Connexin Expression by Human Primary AML Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2147-2147
Author(s):  
Brynjar Foss ◽  
Lars Rune Sæterdal ◽  
Anita Ryningen ◽  
Anne M. Øyan ◽  
Øystein Bruserud
Keyword(s):  
Bone Marrow ◽  
Protein Expression ◽  
Stromal Cells ◽  
Pearson Correlation ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
Monocytic Differentiation ◽  
Membrane Expression ◽  
Hematopoietic Stem

Abstract Abstract 2147 Introduction: Normal hematopoiesis takes place in the bone marrow niches where the hematopoietic stem cells are surrounded by stromal cells and extracellular matrix, and were both soluble mediators as well as cell-cell interactions seem to regulate proliferation and initial maturation. Among the various molecules participating in hematopoietic regulation are gap junctions (GJs) that are formed by connexins (Cxs) and represent intercellular communication channels (reviewed in Foss B et al., Stem Cell Dev. 18(6): 807–812, 2009). Acute myelogenous leukemia (AML) is characterized by bone marrow accumulation of immature leukemic cells, and both disease development as well as chemosensitivity of the leukemic cells seem to be affected neighbouring stromal cells in the bone marrow microenvironment. Studies of leukemic cell lines and animal models suggest that especially Cx43 and possibly Cx32 are involved in regulation of AML cell proliferation and differentiation, but the functional potential of Cxs in AML seems to be wider (reviewed in Foss B, et al., Biochim Biophys Acta. 1798(1):1-8, 2010). Still, the expression profile of Cxs by primary human AML cells are not well characterized. Methods: We characterized the mRNA and protein expression of various Cxs in primary human AML cells. The mRNA expression was investigated in microarray assay (n = 47) and the protein expression in the cell surface membrane by flow cytometry (n = 38). Results: The mRNA levels of Cx32 (mean 0.29, stdv ± 0.16), Cx43 (0.09 ± 0.21) and Cx45 (0.56 ± 0.25) showed very low levels whereas Cx37 showed higher expression (1.66 ± 0.93). The membrane expression was classified as positive (i.e. > 20% of AML cells stained positive) only for a minority of patients especially when investigating Cx32 (5 out of 38 patients examined) but also for Cx37 (13/38) and Cx43 (16/38), whereas the leukemic cells were classified as Cx45 positive for a majority of the patients (21/38). The mean fluorescence intensities (MFI) of the membrane expression for Cx32, Cx37, Cx43 and Cx45 were all significantly correlated (P<0.001). The strongest correlation was observed between Cx43 and Cx45 (Pearson correlation coefficient, r=0.946). The corresponding regression analysis showed R2 = 0.896, clearly suggesting a linear relationship between the membrane expression of Cx43 and Cx45. The membrane expression of Cx43 and Cx45 correlates with the expression of CD14 and CD15, and for Cx45 also with CD11c (all with P<0.05). In addition, the membrane expression of Cx43 and Cx45 were also correlating with cell morphology; cells without signs of differentiation (FAB M0+M1 classification) show less Cx expression than cells with signs of monocytic differentiation (FAB M4+M5, P<0.05, see figure). On the other hand, there was no correlation between the expression of the various Cxs and Flt-3-internal tandem duplication mutation and expression of CD33 and CD34. Conclusions: These results show for the first time that primary AML cells express various Cxs on their cell membranes, but patients are heterogeneous and the expression is seen especially in AML cells with signs of monocytic differentiation. Disclosures: No relevant conflicts of interest to declare.

The Hypoxic Microenvironment in Acute Myelogenous Leukemia: Critical Role of CXCR4 in the Induction of HIF-1α.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1819-1819
Author(s):  
Michael Andreeff ◽  
Martin Dietrich ◽  
Paul Corn ◽  
Sergej Konoplev ◽  
Marina Konopleva ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Critical Role ◽  
Mapk Signaling ◽  
Bone Marrow Microenvironment ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
Hematopoietic Stem ◽  
Hypoxic Conditions

