JAK2 as a surface marker for enrichment of human pluripotent stem cells-derived ventricular cardiomyocytes.

BACKGROUND: Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) hold great promise for cardiac disease modelling, drug discovery and regenerative medicine. Despite the advancement in various differentiation protocols, the heterogeneity of the generated population composed of diverse cardiac subtypes poses a significant challenge to their practical applications. Mixed populations of cardiac subtypes can compromise disease modelling and drug discovery, while transplanting them may lead to undesired arrhythmias as they may not integrate and synchronize with the host tissue's contractility. It is therefore crucial to identify cell surface markers that could enable high purity of ventricular CMs for subsequent applications. METHODS: By exploiting the fact that immature CMs expressing myosin light chain 2A (MLC2A) will gradually express myosin light chain 2 V (MLC2V) protein as they mature towards ventricular fate, we isolated signal regulatory protein alpha (SIRPA)-positive CMs expressing intracellular MLC2A or MLC2V using MARIS (method for analysing RNA following intracellular sorting). Subsequently, RNA sequencing analysis was performed to examine the gene expression profile of MLC2A + and MLC2V + sorted CMs. We identified genes that were significantly up-regulated in MLC2V + samples to be potential surface marker candidates for ventricular specification. To validate these surface markers, we performed immunostaining and western blot analysis to measure MLC2A and MLC2V protein expressions in SIRPA + CMs that were either positive or negative for the putative surface markers, JAK2 (Janus kinase 2) or CD200. We then characterized the electrophysiological properties of surface marker-sorted CMs, using fluo-4 AM, a green-fluorescent calcium indicator, to measure the cellular calcium transient at the single cell level. For functional validation, we investigated the response of the surface marker-sorted CMs to vernakalant, an atrial-selective anti-arrhythmic agent. RESULTS: In this study, while JAK2 and CD200 were identified as potential surface markers for the purification of ventricular-like CMs, the SIRPA+/JAK2+ population showed a higher percentage of MLC2V-expressing cells (~ 90%) compared to SIRPA+/CD200+ population (~ 75%). SIRPA+/JAK2+ sorted CMs exhibited ventricular-like electrophysiological properties, including slower beating rate, slower calcium depolarization and longer calcium repolarization duration. Importantly, vernakalant had limited to no significant effect on the calcium repolarization duration of SIRPA+/JAK2+ population, indicating their enrichment for ventricular-like CMs. CONCLUSION: Our study lays the groundwork for the identification of cardiac subtype surface markers that allow purification of cardiomyocyte sub-populations. Our findings suggest that JAK2 can be employed as a cell surface marker for enrichment of hPSC-derived ventricular-like CMs.

Core-binding factor fusion downregulation of ADAR2 RNA editing contributes to AML leukemogenesis.

Adenosine-to-inosine RNA editing, which is catalyzed by adenosine deaminases acting on RNA (ADAR) family of enzymes, ADAR1 and ADAR2, has been shown to contribute to multiple cancers. However, other than the chronic myeloid leukemia blast crisis, relatively little is known about its role in other types of hematological malignancies. Here, we found that ADAR2, but not ADAR1 and ADAR3, was specifically downregulated in the core-binding factor (CBF) acute myeloid leukemia (AML) with t(8;21) or inv(16) translocations. In t(8;21) AML, RUNX1-driven transcription of ADAR2 was repressed by the RUNX1-ETO additional exon 9a fusion protein in a dominant-negative manner. Further functional studies confirmed that ADAR2 could suppress leukemogenesis specifically in t(8;21) and inv16 AML cells dependent on its RNA editing capability. Expression of 2 exemplary ADAR2-regulated RNA editing targets coatomer subunit α and component of oligomeric Golgi complex 3 inhibits the clonogenic growth of human t(8;21) AML cells. Our findings support a hitherto, unappreciated mechanism leading to ADAR2 dysregulation in CBF AML and highlight the functional relevance of loss of ADAR2-mediated RNA editing to CBF AML.

