Hypoxia-inducible Factor Target Research Articles

Recent developments in targeting breast cancer stem cells (BCSCs): a descriptive review of therapeutic strategies and emerging therapies.

Despite recent advancements in the diagnosis and treatment of breast cancer (BC), patient outcomes in terms of survival, recurrence, and disease progression remain suboptimal. A significant factor contributing to these challenges is the cellular heterogeneity within BC, particularly the presence of breast cancer stem cells (BCSCs). These cells are thought to serve as the clonogenic nexus for new tumor growth, owing to their hierarchical organization within the tumor. This descriptive review focuses on the evolving strategies to target BCSCs, which have become a pivotal aspect of therapeutic development. We explore a variety of approaches, including targeting specific tumor surface markers (CD133 and CD44), transporters, heat shock proteins, and critical signaling pathways like Notch, Akt, Hedgehog, KLF4, and Wnt/β-catenin. Additionally, we discuss the modulation of the tumor microenvironment through the CXCR-12/CXCR4 axis, manipulation of pH levels, and targeting hypoxia-inducible factors, vascular endothelial growth factor, and CXCR1/2 receptors. Further, this review focuses on the roles of microRNA expression, strategies to induce apoptosis and differentiation in BCSCs, dietary interventions, dendritic cell vaccination, oncolytic viruses, nanotechnology, immunotherapy, and gene therapy. We particularly focused on studies reporting identification of BCSCs, their unique properties and the efficacy of various therapeutic modalities in targeting these cells. By dissecting these approaches, we aim to provide insights into the complex landscape of BC treatment and the potential pathways for improving patient outcomes through targeted BCSC therapies.

Abstract 2991: Global analysis of miR-210 and miR-21 dysregulation and direct targeting in thyroid cancer during hypoxia

Abstract Introduction: Hypoxia is frequent in solid tumors and stabilizes Hypoxia Inducible Factor-1 (HIF-1). HIF-1, in turn, activates several cellular pathways that promote tumor growth and resistance to chemotherapy. MicroRNA (miRNA) miR-210 is a direct HIF-1 target. HIF-1 binds to a Hypoxia-Response Element (HRE) in the miR-210 promoter, up-regulating its expression. miR-21 is the most widely reported oncogenic miRNA in cancer. miRNAs complexed with Argonaute-2 (AGO2) bind target mRNAs to inhibit translation. Given that a single miRNA can have hundreds of predicted mRNA targets, we sought to identify mRNA targets of miR-210 and miR-21. Identifying dysregulated target genes of miR-210 and miR-21 may highlight new therapeutic targets. Methods: The SW1736 (anaplastic thyroid cancer) cell line was cultured at 2% (hypoxic) or atmospheric (normoxic) O2 for 24 or 48hrs. HIF-1α accumulation relative to normoxia was analyzed by Western blot. miR-210-3p expression was measured relative to normoxia by RT-qPCR. Small and total RNA-sequencing was performed to identify additional differentially expressed miRNAs and mRNAs in hypoxia after 48hrs. To identify direct miRNA targets, SW1736 cells were cross-linked by UV irradiation after 48hrs of normoxic or hypoxic conditions. AGO2 covalently cross-linked to miRNA and target mRNAs were purified by immunoprecipitation after DNA and RNA digestion. miRNA and target mRNAs protected by AGO2 were ligated to generate chimeric miRNA:mRNA molecules followed by sequencing. miRNA:mRNA chimeras were analyzed using the SCRAP bioinformatic pipeline. Results: miR-210-3p expression was up-regulated by ~11-fold at 24hrs and ~19-fold at 48hrs, which was consistent with increased HIF-1α protein levels assessed by Western blot. miR-210-3p and HIF-1α levels were highest after 48hr of hypoxia relative to normoxia. Small RNA-seq showed that miR-210-3p was the only miRNA significantly up-regulated (>2-fold) in hypoxia (48hr) in SW1736. Further, miRNA:mRNA chimeric sequencing results confirmed that miRNAs primarily interacted with the 3’UTRs and coding sequences of mRNA. miR-210-3p was most abundant in hypoxia and found in chimeras with HIF-1α mRNA as well as some oncogenic and tumor suppressor transcripts involved in metabolism and mRNA processing. miR-21-5p was the most abundant chimera miRNA in both normoxia and hypoxia and was primarily bound to tumor suppressor mRNAs associated with cell cycle arrest, TGFB signaling, and inhibition of tumor invasion and migration. Conclusions: miR-210-3p and miR-21-5p elevated levels and interactions indicate hypoxia and malignancy in solid tumors including breast, thyroid, prostate, and lung. Further analysis of other miRNA:mRNA interactions and functional validation may aid in identifying novel therapies for solid tumors. Citation Format: Bonita H. Powell, William T. Mills, Andrey Turchinovich, Yongchun Wang, Martha A. Zeiger, Christopher B. Umbricht, Mollie K. Meffert, Kenneth W. Witwer. Global analysis of miR-210 and miR-21 dysregulation and direct targeting in thyroid cancer during hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2991.

