Hypoxia Response Pathway as a Target for Novel Therapeutics

Project Leaders
Prof. Johanna Myllyharju, Ph.D., Scientific Director of Biocenter Oulu
Prof. Peppi Karppinen (née) Koivunen, M.D., Ph.D.

Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine

Background and Significance

Oxygen homeostasis is essential for normal development and physiology, many pathological processes being associated with hypoxia. Cells respond to hypoxia by stabilization of the hypoxia-inducible factor (HIF) that induces ~300 genes involved in e.g. hematopoesis, angiogenesis, iron metabolism, glucose utilization, glycolysis, lipid metabolism, inflammation, tumorigenesis and metastasis, and extracellular matrix (ECM) homeostasis (Fig. 1). HIF is an αβ dimer that accumulates in hypoxic cells but is rapidly degraded in normoxia. The stability of HIF is regulated by oxygen and 2-oxoglutarate-dependent prolyl 4-hydroxylases (HIF-P4Hs, also known as PHDs and EGLNs). A transmembrane P4H (P4H-TM) identified by us also acts on HIF, but may have additional substrates and thus novel biological functions. Furthermore, key enzymes involved in collagen synthesis and thus extracellular matrix homeostasis and several chromatin modifying and cancer-associated histone demethylases (HDMs) and the DNA methyl cytosine hydroxylases TETs belong to the same enzyme family with HIF-P4Hs and require common cofactors. Our objective is to understand the roles of these enzymes in disease processes and normal development in detail. Such information on the specific mechanistic and in vivo roles of these enzymes will have a direct translational impact in drug development for pathological conditions associated with hypoxia. Phase 3 clinical trials on the use of a small-molecule HIF-P4H inhibitor to treat anaemia in patients with chronic kidney disease are currently in progress by our collaborator FibroGen Inc. We expect breakthroughs in understanding how the key regulators of the hypoxia response pathway are involved in normal physiological cellular and developmental processes and whether they have therapeutic value in selected common pathological conditions that in addition to individual suffering cause a huge burden to the healthcare system.

Recent Progress

HIF-P4H-2 is the major regulator of HIF. We have previously shown that HIF-P4H-2 hypomorph (Hif-p4h-2gt/gt) mice are protected against cardiac ischemia-reperfusion and infarct injuries. We have now shown that they are also protected against skeletal muscle ischemia-reperfusion injury. The mechanisms involved were mediated via normoxic HIF1a and HIF2a stabilization, increased capillary size but not number in the muscle and improved maintenance of skeletal energy metabolism during ischemia-reperfusion. We have previously shown that the HIF-P4H-2 hypomorph mice have improved glucose and lipid metabolism and are protected against obesity and metabolic dysfunction. Importantly regarding translational aspects, when wild-type (WT) mice were treated with a pharmacological HIF-P4H inhibitor FG-4497 (FibroGen Inc.), the same obesity and metabolic syndrome-opposing phenotype was observed. Furthermore, we have now studied the effect of pharmacologic and genetic HIF-P4H-2 inhibition on the development of atherosclerosis. Treatment of LDL receptor-deficient mice (Lldr-/-) fed with high fat diet with FG-4497 protected them from atherosclerosis development. The mechanisms involved were HIF stabilization-driven changes in gene expression leading to beneficial modifications in glucose and lipid metabolism, reduced adipose tissue inflammation and increased levels of protective autoantibodies.

Unlike mice with conditional broad-spectrum inactivation HIF-P4H-2, the hypomorph HIF-P4H-2 mice do not develop massive erythrocytosis and have no reduction in their life span. However, we have identified extramedullary hematopoiesis in the spleen of the hypomorph mice upon aging resulting in mild erythrocytosis. This was mediated via HIF2a stabilization-driven downregulation of Notch signaling. We also showed that these mice were protected against inflammatory anemia. In addition, we have shown that lack of P4H-TM in mouse results in age-related renal and retinal alterations, the latter with certain characteristics of age-related macular degeneration.

Identification of mutations in the Krebs cycle enzymes succinate dehydrogenase and fumarate hydratase, and in the mitochondrial isocitrate dehydrogenase 2 (IDH2) and its cytosolic isoenzyme IDH1 in human cancers has rekindled the idea that altered cellular metabolism can transform cells. We and others have previously shown that the 2-oxoglutarate-analogues succinate, fumarate and (R)2-hydroxyglutarate (R-2HG), which accumulate in these mutations, respectively, signal at least partly via HIF-P4Hs and HIF. Our recent data in collaboration with Prof. William Kaelin Jr., Dana- Farber Cancer Institute, identified fumarate and succinate also as potent TET inhibitors, and demonstrated that they down-regulated global genomic 5-hydroxymethylcytosine levels and affected gene expression. We have also shown recently that HIF1a accumulation in triple-negative breast cancers is caused by self-inactivation of HIF-P4H-2 (EglN1) in the absence of cysteine. This is caused by excessive secretion of glutamate by these tumours, which blocks the intake of cystine and results in depletion of intracellular cysteine. Cysteine, or vitamin C, were required to prevent auto-oxidation of specific intramolecular cysteine residues in HIF-P4H-2, indicating that in addition to O2 the enzyme can sense cysteine levels.

