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T helper （Tn）-cell subsets, such as TH1 and TH17, mediate inflammation in both peripheral tissues and central nervous system. Here we show that STAT5 is required for T helper-cell pathogenicity in autoimmune neuroinflammation but not in experimental colitis. Although STAT5 promotes regulatory T cell generation and immune suppression, loss of STAT5 in CD4~ T cells resulted in diminished development of experimental autoimmune encephalomyelitis （EAE）, a mouse model of multiple sclerosis. Our results showed that loss of encephalitogenic activity of STAT5-deficient autoreaetive CD4＋ T cells was independent of IFN-7 or interleukin 17 （IL-17） production, but was due to the impaired expression of granulocyte-macrophage colony-stimulating factor （GM-CSF）, a crucial mediator of T-cell pathogenicity. We further showed that IL-7-activated STAT5 promotes the generation of GM-CSF-producing CD4＋ T cells, which were preferentially able to induce more severe EAE than TH17 or TH1 cells. Consistent with GM-CSF-produc- ing cells being a distinct subset of TH cells, the differentiation program of these cells was distinct from that of TH17 or TH1 cells. We further found that IL-3 was secreted in a similar pattern as GM-CSF in this subset of T, cells. In conclusion, the IL-7-STAT5 axis promotes the generation of GM-CSF/IL-3-producing T, cells. These cells display a distinct transcriptional profile and may represent a novel subset of T helper cells which we designate as TH-GM.

Key Points Hypoxia and the cellular hypoxic response have key roles in homeostasis and physiological adaptations, as well as in pathophysiological conditions. The cellular hypoxic response can generate both diversity and specificity in the downstream signalling output, despite a relatively simple core signalling pathway. Hypoxia-inducible factor-α proteins constitute key transcriptional regulators in the cellular hypoxic response, and are subject to various different post-translational modifications. Analysis of the hypoxia transcriptional response has begun to reveal a core hypoxic transcriptional signature in addition to cell type-specific gene activation events. The transcriptional responses to acute and chronic hypoxia are distinct. Likewise, hypoxia-mimicking chemical compounds have a substantially broader transcriptional output than hypoxia. Intersections with other signalling mechanisms, such as Myc and Notch signalling, contribute to modulation of the hypoxic response. In addition to the core hypoxic transcriptional pathway, several mechanisms exist to allow transcriptional diversity and specificity. This Review highlights recent advances in our understanding of the mechanisms and factors that contribute to the tailoring of the appropriate hypoxic transcriptional response. The sensing of oxygen levels and maintenance of oxygen homeostasis is crucial for cells. The hypoxic-sensitive regulation of gene expression allows information about the oxygen status to be converted into appropriate cellular responses. Although there is a core transcriptional pathway, the signalling cascade can be modified to allow diversity and specificity in the transcriptional output. In this Review, we discuss recent advances in our understanding of the mechanisms and factors that contribute to the observed diversity and specificity. A deeper knowledge about how hypoxic signalling is tuned will further our understanding of the cellular hypoxic response in normal physiology and how it becomes derailed in disease.

ObjectivesOesophageal squamous cell carcinoma (OSCC) is an aggressive malignancy and the major histological subtype of oesophageal cancer. Although recent large-scale genomic analysis has improved the description of the genetic abnormalities of OSCC, few targetable genomic lesions have been identified, and no molecular therapy is available. This study aims to identify druggable candidates in this tumour.DesignHigh-throughput small-molecule inhibitor screening was performed to identify potent anti-OSCC compounds. Whole-transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) were conducted to decipher the mechanisms of action of CDK7 inhibition in OSCC. A variety of in vitro and in vivo cellular assays were performed to determine the effects of candidate genes on OSCC malignant phenotypes.ResultsThe unbiased high-throughput small-molecule inhibitor screening led us to discover a highly potent anti-OSCC compound, THZ1, a specific CDK7 inhibitor. RNA-Seq revealed that low-dose THZ1 treatment caused selective inhibition of a number of oncogenic transcripts. Notably, further characterisation of the genomic features of these THZ1-sensitive transcripts demonstrated that they were frequently associated with super-enhancer (SE). Moreover, SE analysis alone uncovered many OSCC lineage-specific master regulators. Finally, integrative analysis of both THZ1-sensitive and SE-associated transcripts identified a number of novel OSCC oncogenes, including PAK4, RUNX1, DNAJB1, SREBF2 and YAP1, with PAK4 being a potential druggable kinase.ConclusionsOur integrative approaches led to a catalogue of SE-associated master regulators and oncogenic transcripts, which may significantly promote both the understanding of OSCC biology and the development of more innovative therapies.

ObjectiveWhile 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). DesignTo 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.ResultsWe 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.ConclusionsBy 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.

