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Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.

Background Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of renal cancer. With currently available therapies, cure of advanced and metastatic ccRCC is achieved only in rare cases. Here, we developed a workflow integrating different - omics technologies to identify ccRCC-specific HLA-presented peptides as potential drug targets for ccRCC immunotherapy. Methods We analyzed HLA-presented peptides by MS-based ligandomics of 55 ccRCC tumors (cohort 1), paired non-tumor renal tissues, and 158 benign tissues from other organs. Pathways enriched in ccRCC compared to its cell type of origin were identified by transcriptome and gene set enrichment analyses in 51 tumor tissues of the same cohort. To retrieve a list of candidate targets with involvement in ccRCC pathogenesis, ccRCC-specific pathway genes were intersected with the source genes of tumor-exclusive peptides. The candidates were validated in an independent cohort from The Cancer Genome Atlas (TCGA KIRC, n  = 452). DNA methylation (TCGA KIRC, n  = 273), somatic mutations (TCGA KIRC, n  = 392), and gene ontology (GO) and correlations with tumor metabolites (cohort 1, n  = 30) and immune-oncological markers (cohort 1, n  = 37) were analyzed to characterize regulatory and functional involvements. CD8 + T cell priming assays were used to identify immunogenic peptides. The candidate gene EGLN3 was functionally investigated in cell culture. Results A total of 34,226 HLA class I- and 19,325 class II-presented peptides were identified in ccRCC tissue, of which 443 class I and 203 class II peptides were ccRCC-specific and presented in ≥ 3 tumors. One hundred eighty-five of the 499 corresponding source genes were involved in pathways activated by ccRCC tumors. After validation in the independent cohort from TCGA, 113 final candidate genes remained. Candidates were involved in extracellular matrix organization, hypoxic signaling, immune processes, and others. Nine of the 12 peptides assessed by immunogenicity analysis were able to activate naïve CD8 + T cells, including peptides derived from EGLN3 . Functional analysis of EGLN3 revealed possible tumor-promoting functions. Conclusions Integration of HLA ligandomics, transcriptomics, genetic, and epigenetic data leads to the identification of novel functionally relevant therapeutic targets for ccRCC immunotherapy. Validation of the identified targets is recommended to expand the treatment landscape of ccRCC.

While adoptive cell therapy has shown success in hematological malignancies, its potential against solid tumors is hindered by an immunosuppressive tumor microenvironment (TME). In recent years, members of the hypoxia-inducible factor (HIF) family have gained recognition as important regulators of T-cell metabolism and function. The role of HIF signalling in activated CD8 T cell function in the context of adoptive cell transfer, however, has not been explored in full depth. Here we utilize CRISPR-Cas9 technology to delete prolyl hydroxylase domain-containing enzymes (PHD) 2 and 3, thereby stabilizing HIF-1 signalling, in CD8 T cells that have already undergone differentiation and activation, modelling the T cell phenotype utilized in clinical settings. We observe a significant boost in T-cell activation and effector functions following PHD2/3 deletion, which is dependent on HIF-1α, and is accompanied by an increased glycolytic flux. This improvement in CD8 T cell performance translates into an enhancement in tumor response to adoptive T cell therapy in mice, across various tumor models, even including those reported to be extremely resistant to immunotherapeutic interventions. These findings hold promise for advancing CD8 T-cell based therapies and overcoming the immune suppression barriers within challenging tumor microenvironments. The hypoxia inducible factor HIF-1α has been shown to regulate T cell metabolism and function. Here authors deleted the prolyl hydroxylase domain-containing enzymes PHD2 and 3, thereby stabilizing HIF-1α, in therapeutic CD8 T cells to achieve better functionality upon adoptive transfer to tumour-bearing mice.

We previously reported that Tibetan-specific variant of prolyl-hydroxylase-2 (PHD2) D4E;C127S protects highlanders from hypoxia-triggered pathologies by destabilizing hypoxia-inducible factor (HIF)-1α. Others have reported that stabilized HIF1α negatively regulates interferon (IFN)-regulating factor (IRF)-3 under hypoxia. We examined the role of PHD2 D4E;C127S variant in IFN synthesis in immune cells during viral infections. Primary monocytes and cells engineered to express the PHD2 D4E;C127S variant displayed protection against dengue virus (DENV)-2 infection by suppressing HIF1α, in turn promoting IRF-3 and IFNα/β synthesis in hypoxia (3% O 2 ) in vitro. However, under normoxia (21% O 2 ), these mutant cells increased reactive oxygen species (ROS) generation following DENV2 infection. Increased ROS then suppressed PHD2 D4E;C127S activity, reflected by reduced hydroxylation and concomitant stabilization of HIF1α, resulting in suppressed IFN synthesis and higher DENV2 replication. The PHD2 WT cells demonstrated the opposite trend. Our data further confirmed the inverse correlation between HIF1α and IFN pathways. CAY10585, a HIF1α-inhibitor, decreased the DENV2 infection by increasing IFN-A/B and IRF-3/7/9 expression. HIF1α-depleted monocytes also showed a similar response to the infection. We extended our findings to COVID-19 infection. The CD4/CD8 T-cells collected from Tibetans with PHD2 D4E;C127S variant and exposed to SARS-CoV-2 infection showed elevated expression of IFN-γ in response to exposure to SARS-CoV-2 receptor-binding domain (RBD) peptide under hypoxia, and a lesser expression under normoxia. The study thus highlights a unique crosstalk of PHD2 D4E;C127S variant with HIF1α-IFN axis under environmental pO 2 in protecting or predisposing Tibetans to viral infections.