Collagen prolyl 4-hydroxylase (C-P4H) is a crucial enzyme for collagen synthesis. It is an α2β2 tetramer, where the α subunits are the catalytic subunits and protein disulfide isomerase (PDI) serves as the β subunit. In collaboration with Prof. Rik Wierenga, Biocenter Oulu, we have previously shown that the α subunit is composed of three functional domains, an N-terminal dimerization domain (DD), a central peptide-substrate-binding domain (PSB) and a C-terminal catalytic domain (CAT). In addition, we have solved the crystal structures of the human C-P4H α subunit DD and PSB domains and of an algal P4H resembling the CAT domain. However, the structure of the C-P4H tetramer has remained elusive and we have now presented for the first time a three-dimensional small-angle X-ray scattering model of the entire human C-P4H isoenzyme I tetramer. C-P4H-I is an elongated, bilobal, symmetric molecule, where the dimerization domains from the two α subunits form a protein-protein dimer interface, that forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β subunits locate at the far ends of the βααβ complex.

Future Goals

Our main interest is targeted to the 2-oxoglutarate dependent dioxygenases that regulate the hypoxia response (HIF-P4Hs and P4H-TM) and extracellular matrix homeostasis (C-P4Hs) and structure of the chromatin (HDMs and TETs). We will continue our studies on the roles of these enzymes in hypoxia-associated diseases, such as cardiac and skeletal ischemia and stroke, atherosclerosis, metabolic syndrome and fatty liver diseases, inflammation and tumor formation and metastasis.

Publications 2016-

Briggs KJ, Koivunen P, Cao S, Backus KM, Olenchock BA, Patel H, Zhang Q, Signoretti S, Gerfen GJ, Richardson AL, Witkiewicz AK, Cravatt BF, Clardy J, Kaelin WG Jr. Paracrine induction of HIF by glutamate in triple-negative breast cancer:  EglN1 senses cysteine. Cell 166:126-139, 2016.

Halt K, Pärssinen H, Junttila S, Saarela U, Sims-Lucas S, Koivunen P, Myllyharju J, Quaggin S, Skovorodkin I, Vainio SJ. CD146+ cells are essential for kidney vasculature development. Kidney Int 90: 311-324, 2016.

Karsikas S, Myllymäki M, Heikkilä M, Sormunen R, Kivirikko KI, Myllyharju J, Serpi R, Koivunen P. HIF-P4H-2 deficiency protects against skeletal muscle ischemia-reperfusion injury. J Mol Med 94:301-310, 2016.

Koivunen P, Fell SM, Lu W, Rabinowitz JD, Kung AL, Schlisio S. The 2-oxoglutarate analog 3-oxoglutarate decreases normoxic hypoxia-inducible factor-1α in cancer cells, induces cell death, and reduces tumor xenograft growth. Hypoxia 4: 15-27, 2016.

Koivunen P, Serpi R, Dimova EY. Hypoxia-inducible factor prolyl 4-hydroxylase inhibition in cardiometabolic diseases. Pharmacol Res 114:265-273, 2016.

Laukka T, Mariani CJ, Ihantola T, Cao JZ, Kaelin WG Jr, Godley LA, Koivunen P. Fumarate and succinate regulate expression of hypoxia-inducible genes via TETs. J Biol Chem 291: 4256-4265, 2016.

Leinonen H*, Rossi M*, Salo AM, Tiainen P, Hyvärinen J, Sormunen R, Miinalainen I, Zhang C, Soininen R, Kivirikko KI, Pitkänen M, Koskelainen A, Tanila H, Myllyharju J#, Koivunen P#. Lack of P4H-TM results in age-related retinal and renal alterations in mice. Hum Mol Genet 25: 3810-3823, 2016. * and # Equal contributions.

Pietilä I, Prunskaite-Hyyryläinen R, Kaisto S, Nicolaou N, van Eerde AM, Salo AM, Garma L, Miinalainen I, Feitz WF, Bongers EMHF, Juffer A, Knoers NVAM, Renkema KY, Myllyharju J, Vainio SJ. Wnt5a deficiency leads to anomalies in ureteric tree development, tubular epithelial cell organization and basement membrane integrity pointing to a role in kidney collecting duct patterning. PLOS One 11:e0147171, 2016.

