Hypoxia inducible factor prolyl hydroxylase inhibition compromises development and progression of acute myeloid leukaemia
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ABSTRACT: Acute myeloid leukaemia (AML) is a largely incurable haematological disease, for which new efficient therapeutic strategies are urgently needed. While leukaemogenesis occurs under hypoxic conditions of the bone marrow, the therapeutic tractability of the hypoxia inducible factor (HIF) system in AML is elusive. Given that inactivation of HIF-1α and HIF-2α promotes leukaemic transformation, a possible therapeutic strategy for AML treatment is to constitutively stabilise HIF- α proteins. This can be achieved by targeting the HIF-prolyl hydroxylases (PHDs), the catalysis of which promotes HIF-1α and HIF-2α degradation. Here, we demonstrate that genetic inactivation of Phd1 or Phd2 compromises initiation and progression of AML, without impacting normal haematopoiesis, thus implying the immense therapeutic potential of targeting PHDs in AML. To pharmacologically inactivate PHDs, we employed clinical grade PHD inhibitors as well as a new selective PHD inhibitor (IOX5), which stabilises HIF-α through a novel mechanism of action. Pharmacological PHD inhibition compromises AML cells in a HIF-α dependent manner to disable pro-leukaemogenic pathways in AML cells, re-program their metabolism, and induce cell death, at least in part via pro-apoptotic BNIP3. Importantly, concurrent inhibition of anti-apoptotic BCL-2 by clinically used Venetoclax potentiates the anti-leukaemic effect of PHD inhibition. Thus PHD inhibition with consequent HIF-α stabilisation is a promising new non-toxic strategy for AML treatment.
Project description:The hypoxia inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an /-heterodimeric transcription factor that regulates the expression of hundreds of genes in a context dependent manner. A hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (FIH). PHD catalysis regulates HIF levels and FIH catalysis regulates HIF activity. How differences in HIF hydroxylation status relate to variations in the induction of HIF target gene transcription is unknown. We report studies using small molecule inhibitors of the HIF hydroxylases to investigate the extent to which HIF target gene upregulation is induced by reduced PHD catalysis. The results reveal substantial differences in the role of prolyl- and asparaginyl-hydroxylation in regulating hypoxia responsive genes in cells. Selective PHD inhibitors with different structural scaffolds behave similarly. However, under the tested conditions, a broad-spectrum 2OG dioxygenase inhibitor is a better mimic of the transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in transcriptional induction of a subset of genes that were not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.
Project description:The hypoxia inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a context dependent manner. A hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (FIH). PHD catalysis regulates HIFα levels and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of HIF target gene transcription is unknown. We report studies using small molecule inhibitors of the HIF hydroxylases to investigate the extent to which HIF target gene upregulation is induced by reduced PHD catalysis. The results reveal substantial differences in the role of prolyl- and asparaginyl-hydroxylation in regulating hypoxia responsive genes in cells. Selective PHD inhibitors with different structural scaffolds behave similarly. However, under the tested conditions, a broad-spectrum 2OG dioxygenase inhibitor is a better mimic of the transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in transcriptional induction of a subset of genes that were not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.
Project description:To investigate the detailed molecular mechanisms for the regulatory role of HIF-2α in experimental colitis, microarray gene expression analysis was performed on colon RNA isolated from 6- to 8-week-old Hif-2αF/F, Hif-2αlΔIE mice treated with 3%DSS for 3 days. Background & Aims: Hypoxic inflammation is characterized by decreased oxygen tension in inflammatory foci, and is a notable feature in inflammatory bowel disease (IBD). Hypoxic response is mediated by transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α, both of which are highly induced in IBD. HIF-1α is a protective factor that limits intestinal barrier dysfunction during inflammation. However, the role of HIF-2α has not been assessed in hypoxic inflammation and IBD. Methods: A hypoxia reporter mouse model was used to test the presence of hypoxia and HIF-2α in dextran sulfate sodium (DSS) and Citrobacter rodentium (C.rod)-induced colitis. The role of HIF-2α in these mouse models of colitis was further assessed in mice with an intestinal epithelium-specific gain- and loss-of-function of HIF-2α. Results: Induction of hypoxia and HIF-2α was confirmed in both murine experimental colitis models and human IBD samples. Disruption of HIF-2α attenuated colonic inflammation whereas stabilization of HIF-2α potentiated inflammation in mouse models of colitis. Interestingly, intestine specific overexpression of HIF-2α but not HIF-1α leads to spontaneous colitis and premature death in mice. Further mechanistic analysis showed that HIF-2α is a driver for pro-inflammatory response and is critical regulator of intestinal epithelial-derived tumor necrosis factor (TNF)-α. Blocking TNF-α completely ameliorated HIF-2α potentiated intestinal inflammation. Conclusions: These data demonstrate that HIF-2α is a critical transcription factor essential in intestinal epithelium elicited inflammatory response. Global gene expression profiling in colon RNAs isolated from 7-week-old Hif-2αF/F (n=6, Shah 007) and Hif-2αΔIE (n=5, Shah 008).
