinations of the KAT7 complex emerge as synergistic lethal and specifically nominate the ING5 PHD domain as a potential therapeutic target when paired with other KAT7 complex member losses. These findings highlight the power of combinatorial screening to provide mechanistic insight and identify therapeutic targets within redundant networks., (© 2023. The Author(s).)

CRISPR-Cas9 genome editing technologies have enabled complex genetic manipulations in situ, including large-scale, pooled screening approaches to probe and uncover mechanistic insights across various biological processes. The RNA-programmable nature of CRISPR-Cas9 greatly empowers tiling mutagenesis approaches to elucidate molecular details of protein function, in particular the interrogation of mechanisms of resistance to small molecules, an approach termed CRISPR-suppressor scanning. In a typical CRISPR-suppressor scanning experiment, a pooled library of single-guide RNAs is designed to target across the coding sequence(s) of one or more genes, enabling the Cas9 nuclease to systematically mutate the targeted proteins and generate large numbers of diverse protein variants in situ. This cellular pool of protein variants is then challenged with drug treatment to identify mutations conferring a fitness advantage. Drug-resistance mutations identified with this approach can not only elucidate drug mechanism of action but also reveal deeper mechanistic insights into protein structure-function relationships. In this article, we outline the framework for a standard CRISPR-suppressor scanning experiment. Specifically, we provide instructions for the design and construction of a pooled sgRNA library, execution of a CRISPR-suppressor scanning screen, and basic computational analysis of the resulting data. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Design and generation of a pooled sgRNA library Support Protocol 1: sgRNA library design using command-line CRISPOR Support Protocol 2: Production and titering of pooled sgRNA library lentivirus Basic Protocol 2: Execution and analysis of a CRISPR-suppressor scanning experiment., (© 2022 Wiley Periodicals LLC.)

The genome can be divided into two spatially segregated compartments, A and B, which partition active and inactive chromatin states. While constitutive heterochromatin is predominantly located within the B compartment near the nuclear lamina, facultative heterochromatin marked by H3K27me3 spans both compartments. How epigenetic modifications, compartmentalization, and lamina association collectively maintain heterochromatin architecture remains unclear. Here we develop Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) to jointly measure chromosome conformation, DNA methylation, and lamina positioning. Through LIMe-Hi-C, we identify topologically distinct sub-compartments with high levels of H3K27me3 and differing degrees of lamina association. Inhibition of Polycomb repressive complex 2 (PRC2) reveals that H3K27me3 is essential for sub-compartment segregation. Unexpectedly, PRC2 inhibition promotes lamina association and constitutive heterochromatin spreading into H3K27me3-marked B sub-compartment regions. Consistent with this repositioning, genes originally marked with H3K27me3 in the B compartment, but not the A compartment, remain largely repressed, suggesting that constitutive heterochromatin spreading can compensate for H3K27me3 loss at a transcriptional level. These findings demonstrate that Polycomb sub-compartments and their antagonism with lamina association are fundamental features of genome structure. More broadly, by jointly measuring nuclear position and Hi-C contacts, our study demonstrates how compartmentalization and lamina association represent distinct but interdependent modes of heterochromatin regulation., (© 2022. The Author(s).)

Targeted protein degradation (TPD) holds immense promise for drug discovery, but mechanisms of acquired resistance to degraders remain to be fully identified. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-suppressor scanning to identify mechanistic classes of drug resistance mutations to molecular glue degraders in GSPT1 and RBM39, neosubstrates targeted by E3 ligase substrate receptors cereblon and DCAF15, respectively. While many mutations directly alter the ternary complex heterodimerization surface, distal resistance sites were also identified. Several distal mutations in RBM39 led to modest decreases in degradation, yet can enable cell survival, underscoring how small differences in degradation can lead to resistance. Integrative analysis of resistance sites across GSPT1 and RBM39 revealed varying levels of sequence conservation and mutational constraint that control the emergence of different resistance mechanisms, highlighting that many regions co-opted by TPD are nonessential. Altogether, our study identifies common resistance mechanisms for molecular glue degraders and outlines a general approach to survey neosubstrate requirements necessary for effective degradation., Competing Interests: The authors declare the following competing financial interest(s): Brian Liau is on the scientific advisory board of H3 Biomedicine., (© 2022 The Authors. Published by American Chemical Society.)