Abstract The bone marrow microenvironment provides functional and structural support for both normal and leukemic hematopoietic stem cells. Importantly, the marrow microenvironment is known to be hypoxic. Given the many critical functions of HIF-1α, we investigated HIF-1α levels in leukemic cell lines and primary AML blasts. Surprisingly, HIF-1α expression was non-detectable in AML cells grown in suspension cultures under normoxic (21% O2) or hypoxic (1% O2) conditions. Normoxic co-cultures with bone marrow-derived mesenchymal stromal (MSC) or MS5 cells likewise did not induce HIF-1α, but hypoxic co-culture conditions induced HIF-1α protein without changes in HIF-1α mRNA levels, suggesting post-transcriptional regulation. Functionality of HIF-1α was confirmed by concomitant increase in the levels of glucose transporter glut-1, the HIF-1α downstream target. Inhibition of stroma-leukemia cell interactions with the small molecule CXCR4 inhibitor AMD3465 (Genzyme/Anormed) at 100 nM completely abrogated the induction of HIF-1α in HL-60 and MOLM13 AML cells. While SDF-1 was unable to induced HIF-1α under normoxic conditions, it did so under both physical (1% O2) and chemical (COCl2) hypoxic conditions, in two different cell lines. Inhibition of P13K (with LY294002) or MEK/ERK signaling (with CI-1040) abrogated HIF-1α induction under hypoxic conditions. Immunohistochemical staining of bone marrow samples from primary AML confirmed the presence of HIF-1α in leukemic cells localized adjacent to bone-lining stromal elements. Results suggest that the bone marrow microenvironment of AML is hypoxic in vivo; in leukemia cells HIF-1α induction under low oxygen tension depends on the presence of stromal cells; HIF-1α induction is dependent on SDF-1/CXCR4 and is mediated by activation of P13K and MAPK signaling. Altogether these findings suggest that SDF-1α/CXCR4 interactions contribute to the survival of leukemic cells via specific induction of HIF-1α signaling by the bone marrow microenvironment. Disruption of these interactions via CXCR4 inhibition strategies may suppress multiple pro-survival HIF-1α targets in leukemic cells.

scholarly journals The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission

Haematologica ◽  
2020 ◽  
Vol 106 (1) ◽  
pp. 111-122 ◽  
Author(s):  
Sandrine Jeanpierre ◽  
Kawtar Arizkane ◽  
Supat Thongjuea ◽  
Elodie Grockowiak ◽  
Kevin Geistlich ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Stromal Cells ◽  
Kinase Inhibitor ◽  
Bmp Signaling ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

Cytokine Profiling of Acute Meylogenous Leukemia and Myelodysplasia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2355-2355
Author(s):  
Steven M. Kornblau ◽  
David McCue ◽  
Kang L. Lu ◽  
Wenjing Chen ◽  
Kevin R. Coombes
Keyword(s):  
Protein Expression ◽  
Expression Profiles ◽  
Pearson Correlation ◽  
Expression Patterns ◽  
Myelogenous Leukemia ◽  
High Expression ◽  
Leukemic Cells ◽  
Proteomic Profiling ◽  
Serum Samples ◽  
Average Linkage

