Oxygen sensing is central to metazoan biology and has implications for human disease. Mammalian cells express multiple oxygen-dependent enzymes called 2-oxoglutarate (OG)-dependent dioxygenases ...(2-OGDDs), but they vary in their oxygen affinities and hence their ability to sense oxygen. The 2-OGDD histone demethylases control histone methylation. Hypoxia increases histone methylation, but whether this reflects direct effects on histone demethylases or indirect effects caused by the hypoxic induction of the HIF (hypoxia-inducible factor) transcription factor or the 2-OG antagonist 2-hydroxyglutarate (2-HG) is unclear. Here, we report that hypoxia promotes histone methylation in a HIF- and 2-HG-independent manner. We found that the H3K27 histone demethylase KDM6A/UTX, but not its paralog KDM6B, is oxygen sensitive. KDM6A loss, like hypoxia, prevented H3K27 demethylation and blocked cellular differentiation. Restoring H3K27 methylation homeostasis in hypoxic cells reversed these effects. Thus, oxygen directly affects chromatin regulators to control cell fate.
Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma with a single driver BRAF rearrangement. Here, we perform scRNAseq in six PAs using methods that enabled ...detection of the rearrangement. When compared to higher-grade gliomas, a strikingly higher proportion of the PA cancer cells exhibit a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibit a progenitor-like phenotype with evidence of proliferation. These express a mitogen-activated protein kinase (MAPK) programme that was absent from higher-grade gliomas. Immune cells, especially microglia, comprise 40% of all cells in the PAs and account for differences in bulk expression profiles between tumor locations and subtypes. These data indicate that MAPK signaling is restricted to relatively undifferentiated cancer cells in PA, with implications for investigational therapies directed at this pathway.
More than 90% of small cell lung cancers (SCLCs) harbor loss-of-function mutations in the tumor suppressor gene
The canonical function of the
gene product, pRB, is to repress the E2F transcription ...factor family, but pRB also functions to regulate cellular differentiation in part through its binding to the histone demethylase KDM5A (also known as RBP2 or JARID1A). We show that KDM5A promotes SCLC proliferation and SCLC's neuroendocrine differentiation phenotype in part by sustaining expression of the neuroendocrine transcription factor ASCL1. Mechanistically, we found that KDM5A sustains ASCL1 levels and neuroendocrine differentiation by repressing NOTCH2 and NOTCH target genes. To test the role of KDM5A in SCLC tumorigenesis in vivo, we developed a CRISPR/Cas9-based mouse model of SCLC by delivering an adenovirus (or an adeno-associated virus AAV) that expresses Cre recombinase and sgRNAs targeting
, and
into the lungs of Lox-Stop-Lox Cas9 mice. Coinclusion of a KDM5A sgRNA decreased SCLC tumorigenesis and metastasis, and the SCLCs that formed despite the absence of KDM5A had higher NOTCH activity compared to
SCLCs. This work establishes a role for KDM5A in SCLC tumorigenesis and suggests that KDM5 inhibitors should be explored as treatments for SCLC.
Celastrol, a pentacyclic triterpene, is the most potent antiobesity agent that has been reported thus far
. The mechanism of celastrol's leptin-sensitizing and antiobesity effects has not yet been ...elucidated. In this study, we identified interleukin-1 receptor 1 (IL1R1) as a mediator of celastrol's action by using temporally resolved analysis of the hypothalamic transcriptome in celastrol-treated DIO, lean, and db/db mice. We demonstrate that IL1R1-deficient mice are completely resistant to the effects of celastrol in leptin sensitization and treatment of obesity, diabetes, and nonalcoholic steatohepatitis. Thus, we conclude that IL1R1 is a gatekeeper for celastrol's metabolic actions.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The immune environment and the potential for neuroendocrine tumors (NETs) to respond to immune checkpoint inhibitors remain largely unexplored. We assessed immune checkpoint marker expression, ...lymphocytic infiltrate, and associated mutational profiles in a cohort of small intestine and pancreatic NETs.
We assessed expression of PDCD1 (PD-1), CD274 (PD-L1), and PDCD1LG2 (PD-L2) in archival tissue from 64 small intestine (SINETs) and 31 pancreatic NETs (pNET). We additionally assessed T-cell infiltrates, categorizing T-cell subsets based on expression of the T-cell markers CD3, CD8, CD45RO (PTPRC), or FOXP3. Finally, we explored associations between immune checkpoint marker expression, lymphocytic infiltrate, and tumor mutational profiles.
Expression of PD-1 or PD-L1 in small intestine or pancreatic NET was rare, whereas expression of PD-L2 was common in both NET subtypes. T-cell infiltrates were more abundant in pNET than in SINET. We found no clear associations between immune checkpoint marker expression, immune infiltrates, and specific mutational profile within each tumor type.
Our findings provide an initial assessment of the immune environment of well-differentiated NETs. Further studies to define the immunologic differences between pNET and SINET, as well as the role of PD-L2 in these tumors, are warranted.
