KDM5 inhibition offers a novel therapeutic strategy for the treatment of KMT2D mutant lymphomas.

Loss-of-function mutations in KMT2D are a striking feature of germinal center (GC) lymphomas, resulting in decreased histone 3 lysine 4 (H3K4) methylation and altered gene expression. We hypothesized that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would reestablish H3K4 methylation and restore the expression of genes repressed on loss of KMT2D. KDM5 inhibition increased H3K4me3 levels and caused an antiproliferative response in vitro, which was markedly greater in both endogenous and gene-edited KMT2D mutant diffuse large B-cell lymphoma cell lines, whereas tumor growth was inhibited in KMT2D mutant xenografts in vivo. KDM5 inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell signaling and altered expression of B-cell lymphoma 2 (BCL2) family members, including BCL2 itself. KDM5 inhibition may offer an effective therapeutic strategy for ameliorating KMT2D loss-of-function mutations in GC lymphomas.

HDAC Inhibition Restores Response to HER2-Targeted Therapy in Breast Cancer via PHLDA1 Induction.

The downregulation of Pleckstrin Homology-Like Domain family A member 1 (PHLDA1) expression mediates resistance to targeted therapies in receptor tyrosine kinase-driven cancers. The restoration and maintenance of PHLDA1 levels in cancer cells thus constitutes a potential strategy to circumvent resistance to inhibitors of receptor tyrosine kinases. Through a pharmacological approach, we identify the inhibition of MAPK signalling as a crucial step in PHLDA1 downregulation. Further ChIP-qPCR analysis revealed that MEK1/2 inhibition produces significant epigenetic changes at the PHLDA1 locus, specifically a decrease in the activatory marks H3Kme3 and H3K27ac. In line with this, we show that treatment with the clinically relevant class I histone deacetylase (HDAC) inhibitor 4SC-202 restores PHLDA1 expression in lapatinib-resistant human epidermal growth factor receptor-2 (HER2)+ breast cancer cells. Critically, we show that when given in combination, 4SC-202 and lapatinib exert synergistic effects on 2D cell proliferation and colony formation capacity. We therefore propose that co-treatment with 4SC-202 may prolong the clinical efficacy of lapatinib in HER2+ breast cancer patients.

Precision medicine and lymphoma.

PURPOSE OF REVIEW: The treatment of the germinal center lymphomas, diffuse large B cell (DLBCL) and follicular lymphoma, has changed little beyond the introduction of immunochemotherapies. However, there exists a substantial group of patients within both diseases for which improvements in care will involve appropriate tailoring of treatment. RECENT FINDINGS: DLBCL consists of two major subtypes with striking differences in their clinical outcomes paralleling their underlying genetic heterogeneity. Recent studies have seen advances in the stratification of germinal center lymphomas, through comprehensive profiling of 1001 DLBCLs alongside refinements in the identification of high-risk follicular lymphoma patients using m7-FLIPI and 23G models. A new wave of novel therapeutic agents is now undergoing clinical trials for germinal center lymphomas, with BCR and EZH2 inhibitors demonstrating preferential benefit in subgroups of patients. The emergence of cell-free DNA has raised the possibility of dynamic disease monitoring to potentially mitigate the complexity of spatial and temporal heterogeneity, whilst predicting tumor evolution in real time. SUMMARY: Altogether knowledge of the genomic landscape of germinal center lymphomas is offering welcome opportunities in patient risk stratification and therapeutics. The challenge ahead is to establish how best to combine upfront or dynamic prognostication with precision therapies, while retaining practicality in clinical trials and the real-world setting.

Long non-coding RNAs in the regulation of the immune response.

It is increasingly clear that long non-coding RNAs (lncRNAs) regulate a variety biological responses, and that they do so by a diverse range of mechanisms. In the field of immunology, recent publications have shown widespread changes in the expression of lncRNAs during the activation of the innate immune response and T cell development, differentiation, and activation. These lncRNAs control important aspects of immunity such as production of inflammatory mediators, differentiation, and cell migration through regulating protein-protein interactions or via their ability to basepair with RNA and DNA. We review the current understanding of the mechanism of action of these immune-related lncRNAs, discuss their impact on physiological and pathological processes, and highlight important areas of inquiry at the intersection between immunology and lncRNA biology.