Abstract Protein expression and activation determines the pathophysiology of leukemic cells in Myelodysplasia (MDS) and Acute Myelogenous Leukemia (AML) and is dependent on endogenous changes (e.g mutation, methylation) and exogenous signals from stromal interactions, cytokines (CTKN) and chemokines. We have previously performed proteomics on primary AML sample (using reverse phase protein arrays) and wanted to assess how cytokines affect protein expression and phosphorylation. Prior studies of CTKN expression in AML and MDS have generally measured individual CTKNs, but not provided an overall assessment of CTKN expression. We measured the level of 26 CTKN (IL-1RA, 1B, 2, 4 5, 6, 7 , 8 , 9, 10,12, 13, 15, 17, Eotaxin, FGFB, G-CSF, GM-CSF, IFNγ, IP10, MCP1, MIP1α, MIP1β, PDGF, TNFα and VEGF) using multiplex cytometry (Bioplex™, Biorad) in serum samples from 176 AML (138 untreated (New), 38 relapsed (REL)) and 114 MDS patients (97 New, 10 post biological therapy, 7 REL) and 19 normal (NL) subjects. Individual CTKN expression was not correlated with clinical features (e.g. age, gender, cytogenetics, FAB, HB, WBC, platelet etc). The levels of IL -1β, 4, 5, 6, 7,10,12, 13, 17, IFNγ, FGFB and MIP1α were significantly lower and IL-8 and 15 higher in AML/MDS compared to NL. The expression profiles of AML and MDS were statistically indistinguishable whether analyzed individually or by unsupervised hierarchical clustering, except for IL-8 and 13 (higher in AML) and VEGF (higher in MDS). When CTKN were evaluated individually in new AML cases higher levels of IL4, 5 and 10 correlated significantly with remission attainment, and higher levels of IL8, Il1Ra, IP-10, Mip1β, VEGF and ILR, correlated significantly with shorter survival. No CTKN predicted remission attainment or survival in MDS. Unsupervised hierarchical bootstrap clustering using Pearson correlation and average linkage of CTKN expression relative to other CTKN expression, where high levels of one CTKN correlated with high expression of the other, revealed 6 highly reproducible expression patterns: IL-1β 4, 7, 10, 12, 13, G-CSF, IFNγ, MIP1α and PDGF IL 1ra, 6, 8 Eotaxin, IP-10, MIP1β and VEGF, IL2, 9, 15 and GMCSF, IL5 IL-7, FGF-Basic, TNFα and MCP1. Similar unsupervised clustering of the samples based on CTKN expression using average linkage also revealed 5 disease clusters and a NL sample cluster (containing all 19 NL samples). Average expression levels of each CTKN in these 5 clusters show diminished expression of most CTKN that had high expression in the NL samples, with each group showing increase in expression in a distinct subset of CTKN relative to NL. Remission attainment was strongly associated with cytokine signature (P=0.005). Additional CTKN are being studied (SCF, TGFβ, IL3). Comparison of CTKN expression patterns with proteomic profiling of expression and phosphorylation status is pending. In summary, this is the largest sample set studied for multiple CTKN expression in AML and MDS and the first assessment of many of these CTKN in these diseases. Most CTKNs showed different expression in AML and MDS compared to NL. Interestingly, CTKN expression in AML and MDS were similar. Many CTKN are predictive of outcome individually. CTKN signatures distinguish groups of patients and are predictive of outcome. Correlation with proteomic profiling may suggest CTKN to target in combination with other targeted therapies to maximally affect activated pathways.

Leukemia-Induced Alterations in Bone Marrow Cytokine and Chemokine Levels Contribute to Altered Stem Cell Lodgment and Impairment of Normal Stem Cell Growth in CML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 962-962
Author(s):  
Bin Zhang ◽  
Yin Wei Ho ◽  
Tessa L. Holyoake ◽  
Claudia S Huettner ◽  
Ravi Bhatia
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mouse Model ◽  
Hematopoietic Stem Cell ◽  
Stromal Cells ◽  
Research Funding ◽  
Mrna Levels ◽  
Hematopoietic Stem ◽  
Tnf Α ◽  
Selective Impairment