Abstract 4131: Combination therapeutic strategy to fight against triple-negative breast cancer

Abstract Triple-negative breast cancers (TNBCs) lack estrogen-and progesteron receptors and human epidermal growth factor receptor-2 (HER2) which limits the therapeutic approaches. The high metastasizing capability of TNBCs is in correlation with the poor oxygenation of the cancerous breast tissue. This leads to the activation of the hypoxia-inducible-factor 1 (HIF-1), which induces multiple target genes that are involved in tumor progression and metastasis formation. This makes HIF-1 an attractive drug target, however, HIF-1 inhibitors failed if applied in monotherapy. Therefore, our aim is to investigate the combined therapeutic effect of HIF-1 inhibitor acriflavine and paclitaxel, a commonly used taxane in TNBC treatment. The synergy assessment of the two drugs was carried out by utilizing Sulforhodamine B assay on TNBC cell lines. The in vivo toxicity and efficacy of the drug combination was tested on NOD SCID mice with orthotopically inoculated MDA-MB-231 cell line. Quantitative and qualitative analysis of multiple genes involved in tumor progression was performed by mRNA and protein expression level assessment with qPCR, western blot and immunohistochemistry methods. HIF-1 stimulation was achieved by either incubating the cells at 1% oxygen level or through CoCl2 treatment. The in vitro mRNA expression studies demonstrated that acriflavin possesses a hypoxic-effect-mitigating property on the expression of metastasis-involved- and HIF-1 target genes. Acriflavine alleviated the epithelial-mesenchymal transition of TNBC cells by increasing the expression of the epithelial marker, E-cadherin. At protein level, we detected decrease in HIF-1alpha and Phospho-Protein kinase B (Akt) and increase in p53 tumor suppressor gene expression in response to acriflavine treatment. We further established MDA-MB-231 xenograft model in mice for investigating in vivo toxicity and efficacy. The drug combination efficiently reduced the tumor size and the macrometastasis formation compared to the control groups. The protein analysis of frozen tumor tissue samples indicated that the combination therapy decreased the HIF-1alpha expression and the amount of Phospho-Akt and Phospho-Mitogen-activated protein kinases (pMAPK/pERK) in CoCl2treated mice. Additionally, expression of epithelial markers increased and that of mesenchymal markers decreased. The combination therapy led to a decrease in the platelet-derived growth factor receptor beta (PDGFRB) as well. To conclude, we identified a novel drug combination which can efficiently combat TNBC by inhibiting HIF-1 signaling, mitigating cell migration and metastasis formation and suppressing the activation of the Phosphoinositide 3-kinase (PI3K) and MAPK pathways. This work was supported by the Hungarian Thematic Excellence Program (TKP2021-EGA-44) and the National Laboratories Program - National Laboratory for Tumor Biology (NLP-17). Citation Format: Laura Svajda, Ivan Ranđelović, Sára Eszter Surguta, Marcell Baranyi, István Kenessey, Mihály Cserepes, József Tóvári. Combination therapeutic strategy to fight against triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4131.