Nuclear FGFR1 promotes pancreatic stellate cell-driven invasion through up-regulation of Neuregulin 1.

Pancreatic stellate cells (PSCs) are key to the treatment-refractory desmoplastic phenotype of pancreatic ductal adenocarcinoma (PDAC) and have received considerable attention as a stromal target for cancer therapy. This approach demands detailed understanding of their pro- and anti-tumourigenic effects. Interrogating PSC-cancer cell interactions in 3D models, we identified nuclear FGFR1 as critical for PSC-led invasion of cancer cells. ChIP-seq analysis of FGFR1 in PSCs revealed a number of FGFR1 interaction sites within the genome, notably NRG1, which encodes the ERBB ligand Neuregulin. We show that nuclear FGFR1 regulates transcription of NRG1, which in turn acts in autocrine fashion through an ERBB2/4 heterodimer to promote invasion. In support of this, recombinant NRG1 in 3D model systems rescued the loss of invasion incurred by FGFR inhibition. In vivo we demonstrate that, while FGFR inhibition does not affect the growth of pancreatic tumours in mice, local invasion into the pancreas is reduced. Thus, FGFR and NRG1 may present new stromal targets for PDAC therapy.

Correction: Genomic profiling reveals spatial intra-tumor heterogeneity in follicular lymphoma.

In the original version of this article the authors noted an omission in the author affiliations where the university details: Queen Mary University of London was not included in the original affiliation for the majority of the authors. The correct affiliations are as follows1. Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK3. Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK6. Evolution and Cancer Laboratory, Barts Cancer Institute, Queen Mary University of London, London, UK.

Knockdown of Nuclear-Located Enhancer RNAs and Long ncRNAs Using Locked Nucleic Acid GapmeRs.

The human genome is widely transcribed outside of protein-coding genes, producing thousands of noncoding RNAs from different subfamilies including enhancer RNAs. Functional studies to determine the role of individual genes are challenging with noncoding RNAs appearing to be more difficult to knockdown than mRNAs. One factor that may have hindered progress is that the majority of noncoding RNAs are thought to be located within the nucleus, where the efficiency of traditional RNA interference techniques is debatable. Here we present an alternative RNA interference technique utilizing Locked Nucleic Acids, which is able to efficiently knockdown noncoding RNAs irrespective of intracellular location.

Follicular lymphoma, a B cell malignancy addicted to epigenetic mutations.

While follicular lymphoma (FL) is exquisitely responsive to immuno-chemotherapy, many patients follow a relapsing remitting clinical course driven in part by a common precursor cell (CPC) population. Advances in next generation sequencing have provided valuable insights into the genetic landscape of FL and its clonal evolution in response to therapy, implicating perturbations of epigenetic regulators as a hallmark of the disease. Recurrent mutations of histone modifiers KMT2D, CREBBP, EP300, EZH2, ARIDIA, and linker histones are likely early events arising in the CPC pool, rendering epigenetic based therapies conceptually attractive for treatment of indolent and transformed FL. This review provides a synopsis of the main epigenetic aberrations and the current efforts in development and testing of epigenetic therapies in this B cell malignancy.

Catalog of Differentially Expressed Long Non-Coding RNA following Activation of Human and Mouse Innate Immune Response.