Abstract Abstract 962 Specialized bone marrow (BM) microenvironmental niches are essential for hematopoietic stem cell (HSC) lodgment and maintenance. However microenvironmental interactions of leukemia stem cells (LSC) are poorly understood. Although chronic myelogenous leukemia (CML) results from HSC transformation by the BCR-ABL gene, the role of the microenvironment in modulating leukemia development is not known. We employed the SCL-tTA-BCR/ABL mouse model of CML to investigate the LSC interactions with the BM microenvironment. In this model, targeted expression of the BCR-ABL gene in murine HSC via a tet-regulated SCL promoter results in development of a chronic phase CML-like disorder. We have reported that LSC capacity is restricted to BCR-ABL+ cells with long-term hematopoietic stem cell (LTHSC) phenotype(LSK Flt3-CD150+CD48-) (Blood 2010 116:1212A). LSC numbers are reduced in the BM but increased in the spleen of CML mice compared with LTHSC from control mice, suggesting that LSC have altered niche interactions. LSC also demonstrate altered trafficking with significant reduction in homing of IV injected LSC to BM, and markedly increased egress of intrafemorally injected LSC to the spleen, potentially related to reduced CXCL12 levels in the BM of CML mice. In addition, levels of several chemokines and cytokines, including MIP1α, MIP1β, MIP2, IL-1α, IL-1β, TNF-α, G-CSF and IL-6, were increased in CML BM, related to increased production by malignant hematopoietic cells. We investigated whether altered chemokine and cytokine expression was associated with altered capacity of the CML BM microenvironment to support LTHSC engraftment. LTHSC from control mice or LSC from CML mice were transplanted into irradiated CML or control recipients. There was reduced engraftment of both control LTHSC and CML LSC in the BM of CML compared to control recipients at 2 weeks after transplantation, associated with reduced homing to CML BM, potentially related to low BM CXCL12 levels. The numbers of control LTHSC in the BM of CML recipient mice remained low at 4 weeks post-transplantation, whereas the numbers of CML LSC increased to numbers similar to those seen in the BM of control recipients. Culture with CML BM supernatants (SN) resulted in impaired growth of control LTHSC compared to control BM SN. In contrast the growth of CML LSC was similar following culture with CML and control BM SN. Culture with individual factors at concentrations similar to those observed in CML BM (16ng/ml MIP1α, 8ng/ml MIP1β, 2.5ng/ml IL-1α, 3.5ng/ml IL-1β, 0.05ng/ml TNF-α) resulted in significantly reduced growth of normal LTHSC compared with CML LSC. These results indicate that diffusible factors produced by leukemic cells in the CML BM environment selectively inhibit normal LTHSC compared to CML LSC growth. Exposure of a murine stromal cell line to CML BM SN resulted in reduced CXCL12 mRNA levels compared to BM SN from control mice. The cytokine G-CSF, which was increased in CML BM SN, has been reported to reduce CXCL12 transcription. We observed significant reduction of CXCL12 mRNA levels in stromal cells cultured with G-CSF (0.2ng/ml), supporting a potential role for increased G-CSF production by leukemia cells in reduced CXCL12 production by CML BM stromal cells and reduced LSC retention in the BM. We evaluated whether defects in microenvironmental function in CML were affected by imatinib treatment. Treatment of CML mice with imatinib (200mg/kg/day, 2 weeks) led to reduction in MIP1α, MIP1β, IL-1β, and IL-6 levels in BM cells. Engraftment of normal LTHSC was significantly enhanced in BM of CML recipients pre-treated with imatinib. Results obtained with the mouse model were validated using specimens obtained from CML patients. CXCL12 mRNA levels were significantly reduced in human CML compared to normal MNCs, whereas expression of MIP1α, MIP-2, IL-1α and IL-1β were increased in CML MNCs, consistent with results obtained with the mouse model. Coculture with CML MNC conditioned medium (CM) resulted in selective impairment of growth of normal CD34+CD38- primitive progenitors compared to CM from normal MNC, but did not inhibit growth of CML progenitors. We conclude that leukemia-induced alterations in BM cytokine and chemokine levels contribute to altered LSC lodgment and to selective impairment of growth of normal LTHSC in the CML BM microenvironment, leading to a relative growth advantage for CML LSC over normal LTHSC and expansion of the leukemic clone. Disclosures: Holyoake: Novartis: Research Funding; Bristol Myers Squibb: Research Funding.

Exosomes Derived from AML/MDS Cells Is Involved in Stromal Dysfunction and Bone Marrow Failure

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4627-4627
Author(s):  
Hiroto Horiguchi ◽  
Masayoshi Kobune ◽  
Shohei Kikuchi ◽  
Wataru Jomen ◽  
Kazuyuki Murase ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Leukemic Cells ◽  
Boyden Chamber ◽  
Hematopoietic Stem ◽  
Coculture System ◽  
Qpcr Array