Project description:Hypoxia Inducible Factor (HIF) prolyl hydroxylase domain (PHD) enzymes catalyse the posttranslational hydroxylation of conserved prolyl residues in the alpha-subunit of the HIF transcription factor. These modifications, which promote the degradation of HIF-alpha subunits by the pVHL E3 ligase complex and impart oxygen-dependent regulation of the HIF transcriptional response, have been extensively characterised at the molecular, cellular and organismal level. Since the discovery of the PHDs, a range of less well-characterised non-HIF substrates have been reported with the potential to confer oxygen-sensitivity to a diverse range of cellular processes. We sought to systematically compare all of the reported non-HIF substrates for their ability to support PHD-catalysed hydroxylation. We performed a comprehensive series of in vitro hydroxylation assays reacting synthetic peptides and full-length protein substrates generated by in vitro transcription and translation with purified recombinant enzyme preparations. Prolyl hydroxylation was assayed directly by mass spectrometry and radiochemical assay for hydroxyproline. Both methods enabled quantitative appraisal of enzyme-catalysed hydroxylation, with liquid chromatography mass spectrometry methods employing NMR-quantified peptide standards to calibrate retention time signatures and determine ionisation efficiencies for each target peptide. Using these approaches we assayed hydroxylation on 23 different proteins. Surprisingly, we did not detect measurable hydroxylation on any of the reported non-HIF substrates using either method. In contrast, control assays with HIF1-alpha substrate supported high stoichiometry (typically >90%) hydroxylation. Our findings suggest that PHD substrates are more restricted than has been reported.
Project description:Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. The oxygen-sensitive Hypoxia Inducible Factor (HIF) transcriptional regulators HIF-1α and HIF-2α are overexpressed in many human NSCLCs, and constitutive HIF-2α activity can promote murine lung tumor progression, suggesting that HIF proteins may be effective NSCLC therapeutic targets. To investigate the consequences of inhibiting HIF activity in lung cancers, we deleted Hif-1α or Hif-2α in an established KrasG12D-driven murine NSCLC model. Deletion of Hif-1α had no obvious effect on tumor growth, whereas Hif-2α deletion resulted in an unexpected increase in tumor burden that correlated with reduced expression of the candidate tumor suppressor gene Scgb3a1 (HIN-1). Here, we identify Scgb3a1 as a direct HIF-2α target gene, and demonstrate that HIF-2α regulates Scgb3a1 expression and tumor formation in human KrasG12D-driven NSCLC cells. AKT pathway activity, reported to be repressed by Scgb3a1, was enhanced in HIF-2α deficient human NSCLC cells and xenografts. Finally, a direct correlation between HIF-2α and SCGB3a1 expression was observed in approximately 70% of human NSCLC samples analyzed. These data suggest that whereas HIF-2α overexpression can contribute to NSCLC progression, therapeutic inhibition of HIF-2α below a critical threshold may paradoxically promote tumor growth by reducing expression of tumor suppressor genes, including Scgb3a1.
