ALBERT

Beschreibung: Increasing evidence suggests that leukemia cells take shelter in the bone marrow (BM) niche, where they hide from chemotherapy and continue to divide. As yet, how leukemia cells alter the BM niche to facilitate their growth and assist them in evading chemotherapy is unclear. In this study, we provide compelling evidences that acute myeloid leukemia (AML), through exosome secretion, transformed the BM niche to facilitate their own growth and suppress normal hematopoiesis. Using AML xenograft and MLL-AF9 knock-in mouse model, we show that leukemia cells as well as AML-derived exosomes stimulate the growth of BM stromal progenitors and blocked the osteolineage development in our stromal compartment analysis. Histological analysis and micro-CT examination confirmed loss or thinning of the bone in both leukemia and leukemic exosome-treated animals. Expression of cell adhesion molecules (NCAM1, VCAM1, CD44, OPN & ICAM1) and factors important for angiogenesis (Angpt1, Angpt2 and VEGF) are upregulated, whereas genes important for HSC maintenance (CXCL12 and SCF), osteoblast (OCN, OSX, Notch3 and IGF1) and chondrocyte (ACAN, SOX9) development are suppressed. While we observed increases in phenotypic LT-HSC in AML-derived exosomes treated mice, these mice show reduced multilineage reconstitution ability, increased cell cycle entry and higher sensitivity to myeloablative stress suggesting that HSCs from exosome-treated mice have lower stem cell activity than their counterparts from normal mice.In addition, leukemia-modified stroma cells exhibit marked reduction in ability to support normal HSCs. Pre-treatment of AML-derived exosome “prime” the animal for leukemia cell invasion and accelerate leukemia progression. Conversely,disruption of exosome secretion by targeting Rab27a in AML cells significantly delays leukemia progression. These data strongly support the notion that leukemia-modified niches favor leukemic cell proliferation and suppress normal hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Key Points Notch1/DII4-mediated signals are normally suppressed by LRF, preventing HSCs from premature T-cell differentiation in the bone marrow. Erythroblastic islands may have the capacity to regulate the fate and function of HSCs.

Beschreibung: Abstract 3467 The microenvironment (niche) cues hematopoietic stem cell (HSC) receives play an important role in the regulation of their decisions between self-renewal and differentiation. However, the cellular constitution of niches remains poorly understood. We identified three adult progenitor populations near the endosteum based on differential expression of cell surface markers, including CD166, CD146 and Sca1. Upon co-transplantation with fetal skeletal progenitors, Sca1+ progenitors can give rise to CD146+ and CD166+ stroma and phenotypically CXCL12 abundant reticular (CAR) cells in marrow. CD146+ and CD166+ progenitors, on the other hand, form bone without marrow cavity. Multiplex single cell qRT-PCR reveals all three progenitors expressed high levels of genes involved in HSC maintenance. In vitro co-culture assay demonstrated that all three progenitors could preserve HSC long-term multi-lineages reconstitution capability. Furthermore, disruption of stem cell factor (SCF) production in Sca1+ progenitor severely limits its ability to support HSC both in culture condition and after transplantation. Our results suggested that Sca1+, CD146+, and CD166+ mesenchymal progenitors and their progeny collaborate to provide supportive environment for hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Increasing evidence suggests that leukemia cells take shelter in the bone marrow (BM) microenvironment (niche), where they hide from chemotherapy and continue to divide. As yet, the identity of niche cells and secreted factors that facilitate leukemia cell growth and assist them in evading chemotherapy is unclear. Further, how leukemia cells alter the bone marrow microenvironment is not known yet. In this study, we provide compelling evidences of a novel role of leukemia-derived exosomes in altering the microenvironment constituents by paracrine mechanisms.As proof-of-concept, we analyzed the cytokines mRNA profiles of primary human and mouse stromal cell co-cultured with primary CD34+CD38- cells from AML patients. Stromal cells co-cultured with leukemia showed increased levels of IL-6, IL-1β, VEGFα, TNF and reduced SDF1 mRNA expression. Similar pattern of gene expression changes were observed from stroma cells co-cultured with leukemia-derived exosomes.By using CFSE labeled exosomes, we observed that leukemia-derived exosomes target marrow stromal and endothelial cells both in-vitro and in-vivo directly. In our in vivo AML model, established using xenografted AML cell lines or primary AML patient samples in Rag2-/- γc-/-mice, we observed expansion of LT-HSC and hematopoietic progenitors compartment. The leukemia animals also showed cellular composition changes in the stromal compartment suggesting osteoblast differentiation was blocked. Interestingly, milder but similar changes were observed in mice treated with leukemia-derived exosomes. Exosomes derived from normal human peripheral blood did not induce significant changes in either hematopoietic or stromal compartments in recipient mice. These data indicate that leukemia cells secrete specialized exosomes to modulate the BM microenvironment. Fluidigm dynamic array analysis of BM stromal cells from leukemic mice revealed that the cell adhesion molecules (NCAM1, VCAM1, CD44, OPN & ICAM1) and factors important for angiogenesis (Angpt1, Angpt 2 &VEGF) were all upregulated in leukemia-modified stromal cells whereas genes important for osteoblast (OCN, OSX), chondrocyte (SOX9) development and HSC maintenance (SDF1 and SCF) were down regulated. These results suggest that leukemia cells can remodel the BM microenvironment by changing the stromal cell composition and influencing expression of important molecular regulators. To evaluate the HSC functions in exosomes-treated mice, we used 5-fluorouracil (5-FU) to suppress hematopoiesis and induce myeloablative stress. Leukemia-derived exosome-pretreated mice succumbed to death earlier compared to the control group (p=0.0001) suggesting that HSCs from leukemia-derived exosome-treated mice may have lower stem cell activity than their counterparts from normal mice. Furthermore, more LT-HSC and hematopoietic progenitors from leukemia-derived exosome-pretreated mice were in active cell cycle (p=0.004 and p=0.01 respectively). These findings support our hypothesis that leukemia cells/exosomes directly or indirectly through leukemia-modified niche, altered the HSCs physiological and quiescence properties. Next we analyzed the ability of leukemia-modified niche to support the normal hematopoiesis. We co-cultured freshly sorted normal CD45.2 LT-HSCs (LSK CD150+CD48-Flk2-) with leukemia cells/exosomes pre-treated stroma cells for 48 hours and transplanted the co-cultured HSC into irradiated CD45.1 mice. 18 weeks after transplantation, we observed a significantly decreased engraftment of the HSCs co-cultured with leukemic cells/exosomes stroma compared with the HSCs co-cultured with normal stroma (p=0.003). Finally, leukemia engrafted better and developed more rapidly (p=0.0026) in mice that received leukemia-derived exosomes pre-treatment. These data suggest that changes induced by leukemia-derived exosomes in the BM niche accelerate leukemia progression and decrease their ability to support HSCs. Collectively, our data demonstrate that the leukemia cells manipulate the bone marrow microenvironment, partly through leukemia-derived exosomes, to suppress the normal hematopoiesis and facilitate growth of the leukemic progeny. Disclosures: No relevant conflicts of interest to declare.