Despite increasing evidence to indicate that long non-coding RNAs (lncRNAs) are novel regulators of immunity, there has been no systematic attempt to identify and characterize the lncRNAs whose expression is changed following the induction of the innate immune response. To address this issue, we have employed next-generation sequencing data to determine the changes in the lncRNA profile in four human (monocytes, macrophages, epithelium, and chondrocytes) and four mouse cell types (RAW 264.7 macrophages, bone marrow-derived macrophages, peritoneal macrophages, and splenic dendritic cells) following exposure to the pro-inflammatory mediators, lipopolysaccharides (LPS), or interleukin-1ß. We show differential expression of 204 human and 210 mouse lncRNAs, with positional analysis demonstrating correlation with immune-related genes. These lncRNAs are predominantly cell-type specific, composed of large regions of repeat sequences, and show poor evolutionary conservation. Comparison within the human and mouse sequences showed less than 1% sequence conservation, although we identified multiple conserved motifs. Of the 204 human lncRNAs, 21 overlapped with syntenic mouse lncRNAs, of which five were differentially expressed in both species. Among these syntenic lncRNA was IL7-AS (antisense), which was induced in multiple cell types and shown to regulate the production of the pro-inflammatory mediator interleukin-6 in both human and mouse cells. In summary, we have identified and characterized those lncRNAs that are differentially expressed following activation of the human and mouse innate immune responses and believe that these catalogs will provide the foundation for the future analysis of the role of lncRNAs in immune and inflammatory responses.

Transcriptional profiling identifies differential expression of long non-coding RNAs in Jo-1 associated and inclusion body myositis.

Myositis is characterised by muscle inflammation and weakness. Although generally thought to be driven by a systemic autoimmune response, increasing evidence suggests that intrinsic changes in the muscle might also contribute to the pathogenesis. Long non-coding RNAs (lncRNAs) are a family of novel genes that regulate gene transcription and translation. To determine the potential role of lncRNAs, we employed next generation sequencing to examine the transcriptome in muscle biopsies obtained from two histologically distinct patient populations, inclusion body myositis (IBM) and anti-Jo-1-associated myositis (Jo-1). 1287 mRNAs and 1068 mRNAs were differentially expressed in the muscle from Jo-1 and IBM patients, respectively. Pathway analysis showed the top canonical pathway in both Jo-1 and IBM was oxidative phosphorylation and mitochondrial dysfunction. We identified 731 known and 325 novel lncRNAs in the muscles biopsies. Comparison with controls showed 55 and 46 lncRNAs were differentially expressed in IBM and Jo-1 myositis, respectively. Of these, 16 lncRNAs were differentially expressed in both IBM and Jo-1 myositis and included upregulated H19, lncMyoD and MALAT1. Given that these are known to regulate muscle proliferation and differentiation, we speculate that changes in lncRNAs might contribute to the phenotypic changes in Jo-1 and IBM myositis.

Automated recording of home cage activity and temperature of individual rats housed in social groups: The Rodent Big Brother project.

Measuring the activity and temperature of rats is commonly required in biomedical research. Conventional approaches necessitate single housing, which affects their behavior and wellbeing. We have used a subcutaneous radiofrequency identification (RFID) transponder to measure ambulatory activity and temperature of individual rats when group-housed in conventional, rack-mounted home cages. The transponder location and temperature is detected by a matrix of antennae in a baseplate under the cage. An infrared high-definition camera acquires side-view video of the cage and also enables automated detection of vertical activity. Validation studies showed that baseplate-derived ambulatory activity correlated well with manual tracking and with side-view whole-cage video pixel movement. This technology enables individual behavioral and temperature data to be acquired continuously from group-housed rats in their familiar, home cage environment. We demonstrate its ability to reliably detect naturally occurring behavioral effects, extending beyond the capabilities of routine observational tests and conventional monitoring equipment. It has numerous potential applications including safety pharmacology, toxicology, circadian biology, disease models and drug discovery.

Epigenetic dysregulation in follicular lymphoma.

The adoption of next-generation sequencing technologies has led to a remarkable shift in our understanding of the genetic landscape of follicular lymphoma. While the disease has been synonymous with the t(14;18), the prevalence of alterations in genes that regulate the epigenome has been established as a pivotal hallmark of these lymphomas. Giant strides are being made in unraveling the biological consequences of these alterations in tumorigenesis opening up new opportunities for directed therapies.