Small molecule drugs form the backbone of modern medicine's therapeutic arsenal. Often less appreciated is the role that small molecules have had in advancing basic biology. In this Review, we highlight how resistance mutations have unlocked the potential of small molecule chemical probes to discover new biology. We describe key instances in which resistance mutations and related genetic variants yielded foundational biological insight and categorize these examples on the basis of their role in the discovery of novel molecular mechanisms, protein allostery, physiology and cell signaling. Next, we suggest ways in which emerging technologies can be leveraged to systematically introduce and characterize resistance mutations to catalyze basic biology research and drug discovery. By recognizing how resistance mutations have propelled biological discovery, we can better harness new technologies and maximize the potential of small molecules to advance our understanding of biology and improve human health., (© 2021. Springer Nature America, Inc.)

Employed for over half a century to study protein synthesis, cycloheximide (CHX, 1 ) is a small molecule natural product that reversibly inhibits translation elongation. More recently, CHX has been applied to ribosome profiling, a method for mapping ribosome positions on mRNA genome-wide. Despite CHX's extensive use, CHX treatment often results in incomplete translation inhibition due to its rapid reversibility, prompting the need for improved reagents. Here, we report the concise synthesis of C13-amide-functionalized CHX derivatives with increased potencies toward protein synthesis inhibition. Cryogenic electron microscopy (cryo-EM) revealed that C13-aminobenzoyl CHX ( 8 ) occupies the same site as CHX, competing with the 3' end of E-site tRNA. We demonstrate that 8 is superior to CHX for ribosome profiling experiments, enabling more effective capture of ribosome conformations through sustained stabilization of polysomes. Our studies identify powerful chemical reagents to study protein synthesis and reveal the molecular basis of their enhanced potency.

Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/β-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. SIGNIFICANCE: Oncogenic G9a abnormalities drive tumorigenesis and the "cold" immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore "hot" tumor immune microenvironments. This article is highlighted in the In This Issue feature, p. 890 ., (©2020 American Association for Cancer Research.)

Understanding the mechanism of small molecules is a critical challenge in chemical biology and drug discovery. Medicinal chemistry is essential for elucidating drug mechanism, enabling variation of small molecule structure to gain structure-activity relationships (SARs). However, the development of complementary approaches that systematically vary target protein structure could provide equally informative SARs for investigating drug mechanism and protein function. Here we explore the ability of CRISPR-Cas9 mutagenesis to profile the interactions between lysine-specific histone demethylase 1 (LSD1) and chemical inhibitors in the context of acute myeloid leukemia (AML). Through this approach, termed CRISPR-suppressor scanning, we elucidate drug mechanism of action by showing that LSD1 enzyme activity is not required for AML survival and that LSD1 inhibitors instead function by disrupting interactions between LSD1 and the transcription factor GFI1B on chromatin. Our studies clarify how LSD1 inhibitors mechanistically operate in AML and demonstrate how CRISPR-suppressor scanning can uncover novel aspects of target biology.

Cycloheximide (CHX) is an inhibitor of eukaryotic translation elongation that has played an essential role in the study of protein synthesis. Despite its ubiquity, few studies have been directed towards accessing synthetic CHX derivatives, even though such efforts may lead to protein synthesis inhibitors with improved or alternate properties. Described here is the total synthesis of CHX and analogues, and the establishment of structure-activity relationships (SAR) responsible for translation inhibition. The SAR studies aided the design of more potent compounds, one of which irreversibly blocks ribosomal elongation, preserves polysome profiles, and may be a broadly useful tool for investigating protein synthesis., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of 'cancer dependencies' not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.