Project description:Disability or death secondary to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron and generation of oxidative stress. Iron chelators bind free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms remain unclear. Here we show that the hypoxia-inducible factor prolylhydroxylase (HIF PHD) family of iron-dependent oxygen sensors is an effector of iron chelation in abrogating ICH-induced death. Molecular reduction of the three HIF PHD isoforms in mouse striatum improves functional recovery following ICH. A low molecular weight, hydroxyquinoline inhibitor of the HIF PHDs, which we call adaptaquin, reduces neuronal death and behavioral deficits following ICH in distinct rodent models. Unexpectedly, adaptaquin protects from oxidative death by suppressing ATF4- dependent prodeath gene expression rather than by activating a HIF-dependent prosurvival pathway. Adaptaquin treated neurons
Project description:To investigate the detailed molecular mechanisms for the regulatory role of HIF-2α in experimental colitis, microarray gene expression analysis was performed on colon RNA isolated from 6- to 8-week-old Hif-2αF/F, Hif-2αlΔIE mice treated with 3%DSS for 3 days. Background & Aims: Hypoxic inflammation is characterized by decreased oxygen tension in inflammatory foci, and is a notable feature in inflammatory bowel disease (IBD). Hypoxic response is mediated by transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α, both of which are highly induced in IBD. HIF-1α is a protective factor that limits intestinal barrier dysfunction during inflammation. However, the role of HIF-2α has not been assessed in hypoxic inflammation and IBD. Methods: A hypoxia reporter mouse model was used to test the presence of hypoxia and HIF-2α in dextran sulfate sodium (DSS) and Citrobacter rodentium (C.rod)-induced colitis. The role of HIF-2α in these mouse models of colitis was further assessed in mice with an intestinal epithelium-specific gain- and loss-of-function of HIF-2α. Results: Induction of hypoxia and HIF-2α was confirmed in both murine experimental colitis models and human IBD samples. Disruption of HIF-2α attenuated colonic inflammation whereas stabilization of HIF-2α potentiated inflammation in mouse models of colitis. Interestingly, intestine specific overexpression of HIF-2α but not HIF-1α leads to spontaneous colitis and premature death in mice. Further mechanistic analysis showed that HIF-2α is a driver for pro-inflammatory response and is critical regulator of intestinal epithelial-derived tumor necrosis factor (TNF)-α. Blocking TNF-α completely ameliorated HIF-2α potentiated intestinal inflammation. Conclusions: These data demonstrate that HIF-2α is a critical transcription factor essential in intestinal epithelium elicited inflammatory response.
Project description:Mutational inactivation of VHL is the earliest genetic event in the majority of ccRCCs, leading to activation of the HIF-1α and HIF-2α transcription factors. While correlative studies of human ccRCCs and functional studies using human ccRCC cell lines have implicated HIF-1α as an inhibitor and HIF-2α as a promoter of aggressive tumour behaviours, their roles in tumour onset have not been functionally addressed. Using an autochthonous ccRCC model, we show genetically that Hif1a is necessary for tumour formation whereas Hif2a deletion has only minor effects on tumour initiation and growth. Both HIF-1α and HIF-2α are necessary for the clear cell phenotype. Transcriptomic and proteomic analyses revealed that HIF-1α regulates glycolysis while HIF-2α regulates genes associated with lipoprotein metabolism, ribosome biogenesis and E2F and MYC transcriptional activities. Deficiency of HIF-2α increased CD8+ T cell infiltration and activation. These studies reveal different functions of HIF-1α and HIF-2α in ccRCC. SIGNIFICANCE The roles of HIF-1α and HIF-2α in ccRCC pathogenesis remain unclear. Using a mouse genetic approach we show that HIF-1α but not HIF-2α is important for tumour formation, contrary to predictions from studies of human ccRCC. We show that HIF-1α and HIF-2α transcriptionally regulate different aspects of metabolism and identify HIF-2α as a suppressor of immune cell infiltration and activation.
Project description:Disability or death secondary to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron and generation of oxidative stress. Iron chelators bind free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms remain unclear. Here we show that the hypoxia-inducible factor prolylhydroxylase (HIF PHD) family of iron-dependent oxygen sensors is an effector of iron chelation in abrogating ICH-induced death. Molecular reduction of the three HIF PHD isoforms in mouse striatum improves functional recovery following ICH. A low molecular weight, hydroxyquinoline inhibitor of the HIF PHDs, which we call adaptaquin, reduces neuronal death and behavioral deficits following ICH in distinct rodent models. Unexpectedly, adaptaquin protects from oxidative death by suppressing ATF4- dependent prodeath gene expression rather than by activating a HIF-dependent prosurvival pathway.
Project description:Hypoxia inducible factors (HIFs) are critical to the development and homeostasis of the intervertebral disc. While HIF-2α, one of the main HIF-α isoforms is expressed in nucleus pulposus (NP) cells, its functions remain unknown. We used microarrays to explore the transcriptomics of differentially expressed genes between control and HIF-2α cKO NP cells in 6M and 14M mice.