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. HIF 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 JmjC histone lysine demethylases (KDMs) 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 small-molecule HIF-P4H inhibitors to treat anaemia in patients with chronic kidney disease are currently in progress by several pharmaceutical companies, including 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 mice are protected against cardiac and skeletal muscle ischemic injury. The mechanisms involved HIF1 and HIF2 stabilization, increased capillary size but not number and improved maintenance of energy metabolism. We have also shown that the HIF-P4H-2 hypomorph mice have improved glucose and lipid metabolism and are protected against obesity and metabolic dysfunction, the same protective phenomenon being obtained with a pharmacological HIF-P4H inhibitor FG-4497 (FibroGen Inc.). Furthermore, we have shown that both pharmacologic and genetic HIF-P4H-2 inhibition protected mice from atherosclerosis development via HIF stabilization-driven changes in gene expression resulting in beneficial changes in glucose and lipid metabolism, reduced adipose tissue inflammation and increased levels of protective autoantibodies. In collaboration with scientists from the Medical Research Center Oulu and Harvard Medical School we showed that remote ischemic preconditioning in a pig model of hypothermic circulatory arrest reduced brain damage, lowered serum lactate levels, improved cardiac index and brain antioxidant response and modulated plasma proteome and metabolies.

Unlike mice with conditional broad-spectrum inactivation of 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 HIF2 stabilization-driven downregulation of Notch signaling. We also showed that these mice were protected against inflammatory anemia.

Of the HIF-P4Hs, HIF-P4H-1 in particular, has been previously shown to be involved in the regulation of inflammatory and apoptotic pathways. We have shown that HIF-P4H-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 pathways and observed decreased inflammation and increased apoptosis in a skin inflammation model in HIF-P4H-1 null mice. Furthermore, we showed that HIF-P4H-1 inactivation increases p53 activity and stability and hydroxylation of proline 142 in p53 had an important role in this regulation.

Hypoxia and loss of cell polarity are common features of cancer and increased HIF1 signaling has been shown to be associated with epithelial-mesenchymal transformation. Using a three-dimensional Madin–Darby canine kidney epithelial cell culture system we have shown in collaboration with Aki Manninen, Biocenter Oulu, that hypoxia disrupts polarized epithelial morphogenesis in a HIF1-dependent manner by modulating the TGFβ signaling pathway. We identified the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) as a novel mediator of HIF1-driven activation of TGFβ signaling and showed that similarly to HIF1 depletion, inactivation of BAMBI rescues MDCK cystogenesis during hypoxia.

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 show that the cancer-associated 2-oxoglutarate analogues also inhibit KDMs and therefore modify histone methylation. R-2HG was the most efficient inhibitor for KDMs 6A, 4A and 4B followed by S-2HG, which has been shown to accumulate under hypoxic conditions. In agreement, treatment of cells with the cell-permeable forms of these compounds accumulated histone H3K9me3 and H3K27me3 marks. Altogether, our data suggest that inhibition of KDMs by the 2-oxoglutarate analogues is involved in the disease mechanism in cancer where these compounds accumulate.

Collagen prolyl 4-hydroxylase (C-P4H) is a crucial enzyme for collagen synthesis. In collaboration with Prof. Carsten Bönnemann, National Institutes of Health, USA, we have identified a first human connective tissue disorder caused by bi-allelic P4HA1 mutations. The disease manifests as as early-onset joint hypermobility, joint contractures, muscle weakness and bone dysplasia as well as high myopia, with evidence of clinical improvement of motor function over time. The patients were compound heterozygous for frameshift and splice site mutations leading to reduced, but not absent, P4HA1 protein and total C-P4H activity, and reduced thermal stability of collagen. Using collagen with reduced hydroxylation isolated from our C-P4H mutant mice as well as synthetic collagen-like peptides we have shown in collaboration with Prof. Jyrki Heino, University of Turku, that lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via local structural destabilization of the triple helix. With Prof. Rik Wierenga, Biocenter Oulu, we have for the first time generated a three-dimensional small-angle X-ray scattering model of the entire human C-P4H isoenzyme I tetramer. The data showed that C-P4H-I is an elongated, bilobal, symmetric βααβ molecule, where N-terminal dimerization domains from the two catalytic α subunits form a protein-protein dimer interface and a thin waist in the bilobal tetramer. The two peptide-substrate-binding/catalytic regions of the α subunits make a complex with the PDI/β subunit and form two bulky lobes pointing outward from the waist region so that the PDI/β subunits locate at the far ends of the 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 (KDMs and TETs). We will continue our studies on the roles of these enzymes in various hypoxia-associated diseases, such as metabolic syndrome and fatty liver diseases, inflammation and tumor formation and metastasis.

International publications 2017-

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 474:751-769, 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.

Sharma U*, Carrique L*, Vadon-Le Goff S*, Mariano N, Georges RN, Delolme F, Koivunen P, Myllyharju J, Moali C, Aghajari N#, Hulmes DJS#: Why collagen I is normally a heterotrimer. Nat Commun 8: 14671, 2017. * and #, Equal contributions.

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 5: 11-20, 2017.

Herajärvi J, Anttila T, Dimova EY, Laukka T, Myllymäki M, Haapanen H, Olenchock BA, Tuominen H, Puistola U, Karihtala P, Kiviluoma K, Koivunen P, Anttila V, Juvonen T. Exploring effects of remote ischemic preconditioning in a pig model of hypothermic circulatory arrest. Scand Cardiovasc J 51:233-241, 2017.

