Identification of Renin Progenitors in the Mouse Bone Marrow That Give Rise to B-Cell Leukaemia

ARTICLE Received 10 Aug 2013 | Accepted 16 Jan 2014 | Published 18 Feb 2014 DOI: 10.1038/ncomms4273 OPEN Identification of renin progenitors in the mouse bone marrow that give rise to B-cell leukaemia Brian C. Belyea1, Fang Xu1, Ellen S. Pentz1, Silvia Medrano1, Minghong Li1, Yan Hu1, Stephen Turner2, Robin Legallo3, Craig A. Jones4, Joseph D. Tario4, Ping Liang5, Kenneth W. Gross4, Maria Luisa S. Sequeira-Lopez1 & R. Ariel Gomez1 The cell of origin and triggering events for leukaemia are mostly unknown. Here we show that the bone marrow contains a progenitor that expresses renin throughout development and possesses a B-lymphocyte pedigree. This cell requires RBP-J to differentiate. Deletion of RBP-J in these renin-expressing progenitors enriches the precursor B-cell gene programme and constrains lymphocyte differentiation, facilitated by H3K4me3 activating marks in genes that control the pre-B stage. Mutant cells undergo neoplastic transformation, and mice develop a highly penetrant B-cell leukaemia with multi-organ infiltration and early death. These renin- expressing cells appear uniquely vulnerable as other conditional models of RBP-J deletion do not result in leukaemia. The discovery of these unique renin progenitors in the bone marrow and the model of leukaemia described herein may enhance our understanding of normal and neoplastic haematopoiesis. 1 Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. 2 Department of Bioinformatics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. 3 Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. 4 Roswell Park Cancer Institute, Buffalo, New York 14263, USA. 5 Department of Biological Sciences, Brock University, St Catharines, Ontario, L2S 3A1, Canada. Correspondence and requests for materials should be addressed to R.A.G. (email: [email protected]). NATURE COMMUNICATIONS | 5:3273 | DOI: 10.1038/ncomms4273 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4273 enin cells have traditionally been associated with the (Fig. 1e), lymph node tumour formation (74% of mutant mice regulation of blood pressure and fluid-electrolyte home- developed axillary or retroperitoneal tumours) (Fig. 1f), and Rostasis. Pursuing the mechanisms that regulate the identity succumbed at an early age with a median survival of 269 days of these cells, we uncovered an unexpected property of renin cells (Fig. 1g). in the bone marrow with relevance to the development of Blood smears from mutant mice demonstrated a predomi- malignancy. Renin progenitors appear early in the embryo and nance of lymphoblasts (Fig. 2a). Flow cytometry of bone marrow give rise to many different cell types throughout the body1. and tumours confirmed the presence of an aberrant B-lymphoid Whereas the function of renin cells in extra renal tissues is (B220dim/CD19 þ ) immunophenotype at the expense of myeloid unclear, the ontogeny and function of renin cells in the kidney are cells compared with control mice (Fig. 2b). The phenotype was better understood2–5. In the embryonic kidney, renin precursors indicative of precursor B-cell leukaemia evidenced by the lack of are distributed extensively along nephrovascular units and IgM and IgD expressions (Fig. 2c). Similar findings were found in participate in the assembly and branching morphogenesis of the the spleen and peripheral blood of leukaemic animals kidney arterioles. As development of the kidney proceeds, renin (Supplementary Fig. 1a–c). precursors differentiate in situ into arteriolar smooth muscle cells, Given the presence of lymphoblasts within the blood and bone glomerular mesangial cells and interstitial pericytes. Thus, in the marrow and the extensive organomegaly, we performed a adult only a few cells at the tip of the renal arterioles near the comprehensive histologic evaluation of mutant and control mice. glomeruli, the juxtaglomerular (JG) cells, retain the ability to The spleens of mutant mice were massively infiltrated with synthesize and secrete renin upon physiological demands1. Under lymphoblasts resulting in defacement of their normal architecture normal conditions, those cells suffice to regulate blood pressure without recognizable red or white pulp (Fig. 2d). Lymphoblasts and fluid-electrolyte homeostasis. However, if such adult animal invaded the liver in a periportal and perisinusoidal distribution is subjected to a homeostatic threat (such as hypotension, (Fig. 2e) and kidney interstitium (Supplementary Fig. 2a). Mutant dehydration, sodium depletion or administration of renin– mice developed skin lesions (characterized by loss of fur, nodules angiotensin inhibitors), there is an increase in the number of and ulcerations