A40 THE ROLE OF HYPOXIA-INDUCIBLE FACTOR IN CELLULAR METABOLIC ADAPTATION UPON GIARDIA INFECTION

Abstract Background Recent findings have highlighted the integral role of oxygen tension in gastrointestinal (GI) homeostasis. During hypoxia when oxygen supply is limited, mammalian cells utilize the Hypoxia-Inducible Factor (HIF) complex to activate genes that promote cell survival and alter glucose metabolism. The GI system exists in a state of physiologic hypoxia, subject to further alterations by pathogens. Although tissue and blood parasites are known to influence tissue oxygen tension, little is known regarding the modulation of hypoxia and HIF by enteric parasites. Aims This project aims to characterize the effect of Giardia duodenalis, a common enteric protozoan that perturbs gastrointestinal function leading to diarrheal disease, on the cellular hypoxic response. We hypothesize that hypoxia-associated genes are activated upon Giardia infection, leading to an adaptive metabolic response with increased glycolytic flux to maintain cellular bioenergetic homeostasis. Methods Caco-2 colonic epithelial cells were infected with Giardia isolate GS/M (MOI 10) for 1.5 or 4.5 hours to capture early and peak HIF activation, under normoxic (21% O2) or hypoxic (1%O2, STEMCELL hypoxia incubator) conditions. RNA was extracted for RT-qPCR to assess transcriptional changes of known HIF-target genes. Intracellular metabolite analysis via liquid-chromatography mass-spectrometry (hydrophilic-interaction chromatography method) was performed to assess HIF-mediated metabolic changes in uninfected and Giardia-infected cells (MOI 10) exposed to the hypoxia mimetic DMOG or the HIF inhibitor PX-478. Results Under normoxic conditions, genes associated with compensatory cellular stress responses such as VEGFA, ANKRD37, GADD45A, and glycolysis-associated genes HK2, and LDHA are upregulated in Giardia-infected cells in a time-dependent manner (pampersand:003C0.05). Under hypoxic conditions, fewer HIF-target genes are upregulated (e.g., VEGFA, GADD45A, PGK1; pampersand:003C0.05), suggesting Giardia-infected cells behave similarly to uninfected cells with constant HIF activation. Analysis of the Caco-2 intracellular metabolome indicates HIF-dependent changes in amino acid (e.g., glycine, threonine) and nucleic acid (e.g., guanosine, inosine, uracil) metabolism. DMOG treatment altered the production of glycolytic intermediates (e.g., DHAP, PEP), confirming the promotion of glycolytic flux by HIF. Conclusions Both cell-stress-related and glucose-metabolism HIF target genes are activated upon Giardia infection. As well, some metabolic changes are dependent on HIF activation. These novel findings indicate Giardia promotes a hypoxic state in human intestinal cells, highlighting a metabolic cell rescue mechanism in response to enteropathogens. Furthermore, understanding the role of hypoxia in enteric parasitic infections will shed light on the potential of HIF as a therapeutic target. Funding Agencies Natural Sciences and Engineering Research Council of Canada (NSERC)

Therapeutic Targeting of Hypoxia-Inducible Factors in Cancer.

In the realm of cancer therapeutics, targeting the hypoxia-inducible factor (HIF) pathway has emerged as a promising strategy. This study delves into the intricate web of HIF-associated mechanisms, exploring avenues for future anticancer therapies. Framing the investigation within the broader context of cancer progression and hypoxia response, this article aims to decipher the pivotal role played by HIF in regulating genes influencing angiogenesis, cell proliferation, and glucose metabolism. Employing diverse approaches such as HIF inhibitors, anti-angiogenic therapies, and hypoxia-activated prodrugs, the research methodologically intervenes at different nodes of the HIF pathway. Findings showcase the efficacy of agents like EZN-2968, Minnelide, and Acriflavine in modulating HIF-1α protein synthesis and destabilizing HIF-1, providing preliminary proof of HIF-1α mRNA modulation and antitumor activity. However, challenges, including toxicity, necessitate continued exploration and development, as exemplified by ongoing clinical trials. This article concludes by emphasizing the potential of targeted HIF therapies in disrupting cancer-related signaling pathways.

High glucose promotes osteogenic differentiation of human lens epithelial cells through hypoxia-inducible factor (HIF) activation.

Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- tofivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphataseand osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1αand increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.

Targeting Hypoxia-Inducible Factor-1 (HIF-1) in Cancer: Emerging Therapeutic Strategies and Pathway Regulation.

Hypoxia-inducible factor-1 (HIF-1) is a key regulator for balancing oxygen in the cells. It is a transcription factor that regulates the expression of target genes involved in oxygen homeostasis in response to hypoxia. Recently, research has demonstrated the multiple roles of HIF-1 in the pathophysiology of various diseases, including cancer. It is a crucial mediator of the hypoxic response and regulator of oxygen metabolism, thus contributing to tumor development and progression. Studies showed that the expression of the HIF-1α subunit is significantly upregulated in cancer cells and promotes tumor survival by multiple mechanisms. In addition, HIF-1 has potential contributing roles in cancer progression, including cell division, survival, proliferation, angiogenesis, and metastasis. Moreover, HIF-1 has a role in regulating cellular metabolic pathways, particularly the anaerobic metabolism of glucose. Given its significant and potential roles in cancer development and progression, it has been an intriguing therapeutic target for cancer research. Several compounds targeting HIF-1-associated processes are now being used to treat different types of cancer. This review outlines emerging therapeutic strategies that target HIF-1 as well as the relevance and regulation of the HIF-1 pathways in cancer. Moreover, it addresses the employment of nanotechnology in developing these promising strategies.

Targeting hypoxia-inducible factors: therapeutic opportunities and challenges.

Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that are crucial for adaptation of metazoans to limited oxygen availability. Recently, HIF activation and inhibition have emerged as therapeutic targets in various human diseases. Pharmacologically desirable effects of HIF activation include erythropoiesis stimulation, cellular metabolism optimization during hypoxia and adaptive responses during ischaemia and inflammation. By contrast, HIF inhibition has been explored as a therapy for various cancers, retinal neovascularization and pulmonary hypertension. This Review discusses the biochemical mechanisms that control HIF stabilization and the molecular strategies that can be exploited pharmacologically to activate or inhibit HIFs. In addition, we examine medical conditions that benefit from targeting HIFs, the potential side effects of HIF activation or inhibition and future challenges in this field.

Therapeutic targeting of hypoxia inducible factor in acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is characterized by bilateral chest infiltration and acute hypoxic respiratory failure. ARDS carries significant morbidity and mortality despite advancements in medical management, calling for the development of novel therapeutic targets. Hypoxia-inducible factor (HIF) is a heterodimeric protein involved in various essential pathways, including metabolic reprogramming, immune modulation, angiogenesis and cell cycle regulation. HIF is routinely degraded in homeostasis conditions via the prolyl hydroxylase domain/von Hippel-Lindau protein pathway. However, HIF is stabilized in ARDS via various mechanisms (oxygen-dependent and independent) as an endogenous protective pathway and plays multifaceted roles in different cell populations. This review focuses on the functional role of HIF and its target genes during ARDS, as well as how HIF has evolved as a therapeutic target in current medical management.

Alternatively Spliced ATP6V1H induces Inflammation in Polycythemia Vera and Essential Thrombocythemia

Polycythemia vera (PV) and essential thrombocythemia (ET) are clonal myeloproliferative neoplasm originating from a hematopoietic stem cell. PV is characterized by increased hemoglobin; ET is characterized by high platelets due to gain-of-function mutations of the tyrosine kinase JAK2 gene, with JAK2V617F being the most common phenotype defining mutation. Thrombosis remains a major cause of PV and ET morbidity and mortality. Clonal myelopoiesis in PV is further enhanced by inflammation, contributing to PV pathogenesis. The molecular mechanisms underlying the augmented inflammation in PV remain incompletely characterized but neutrophils play a major role in inflammatory and prothrombotic complications in PV and ET. Our whole transcriptome analysis using 46 PV neutrophils and 10 healthy controls showed that 1833 genes in PV granulocytes (adjusted p-value <0.05 and log2 fold changes >0.5) were dysregulated. Gene set enrichment analysis showed that genes involved in inflammatory response were upregulated in PV (FDR: 0.047, enrichment score: 0.578). We then interrogated in our transcriptome set differential exon usage and identified a differentially spliced exon of ATP6V1H (AS-ATP6V1H) in PV. ATP6V1H encodes ATPase H+ transporting V1 (v-ATPase) subunit H. Inhibition of v-ATPase augments the production, release, and expression of inflammatory genes. We found higher expression levels of AS-ATP6V1H (ENST00000522849) in PV neutrophil transcriptome (Figure B). The exon in the black box in Figure A is only present in AS-ATP6V1H (ENST00000522849) and its expression was higher in PV. This transcript undergoes nonsense-mediated decay, resulting in decreased ATP6V1H expression in PV (Figure C). This finding was then verified using 46 patients with PV, 24 with ET and 29 healthy controls. AS-ATP6V1H was upregulated both in PV and ET neutrophils (Figures D). However, these expression differences were not detected in platelets (Figure E). AS-ATP6V1H transcript levels demonstrated a positive correlation with genes that are upregulated in inflammation - CXCL8, CD55, IRAK1, and IL1RAP, while showing an inverse correlation with genes downregulated in inflammation, including IGHG1, CD40LG, and TNFRSF4.These transcript levels showed a positive correlation with white blood cell (r=0.4677, p<0001) and neutrophil counts (r=0.5268, p<0001), indicating an association with heightened inflammation in PV and ET. PV and ET patients with a history of thrombosis had higher AS-ATP6V1H transcript levels compared to those without thromboses (p=0.0427). AS-ATP6V1H transcript levels positively correlated with the levels of thrombotic genes tissue factor ( F3) and p-selectin ( SELP) and inversely correlated with an anti-thrombotic gene, KLF2 (Song, Blood Advances, 2023). These results suggest that elevated AS-ATP6V1H may also be associated with an increased risk of thrombosis. The expression of inflammatory and thrombotic genes is influenced by hypoxia inducible factors (HIFs) (Gangaraju, Blood Advances, 2020). Reduced ATP6V1H increases HIF-1a protein levels and its transcriptional activities (Yambire, elife, 2019). We also measured the expression levels of HIF target genes in neutrophils ( EDN1, LDHA, SLC29A1, CKB, VEGFA, and SLC2A1), and these levels positively correlated with AS-ATP6V1H transcript levels. Since inflammation is also associated with increasing JAK2V617F variant allele frequency (VAF), we also tested the correlation with JAK2V617F VAF. We observed a positive correlation of JAK2V617F VAF with AS-ATP6V1H transcript levels (r=0.74, p<0001), suggesting that hyperactive JAK2 activity might induce an mRNA splicing event in ATP6V1H. In conclusion, our study showed a relationship between AS-ATP6V1H and inflammation in PV and ET. Through whole transcriptome analysis, we identified AS-ATP6V1H as a candidate gene, and its upregulation in PV and ET suggests its potential involvement in increased inflammation and clonal myelopoiesis. We show that AS-ATP6V1H transcript levels correlated with the expression levels of genes involved in inflammation and thrombosis and that AS-ATP6V1H may be regulated by HIFs and JAK2 activity. Further investigations should demonstrate the molecular mechanisms underlying AS-ATP6V1H's role in inflammation and thrombosis, which could lead to novel therapeutic approaches for managing PV and ET.