Chromophobe renal cell carcinoma (ChRCC) accounts for 5% of all sporadic renal cancers and can also occur in genetic syndromes including Birt–Hogg–Dube (BHD) and tuberous sclerosis complex (TSC). ...ChRCC has a distinct accumulation of abnormal mitochondria, accompanied by characteristic chromosomal imbalances and relatively few “driver” mutations. Metabolomic profiling of ChRCC and oncocytomas (benign renal tumors that share pathological features with ChRCC) revealed both similarities and differences between these tumor types, with principal component analysis (PCA) showing a distinct separation. ChRCC have a striking decrease in intermediates of the glutathione salvage pathway (also known as the gamma-glutamyl cycle) compared with adjacent normal kidney, as well as significant changes in glycolytic and pentose phosphate pathway intermediates. We also found that gamma glutamyl transferase 1 (GGT1), the key enzyme of the gamma-glutamyl cycle, is expressed at ∼100-fold lower levels in ChRCC compared with normal kidney, while no change in GGT1 expression was found in clear cell RCC (ccRCC). Significant differences in specific metabolite abundance were found in ChRCC vs. ccRCC, including the oxidative stress marker ophthalmate. Down-regulation of GGT1 enhanced the sensitivity to oxidative stress and treatment with buthionine sulfoximine (BSO), which was associated with changes in glutathione-pathway metabolites. These data indicate that impairment of the glutathione salvage pathway, associated with enhanced oxidative stress, may have key therapeutic implications for this rare tumor type for which there are currently no specific targeted therapies.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
RNA sequencing has become a ubiquitous technology used throughout life sciences as an effective method of measuring RNA abundance quantitatively in tissues and cells. The increase in use of RNA-seq ...technology has led to the continuous development of new tools for every step of analysis from alignment to downstream pathway analysis. However, effectively using these analysis tools in a scalable and reproducible way can be challenging, especially for non-experts.
Using the workflow management system Snakemake we have developed a user friendly, fast, efficient, and comprehensive pipeline for RNA-seq analysis. VIPER (Visualization Pipeline for RNA-seq analysis) is an analysis workflow that combines some of the most popular tools to take RNA-seq analysis from raw sequencing data, through alignment and quality control, into downstream differential expression and pathway analysis. VIPER has been created in a modular fashion to allow for the rapid incorporation of new tools to expand the capabilities. This capacity has already been exploited to include very recently developed tools that explore immune infiltrate and T-cell CDR (Complementarity-Determining Regions) reconstruction abilities. The pipeline has been conveniently packaged such that minimal computational skills are required to download and install the dozens of software packages that VIPER uses.
VIPER is a comprehensive solution that performs most standard RNA-seq analyses quickly and effectively with a built-in capacity for customization and expansion.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Lack of insight into mechanisms governing breast cancer metastasis has precluded the development of curative therapies. Metastasis-initiating cancer cells (MICs) are uniquely equipped to establish ...metastases, causing recurrence and therapeutic resistance. Using various metastasis models, we discovered that certain primary tumours elicit a systemic inflammatory response involving interleukin-1β (IL-1β)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1β maintains MICs in a ZEB1-positive differentiation state, preventing MICs from generating highly proliferative E-cadherin-positive progeny. Thus, when the inherent plasticity of MICs is impeded, overt metastases cannot be established. Ablation of the pro-inflammatory response or inhibition of the IL-1 receptor relieves the differentiation block and results in metastatic colonization. Among patients with lymph node-positive breast cancer, high primary tumour IL-1β expression is associated with better overall survival and distant metastasis-free survival. Our data reveal complex interactions that occur between primary tumours and disseminated MICs that could be exploited to improve patient survival.
Regulatory T cells (Tregs) are vital for maintaining immune self-tolerance, and their impaired function leads to autoimmune disease. Mutations in FoxP3, the master transcriptional regulator of Tregs, ...leads to immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome in humans and the early lethal “scurfy” phenotype with multi-organ autoimmune disease in mice. We recently identified serine/arginine-rich splicing factor 1 (SRSF1) as an indispensable regulator of Treg homeostasis and function. Intriguingly, Treg-conditional SRSF1-deficient mice exhibit early lethal systemic autoimmunity with multi-organ inflammation reminiscent of the scurfy mice. Importantly, SRSF1 is decreased in T cells from patients with the autoimmune disease systemic lupus erythematosus (SLE), and low SRSF1 levels inversely correlate with disease severity. Given that the Treg-specific deficiency of SRSF1 causes similarly profound autoimmune disease outcomes in mice as the deficiency/mutation in FoxP3, we aimed to evaluate the genes and molecular pathways controlled by these two indispensable regulatory proteins. We performed comparative bioinformatic analyses of transcriptomic profiles of Tregs from Srsf1-knockout mice and two Foxp3 mutant mice––the FoxP3-deficient ΔFoxp3 and the Foxp3 M370I mutant mice. We identified 132 differentially expressed genes (DEGs) unique to Srsf1-ko Tregs, 503 DEGs unique to Foxp3 M370I Tregs, and 1367 DEGs unique to ΔFoxp3 Tregs. Gene set enrichment and pathway analysis of DEGs unique to Srsf1-ko Tregs indicate that SRSF1 controls cytokine and immune response pathways. Conversely, FoxP3 controls pathways involved in DNA replication and cell cycle. Besides the distinct gene signatures, we identified only 30 shared genes between all three Treg mutants, mostly contributing to cytokine and immune defense pathways. Prominent genes included the chemokines CXCR6 and CCL1 and the checkpoint inhibitors FASLG and PDCD1. Thus, we demonstrate that SRSF1 and FoxP3 control common and distinct molecular pathways implicated in autoimmunity. Our analyses suggest that SRSF1 controls crucial immune functions in Tregs contributing to immune tolerance, and perturbations in its levels lead to systemic autoimmunity via mechanisms that are largely distinct from FoxP3.
•SRSF1- and FoxP3-deficient mice share similar autoimmune disease phenotypes.•SRSF1 controls gene programs in regulatory T cells discrete from FoxP3.•SRSF1 regulates cytokine signaling and leukocyte differentiation pathways in regulatory T cells.•SRSF1 and FoxP3 control shared and distinct molecular pathways to autoimmunity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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