Oxygen-dependent Degradation Domain Research Articles

Characterizing OXPHOS inhibitor-mediated alleviation of hypoxia using high-throughput live cell-imaging

BackgroundHypoxia is a common feature of many solid tumors and causes radiotherapy and immunotherapy resistance. Pharmacological inhibition of oxidative phosphorylation (OXPHOS) has emerged as a therapeutic strategy to reduce hypoxia. However, the OXPHOS inhibitors tested in clinical trials caused only moderate responses in hypoxia alleviation or trials were terminated due to dose-limiting toxicities. To improve the therapeutic benefit, FDA approved OXPHOS inhibitors (e.g. atovaquone) were conjugated to triphenylphosphonium (TPP+) to preferentially target cancer cell’s mitochondria. In this study, we evaluated the hypoxia reducing effects of several mitochondria-targeted OXPHOS inhibitors and compared them to non-mitochondria-targeted OXPHOS inhibitors using newly developed spheroid models for diffusion-limited hypoxia.MethodsB16OVA murine melanoma cells and MC38 murine colon cancer cells expressing a HIF-Responsive Element (HRE)-induced Green Fluorescent Protein (GFP) with an oxygen-dependent degradation domain (HRE-eGFP-ODD) were generated to assess diffusion-limited hypoxia dynamics in spheroids. Spheroids were treated with IACS-010759, atovaquone, metformin, tamoxifen or with mitochondria-targeted atovaquone (Mito-ATO), PEGylated mitochondria-targeted atovaquone (Mito-PEG-ATO) or mitochondria-targeted tamoxifen (MitoTam). Hypoxia dynamics were followed and quantified over time using the IncuCyte Zoom Live Cell-Imaging system.ResultsHypoxic cores developed in B16OVA.HRE and MC38.HRE spheroids within 24 h hours after seeding. Treatment with IACS-010759, metformin, atovaquone, Mito-PEG-ATO and MitoTam showed a dose-dependent reduction of hypoxia in both B16OVA.HRE and MC38.HRE spheroids. Mito-ATO only alleviated hypoxia in MC38.HRE spheroids while tamoxifen was not able to reduce hypoxia in any of the spheroid models. The mitochondria-targeted OXPHOS inhibitors demonstrated stronger anti-hypoxic effects compared to the non-mito-targeted OXPHOS inhibitors.ConclusionsWe successfully developed a high-throughput spheroid model in which hypoxia dynamics can be quantified over time. Using this model, we showed that the mitochondria-targeted OXPHOS inhibitors Mito-ATO, Mito-PEG-ATO and MitoTam reduce hypoxia in tumor cells in a dose-dependent manner, potentially sensitizing hypoxic tumor cells for radiotherapy.

Increased HIF-2α activity in the nucleus pulposus causes intervertebral disc degeneration in the aging mouse spine.

Hypoxia-inducible factors (HIFs) are essential to the homeostasis of hypoxic tissues. Although HIF-2α, is expressed in nucleus pulposus (NP) cells, consequences of elevated HIF-2 activity on disc health remains unknown. We expressed HIF-2α with proline to alanine substitutions (P405A; P531A) in the Oxygen-dependent degradation domain (HIF-2αdPA) in the NP tissue using an inducible, nucleus pulposus-specific K19CreERT allele to study HIF-2α function in the adult intervertebral disc. Expression of HIF-2α in NP impacted disc morphology, as evident from small but significantly higher scores of degeneration in NP of 24-month-old K19CreERT; HIF-2αdPA (K19-dPA) mice. Noteworthy, comparisons of grades within each genotype between 14 months and 24 months indicated that HIF-2α overexpression contributed to more pronounced changes than aging alone. The annulus fibrosus (AF) compartment in the 14-month-old K19-dPA mice exhibited lower collagen turnover and Fourier transform-infrared (FTIR) spectroscopic imaging analyses showed changes in the biochemical composition of the 14- and 24-month-old K19-dPA mice. Moreover, there were changes in aggrecan, chondroitin sulfate, and COMP abundance without alterations in NP phenotypic marker CA3, suggesting the overexpression of HIF-2α had some impact on matrix composition but not the cell phenotype. Mechanistically, the global transcriptomic analysis showed enrichment of differentially expressed genes in themes closely related to NP cell function such as cilia, SLIT/ROBO pathway, and HIF/Hypoxia signaling at both 14- and 24-month. Together, these findings underscore the role of HIF-2α in the pathogenesis of disc degeneration in the aged spine.