FEZ1 participates in human embryonic brain development by modulating neuronal progenitor subpopulation specification and migrations.

Mutations in the human fasciculation and elongation protein zeta 1 (FEZ1) gene are found in schizophrenia and Jacobsen syndrome patients. Here, using human cerebral organoids (hCOs), we show that FEZ1 expression is turned on early during brain development and is detectable in both neuroprogenitor subtypes and immature neurons. FEZ1 deletion disrupts expression of neuronal and synaptic development genes. Using single-cell RNA sequencing, we detected abnormal expansion of homeodomain-only protein homeobox (HOPX)- outer radial glia (oRG), concurrent with a reduction of HOPX+ oRG, in FEZ1-null hCOs. HOPX- oRGs show higher cell mobility as compared to HOPX+ oRGs. Ectopic localization of neuroprogenitors to the outer layer is seen in FEZ1-null hCOs. Anomalous encroachment of TBR2+ intermediate progenitors into CTIP2+ deep layer neurons further indicated abnormalities in cortical layer formation these hCOs. Collectively, our findings highlight the involvement of FEZ1 in early cortical brain development and how it contributes to neurodevelopmental disorders.

Robust generation of human-chambered cardiac organoids from pluripotent stem cells for improved modelling of cardiovascular diseases.

BACKGROUND: Tissue organoids generated from human pluripotent stem cells are valuable tools for disease modelling and to understand developmental processes. While recent progress in human cardiac organoids revealed the ability of these stem cell-derived organoids to self-organize and intrinsically formed chamber-like structure containing a central cavity, it remained unclear the processes involved that enabled such chamber formation. METHODS: Chambered cardiac organoids (CCOs) differentiated from human embryonic stem cells (H7) were generated by modulation of Wnt/ß-catenin signalling under fully defined conditions, and several growth factors essential for cardiac progenitor expansion. Transcriptomic profiling of day 8, day 14 and day 21 CCOs was performed by quantitative PCR and single-cell RNA sequencing. Endothelin-1 (EDN1) known to induce oxidative stress in cardiomyocytes was used to induce cardiac hypertrophy in CCOs in vitro. Functional characterization of cardiomyocyte contractile machinery was performed by immunofluorescence staining and analysis of brightfield and fluorescent video recordings. Quantitative PCR values between groups were compared using two-tailed Student's t tests. Cardiac organoid parameters comparison between groups was performed using two-tailed Mann-Whitney U test when sample size is small; otherwise, Welch's t test was used. Comparison of calcium kinetics parameters derived from the fluorescent data was performed using two-tailed Student's t tests. RESULTS: Importantly, we demonstrated that a threshold number of cardiac progenitor was essential to line the circumference of the inner cavity to ensure proper formation of a chamber within the organoid. Single-cell RNA sequencing revealed improved maturation over a time course, as evidenced from increased mRNA expression of cardiomyocyte maturation genes, ion channel genes and a metabolic shift from glycolysis to fatty acid ß-oxidation. Functionally, CCOs recapitulated clinical cardiac hypertrophy by exhibiting thickened chamber walls, reduced fractional shortening, and increased myofibrillar disarray upon treatment with EDN1. Furthermore, electrophysiological assessment of calcium transients displayed tachyarrhythmic phenotype observed as a consequence of rapid depolarization occurring prior to a complete repolarization. CONCLUSIONS: Our findings shed novel insights into the role of progenitors in CCO formation and pave the way for the robust generation of cardiac organoids, as a platform for future applications in disease modelling and drug screening in vitro.

Pan-cancer pervasive upregulation of 3' UTR splicing drives tumourigenesis.