Abstract The failure of normal hematopoiesis in myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) could be induced by a variety of mechanism such as the alteration of property of hematopoietic stem cells and stem cell niche. However, it has not yet been clarified precise mechanism how MDS stem/progenitor cells could replace normal hematopoietic stem/progenitor cells especially regarding involvement of mesenchymal stromal cells (MSCs). To gain insight into the mechanism of stromal dysfunction, comparative analyses of transcriptomes were conducted between normal and MDS/AML-derived MSCs. Further, we attempted to identify certain effectors originated from MDS/AML cells could alter the function of bone marrow (BM) MSCs. The MSCs derived from healthy volunteer (HV)-derived (normal) and MDS/AML-derived stromal cells were established and analyzed mRNA expression by quantitive PCR (qPCR) array. Additionally, the supporting activity of MSCs for BM CD34+ progenitor/stem cells was examined using serum free coculture system. The interaction between MDS/AML cells and MSCs were evaluated by using Boyden Chamber and the changes of mRNA expression were analyzed. The results of qPCR array revealed that the expression of hematopoietic factors was drastically altered in MDS/AML-derived MSCs as compared with normal MSCs. Among these factors, the expression of SCF and JAG1 mRNA were significantly and consistently reduced in all MDS/AML patients examined. Functional assay of these MSCs demonstrated that the number of colony-forming units (CFU) mixed cells (MIXs) and cobblestone area-forming cells (CAFCs) derived from CD34+ cells was significantly reduced after coculture with MDS/AML-derived MSCs as compared with normal MSCs. Even non-contact culture using Boyden Chamber between leukemic cells and MSCs induced the reduction of SCF and JAG1 mRNA, indicating that certain inducers could be soluble factors. Interestingly, this effect of transcriptomes alteration was negated by nSMase2 inhibitor (GW4869). Exosome transfer assay using Boyden Chamber revealed that GFP and PKH26 in leukemic cells transmit onto MSCs in non-contact coculture system and this transfer of exosome was significantly inhibited by GW4869 or nSMase siRNA. The multiple type of microRNA in exosome derived from MDS/AML cells was transferred into MSCs, suggesting that exosome could contribute to the alteration of mRNA expression in stromal cells. Collectively, these results indicated that exosome derived from MDS/AML cells could be involved in the reduction of SCF/JAG mRNA and the stromal supporting activity of normal hematopoietic stem/progenitor cells. Disclosures No relevant conflicts of interest to declare.

Bone Marrow Stromal Cell Remodeling Is a Common Feature of Diverse Fibrotic Myeloproliferative Neoplasm Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 25-25
Author(s):  
Timothy B Campbell ◽  
Si Yi Zhang ◽  
Alexander Valencia ◽  
Emmanuelle Passegue
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Critical Role ◽  
Myeloproliferative Neoplasm ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Cell Numbers

Abstract Myeloproliferative neoplasms (MPN) are blood cancers initiated by driver mutations that transform hematopoietic stem cells. MPN exhibit gross pathologic bone marrow (BM) stromal remodeling, including damaging myelofibrotic change that leads to dependence on extramedullary hematopoiesis and more severe clinical diseases. Therapies targeting fibrotic change would have broad appeal in the treatment of these diseases. We previously demonstrated a critical role for malignant myeloid cells in remodeling endosteal mesenchymal stromal cells (MSC) into myelofibrotic osteoblast-lineage cells (OBC) in a model of chronic myelogenous leukemia (CML) driven by BCR/ABL (Schepers et al., Cell Stem Cell, 2013). In a separate study in a fibrotic MPN model driven by Jak2V617F, neuropathy and nestin-positive MSC cell death were found critical to disease progression but their involvement in myelofibrosis was not investigated (Arranz et al. Nature. 2014). Our goal is to characterize the type of BM stromal remodeling occurring in non-CML MPN models driven by various mutations and representing a spectrum of disease severity and fibrosis. This includes a minimally fibrotic transgenic Jak2V617F alone model (Jak2V617F model, Xing et al., Blood, 2008) and more advanced fibrotic models driven by MPLW515L expression (MPLW515L model, Pikman et al., PLoS Med, 2006) or combined transgenic Jak2V617F expression with conditional deletion of the polycomb gene EZH2 (Jak2V617F/EZH2-/- model, Sashida et al., JEM, 2016). We found common blood and BM hematopoietic changes in all three models, including thrombocytosis and expansion of myeloid-biased multipotent progenitor BM cells and confirmed the degree of fibrosis using picrosirius red staining of bone sections. Both MPLW515L and Jak2V617F/EZH2-/- heavily fibrotic models demonstrated inhibition of total endosteal MSC, OBC and endothelial cell (EC) numbers during disease development - in most cohorts a greater than 50% decrease in absolute stromal cell numbers was found. In addition, we observed that whole BM cells from Jak2V617F/EZH2-/-mice contained a significantly lower number of totalfibroblast colony forming cells (CFU-F). In co-culture experiments designed to measure direct MSC remodeling induced by malignant cells, both MPLW515L and Jak2V617F/EZH2-/- BM cells inhibited healthy endosteal MSC colony formation over time. In contrast, we found no inhibition of stromal cell numbers or co-culture MSC growth in the minimal fibrotic Jak2V617F model. In initial experiments measuring rare central marrow perivascular MSC, we found reduced LepR+ MSC (Ding et al., Nature, 2012) in both MPLW515L and Jak2V617F/EZH2-/- long bone sections using immunofluorescence. Our results show that fibrotic development in non-CML MPN inhibits stromal cell numbers and function likely via direct effects of malignant hematopoietic cells. This is in contrast to fibrotic CML development where myelofibrotic endosteal stromal cells are expanded. This difference could be partly explained by the type and localization of fibrosis in these various models. The CML model has focal endosteal collagen-I fibrosis which is heavily reliant on osteoblast remodeling, while the MPLW515L and Jak2V617F/EZH2-/- models have more diffuse reticulin central marrow fibrosis which may be produced through a process of stromal cell senescence or differentiation. Overall, this study underscores that a “one size fits all“ approach to understanding myelofibrosis is insufficient. To tease out these differences, we are examining qualitative and quantitative changes in additional central marrow MSC populations, including PDGFR+, Sca-1+ and Gli-1+ MSC, during MPN development as well as assaying the molecular mediators of stromal remodeling. Our long-term goal is to identify therapies that can restore a more normal BM stroma and support healthy hematopoiesis in MPN. Disclosures No relevant conflicts of interest to declare.

