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Unique Molecular Identifiers (UMIs) are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are the main source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing UMIs using homotrimeric nucleotide blocks provides an error correcting solution, that allows absolute counting of sequenced molecules.

Chordoma RNAseq_all cell lines_gene lists

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Hypoxia is a common phenomenon in solid tumors and is strongly linked to hallmarks of cancer. Recent evidence has shown that hypoxia promotes local immune suppression. Type I IFN supports cytotoxic T lymphocytes by stimulating the maturation of dendritic cells and enhancing their capacity to process and present antigens. However, little is known about the relationship between hypoxia and the type I IFN pathway, which comprises the sensing of double-stranded RNA and DNA (dsRNA/dsDNA) followed by IFNα/β secretion and transcriptional activation of IFN-stimulated genes (ISG). In this study, we determined the effects of hypoxia on the type I IFN pathway in breast cancer and the mechanisms involved. In cancer cell lines and xenograft models, mRNA and protein expressions of the type I IFN pathway were downregulated under hypoxic conditions. This pathway was suppressed at each level of signaling, from the dsRNA sensors RIG-I and MDA5, the adaptor MAVS, transcription factors IRF3, IRF7, and STAT1, and several ISG including RIG-I, IRF7, STAT1, and ADAR-p150. Importantly, IFN secretion was reduced under hypoxic conditions. HIF1α- and HIF2α-mediated regulation of gene expression did not explain most of the effects. However, ATAC-seq data revealed in hypoxia that peaks with STAT1 and IRF3 motifs had decreased accessibility. Collectively, these results indicate that hypoxia leads to an overall downregulation of the type I IFN pathway due to repressed transcription and lower chromatin accessibility in an HIF1/2α-independent manner, which could contribute to immunosuppression in hypoxic tumors.Significance:These findings characterize a new mechanism of immunosuppression by hypoxia via downregulation of the type I IFN pathway and its autocrine/paracrine effects on tumor growth.

Expression of the transcription factor brachyury (TBXT) is normally restricted to the embryo, and its silencing is epigenetically regulated. TBXT promotes mesenchymal transition in a subset of common carcinomas, and in chordoma, a rare cancer showing notochordal differentiation, TBXT acts as a putative oncogene. We hypothesized that TBXT expression is controlled through epigenetic inhibition to promote chordoma cell death. Screening of five human chordoma cell lines revealed that pharmacologic inhibition of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (JMJD3) leads to cell death. This effect was phenocopied by dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Inhibition of KDM6A/B with a novel compound KDOBA67 led to a genome-wide increase in repressive H3K27me3 marks with concomitant reduction in active H3K27ac, H3K9ac, and H3K4me3 marks. TBXT was a KDM6A/B target gene, and chromatin changes at TBXT following KDOBA67 treatment were associated with a reduction in TBXT protein levels in all models tested, including primary patient-derived cultures. In all models tested, KDOBA67 treatment downregulated expression of a network of transcription factors critical for chordoma survival and upregulated pathways dominated by ATF4-driven stress and proapoptotic responses. Blocking the AFT4 stress response did not prevent suppression of TBXT and induction of cell death, but ectopic overexpression of TBXT increased viability, therefore implicating TBXT as a potential therapeutic target of H3K27 demethylase inhibitors in chordoma. Our work highlights how knowledge of normal processes in fetal development can provide insight into tumorigenesis and identify novel therapeutic approaches.Significance:Pharmacologic inhibition of H3K27-demethylases in human chordoma cells promotes epigenetic silencing of oncogenic TBXT, alters gene networks critical to survival, and represents a potential novel therapy.

SummaryEndolysosomes (EL) are known for their role in regulating both intracellular trafficking and proteostasis. EL help facilitate elimination of damaged membrane and cytosolic proteins, protein aggregates, membranous organelles and also play an important role in calcium signalling. Despite the importance of EL, their specific role in cardiovascular disease is not well understood. In particular, it’s unclear how EL contribute to atrial pathology over longer time frames. To shed light on this question, we conducted a comprehensive analysis that involved proteomics, transcriptomics, integrated analysis, electron tomography, western blotting, and enzyme assays. To identify the role of EL in atrial fibrillation (AF), we applied a recently published organelle protein isolation method. We used this method to study biopsies from AF goat model and analyse the EL-specific proteins and pathways involved in this condition. Our results revealed the upregulation of the AMPK pathway and the expression of EL-specific proteins that were not found in whole tissue lysates (TL), including GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle-specific HSPB2. We also observed structural anomalies, such as autophago-vacuole formation, irregularly shaped mitochondria, and glycogen deposition, which provide insights into the EL’s contribution to AF and related pathways and molecular mechanisms. Overall, our findings suggest that EL play an important role in the development of AF over longer time frames, and provide a more detailed understanding of the underlying molecular processes involved.