Most mammalian genes generate messenger RNAs with variable untranslated regions (UTRs) that are important post-transcriptional regulators. In cancer, shortening at 3' UTR ends via alternative polyadenylation can activate oncogenes. However, internal 3' UTR splicing remains poorly understood as splicing studies have traditionally focused on protein-coding alterations. Here we systematically map the pan-cancer landscape of 3' UTR splicing and present this in SpUR ( http://www.cbrc.kaust.edu.sa/spur/home/ ). 3' UTR splicing is widespread, upregulated in cancers, correlated with poor prognosis and more prevalent in oncogenes. We show that antisense oligonucleotide-mediated inhibition of 3' UTR splicing efficiently reduces oncogene expression and impedes tumour progression. Notably, CTNNB1 3' UTR splicing is the most consistently dysregulated event across cancers. We validate its upregulation in hepatocellular carcinoma and colon adenocarcinoma, and show that the spliced 3' UTR variant is the predominant contributor to its oncogenic functions. Overall, our study highlights the importance of 3' UTR splicing in cancer and may launch new avenues for RNA-based anti-cancer therapeutics.

Multilayered control of splicing regulatory networks by DAP3 leads to widespread alternative splicing changes in cancer.

The dynamic regulation of alternative splicing requires coordinated participation of multiple RNA binding proteins (RBPs). Aberrant splicing caused by dysregulation of splicing regulatory RBPs is implicated in numerous cancers. Here, we reveal a frequently overexpressed cancer-associated protein, DAP3, as a splicing regulatory RBP in cancer. Mechanistically, DAP3 coordinates splicing regulatory networks, not only via mediating the formation of ribonucleoprotein complexes to induce substrate-specific splicing changes, but also via modulating splicing of numerous splicing factors to cause indirect effect on splicing. A pan-cancer analysis of alternative splicing across 33 TCGA cancer types identified DAP3-modulated mis-splicing events in multiple cancers, and some of which predict poor prognosis. Functional investigation of non-productive splicing of WSB1 provides evidence for establishing a causal relationship between DAP3-modulated mis-splicing and tumorigenesis. Together, our work provides critical mechanistic insights into the splicing regulatory roles of DAP3 in cancer development.

ADARs act as potent regulators of circular transcriptome in cancer.

Circular RNAs (circRNAs) are produced by head-to-tail back-splicing which is mainly facilitated by base-pairing of reverse complementary matches (RCMs) in circRNA flanking introns. Adenosine deaminases acting on RNA (ADARs) are known to bind double-stranded RNAs for adenosine to inosine (A-to-I) RNA editing. Here we characterize ADARs as potent regulators of circular transcriptome by identifying over a thousand of circRNAs regulated by ADARs in a bidirectional manner through and beyond their editing function. We find that editing can stabilize or destabilize secondary structures formed between RCMs via correcting A:C mismatches to I(G)-C pairs or creating I(G).U wobble pairs, respectively. We provide experimental evidence that editing also favors the binding of RNA-binding proteins such as PTBP1 to regulate back-splicing. These ADARs-regulated circRNAs which are ubiquitously expressed in multiple types of cancers, demonstrate high functional relevance to cancer. Our findings support a hitherto unappreciated bidirectional regulation of circular transcriptome by ADARs and highlight the complexity of cross-talk in RNA processing and its contributions to tumorigenesis.

PLK1 inhibition selectively induces apoptosis in ARID1A deficient cells through uncoupling of oxygen consumption from ATP production.

Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2/M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knock-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of specific subunits of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer.

p53-NEIL1 co-abnormalities induce genomic instability and promote synthetic lethality with Chk1 inhibition in multiple myeloma having concomitant 17p13(del) and 1q21(gain).