Rahtu-Korpela L, Määttä J, Dimova EY, Hörkkö S, Gylling H, Walkinshaw G, Hakkola J, Kivirikko KI, Myllyharju J, Serpi R, Koivunen P. Hypoxia-inducible factor prolyl 4-hydroxylase-2 inhibition protects against development of atherosclerosis. Arterioscler Thromb Vasc Biol 36: 608-617, 2016.

Koski MK, Anantharajan J, Kursula P, Dhavala P, Murthy, AV, Bergmann U, Myllyharju J, Wierenga RK. The elongated collagen prolyl 4-hydroxylase α2β2 tetramer is assembled around a central central α2 dimer. Biochem J 474(5):751-769, 2017.

Myllymäki MNM, Määttä J, Dimova EY, Izzi V, Väisänen T, Myllyharju J, Koivunen P*, Serpi R*. Notch downregulation and extramedullary erythrocytosis in HIF prolyl 4-hydroxylase-2-deficient mice. Mol Cell Biol 37: e00529-16, 2017. * Equal contribution.

Ratcliffe P, Koivunen P, Myllyharju J, Ragoussis J, Bovée J, Batinic-Haberle I, Vinatier C, Trichet V, Robriquet F, Oliver L, Gardie B. Update on hypoxia-inducible factors and hydroxylases in oxygen regulatory pathways: from physiology to therapeutics. Hypoxia 2017, in press.

Doctoral Theses 2016

Mikko Myllymäki: Hypoxia-inducible factor prolyl 4-hydroxylase-2 in Tibetan high-altitude adaptation, extramedullary erythropoiesis and skeletal muscle ischemia. Acta Universitatis Ouluensis, Series D, Medica 1366, ISBN 978-952-62-1220-3

Research Group Members

Project Leaders:
Johanna Myllyharju, Ph.D., Professor (University of Oulu and Biocenter Oulu)
Peppi Karppinen (née Koivunen), M.D., Ph.D., Professor (University of Oulu)

Senior and Post-doctoral Investigators:
Kari I. Kivirikko, M.D., Ph.D., Professor (Academy of Finland, emeritus)
Nadiya Byts, Ph.D. (Academy of Finland)
Elitza Dimova, Ph.D. (Jane & Aatos Erkko Foundation)
Joni Mäki, Ph.D. (Academy of Finland)
Antti Railo, Ph.D. (Academy of Finland)
Irina Raykhel, Ph.D. (Biocenter Oulu and Academy of Finland)
Antti Salo, Ph.D. (Biocenter Oulu and Jane & Aatos Erkko Foundation)
Raisa Serpi, Ph.D., Adjunct professor (Academy of Finland)

Ph.D. Students:
Kati Drushinin, M.Sc. (Biocenter Oulu Doctoral Programme)
Anna Laitakari, M.Sc. (Biocenter Oulu Doctoral Programme)
Tuomas Laukka, Med.Cand. (Sigrid Jusélius Foundation)
Jari Matinlauri, Med. Stud. (Sigrid Jusélius Foundation
Fazeh Moafi, M.Sc. (Biocenter Oulu Doctoral Programme)
Jenni Määttä, Med.Cand. (Sigrid Jusélius Foundation)
Teemu Ollonen, Med. Cand. (Sigrid Jusélius Foundation)
Mia Raasakka, Med.Cand. (Sigrid Jusélius Foundation)
Ann-Helen Rosendahl, M.D. (Sigrid Jusélius Foundation)
Niina Sissala, B.Sc., Med. Stud. (Sigrid Jusélius Foundation)
Joona Tapio, B.Sc., Med. Stud. (Sigrid Jusélius Foundation)
Jussi-Pekka Tolonen, Med.Cand. (Sigrid Jusélius Foundation)
Karim Ullah, M.Sc. (Academy of Finland)

Laboratory Technicians, 5 (University of Oulu, Academy of Finland, Jane & Aatos Erkko Foundation and FibroGen Inc.)

Main source of salary in brackets.

Foreign Scientists, 5

National and International Activities

Centre of Excellence in Cell-Extracellular Matrix Research, Academy of Finland Program for 2012–2017

Taina Pihlajaniemi, Director, Johanna Myllyharju, Vice director, other Group leaders Lauri Eklund, Aki Manninen, Seppo Vainio and Robert Winqvist

Group Members Who Spent More Than Two Weeks in Foreign Laboratories During 2016

Kari I. Kivirikko, M.D., Ph.D.: FibroGen Inc., San Francisco, CA, USA
Jussi-Pekka Tolonen, Med. Cand., Ann Arbor, Michigan, USA

Co-operation With Finnish and Foreign Companies

FibroGen Inc., San Francisco, CA, USA
FibroGen Europe, Helsinki, Finland

Last updated: 25/4/2017