Published OnlineFirst August 23, 2018; DOI: 10.1158/2159-8290.CD-17-1203 RESEARCH ARTICLE Pathobiological Pseudohypoxia as a Putative Mechanism Underlying Myelodysplastic Syndromes Yoshihiro Hayashi 1 , Yue Zhang 2 , Asumi Yokota 1 , Xiaomei Yan 1 , Jinqin Liu 2 , Kwangmin Choi 1 , Bing Li 2 , Goro Sashida3 , Yanyan Peng 4 , Zefeng Xu 2 , Rui Huang 1 , Lulu Zhang 1 , George M. Freudiger 1 , Jingya Wang 2 , Yunzhu Dong1 , Yile Zhou 1 , Jieyu Wang 1 , Lingyun Wu 1 , 5 , Jiachen Bu 1 , 6 , Aili Chen 6 , Xinghui Zhao 1 , Xiujuan Sun 2 , Kashish Chetal7 , Andre Olsson 8 , Miki Watanabe 1 , Lindsey E. Romick-Rosendale 1 , Hironori Harada 9 , Lee-Yung Shih10 , William Tse 11 , James P. Bridges 12 , Michael A. Caligiuri 13 , Taosheng Huang 4 , Yi Zheng1 , David P. Witte 1 , Qian-fei Wang 6 , Cheng-Kui Qu 14 , Nathan Salomonis 7 , H. Leighton Grimes 1 , 8 , Stephen D. Nimer15 , Zhijian Xiao 2 , and Gang Huang 1 , 2 ABSTRACT Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic disorders that are incurable with conventional therapy. Their incidence is increasing with global population aging. Although many genetic, epigenetic, splicing, and metabolic aberrations have been identifi ed in patients with MDS, their clinical features are quite similar. Here, we show that hypoxia-independent activation of hypoxia-inducible factor 1α (HIF1A) signaling is both necessary and suffi cient to induce dysplastic and cytopenic MDS phenotypes. The HIF1A transcriptional signature is generally activated in MDS patient bone marrow stem/progenitors. Major MDS-associated mutations (Dnmt3a, Tet2, Asxl1, Runx1, and Mll1 ) activate the HIF1A signature. Although inducible activation of HIF1A signaling in hematopoietic cells is suffi cient to induce MDS phenotypes, both genetic and chemi- cal inhibition of HIF1A signaling rescues MDS phenotypes in a mouse model of MDS. These fi ndings reveal HIF1A as a central pathobiologic mediator of MDS and as an effective therapeutic target for a broad spectrum of patients with MDS. SIGNIFICANCE: We showed that dysregulation of HIF1A signaling could generate the clinically relevant diversity of MDS phenotypes by functioning as a signaling funnel for MDS driver mutations. This could resolve the disconnection between genotypes and phenotypes and provide a new clue as to how a vari- ety of driver mutations cause common MDS phenotypes. Cancer Discov; 8(11); 1438–57. ©2018 AACR. See related commentary by Chen and Steidl, p. 1355. 13 1 Divisions of Pathology and Experimental Hematology and Cancer Biol- Ohio. The Ohio State University Comprehensive Cancer Center, Columbus, 14 ogy, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio. 2 State Ohio. Division of Hematology/Oncology, Afl ac Cancer and Blood Disorders 15 Key Laboratory of Experimental Hematology, Institute of Hematology Center, Emory University School of Medicine, Atlanta, Georgia. Sylvester and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Comprehensive Cancer Center, University of Miami, Miami, Florida. Peking Union Medical College, Tianjin, China. 3 International Research Note: Supplementary data for this article are available at Cancer Discovery Center for Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Online (http://cancerdiscovery.aacrjournals.org/). Japan. 4 Division of Human Genetics, Cincinnati Children’s Hospital Medi- Current address for Y. Hayashi: Laboratory of Oncology, School of Life 5 cal Center, Cincinnati, Ohio. Department of Hematology, Sixth Hospital Science, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan; 6 Affi liated to Shanghai Jiaotong University, Shanghai, China. Key Labora- and current address for Y. Zhang: Henan University of Chinese Medicine, tory of Genomic and Precision Medicine, Collaborative Innovation Center Henan, China. of Genetics and Development, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China. 7 Division of Biomedical Informatics, Z. Xiao and G. Huang jointly supervised this work. Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio. 8 Division of Y. Hayashi, Y. Zhang, and A. Yokota contributed equally to this article. Immunobiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Corresponding Authors: Gang Huang, Cincinnati Children’s Hospital Medi- 9 Ohio. Laboratory of Oncology, School of Life Science, Tokyo University of cal Center, 3333 Burnet Avenue, Room S7.607, Cincinnati, OH 45229. 10 Pharmacy and Life Sciences, Tokyo, Japan. Department of Hematology and Phone: 513-636-3214; E-mail: [email protected] ; and Zhijian Xiao, Oncology, Chang Gung Memorial Hospital-Linkou and Chang Gung University [email protected] College of Medicine, Taoyuan, Taiwan. 