Recurrent cytogenetic abnormalities are the main hallmark of multiple myeloma (MM) and patients having 2 or more high-risk prognostic events are associated with extremely poor outcome. 17p13(del) and 1q21(gain) are critical and independent high-risk cytogenetic markers, however, the biological significance underlying the poor outcome in MM patients having co-occurrence of both these chromosomal aberrations has never been interrogated. Herein, we identified that patients harbouring concomitant 17p13(del) with 1q21(gain) demonstrated the worst prognosis as compared to patients with single- (either 17p13(del) or 1q21(gain)) and with no chromosomal events (WT for both chromosomal loci); and they are highly enriched for genomic instability (GI) signature. We discovered that the GI feature in the patients with concomitant 17p13(del)-1q21(gain) was recapitulating the biological properties of myeloma cells with co-existing p53-deficiency and NEIL1 mRNA-hyper-editing (associated with chromosome 17p and 1q, respectively) that have inherent DNA damage response (DDR) and persistent activation of Chk1 pathway. Importantly, this became a vulnerable point for therapeutic targeting whereby the cells with this co-abnormalities demonstrated hyper-sensitivity to siRNA- and pharmacological-mediated-Chk1 inhibition, as observed at both the in vitro and in vivo levels. Mechanistically, this was attributable to the synthetic lethal relationship between p53-NEIL1-Chk1 abnormalities. The Chk1 inhibitor (AZD7762) tested showed good synergism with standard-of-care myeloma drugs, velcade and melphalan, thus further reinforcing the translational potential of this therapeutic approach. In summary, combination of NEIL1-p53 abnormalities with an ensuing Chk1 activation could serve as an Achilles heel and predispose MM cells with co-existing 1q21(gain) and 17p13(del) to therapeutic vulnerability for Chk1 inhibition.

T and NK cell lymphoma cell lines do not rely on ZAP-70 for survival.

B-cell receptor (BCR) signalling is critical for the survival of B-cell lymphomas and is a therapeutic target of drugs such as Ibrutinib. However, the role of T-cell receptor (TCR) signalling in the survival of T/Natural Killer (NK) lymphomas is not clear. ZAP-70 (zeta associated protein-70) is a cytoplasmic tyrosine kinase with a critical role in T-cell receptor (TCR) signalling. It has also been shown to play a role in normal NK cell signalling and activation. High ZAP-70 expression has been detected by immunohistochemistry in peripheral T cell lymphoma (PTCL) and NK cell lymphomas (NKTCL). We therefore, studied the role of TCR pathways in mediating the proliferation and survival of these malignancies through ZAP-70 signalling. ZAP-70 protein was highly expressed in T cell lymphoma cell lines (JURKAT and KARPAS-299) and NKTCL cell lines (KHYG-1, HANK-1, NK-YS, SNK-1 and SNK-6), but not in multiple B-cell lymphoma cell lines. siRNA depletion of ZAP-70 suppressed the phosphorylation of ZAP-70 substrates, SLP76, LAT and p38MAPK, but did not affect cell viability or induce apoptosis in these cell lines. Similarly, while stable overexpression of ZAP-70 mediates increased phosphorylation of target substrates in the TCR pathway, it does not promote increased survival or growth of NKTCL cell lines. The epidermal growth factor receptor (EGFR) inhibitor Gefitinib, which has off-target activity against ZAP-70, also did not show any differential cell kill between ZAP-70 overexpressing (OE) or knockdown (KD) cell lines. Whole transcriptome RNA sequencing highlighted that there was very minimal differential gene expression in three different T/NK cell lines induced by ZAP-70 KD. Importantly, ZAP-70 KD did not significantly enrich for any downstream TCR related genes and pathways. Altogether, this suggests that high expression and constitutive signalling of ZAP-70 in T/NK lymphoma is not critical for cell survival or downstream TCR-mediated signalling and gene expression. ZAP-70 therefore may not be a suitable therapeutic target in T/NK cell malignancies.

Upregulation of the JAK-STAT pathway promotes maturation of human embryonic stem cell-derived cardiomyocytes.