Evolution of a novel regulatory mechanism of hypoxia inducible factor in hypoxia-tolerant electric fishes

Hypoxia is a significant source of metabolic stress that activates many cellular pathways involved in cellular differentiation, proliferation, and cell death. Hypoxia is also a major component in many human diseases and a known driver of many cancers. Despite the challenges posed by hypoxia, there are animals that display impressive capacity to withstand lethal levels of hypoxia for prolonged periods of time and thus offer a gateway to a more comprehensive understanding of the hypoxic response in vertebrates. The weakly electric fish genus Brachyhypopomus inhabits some of the most challenging aquatic ecosystems in the world, with some species experiencing seasonal anoxia, thus providing a unique system to study the cellular and molecular mechanisms of hypoxia tolerance. In this study, we use closely related species of Brachyhypopomus that display a range of hypoxia tolerances to probe for the underlying molecular mechanisms via hypoxia inducible factors (HIFs)-transcription factors known to coordinate the cellular response to hypoxia in vertebrates. We find that HIF1⍺ from hypoxia tolerant Brachyhypopomus species displays higher transactivation in response to hypoxia than that of intolerant species, when overexpressed in live cells. Moreover, we identified two SUMO-interacting motifs near the oxygen-dependent degradation and transactivation domains of the HIF1⍺ protein that appear to boost transactivation of HIF1, regardless of the genetic background. Together with computational analyses of selection, this shows that evolution of HIF1⍺ are likely to underlie adaptations to hypoxia tolerance in Brachyhypopomus electric fishes, with changes in two SUMO-interacting motifs facilitating the mechanism of this tolerance.

Protein kinase B/AKT phosphorylates hypoxia-inducible factor-3α1 in response to insulin, promoting cell growth and migration.

Hypoxia-inducible factors (HIFs) are best known for their roles in the adaptation to low oxygen environments. Besides hypoxia, HIF-1/2 α-subunits are also regulated by various non-hypoxic stimuli including insulin which can act via the PI3K/protein kinase B (PKB) signaling pathway. However, with respect to insulin little is known about HIF-3α. We aimed to investigate this relationship and found that insulin stimulates HIF-3α expression under both normal and low oxygen conditions. Blocking PKB activity reversed the effects of insulin, indicating that HIF-3α is a direct target of PKB. We identified serine 524, located in the oxygen-dependent degradation domain of HIF-3α, as a phosphorylation site of PKB. Mutating serine 524 impaired binding of PKB to HIF-3α and its ubiquitination, suggesting that PKB regulates HIF-3α stability through phosphorylation, thereby affecting important cellular processes such as cell viability and cell adhesion. Importantly, we discovered that this phosphorylation site also influenced insulin-dependent cell migration. These findings shed light on a novel mechanism by which insulin affects PKB-dependent HIF-3α expression and activity, with potential implications in metabolic diseases and cancer.

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.

Hypoxia-Inducible Factors-Based Single Nucleotide Polymorphism in Breast Cancer with More Cancer Susceptibility.

Hypoxia-inducible factors (HIFs) are a collection of transcriptional factors that engage in the regulation of oxygen homeostasis. They are hypoxia-responsive stress factors whose expression is linked to tumor growth and angiogenesis. HIF is a crucial player in the progression of breast cancer. Patients with high levels of hypoxia-inducible HIFs in their primary tumor biopsies had a higher chance of metastasis, the leading cause of breast cancer-related death. HIF polymorphisms have been shown in several epidemiological studies to influence breast cancer susceptibility. In the oxygendependent degradation domain, several short nucleotide polymorphisms (SNPs) of the HIF gene have been connected with higher HIF activity. To find SNP that make up the genetic diversity that underpins the phenotypic difference found between individuals in their susceptibility to cancer and the course of their disease, researchers used a variety of potential pathway-based approaches.

Overactivated NRF2 induces pseudohypoxia in hepatocellular carcinoma by stabilizing HIF-1α.