scholarly journals 5-Fluorouracil Treatment Leads to Activation of Stem Cell Niche By Reconstructing Mesenchymal Stromal Cells and Exert a Distinct Microenvironmental Impact on Normal and Leukemic Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1197-1197
Author(s):  
Seon-Yeong Jeong ◽  
Jin-A Kim ◽  
Il-Hoan Oh
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Mesenchymal Cells ◽  
Leukemic Cells ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Cellular Changes ◽  
Rapid Emergence

Abstract Reactivation of endogenous hematopoietic stem cells (HSCs) are initiated by stimulation of bone marrow niche triggered by various injury signals. Here, we show that treatment with 5-fluorouracil (5-FU) leads to reconstruction of bone marrow (BM) microenvironment to establish an activated niche stimulating hematopoietic stem cells (HSCs). First, we show that pre-treatment with 5-FU leads to engraftment of donor cells in non-irradiated recipient mice without affecting the homing efficiency of HSCs into BM. The HSC activation effects were reproduced in-vitro by co-culturing hematopoietic cells with CD45-Ter119- stromal cells derived from 5-FU treated BM, but not by co-culture with CD45+ cells or stromal cells obtained from enzymatic digestion of bone from the same mice. Examination of BM mesenchymal cells after 5-FU treatment revealed a rapid emergence of high-proliferating mesenchymal progenitors exhibiting large size colony (CFU-F) and higher self-renewal of colonogenic cells 3-5 days after 5-FU treatment, which was concomitantly associated with regeneration of CD34+Lin-Sca-1+c-kit+ (LSK) cells in the same BM. The cellular changes in mesenchymal stroma was associated with rapid emergence of characteristic mesenchymal cell populations (PDGFR-a+/Leptin receptor+/SSEA-3+: PLS) with 650-folds increase of the PLS cells in BM in 3 days after 5-FU treatment. However, the increase of these PLS mesenchymal cells were not associated with increase in mitotic activity of mesenchymal cells (<5% BrdU+ cells), indicating phenotypic conversion of subpopulation in BM. Moreover, cellular changes in mesenchymal niche were associated with rapid increase of mesenchymal cells expressing cross-talk molecules such as CXCL-12 (20-folds), Jagged-1 (13-folds) and DLL-1 (15-folds). Furthermore, in-vivo administration of chemicals blocking CXCL-12 and notch signaling during the recovery from the 5-FU treatment led to the significant loss of LSK-SLAM cells in the regenerated BM. Interestingly, the BM niche activated by 5-FU exerted a distinct effect on normal and leukemic cells in a manner that it provide higher support on the primitive state of normal HSCs than for MN-1 induced leukemia cells. Thus, leukemic mice engrafted with MN-1 cells exhibited a decrease in primitive leukemic cell (Lin-c-kit+) and higher survival by 5-FU treatment than those treated by radiation. Taken together, our study reveals the cellular reconstruction of mesenchymal niche in BM during stimulus-induced niche activation and provides an insight on the selective niche targeting as a novel therapeutic strategies for hematological diseases. Disclosures No relevant conflicts of interest to declare.

