Hypoxia-inducible Factor Target Genes Research Articles

Design, synthesis, and target identification of new hypoxia-inducible factor 1 (HIF-1) inhibitors containing 1-alkyl-1H-pyrazole-3-carboxamide moiety

Hypoxia-inducible factor 1 (HIF-1) is a promising drug target for cancer chemotherapy. In our screening program aimed at identifying new HIF-1 inhibitors by using a hypoxia-responsive luciferase reporter gene assay, KUSC-5001 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety was found as a potential hit molecule. During an extensive structure–activity relationship (SAR) study, we developed a more effective HIF-1 inhibitor KUSC-5037 (IC50 = 1.2 μM). Under hypoxic conditions, KUSC-5037 suppressed the HIF-1α (a regulatory subunit of HIF-1) mRNA, causing decreases in the gene expression of HIF-1 target genes such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) genes. Furthermore, by applying our fluorescent and bifunctional probes, ATP5B, a catalytic β subunit of mitochondrial FoF1-ATP synthase, was identified as a target protein of KUSC-5037. These results indicate that the derivatives of KUSC-5037 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety are promising lead molecules for the inhibition of HIF-1 signaling via FoF1-ATP synthase suppression.

Targeting hypoxia-inducible factor 1 (HIF-1) signaling with natural products toward cancer chemotherapy.

Tumor cells are often exposed to hypoxia because of the lower oxygen supply deep inside the tumor tissues. However, tumor cells survive in these severe conditions by adapting to hypoxic stress through the induction of hypoxia-inducible factor 1 (HIF-1) signaling. HIF-1 activation is responsible for the expression of numerous HIF-1 target genes, which are related to cell survival, proliferation, angiogenesis, invasion, metastasis, cancer stemness, and metabolic reprogramming. Therefore, HIF-1 is expected to be a potential pharmacological target for cancer therapy. Small molecules derived from natural products (microbial origin, plant-derived, or marine organisms) have been shown to have unique chemical structures and biological activities, including HIF-1 inhibition. Several studies identified HIF-1 inhibitors from natural products. In this review, we summarize the current HIF-1 signaling inhibitors originating from natural products with a variety of modes of action, mainly focusing on microbial metabolites.

Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors.

Angiogenesis is essential for the sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Therefore, understanding the mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1α in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1α at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases to bind and hydroxylate HIF-1α, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Moreover, phosphorylation of the analogous site in HIF-2α (S435) stabilizes the protein through the same mechanism, indicating post-translational modification within the oxygen-dependent degradation domain as a mechanism of regulating the HIF-α subunits. In vitro and in vivo models demonstrate that expression of PIM1 is sufficient to stabilize HIF-1α and HIF-2α in normoxia and stimulate angiogenesis in a HIF-1-dependent manner. CRISPR mutants of HIF-1α (Thr455D) promoted increased tumor growth, proliferation and angiogenesis. Moreover, HIF-1α-T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.

Expression and Roles of Individual HIF Prolyl 4-Hydroxylase Isoenzymes in the Regulation of the Hypoxia Response Pathway along the Murine Gastrointestinal Epithelium.

The HIF prolyl 4-hydroxylases (HIF-P4H) control hypoxia-inducible factor (HIF), a powerful mechanism regulating cellular adaptation to decreased oxygenation. The gastrointestinal epithelium subsists in “physiological hypoxia” and should therefore have an especially well-designed control over this adaptation. Thus, we assessed the absolute mRNA expression levels of the HIF pathway components, Hif1a, HIF2a, Hif-p4h-1, 2 and 3 and factor inhibiting HIF (Fih1) in murine jejunum, caecum and colon epithelium using droplet digital PCR. We found a higher expression of all these genes towards the distal end of the gastrointestinal tract. We detected mRNA for Hif-p4h-1, 2 and 3 in all parts of the gastrointestinal tract. Hif-p4h-2 had significantly higher expression levels compared to Hif-p4h-1 and 3 in colon and caecum epithelium. To test the roles each HIF-P4H isoform plays in the gut epithelium, we measured the gene expression of classical HIF target genes in Hif-p4h-1−/−, Hif-p4h-2 hypomorph and Hif-p4h-3−/− mice. Only Hif-p4h-2 hypomorphism led to an upregulation of HIF target genes, confirming a predominant role of HIF-P4H-2. However, the abundance of Hif-p4h-1 and 3 expression in the gastrointestinal epithelium implies that these isoforms may have specific functions as well. Thus, the development of selective inhibitors might be useful for diverging therapeutic needs.

