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High-altitude polycythemia (HAPC) is a common chronic altitude disease caused by living in low-pressure and low-oxygen environment. At present, there is still no effective cure for HAPC. HIF-2α may play an important role in the development of HAPC in regulating the increased red blood cell excessively induced by HIF-EPO and the blood vessel formation induced by VEGF-VEGFR. Here, we established a rat HAPC model and treated it with the HIF-2α inhibitor PT2385. We mainly evaluated the therapeutic effect of PT2385 on HAPC rats by observing the changes in rat phenotype, tissue and organ damage, red blood cell and hemoglobin content, angiogenesis, lipid peroxidation reaction, and inflammatory factors. The results showed that PT2385 treatment improved the congestion phenotype characteristics, inhibited increased erythrocytes and hemoglobin, reduced blood vessel formation, lipid peroxidation, and inflammation, and reduced tissue and organ damage in HAPC rats. This study preliminarly explains the physiological, pathological, and immunological effects of PT2385 treatment for HAPC. It provides a new idea, a reliable experimental basis, and theoretical support for the clinical prevention and treatment of HAPC.

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Hypoxia-inducible-factors (HIF) are transcription factors that regulate cellular adaptation to hypoxic conditions, enabling cells to survive in low-oxygen environments. Viruses have evolved to stabilize this pathway to promote successful viral infection, therefore modulation of HIFs could represent a novel antiviral strategy. In previous in vitro studies, we found that respiratory syncytial virus (RSV), a leading cause of respiratory illness, stabilizes HIFs under normoxic conditions, with inhibition of HIF-1α resulting in reduced viral replication. Despite several HIF modulating compounds being tested/approved for use in other non-infectious models, little is known about their efficacy against respiratory viruses using relevant animal models. This study aimed to characterize the disease modulating properties and antiviral potential of anti-HIF-1α (PX478) and anti-HIF-2α (PT2385) in RSV-infected BALB/c mice. We found that inhibition of HIF-1α worsen clinical disease parameters, while simultaneously improving airway function. Additionally, anti-HIF-1α results in significantly reduced viral titer at early and peak time points of RSV replication, followed by a loss in viral clearance when given every day, but not every-other-day. In contrast, inhibition of HIF-2α was associated with improved clinical parameters, with no changes in airway function, and amelioration of interstitial pneumonia. Furthermore, anti-HIF-2α reduced early and peak lung viral replication, with no impairment of viral clearance. Analysis of lung cells found significant modification in the T cell compartment that correlated with changes in lung pathology and viral titers in response to each HIF inhibitor administration. These data underscore the complex role of HIFs in RSV infection and highlight the need for careful therapeutic consideration.

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A number of erythropoiesis stimulants are entering the final stage of clinical trials due to recent scientific progress in hypoxia-regulated erythropoiesis. Considering how erythropoiesis-stimulating compounds enhance the capacity of the organism to transport oxygen, they pose a great risk of being misused as performance enhancers. In this paper, we report the metabolic fate of three popular hypoxia-inducible factor-prolyl hydroxylase Inhibitors (HIF-PHI) compounds, namely, BAY 87-2243, MK-8617, and PT-2385 in equine liver microsomes using Q-Exactive high-resolution mass spectrometry. This study found 22 metabolites for BAY 87-2243 (19 phase I and three phase II), three metabolites for MK-8617 (all phase I), and five metabolites for PT-2385 (two phase I and three phase II). The major findings of the present study are as follows: (1) all three potential HIF-PHI drug candidates, namely, BAY 87-2243, MK-8617, and PT-2385 are susceptible to oxidation, producing their corresponding hydroxylated metabolites; (2) the ring dissociated metabolites were detected for BAY 87-2243 and PT-2385; (3) in the case of BAY 87-2243 and PT-2385, glucuronic acid conjugated metabolites were detected; and (4) none of the drugs produced sulfonic acid conjugated metabolites.

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BACKGROUND AND PURPOSE: Recent studies reported therapeutic effects of monotherapy with either tumour suppressor p53 (p53) agonist or hypoxia-inducible factor 2α (HIF-2α) antagonist for pulmonary hypertension (PH). This study investigated whether a combined treatment of p53 agonist, Nutlin3a, and HIF-2α antagonist, PT2385, would be more effective than monotherapy, based on the cell type-divergent regulation of p53 in pulmonary arterial smooth muscle cells (PASMC) and endothelial cells (PAEC) in patients and animals with PH. EXPERIMENTAL APPROACH: The SU5416/hypoxia-induced PH (SuHx-PH) rat model was used, along with cultured human PASMC and PAEC. Western blot, RT-PCR, siRNA and immunohistochemical methods were used along with echocardiography and studies with isolated pulmonary arteries. KEY RESULTS: Hypoxia-induced proliferation of PASMC is associated with decreased p53, whereas hypoxia-induced PAEC apoptosis is associated with increased p53, via a HIF-2α-dependent mechanism. Combined treatment with Nutlin3a and PT2385 is more effective by simultaneously inhibiting the hypoxia-induced PASMC proliferation and PAEC apoptosis, overcoming the side-effects of monotherapy. These are (i) Nutlin3a exacerbates hypoxia-induced PAEC apoptosis by inducing p53 in PAEC and (ii) PT2385 inhibits PAEC apoptosis because HIF-2α is predominantly expressed in PAEC but lacks direct effects on the hypoxia-induced PASMC proliferation. In rats, combination treatment is more effective than monotherapy in reversing established SuHx-PH, especially in protecting pulmonary arterial vasculature, by normalizing smooth muscle thickening, protecting against endothelial damage and improving function. CONCLUSION AND IMPLICATIONS: Combination treatment confers greater therapeutic efficacy against PH through a selective modulation of p53 and HIF-2α in PASMC and PAEC.