Do Microenvironmental Factors Play Important Role in Supporting the Maintenance of Leukemic Stem Cells of Chronic Myelogenous Leukemia?

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4219-4219
Author(s):  
Yeon Hee Cho ◽  
Dong Hwan Kim ◽  
Jun Ho Jang ◽  
Seok Jin Kim ◽  
Kihyun Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Notch Pathway ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Background : In the era of tyrosine kinase inhibitor(TKI) for chronic myelogenou leukemia(CML), it is becoming more important to develop the way to get rid of the leukemic stem cells(LSC), as there is frequent relapse when TKI therapy is stopped even when molecular remission is achieved. Some researchers reported macrophages and Wnt signaling in bone marrow microenvironment might play significant role in the development and the progression of CML. Methods : We isolated CD34+CD38− LSC from bone marrow cells from 8 patients with CML chronic phase and also CD34+CD38− normal hematopoietic stem cell from 7 normal controls using magnetic cell separation system. CD34+CD38− cells were cultured on stromal cells for 5 weeks and transfered to methylcellulose media for long-term culture initiating cell(LTC-IC) assay. We checked whether there is a difference in LTC-IC capacity of LSC when cultured on leukemic or normal stromal feeder cells. CD45− stromal supporting cells were established by 3-week culture after 12Gy irradiation of CD34− bone marrow cells. We studied the expression of VCAM, ICAM, E-selectin in stromal cells and the presence of BCR-ABL transcript. Wnt and β-catenin, frizzled, Notch1 were analyzed in the LSCs. CD14+ cells were isolated and added in LTC-IC assay whether the presence of leukemic or normal CD14+ cells would make a change in the maintenance of LSCs. Results : The number of CD34+CD38− cells in CML was in the range of 1.3 × 103 to 9.2 × 104, that we could do only two sets of 2 × 2 LTC-IC assay that statistical analysis was impossible. LTC-IC frequency of LSCs was about 5-times higher compared to that of normal stem cells irrespective of the kind of stromal cells used. CD14+ cell co-culture did not changed significantly the LTC-IC frequency both in normal and LSCs. Wnt 2B, 5A and 10B were highly expressed in LSCs irrespective of the stromal cells. β-catenin was also strongly expressed in LSCs compared to normal stem cells. Notch1 was expressed only in CD34+CD38− LSCs but not in normal stem cells. The kind of stromal cells or the addition of CD14+ cells in LTC-IC assay did not affected the Notch1 expression in LSCs. Conclusion : CML CD34+CD38− LSCs seem to have more LTC-ICs and wnt and notch pathway may play important role in the maintenance of LSCs that could be therapeutic targets.

scholarly journals Bone Marrow Microenvironment Is a New Therapeutic Target of Ruxolitinib in Myeloproliferative Neoplasm

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2930-2930
Author(s):  
Kaimin Hu ◽  
Lou Lixia ◽  
Lizhen Liu ◽  
Binsheng Wang ◽  
Shan Fu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protective Effect ◽  
Stromal Cells ◽  
Osteoblast Differentiation ◽  
Myeloproliferative Neoplasms ◽  
Myelogenous Leukemia ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Hematopoietic Stem ◽  
Qrt Pcr