The immature characteristics and metabolic phenotypes of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) restrict their applications for disease modeling, drug discovery, and cell-based therapy. Leveraging on the metabolic shifts from glycolysis to fatty acid oxidation as CMs mature, a human hexokinase1-GFP metabolic reporter cell line (H7 HK1-GFP) was generated to facilitate the isolation of fetal or more matured hPSC-CMs. RNA sequencing of fetal versus more matured CMs uncovered a potential role of interferon-signaling pathway in regulating CM maturation. Indeed, IFN-γ-treated CMs resulted in an upregulation of the JAK-STAT pathway, which was found to be associated with increased expression of CM maturation genes, shift from MYH6 to MYH7 expression, and improved sarcomeric structure. Functionally, IFN-γ-treated CMs exhibited a more matured electrophysiological profile, such as increased calcium dynamics and action potential upstroke velocity, demonstrated through calcium imaging and MEA. Expectedly, the functional improvements were nullified with a JAK-STAT inhibitor, ruxolitinib.

Antisense RNAs Influence Promoter Usage of Their Counterpart Sense Genes in Cancer.

Multiple noncoding natural antisense transcripts (ncNAT) are known to modulate key biological events such as cell growth or differentiation. However, the actual impact of ncNATs on cancer progression remains largely unknown. In this study, we identified a complete list of differentially expressed ncNATs in hepatocellular carcinoma. Among them, a previously undescribed ncNAT HNF4A-AS1L suppressed cancer cell growth by regulating its sense gene HNF4A, a well-known cancer driver, through a promoter-specific mechanism. HNF4A-AS1L selectively activated the HNF4A P1 promoter via HNF1A, which upregulated expression of tumor suppressor P1-driven isoforms, while having no effect on the oncogenic P2 promoter. RNA-seq data from 23 tissue and cancer types identified approximately 100 ncNATs whose expression correlated specifically with the activity of one promoter of their associated sense gene. Silencing of two of these ncNATs ENSG00000259357 and ENSG00000255031 (antisense to CERS2 and CHKA, respectively) altered the promoter usage of CERS2 and CHKA. Altogether, these results demonstrate that promoter-specific regulation is a mechanism used by ncNATs for context-specific control of alternative isoform expression of their counterpart sense genes. SIGNIFICANCE: This study characterizes a previously unexplored role of ncNATs in regulation of isoform expression of associated sense genes, highlighting a mechanism of alternative promoter usage in cancer.

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness.

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.

"3G" Trial: An RNA Editing Signature to Guide Gastric Cancer Chemotherapy.

Gastric cancer cases are often diagnosed at an advanced stage with poor prognosis. Platinum-based chemotherapy has been internationally accepted as first-line therapy for inoperable or metastatic gastric cancer. To achieve greater benefits, selection of patients eligible for this treatment is critical. Although gene expression profiling has been widely used as a genomic classifier to identify molecular subtypes of gastric cancer and to stratify patients for different chemotherapy regimens, its prediction accuracy can be improved. Adenosine-to-inosine (A-to-I) RNA editing has emerged as a new player contributing to gastric cancer development and progression, offering potential clinical utility for diagnosis and treatment. Using a systematic computational approach followed by both in vitro validations and in silico validations in The Cancer Genome Atlas (TCGA), we conducted a transcriptome-wide RNA editing analysis of a cohort of 104 patients with advanced gastric cancer and identified an RNA editing (GCRE) signature to guide gastric cancer chemotherapy. RNA editing events stood as a prognostic and predictive biomarker in advanced gastric cancer. A GCRE score based on the GCRE signature consisted of 50 editing sites associated with 29 genes, predicting response to chemotherapy with a high accuracy (84%). Of note, patients demonstrating higher editing levels of this panel of sites presented a better overall response. Consistently, gastric cancer cell lines with higher editing levels showed higher chemosensitivity. Applying the GCRE score on TCGA dataset confirmed that responders had significantly higher levels of editing in advanced gastric cancer. Overall, this newly defined GCRE signature reliably stratifies patients with advanced gastric cancer and predicts response from chemotherapy. SIGNIFICANCE: This study describes a novel A-to-I RNA editing signature as a prognostic and predictive biomarker in advanced gastric cancer, providing a new tool to improve patient stratification and response to therapy.