Aberrant miRNA expression has been associated with a large number of human diseases. Therefore, targeting miRNAs to regulate their expression levels has become an important therapy against diseases that stem from the dysfunction of pathways regulated by miRNAs. In recent years, small molecules have demonstrated enormous potential as drugs to regulate miRNA expression (i.e., SM-miR). A clear understanding of the mechanism of action of small molecules on the upregulation and downregulation of miRNA expression allows precise diagnosis and treatment of oncogenic pathways. However, outside of a slow and costly process of experimental determination, computational strategies to assist this on an ad hoc basis have yet to be formulated. In this work, we developed, to the best of our knowledge, the first cross-platform prediction tool, DeepsmirUD, to infer small-molecule-mediated regulatory effects on miRNA expression (i.e., upregulation or downregulation). This method is powered by 12 cutting-edge deep-learning frameworks and achieved AUC values of 0.843/0.984 and AUCPR values of 0.866/0.992 on two independent test datasets. With a complementarily constructed network inference approach based on similarity, we report a significantly improved accuracy of 0.813 in determining the regulatory effects of nearly 650 associated SM-miR relations, each formed with either novel small molecule or novel miRNA. By further integrating miRNA–cancer relationships, we established a database of potential pharmaceutical drugs from 1343 small molecules for 107 cancer diseases to understand the drug mechanisms of action and offer novel insight into drug repositioning. Furthermore, we have employed DeepsmirUD to predict the regulatory effects of a large number of high-confidence associated SM-miR relations. Taken together, our method shows promise to accelerate the development of potential miRNA targets and small molecule drugs.

The molecular and cellular basis of health in human tendons remains poorly understood. Amongst human tendons, the hamstrings are the least likely to be injured or degenerate, providing a prototypic healthy tendon reference. The aim of this study was to define the transcriptome and location of all cell types in healthy hamstring tendon. We profiled the transcriptomes of 10,533 nuclei from 4 healthy donors using single-nucleus RNA sequencing (snRNA-seq) and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and revealed the presence of cell types previously unreported for tendon sites, including different skeletal muscle cell types, satellite cells, adipocytes, and nerve cells, which are undefined nervous system cells. Location of these cell types within tendon was defined using spatial transcriptomics and imaging, and transcriptional networks and cell-cell interactions were identified. We demonstrate that fibroblasts have a high number of potential cell-cell interactions, are present throughout the whole tendon tissue, and play an important role in the production and organisation of extracellular matrix, thus confirming their role as key regulators of hamstring tendon tissue homeostasis. Overall, our findings highlight the highly complex cellular networks underpinning tendon function and underpins the importance of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

Here we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line., Single-cell transcriptomes are accurately sequenced with error-tolerant barcodes.

Aberrant miRNA expression has pervasively been found to relate to a growing number of human diseases. Therefore, targeting miRNAs to regulate their expression levels has become an important therapy against diseases that stem from the dysfunction of oncogenic pathways regulated by the miRNAs. In recent years, small molecule compounds have demonstrated enormous potential as drugs to regulate miRNA expression (i.e., SM-miR). A clear understanding of the mechanism of action of small molecules on down- and up-regulating miRNA expression allows precise diagnosis and treatment of oncogenic pathways. However, outside of a slow and costly process of experimental determination, computational strategies to assist this in an ad hoc manner have still not been enabled. In this work, we develop, to the best of our knowledge, the first prediction tool, DeepsmirUD, to infer small molecule-mediated regulatory effects on miRNA expression. This method is powered by an ensemble of 12 cutting-edged deep learning frameworks and achieves state-of-the-art performance with AUC values of 0.840/0.969 and AUCPR values of 0.866/0.983 on two independent test datasets. With a complementarily constructed network inference approach based on similarity, we report a significantly improved accuracy of 0.813 in determining regulatory effects of nearly 650 SM-miR relations formed with either novel small molecules or novel miRNAs. By further integrating miRNA-cancer relations, we established a database of potentially pharmaceutical drugs to aid in understanding the drug mechanisms of action in diseases and to offer novel insight into drug repositioning. Taken together, our method shows great promise to assist and accelerate the therapeutic development of potential miRNA targets and small molecule drugs. Furthermore, we have employed DeepsmirUD to predict regulatory effects of a large number of high-confidence SM-miR relations curated from Psmir, which are publicly available through https://github.com/2003100127/deepsmirud and https://rujinlong.github.io/deepsmirud/ alongside the DeepsmirUD standalone package.

Epigenetic reprogramming to pluripotency requires extensive remodeling of chromatin landscapes to silence existing cell-type-specific genes and activate pluripotency genes. ATP-dependent chromatin remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of recently identified Bromodomain-containing protein 9 (BRD9) and the associated non-canonical BRG1-associated factors (ncBAF) complex in reprogramming remains unknown. Here, we show that genetic or chemical inhibition of BRD9, as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase human somatic cell reprogramming efficiency and can replace KLF4 and c-MYC. We find that BRD9 is dispensable for human induced pluripotent stem cells under primed but not under naive conditions. Mechanistically, BRD9 inhibition downregulates fibroblast-related genes and decreases chromatin accessibility at somatic enhancers. BRD9 maintains the expression of transcriptional regulators MN1 and ZBTB38, both of which impede reprogramming. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming., Scientific and Technological Research Council of Turkey (TÜBİTAK); BİDEB Scholarship; Newton Advanced Fellowship; Arthritis Research UK; Cancer Research UK; LEAN project of the Leducq Foundation; European Union (EU); Seventh Frame-work Program; Marie Curie Actions