Abstract Deregulation of both hematopoietic stem cell (HSC) activity and bone marrow (BM) microenvironment is pivotal in the development of myeloproliferative neoplasms (MPNs). Previous studies indicate that, in addition to HSCs, myeloid malignancies also affect the function of BM microenvironment. MPNs progressively remodel endosteal BM niche into a self-reinforcing leukemic niche and contribute to BM fibrosis, indicating that BM microenvironment should not be underestimated in MPN treatment. Until now, treatment options for MPNs are still limited. Ruxolitinib (Rux), an inhibitor of JAK 1 and 2, has significant clinical efficacy in myelofibrosis. Recent evidence reveals that combination of Rux and tyrosine kinase inhibitor (TKI) Nilotinib contributes to elimination of CD34+ cells in chronic myelogenous leukemia (CML), a subtype of MPNs, both in vitro and in vivo. However, treatment targeting BM microenvironment in MPNs remains poorly understood. Therefore, we aim to characterize the role of Rux in mesenchymal stromal cells (MSCs), which are key stromal cells in hematopoietic support of BM microenvironment. Our results showed that Rux with concentration gradients from 0.1 to 5µM inhibited proliferation of MSCs in a dose-dependent manner, without increasing apoptosis rate (P£¾0.05) (Figure 1A). Compared with control cells, MSCs exposed to 5µM Rux displayed similar morphology, and expressed comparable levels of CD73, CD90, CD105, CD34, CD45 and HLA-DR surface antigens (Figure 1B). As previously reported, leukemic myeloid cells stimulated MSCs to overproduce functionally altered osteoblastic lineage cells (OBCs). Thus we assessed the effect of Rux on osteogenic differentiation of MSCs. We found that 1µM Rux significantly inhibited osteoblast differentiation evidenced by reduced mineralization with Alizarin red staining on day 14, while 3µM Rux exhibited a relatively mild inhibitory ability. To further confirm the effect of Rux on this process, we analyzed the expression of osteoblast-specific transcription factors (Runx2 and Osterix) and osteoblastic markers (IBSP and BGLAP) by qrt-PCR. Consistently, the expression of both early (Runx2 and IBSP) and late (Osterix and BGLAP) osteoblast differentiation-related genes were significantly suppressed by Rux (Figure 1C). Since Rux showed a synergistic effect with nilotinib on CML and leukemia cells obtained cytotoxic drug resistance when co-cultured with MSCs, we explored whether Rux can reverse this protective effect. We pre-treated MSCs with 1 and 3µM Rux for 3 days, washed the drug away, and then co-cultured them with K562 cells at a ratio of 1:10. Our results demonstrated that when treated with Nilotinib, K562 cell survival was significantly increased in presence of MSCs (48.5±1.7% v.s. 69.4±4.6%), while MSCs pretreated with Rux exhibited a remarkably reduced protective effect (with 1µM Rux 61.9±4.8%; 3µM Rux 60.9±4.1%) (Figure 1D). To investigate the possible mechanisms engaged, we conducted western blot and qrt-PCR analysis. As shown in figure 1E, a decreased phosphorylation level of STAT3 and Akt but not Erk was detected in Rux conditioned MSCs. And qrt-PCR results confirmed that Rux down-regulated JAK2/STAT3 targeted genes whose levels were decreased by one third (C-myc), one half (STAT3) and three quarters (Cyclin D1). In conclusion, these data indicated that Rux, a specific antagonist of JAK2, exerted a negative effect on MSCs proliferation and osteoblast differentiation, which were pivotal in the sinister hematopoietic-stromal symbiosis in MPN, especially in CML. Rux also significantly weakened the protective effect of MSCs on CML against nilotinib through blocking JAK2/STAT3 signaling. Collectively, Rux provides further hope that therapeutic targeting on both malignant hematopoietic cells and the BM microenvironment could eliminate malignant cells and regenerate normal microenvironment with resolved myelofibrosis that supports normal hematopoiesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Aging of Bone Marrow Mesenchymal Stromal Cells: Hematopoiesis Disturbances and Potential Role in the Development of Hematologic Cancers

Cancers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 68
Author(s):  
Fulvio Massaro ◽  
Florent Corrillon ◽  
Basile Stamatopoulos ◽  
Nathalie Meuleman ◽  
Laurence Lagneaux ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Progression ◽  
Cancer Progression ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Cell Communication ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Wide Range ◽  
Major Player

Aging of bone marrow is a complex process that is involved in the development of many diseases, including hematologic cancers. The results obtained in this field of research, year after year, underline the important role of cross-talk between hematopoietic stem cells and their close environment. In bone marrow, mesenchymal stromal cells (MSCs) are a major player in cell-to-cell communication, presenting a wide range of functionalities, sometimes opposite, depending on the environmental conditions. Although these cells are actively studied for their therapeutic properties, their role in tumor progression remains unclear. One of the reasons for this is that the aging of MSCs has a direct impact on their behavior and on hematopoiesis. In addition, tumor progression is accompanied by dynamic remodeling of the bone marrow niche that may interfere with MSC functions. The present review presents the main features of MSC senescence in bone marrow and their implications in hematologic cancer progression.

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