Genetic and pharmaceutical targeting of HIF1α allows combo-immunotherapy to boost graft vs. leukemia without exacerbation graft vs. host disease

Despite potential impact on the graft vs. leukemia (GVL) effect, immunotherapy targeting CTLA-4 and/or PD-1 has not been successfully combined with bone marrow transplant (BMT) because it exacerbates graft vs. host disease (GVHD). Here, using models of GVHD and leukemia, we demonstrate that targeting hypoxia-inducible factor 1α (HIF1α) via pharmacological or genetic approaches reduces GVHD by inducing PDL1 expression on host tissue while selectively inhibiting PDL1 in leukemia cells to enhance the GVL effect. More importantly, combination of HIF1α inhibition with anti-CTLA-4 antibodies allows simultaneous inhibition of both PDL1 and CTLA-4 checkpoints to achieve better outcomes in models of mouse and human BMT-leukemia settings. These findings provide an approach to enhance the curative effect of BMT for leukemia and broaden the impact of cancer immunotherapy.

ABCE1 selectively promotes HIF-1α transactivation of angiogenic gene expression

BackgroundCopper (Cu), by inhibiting the factor inhibiting HIF-1 (FIH-1), promotes the transcriptional activity of hypoxia-inducible factor-1 (HIF-1). ObjectiveThe present study was undertaken to understand the molecular mechanism by which Cu inhibits FIH-1. MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with dimethyloxalylglycine (DMOG) resulting in HIF-1α accumulation and the FIH-1 protein complexes were pulled down for candidate protein analysis. The metal binding sites were predicted by both MetalDetector V2.0 and Metal Ion-Binding Site Prediction Server, and then the actual ability to bind to Cu in vitro was tested by both Copper-Immobilized metal affinity chromatography (Cu-IMAC) and Isothermal Titration Calorimetry (ITC). Subsequently, subcellular localization was monitored by immunocytochemistry, GFP-fusion protein expression plasmid and Western blotting in the nuclear extract. The interaction of candidate protein with HIF-1α and FIH-1 was validated by Co-Immunoprecipitation (Co-IP). Finally, the effect of candidate protein on the FIH-1 structure and HIF-1α transcriptional activity was analyzed by the InterEvDock3 web server and real-time quantitative RT-PCR. ResultsATP-binding cassette E1 (ABCE1) was present in the FIH-1 complexes and identified as a leading Cu-binding protein as indicated by a number of possible Cu binding sites. The ability of ABCE1 to bind Cu was demonstrated in vitro. ABCE1 entered the nucleus along with FIH-1 under hypoxic conditions. Protein interaction analysis revealed that ABCE1 prevented FIH-1 to bind iron ions, inhibiting FIH-1 enzymatic activity. ABCE1 silencing suppressed the expression of Cu-dependent HIF-1 target gene BNIP3, not that of Cu-independent IGF-2. ConclusionThe results demonstrate that ABCE1, as a Cu-binding protein, enters the nucleus under hypoxic conditions and inhibits FIH-1degradation of HIF-1α, thus promoting HIF-1 transactivation of angiogenic gene expression.