Low oxygen tension or hypoxia is a determining factor in the course of many different processes in animals, including when tissue expansion and cellular metabolism result in high oxygen demands that exceed its supply. This is mainly happening when cells actively proliferate and the proliferating mass becomes distant from the blood vessels, such as in growing tumors. Metabolic alterations in response to hypoxia can be triggered in a direct manner, such as the switch from oxidative phosphorylation to glycolysis or inhibition of fatty acid desaturation. However, as the modulated action of hypoxia-inducible factors or the oxygen sensors (prolyl hydroxylase domain-containing enzymes) can also lead to changes in enzyme expression, these metabolic changes can also be indirect. With this review, we want to summarize our current knowledge of the hypoxia-induced changes in metabolism during cancer development, how they are affected in the tumor cells and in the cells of the microenvironment, most prominently in immune cells.

Background Recent studies have revealed that CD8 + T cells can be activated via genetic upregulation of HIF-1α, thereby augmenting antitumor effector functions. HIF-1α upregulation can be attained by inhibiting HIF-prolyl hydroxylase (HIF-PH) under normoxic conditions, termed pseudohypoxia. This study investigated whether pseudohypoxia induced by HIF-PH inhibitors suppresses Microsatellite stable (MSS) colorectal cancer (CRC) by affecting tumor immune response. Methods The HIF-PH inhibitors Roxadustat and Vadadustat were utilized in this study. In vitro, we assessed the effects of HIF-PH inhibitors on human and murine colon cancer cell lines (SW480, HT29, Colon26) and murine T cells. In vivo experiments were performed with mice bearing Colon26 tumors to evaluate the effect of these inhibitors on tumor immune responses. Tumor and spleen samples were analyzed using immunohistochemistry, RT-qPCR, and flow cytometry to elucidate potential mechanisms. Results HIF-PH inhibitors demonstrated antitumor effects in vivo but not in vitro. These inhibitors enhanced the tumor immune response by increasing the infiltration of CD8 + and CD4 + tumor-infiltrating lymphocytes (TILs). HIF-PH inhibitors induced IL-2 production in splenic and intratumoral CD4 + T cells, promoting T cell proliferation, differentiation, and immune responses. Roxadustat synergistically enhanced the efficacy of anti-PD-1 antibody for MSS cancer by increasing the recruitment of TILs and augmenting effector-like CD8 + T cells. Conclusion Pseudohypoxia induced by HIF-PH inhibitors activates antitumor immune responses, at least in part, through the induction of IL-2 secretion from CD4 + T cells in the spleen and tumor microenvironment, thereby enhancing immune efficacy against MSS CRC.

Late-stage lung adenocarcinoma (LUAD) frequently results in distant metastasis, with liver metastasis indicating the poorest prognosis. To successfully colonize the liver, metastatic LUAD cells must overcome its relatively hypoxic microenvironment. This study explores the metabolic adaptations that facilitate LUAD liver metastasis, identifying Spindle and Kinetochore Associated Protein 3 (SKA3) as a critical mediator. Under hypoxic conditions, SKA3 expression is significantly upregulated, driving glucose metabolic reprogramming in LUAD cells to enable survival within the liver’s hypoxic niche. Mechanistically, SKA3 competitively binds to prolyl hydroxylase domain-containing protein 2 (PHD2), disrupting its interaction with hypoxia-inducible factor 1-alpha (HIF-1α). Consequently, stabilized HIF-1α further enhances glycolytic enzyme transcription, amplifying glycolysis and enabling adaptation to liver hypoxia. Furthermore, hypoxia upregulates the E3 ubiquitin ligase MDM2, promoting p53 ubiquitination and degradation, thereby relieving p53-mediated repression of SKA3 and further reinforcing the SKA3/HIF-1α axis. Interestingly, HIF-1α directly binds to the hypoxia response element (HRE) in the SKA3 promoter, creating a positive feedback loop to maintain high SKA3 expression. Thus, SKA3-mediated metabolic reprogramming significantly contributes to LUAD cells colonization and proliferation in the liver. Finally, our findings demonstrated that the SKA3/HIF-1α axis was critical for establishing hypoxia tolerance in LUAD cells, underscoring its potential as a therapeutic target for treating liver metastasis in LUAD.