RNA editing mediates the functional switch of COPA in a novel mechanism of hepatocarcinogenesis.

BACKGROUND & AIMS: RNA editing introduces nucleotide changes in RNA sequences. Recent studies have reported that aberrant adenosine-to-inosine RNA editing is implicated in cancers. Until now, very few functionally important protein-recoding editing targets have been discovered. Here, we investigated the role of a recently discovered protein-recoding editing target COPA (coatomer subunit α) in hepatocellular carcinoma (HCC). METHODS: Clinical implication of COPA editing was studied in a cohort of 125 HCC patients. CRISPR/Cas9-mediated knockout of the editing site complementary sequence (ECS) was used to delete edited COPA transcripts endogenously. COPA editing-mediated change in its transcript or protein stability was investigated upon actinomycin D or cycloheximide treatment, respectively. Functional difference in tumourigenesis between wild-type and edited COPA (COPAWTvs. COPAI164V) and the exact mechanisms were also studied in cell models and mice. RESULTS: ADAR2 binds to double-stranded RNA formed between edited exon 6 and the ECS at intron 6 of COPA pre-mRNA, causing an isoleucine-to-valine substitution at residue 164. Reduced editing of COPA is implicated in the pathogenesis of HCC, and more importantly, it may be involved in many cancer types. Upon editing, COPAWT switches from a tumour-promoting gene to a tumour suppressor that has a dominant-negative effect. Moreover, COPAI164V may undergo protein conformational change and therefore become less stable than COPAWT. Mechanistically, COPAI164V may deactivate the PI3K/AKT/mTOR pathway through downregulation of caveolin-1 (CAV1). CONCLUSIONS: We uncover an RNA editing-associated mechanism of hepatocarcinogenesis by which downregulation of ADAR2 caused the loss of tumour suppressive COPAI164V and concurrent accumulation of tumour-promoting COPAWT in tumours; a rapid degradation of COPAI164V protein and hyper-activation of the PI3K/AKT/mTOR pathway further promote tumourigenesis. LAY SUMMARY: RNA editing is a process in which RNA is changed after it is made from DNA, resulting in an altered gene product. In this study, we found that RNA editing of a gene known as coatomer subunit α (COPA) is lower in tumour samples and discovered that this editing process changes COPA protein from a tumour-promoting form to a tumour-suppressive form. Loss of the edited COPA promotes the development of liver cancer.

CSI NGS Portal: An Online Platform for Automated NGS Data Analysis and Sharing.

Next-generation sequencing (NGS) has been a widely-used technology in biomedical research for understanding the role of molecular genetics of cells in health and disease. A variety of computational tools have been developed to analyse the vastly growing NGS data, which often require bioinformatics skills, tedious work and a significant amount of time. To facilitate data processing steps minding the gap between biologists and bioinformaticians, we developed CSI NGS Portal, an online platform which gathers established bioinformatics pipelines to provide fully automated NGS data analysis and sharing in a user-friendly website. The portal currently provides 16 standard pipelines for analysing data from DNA, RNA, smallRNA, ChIP, RIP, 4C, SHAPE, circRNA, eCLIP, Bisulfite and scRNA sequencing, and is flexible to expand with new pipelines. The users can upload raw data in FASTQ format and submit jobs in a few clicks, and the results will be self-accessible via the portal to view/download/share in real-time. The output can be readily used as the final report or as input for other tools depending on the pipeline. Overall, CSI NGS Portal helps researchers rapidly analyse their NGS data and share results with colleagues without the aid of a bioinformatician. The portal is freely available at: https://csibioinfo.nus.edu.sg/csingsportal.

Suppression of adenosine-to-inosine (A-to-I) RNA editome by death associated protein 3 (DAP3) promotes cancer progression.

