RESEARCH ARTICLE Sphingosine-1-Phosphate Receptor 3 Potentiates Inflammatory Programs in Normal and Leukemia Stem Cells to Promote Differentiation Stephanie Z. Xie1, Kerstin B. Kaufmann1, Weijia Wang2, Michelle Chan-Seng-Yue1,3, Olga I. Gan1, Elisa Laurenti1,4, Laura Garcia-Prat1, Shin-ichiro Takayanagi1,5, Stanley W.K. Ng6, ChangJiang Xu7,8, Andy G.X. Zeng1,9, Liqing Jin1, Jessica McLeod1, Elvin Wagenblast1, Amanda Mitchell1, James A. Kennedy1,10,11, Qiang Liu1, Héléna Boutzen1, Melissa Kleinau1, Joseph Jargstorf1, Gareth Holmes1, Yang Zhang2, Veronique Voisin7,8, Gary D. Bader7,8, Jean C.Y. Wang1,10,11, Yusuf A. Hannun12, Chiara Luberto13, Timm Schroeder2, Mark D. Minden1,10,11,14, and John E. Dick1,9 ABSTRACT Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature mye- loid cells and influences stemness programs and lineage determination in HSCs by activating stress myelopoiesis, such roles in LSCs are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator that drives myeloid differentiation and activates inflammatory programs in both HSCs and LSCs. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across cohorts of patients with AML, each with distinct phenotypic and clinical properties. S1PR3 was high in LSCs and blasts of mature myeloid samples with linkages to chemosensitivity, whereas S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset. SIGNIFICANCE: S1PR3 is a novel regulator of myeloid fate in normal hematopoiesis that is heterogene- ously expressed in AML. S1PR3 marks a subset of less primitive AML cases with a distinct inflamma- tory signature and therefore has clinical implications as both a therapeutic target and a biomarker to distinguish primitive from mature AML. See related commentary by Yang et al. 1Princess Margaret Cancer Centre, University Health Network, Toronto, Medicine, Stony Brook, New York. 14Department of Medical Biophysics, Ontario, Canada. 2Department of Biosystems Science and Engineering, University of Toronto, Toronto, Ontario, Canada. 3 ETH Zürich, Basel, Switzerland. PanCuRx Translational Research Initia- Note: Supplementary data for this article are available at Blood Cancer tive, Ontario Institute for Cancer Research, Toronto, Ontario, Canada. Discovery Online (https://bloodcancerdiscov.aacrjournals.org/). 4Wellcome Trust - Medical Research Council Cambridge Stem Cell Insti- tute, Department of Haematology, University of Cambridge, Cambridge, K.B. Kaufmann, W. Wang, and M. Chan-Seng-Yue contributed equally to United Kingdom. 5Cell Therapy Project, R&D Division, Kirin Holdings Com- this article. pany, Limited , Kanagawa, Japan. 6Institute of Biomaterials and Biomedical Corresponding Authors: John E. Dick, Princess Margaret Cancer Centre, Engineering, University of Toronto, Toronto, Ontario, Canada. 7The Don- University Health Network, University of Toronto, Princess Margaret nelly Centre, University of Toronto, Toronto, Ontario, Canada. 8Department Cancer Research Tower, 101 College Street, Toronto, Ontario M5G 1L7, of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. Canada. Phone: 416-581-7472; Fax: 416-581-7476; E-mail: John.Dick@ 9Department of Molecular Genetics, University of Toronto, Toronto, uhnresearch.ca; and Stephanie Z. Xie. Phone: 416-581-7470; Fax: 416- Ontario, Canada. 10Division of Medical Oncology and Hematology, Depart- 581-7476; E-mail: [email protected] ment of Medicine, University Health Network, Toronto, Ontario, Canada. Blood Cancer Discov 2021;2:1–22 11Department of Medicine, University of Toronto, Toronto, Ontario, Can- ada. 12Stony Brook Cancer Center and Departments of Medicine, Bio- doi: 10.1158/2643-3230.BCD-20-0155 chemistry, and Pathology, Stony Brook University, Stony Brook, New ©2020 American Association for Cancer Research. York. 13Department of Physiology and Biophysics, Stony Brook School of OF1 | BLOOD CANCER DISCOVERY JANUARY 2021 AACRJournals.org Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 30, 2021. Copyright 2020 American Copyright 2020 by AssociationAmerican for Association Cancer Research. for Cancer Research. INTRODUCTION as a hallmark of cancer and is known to be distinct between HSCs and their downstream progenitors (12, 13). We recently Acute myeloid leukemia (AML) is a heterogeneous disease showed that sphingolipid composition is diverse across the characterized by impaired myeloid differentiation that is hier- human hematopoietic hierarchy and uncovered a novel role archically organized akin to the normal blood system (1–4). for sphingolipid metabolism