Honkanen T, Wilenius E, Koivunen P, Koivunen JP. HER2 regulates cancer stem-like cell phenotype in ALK translocated NSCLC. Int J Oncol 51:599-606, 2017.

Zou Y*, Donkervoort S*, Salo AM*, Barnes AM,  Hu Y, Reghan Foley A, Makareeva E, Leach ME, Dastgir J, Cohn RD, DiNonno W, Leikin S, Marini JC, Myllyharju J & Bönnemann CG. P4HA1 mutations cause a unique congenital disorder of connective tissue involving tendon, bone, muscle and the eye. Hum Mol Genet 26: 2207-2217, 2017. *Equal contribution.

Rachman-Tzemah C, Zaffryar-Eilot S, Grossman M, Ribero D, Timaner M, Mäki JM, Myllyharju J, Bertolini F, Hershkovitz D, Sagi I, Hasson P* & Shaked Y*. Blocking surgically-induced lysyl oxidase activity reduces risk of lung metastases. Cell Rep 19:774-784, 2017. *Equal contribution.

Ullah K, Rosendahl A-H, Izzi V, Bergmann U, Pihlajaniemi T, Mäki JM & Myllyharju J. Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways. ptosis via NF-κB and p53 signaling pathways. Sci Rep 7:17220, 2017.

Koivunen P, Myllyharju J. Kinetic analysis of HIF prolyl hydroxylases. Methods Mol Biol 1742:15-25, 2018.

Koivunen P, Laukka T. The TET enzymes. Cell Mol Life Sci 2018, in press. doi: 10.1007/s00018-017-2721-8.

Richter K, Paakkola T, Mennerich D, Kubaichuk K, Konzack A, Kippari HA, Kozlova N, Koivunen P, Haapasaari KM, Jukkola-Vuorinen A, Teppo HR, Dimova EY, Bloigu R, Szabo Z, Kerkelä R & Kietzmann T. USP28 deficiency promotes breast and liver carcinogenesis as well as tumor angiogenesis in a HIF-independent manner. Mol Cancer Res 2018, in press.

Sipilä KH, Drushinin K, Rappu P, Jokinen J, Salminen T, Salo A, Käpylä J, Myllyharju J & Heino J. Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms. J Biol Chem 293: 7645-7658, 2018.

Raykhel I, Moafi F, Myllymäki SM, Manninen A* & Myllyharju J*. BAMBI is a novel HIF1-dependent modulator of TGFβ-mediated disruption of cell polarity in hypoxia. J Cell Sci 131: jcs210906, 2018. *Equal contribution.

Aviram R, Zaffryar-Eilot S, Hubmacher D, Mäki JM, Myllyharju J, Apte SS & Hasson P. Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families. Matrix Biol 2018, in press.

Laukka T, Myllykoski M, Looper RE & Koivunen P. Cancer-associated 2-oxoglutarate analogues modify histone methylation by inhibiting histone lysine demethylases. J Mol Biol, in press.

Murthy AV, Sulu R, Koski K, Tu H, Ananthanarajan J, Sah-Teli SK, Myllyharju J & Wierenga RK. Crystal structures of the PSB domain of human collagen prolyl 4-hydroxylase II complexed with proline-rich peptides suggest a novel PxGP binding motif. Prot Sci 2018, in press.

Other publications 2017-

Karppinen P (2017): Lasker Award –palkinto hypoksiavastereitin selvittäjille – reitin lääkkeet edenneet kliinisissä kokeissa viimeiseen vaiheeseen. Duodecim, 133: 9.

Karppinen P (2017): Tekoäly vastaan dermatologi ihosyöpien diagnosoinnissa. Duodecim, 133: 915.

Karppinen P (2017): Liikkumisen maailmanlaajuinen epäsuhtaisuus. Duodecim, 133: 1863.

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., Academy Professor emeritus
Nadiya Byts, Ph.D. (University of Oulu and Jane & Aatos Erkko Foundation)
Elitsa Dimova, Ph.D., Adjunct Professor (Jane & Aatos Erkko Foundation)
Joni Mäki, Ph.D. (Academy of Finland)
Matti Myllykoski, Ph.D. (Biocenter Oulu and University of Oulu)
Antti Railo, Ph.D. (Academy of Finland)
Irina Raykhel, Ph.D. (Sigrid Jusélius Foundation)
Antti Salo, Ph.D. (Biocenter Oulu and University of Oulu)
Raisa Serpi, Ph.D., Adjunct professor (Academy of Finland)

Ph.D. Students:
Kati Drushinin, M.Sc. (Acadeny of Finland and FibroGen)
Anna Laitakari, M.Sc. (University of Oulu Health and Biosciences Doctoral Programme)
Tuomas Laukka, Med.Cand. (Sigrid Jusélius Foundation)
Jari Matinlauri, Med. Stud. (Sigrid Jusélius Foundation)
Fazeh Moafi, M.Sc. (Academy of Finland and Jane & Aatos Erkko Foundation)
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. (Academy of Finland and Finnish Cultural Foundation)
Tapio Röning, Med. Stud. (Sigrid Jusélius Foundation)
Subodh Sharma (University of Oulu Health and Biosciences Doctoral Programme)
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 and Jane & Aatos Erkko Foundation)

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

Main source of salary in brackets.

Foreign Scientists, 6

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 2017
Kari I. Kivirikko, M.D., Ph.D.: FibroGen Inc., San Francisco, CA, USA

Co-operation With Finnish and Foreign Companies
FibroGen Inc., San Francisco, CA, USA
FibroGen Europe, Helsinki, Finland

Last updated: 15.10.2018