Abstract A002: Targeting hypoxia inducible factor (HIF)-2α with AB521, a novel and potent small molecule HIF-2α inhibitor, for the treatment of clear cell renal cell carcinoma

Abstract Cells in the solid tumor microenvironment are frequently exposed to hypoxic or pseudohypoxic conditions, necessitating molecular adaptations such as induction of transcriptional programs for survival. HIF-2α is a transcriptional regulator of hypoxia-induced genes that is regulated post-translationally in an oxygen-dependent manner via hydroxylation by prolyl hydroxylase enzymes - a modification required for recognition by pVHL for subsequent degradation. Therefore, under hypoxic conditions or in the case of pVHL inactivation (pseudohypoxia), HIF-2α is stabilized and translocates to the nucleus where it forms a heterodimer with ARNT to initiate transcription of its target genes. HIF-2α has been shown to be an oncogenic driver in clear cell renal cell carcinoma (ccRCC) where VHL mutations occur frequently, resulting in a pseudohypoxic state and stable HIF-2α expression. There is both preclinical and clinical evidence in ccRCC demonstrating that inhibiting HIF-2α is a valid therapeutic approach in blocking tumor progression. AB521 is a clinical-stage novel and potent small-molecule HIF-2α inhibitor. In vitro, AB521 effectively inhibits transcription of pro-tumorigenic HIF-2α target genes in multiple human cell types relevant to the tumor microenvironment, including cancer cells, primary endothelial cells, and pro-tumorigenic M2-polarized macrophages. AB521 also blocks HIF-2α-mediated gene transcription in vivo, as detected using both intratumoral and peripheral markers, an activity that is accompanied by a marked reduction in tumor burden in two VHL-mutant ccRCC xenograft models. Supporting a potential combinatorial treatment approach for HIF-2α inhibition in ccRCC, AB521 combines favorably with standard-of-care tyrosine kinase inhibitor cabozantinib in mouse models, further enhancing tumor efficacy in VHL-mutant ccRCC compared to either single agent alone. In healthy human volunteers, AB521 exhibits favorable pharmacokinetic (PK) and pharmacodynamic (PD) properties including a half-life of 18-24 hours, supportive of once-daily dosing, dose-dependent reductions in serum erythropoietin (EPO) following a single dose of 10 to 100 mg, and sustained EPO reduction after multiple daily dosing. In summary, AB521 is a potent and selective HIF-2α inhibitor that has been thoroughly characterized in a suite of in vitro assays, reduces tumor burden in ccRCC xenografts both as a single agent and in combination with cabozantinib, and exhibits favorable PK/PD properties in human healthy volunteers. Phase I clinical evaluation of AB521 in subjects with ccRCC and other solid tumors has been initiated and is currently ongoing (NCT05536141). Citation Format: Dana Piovesan, Soonweng Cho, Kenneth V. Lawson, Kai H. Liao, Paul G. Foster, Tzuling Cheng, Matthew J. Walters, Kelsey E. Sivick Gauthier. Targeting hypoxia inducible factor (HIF)-2α with AB521, a novel and potent small molecule HIF-2α inhibitor, for the treatment of clear cell renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A002.

Structural basis for binding of the renal carcinoma target hypoxia-inducible factor 2α to prolyl hydroxylase domain 2.

The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen-dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C-terminal HIF2α-ODD in the presence of its 2-oxoglutarate cosubstrate or N-oxalylglycine inhibitor. Combined with the reported PHD2.HIFα-ODD structures and biochemical studies, the results inform on the different PHD.HIFα-ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform-selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment.

Targeting hypoxia-inducible factors with 32-134D safely and effectively treats diabetic eye disease in mice.

Many patients with diabetic eye disease respond inadequately to anti-VEGF therapies, implicating additional vasoactive mediators in its pathogenesis. We demonstrate that levels of angiogenic proteins regulated by HIF-1 and -2 remain elevated in the eyes of people with diabetes despite treatment with anti-VEGF therapy. Conversely, by inhibiting HIFs, we normalized the expression of multiple vasoactive mediators in mouse models of diabetic eye disease. Accumulation of HIFs and HIF-regulated vasoactive mediators in hyperglycemic animals was observed in the absence of tissue hypoxia, suggesting that targeting HIFs may be an effective early treatment for diabetic retinopathy. However, while the HIF inhibitor acriflavine prevented retinal vascular hyperpermeability in diabetic mice for several months following a single intraocular injection, accumulation of acriflavine in the retina resulted in retinal toxicity over time, raising concerns for its use in patients. Conversely, 32-134D, a recently developed HIF inhibitor structurally unrelated to acriflavine, was not toxic to the retina, yet effectively inhibited HIF accumulation and normalized HIF-regulated gene expression in mice and in human retinal organoids. Intraocular administration of 32-134D prevented retinal neovascularization and vascular hyperpermeability in mice. These results provide the foundation for clinical studies assessing 32-134D for the treatment of patients with diabetic eye disease.