High drug delivery efficiency, desirable therapeutic effects, and low toxicity have become crucial to develop nanotherapeutics. Natural nanoparticles (NPs) from edible plants contain a large quantity of bioactive small molecules, proteins, glycolipids, and microRNAs. The development of these NPs has rapidly attracted increasing attention due to their merits of green production, excellent biocompatibility, anti-inflammatory activities, and antitumor capacities.Here, we introduce the extraction, purification, and construction strategies of plant-derived exosome-like NPs (PDENs) and expound on their physicochemical properties, biomedical functions, and therapeutic effects against various diseases. We also recapitulate future directions and challenges of the emerging nanotherapeutics.PDENs have been used as natural nanotherapeutics and nanocarriers. The challenges of applying PDENs primarily stem from the lack of understanding of the mechanisms that drive the tissue-specific targeting properties. Elucidating the underlying targeting mechanisms is one of the major focuses in this review, which helps to gain new research opportunities for the development of natural nanotherapeutics. Despite excellent biosafety and therapeutic effects in the treatment of various diseases, the medical translation of these NPs has still been limited by low yields and cold-chain dependence. Therefore, exploiting new techniques will be required for their massive production and storage.

Expression of the transcription factor brachyury (TBXT) is normally restricted to the embryo, and its silencing is epigenetically regulated. TBXT promotes mesenchymal transition in a subset of common carcinomas, and in chordoma, a rare cancer showing notochordal differentiation, TBXT acts as a putative oncogene. We hypothesized that TBXT expression is controlled through epigenetic inhibition to promote chordoma cell death. Screening of five human chordoma cell lines revealed that pharmacologic inhibition of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (JMJD3) leads to cell death. This effect was phenocopied by dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Inhibition of KDM6A/B with a novel compound KDOBA67 led to a genome-wide increase in repressive H3K27me3 marks with concomitant reduction in active H3K27ac, H3K9ac, and H3K4me3 marks. TBXT was a KDM6A/B target gene, and chromatin changes at TBXT following KDOBA67 treatment were associated with a reduction in TBXT protein levels in all models tested, including primary patient-derived cultures. In all models tested, KDOBA67 treatment downregulated expression of a network of transcription factors critical for chordoma survival and upregulated pathways dominated by ATF4-driven stress and proapoptotic responses. Blocking the AFT4 stress response did not prevent suppression of TBXT and induction of cell death, but ectopic overexpression of TBXT increased viability, therefore implicating TBXT as a potential therapeutic target of H3K27 demethylase inhibitors in chordoma. Our work highlights how knowledge of normal processes in fetal development can provide insight into tumorigenesis and identify novel therapeutic approaches. Significance: Pharmacologic inhibition of H3K27-demethylases in human chordoma cells promotes epigenetic silencing of oncogenic TBXT, alters gene networks critical to survival, and represents a potential novel therapy.

Significance Fibrosis remains a major unmet medical need as therapeutic targets discovered in animal models have failed to translate. A major challenge for identifying novel targets is the limited availability of early-stage human disease tissue. Here, we utilize Dupuytren’s disease (DD), a common localized fibrotic disorder, to evaluate the impact of epigenetic regulation of myofibroblasts and identify potential tractable targets in human fibrosis. We demonstrate that the epigenetic regulator CREBBP/EP300 is a critical determinant of the profibrotic phenotype. Furthermore, we identify collagen VI to be a key downstream target of CREBBP/EP300 and reveal valuable insights in the role it plays in key profibrotic functions, including contractile force, chemotaxis, and wound healing, and hence its potential as a therapeutic target., Fibrotic diseases remain a major cause of morbidity and mortality, yet there are few effective therapies. The underlying pathology of all fibrotic conditions is the activity of myofibroblasts. Using cells from freshly excised disease tissue from patients with Dupuytren’s disease (DD), a localized fibrotic disorder of the palm, we sought to identify new therapeutic targets for fibrotic disease. We hypothesized that the persistent activity of myofibroblasts in fibrotic diseases might involve epigenetic modifications. Using a validated genetics-led target prioritization algorithm (Pi) of genome wide association studies (GWAS) data and a broad screen of epigenetic inhibitors, we found that the acetyltransferase CREBBP/EP300 is a major regulator of contractility and extracellular matrix production via control of H3K27 acetylation at the profibrotic genes, ACTA2 and COL1A1. Genomic analysis revealed that EP300 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, and broad transcriptomic and proteomic profiling of CREBBP/EP300 inhibition by the chemical probe SGC-CBP30 identified collagen VI (Col VI) as a prominent downstream regulator of myofibroblast activity. Targeted Col VI knockdown results in significant decrease in profibrotic functions, including myofibroblast contractile force, extracellular matrix (ECM) production, chemotaxis, and wound healing. Further evidence for Col VI as a major determinant of fibrosis is its abundant expression within Dupuytren’s nodules and also in the fibrotic foci of idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent a tractable therapeutic target across a range of fibrotic disorders.