Hypoxia-inducible factor-1α (HIF-1α) is highly expressed/activated in most hypoxic tumors including hepatocellular carcinoma (HCC). Another key transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2), is also constitutively overactivated in HCC. In an attempt to determine whether HIF-1α and NRF2 could play complementary roles in HCC growth and progression, we investigated the crosstalk between these two transcription factors and underlying molecular mechanisms in cultured HCC cells and experimentally induced hepatocarcinogenesis as well as clinical settings. While silencing of HIF-1α in HepG2 human hepatoma cells did not alter the protein expression of NRF2, NRF2 knockdown markedly reduced the nuclear accumulation of HIF-1α without influencing its mRNA expression. In diethylnitrosamine-induced hepatocarcinogenesis in wild type mice, there was elevated NRF2 expression with concomitant upregulation of HIF-1α. However, this was abolished in Nrf2 knockout mice. NRF2 and HIF-1α co-localized and physically interacted with each other as assessed by in situ proximity ligation and immunoprecipitation assays. In addition, the interaction between NRF2 and HIF-1α as well as their overexpression was found in tumor specimens obtained from HCC patients. In normoxia, HIF-1α undergoes hydroxylation by a specific HIF-prolyl hydroxylase domain protein (PHD), which facilitates ubiquitination and proteasomal degradation of HIF-1α. NRF2 contributes to pseudohypoxia, by directly binding to the oxygen-dependent degradation (ODD) domain of HIF-1α, which hampers the PHD2-mediated hydroxylation, concomitant recruitment of von-Hippel-Lindau and ubiquitination of HIF-1α.

Novel Oxygen-Dependent Degradable Immunotoxin Regulated by the Ubiquitin-Proteasome System Reduces Nonspecific Cytotoxicity.

The use of bacterial toxins as antitumor agents has received considerable attention. Immunotoxins based on antigen recognition of single-chain antibodies have been widely explored for cancer therapy. Despite their impressive killing effect on tumor cells, immunotoxins still display unspecific toxicity with undesired side effects. High levels of hypoxia-inducible factor 1α (HIF-1α) are well-known indicators of hypoxia in cancer cells. In this study, different linkers were employed to fuse the immunotoxin DAB389-4D5 scFv (DS) with the oxygen-dependent degradation domain (ODDD) of HIF-1α, a domain selectively facilitating the accumulation of HIF-1α under hypoxia, to construct the oxygen-dependent degradable immunotoxin DS-ODDD (DSO). The engineered fusion protein DSO-2 containing a linker (G4S)3 possesses the best killing effect on cancer cells under hypoxia and displayed considerably reduced nonspecific toxicity to normal cells under normoxic conditions. Flow cytometry, immunofluorescence, and immunoblot analyses demonstrated that DSO-2 was degraded via the ubiquitin-proteasome pathway regulated by the oxygen-sensitive mechanism. Western blot analysis indicated that the degradation of DSO-2 significantly decreased the activation of apoptosis-related molecules in normal cells. The engineered immunotoxin with oxygen-sensing properties developed herein is a potential therapeutic agent for cancer treatment.

Ocular Hypertension Results in Hypoxia within Glia and Neurons throughout the Visual Projection.

The magnitude and duration of hypoxia after ocular hypertension (OHT) has been a matter of debate due to the lack of tools to accurately report hypoxia. In this study, we established a topography of hypoxia in the visual pathway by inducing OHT in mice that express a fusion protein comprised of the oxygen-dependent degradation (ODD) domain of HIF-1α and a tamoxifen-inducible Cre recombinase (CreERT2) driven by a ubiquitous CAG promoter. After tamoxifen administration, tdTomato expression would be driven in cells that contain stabilized HIF-1α. Intraocular pressure (IOP) and visual evoked potential (VEP) were measured after OHT at 3, 14, and 28 days (d) to evaluate hypoxia induction. Immunolabeling of hypoxic cell types in the retina and optic nerve (ON) was performed, as well as retinal ganglion cell (RGC) and axon number quantification at each time point (6 h, 3 d, 14 d, 28 d). IOP elevation and VEP decrease were detected 3 d after OHT, which preceded RGC soma and axon loss at 14 and 28 d after OHT. Hypoxia was detected primarily in Müller glia in the retina, and microglia and astrocytes in the ON and optic nerve head (ONH). Hypoxia-induced factor (HIF-α) regulates the expression of glucose transporters 1 and 3 (GLUT1, 3) to support neuronal metabolic demand. Significant increases in GLUT1 and 3 proteins were observed in the retina and ON after OHT. Interestingly, neurons and endothelial cells within the superior colliculus in the brain also experienced hypoxia after OHT as determined by tdTomato expression. The highest intensity labeling for hypoxia was detected in the ONH. Initiation of OHT resulted in significant hypoxia that did not immediately resolve, with low-level hypoxia apparent out to 14 and 28 d, suggesting that continued hypoxia contributes to glaucoma progression. Restricted hypoxia in retinal neurons after OHT suggests a hypoxia management role for glia.