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Renal cell carcinoma (RCC) encompasses a heterogenous group of tumors, but representative preclinical models are lacking. We previously showed that patient-derived tumorgraft (TG) models recapitulate the biology and treatment responsiveness. Through systematic orthotopic implantation of tumor samples from 926 ethnically diverse individuals into non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice, we generate a resource comprising 172 independently derived, stably engrafted TG lines from 148 individuals. TG lines are characterized histologically and genomically (whole-exome [n = 97] and RNA [n = 102] sequencing). The platform features a variety of histological and oncogenotypes, including TCGA clades further corroborated through orthogonal metabolomic analyses. We illustrate how it enables a deeper understanding of RCC biology; enables the development of tissue- and imaging-based molecular probes; and supports advances in drug development.

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PT-2385 is currently regarded as a potent and selective inhibitor of hypoxia-inducible factor-2α (HIF-2α), with potential antineoplastic activity. However, the membrane ion channels changed by this compound are obscure, although it is reasonable to assume that the compound might act on surface membrane before entering the cell´s interior. In this study, we intended to explore whether it and related compounds make any adjustments to the plasmalemmal ionic currents of pituitary tumor (GH3) cells and human 13-06-MG glioma cells. Cell exposure to PT-2385 suppressed the peak or late amplitude of delayed-rectifier K+ current (IK(DR)) in a time- and concentration-dependent manner, with IC50 values of 8.1 or 2.2 µM, respectively, while the KD value in PT-2385-induced shortening in the slow component of IK(DR) inactivation was estimated to be 2.9 µM. The PT-2385-mediated block of IK(DR) in GH3 cells was little-affected by the further application of diazoxide, cilostazol, or sorafenib. Increasing PT-2385 concentrations shifted the steady-state inactivation curve of IK(DR) towards a more hyperpolarized potential, with no change in the gating charge of the current, and also prolonged the time-dependent recovery of the IK(DR) block. The hysteretic strength of IK(DR) elicited by upright or inverted isosceles-triangular ramp voltage was decreased during exposure to PT-2385; meanwhile, the activation energy involved in the gating of IK(DR) elicitation was noticeably raised in its presence. Alternatively, the presence of PT-2385 in human 13-06-MG glioma cells effectively decreased the amplitude of IK(DR). Considering all of the experimental results together, the effects of PT-2385 on ionic currents demonstrated herein could be non-canonical and tend to be upstream of the inhibition of HIF-2α. This action therefore probably contributes to down-streaming mechanisms through the changes that it or other structurally resemblant compounds lead to in the perturbations of the functional activities of pituitary cells or neoplastic astrocytes, in the case that in vivo observations occur.

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BACKGROUND: Hypoxia-inducible factor (HIF)-2α associates with poor outcome in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1 gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2α PAS-B domain with PT2385 have demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, we investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity. METHODS: Neuroblastoma patient-derived xenograft (PDX) cells were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2α protein localization, response to chemotherapy and orthotopic tumor growth in vivo. Two-sided student t-test was used. RESULTS: We detected high levels of HIF-2α protein in perivascular niches in neuroblastoma PDXs in vivo and at oxygenated conditions in PDX-derived cell cultures in vitro, particularly in the cytoplasmic fraction. Nuclear HIF-2α expression was reduced following PT2385 treatment, but surprisingly, virtually no effects on tumor growth in vivo or expression of canonical HIF downstream target genes in vitro were observed. In coherence, RNA sequencing of PT2385-treated PDX cells revealed a virtually unaffected transcriptome. Treatment with PT2385 did not affect cellular response to chemotherapy. In contrast, HIF-2α protein knockdown resulted in profound downregulation of target genes. CONCLUSIONS: The lack of effect from PT2385 treatment in combination with high cytoplasmic HIF-2α expression at normoxia suggest that HIF-2α have additional roles than acting as an ARNT dependent transcription factor. It is important to further unravel the conditions at which HIF-2α has transcriptional and non-transcriptional roles in neuroblastoma.

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Two recent independent studies published in Nature show robust responses of clear cell renal cell carcinoma (ccRCC) cell lines, preclinical ccRCC xenograft models and, remarkably, a patient with progressive ccRCC despite receiving multiple lines of treatment, to the long-awaited, recently developed inhibitors of hypoxia-inducible factor 2-alpha (HIF2α). This commentary published in Endocrine-Related Cancer is based on the recognition of similar molecular drivers in ccRCC and the endocrine neoplasias pheochromocytomas and paragangliomas (PPGLs), ultimately leading to stabilization of HIFs. HIF-stabilizing mutations have been detected in the von Hippel-Lindau (VHL) gene, as well as in other genes, such as succinate dehydrogenase (SDHx), fumarate hydratase (FH) and transcription elongation factor B subunit 1 (TCEB1), as well as the gene that encodes HIF2α itself: EPAS1HIF2α Importantly, the recent discovery of EPAS1 mutations in PPGLs and the results of comprehensive in vitro and in vivo studies revealing their oncogenic roles characterized a hitherto unknown direct mechanism of HIF2α activation in human cancer. The now available therapeutic opportunity to successfully inhibit HIF2α pharmacologically with PT2385 and PT2399 will certainly spearhead a series of investigations in several types of cancers, including patients with SDHB-related metastatic PPGL for whom limited therapeutic options are currently available. Future studies will determine the efficacy of these promising drugs against the hotspot EPAS1 mutations affecting HIF2α amino acids 529-532 (in PPGLs) and amino acids 533-540 (in erythrocytosis type 4), as well as against HIF2α protein activated by VHL, SDHx and FH mutations in PPGL-derived chromatin cells.

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