Structural Basis of Prolyl Hydroxylase Domain Inhibition by Molidustat.

Human prolyl‐hydroxylases (PHDs) are hypoxia‐sensing 2‐oxoglutarate (2OG) oxygenases, catalysis by which suppresses the transcription of hypoxia‐inducible factor target genes. PHD inhibition enables the treatment of anaemia/ischaemia‐related disease. The PHD inhibitor Molidustat is approved for the treatment of renal anaemia; it differs from other approved/late‐stage PHD inhibitors in lacking a glycinamide side chain. The first reported crystal structures of Molidustat and IOX4 (a brain‐penetrating derivative) complexed with PHD2 reveal how their contiguous triazole, pyrazolone and pyrimidine/pyridine rings bind at the active site. The inhibitors bind to the active‐site metal in a bidentate manner through their pyrazolone and pyrimidine nitrogens, with the triazole π‐π‐stacking with Tyr303 in the 2OG binding pocket. Comparison of the new structures with other PHD inhibitor complexes reveals differences in the conformations of Tyr303, Tyr310, and a mobile loop linking β2–β3, which are involved in dynamic substrate binding/product release.

Hypoxia compromises the mitochondrial metabolism of Alzheimer's disease microglia via HIF1.

Genetic Alzheimer's disease (AD) risk factors associate with reduced defensive amyloid β plaque-associated microglia (AβAM), but the contribution of modifiable AD risk factors to microglial dysfunction is unknown. In AD mouse models, we observe concomitant activation of the hypoxia-inducible factor 1 (HIF1) pathway and transcription of mitochondrial-related genes in AβAM, and elongation of mitochondria, a cellular response to maintain aerobic respiration under low nutrient and oxygen conditions. Overactivation of HIF1 induces microglial quiescence in cellulo, with lower mitochondrial respiration and proliferation. In vivo, overstabilization of HIF1, either genetically or by exposure to systemic hypoxia, reduces AβAM clustering and proliferation and increases Aβ neuropathology. In the human AD hippocampus, upregulation of HIF1α and HIF1 target genes correlates with reduced Aβ plaque microglial coverage and an increase of Aβ plaque-associated neuropathology. Thus, hypoxia (a modifiable AD risk factor) hijacks microglial mitochondrial metabolism and converges with genetic susceptibility to cause AD microglial dysfunction.

TGF-β1 can be regulated by NDRG2 via the NF-κB pathway in hypoxia-induced liver fibrosis

BackgroundThe identification of the important elements that control hepatic stellate cell (HSC) activation will expand our understanding of the mechanism of liver fibrosis induced by hypoxia and affect the outcome of clinical treatment. A previous research demonstrated that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of fibrosis and a downstream target gene of hypoxia-inducible factor 1 (HIF-1). In this research, we studied the expression and function of NDRG2 in liver fibrosis induced by hypoxia.MethodsLX-2 cells/NF-κB-silenced LX-2 cells were exposed to hypoxic conditions (1% O2) to activate HSCs in vitro. The protein and mRNA expression levels of NDRG2, α-SMA and transforming growth factor beta 1 (TGF-β1) were evaluated by western blotting and real-time polymerase chain reaction (RT-PCR), respectively. Functional studies were performed using adenovirus-mediated gene upregulation.ResultsThe NDRG2 mRNA and protein levels were reduced under hypoxic conditions in LX-2 cells and overexpression of NDRG2 resulted in a decrease in the expression of TGF-β1 and α-SMA. Interestingly, no relationship was observed between NDRG2 and TGF-β1 when the NF-κB pathway was blocked, which indicates that NDRG2 can regulate the expression of TGF-β1 in LX-2 cells via the NF-κB pathway under hypoxic conditions.ConclusionsNDRG2 may regulate the expression of TGF-β1 via the NF-κB pathway and may be a novel therapeutic target for liver fibrosis induced by hypoxia.

