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Hypoxemia is a defining feature of acute respiratory distress syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, yet the resulting tissue hypoxia, and its impact on immune responses, is often neglected. In the present study, we have shown that ARDS patients were hypoxemic and monocytopenic within the first 48 h of ventilation. Monocytopenia was also observed in mouse models of hypoxic acute lung injury, in which hypoxemia drove the suppression of type I interferon signaling in the bone marrow. This impaired monopoiesis resulted in reduced accumulation of monocyte-derived macrophages and enhanced neutrophil-mediated inflammation in the lung. Administration of colony-stimulating factor 1 in mice with hypoxic lung injury rescued the monocytopenia, altered the phenotype of circulating monocytes, increased monocyte-derived macrophages in the lung and limited injury. Thus, tissue hypoxia altered the dynamics of the immune response to the detriment of the host and interventions to address the aberrant response offer new therapeutic strategies for ARDS.

Hypoxia-inducible factor (HIF) and aryl hydrocarbon receptor (AHR) are members of the bHLH-PAS family of transcription factors that underpin cellular responses to oxygen and to endogenous and exogenous ligands, respectively, and have central roles in the pathogenesis of renal cancer. Composed of heterodimers, they share a common HIF-1β/ARNT subunit and similar DNA-binding motifs, raising the possibility of crosstalk between the two transcriptional pathways. Here, we identify both general and locus-specific mechanisms of interaction between HIF and AHR that act both antagonistically and cooperatively. Specifically, we observe competition for the common HIF-1β/ARNT subunit, in cis synergy for chromatin binding, and overlap in their transcriptional targets. Recently, both HIF and AHR inhibitors have been developed for the treatment of solid tumours. However, inhibition of one pathway may promote the oncogenic effects of the other. Therefore, our work raises important questions as to whether combination therapy targeting both of these pro-tumourigenic pathways might show greater efficacy than targeting each system independently.

The Jumonji domain–containing protein JMJD6 is a 2-oxoglutarate–dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid–liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia.

Acute Respiratory Distress Syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, has no cure. Hypoxemia is a defining feature, yet its impact on inflammation is often neglected. Patients with ARDS are monocytopenic early in the onset of the disease. Endotoxin or Streptococcus pneumoniae acute lung injury (ALI) in the context of hypoxia replicates this finding, through hypoxia-driven suppression of type I interferon signalling. This results in failed lung monocyte-derived interstitial macrophages (IM) niche expansion and unchecked neutrophilic inflammation. Administration of colony stimulating factor 1 (CSF1) rescues the monocytopenia, alters the circulating classical monocyte phenotype in hypoxic endotoxin-driven ALI and enables lung IM population expansion, thus limiting lung injury in endotoxin- and virally-induced hypoxic ALI. Hypoxia directly alters immune dynamics to the detriment of the host and manipulation of this aberrant response offers new therapeutic strategies for ARDS.

The 2‐oxoglutarate‐dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure–activity relationship and crystallographic studies on a promising class of 4‐hydroxypyrimidine‐containing PHD inhibitors., PHD candidates: The 2‐oxoglutarate‐dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late‐stage clinical trials. However, the number of reported templates for PHD inhibition is limited. We report structure‐activity relationship and crystallographic studies on a promising class of 4‐hydroxypyrimidine‐containing PHD inhibitors.

Hypoxia inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways orchestrate responses to oxygen and nutrient availability. These pathways are frequently dysregulated in cancer, but their interplay is poorly understood, in part because of difficulties in simultaneous measurement of global and mRNA-specific translation. Here, we describe a workflow for measurement of ribosome load of mRNAs resolved by their transcription start sites (TSSs). Its application to kidney cancer cells reveals extensive translational reprogramming by mTOR, strongly affecting many metabolic enzymes and pathways. By contrast, global effects of HIF on translation are limited, and we do not observe reported translational activation by HIF2A. In contrast, HIF-dependent alterations in TSS usage are associated with robust changes in translational efficiency in a subset of genes. Analyses of the interplay of HIF and mTOR reveal that specific classes of HIF1A and HIF2A transcriptional target gene manifest different sensitivity to mTOR, in a manner that supports combined use of HIF2A and mTOR inhibitors in treatment of kidney cancer.

Hypoxia inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways orchestrate responses to oxygen and nutrient availability. These pathways are frequently dysregulated in cancer, but their interplay is poorly understood, in part because of difficulties in simultaneous measurement of global and mRNA-specific translation. Here we describe a workflow for measurement of absolute translational efficiency of mRNAs resolved by their transcription start sites (TSSs). Its application to kidney cancer cells revealed a remarkable extent of translational reprogramming by mTOR, strongly affecting many metabolic enzymes and pathways. By contrast, global effects of HIF on translation were limited, and we did not observe reported translational activation by HIF2A. In contrast, HIF-dependent alterations in TSS usage were associated with robust changes in translational efficiency in a subset of genes. Analyses of the interplay of HIF and mTOR revealed that specific classes of HIF1A and HIF2A transcriptional target gene manifest markedly different sensitivity to mTOR, in a manner that supports combined use of HIF2A and mTOR inhibitors in treatment of kidney cancer.