Novel mutations in EPO-R and oxygen-dependent degradation (ODD) domain of EPAS1 genes-a causative reason for Congenital Erythrocytosis

Congenital Erythrocytosis (CE) can be primary or secondary due to the mutations in genes involved in the erythropoietin receptor and oxygen sensing pathway. In this study, 42 patients with 38 unrelated patients and one family (4 patients) who were JAK-2 mutation (both exon 12 and exon 14) negative with high haematocrit values were investigated. The Endogenous Erythroid colony (EEC) assay was performed in all patients, interestingly EEC colonies were high in EPAS1 and EPOR mutated patients compared to non-mutated patients. The sequence analysis of EPAS1 (exon 12), EPO-R (exon-8), VHL (exon-3), and EGLN1 (exon-1) genes in all these patients showed 19% of patients (8/42) had mutations, in exon12 of EPAS1 and exon 8 of EPO-R genes. Two novel missense mutations MW_600850:c.1183G>C, MW_600851:c.1028A>C in EPO-R gene were observed in the study group. One new MW_600849:c.1969C>T nonsense mutation and five MW_619914:c.1715A>G, MW_619915:c.1694G>T, MW_619916:c.1634T>C, MW_600852:c.1771C>G, MW_600848:c.1859G>A novel missense mutations were observed in the EPAS1 gene. Among them, 4 mutations p. (Gln572Arg), p. (Ser565Ile), p. (Ile545Thr), p. (Gln591Glu) in the ODD (Oxygen-dependent degradation) domain of HIF2α, all these variations contributed to the formation of non-functional HIF2α. No mutations were observed in VHL and EGLN1 genes. Using in silico analysis we observed that these mutations contributed to major conformational changes in the HIF2α protein making it non-functional. The mutations in the EPAS1 gene were heterozygous and show autosomal dominant inheritance patterns and we observed in one family. These novel mutations in the EPAS1 (75% (6/8)) and 25% (2/8) EPO-R genes correlating with EEC positivity were observed for the first time in India in CE patients.

Endothelial PPARδ facilitates the post-ischemic vascular repair through interaction with HIF1α.

Rationale: Restoration of vascular perfusion in peripheral arterial disease involves a combination of neovessel formation and the functional restoration of vascular endothelium. Previous studies indicated that ligand-dependent PPARδ activation enhances angiogenesis. However, how PPARδ is triggered by hypoxia and its downstream effects during post-ischemic vascular repair was not well understood.Methods: We induced experimental hindlimb ischemia in endothelial cell selective Ppard knockout induced by Cdh5-Cre mediated deletion of floxed Ppard allele in mice and their wild type control and observed blood perfusion, capillary density, vascular relaxation, and vascular leakage.Results: Deletion of endothelial Ppard delayed perfusion recovery and tissue repair, accompanied by delayed post-ischemic angiogenesis, impaired restoration of vascular integrity, more vascular leakage and enhanced inflammatory responses. At the molecular level, hypoxia upregulated and activated PPARδ in endothelial cells, whereas PPARδ reciprocally stabilized HIF1α protein to prevent its ubiquitin-mediated degradation. PPARδ directly bound to the oxygen-dependent degradation domain of HIF1α at the ligand-dependent domain of PPARδ. Importantly, this HIF1α-PPARδ interaction was independent of PPARδ ligand. Adeno-associated virus mediated endothelium-targeted overexpression of stable HIF1α in vivo improved perfusion recovery, suppressed vascular inflammation, and enhanced vascular repair, to counteract with the effect of Ppard knockout after hindlimb ischemia in mice.Conclusions: In summary, hypoxia-induced, ligand-independent activation of PPARδ in ECs stabilizes HIF1α and serves as a critical regulator for HIF1α activation to facilitate the post-ischemic restoration of vascular homeostasis.