HIF Prolyl Hydroxylase Inhibitors for COVID-19 Treatment: Pros and Cons.

The review analyzes the potential advantages and problems associated with using HIF prolyl hydroxylase inhibitors as a treatment for COVID-19. HIF prolyl hydroxylase inhibitors are known to boost endogenous erythropoietin (Epo) and activate erythropoiesis by stabilizing and activating the hypoxia inducible factor (HIF). Recombinant Epo treatment has anti-inflammatory and healing properties, and thus, very likely, will be beneficial for moderate to severe cases of COVID-19. However, HIF PHD inhibition may have a significantly broader effect, in addition to stimulating the endogenous Epo production. The analysis of HIF target genes reveals that some HIF-targets, such as furin, could play a negative role with respect to viral entry. On the other hand, HIF prolyl hydroxylase inhibitors counteract ferroptosis, the process recently implicated in vessel damage during the later stages of COVID-19. Therefore, HIF prolyl hydroxylase inhibitors may serve as a promising treatment of COVID-19 complications, but they are unlikely to aid in the prevention of the initial stages of infection.

Melatonin Lowers HIF-1α Content in Human Proximal Tubular Cells (HK-2) Due to Preventing Its Deacetylation by Sirtuin 1.

Although melatonin is widely known for its nephroprotective properties, there are no reports clearly pointing at its impact on the activity of hypoxia-inducible factor-1 (HIF-1), the main mediator of metabolic responses to hypoxia, in kidneys. The aim of the present study was to elucidate how melatonin affects the expression of the regulatory subunit HIF-1α in renal proximal tubules. HK-2 cells, immortalized human proximal tubular cells, were cultured under hypoxic conditions (1% O2). Melatonin was applied at 100 μM concentration. Protein and mRNA contents were determined by Western blot and RT-qPCR, respectively. HIF-1α acetylation level was established by means of immunoprecipitation followed by Western blot. Melatonin receptors MT1 and MT2 localization in HK-2 cells was visualized using immunofluorescence confocal analysis. It was found that melatonin in HK-2 cells (1) lowered HIF-1α protein, but not mRNA, content; (2) attenuated expression of HIF-1 target genes; (3) increased HIF-1α acetylation level; and (4) diminished sirtuin 1 expression (both protein and mRNA). Sirtuin 1 involvement in the regulation of HIF-1α level was confirmed applying cells with silenced Sirt1 gene. Moreover, the presence of membrane MT1 and MT2 receptors was identified in HK-2 cells and their ligand, ramelteon, turned out to mimic melatonin action on both HIF-1α and sirtuin 1 levels. Thus, it is concluded that the mechanism of melatonin-evoked decline in HIF-1α content in renal proximal tubular cells involves increased acetylation of this subunit which results from the attenuated expression of sirtuin 1, an enzyme reported to deacetylate HIF-1α. This observation provides a new insight to the understanding of melatonin action in kidneys.

Hypoxia-induced CREB cooperates MMSET to modify chromatin and promote DKK1 expression in multiple myeloma

Myeloma cells produce excessive levels of dickkopf-1 (DKK1), which mediates the inhibition of Wnt signaling in osteoblasts, leading to multiple myeloma (MM) bone disease. Nevertheless, the precise mechanisms underlying DKK1 overexpression in myeloma remain incompletely understood. Herein, we provide evidence that hypoxia promotes DKK1 expression in myeloma cells. Under hypoxic conditions, p38 kinase phosphorylated cAMP-responsive element-binding protein (CREB) and drove its nuclear import to activate DKK1 transcription. In addition, high levels of DKK1 were associated with the presence of focal bone lesions in patients with t(4;14) MM, overexpressing the histone methyltransferase MMSET, which was identified as a downstream target gene of hypoxia-inducible factor (HIF)-1α. Furthermore, we found that CREB could recruit MMSET, leading to the stabilization of HIF-1α protein and the increased dimethylation of histone H3 at lysine 36 on the DKK1 promoter. Knockdown of CREB in myeloma cells alleviated the suppression of osteoblastogenesis by myeloma-secreted DKK1 in vitro. Combined treatment with a CREB inhibitor and the hypoxia-activated prodrug TH-302 (evofosfamide) significantly reduced MM-induced bone destruction in vivo. Taken together, our findings reveal that hypoxia and a cytogenetic abnormality regulate DKK1 expression in myeloma cells, and provide an additional rationale for the development of therapeutic strategies that interrupt DKK1 to cure MM.

