Author Manuscript Published OnlineFirst on August 23, 2018; DOI: 10.1158/2159-8290.CD-17-1203 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Title: Pathobiologic Pseudohypoxia as a Putative Mechanism Underlying Myelodysplastic Syndromes 2 3 Running title: Activation of HIF1A Signaling by Pseudohypoxia in MDS 4 5 Yoshihiro Hayashi1,16*, Yue Zhang2,17*, Asumi Yokota1*, Xiaomei Yan1, Jinqin Liu2, Kwangmin Choi1, 6 Bing Li2, Goro Sashida3, Yanyan Peng4, Zefeng Xu2, Rui Huang1, Lulu Zhang1, George M. Freudiger1, 7 Jingya Wang2, Yunzhu Dong1, Yile Zhou1, Jieyu Wang1, Lingyun Wu1,5, Jiachen Bu1,6, Aili Chen6, 8 Xinghui Zhao1, Xiujuan Sun2, Kashish Chetal7, Andre Olsson8, Miki Watanabe1, Lindsey E. Romick- 9 Rosendale1, Hironori Harada9, Lee-Yung Shih10, William Tse11, James P. Bridges12, Michael A. 10 Caligiuri13, Taosheng Huang4, Yi Zheng1, David P. Witte1, Qian-fei Wang6, Cheng-Kui Qu14, Nathan 11 Salomonis7, H. Leighton Grimes1,8, Stephen D. Nimer15, Zhijian Xiao2,18, and Gang Huang1,2,18 12 13 1 Divisions of Pathology and Experimental Hematology and Cancer Biology, Cincinnati Children’s 14 Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA 15 2 State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases 16 Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, 17 China 18 3 International Research Center for Medical Sciences, Kumamoto University, 2-2-1 Honjo, Chuo-ku, 19 Kumamoto 860-0811, Japan 20 4 Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, 21 Cincinnati, OH 45229, USA 22 5 Department of Hematology, Sixth Hospital Affiliated to Shanghai Jiaotong University, Shanghai 23 200233, China 1 Downloaded from cancerdiscovery.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2018; DOI: 10.1158/2159-8290.CD-17-1203 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 24 6 Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and 25 Development, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China. 26 7 Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, 3333 Burnet 27 Avenue, Cincinnati, Ohio 45229, USA 28 8 Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, 29 Cincinnati, Ohio 45229, USA 30 9 Laboratory of Oncology, School of Life Science, Tokyo University of Pharmacy and Life Sciences, 31 Tokyo 192-0392, Japan 32 10 Department of Hematology and Oncology, Chang Gung Memorial Hospital-Linkou and Chang Gung 33 University College of Medicine, Taoyuan, Taiwan 34 11 James Graham Brown Cancer Center, University of Louisville Hospital, Louisville, Kentucky 40202, 35 USA 36 12 Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, 37 Cincinnati, Ohio 45229, USA 38 13 The Ohio State University Comprehensive Cancer Center, Columbus, Ohio 43201, USA 39 14 Division of Hematology/Oncology, Aflac Cancer and Blood Disorders Center, Emory University 40 School of Medicine, Atlanta, GA 30322, USA 41 15 Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL 33136, USA 42 16 Present address: Laboratory of Oncology, School of Life Science, Tokyo University of Pharmacy and 43 Life Sciences, Tokyo 192-0392, Japan 44 17 Present address: Henan University of Chinese Medicine, Henan 450046, China 45 18 These authors jointly supervised this work. 46 * These authors contributed equally to this work. 2 Downloaded from cancerdiscovery.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2018; DOI: 10.1158/2159-8290.CD-17-1203 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 47 48 The authors declare no potential conflicts of interest. 49 50 CORRESPONDING AUTHOR: 51 Correspondence and requests for materials should be addressed to ZJ.X. ([email protected]) or G.H. 52 ([email protected]). 53 54 LEAD CONTACT : Gang Huang, Ph.D. 55 Divisions of Pathology and Experimental Hematology and Cancer Biology, 56 Cincinnati Children's Hospital Medical Center, 57 3333 Burnet Avenue, Room S7.607, Cincinnati, Ohio 45229-3039, USA 58 Phone: (513) 636-3214, Fax: (513) 636-3768, Email: [email protected] 59 60 61 ABSTRACT: 62 Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic disorders that are incurable with 63 conventional therapy. The incidence is increasing with global population ageing. Although many genetic, 64 epigenetic, splicing, and metabolic aberrations have been identified in MDS patients, their clinical 65 features are quite similar. Here we show that hypoxia-independent activation of hypoxia-inducible factor 66 1α (HIF1A) signaling is both necessary and sufficient to induce dysplastic and cytopenic MDS 67 phenotypes. The HIF1A transcriptional signature is generally activated in MDS-patient bone-marrow 68 stem/progenitors. Major MDS-associated mutations (Dnmt3a, Tet2, Asxl1, Runx1, and Mll1) activate the 69 HIF1A signature. While inducible activation of HIF1A signaling in hematopoietic cells is sufficient to 3 Downloaded from cancerdiscovery.