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment and one important factor to consider is oxygen tension, where hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its Spike glycoprotein binding to angiotensin-converting enzyme (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via a HIF-1α dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication showing that post-entry steps in the viral life cycle are oxygen-sensitive. This study highlights the importance of HIF signalling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19., Graphical Abstract, Wing et al. demonstrate that key aspects of the SARS-CoV-2 life cycle are dependent on cellular oxygen tension. Activation of the cellular oxygen sensing pathway inhibits SARS-CoV-2 infection, highlighting a key cellular pathway that could be exploited as a potential therapeutic avenue for COVID-19.

Hypoxia is a key feature of the solid tumour microenvironment, in which it is associated with poor prognosis and resistance to therapy. Hypoxia-inducible factor (HIF) is the central mediator of cellular responses to hypoxia and drives a transcriptional program with central roles in tumorigenesis. Clear cell renal cell cancers (ccRCC) exhibit the most direct and profound upregulation of the HIF pathway - through bi-allelic inactivation of the VHL tumour suppressor, a ubiquitin ligase that targets HIF-alpha subunits for degradation under normal oxygenated conditions. HIF-alpha proteins are members of the bHLH-PAS family and interact with HIF-1beta (also known as ARNT) to form functional heterodimers binding chromatin. However, the aryl hydrocarbon receptor (AHR), another bHLH-PAS protein that dimerises with HIF-1beta, is also up-regulated in ccRCC, raising the possibility of crosstalk between these two transcriptional pathways. Previous studies support an antagonistic relationship between HIF and AHR, with evidence of a context-dependent and gene-specific crosstalk. Here, we investigate the pan-genomic and precise relationship between the HIF and AHR transcription factors in ccRCC. Using immunoprecipitation, we show direct competition for HIF-1beta binding between the HIF-alpha and AHR proteins in renal cancer cell lines. Pan-genomic analysis of HIF-1alpha, HIF-2alpha, HIF-1beta and AHR chromatin binding by ChIP-seq analysis reveals a widespread competitive interaction between HIF and AHR. In RCC4 cells, this interaction is bidirectional, with induction of HIF leading to a reduction in AHR binding, while induction of AHR reduces binding of both HIF-1alpha and HIF-2alpha to chromatin. However, this competition is diminished at sites bound by both HIF and AHR, indicating a cooperative interaction between the two transcription factors in cis. Thus, while the HIF-alpha and AHR proteins compete for the common binding partner HIF-1beta, they appear to form a cooperative interaction when bound to neighbouring sites on chromatin. Linking these findings to changes in gene expression by RNA-seq analysis, we show the global downstream effects of the competition between HIF and AHR, but also reveal a degree of overlap and cooperativity between their transcriptional programs. Furthermore, we provide evidence of this antagonistic relationship in ccRCC tumours (TCGA database), in which we observe a negative correlation between HIF and AHR target gene expression. These findings shed light on the complex crosstalk between the HIF and AHR transcriptional pathways, which encompasses both a widespread inhibitory interaction and specific cooperative events and highlight a role for AHR in modifying the HIF transcriptional output in ccRCC. Citation Format: Véronique N. Lafleur, Silvia Halim, Peter J. Ratcliffe, David R. Mole. Bi-directional crosstalk between the HIF and AHR transcription factors in clear cell kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 677.