De-Chen Lin, Ming-Rong Wang and colleagues report exome sequencing, RNA sequencing, and copy number analyses of esophageal squamous cell carcinoma. They identified recurrent mutations in FAT1 , FAT2 , ZNF750 , EP300 and KMT2D . Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide and particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified previously uncharacterized mutated genes such as FAT1 , FAT2 , ZNF750 and KMT2D, in addition to those already known ( TP53, PIK3CA and NOTCH1 ). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Our approaches also uncovered many druggable candidates, and XPO1 was further explored as a therapeutic target because it showed both gene mutation and protein overexpression. Our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets.

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. RNA editing and circRNAs are involved in tumorigenesis. Here the authors report that ADARs regulate the circular transcriptome in a bidirectional manner through and beyond their editing function in multiple cancer cells.

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. RNA editing and RNA splicing are involved in tumorigenesis. Here the authors report crosstalk between RNA editing and splicing by identifying ADAR1/2-dependent splicing events in esophageal squamous carcinoma cells.

Somatic mutations in the spliceosome gene ZRSR2 —located on the X chromosome—are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3′-splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here we characterize ZRSR2 as an essential component of the minor spliceosome (U12 dependent) assembly. shRNA-mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns and RNA-sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns, while splicing of the U2-type introns remain mostly unaffected. ZRSR2-deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro . These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS. Somatic mutations in components of the core RNA splicing machinery, including ZRSR2, have been implicated in myelodysplastic syndrome (MDS). Here, Madan et al. show that ZRSR2 plays a pivotal role in splicing of the U12-type introns, while the U2-dependent splicing is largely unaffected in ZRSR2 mutant MDS bone marrow.

De-Chen Lin and colleagues report whole-exome sequencing, targeted sequencing and SNP array analysis of 128 cases of nasopharyngeal carcinoma (NPC), a tumor type connected to Epstein-Barr virus infection. Their results identify a distinct mutational signature with nine significantly mutated genes and mutations enriched in cellular processes, including chromatin modification and autophagy. Nasopharyngeal carcinoma (NPC) has extremely skewed ethnic and geographic distributions, is poorly understood at the genetic level and is in need of effective therapeutic approaches. Here we determined the mutational landscape of 128 cases with NPC using whole-exome and targeted deep sequencing, as well as SNP array analysis. These approaches revealed a distinct mutational signature and nine significantly mutated genes, many of which have not been implicated previously in NPC. Notably, integrated analysis showed enrichment of genetic lesions affecting several important cellular processes and pathways, including chromatin modification, ERBB-PI3K signaling and autophagy machinery. Further functional studies suggested the biological relevance of these lesions to the NPC malignant phenotype. In addition, we uncovered a number of new druggable candidates because of their genomic alterations. Together our study provides a molecular basis for a comprehensive understanding of, and exploring new therapies for, NPC.

Metabolic reprogramming is widely known as a hallmark of cancer cells to allow adaptation of cells to sustain survival signals. In this report, we describe a novel oncogenic signaling pathway exclusively acting in mutated epidermal growth factor receptor (EGFR) non‐small cell lung cancer (NSCLC) with acquired tyrosine kinase inhibitor (TKI) resistance. Mutated EGFR mediates TKI resistance through regulation of the fatty acid synthase (FASN), which produces 16‐C saturated fatty acid palmitate. Our work shows that the persistent signaling by mutated EGFR in TKI‐resistant tumor cells relies on EGFR palmitoylation and can be targeted by Orlistat, an FDA‐approved anti‐obesity drug. Inhibition of FASN with Orlistat induces EGFR ubiquitination and abrogates EGFR mutant signaling, and reduces tumor growths both in culture systems and in vivo . Together, our data provide compelling evidence on the functional interrelationship between mutated EGFR and FASN and that the fatty acid metabolism pathway is a candidate target for acquired TKI‐resistant EGFR mutant NSCLC patients. Synopsis In EGFR mutated Non‐Small Cell Lung Carcinoma with acquired Tyrosine Kinase Inhibitors resistance, FASN mediates EGFR palmitoylation and supports tumor growth. With limited effective therapeutics, these data show that FASN is a candidate target for acquired TKI‐resistant EGFR mutant NSCLC. Identification of a positive feedback loop involving mutated EGFR and FASN in EGFR mutated NSCLC with acquired TKI resistance. FASN‐mediated EGFR palmitoylaton is crucial for survival of NSCLC cells, and influences its translocation to the nucleus. FASN inhibition suppresses tumor growth in both cell culture systems and in vivo models, highlighting its potential as a target for therapeutic intervention. Graphical Abstract In EGFR mutated Non‐Small Cell Lung Carcinoma with acquired Tyrosine Kinase Inhibitors resistance, FASN mediates EGFR palmitoylation and supports tumor growth. With limited effective therapeutics, these data show that FASN is a candidate target for acquired TKI‐resistant EGFR mutant NSCLC.