Current Drugs and their Therapeutic Targets for Hypoxia-inducible Factors in Cancer.

Hypoxia, a prevalent characteristic of both solid and liquid malignancies, is found to regulate how genes are expressed in a way that promotes cellular adaptability and survival. Metastasis is controlled by hypoxia-inducible factors (HIFs). HIFs are dimeric protein molecules made up of an oxygen (O2) responsive HIF-1α, HIF-2α, or HIF-3α domain and a periodically produced HIF-1β portion (also known as ARNT). Nevertheless, it is important to note that HIFs degrade under normoxic conditions. A large multitude of different biological operations, including vessels generation, oxygen delivery, stemness, pluripotency, multiplication, epithelial to mesenchymal shift, metastatic prevalence and intrusion, redox equilibrium, and programmed cell death, are strictly controlled by over 70 immediate HIF target genes that have been reported. Metabolic reprogramming, which modulates cellular energy generation aside from oxidative phosphorylation and concerning glycolysis, is among the core tasks of HIF target genes. As a result, choosing HIFs as a primary target in the treatment of various tumors is essential. We have a very limited understanding of this extremely complex topic, which is characterised by hypoxia- induced resistance. In order to combat this, scientists are investigating numerous cutting-edge approaches. Traditional chemotherapeutic drugs used to treat cancer are frequently linked to unfavourable side effects and the development of chemoresistance. The use of natural compounds in conjunction with chemotherapy drugs is rising as a result of their capacity to alter a number of molecular practices with a lower detrimental impact. Experimental and clinical research is accumulating evidence that phytochemicals can influence the genesis and progression of cancer by favourably modulating a number of signalling pathways. Combinations of phytochemicals are potent cancer treatment options because they incentivise apoptosis, limit cell prevalence, make cancerous cells more susceptible, and escalate immunity. Despite being characterised, HIF-1-independent mechanisms for medication resistance in hypoxia are still infrequently reported. The prime aim of the article is to summarise the most recent research on the molecular basis of hypoxia-induced chemoresistance and how chemotherapy and phytochemicals can be used to treat cancer patients who are resistant to drugs.

No benefit of HIF prolyl hydroxylase inhibition for hypertensive renal damage in renovascular hypertensive rats.

Introduction: We previously reported that malignant hypertension is associated with impaired capillary density of target organs. Here, we tested the hypothesis that stabilization of hypoxia-inducible factor (HIF) in a modified "preconditioning" approach prevents the development of malignant hypertension. To stabilize HIF, we employed pharmacological inhibition of HIF prolyl hydroxylases (PHD), that profoundly affect HIF metabolism. Methods: Two-kidney, one-clip renovascular hypertension (2K1C) was induced in rats; controls were sham operated. 2K1C rats received either intermittent injections of the PHD inhibitor ICA (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate) or placebo. Thirty-five days after clipping, the frequency of malignant hypertension was assessed (based on weight loss and the occurrence of characteristic vascular lesions). In addition, kidney injury was compared between all ICA treated versus all placebo treated 2K1C, regardless of the occurrence of malignant hypertension. HIF stabilization was evaluated by immunohistochemistry, and HIF target gene expression by RT-PCR. Results: Blood pressure was elevated to the same degree in ICA- and placebo-treated 2K1C compared to control rats. ICA treatment did not affect the frequency of malignant hypertension or the extent of kidney tissue fibrosis, inflammation, or capillary density. There was a trend towards higher mortality and worse kidney function in ICA-treated 2K1C rats. ICA increased the number of HIF-1α-positive renal tubular cell nuclei and induced several HIF-1 target genes. In contrast, expression of HIF-2α protein as well as HIF-2 target genes were markedly enhanced by 2K1C hypertension, irrespective of ICA treatment. Discussion: We conclude that intermittent PHD inhibition did not ameliorate severe renovascular hypertension in rats. We speculate that the unexpected strong renal accumulation of HIF-2α in renovascular hypertension, which could not be further augmented by ICA, may contribute to the lack of a benefit from PHD inhibition.

Regulatory Functions of Hypoxia in Host-Parasite Interactions: A Focus on Enteric, Tissue, and Blood Protozoa.