HIF-1α Hydroxyprolines Modulate Oxygen-Dependent Protein Stability Via Single VHL Interface With Comparable Effect on Ubiquitination Rate

The basic molecular mechanism underlying mammalian oxygen-dependent regulation of hypoxia-inducible factor (HIF) via the von Hippel-Lindau E3 ubiquitin ligase is well established. The principal step in this critical cellular process is the hydroxylation of either or both of the two conserved proline residues P402 and P564 within the oxygen-dependent degradation domain (ODD) of HIF-1α subunit via prolyl hydroxylases, which is necessary for binding VHL. However, the significance of the two prolines has remained unclear considering that only one hydroxyproline is sufficient for the recruitment of VHL. Here, we show using biophysical analyses that both hydroxyprolines bind to the same interface on VHL with similar affinity; VHL binding affinity to HIF-1α ODD remains relatively unchanged regardless of whether the ODD contains one or two hydroxyprolines; ODD with two hydroxyprolines can accommodate two VHLs; and the rate of in vitro ubiquitination of ODD with one hydroxyproline via VHL E3 ligase is comparable to the rate observed with ODD containing two hydroxyprolines. However, the two hydroxyprolines show distinct contributions to the intracellular stability of HIF-1α ODD. These results demonstrate for the first time that the graduated HIF-1α stability profile observed over a range of oxygen tension is not attributed to the binding of or ubiquitination via VHL per se, but is likely due to the preceding events such as the efficacy of oxygen-dependent prolyl hydroxylase-mediated hydroxylation of HIF-1α.

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.

Abstract 1206: Discovery and characterization of AB521, a novel, potent, and selective hypoxia-inducible factor (HIF)-2α inhibitor

Abstract A hypoxic environment is a common characteristic of solid tumors. Cancer cells adapt to hypoxia and become more aggressive by up-regulation of genes associated with metabolism, growth, proliferation, angiogenesis, and erythropoiesis. Hypoxia-inducible factors (HIFs) are the central driving force for the cellular response to hypoxia and regulate a vast array of these genes. HIFs are heterodimers composed of an oxygen-sensitive HIF-α subunit (HIF-1α, HIF-2α, and HIF-3α) and a constitutively expressed HIF-1β subunit. HIF-2α is constitutively synthesized and regulated in an oxygen-dependent manner. Proline residues present in the oxygen-dependent degradation domain of the HIF-2α subunit are hydroxylated and subject to ubiquitination via the von Hippel-Lindau (pVHL) E3 ligase complex for subsequent proteasomal degradation. Under hypoxia or pseudohypoxia, caused by loss of VHL function, this process is inhibited, allowing HIF-2α translocation to the nucleus where, in complex with HIF-1β/ARNT, it promotes transcription of various pro-tumorigenic gene sets. Inhibition of HIF-2α has been demonstrated to be an effective strategy to mitigate tumor growth in the clinic, particularly in clear cell renal cell carcinoma (ccRCC). Applying a pharmacophore mapping and structure-based design approach, we identified multiple novel series of small molecule HIF-2α inhibitors which avidly bind the HIF-2α PAS-B domain and disrupt dimerization with HIF-1β, preventing HIF-2α-mediated gene transcription. Interrogation of structure activity relationships and pharmacokinetic trends yielded highly optimized inhibitors, including AB521, which potently inhibits HIF-2α reporter transcription and VEGF secretion in a human ccRCC line. AB521 was confirmed to bind HIF-2α via multiple biochemical assays and inhibited HIF-2α-, but not HIF-1α-, mediated gene expression in a hepatocellular carcinoma cell line. Furthermore, AB521 is characterized by a favorable preclinical pharmacokinetic profile with high stability to human hepatocytes and no significant direct or time-dependent inhibition of the major CYP450 drug metabolizing enzymes. AB521 exhibits high bioavailability across preclinical species and is projected to possess a human pharmacokinetic profile suitable for once-daily dosing. In conclusion, we have discovered and extensively characterized a series of novel HIF-2α inhibitors, exemplified by AB521, which potently and selectively inhibits HIF-2α and possesses a favorable preclinical pharmacokinetic profile. The data described herein provides further support for the development of novel HIF-2α inhibitors for cancer therapy. Citation Format: Kenneth V. Lawson, Kelsey E. Sivick Gauthier, Artur K. Mailyan, Jeremy T. Fournier, Joel W. Beatty, Samuel L. Drew, Jarek Kalisiak, Balint Gal, Guillaume Mata, Zhang Wang, Xuelei Yan, Lixia Jin, Elaine Ginn, Dana Piovesan, Jennifer Au, Cesar A. Meleza, Stephen W. Young, Matthew J. Walters, Manmohan Leleti, Jay P. Powers. Discovery and characterization of AB521, a novel, potent, and selective hypoxia-inducible factor (HIF)-2α inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1206.