Divergent expression of hypoxia response systems under deoxygenation in reef-forming corals aligns with bleaching susceptibility.

Exposure of marine life to low oxygen is accelerating worldwide via climate change and localized pollution. Mass coral bleaching and mortality have recently occurred where reefs have experienced chronic low oxygen events. However, the mechanistic basis of tolerance to oxygen levels inadequate to sustain normal functioning (i.e. hypoxia) and whether it contributes to bleaching susceptibility, remain unknown. We therefore experimentally exposed colonies of the environmentally resilient Acropora tenuis, a common reef-building coral from the Great Barrier Reef, to deoxygenation-reoxygenation stress that was aligned to their natural night-day light cycle. Specifically, the treatment involved removing the 'night-time O2 buffer' to challenge the inherent hypoxia thresholds. RNA-Seq analysis revealed that coral possess a complete and active hypoxia-inducible factor (HIF)-mediated hypoxia response system (HRS) homologous to other metazoans. As expected, A. tenuis exhibited bleaching resistance and showed a strong inducibility of HIF target genes in response to deoxygenation stress. We applied this same approach in parallel to a colony of Acropora selago, known to be environmnetally susceptible, which conversely exhibited a bleaching phenotype response. This phenotypic divergence of A. selago was accompanied by contrasting gene expression profiles indicative of varied effectiveness of their HIF-HRS. Based on our RNA-Seq analysis, we propose (a) that the HIF-HRS is central for corals to manage deoxygenation stress and (b) that key genes of this system (and the wider gene network) may contribute to variation in coral bleaching susceptibility. Our analysis suggests that heat shock protein (hsp) 70 and 90 are important for low oxygen stress tolerance and further highlights how hsp90 expression might also affect the inducibility of coral HIF-HRS in overcoming a metabolic crisis under deoxygenation stress. We propose that differences in coral HIF-HRS could be central in regulating sensitivity to other climate change stressors-notably thermal stress-that commonly drive bleaching.

CHD4 Promotes Breast Cancer Progression as a Coactivator of Hypoxia-Inducible Factors.

Recruitment of RNA polymerase II to hypoxia-inducible factor (HIF) target genes under normoxia is a prerequisite for HIF-mediated transactivation. However, the underlying mechanism of this recruitment remains unknown. Here we report that chromodomain helicase DNA-binding protein 4 (CHD4) physically interacts with α and β subunits of HIF1 and HIF2 and enhances HIF-driven transcriptional programs to promote breast cancer progression. Loss of HIF1/2α abolished CHD4-mediated breast tumor growth in mice. In breast cancer cells under normoxia, CHD4 enrichment at HIF target gene promoters increased RNA polymerase II loading through p300. Hypoxia further promoted CHD4 binding to the chromatin via HIF1/2α, where CHD4 in turn enhanced recruitment of HIF1α, leading to HIF target gene transcription. CHD4 was upregulated and correlated with HIF target gene expression in human breast tumors; upregulation of CHD4 and other known HIF coactivators in human breast tumors was mutually exclusive. Furthermore, CHD4 was associated with poor overall survival of patients with breast cancer. Collectively, these findings reveal a new fundamental mechanism of HIF regulation in breast cancer, which has clinical relevance. SIGNIFICANCE: This study identifies CHD4 as a HIF coactivator and elucidates the fundamental mechanism underlying CHD4-mediated HIF transactivation in breast tumors.

An organoid-based drug screening identified a menin-MLL inhibitor for endometrial cancer through regulating the HIF pathway.