Body tissues are subjected to various oxygenic gradients and fluctuations and hence can become transiently hypoxic. Hypoxia-inducible factor (HIF) is the master transcriptional regulator of the cellular hypoxic response and is capable of modulating cellular metabolism, immune responses, epithelial barrier integrity, and local microbiota. Recent reports have characterized the hypoxic response to various infections. However, little is known about the role of HIF activation in the context of protozoan parasitic infections. Growing evidence suggests that tissue and blood protozoa can activate HIF and subsequent HIF target genes in the host, helping or hindering their pathogenicity. In the gut, enteric protozoa are adapted to steep longitudinal and radial oxygen gradients to complete their life cycle, yet the role of HIF during these protozoan infections remains unclear. This review focuses on the hypoxic response to protozoa and its role in the pathophysiology of parasitic infections. We also discuss how hypoxia modulates host immune responses in the context of protozoan infections.

ZBTB2 is Recruited to a Specific Subset of HIF-1 Target Loci to Facilitate Full Gene Expression Under Hypoxia

The cellular response to hypoxia is mainly governed by a transcription factor, hypoxia-inducible factor 1 (HIF-1). Although upregulation of HIF-1 target genes has been hypothesized to require interaction of HIF-1 with other coactivators, much remains to be elucidated regarding the underlying mechanisms. Here, we demonstrate that zinc finger and BTB domain-containing protein 2 (ZBTB2) enhances the expression of certain HIF-1 target genes under hypoxia. ChIP-Seq analysis showed that there is a subset of HIF-1 target genes with overlapping HIF-1 and ZBTB2 peaks. Examination of a representative gene, EGFR antisense RNA 1 (EGFR-AS1), showed that HIF-1 binding to the consensus hypoxia-responsive element (HRE) sequence resulted in the recruitment of ZBTB2 to the gene locus and increased p300-mediated histone acetylation, leading to enhanced gene expression under hypoxia. In contrast, expression of HIF-1 target genes lacking ZBTB2 peaks, such as carbonic anhydrase 9 (CA9), was not upregulated by ZBTB2. These findings demonstrate that ZBTB2 is a novel factor that can be recruited to the vicinity of HREs on a subset of HIF-1 target gene loci, and is required for their full expression under hypoxia.

STRUCTURAL MODIFICATIONS OF BUTYRATE REVEAL TEMPLATES FOR INTESTINAL EPITHELIAL HIF STABILIZERS

Microbiota-derived short chain fatty acids, particularly butyrate (BA), show multiple beneficial influences on health. In the colon, BA ranges from 10-20 mM and up to 99% is utilized as a metabolic fuel by the mucosa. BA plays a key role in epithelial barrier regulation, reduces inflammation, and regulates cell growth and differentiation. There are multiple mechanisms by which BA contributes to gut health, many due to its regulatory capacity for gene expression. Our group reported (PMID: 34190032) a direct influence of BA on the stabilization of the transcription factor hypoxia-inducible factor (HIF). It is known that HIF stabilization is essential for appropriate mucosal barrier regulation and the coordination of regenerative capacity in the intestine. However, BA is constantly metabolized limiting its HIF stabilization effect. This observation led to the design and investigation of BA-mimicking compounds that stabilize HIF, but may not be involved in metabolism. We hypothesize that structural modifications of BA yield analogues that stabilize HIF with better efficacy and a longer biological half-life. A library of BA derivatives was screened in search of a non-endogenous analogue with higher potency and/or longer half-life for HIF stabilization. In vitro screenings at physiologically-relevant concentrations (5 mM) were performed using intestinal epithelial cell lines (T84 and CaCO2) in normoxia to determine HIF-1a protein abundance. Various analogues stabilized HIF, with 4-mercapto-butyrate (MBA) being the most promising candidate. The best analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CAIX by q-PCR. MBA exhibited higher induction of these targets compared to BA. Time-course studies revealed that MBA exhibits significantly longer half-life as observed in the stabilization of HIF and induction of gene targets for up to 72 h compared to BA (24 h). Loss of gene induction was observed in MBA treated cells expressing lentiviral shRNA against HIF1b (HIF1b KD), supporting a HIF-dependent transcription. Furthermore, it’s been widely reported that BA enhances epithelial barrier, primarily through HIF coordinated barrier protection. Among several other BA derivatives, MBA enhanced and prolonged epithelial barrier function in intestinal epithelial cells. In vivo studies in C57BL/6 mice explored the induction of HIF targets in tissue and specifically erythropoietin (EPO) in circulation following administration of BA and MBA at similar doses. These studies revealed that induction of EPO with a single dose of MBA exceeded that of native BA. In colonic and kidney tissue, GLUT1 and BNIP3 were moderately induced in vivo. Thus, a non-endogenous BA derivative (MBA) stabilizes HIF at higher potency and longer half-life than native BA and will be used as a template for the development of HIF stabilizing agents.