Significance The early life of an individual mouse is characterized by the generation of a population of long-lived and frequently autoreactive B cells, called B1 cells, which are largely developed in the fetal liver, whilst adult life is characterized by continuous B cell production in the bone marrow, the development of normally short-lived B cells and the stringent elimination of any immature B cells that are dangerously autoreactive. In this study, we show that the change from positive to negative selection in response to self-proteins requires a Lin28b to Let-7 microRNA-dependent switch, which coincides with the transition from fetal liver to bone marrow B cell development., Developing B cells can be positively or negatively selected by self-antigens, but the mechanisms that determine these outcomes are incompletely understood. Here, we show that a B cell intrinsic switch between positive and negative selection during ontogeny is determined by a change from Lin28b to let-7 gene expression. Ectopic expression of a Lin28b transgene in murine B cells restored the positive selection of autoreactive B-1 B cells by self-antigen in adult bone marrow. Analysis of antigen-specific immature B cells in early and late ontogeny identified Lin28b-dependent genes associated with B-1 B cell development, including Arid3a and Bhleh41, and Lin28b-independent effects are associated with the presence or absence of self-antigen. These findings identify cell intrinsic and extrinsic determinants of B cell fate during ontogeny and reconcile lineage and selection theories of B cell development. They explain how changes in the balance of positive and negative selection may be able to adapt to meet the immunological needs of an individual during its lifetime.

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast–cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their ‘native’ tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.

Biomaterial augmentation of surgically repaired rotator cuff tendon tears aims to improve the high failure rates (∼40%) of traditional repairs. Biomaterials that can alter cellular phenotypes through the provision of microscale topographical cues are now under development. We aimed to systematically evaluate the effect of topographic architecture on the cellular phenotype of fibroblasts from healthy and diseased tendons. Electrospun polydioxanone scaffolds with fiber diameters ranging from 300 to 4000 nm, in either a highly aligned or random configuration, were produced. Healthy tendon fibroblasts cultured for 7 days on scaffolds with highly aligned fibers demonstrated a distinctive elongated morphology, whilst those cultured on randomly configured fibers demonstrated a flattened and spread morphology. The effect of scaffold micro-architecture on the transcriptome of both healthy and diseased tendon fibroblasts was assessed with bulk RNA-seq. Both healthy (n = 3) and diseased tendon cells (n = 3) demonstrated a similar transcriptional response to architectural variants. Gene set enrichment analysis revealed that large diameter (≥2000 nm) aligned scaffolds induced an upregulation of genes involved in cellular replication and a downregulation of genes defining inflammatory responses and cell adhesion. Similarly, PDPN and CD248, markers of inflammatory or “activated” fibroblasts, were downregulated during culture of both healthy and diseased fibroblasts on aligned scaffolds with large (≥2000 nm) fiber diameters. In conclusion scaffold architectures resembling that of disordered type III collagen, typically present during the earlier phases of wound healing, resulted in tendon fibroblast activation. Conversely, scaffolds mimicking aligned diameter collagen I fibrils, present during tissue remodelling, did not activate tendon derived fibroblasts. This has implications for the design of scaffolds used during rotator cuff repair augmentation.

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a 'chromatin or histone code'. Proteins that con-tain 'reader' domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyl-lysine/arginine reader domains and was identified as a putative onco-gene and transcriptional co-activator. Here we report a SPIN1 chemi-cal probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, iden-tified genes which are transcriptionally regulated by SPIN1 in squa-mous cell carcinoma and suggest that SPIN1 may have a roll in cancer related inflammation and/or cancer metastasis.

Epigenetic reprogramming requires extensive remodeling of chromatin landscapes to silence cell-type specific gene expression programs. ATP-dependent chromatin-remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of Bromodomain-containing protein 9 (BRD9) and the associated ncBAF (non-canonical BRG1-associated factors) complex in reprogramming remains unknown. Here, we show that genetic suppression of BRD9 as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase the efficiency by which induced pluripotent stem cells (iPSCs) can be generated from human somatic cells. Chemical inhibition and acute degradation of BRD9 phenocopied this effect. Interestingly, we find that BRD9 is dispensable for establishment and maintenance of human pluripotency but required for mesendodermal lineage commitment during differentiation. Mechanistically, BRD9 inhibition downregulates somatic cell type-specific genes and decreases chromatin accessibility at somatic enhancers. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design and personalized medicine approaches for COVID-19., Graphical Abstract, A multi-omic analysis of patient blood samples reveals both similarities and specific features of COVID-19 when compared with samples obtained from sepsis or influenza patients which could yield better targetted therapies for severe COVID-19.