Long Intergenic Noncoding RNAs Mediate the Human Chondrocyte Inflammatory Response and Are Differentially Expressed in Osteoarthritis Cartilage.

OBJECTIVE: To identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA. METHODS: OA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non-OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase-polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex. RESULTS: RNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin-1ß (IL-1ß) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50-associated cyclooxygenase 2-extragenic RNA (PACER) and 2 novel chondrocyte inflammation-associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non-OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL-1-stimulated secretion of proinflammatory cytokines. CONCLUSION: The inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role in mediating inflammation-driven cartilage degeneration in OA.

Reduced Expression of Histone Methyltransferases KMT2C and KMT2D Correlates with Improved Outcome in Pancreatic Ductal Adenocarcinoma.

Genes encoding the histone H3 lysine 4 methyltransferases KMT2C and KMT2D are subject to deletion and mutation in pancreatic ductal adenocarcinoma (PDAC), where these lesions identify a group of patients with a more favorable prognosis. In this study, we demonstrate that low KMT2C and KMT2D expression in biopsies also defines better outcome groups, with median survivals of 15.9 versus 9.2 months (P = 0.029) and 19.9 versus 11.8 months (P = 0.001), respectively. Experiments with eight human pancreatic cell lines showed attenuated cell proliferation when these methyltransferases were depleted, suggesting that this improved outcome may reflect a cell-cycle block with diminished progression from G0-G1 RNA-seq analysis of PDAC cell lines following KMT2C or KMT2D knockdown identified 31 and 124 differentially expressed genes, respectively, with 19 genes in common. Gene-set enrichment analysis revealed significant downregulation of genes related to cell-cycle and growth. These data were corroborated independently by examining KMT2C/D signatures extracted from the International Cancer Genome Consortium and The Cancer Genome Atlas datasets. Furthermore, these experiments highlighted a potential role for NCAPD3, a condensin II complex subunit, as an outcome predictor in PDAC using existing gene expression series. Kmt2d depletion in KC/KPC cell lines also led to an increased response to the nucleoside analogue 5-fluorouracil, suggesting that lower levels of this methyltransferase may mediate the sensitivity of PDAC to particular treatments. Therefore, it may also be therapeutically beneficial to target these methyltransferases in PDAC, especially in those patients demonstrating higher KTM2C/D expression. Cancer Res; 76(16); 4861-71. ©2016 AACR.

Divergent signalling pathways regulate lipopolysaccharide-induced eRNA expression in human monocytic THP1 cells.

Recent studies have indicated that non-coding RNAs transcribed from enhancer regions are important regulators of enhancer function and gene expression. In this report, we have characterised the expression of six enhancer RNAs (eRNAs) induced in human monocytic THP1 cells following activation of the innate immune response by lipopolysaccharide (LPS). Specifically, we have demonstrated that LPS-induced expression of individual eRNAs is mediated through divergent intracellular signalling pathways that includes NF-κB and the mitogen activated protein kinases, extracellular regulated kinase-1/2 and p38.

Long non-coding RNAs and enhancer RNAs regulate the lipopolysaccharide-induced inflammatory response in human monocytes.

Early reports indicate that long non-coding RNAs (lncRNAs) are novel regulators of biological responses. However, their role in the human innate immune response, which provides the initial defence against infection, is largely unexplored. To address this issue, here we characterize the long non-coding RNA transcriptome in primary human monocytes using RNA sequencing. We identify 76 enhancer RNAs (eRNAs), 40 canonical lncRNAs, 65 antisense lncRNAs and 35 regions of bidirectional transcription (RBT) that are differentially expressed in response to bacterial lipopolysaccharide (LPS). Crucially, we demonstrate that knockdown of nuclear-localized, NF-κB-regulated, eRNAs (IL1ß-eRNA) and RBT (IL1ß-RBT46) surrounding the IL1ß locus, attenuates LPS-induced messenger RNA transcription and release of the proinflammatory mediators, IL1ß and CXCL8. We predict that lncRNAs can be important regulators of the human innate immune response.