Clear cell renal cell carcinoma (ccRCC) is driven by the biallelic inactivation of the von Hippel Lindau tumor suppressor (VHL) gene, which results directly in the normoxic stabilization of hypoxia inducible factors (HIF-1 and -2). While VHL is expressed ubiquitously, its inactivation is tumorigenic only in specific tissues. We hypothesize that this tissue-specificity arises from a negative selective pressure on VHL KO cells that is tolerated in certain contexts. To assess this phenomenon, we induced Vhl deletion in vivo simultaneously across the mouse body and observed cell fate with single-cell resolution. Here we show that inducing widespread inter-organ Vhl deletion results in a massive removal of affected cells from tissue. Our study required technology that allowed us to visualize and isolate Vhl-recombined cells from tissue. For this, we developed a novel lineage marking model in which Cre-mediated excision of Vhl is coupled to knock-in of a tdTomato reporter cassette under the endogenous Vhl promoter. We used this model in conjunction with the ubiquitously-expressed UBC-CreERT2 to induce marked biallelic (KO/KO) or monoallelic (wt/KO) inactivation of Vhl. One week after inactivation was induced by administration of tamoxifen, there were equal numbers of tdTomato-positive cells in both genotypes, implying that initial recombination was similar and that Vhl KO did not affect immediate cell survival. Surprisingly, there was a massive reduction in the number of tdTomato-positive cells in multiple KO/KO organs after four weeks. This loss was accompanied by proliferation of tdTomato-negative cells, as assayed by BrdU incorporation, suggesting that tissues can at least partially compensate for loss of Vhl-inactivated cells. Finally, we found that the removal of cells was preceded by signs of cellular damage and stress, such as accumulation of p27, cytoplasmic translocation of HMGB1, and TUNEL-positivity in the kidney and liver. Taken together, we conclude that Vhl inactivation is inherently deleterious to cells and entrains a negative cell selection process in multiple organs. One corollary is that there must exist some conditions under which such selection might not occur; secondary mutations found in ccRCC, such as in Pbrm1, or loss of individual HIF isoforms, might provide such conditions to Vhl KO cells. Currently we are investigating the role of these additional perturbations in modifying the cellular response to Vhl loss. Our study will provide new insights into the genesis of ccRCC by redefining the mechanisms underlying the creation of pro-tumorigenic niches following Vhl inactivation. Citation Format: Samvid Kurlekar, Joanna D C Lima, Chris W. Pugh, Julie Adam, Peter J. Ratcliffe. Negative cellular outcomes following acute in vivo Vhl inactivation in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 862.

Kidney cancer is the seventh most common malignancy in the Western world and 80% of these are clear cell renal cell cancers (ccRCC). ccRCC arises following inactivation of the VHL gene, which negatively regulates HIF (hypoxia-inducible factor) transcription factors. Therefore, in ccRCC, HIF is constitutively active and drives a gene expression program that is central to tumorigenesis. Whilst this is necessary for ccRCC formation, it is not sufficient, and other mutations must also occur in combination with VHL inactivation to drive ccRCC tumorigenesis. The mechanisms underlying their synergy with VHL inactivation remain poorly understood. These secondary driver mutations are often subclonal in nature, suggesting the presence of phenotypically distinct malignant cells within an individual tumor. This intratumor heterogeneity renders it difficult to study the functional impact of driver mutations in ccRCC by conventional ‘bulk’ analyses such as RNA-seq. Consequently, the molecular mechanisms underlying ccRCC initiation are not known. Furthermore, non-malignant stromal cells (including the vasculature and immune response) can support tumor growth, and little is known as to how they interact with malignant cells in ccRCC. This is particularly important since current therapeutic strategies in kidney cancer target the stromal compartment. Therefore, there is an unmet need to better-understand transcriptional heterogeneity and cellular diversity in ccRCC. To that end, we have sampled multiple regions from ccRCC tumors (and tumor-adjacent kidney tissue) and performed single cell RNA-seq (10x Genomics) on approximately 150,000 individual cells. These encompass malignant cells as well as non-malignant cells (including fibroblasts, endothelial cells, pericytes and immune cells), both of which exhibit pronounced transcriptional heterogeneity within and between tumors. Analysis of these transcriptomes has identified evolutionary gene expression programs underlying cancer cell dedifferentiation. In addition, we observe concurrent heterogeneity in angiogenic signaling between malignant cells and non-malignant blood vessel cells within these tumors. Lastly, we have explored the effect of microenvironmental hypoxia across different ccRCC tumor cell types. In summary, we present a single cell atlas derived from multi-region heterogenous ccRCC samples. This sheds light on the mechanisms underlying tumor evolution and progression, as well as angiogenic heterogeneity (which may contribute to the variable response to receptor tyrosine kinase inhibition) and the effect of hypoxia on ccRCC biology. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Peter J. Ratcliffe, David R. Mole. Multi region single cell sequencing reveals dedifferentiation programs, angiogenic heterogeneity and the effect of microenvironmental hypoxia in ccRCC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1587.