Tumor organoids recapitulate pathological properties and would serve as an excellent ex vivo model for drug discovery. Here, we performed an unbiased drug screening on drivers-defined tumor organoids from mouse endometrial cancer, the most prevalent gynecological malignancy in human, with a small molecule library targeting epigenetic factors. Among them, menin-MLL inhibitors MI-136 and MI-463 scored. The therapeutic capacity of MI-136 was further validated in tumor organoids in vitro and an orthotopic model in vivo. CRISPR/cas9-mediated mutations of major components of the menin-MLL complex, Men1, Kmt2a and Ash2l, inhibited the growth of tumor organoids, suggesting that the complex was the target of MI-136. Transcriptome analysis showed that the hypoxia-inducible factor (HIF) pathway was the most significantly downregulated pathway by MI-136 treatment. Consistently, Men1, Kmt2a, and Ash2l knockout also repressed the expressions of the HIF target genes. Loss of Hif1a or Hif1b partially phenocopied the inhibition of the menin-MLL complex by MI-136 or mutations in term of tumor organoid growth. Further, we found that MEN1 was upregulated in human endometrial cancers, which were tightly correlated with the expression levels of HIF1A, and associated with poor prognosis. Importantly, MI-136 also significantly inhibited the growth of endometrial cancer organoids derived from patients. Thus, our study identified MI-136 as a potential inhibitor for endometrial cancer through regulating the HIF pathway, a novel molecular mechanism distinguished from those in AML and prostate cancer.

Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate.

Sphingosine kinase 2 (SphK2) is known to phosphorylate the nuclear sphingolipid metabolite to generate sphingosine-1-phosphate (S1P). Nuclear S1P is involved in epigenetic regulation of gene expression; however, the underlying mechanisms are not well understood. In this work, we have identified the role of nuclear S1P and SphK2 in regulating hypoxia-responsive master transcription factors hypoxia-inducible factor (HIF)-1α/2α, and their functions in breast cancer, with a focus on triple-negative breast cancer (TNBC). We have shown SphK2 is associated with HIF-1α in protein complexes, and is enriched at the promoters of HIF target genes, including vascular endothelial growth factor (VEGF), where it enhances local histone H3 acetylation and transcription. S1P specifically binds to the PAS domains of HIF-1α. SphK2, and HIF-1α expression levels are elevated in metastatic estrogen receptor-positive (ER+) and TNBC clinical tissue specimens compared to healthy breast tissue samples. To determine if S1P formation in the nucleus by SphK2 is a key regulator of HIF functions, we found using a preclinical TNBC xenograft mouse model, and an existing selective SphK2 inhibitor K-145, that nuclear S1P, histone acetylation, HIF-1α expression, and TNBC tumor growth were all reduced in vivo. Our results suggest that S1P and SphK2 in the nucleus are linked to the regulation of HIF-1α/2α functions associated with breast cancer progression, and may provide potential therapeutic targets.

Direct Phosphorylation and Stabilization of HIF-1α By PIM1 Kinase Drives Angiogenesis in Solid Tumors

Angiogenesis is essential for sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Thus, understanding mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1α in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1α at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases (PHDs) to bind and hydroxylate HIF-1α, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Overexpression of PIM1 is sufficient to stabilize HIF-1α in normoxia and stimulate angiogenesis in a HIF-1-dependent manner in vivo. CRISPR mutants of HIF-1α (Thr455D) showed increased tumor growth, proliferation and angiogenesis. Moreover, T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.