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2018; DOI: 10.1158/2159-8290.CD-17-1203 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 70 induce MDS phenotypes, both genetic and chemical inhibition of HIF1A signaling rescues MDS 71 phenotypes in a mouse model of MDS. These findings reveal HIF1A as a central pathobiologic mediator 72 of MDS, and as an effective therapeutic target for a broad spectrum of MDS patients. 73 74 SIGNIFICANCE: 75 We showed that dysregulation of HIF1A signaling could generate the clinically-relevant diversity of 76 MDS phenotypes by functioning as a signaling funnel for MDS driver-mutations. This could resolve the 77 disconnection between genotypes and phenotypes, and provide a new clue as to how a variety of driver- 78 mutations cause common MDS phenotypes. 79 80 INTRODUCTION: 81 MDS are a group of heterogeneous clonal disorders which are characterized by ineffective 82 hematopoiesis and uni- or multi-lineage dysplasia (1,2). Because of its diversity and complexity, the 83 pathogenesis of MDS remains to be elucidated. Limited preclinical models are available for dissecting 84 the pathogenesis and testing new drugs, and each model has its limitation (3,4). Stem cell transplantation 85 is a curative strategy for MDS, however, few patients are eligible for transplantation. Further elucidation 86 of the pathogenesis of MDS and development of novel therapeutic strategies are needed. MDS are 87 associated with mutations in chromatin-modifying enzymes, splicing factors, transcription factors, 88 cohesin complex, and metabolic enzymes that regulate hematopoietic stem cell (HSC) self-renewal, 89 survival, and differentiation. Cooperating genetic lesions occur, involving signaling molecules that 90 regulate cell growth and proliferation (1,2,5). Although a number of heterogeneous genomic aberrations 91 have been identified in MDS patients (6,7), their key clinical phenotypes are similar. Therefore, we 92 hypothesized that driver mutations activate common underlying mechanisms involved in MDS 4 Downloaded from cancerdiscovery.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2018; DOI: 10.1158/2159-8290.CD-17-1203 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 93 phenotypes. Identification of these key mediators would reveal fundamental insights into MDS 94 pathogenesis, and present novel opportunities for therapeutic intervention beyond specific mutations or 95 aberrance for MDS. 96 Hypoxia inducible factor-1α (HIF1A) is a critical transcription factor for the hypoxic response, 97 angiogenesis, normal HSC regulation, and cancer development (8,9). Importantly, HIF1A is also 98 essential for the activation of innate and adaptive immunity (10). HIF1A is regulated by both oxygen- 99 dependent and oxygen-independent ways (11). HSCs and progenitor cells (HSPCs) isolated from MDS 100 patients display abnormal self-renewal and differentiation, and accumulating clinical and research 101 evidence suggests an important role for systemic inflammation and immune activation in MDS 102 pathogenesis (12). Thus, we tested the impact of HIF1A signaling in MDS. 103 104 RESULTS: 105 Activated HIF1A Pathway in the Broad Spectrum of MDS Patients 106 HIF1A is mainly regulated at translational and protein levels. Thus, analyzing downstream HIF1A- 107 signature-gene expression is a reliable approach to measure HIF1A activation. To determine whether 108 MDS patients have an activated HIF1A gene signature, we analyzed a published cohort of CD34+ BM 109 cells isolated from healthy donors (n = 17) or from MDS patients (n = 183) (13). This MDS cohort 110 contains patients with refractory anemia (RA) (n = 55), refractory anemia with ring sideroblasts (RARS) 111 (n = 48), refractory anemia with excess blast type 1 (RAEB1) (n = 37), or type 2 (RAEB2) (n = 43). 112