Low oxygen availability (hypoxia) is a common feature of many solid tumors and is associated with poor prognosis and resistance to therapy. The main regulator of cellular responses to hypoxia is the transcription factor HIF (hypoxia-inducible factor). In addition, several histone lysine demethylases (KDMs) are 2-OG dependent dioxygenases and require oxygen for their activity. When oxygen availability is limited, the activity of these KDMs is inhibited leading to global histone hypermethylation, potentially altering gene expression. However, some KDMs are also known to be direct transcriptional targets of HIF, increasing their expression in hypoxia and counteracting this histone hypermethylation. Furthermore, HIF can facilitate recruitment of histone methyltransferase activity to chromatin in cis, leading to local increases in methylation status. Histone methylation can be associated with both gene activation and gene repression, and it remains unclear how the balance of these widespread changes relates to locus-specific regulation of gene expression. Furthermore, previous attempts to analyze regulation of histone methylation by ChIP-seq in hypoxia have not employed normalization methods that take into account global changes in histone modification. We have performed ChIP-seq analysis of histone trimethylation (H3K4me3, H3K9me3, H3K27me3 and H3K36me3) in PC3 prostate cancer cells incubated in normoxia or (16hrs, 1%) hypoxia. We have then examined the effects of the HIF-transcriptional pathway on these marks using the same cell line following deletion of HIF-1b to block the HIF response. We employ an integrated spike-in with drosophila chromatin to provide a normalization control. We then relate our findings to changes in gene expression using RNA-seq analysis, again normalized to a spike-in control. We observe global increases in all 4 histone trimethylations across the genome in hypoxia. In particular, we observe hypoxic increases in histone trimethylation at all gene loci. However, while basal levels of histone trimethylation correlated with basal transcript levels as previously reported, hypoxic increases in promoter-associated histone H3K4me3 trimethylation bore no relation to either the direction or magnitude of gene regulation in hypoxia. Although, more subtle increases were observed across the gene body at gene loci induced by hypoxia. These findings question the currently accepted model that changes in H3K4me3 instruct changes in transcription in hypoxia and suggest that the overall effect of histone trimethylation is a more subtle balance of competing effects. Citation Format: Jessica D. Kindrick, Peter J. Ratcliffe, WIlliam Doug Figg, David R. Mole. Correlating locus-specific changes in histone trimethylation and gene expression in hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3766.

Low levels of tissue oxygen, or intra-tumor hypoxia, is a common feature of many solid cancers in which dysregulated cell proliferation often outstrips the ability to develop new functional blood vessels. Furthermore, hypoxia pathways may also be activated in response to mutation in key tumor suppressor genes or oncogenes. Importantly, in many types of cancer, activation of hypoxia pathways is associated with a clinically aggressive phenotype as well as with resistance to therapy. Indeed, hypoxia drives many of the “hallmarks” of cancer, including cell proliferation, apoptosis, metabolism, immune responses, genomic instability, vascularization, invasion and metastasis. However, the full-range of genes activated by hypoxia varies greatly between cell-types and for many this remains uncharacterized. The key mediator of the cellular transcriptional response to hypoxia is the HIF (hypoxia-inducible factor) family of transcription factors. Here, we generate pan-genomic datasets encompassing HIF ChIP-seq and RNA-seq across cancer cell lines from diverse common tumor types. We then employ integrated analysis of our 72 transcriptome and whole-genome-binding datasets to define a core set of HIF-target genes that are conserved across multiple cell types. We show that these genes can be used to identify HIF activation and tumor hypoxia in bulk RNA-seq analysis of solid tumors, and that this can be applied to large tumor databases (TCGA). We then leverage this analysis to identify tumor-type-specific HIF-associated genes in 32 different cancer types, including those lacking cell-culture models. We further show that these conserved HIF-target genes can be applied to single cell RNA-seq datasets from solid tumors and used to examine intra-tumor heterogeneity in HIF-pathway activation within individual cancer samples. Importantly, using a panel of genes reduces the phenomenon of “drop-out” typically observed in gene-level expression in these datasets. Lastly, applying this gene signature to single cell data allows the hypoxia response to be deconvoluted across diverse cell-types in heterogenous tumor samples. This includes not only the identification of HIF-activity in cancer cells, but also in non-cancer cells within the tumor, thereby allowing the effects of intra-tumor hypoxia on stromal responses (such as tumor angiogenesis) to be studied in vivo in solid tumors. Citation Format: Olivia Lombardi, Ran Li, Silvia Halim, Peter J. Ratcliffe, David R. Mole. Pan-cancer analysis of HIF pathways defines a robust molecular signature that reflects tissue and cellular hypoxia in bulk and single-cell RNA-seq analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 119.