due to epidermal and dermal lymphoblastic renin-expressing cells along the arterioles, glomeruli and infiltration) and lymphoid tumours (Supplementary Fig. 2b,c). interstitium, resembling the embryonic pattern described Finally, bone marrow from leukaemic animals was hypercellular above6,7. This phylogenetically conserved process occurs by compared with controls and consisted of a homogenous blast re-transformation of arteriolar smooth muscle cells, mesangial population that occupied over 90% of marrow space (Fig. 2f). cells and pericytes into renin-expressing cells1,8. These blasts replaced other haematopoietic cells explaining the Because renin cells contain all the components of the Notch marked anaemia found in mutant animals. Lymphoblasts pathway including RBP-J, the final transcriptional effector of all displayed diffuse and strong immunoreactivity for PAX5 and the Notch receptors9, and Notch/RBP-J is known to regulate cell CD79a, further confirming a B-cell lineage and expressed TdT fate, we previously examined whether deletion of RBP-J and CD34 supporting a B-cell precursor maturational stage regulates the identity and plasticity of kidney renin cells (Supplementary Fig. 2d–g). during normal development and in response to a physiological To follow the distribution of mutant cells, we bred our mutant threat. Conditional deletion of RBP-J in renin-expressing cells and control mice with mT/mG reporter mice, which normally resulted in a decrease in the number of renin-positive JG cells in express RFP in all cells but GFP only in cells that express Cre the kidney and an inability of smooth muscle cells along the recombinase. Therefore, cells harbouring the RBP-J deletion are kidney vasculature to regain the renin phenotype10. GFP þ , whereas non-mutant leukocytes are RFP þ 12. The Unexpectedly, as these mice aged beyond 6 months, they number of GFP þ cells was markedly elevated in the blood and developed signs and symptoms of a highly penetrant and organs of mutant mice (Fig. 2g), indicating that deletion of RBP-J fulminant form of precursor B-lymphoblastic leukaemia. Given causes B-cell leukaemia with expansion of lymphoblasts to the the potential medical relevance of this finding, in this study we bloodstream and invasion and morphological distortion of perform an extensive series of experiments to fully characterize peripheral organs. this mouse model of leukaemia, including its natural history and Following the current classification of murine lymphoid the genomic and epigenetic events underlying its development. neoplasms, the majority (92%) of mutant mice (n ¼ 22) We also set out to identify, and characterize in detail, which cells developed precursor B-cell lymphoblastic lymphoma/leukaemia in the bone marrow are capable of producing renin under (pre-B LBL)13. A minority (8%) of mice (n ¼ 2) was affected by a normal circumstances and whether those cells may be the origin myeloproliferative or mixed lymphoid/myeloid disorder and of this striking model of leukaemia. Finally, we ascertain three animals developed mild disease. Given the striking whether mutations in the RBP-J gene are associated with predominance of the pre-B LBL, we focused our efforts on a leukaemia in humans as well. We find that renin is expressed by detailed characterization of this phenotype. a subset of B-cell progenitors in the mouse bone marrow, and We performed multiple experiments to investigate if this that these renin-expressing cells are the cell of origin for B-cell leukaemia model represents a clonal process and if the leukaemic leukaemia when RBP-J is deleted. cells can grow in a cell-autonomous manner in culture and transplant systems. First, analysis of immunoglobulin gene rearrangements was performed to determine the clonal nature Results of leukaemic cells. These studies demonstrated that B cells from Deletion of RBP-J in renin cells results in B-cell leukaemia.We the spleen and bone marrow of mutant animals had unique deleted RBP-J in cells of the renin-lineage by crossing mice that patterns of V(D)JH rearrangements when compared with wild- express Cre recombinase under control of the renin locus with type animals, with a predominant recombination indicating RBP-Jfl/fl mice1,10,11. Mutant mice (RBP-Jfl/fl; Ren1dcre/ þ ) mono- or oligo-clonal IgH patterns (Supplementary Fig. 3a–c). developed progressive abdominal distension and poor overall Next, we performed colony-forming assays from bone marrow health by B6 months of age (Fig. 1a). Necropsy demonstrated cells of mutant mice to determine the clonogenic potential of massive hepatosplenomegaly (spleen and liver weights were leukaemic cells. Whereas wild-type bone marrow did not form 15-

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