Increased interleukin (IL)-17A has been identified in joints affected by osteoarthritis (OA), but it is unclear how IL-17A, and its family members IL-17AF and IL-17F, can contribute to human OA pathophysiology. Therefore, we aimed to evaluate the gene expression and signalling pathway activation effects of the different IL-17 family members in chondrocytes and synovial fibroblasts derived from cartilage and synovium of patients with end-stage knee OA. Immunohistochemistry staining confirmed that IL-17 receptor A (IL-17RA) and IL-17RC are expressed in end-stage OA-derived cartilage and synovium. Chondrocytes and synovial fibroblasts derived from end-stage OA patients were treated with IL-17A, IL-17AF, or IL-17F, and gene expression was assessed with bulk RNA-Seq. Hallmark pathway analysis showed that IL-17 cytokines regulated several OA pathophysiology-related pathways including immune-, angiogenesis-, and complement-pathways in both chondrocytes and synovial fibroblasts derived from end-stage OA patients. While overall IL-17A induced the strongest transcriptional response, followed by IL-17AF and IL-17F, not all genes followed this pattern. Disease-Gene Network analysis revealed that IL-17A-related changes in gene expression in these cells are associated with experimental arthritis, knee arthritis, and musculoskeletal disease gene-sets. Western blot analysis confirmed that IL-17A significantly activates p38 and p65 NF-κB. Incubation of chondrocytes and synovial fibroblasts with anti-IL-17A monoclonal antibody secukinumab significantly inhibited IL-17A-induced gene expression. In conclusion, the association of IL-17-induced transcriptional changes with arthritic gene-sets supports a role for IL-17A in OA pathophysiology. Future studies should further investigate the role of IL-17A in the OA joint to establish whether anti-IL-17 treatment could be a potential therapeutic option in OA patients with an inflammatory phenotype.

Summary ParagraphWe identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone1, a compound in clinical trials for anti-fibrotic and anti-inflammatory applications2, as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry3. We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir4. Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone’s translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

Droplet-based single-cell sequencing techniques have provided unprecedented insight into cellular heterogeneities within tissues. However, these approaches only allow for the measurement of the distal parts of a transcript following short-read sequencing. Therefore, splicing and sequence diversity information is lost for the majority of the transcript. The application of long-read Nanopore sequencing to droplet-based methods is challenging because of the low base-calling accuracy currently associated with Nanopore sequencing. Although several approaches that use additional short-read sequencing to error-correct the barcode and UMI sequences have been developed, these techniques are limited by the requirement to sequence a library using both short- and long-read sequencing. Here we introduce a novel approach termed single-cell Barcode UMI Correction sequencing (scBUC-seq) to efficiently error-correct barcode and UMI oligonucleotide sequences synthesized by using blocks of dimeric nucleotides. The method can be applied to correct either short-read or long-read sequencing, thereby allowing users to recover more reads per cell and permits direct single-cell Nanopore sequencing for the first time. We illustrate our method by using species-mixing experiments to evaluate barcode assignment accuracy and evaluate differential isoform usage and fusion transcripts using myeloma and sarcoma cell line models.

BackgroundIDH1/2-mutant gliomas are primary brain tumors for which curative treatments are lacking. Mutant IDH-dependent 2-hydroxyglutarate (2-HG) accumulation leads to DNA and histone hypermethylation. Based on this distinct phenotype, we interrogated epigenetic dependencies of IDH-mutant glioma that can be targeted therapeutically.MethodsWe conducted a chemical screen targeting chromatin modifiers in patient derived IDH1-mutant GBM cells. We investigated mechanisms of action of compound hits and their combinations through cell-based functional assays, live-cell imaging, Western blot, CRISPR knockout, RNA-seq and ChIP experiments. The therapeutic concept was validated in vivo using chemical inhibitors GSK-J4 and Belinostat in an orthotopic GBM model.ResultsWe identified the H3K27me3 demethylase (KDM6) inhibitor GSK-J4 and histone deacetylase inhibitor Belinostat as potent, genotype-selective agents against IDH1-mutant glioma. RNA-sequencing on paired wild-type and IDH1R132H cells revealed inhibition of cholesterol biosynthesis and activation of cellular stress in IDH1R132H cells, which were reversible with a mutant IDH1 inhibitor. GSK-J4 caused further repression of cholesterol biosynthesis pathway genes through H3K27me3 deposition and exacerbated the ATF4-mediated integrated stress response. Belinostat inhibited anti-apoptotic pathways through activation of TGF-β signaling and induced cell cycle arrest. Together, the GSK-J4 and Belinostat combination activated DDIT3/CHOP-dependent apoptosis in IDH1-mutant cells and extended survival in an IDH1-mutant orthotopic model in vivo.ConclusionsThese results provide a possible therapeutic approach that exploits epigenetic vulnerabilities of IDH-mutant gliomas.Key points-Combination of GSK-J4 and Belinostat selectively targets IDH1-mutant cells.-GSK-J4 downregulates cholesterol biosynthesis and activates an ATF4-mediated stress response.-Belinostat activates the TGFβ pathway, induces G2/M arrest and inhibits anti-apoptotic pathways.Importance of the studyIDH1/2 genes are frequently mutated in low grade glioma and secondary glioblastoma. These tumors exhibit a distinct epigenomic signature with increased DNA and histone methylation; therefore, identifying and exploiting their epigenetic vulnerabilities may lead to effective therapies. We discovered that targeting of KDM6A/6B together with HDACs provides a promising therapeutic approach for IDH1-mutant glioma.

Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

Hypoxia is a common phenomenon in solid tumors and is strongly linked to hallmarks of cancer. Recent evidence has shown that hypoxia promotes local immune suppression. Type I IFN supports cytotoxic T lymphocytes by stimulating the maturation of dendritic cells and enhancing their capacity to process and present antigens. However, little is known about the relationship between hypoxia and the type I IFN pathway, which comprises the sensing of double-stranded RNA and DNA (dsRNA/dsDNA) followed by IFNα/β secretion and transcriptional activation of IFN-stimulated genes (ISG). In this study, we determined the effects of hypoxia on the type I IFN pathway in breast cancer and the mechanisms involved. In cancer cell lines and xenograft models, mRNA and protein expressions of the type I IFN pathway were downregulated under hypoxic conditions. This pathway was suppressed at each level of signaling, from the dsRNA sensors RIG-I and MDA5, the adaptor MAVS, transcription factors IRF3, IRF7, and STAT1, and several ISG including RIG-I, IRF7, STAT1, and ADAR-p150. Importantly, IFN secretion was reduced under hypoxic conditions. HIF1α- and HIF2α-mediated regulation of gene expression did not explain most of the effects. However, ATAC-seq data revealed in hypoxia that peaks with STAT1 and IRF3 motifs had decreased accessibility. Collectively, these results indicate that hypoxia leads to an overall downregulation of the type I IFN pathway due to repressed transcription and lower chromatin accessibility in an HIF1/2α-independent manner, which could contribute to immunosuppression in hypoxic tumors. Significance: These findings characterize a new mechanism of immunosuppression by hypoxia via downregulation of the type I IFN pathway and its autocrine/paracrine effects on tumor growth.

Background Tendon injuries occur frequently in human and equine athletes. Treatment options are limited, and the prognosis is often poor with functionally deficient scar tissue resulting. Fetal tendon injuries in contrast are capable of healing without forming scar tissue. Embryonic stem cells (ESCs) may provide a potential cellular therapeutic to improve adult tendon regeneration; however, whether they can mimic the properties of fetal tenocytes is unknown. To this end, understanding the unique expression profile of normal adult and fetal tenocytes is crucial to allow validation of ESC-derived tenocytes as a cellular therapeutic. Methods Equine adult, fetal and ESC-derived tenocytes were cultured in a three-dimensional environment, with histological, morphological and transcriptomic differences compared. Additionally, the effects on gene expression of culturing adult and fetal tenocytes in either conventional two-dimensional monolayer culture or three-dimensional culture were compared using RNA sequencing. Results No qualitative differences in three-dimensional tendon constructs generated from adult, fetal and ESCs were found using histological and morphological analysis. However, genome-wide transcriptomic analysis using RNA sequencing revealed that ESC-derived tenocytes’ transcriptomic profile more closely resembled fetal tenocytes as opposed to adult tenocytes. Furthermore, this study adds to the growing evidence that monolayer cultured cells’ gene expression profiles converge, with adult and fetal tenocytes having only 10 significantly different genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in 3D, large distinctions in gene expression between these two developmental stages were found, with 542 genes being differentially expressed. Conclusion The information provided in this study makes a significant contribution to the investigation into the differences between adult reparative and fetal regenerative cells and supports the concept of using ESC-derived tenocytes as a cellular therapy. Comparing two- and three-dimensional culture also indicates three-dimensional culture as being a more physiologically relevant culture system for determining transcriptomic difference between the same cell types from different developmental stages.

We profiled gene expression signatures to distinguish rheumatoid arthritis (RA) from non-inflammatory arthralgia (NIA), self-limiting arthritis (SLA), and undifferentiated arthritis (UA) as compared to healthy controls as novel potential biomarkers for therapeutic responsiveness. Global gene expression profiles of PBMCs from 43 drug-naïve patients presenting with joint symptoms were evaluated and differentially expressed genes identified by comparative analysis with 24 healthy volunteers. Patients were assessed at presentation with follow up at 6 and 12 months. Gene ontology and network pathway analysis were performed using DAVID Bioinformatics Resources v6.7. Gene expression profiles were also determined after disease-modifying anti-rheumatic drug (DMARD) treatment in the inflammatory arthritis groups (i.e. RA and UA) and confirmed by qRT-PCR. Receiver operating characteristic (ROC) curves analysis and Area Under the Curve (AUC) estimation were performed to assess the diagnostic value of candidate gene expression signatures. A type I interferon (IFN) gene signature distinguished DMARD-naïve patients who will subsequently develop persistent inflammatory arthritis (i.e. RA and UA) from those with NIA. In patients with RA, the IFN signature is characterised by up-regulation of SIGLEC1 (p = 0.00597) and MS4A4A (p = 0.00000904). We also identified, EPHB2 (p = 0.000542) and PDZK1IP1 (p = 0.0206) with RA-specific gene expression profiles and elevated expression of the ST6GALNAC1 (p = 0.0023) gene in UA. ROC and AUC risk score analysis suggested that MSA4A (AUC: 0.894, 0.644, 0.720), PDZK1IP1 (AUC: 0.785, 0.806, 0.977), and EPHB2 (AUC: 0.794, 0.723, 0.620) at 0, 6, and 12 months follow-up can accurately discriminate patients with RA from healthy controls and may have practical value for RA diagnosis. In patients with early inflammatory arthritis, ST6GALNAC1 is a potential biomarker for UA as compared with healthy controls whereas EPHB2, MS4A4A, and particularly PDZK1IP1 may discriminate RA patients. SIGLEC1 may also be a useful marker of disease activity in UA.