Clear cell renal cell carcinoma (ccRCC) is driven by biallelic inactivation of von Hippel Lindau (VHL) tumour suppressor gene. While ccRCC genesis has been associated with several signaling and metabolic pathways, the exact process by which VHL inactivation becomes tumorigenic remains unknown. We surmised that observing the behaviour and gene expression profiles of Vhl-inactivated cells in vivo with spatiotemporal resolution would help define cell types and conditions in which this genetic lesion is on balance tumorigenic. Here we report a novel lineage marking model of in vivo Vhl inactivation and describe the immediate consequences of Vhl loss in different nephron segments at cellular resolution. Studying Vhl inactivation with single-cell resolution requires a system in which Vhl-inactivated cells can be accurately identified. We developed a mouse model in which Vhl recombination is structurally linked to the expression of a tdTomato reporter cassette. This allows recombined cells to be visualised and isolated based on tdTomato expression. We coupled this model with the renal tubular epithelium (RTE) restricted Pax8-CreERT2 to induce biallelic (KO/KO) or monoallelic (wt/KO) inactivation of Vhl and observed the immediate fates of affected cells. Vhl is thought to be haplosufficient; thus, the wt/KO mice act as lineage-marked Vhl-competent controls to lineage-marked Vhl-null cells. We first observed the behavior of Vhl KO cells in the RTE over time. Vhl was inactivated by a controlled dose of tamoxifen that induced recombination in a subset of cells across the nephron. Three weeks after recombination, Vhl KO cells were morphologically normal but had stabilized HIF-2, confirming a reported ‘HIF-isoform’ switch. There were equal numbers of tdTomato-positive cells in the KO/KO and wt/KO kidneys, suggesting that initial recombination is equal between the genotypes and that there is no immediate distortion in cell fates following Vhl KO. However, four months after recombination, there were fewer tdTomato-positive cells in the KO/KO kidneys, suggesting a gradual removal or loss of Vhl KO from tissue. This was most prominent in the renal papilla and cortex. Additionally, some Vhl KO cells presented with morphological signs of cellular stress and altered differentiation. Taken together, we believe Vhl KO cells are subjected to profound selection in the nephron and that cell-type-specific, negative, and heterogenous consequences of Vhl KO impact on cell survival. One corollary is that many cells which ultimately survive in Vhl KO organs are in fact Vhl-competent. By intrinsically coupling Vhl-inactivation and lineage marking, our model allows these populations to be distinguished and followed over time. We are now studying the differential gene expression patterns of Vhl KO cells in cells of the RTE and aim to define genetic signatures for contexts in which Vhl KO cells survive and contribute to ccRCC genesis. Citation Format: Joanna D C Lima, Samvid Kurlekar, Chris W. Pugh, Julie Adam, Peter J. Ratcliffe. A novel lineage-tracing model of Vhldeletion reveals time-dependent cellular changes in the kidney [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 939.

Rationale: Endothelial injury may provoke emphysema, but molecular pathways of disease development require further discernment. Emphysematous lungs exhibit decreased expression of HIF-2α (hypoxia-inducible factor-2α)-regulated genes, and tobacco smoke decreases pulmonary HIF-2α concentrations. These findings suggest that decreased HIF-2α expression is important in the development of emphysema. Objectives: The objective of this study was to evaluate the roles of endothelial-cell (EC) HIF-2α in the pathogenesis of emphysema in mice. Methods: Mouse lungs were examined for emphysema after either the loss or the overexpression of EC Hif-2α. In addition, SU5416, a VEGFR2 inhibitor, was used to induce emphysema. Lungs were evaluated for HGF (hepatocyte growth factor), a protein involved in alveolar development and homeostasis. Lungs from patients with emphysema were measured for endothelial HIF-2α expression. Measurements and Main Results: EC Hif-2α deletion resulted in emphysema in association with fewer ECs and pericytes. After SU5416 exposure, EC Hif-2α–knockout mice developed more severe emphysema, whereas EC Hif-2α–overexpressing mice were protected. EC Hif-2α–knockout mice demonstrated lower levels of HGF. Human emphysema lung samples exhibited reduced EC HIF-2α expression. Conclusions: Here, we demonstrate a unique protective role for pulmonary endothelial HIF-2α and how decreased expression of this endogenous factor causes emphysema; its pivotal protective function is suggested by its ability to overcome VEGF antagonism. HIF-2α may maintain alveolar architecture by promoting vascular survival and associated HGF production. In summary, HIF-2α may be a key endogenous factor that prevents the development of emphysema, and its upregulation has the potential to foster lung health in at-risk patients.

Despite a general role for the HIF hydroxylase system in cellular oxygen sensing and tumour hypoxia, cancer-associated mutations of genes in this pathway, including PHD2, PHD1, EPAS1 (encoding HIF-2α) are highly tissue-restricted, being observed in pseudohypoxic pheochromocytoma and paraganglioma (PPGL) but rarely, if ever, in other tumours. In an effort to understand that paradox and gain insights into the pathogenesis of pseudohypoxic PPGL, we constructed mice in which the principal HIF prolyl hydroxylase, Phd2, is inactivated in the adrenal medulla using TH-restricted Cre recombinase. Investigation of these animals revealed a gene expression pattern closely mimicking that of pseudohypoxic PPGL. Spatially resolved analyses demonstrated a binary distribution of two contrasting patterns of gene expression among adrenal medullary cells. Phd2 inactivation resulted in a marked shift in this distribution towards a Pnmt−/Hif-2α+/Rgs5+ population. This was associated with morphological abnormalities of adrenal development, including ectopic TH+ cells within the adrenal cortex and external to the adrenal gland. These changes were ablated by combined inactivation of Phd2 with Hif-2α, but not Hif-1α. However, they could not be reproduced by inactivation of Phd2 in adult life, suggesting that they arise from dysregulation of this pathway during adrenal development. Together with the clinical observation that pseudohypoxic PPGL manifests remarkably high heritability, our findings suggest that this type of tumour likely arises from dysregulation of a tissue-restricted action of the PHD2/HIF-2α pathway affecting adrenal development in early life and provides a model for the study of the relevant processes.

