Acute Myeloid Leukemia Alters The Mesenchymal Stem Cell Potential Of The HSC Niche: Evidence For Modulation By β-Adrenergic Signals

Hematopoietic stem cells (HSC) reside in specific bone marrow niches comprised of perisinusoidal Nestin-GFP⁺ (Nes-GFP⁺) and leptin receptor (LepR)⁺ stromal cells which highly overlap with each other, as well as osteolineage and endothelial cells. These different cellular constituents regulate HSC maintenance and retention in the bone marrow (BM). Recently, our laboratory further identified rare periarteriolar Nes-GFP^bright cells which have been identified to be pericytes and functionally crucial for HSC quiescence and maintenance, from the more abundant reticular Nes-GFP^dim population (Kunisaki et al, unpublished data). The function of Nes-GFP⁺ niche cells is tightly regulated by the sympathetic nervous system (SNS) via β₂- and β₃-adrenergic receptors. In acute myeloid leukemia (AML), BM infiltration by leukemic blasts is known to lead to hematopoietic failure. Although cytopenias are thought to result from the BM occupation by AML, the mechanisms remain unclear. In this study, we investigated the impact of AML on BM niche constituents, and evaluated the influence of SNS signals in AML progression using a syngeneic murine MLL-AF9 transplantation model. We observed a ∼4.4-fold expansion of Nes-GFP⁺ stromal cells (p<0.05) in leukemic BM, which positively correlated with the degree of leukemic marrow infiltration (r=0.49, p<0.01). However, while we observed a massive expansion of reticular Nes-GFP^dim cells, we detected a ∼2.2-fold reduction in pericytic Nes-GFP^bright cells (p<0.05). Phenotypically, Nes-GFP^dim cells showed similar expression of LepR, PDGFRα and CD51 to healthy controls, markers enriching for BM mesenchymal stem cells. Interestingly, Nes-GFP⁺ cells in leukemic BM had a ∼1.5-fold higher colony-forming units-fibroblast (CFU-F) and ∼2.6-fold higher CFU-Osteoblast capacity. The gene expression profile of Nes-GFP⁺ cells using Affymetrix Gene 1.0 ST microarrays revealed a profound dysregulation when the BM was infiltrated with AML, including most notably activated TGFβ signaling pathways and upregulation in the expression of inflammatory response genes. Nes-GFP⁺ niche cells were previously shown to be highly enriched for HSC-maintenance gene expression. In leukemic BM, Nes-GFP⁺ cells showed a significant reduction in the expression of Cxcl12, Scf and Vcam1 (p<0.01), while no significant differences were observed for Opn. In line with these data, we observed a significant reduction of HSC-enriched Lin⁻Sca1⁺c-Kit⁺Flt3⁻ and progenitor cells in the BM (p<0.05, except for granulocyte-macrophage progenitors) and their mobilization into peripheral blood and spleen (p<0.01). To further analyze if Nes-GFP⁺ niche cells in leukemic BM still depended on adrenergic signals, we performed in vivo chemical sympathetic denervation with 6-hydroxydopamine. Sympathectomized leukemic mice showed a ∼1.5-fold increase in Nes-GFP⁺ cells, a ∼1.8-fold increase of IL-7R^-Lin^-c-Kit^hiCD34⁺FcγRII/III^hi leukemic stem cells (LSC, p<0.05) which was associated with significantly shorter survival (p=0.017). Denervated mice did not show differences in homing, proliferation or apoptosis of leukemic cells. We confirmed our findings in a xenograft model, in which sympathectomized mice were transplanted with primary human AML samples and showed a higher leukemic BM infiltration (p<0.05). Further analyses using specific adrenergic β2- and β3-receptor antagonists (ICI118,551 hydrochloride and SR59230A) suggested that adrenergic signals were predominantly mediated by the β2-adrenergic receptor. Preliminary data indicate that activation of the β2-adrenergic receptor leads to decreased leukemia burden. In summary, our data identify that leukemic cells severely transform the BM niche by gradually expanding and directing mesenchymal stem cell differentiation into the osteoblastic lineage and simultaneously decreasing numbers of pericytic niche cells. Ultimately, this creates a microenvironment with impaired HSC maintenance capacity and favors myeloid expansion. This transformed niche, however, remains regulated by signals from the SNS which in turn modulates leukemic BM infiltration. These results thus uncover a novel approach for niche-targeted therapeutic strategies in AML.