The discovery of how cells sense and adapt to oxygen availability

Oxygen is ubiquitous on the Earth’s surface and essential for the survival of almost all eukaryotes. Nearly all living organisms require molecular oxygen for the metabolism of the macro-molecules to obtain energy for growth, development and reproduction. It is the subject of immense physiological, biochemical and clinical literature. In response to inadequate oxygen availability, eukaryotic body or cells undergo adaptive changes in gene expression program that either enhance oxygen delivery or promote survival in a low oxygen environment. The discovery of the mechanism by which cells sense and adapt to oxygen availability, which is awarded the 2019 Nobel Prize in Physiology or Medicine, lays the foundation for understanding how oxygen affects multiple physiological and pathological process. Hypoxia-inducible factor 1 (HIF-1) lies at the center of the hypoxia signal transduction pathway. HIF-1 was identified in a search for molecules that enhance erythropoietin (EPO) expression in anemia or hypoxia. HIF-1 is a heterodimer composed of α and β subunits, each containing basic helix-loop-helix-PAS (bHLH-PAS) domains. HIF-1β is constitutively expressed, while HIF-1α expression depends on oxygen availability and only a limited amount of protein is stabilized in the cytoplasm. Under hypoxic conditions, expressed cytosolic HIF-1α is stabilized and translocated into the nucleus to form heterodimers with HIF-1β, then binds to hypoxia response elements (HREs) to initiate transcription of target genes. More than 1000 genes have been identified to be directly transcriptionally activated by HIF-1. The proteins encoded by HIF-1 target genes are usually divided into 2 categories: one is those proteins that increase oxygen delivery like <italic>EPO</italic>, vascular endothelial growth factor (<italic>VEGF</italic>) et al<italic>, </italic>and the other is those proteins that decrease oxygen consumption like lactate dehydrogenase A (<italic>LDHA</italic>), pyruvate dehydrogenase kinase 1 (<italic>PDK1</italic>) and so on. Under normoxic conditions, prolyl hydroxylase 2 (PHD2), which serves as the sensor of cellular oxygen levels, hydroxylates the conserved proline residues in the α subunit of HIF-1. Hydroxylated HIF-1α is recognized and bound by the ubiquitin ligase von Hippel-Lindau (VHL)/Elongin B/Elongin C/Cullin-2 complex, leading to its rapid degradation by the proteasome. HIF-1α transcription activity is negatively regulated by factor inhibiting HIF-1 (FIH-1) via FIH-1-dependent asparaginyl hydroxylation using oxygen and a-ketoglutarate as substrates, which blocks the association of the coactivators CBP (CREB-binding protein) and p300. Abnormal hypoxia signal transduction contributes to the pathogenesis of many kinds of diseases like hypertension, ocular neovascularization, cancer, erythrocytosis and so on. Currently, extensive efforts are ongoing to develop drugs for clinical treatment of hypoxia-related diseases.

Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.