Hypoxia-inducible transcription factors (HIFs) are fundamental to the cellular adaptation to low oxygen levels but how they interact with chromatin and efficiently activate their target genes is unclear. Using genome-wide mutagenesis in human cancer cells, we define genes required for HIF transcriptional activation, and identify a requirement for the Histone 3 lysine 4 (H3K4) methyltransferase SET1B. Loss of SET1B leads to a selective reduction in HIF transcriptional activity in hypoxia, with SET1B driving expression of genes involved in angiogenesis rather than glycolysis, resulting in impaired tumour establishment in SET1B deficient xenografts. Mechanistically, we show that SET1B is itself oxygen regulated, accumulates on chromatin in hypoxia, and is recruited to HIF target genes through HIF-1α. Accordingly, we show that the hypoxic induction of H3K4me3 at specific HIF targets is both HIF and SET1B dependent, and when impaired, decreases promoter acetylation and gene expression. Together, these findings reveal SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.

Hypoxia-inducible transcription factors (HIFs) are fundamental to the cellular adaptation to low oxygen levels but it is unclear how they interact with chromatin and activate their target genes. Here we use genome-wide mutagenesis to identify genes involved in HIF transcriptional activity, and define a requirement for the histone H3 lysine 4 (H3K4) methyltransferase SET1B. SET1B loss leads to a selective reduction in transcriptional activation of HIF target genes, resulting in impaired cell growth, angiogenesis, and tumor establishment in SET1B-deficient xenografts. Mechanistically, we show that SET1B accumulates on chromatin in hypoxia, and is recruited to HIF target genes by the HIF complex. The selective induction of H3K4 trimethylation at HIF target loci is both HIF- and SET1B-dependent, and when impaired correlates with decreased promoter acetylation and gene expression. Together, these findings reveal SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.

Prolyl hydroxylation is a post-translational modification that is known to regulate several key cell functions including translation and protein stability. Enzymes that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase (2OGDO) family. A newly-identified member of the 2OGDO enzyme family, 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (Ogfod1), catalyzes prolyl hydroxylation of ribosomal protein s23 (Rps23), a component of the 40S ribosome. To establish an in vivo role for Ogfod1, we ablated Ogfod1 in mice and subjected isolated perfused hearts to ischemia and reperfusion and found Ogfod1 ablation to be cardioprotective. The mechanisms by which these changes occur are currently unknown, so we investigated Ogfod1 regulation and mechanisms of cardioprotection. Previous work has shown that Ogfod1-mediated Rps23 prolyl hydroxylation occurs in the nucleus, where ribosomes are assembled. Additionally, Ogfod1 localizes to cytoplasmic stress granules in transformed cells exposed to endoplasmic reticulum (ER) stress. Based on these studies, we tested the hypothesis that Ogfod1 localization, and subsequently its function, change in response to stress. We induced ER stress by treating isolated adult cardiomyocytes with thapsigargin, and monitored Ogfod1 localization using immunofluorescence. Thapsigargin inhibits sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) which activates the unfolded protein response. In the absence of thapsigargin, Ogfod1 localized to the nucleus, peri-nuclear space, cytoplasm, and cell membrane. After stress induction, Ogfod1 nuclear- and perinuclear-localization decreased. This suggests that in murine adult cardiomyocytes subjected to ER stress, Ogfod1 is exported from the nucleus, potentially as a mechanism for down-regulating its activity. As Ogfod1 ablation has been found to be cardioprotective in mice, understanding Ogfod1 localization and regulation in the cardiomyocyte stress response may provide mechanistic insight that will be useful in developing treatments for stressors such as heart failure.

The emergence of the novel coronavirus SARS-CoV-2 has led to a pandemic infecting more than two million people worldwide in less than four months, posing a major threat to healthcare systems. This is compounded by the shortage of available tests causing numerous healthcare workers to unnecessarily self-isolate. We provide a roadmap instructing how a research institute can be repurposed in the midst of this crisis, in collaboration with partner hospitals and an established diagnostic laboratory, harnessing existing expertise in virus handling, robotics, PCR, and data science to derive a rapid, high throughput diagnostic testing pipeline for detecting SARS-CoV-2 in patients with suspected COVID-19. The pipeline is used to detect SARS-CoV-2 from combined nose-throat swabs and endotracheal secretions/ bronchoalveolar lavage fluid. Notably, it relies on a series of in-house buffers for virus inactivation and the extraction of viral RNA, thereby reducing the dependency on commercial suppliers at times of global shortage. We use a commercial RT-PCR assay, from BGI, and results are reported with a bespoke online web application that integrates with the healthcare digital system. This strategy facilitates the remote reporting of thousands of samples a day with a turnaround time of under 24 hours, universally applicable to laboratories worldwide.