CaMKIIα Expressing Neurons to Report Activity-Related Endogenous Hypoxia upon Motor-Cognitive Challenge.

We previously introduced the brain erythropoietin (EPO) circle as a model to explain the adaptive ‘brain hardware upgrade’ and enhanced performance. In this fundamental circle, brain cells, challenged by motor-cognitive tasks, experience functional hypoxia, triggering the expression of EPO among other genes. We attested hypoxic cells by a transgenic reporter approach under the ubiquitous CAG promoter, with Hif-1α oxygen-dependent degradation-domain (ODD) fused to CreERT2-recombinase. To specifically focus on the functional hypoxia of excitatory pyramidal neurons, here, we generated CaMKIIα-CreERT2-ODD::R26R-tdTomato mice. Behavioral challenges, light-sheet microscopy, immunohistochemistry, single-cell mRNA-seq, and neuronal cultures under normoxia or hypoxia served to portray these mice. Upon complex running wheel performance as the motor-cognitive task, a distinct increase in functional hypoxic neurons was assessed immunohistochemically and confirmed three-dimensionally. In contrast, fear conditioning as hippocampal stimulus was likely too short-lived to provoke neuronal hypoxia. Transcriptome data of hippocampus under normoxia versus inspiratory hypoxia revealed increases in CA1 CaMKIIα-neurons with an immature signature, characterized by the expression of Dcx, Tbr1, CaMKIIα, Tle4, and Zbtb20, and consistent with accelerated differentiation. The hypoxia reporter response was reproduced in vitro upon neuronal maturation. To conclude, task-associated activity triggers neuronal functional hypoxia as a local and brain-wide reaction mediating adaptive neuroplasticity. Hypoxia-induced genes such as EPO drive neuronal differentiation, brain maturation, and improved performance.

Hippocampal neurons respond to brain activity with functional hypoxia

Physical activity and cognitive challenge are established non-invasive methods to induce comprehensive brain activation and thereby improve global brain function including mood and emotional well-being in healthy subjects and in patients. However, the mechanisms underlying this experimental and clinical observation and broadly exploited therapeutic tool are still widely obscure. Here we show in the behaving brain that physiological (endogenous) hypoxia is likely a respective lead mechanism, regulating hippocampal plasticity via adaptive gene expression. A refined transgenic approach in mice, utilizing the oxygen-dependent degradation (ODD) domain of HIF-1α fused to CreERT2 recombinase, allows us to demonstrate hypoxic cells in the performing brain under normoxia and motor-cognitive challenge, and spatially map them by light-sheet microscopy, all in comparison to inspiratory hypoxia as strong positive control. We report that a complex motor-cognitive challenge causes hypoxia across essentially all brain areas, with hypoxic neurons particularly abundant in the hippocampus. These data suggest an intriguing model of neuroplasticity, in which a specific task-associated neuronal activity triggers mild hypoxia as a local neuron-specific as well as a brain-wide response, comprising indirectly activated neurons and non-neuronal cells.

Transcriptional modulation patterns of abalone Haliotis discus hannai hypoxia inducible factor-1α (HIF-1α) in interdependent crosstalk between hypoxia, infection, and environmental stresses

Hypoxia-inducible factor-1α (HIF-1α) is a key transcription factor for regulating oxygen-dependent homeostasis. Here, we characterized HIF-1α in Pacific abalone (Haliotis discus hannai), an aquaculture-relevant mollusk species, and its transcriptional regulation patterns in response to oxygen-dependent and oxygen-independent stimuli were investigated. The molecular cloning and sequence analysis revealed that the abalone HIF-1α shared conversed features in its domain organization such as basic helix–loop–helix (bHLH) domain, Per-Arnt-Sim (PAS) domain, oxygen-dependent degradation domains (ODDDs), and C-terminal transactivation domain (C-TAD) with metazoan orthologs. The abalone HIF-1α was proven to be transcriptionally active in multiple tissues according to dissolved oxygen contents, with the most sensitive responsiveness in the gill. Under non-hypoxic conditions, the tissue-dependent transcriptional regulation patterns of the abalone HIF-1α were closely correlated and intertwined with those of HSP90 and MT in response to thermal challenge and heavy metal exposure, respectively. The abalone HIF-1α expression was significantly modulated under normoxic conditions in response to immune challenges with lipopolysaccharide and Vibrio harveyi with the highest upregulation in the hemocytes. However, the combined treatment of hypoxia and immune stimulation exhibited no synergistic activation for the induction of HIF-1α mRNA. Collectively, dynamic transcriptional regulation of the abalone HIF-1α with the tissue specificity in response to oxygen-dependent and oxygen-independent stimulatory challenges suggests that HIF-1α plays crucial roles in interdependent crosstalk between hypoxia, innate immunity, and environmental stresses, which can be a useful basis to investigate cellular and physiological responses of farmed abalones to oxygen depletion and other-related stress factors.