Hypoxia Inducible Factors' Transcriptional Activity in Polycythemia Vera

Polycythemia vera (PV), a myeloproliferative neoplasms (MPN), is characterized by high red cell mass due to excessive erythrocytosis and by clonal proliferation of myeloid cells. The major PV complication, increased risk of thrombosis, is also seen in Chuvash erythrocytosis (CE), a disorder of upregulated hypoxia sensing. Hypoxia inducible factor (HIF)-1 and HIF-2 are the principle regulators of the hypoxic response by regulating the transcription of hypoxia responsive genes. We previously showed that MPN patients have upregulation of some HIF target genes in granulocytes and platelets along with increased glucose uptake (Sana, et al., Blood 2013 122:1604). Further, stem cell factor and thrombopoietin signaling increase HIF-1 activity (Kirito et al, Blood 2005); however, the role of HIFs in PV is unknown. We evaluated HIF transcriptional activity in PV by unbiased RNA sequencing of JAK2V617F positive PV granulocytes (22 patients and 10 controls) and platelets (24 patients and 4 controls). Differential gene expression was determined by DEseq2 and data stratified by p&lt;0.05 and Log2fold change&gt;1. BaseSpace Correlation Engine (Illumina Inc.) was used to identify genes regulated by HIF. Fifty-seven HIF target genes were dysregulated in PV granulocytes. There were 44 upregulated genes involved in apoptosis, metabolism, cellular proliferation, intracellular signal transduction, and ion transport. Thirteen genes were downregulated in PV, all associated with Ras and Rap1 signaling. Thirty-five genes were correlated with JAK2V617F allele burden; 27 genes were positively correlated while 8 genes were inversely correlated. SOCS2, FLT1, PDLIM1, ALDH1A2, NPR3, and BATF3 were regulated by both STAT5 and HIF suggesting that HIFs and the JAK-STAT pathway may cross-talk. CAV1, PLOD2, NRIP3, NGFRAP1, and UBXN10 inversely correlated with the white blood cell counts. Eighty-seven HIF target genes were differentially expressed in PV platelets. Seventy-two genes were upregulated and 15 genes were downregulated. Upregulated genes were associated with apoptosis, proliferation, and metabolism. Unlike in PV granulocytes, some of these upregulated genes were associated with immune responses including the TNF and NF-kB signaling pathways; NF-kB and HIF-1 co-stimulate each other. Twenty genes positively correlated with JAK2V617F allele burden but BCL2 and PNOC inversely correlated. There were 13 genes regulated by both STAT5 and HIF. DOT1L, GYG1, HOXA1, PRELID1, and RPIA transcript levels inversely correlated with the platelet counts. In both granulocytes and platelets, 17 genes were dysregulated. CAV1, CHAD, and SOCS2 are associated with negative regulation of receptor signaling via JAK-STAT. CHAD was downregulated while CAV1 and SOCS2 were upregulated in both granulocytes and platelets. ALDH1A2, CAV1, KCNMA1, and MMP9 genes were upregulated and associated with apoptosis. We also observed an increased transcript of inflammatory genes in PV. PV granulocytes had 82 upregulated and 11 downregulated inflammatory genes. Among these upregulated genes, MGLL, PROK2, and MAP2K6 were also HIF target genes. In platelets, 34 genes were dysregulated (31 genes were upregulated and 3 genes were downregulated). It is of interest that 2 prothrombotic genes are reported to be regulated by HIFs, tissue factor encoded by F3 (Giannarelli, et al., Circulation 2014) and protein S (encoded by PROS1). We found F3 transcript to be upregulated also in PV and CE granulocytes. PROS1 was reported to be negatively regulated by HIF-1 as detected by decreased protein S in mouse plasma and by decreased PROS1mouse liver transcript and human cell line HepG2 (Pilli, et al., Blood 2018). We found PROS1 to be also expressed in human granulocytes and platelets; however, it was overexpressed in PV and CP granulocytes but not in platelets. We demonstrate here high HIF transcriptional activity in granulocytes and platelets of PV; some of these HIF-regulated genes are also associated with immune response genes. Dysregulated HIF target genes were involved in apoptosis and cell proliferation pathways. Most HIF target gene transcription correlated with JAK2V617F allele burden, platelet counts, and white blood cell counts. These results suggest that HIF might be a therapeutic target of PV. Of drugs in clinical use, digoxin is a potent HIF-1 inhibitor (Zhang, et al., PNAS 2008); its potential role in PV and CE is being explored. Disclosures Gordeuk: Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding; Pfizer: Research Funding; Modus Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding.

A Hypoxia-Inducible HIF1-GAL3ST1-Sulfatide Axis Enhances ccRCC Immune Evasion via Increased Tumor Cell-Platelet Binding.

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and the major cause of mortality for individuals with von Hippel-Lindau (VHL) disease. ccRCC is characterized most frequently by inactivation of VHL tumor suppressor protein that mediates degradation of the alpha subunit of the hypoxia-inducible factor (HIF) transcription factor family. HIF has been implicated in disease progression and the aim of this study was to identify novel HIF target genes that may contribute to ccRCC. We show that GAL3ST1, an enzyme that catalyzes the sulfonation of the plasma membrane sulfolipid sulfatide, is among the top 50 upregulated genes in ccRCC tissue relative to matched normal tissue. Increased expression of GAL3ST1 in primary ccRCC correlates with decreased survival. We show that GAL3ST1 is a HIF target gene whose expression is induced upon VHL loss leading to the accumulation of its enzymatic product sulfatide. Notably, platelets bind more efficiently to renal cancer cells with high GAL3ST1-sulfatide expression than to GAL3ST1-sulfatide-negative counterparts, which protects ccRCC cells against natural killer cell-mediated cytotoxicity. These results suggest that GAL3ST1 is a HIF-responsive gene that may contribute to ccRCC development via promoting cancer cell evasion of immune surveillance. IMPLICATIONS: Cancer development is in part dependent on evasion of immune response. We identify a HIF target gene product GAL3ST1 that may play a role in this critical process.

Mechanisms of hypoxia signalling: new implications for nephrology.

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 catalyse 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, 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.