McClary et al. (2017) identify the eukaryotic ribosome as a cellular target of agelastatin A, resolving the long-standing mystery surrounding the cytotoxic natural product's mechanism of action. Structural and modeling studies further pinpointed the molecule's binding site to the ribosome peptidyl transferase center, revealing key molecular interactions that drive binding., (Copyright © 2017. Published by Elsevier Ltd.)

Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show that GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors. In this state, GSCs upregulate primitive developmental programs and are dependent upon Notch signaling. This transition is accompanied by widespread redistribution of repressive histone methylation. Accordingly, persister GSCs upregulate, and are dependent on, the histone demethylases KDM6A/B. Slow-cycling cells with high Notch activity and histone demethylase expression are present in primary glioblastomas before treatment, potentially contributing to relapse. Our findings illustrate how cancer cells may hijack aspects of native developmental programs for deranged proliferation, adaptation, and tolerance. They also suggest strategies for eliminating refractory tumor cells by targeting epigenetic and developmental pathways., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor-initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH.

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.

A unique subset of the Lycopodium alkaloid natural products share a 7-membered-ring substructure and may potentially arise from a common biosynthetic precursor. To both explore and exploit these structural relationships, we sought to develop a unified biosynthetically inspired strategy to efficiently access these complex polycyclic alkaloids through the use of a cascade sequence. In pursuit of these goals, the first total synthesis of (+)-fastigiatine (2) was accomplished via a series of cascade reactions; we describe herein a full account of our efforts. Insight from these endeavors led to critical modifications of our synthetic strategy, which enabled the first total syntheses of (-)-himeradine A (1), (-)-lycopecurine (3), and (-)-dehydrolycopecurine (4), as well as the syntheses of (+)-lyconadin A (5) and (-)-lyconadin B (6). Our approach features a diastereoselective one-pot sequence for constructing the common 7-membered-ring core system, followed by either a biomimetic transannular Mannich reaction to access himeradine A (1), lycopecurine (3), and dehydrolycopecurine (4) or an imine reduction for lyconadins A (5) and B (6). This strategy may potentially enable access to all 7-membered-ring-containing Lycopodium alkaloids and provides additional insight into their biosynthetic origin.

Total syntheses of HMP-Y1, atrop-HMP-Y1, hibarimicinone, atrop-hibarimicinone, and HMP-P1 are described using a two-directional synthesis strategy. A novel benzyl fluoride Michael-Claisen reaction sequence was developed to construct the complete carbon skeleton of HMP-Y1 and atrop-HMP-Y1 via a symmetrical, two-directional, double annulation. Through efforts to convert HMP-Y1 derivatives to hibarimicinone and HMP-P1, a biomimetic mono-oxidation to desymmetrize protected HMP-Y1 was realized. A two-directional unsymmetrical double annulation and biomimetic etherification was developed to construct the polycyclic and highly oxidized skeleton of hibarimicinone, atrop-hibarimicinone, and HMP-P1. The use of a racemic biaryl precursor allowed for the synthesis of both hibarimicinone atropisomers and provides the first confirmation of the structure of atrop-hibarimicinone. Additionally, this work documents the first reported full characterization of atrop-hibarimicinone, HMP-Y1, atrop-HMP-Y1, and HMP-P1. Last, a pH-dependent rotational barrier about the C2-C2' bond of hibarimicinone was discovered, which provides valuable information necessary to achieve syntheses of the glycosylated congeners of hibarimicinone.

A gram-scale enantiospecific synthesis of the A'B'-subunit of angelmicin B is reported. The synthesis involves a Lewis acid catalyzed contrasteric Diels-Alder reaction and a tandem silyl zincate 1,6-addition/enolate oxidation sequence.

The first total synthesis of the Lycopodium alkaloid (+)-fastigiatine has been accomplished in 15 steps and 30% overall yield from known compounds. Noteworthy transformations include a convergent fragment coupling via a nucleophilic cyclopropane opening, a highly diastereoselective formal [3 + 3]-cycloaddition, and a transannular Mannich reaction to construct the core of the natural product.

An analysis is presented to explain the faster rate of ring formation 5 --> 7 as compared to 6 --> 8.