Niche-mediated depletion of the normal hematopoietic stem cell reservoir by Flt3-ITD-induced myeloproliferation.

Adam J Mead,Petter Woll,Emmanouela Repapi,Cristina Di Genua,Wen Hao Neo,Nikolaos Barkas,Deborah Atkinson,Claus Nerlov,Toshio Suda,Nicholas Fordham,Tiphaine Bouriez,Supat Thongjuea,Christopher A.G Booth,Alice Giustacchini,Shabnam Kharazi,Onima Chowdhury,Sten Eirik W Jacobsen,R Facchini ,Nicola E Gray ,Lauren E Jamieson ,Clark S-A ,Satoshi Matsuoka

doi:10.1084/jem.20161418

Abstract

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD-driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.

Highlights

Suppression and collapse of normal blood cell replenishment underlies the severe morbidity and high mortality accompanying many hematologic malignancies, including acute myeloid leukemia (AML; Löwenberg et al, 1999)
Gene expression analysis of 120 single LSKCD150+48− cells and 126 single LSK cells not residing in the LSKCD150+48− hematopoietic stem cell (HSC) compartment showed that as many as 72% of LSKCD150+48− cells did not express any detectable FMS-like tyrosine kinase 3 (Flt3) at the single-cell level as opposed to only 13% of other (MPP) LSK cells (Fig. 1 C; P < 0.001)
Gene expression of single LSKCD150+48− cells captured in nanofluidic reaction chambers (Fig. 1 G), with sensitivity for gene expression detection to the single-molecule level (Wu et al, 2014), showed that 12 of 17 (71%) captured LSKCD150+48− cells did not express any Flt3 mRNA, supporting that the absence of Flt3 expression is unlikely to represent a lack of sensitivity of the assay

Summary

Introduction

Suppression and collapse of normal blood cell replenishment underlies the severe morbidity and high mortality accompanying many hematologic malignancies, including acute myeloid leukemia (AML; Löwenberg et al, 1999). Those results highlight the well-recognized heterogeneity of the phenotypic HSC compartment, which in addition to genuine HSCs contains non-HSC progenitors, marked in part by expression of Flt transcript (Purton and Scadden, 2007; Boyer et al, 2011; Buza-Vidas et al, 2011)

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