The mechanistic basis of the marked oxygen sensitivity of glomus cells in the carotid body has long puzzled physiologists. In this issue of Science Signaling, Moreno-Dominguez et al. show the critical importance of high levels of hypoxia-inducible factor, HIF2α/EPAS1, and the nuclear-encoded mitochondrial cytochrome c oxidase subunit, COX4I2, in glomus cell sensitivity to hypoxia.

Key points The carotid body is a peripheral arterial chemoreceptor that regulates ventilation in response to both acute and sustained hypoxia.Type I cells in this organ respond to low oxygen both acutely by depolarization and dense core vesicle secretion and, over the longer term, via cellular proliferation and enhanced ventilatory responses.Using lineage analysis, the present study shows that the Type I cell lineage itself proliferates and expands in response to sustained hypoxia.Inactivation of HIF‐2α in Type I cells impairs the ventilatory, proliferative and cell intrinsic (dense core vesicle) responses to hypoxia.Inactivation of PHD2 in Type I cells induces multilineage hyperplasia and ultrastructural changes in dense core vesicles to form paraganglioma‐like carotid bodies.These changes, similar to those observed in hypoxia, are dependent on HIF‐2α.Taken together, these findings demonstrate a key role for the PHD2–HIF‐2α couple in Type I cells with respect to the oxygen sensing functions of the carotid body. Abstract The carotid body is a peripheral chemoreceptor that plays a central role in mammalian oxygen homeostasis. In response to sustained hypoxia, it manifests a rapid cellular proliferation and an associated increase in responsiveness to hypoxia. Understanding the cellular and molecular mechanisms underlying these processes is of interest both to specialized chemoreceptive functions of that organ and, potentially, to the general physiology and pathophysiology of cellular hypoxia. We have combined cell lineage tracing technology and conditionally inactivated alleles in recombinant mice to examine the role of components of the HIF hydroxylase pathway in specific cell types within the carotid body. We show that exposure to sustained hypoxia (10% oxygen) drives rapid expansion of the Type I, tyrosine hydroxylase expressing cell lineage, with little transdifferentiation to (or from) that lineage. Inactivation of a specific HIF isoform, HIF‐2α, in the Type I cells was associated with a greatly reduced proliferation of Type I cells and hypoxic ventilatory responses, with ultrastructural evidence of an abnormality in the action of hypoxia on dense core secretory vesicles. We also show that inactivation of the principal HIF prolyl hydroxylase PHD2 within the Type I cell lineage is sufficient to cause multilineage expansion of the carotid body, with characteristics resembling paragangliomas. These morphological changes were dependent on the integrity of HIF‐2α. These findings implicate specific components of the HIF hydroxylase pathway (PHD2 and HIF‐2α) within Type I cells of the carotid body with respect to the oxygen sensing and adaptive functions of that organ., Key points The carotid body is a peripheral arterial chemoreceptor that regulates ventilation in response to both acute and sustained hypoxia.Type I cells in this organ respond to low oxygen both acutely by depolarization and dense core vesicle secretion and, over the longer term, via cellular proliferation and enhanced ventilatory responses.Using lineage analysis, the present study shows that the Type I cell lineage itself proliferates and expands in response to sustained hypoxia.Inactivation of HIF‐2α in Type I cells impairs the ventilatory, proliferative and cell intrinsic (dense core vesicle) responses to hypoxia.Inactivation of PHD2 in Type I cells induces multilineage hyperplasia and ultrastructural changes in dense core vesicles to form paraganglioma‐like carotid bodies.These changes, similar to those observed in hypoxia, are dependent on HIF‐2α.Taken together, these findings demonstrate a key role for the PHD2–HIF‐2α couple in Type I cells with respect to the oxygen sensing functions of the carotid body.

Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from poly some formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 1 million fatalities to date. Understanding how host factors modify the viral life cycle could inform susceptibility to viral infection and the design of new therapies. Viral replication is shaped by the cellular microenvironment and one important factor is local oxygen tension, where hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its Spike glycoprotein binding to angiotensin-converting enzyme (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat (FG-4592) reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via a HIF-1α dependent signalling pathway. Further, hypoxia and Roxadustat inhibit viral replication in SARS-CoV-2 infected cells, showing that post-entry steps in the viral life cycle are oxygen-sensitive. This study highlights the importance of hypoxia and HIF signalling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention and/or treatment of COVID-19. Funding: The McKeating laboratory is funded by a Wellcome Investigator Award (IA) 200838/Z/16/Z, UK Medical Research Council (MRC) project grant MR/R022011/1 and Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant number: 2018-I2M-2-002). The Ratcliffe laboratory is funded by the Oxford Branch of the Ludwig Institute for Cancer Research; Wellcome IA 106241/Z/14/Z; the Francis Crick Institute, which receives core funding from Cancer Research UK (FC001501), UK MRC (FC001501) and Wellcome (FC001501); the Paradifference Foundation. PJR, EJH and TB are additionally funded by the COVID-19 Research Response Fund, University of Oxford. SK is funded by the Clarendon Scholarships Fund and the Christopher Welch Trust. The Davis laboratory is funded by Wellcome IA 209412/Z/17/Z and Wellcome Strategic Awards 091911/B/10/Z and 107457/Z/15/Z. JYL is funded by the Medial Sciences Graduate Studentship, University of Oxford. The Hinks laboratory is funded by grants from the Wellcome (104553/z/14/z, 211050/Z/18/z) and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre; the views expressed are those of the authors and not those of the NHS or NIHR. Conflict of Interest: EJH is employed under the Cambridge Experimental Medicine Initiative, which is partly funded by AstraZeneca although they have not been involved in this project. The other authors declare no financial interests. Ethical Approval: The study was reviewed by the Oxford Research Ethics Committee B (18/SC/0361).

Emerging evidence suggests that dysregulation of oncogenic pathways requires precise tuning in order for cancer to develop. To test this, we examined the overlap between cis-acting elements of the hypoxia-inducible factor (HIF) pathway and cancer-susceptibility polymorphisms as defined in genome-wide association studies (GWAS). In renal cancer, where HIF is constitutively and un-physiologically activated by mutation of the von Hippel-Lindau tumour suppressor, we observed marked excess overlap, which extended to potential susceptibility polymorphisms that are below the conventional threshold applied in GWAS. In contrast, in other cancers where HIF is upregulated by different mechanisms, including micro-environmental hypoxia, we observed no excess in overlap. Our findings support a ‘pathway tuning’ model of cancer, whereby precise modulation of multiple outputs of specific, activated pathways is important in oncogenesis. This implies that selective pressures to modulate such pathways operate during cancer development and should focus attempts to identify their nature and consequences.

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer and there is great interest in applying inhibitors of HIF as anti-cancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors impact on physiological responses to hypoxia at relevant doses. Here we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumour growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatisation and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerisation with HIF-1β, did not respond to PT2385 indicating that these effects are on target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerisation with HIF-1β. Although PT2385 was well-tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.

Studies of the regulation of erythropoietin (EPO) production by the liver and kidneys, one of the classical physiological responses to hypoxia, led to the discovery of human oxygen-sensing mechanisms, which are now being targeted therapeutically. The oxygen-sensitive signal is generated by 2-oxoglutarate-dependent dioxygenases that deploy molecular oxygen as a co-substrate to catalyze the post-translational hydroxylation of specific prolyl and asparaginyl residues in hypoxia-inducible factor (HIF), a key transcription factor that regulates transcriptional responses to hypoxia. Hydroxylation of HIF at different sites promotes both its degradation and inactivation. Under hypoxic conditions, these processes are suppressed, enabling HIF to escape destruction and form active transcriptional complexes at thousands of loci across the human genome. Accordingly, HIF prolyl hydroxylase inhibitors stabilize HIF and stimulate expression of HIF target genes, including the EPO gene. These molecules activate endogenous EPO gene expression in diseased kidneys and are being developed for, or are already in clinical use for, the treatment of renal anaemia. In this Review, we summarize information on the molecular circuitry of hypoxia signalling pathways underlying these new treatments and highlight some of the outstanding questions relevant to their clinical use.

Hypoxia-inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long noncoding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long noncoding RNA Noncoding Intergenic Co-Induced transcript (NICI) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knockdown or knockout of NICI attenuated hypoxic induction of SLC2A3, indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR-mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than posttranscriptional mechanisms because knockout of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the von Hippel-Lindau (VHL) tumor suppressor and is highly expressed in clear cell renal cell carcinoma (ccRCC), where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.

In human cells oxygen levels are 'sensed' by a set of ferrous iron and 2-oxoglutarate dependent dioxygenases. These enzymes regulate a broad range of cellular and systemic responses to hypoxia by catalysing the post-translational hydroxylation of specific residues in the alpha subunits of hypoxia inducible factor (HIF) transcriptional complexes. The HIF hydroxylases are now the subject of pharmaceutical targeting by small molecule inhibitors that aim to activate or augment the endogenous HIF transcriptional response for the treatment of anaemia and other hypoxic human diseases. Here we consider the rationale for this therapeutic strategy from the biochemical, biological and medical perspectives. We outline structural and mechanistic considerations that are relevant to the design of HIF hydroxylase inhibitors, including likely determinants of specificity, and review published reports on their activity in pre-clinical models and clinical trials.