32 Poster Discussion - Discovery and characterization of novel, potent, and selective hypoxiainducible factor (HIF)-2α inhibitors

The microenvironment of solid tumors is known to be hypoxic and requires induction of genes associated with metabolism, growth, proliferation, angiogenesis, and erythropoiesis for tumor cells to survive and metastasize. Hypoxia-inducible factors (HIFs) are the master driving force for the cellular response to hypoxia. HIFs are heterodimers composed of an oxygen-sensitive HIF-α subunit (HIF-1α, HIF-2α, and HIF-3α) and a constitutively expressed HIF-1β subunit. Under normoxic conditions, proline residues present in the oxygen-dependent degradation domain of the HIF-α subunits are hydroxylated and subject to recognition and proteasomal degradation via the von Hippel-Lindau (pVHL) E3-ubiquitin ligase complex. When exposed to low oxygen conditions or loss of pVHL function, via mutation or silencing, this process is inhibited, allowing HIF-2α translocation to the nucleus where, in complex with HIF-1β, it promotes transcription of various pro-tumorigenic gene sets. In patients, overexpression of HIF-2α is associated with poor prognosis, and both preclinical and clinical evidence is mounting that suggests inhibiting HIF-2α is an effective strategy to mitigate tumor growth, particularly in clear cell renal cell carcinoma, warranting further development of HIF-2α inhibitors.

Abstract 1482: PIM1 promotes angiogenesis via phosphorylation and stabilization of HIF-1α

Abstract An essential process for the growth and dissemination of solid tumors is angiogenesis, or the formation of new blood vessels. Overexpression of Proviral Integration site for Moloney murine leukemia virus (PIM)-1, a serine-threonine kinase, has been implicated as a driver of aggressive prostate and colon cancer. PIM1 is known to promote tumor growth and survival, and we recently uncovered a role for PIM1 in promoting resistance to anti-angiogenic agents. Here, we identify a novel signaling pathway that directly links PIM to tumor angiogenesis. Immunohistochemical staining of prostate cancer tissue revealed a significant positive correlation between PIM1 and CD31 expression, a marker of endothelial cells. Using prostate cancer as a model, in vitro models of endothelial tube formation and DCE-MRI confirmed that PIM1 overexpression increases tumor angiogenesis. Gene expression analysis of PIM1 overexpressing cells showed that PIM kinases regulates the expression levels of many hypoxia inducible factor-1α (HIF-1α) target genes in normoxic conditions. In vitro, in vivo and kinase assays discovered a novel phosphorylation site within the oxygen-dependent degradation domain (ODDD) of HIF-1α that is a direct target of PIM1. Biochemical analysis demonstrates that phosphorylation of this site increases HIF-1α stability by decreasing PHD binding, hydroxylation, and subsequent proteasomal degradation of HIF-1α. Two CRISPR-generated cell lines with point mutations that mimic phosphorylation of this site were used in xenograft models of colon cancer to confirm the significance of this site to the identified PIM-HIF signaling and tumor angiogenesis. Importantly, we show that the anti-angiogenic and cytotoxic effects of PIM inhibitors are largely dependent on their ability to downregulate HIF-1α signaling. In summary, we uncovered a novel role for PIM1 in promoting tumor angiogenesis and identified a novel phosphorylation site that controls the stability of HIF-1α in normoxic and hypoxic conditions.This research yields important insight into the role of PIM signaling in solid tumors and provides preclinical evidence that to improve the translation of PIM kinase inhibitors in solid tumors. Citation Format: Andrea L. Casillas, Shailender S. Chauhan, Rachel K. Toth, Corbin C. Jensen, Noel A. Warfel. PIM1 promotes angiogenesis via phosphorylation and stabilization of HIF-1α [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1482.

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.