Mitochondrial impairment induced by oxidative stress is a main characteristic of intrinsic cell death pathways in neurons underlying the pathology of neurodegenerative diseases. Therefore, protection of mitochondrial integrity and function is emerging as a promising strategy to prevent neuronal damage. Here, we show that pharmacological inhibition of hypoxia-inducible factor prolyl-4-hydroxylases (HIF-PHDs) by adaptaquin inhibits lipid peroxidation and fully maintains mitochondrial function as indicated by restored mitochondrial membrane potential and ATP production, reduced formation of mitochondrial reactive oxygen species (ROS) and preserved mitochondrial respiration, thereby protecting neuronal HT-22 cells in a model of glutamate-induced oxytosis. Selective reduction of PHD1 protein using CRISPR/Cas9 technology also reduced both lipid peroxidation and mitochondrial impairment, and attenuated glutamate toxicity in the HT-22 cells. Regulation of activating transcription factor 4 (ATF4) expression levels and related target genes may mediate these beneficial effects. Overall, these results expose HIF-PHDs as promising targets to protect mitochondria and, thereby, neurons from oxidative cell death.

The tumor microenvironment in colorectal cancer (CRC) is marked by a diverse and abundant population of cancer-associated fibroblasts (CAFs), which play a crucial role in radioresistance. Nonetheless, the mechanisms through which CAFs contribute to radioresistance remain unclear. In this study, we demonstrate that CAF R , a specific subset of CAFs derived from radioresistant CRC patients, produces higher levels of transforming growth factor-β1 (TGF-β1) compared to CAFs isolated from radiosensitive CRC patients. Through long noncoding RNA (lncRNA) profiling of tumor cells treated with CAF-conditioned medium (CAF-CM), we identify WARS2-IT1 (WARS2 intronic transcript 1), whose expression is directly stimulated by TGF-β1 signaling. This lncRNA serves as a key player in promoting radioresistance and is essential for the TGFβ1-induced radioresistance pathway. Mechanistically, WARS2-IT1 interferes with the interaction between prolyl hydroxylase domain 2 (PHD2) and hypoxia-inducible factor-1α (HIF-1α), preventing the hydroxylation and subsequent degradation of HIF-1α. This process leads to the activation of glycolytic pathways, thereby enhancing radioresistance. Our findings underscore the potential of targeting CAF-driven WARS2-IT1 as a promising strategy to counteract tumor radioresistance in CRC.

Key Points Mucosal hypoxia is an integral component of IBD Hypoxia-induced signalling by hypoxia-inducible factors and nuclear factor-κB can promote or counteract the intestinal inflammatory response, depending on the context and cell type studied Oxygen-sensitive prolyl hydroxylases (PHDs) tightly regulate hypoxia-induced signalling pathways and have been identified as promising therapeutic targets in IBD Pan-hydroxylase inhibitors are in an advanced stage of development for the treatment of anaemia related to chronic kidney disease and are in phase I trials for the treatment of ulcerative colitis The use of orally administered and isotype-specific PHD inhibitors might reduce systemic exposure and the risk of unwanted side-effects IBD is associated with markedly reduced intestinal mucosal oxygen levels. In this Review, the authors discuss the role of mucosal hypoxia and hypoxia-induced signalling in IBD and identify potential targets for therapies, focusing on the cell-specific functions of hypoxia-inducible factors, prolyl hydroxylases and nuclear factor-κB. Tissue hypoxia occurs when local oxygen demand exceeds oxygen supply. In chronic inflammatory conditions such as IBD, the increased oxygen demand by resident and gut-infiltrating immune cells coupled with vascular dysfunction brings about a marked reduction in mucosal oxygen concentrations. To counter the hypoxic challenge and ensure their survival, mucosal cells induce adaptive responses, including the activation of hypoxia-inducible factors (HIFs) and modulation of nuclear factor-κB (NF-κB). Both pathways are tightly regulated by oxygen-sensitive prolyl hydroxylases (PHDs), which therefore represent promising therapeutic targets for IBD. In this Review, we discuss the involvement of mucosal hypoxia and hypoxia-induced signalling in the pathogenesis of IBD and elaborate in detail on the role of HIFs, NF-κB and PHDs in different cell types during intestinal inflammation. We also provide an update on the development of PHD inhibitors and discuss their therapeutic potential in IBD.