11 James Graham Brown Cancer Center, University of Louisville Hospital, Louisville, Kentucky. 12 Division of Pul- doi: 10.1158/2159-8290.CD-17-1203 monary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, © 2018 American Association for Cancer Research. 1438 | CANCER DISCOVERY NOVEMBER 2018 www.aacrjournals.org Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 23, 2018; DOI: 10.1158/2159-8290.CD-17-1203 Activation of HIF1A Signaling by Pseudohypoxia in MDS RESEARCH ARTICLE INTRODUCTION reveal fundamental insights into MDS pathogenesis and pre- sent novel opportunities for therapeutic intervention beyond Myelodysplastic syndromes (MDS) are a group of hetero- specific mutations for MDS. geneous clonal disorders that are characterized by ineffective Hypoxia-inducible factor-1α (HIF1A) is a critical transcrip- hematopoiesis and unilineage or multilineage dysplasia (1, 2). tion factor for the hypoxic response, angiogenesis, normal Because of its diversity and complexity, the pathogenesis of HSC regulation, and cancer development (8, 9). Importantly, MDS remains to be elucidated. Limited preclinical models HIF1A is also essential for the activation of innate and are available for dissecting the pathogenesis and testing new adaptive immunity (10). HIF1A is regulated by both oxygen- drugs, and each model has its limitations (3, 4). Stem-cell dependent and oxygen-independent mechanisms (11). HSCs transplantation is a curative strategy for MDS; however, few and progenitor cells (HSPC) isolated from patients with MDS patients are eligible for transplantation. Further elucida- display abnormal self-renewal and differentiation, and accu- tion of the pathogenesis of MDS and development of novel mulating clinical and research evidence suggests an impor- therapeutic strategies are needed. MDS are associated with tant role for systemic inflammation and immune activation mutations in chromatin-modifying enzymes, splicing fac- in MDS pathogenesis (12). Thus, we tested the impact of HIF1A tors, transcription factors, cohesin complex, and metabolic sig naling in MDS. enzymes that regulate hematopoietic stem cell (HSC) self- renewal, survival, and differentiation. Cooperating genetic lesions occur, involving signaling molecules that regulate RESULTS cell growth and proliferation (1, 2, 5). Although a number of heterogeneous genomic aberrations have been identified Activated HIF1A Pathway in a Broad in patients with MDS (6, 7), their key clinical phenotypes are Spectrum of Patients with MDS similar. Therefore, we hypothesized that driver mutations HIF1A is mainly regulated at translational and protein activate common underlying mechanisms involved in MDS levels. Thus, analyzing downstream HIF1A signature gene phenotypes. Identification of these key mediators would expression is a reliable approach to measure HIF1A activation. NOVEMBER 2018 CANCER DISCOVERY | 1439 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 23, 2018; DOI: 10.1158/2159-8290.CD-17-1203 RESEARCH ARTICLE Hayashi et al. To determine whether patients with MDS have an activated achieved doxycycline-inducible expression of both a stable HIF1A gene signature, we analyzed a published cohort of and constitutively active human HIF1A triple-point-mutant CD34+ bone marrow (BM) cells isolated from healthy donors (TPM; ref. 16) and wild-type (WT) ARNT (also known as (n = 17) or from patients with MDS (n = 183; ref. 13). This HIF1B, a subunit for dimerization with HIF1A; tet-on-TPM/ MDS cohort contains patients with refractory anemia (RA; ARNT; B6J/129 × 1 SvJ; Fig. 2A). After doxycycline admin- n = 55), refractory anemia with ring sideroblasts (RARS; n = istration, we found increased HIF1A protein expression 48), or refractory anemia with excess blast type 1 (RAEB1; n = in the c-KIT+ BM cells from Vav1-Cre/TPM mice (Fig. 2B). 37) or type 2 (RAEB2; n = 43). HIF1A regulates many genes, The resulting HIF1A protein expression level was 1.5-fold which could be either HIF1A direct targets or indirect tar- higher than in MllPTD/WT cells (Fig. 2C). To identify gene- gets (such as those regulated by HIF1A-regulated miRNAs). expression changes, we performed RNA-sequencing (RNA- The number of HIF1A-regulated genes exceeds 1,000 and seq) analysis of Vav1-Cre/TPM and wild-type c-KIT+ BM continues to increase (11). Notably, there are unique sets of cells. Pathway enrichment analysis revealed that a number target genes activated by HIF1A signaling in individual cell of HIF1A-related gene sets were significantly upregulated types (different lineage, at different maturation stage; ref. in the c-KIT+ BM cells from Vav1-Cre/TPM mice