in determining HSC fate (14). Leukemia stem cells (LSC) possess properties of self-renewal Sphingosine-1-phosphate (S1P) is a potent bioactive sphin- and lineage differentiation and are responsible for long-term golipid crucial for systemic health, particularly in disorders clonal propagation in AML, similar to their normal long-term associated with inflammation (15–17). As an obligate ligand hematopoietic stem cell (LT-HSC) counterpart that sustains for a family of five G-protein–coupled receptors (S1PR1–5), lifelong blood production (2). LSCs are often resistant to S1P plays pleiotropic roles in cellular proliferation, survival, standard chemotherapy and are responsible for clinical relapse and migration—processes that are dysregulated in inflamma- (2, 5–7). AML is a cellular hierarchy, driven by LSCs that share tory diseases (15, 16, 18, 19). Notably, within the murine sys- many LT-HSC stemness properties but remain impaired for tem, S1P signaling via S1PR1 regulates B lymphopoiesis and normal myeloid lineage development (3, 4, 7). How myeloid neuroinflammation (20). In humans, S1P receptor modula- fate networks at the stem cell level become perturbed during tors are used to target immune cells to treat multiple sclero- leukemogenesis remains poorly understood. Most studies of sis, a chronic inflammatory disease (18, 21–23). Aside from how hematopoietic stem cells (HSC) choose to differentiate mature lineage cells, inflammation has pleiotropic effects into one lineage versus another have focused on the role of within primitive hematopoietic cells, governing both lineage cytokines and the transcriptional factor networks that they determination and HSC stemness functions (10, 11). There engage (8, 9). In parallel, investigations of AML primarily focus is also a role in hematopoietic malignancies, although stud- on how cytokines and their signaling networks are impaired by ies exploring inflammation across the individual cells that AML driver oncogenes (3, 8, 10, 11). However, compelling evi- make up the leukemia hierarchy are limited (11). Aging is dence is emerging that lineage determination is not solely the also associated with increased inflammation, dysregulated consequence of lineage-specific cytokine exposure and signal- lineage determination, and myeloid skewing, and is a major ing, but rather that metabolites generated from many path- risk factor for developing AML (24, 25). Some reports sug- ways can play a role (12). Cellular metabolism is recognized gest that both complex membrane sphingolipids, such as JANUARY 2021 BLOOD CANCER DISCOVERY | OF2 Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 30, 2021. Copyright 2020 American Association for Cancer Research. RESEARCH ARTICLE Xie et al. sphingomyelin, that are catabolized for S1P production and 10 S1P-related genes, including its five receptors (S1PR1–5) levels of S1P itself are dysregulated during aging (26–28). Over- and enzymes involved in its synthesis/degradation across all, the role of S1P signaling in the interplay between inflam- six human hematopoietic stem and progenitor cell (HSPC) mation and normal hematopoiesis or in the development of populations and seven mature lineages from cord blood stem cell malignancies like AML is poorly understood. (CB; 37). Expression of S1PR1, 3, and 5 was most enriched Dysregulated myeloid cells are the linchpin of inflamma- in specific mature lineages (Fig. 1A).S1PR1 was most highly tion within many chronic human conditions associated with expressed in T- and B-lymphoid, natural killer (NK), and den- aging, including clonal hematopoiesis and atherosclerotic dritic cell (DC) populations, whereas S1PR5 was restricted cardiovascular disease (29–31). TNFα and IL6 are two key to NK and DC lineages, consistent with published reports inflammatory cytokines linked to chronic inflammatory dis- primarily in the murine system (16, 20). S1PR3 was of par- eases, myeloid dysregulation, and stem cell function (11, 23, ticular interest, as its expression was specific to monocytes 32). Moreover, a large body of literature points to NF-κB, and granulocytes, and it had not been previously implicated originally described as a gatekeeper for inflammatory con- in myeloid fate specification in either normal or malignant trol of immune cell responses, as a key transcriptional factor human hematopoiesis. Flow cytometric analysis confirmed in the regulatory network activated by these inflammatory that S1PR3 protein was highly expressed on the surface of cytokines and other