RNA editing introduces nucleotide changes in RNA sequences. Recent studies have reported that aberrant A-to-I RNA editing profiles are implicated in cancers. Albeit changes in expression and activity of ADAR genes are thought to have been responsible for the dysregulated RNA editome in diseases, they are not always correlated, indicating the involvement of secondary regulators. Here, we uncover DAP3 as a potent repressor of editing and a strong oncogene in cancer. DAP3 mainly interacts with the deaminase domain of ADAR2 and represses editing via disrupting association of ADAR2 with its target transcripts. PDZD7, an exemplary DAP3-repressed editing target, undergoes a protein recoding editing at stop codon [Stop →Trp (W)]. Because of editing suppression by DAP3, the unedited PDZD7WT, which is more tumorigenic than edited PDZD7Stop518W, is accumulated in tumors. In sum, cancer cells may acquire malignant properties for their survival advantage through suppressing RNA editome by DAP3.

Cis- and trans-regulations of pre-mRNA splicing by RNA editing enzymes influence cancer development.

RNA editing and splicing are the two major processes that dynamically regulate human transcriptome diversity. Despite growing evidence of crosstalk between RNA editing enzymes (mainly ADAR1) and splicing machineries, detailed mechanistic explanations and their biological importance in diseases, such as cancer are still lacking. Herein, we identify approximately a hundred high-confidence splicing events altered by ADAR1 and/or ADAR2, and ADAR1 or ADAR2 protein can regulate cassette exons in both directions. We unravel a binding tendency of ADARs to dsRNAs that involves GA-rich sequences for editing and splicing regulation. ADAR1 edits an intronic splicing silencer, leading to recruitment of SRSF7 and repression of exon inclusion. We also present a mechanism through which ADAR2 binds to dsRNA formed between GA-rich sequences and polypyrimidine (Py)-tract and precludes access of U2AF65 to 3' splice site. Furthermore, we find these ADARs-regulated splicing changes per se influence tumorigenesis, not merely byproducts of ADARs editing and binding.

Master transcription factors form interconnected circuitry and orchestrate transcriptional networks in oesophageal adenocarcinoma.

OBJECTIVE: While oesophageal squamous cell carcinoma remains infrequent in Western populations, the incidence of oesophageal adenocarcinoma (EAC) has increased sixfold to eightfold over the past four decades. We aimed to characterise oesophageal cancer-specific and subtypes-specific gene regulation patterns and their upstream transcription factors (TFs). DESIGN: To identify regulatory elements, we profiled fresh-frozen oesophageal normal samples, tumours and cell lines with chromatin immunoprecipitation sequencing (ChIP-Seq). Mathematical modelling was performed to establish (super)-enhancers landscapes and interconnected transcriptional circuitry formed by master TFs. Coregulation and cooperation between master TFs were investigated by ChIP-Seq, circularised chromosome conformation capture sequencing and luciferase assay. Biological functions of candidate factors were evaluated both in vitro and in vivo. RESULTS: We found widespread and pervasive alterations of the (super)-enhancer reservoir in both subtypes of oesophageal cancer, leading to transcriptional activation of a myriad of novel oncogenes and signalling pathways, some of which may be exploited pharmacologically (eg, leukemia inhibitory factor (LIF) pathway). Focusing on EAC, we bioinformatically reconstructed and functionally validated an interconnected circuitry formed by four master TFs-ELF3, KLF5, GATA6 and EHF-which promoted each other's expression by interacting with each super-enhancer. Downstream, these master TFs occupied almost all EAC super-enhancers and cooperatively orchestrated EAC transcriptome. Each TF within the transcriptional circuitry was highly and specifically expressed in EAC and functionally promoted EAC cell proliferation and survival. CONCLUSIONS: By establishing cancer-specific and subtype-specific features of the EAC epigenome, our findings promise to transform understanding of the transcriptional dysregulation and addiction of EAC, while providing molecular clues to develop novel therapeutic modalities against this malignancy.