Understanding multicellular physiology and pathobiology requires analysis of the relationship between genotype, chromatin organisation and phenotype. In the multi-omics era, many methods exist to investigate biological processes across the genome, transcriptome, epigenome, proteome and metabolome. Until recently, this was only possible for populations of cells or complex tissues, creating an averaging effect that may obscure direct correlations between multiple layers of data. Single-cell sequencing methods have removed this averaging effect, but computational integration after profiling distinct modalities separately may still not completely reflect underlying biology. Multiplexed assays resolving multiple modalities in the same cell are required to overcome these shortcomings and have the potential to deliver unprecedented understanding of biology and disease.

The Human Cell Atlas (HCA) project aims to map tissues and organs during development, maturation and pathology at single cell resolution. The musculoskeletal HCA network is a community for fostering collaboration and shared expertise to help develop the therapeutic approaches needed to address the high global burden of musculoskeletal disorders.

Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that charge tRNAs with their cognate amino acids. Recent studies have revealed that the expression of aaRSs is dynamically altered in response to cellular stresses, and that specific isoforms play pivotal roles in carcinogenesis and tumor progression. Despite emerging interest in aaRSs in cancer, the biologic sequelae of aaRSs in multiple myeloma (MM) have not yet been elucidated. We first examined RNA expression levels of aaRSs in tumor cells from MM patients. Gene expression analyses revealed that most aaRSs were upregulated in MM patients compared to healthy donors. Importantly, expression of 4 aaRSs, including glutamyl-prolyl-tRNA synthetase (EPRS), correlated with worse clinical prognosis: high EPRS expression correlated with disease progression (GSE6477; P< 0.001) and shorter survival (CoMMpass; P = 0.007). Consistent with clinical data, ERPS knockdown in MM cells (AMO1, RPMI 8226, and MM.1S) triggered significant cell growth inhibition, suggesting that EPRS is a growth and survival factor in MM cells. Halofuginone (HF) is an inhibitor of the prolyl-tRNA-synthetase (PRS) activity of EPRS and potently inhibits MM growth. However, HF is proline (Pro) competitive and increased levels of Pro abrogates the effect of HF, including the Pro-rich tumor microenvironment. To overcome this limitation, we developed a novel ATP competitive and Pro non-competitive PRS inhibitor, NCP26. NCP26 potently decreased growth in MM cell lines, including drug (Dox, Dex, PI, IMiDs) resistant isogenic derivatives, and patient MM cells (EC50 ≈ 500 nM), without significant toxicity to peripheral blood mononuclear cells. Neither anti-apoptotic cytokines (i.e., IL-6) nor co-culture with bone marrow (BM) stromal cells abrogated NCP26-induced MM cell death. Importantly, unlike HF, NCP26-induced cell growth inhibition was not altered by an excess of exogenous Pro. Since aaRSs inhibition results in accumulation of uncharged tRNAs sensed by the amino acid response (AAR), we next examined whether NCP26 activates the AAR pathway in MM cells. As expected, NCP26 increased both p-GCN2 and p-eIF2α, associated with upregulation of ATF4 and CHOP expression. Moreover, NCP26 treatment induced G0/G1 cell cycle arrest followed by apoptotic cell death, evidenced by cleavage of caspases and PARP. We also evaluated in vivo efficacy of NCP26 in the AMO1 xenograft mouse model: NCP26 (10 mg/kg, q.d.) significantly inhibited tumor growth (P = 0.005) and prolonged host survival (P < 0.001) compared to control, without affecting body weight. To further characterize the biological impact of NCP26 in MM cells, we performed single-cell RNA-seq using BM samples from MM patients. While both HF and NCP26 treatment reduced MM, NCP26 triggered more sustained and robust activation of integrated stress response, evidenced by induction of GCN2, CHOP, and IRE1, compared to HF. Gene set enrichment analysis also revealed significant alteration in chromatin processes, besides proteostasis, such as translation, signal peptide, and stress response, after NCP26 treatment. Since previous studies suggested that translation of genes bearing high Pro-Pro (PP) motifs is more susceptible to EPRS inhibition than non-PP-bearing genes, we next performed a quantitative proteomic analysis in AMO1 cells treated with NCP26: of 7,366 proteins, 84 were upregulated and 363 were downregulated after 6-hour treatment. We also performed in silico bioinformatic analysis to uncover PP-bearing genes, and incorporated CRISPR-screening data to identify genetic dependencies in MM. By overlapping the three datasets, we identified a group of 55 PP motif-containing proteins which play essential roles in MM cell pathogenesis, including MYC, PIM2, and CCND1/2. We also confirmed that PP motif-containing proteins were significantly downregulated by NCP26 treatment in MM cells both in vitro and in vivo. In conclusion, our results identify EPRS as a biomarker of poor prognosis and a novel therapeutic target in MM. NCP26, a novel ATP-competitive and Pro non-competitive EPRS inhibitor, induces MM cytotoxicity both in vitro and in vivo by triggering AAR and EPRS-Pro-rich proteins pathways. Our data provide the preclinical rationale for development of NCP26-based clinical candidates to improve patient outcomes in MM. Disclosures Gooding: Bristol Myers Squibb: Research Funding. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ramasamy: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Celgene (BMS): Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive biotech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Introduction Germinal centre (GC) lymphomas Follicular Lymphoma (FL) and Diffuse Large B Cell Lymphoma (DLBCL) are strikingly dependent on mutations affecting chromatin regulators. The majority of FL tumours harbour multiple mutated chromatin-regulatory genes, including loss-of-function mutations in KMT2D (80%), CREBBP (60%) and gain-of-function mutations in EZH2 (25%), whilst these are also frequently mutated within the GC B-cell (GCB) subtype of DLBCL. The FDA approval of EZH2-inhibitor Tazemetostat for relapsed/refractory FL exemplifies the potential and the need to broaden our search for novel avenues targeting epigenetic reprogramming in GC lymphomas. Hence, in this study, we sought to identify novel epigenetic vulnerabilities and biological interactions in GC B cell lymphomas. Methods and Results: 25 compounds targeting 18 histone binding/modifying enzymes were tested across 12 GCB-DLBCL cell lines harbouring differing degrees of epigenetic dysregulation. The two most potent cytotoxic compounds (GSK-J4 and KDOBA67) both target the H3K27me3 demethylase, KDM6. The KDM6 family consists of KDM6A (UTX) and KDM6B (JMJD3), both of which demethylate H3K27me3/H3K27me2 to lower methylation states, and KDM6C (UTY) which has negligible enzymatic activity. Indeed, KDM6 inhibition (KDM6i) with GSK-J4 increased levels of the inhibitory chromatin mark H3K27me3 and simultaneously reduced H3K27me1/me2, while we observed no global changes in H3K4me3. The induction of apoptosis in the cell lines following either inhibition of the 'eraser' KDM6 (leading to increased H3K27me3) or the opposing 'writer' EZH2 (lowering H3K27me3) appears paradoxical at first glance, given the EZH2 gain-of-function mutations seen in FL/DLBCL and known efficacy of EZH2 inhibitors. However, RNA-sequencing analysis of GSK-J4-treated cells revealed no significant overlap with published EZH2 mutation or EZH2 inhibition signatures from GC lymphoma models, implying that KDM6i-regulated H3K27me3 peaks do not overlay gene promoters targeted by EZH2 in the GC reaction. The most significant gene regulation by KDM6i was a significant up-regulation of 6 members of the metallothionein (MT) family within 4-hours of treatment, which was sustained for 72-hours (MT1E, MT1F, MT1G, MT1H, MT1X and MT2A at 4 hours log2FoldChange 5.1-12.1, average p adj 3.4E-06). These metal-binding proteins cluster on chromosome 16 and their up-regulation correlated with up-regulation of the zinc transporter SLC30A1, suggesting a potential biological relationship between KDM6 enzymes and zinc homeostasis, with both isoforms containing a zinc-binding domain. Targeted validation across 6 DLBCL cell lines also showed MT up-regulation correlated with sensitivity to GSK-J4, whilst siRNA-mediated knockdown of KDM6A or KDM6B individually recapitulated the up-regulation of MT genes, albeit to a lower extent. ChIP-qPCR profiling at the MT loci for H3K27me3 and H3K4me3 revealed no significant changes at 4 or 24 hours; considering that expression of MT genes increase within 60 minutes of drug exposure, suggesting these changes may be induced by an indirect mechanism of KDM6A and/or KDM6B activity. Using ENCODE data of 4 cell lines included in our series (DOHH-2, OCI-LY-1, SU-DHL-6, KARPAS-422), we observe a link between GSK-J4 sensitivity, up-regulation of MT genes and significantly lower basal enrichment for H3K27ac at the MT loci. This distinction may reflect altered 3D chromatin conformation that render only sensitive cell lines permissive to regulation by KDM6i, with H3K27ac having recently been shown to modulate enhancer positioning and interactions in DLBCL cell lines. Conclusion: Screening epigenetic-targeting compounds in GC lymphomas has identified KDM6 as a particularly promising candidate target. Overall, we describe up-regulation of MT genes as one potential mechanism of action, which correlates with response in DLBCL cell lines and suggests that KDM6 members may regulate H3K27me3 in a manner different from EZH2. In our current model we propose, that in GC-lymphomas KDM6A may have tumour suppressive roles whilst KDM6B is required for cell survival. Given KDM6A is frequently lost in cancer and Kabuki syndrome, a congenital developmental disorder caused by germline mutations in KMT2D or KDM6A, our results highlight the importance of establishing what roles the KDM6 family play in lymphoma pathogenesis. Disclosures Fitzgibbon: Epizyme: Research Funding.