scholarly journals Osterix Marks a Distinct Population of Circulatory Bone Marrow Mesenchymal Stem Cells That Is Increased upon Oncogenic Stress

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-1
Author(s):  
Rafael Heinz Montoya ◽  
Rasoul Pourebrahim ◽  
Zoe Alaniz ◽  
Lauren B Ostermann ◽  
Jared K. Burks ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fluorescence Microscopy ◽  
Stromal Cells ◽  
Research Funding ◽  
Osteoblast Differentiation ◽  
Mesenchymal Progenitor Cells ◽  
Flow Cytometric ◽  
Reporter Mice ◽  
Marrow Mesenchymal Stem Cells ◽  
Osteoblast Lineage

Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that can differentiate into a variety of lineages and play a critical role in tissue homeostasis upon injury and repair. Previous studies using Osx-Cre transgenic mice have demonstrated that the expression of Sp7 (Osterix) marks a population of mesenchymal progenitor cells that can differentiate to osteoblasts as well as bone marrow stromal cells (Mizoguchi et al., 2014). Using a lineage-tracing system, we show that in addition to marking mesenchymal progenitor cells in the bone marrow, Osx-Cre also marks a population of BM-MSCs that circulate throughout the body and home in different tissues such as lung, spleen, intestine and muscle. Osx-Cre mice crossed with R26-mTmG reporter mice were analyzed at E14.5 and adulthood by fluorescence microscopy and flow cytometry. At E14.5, GFP+ cells were exclusively located in bone tissues. At two months of age, GFP+ cells were detectable in blood and many other tissues such as lung, liver, spleen and intestine. Flow cytometric profiling indicated that the GFP+ cells were positive for BM-MSC markers CD105, CD73 and CD140a. In order to exclude the possibility of non-specific recombination of the reporter in the non-osteoblast-lineage, as previously reported, we performed a pulse chase experiment utilizing Osx-CreER;mTmG mice. Fluorescence microscopy of the bone marrow upon Tamoxifen injection revealed that Cre activity was primarily limited to the osteoblast-lineage and bone tissue, whereas GFP+ cells were undetectable in lung and spleen, indicating that the GFP+ cells in the lung migrated from the bone marrow. Given that previous reports identified p53 as a negative regulator of osteoblast differentiation (Lengner et al., 2006), we further sought to determine the effect of p53 on circulatory BM-MSCs. Flow cytometric analysis of Osx-Cre;p53Fx/Fx;mTmG peripheral blood cells revealed a significant reduction of circulatory GFP+ cells as compared to p53 wild type mice (p<0.0001) suggesting a role for p53 in expansion of circulatory BM-MSCs. To further characterize the population of circulatory BM-MSCs in a cancer model, we analyzed the population of GFP+ cells in a syngeneic leukemia using fluorescence microscopy and flow cytometry. We transplanted p53 wildtype (Osx-Cre;mTmG) and p53 mutant (Osx-Cre;p53Fx/R172H;mTmG) reporter mice with AML-ETO-Turquoise leukemia cells and the population of GFP+ cells were analyzed three weeks after transplant. The population of GFP+MSCs were significantly increased in bone marrow and spleen, indicating the recruitment of circulatory BM-MSCs. Conclusion: We present the Osx-Cre;mTmG mouse as a faithful model to study circulatory BM-MSCs in vivo and identified a role for p53 in the regulation of circulatory BM-MSCs. We previously reported that BM-derived MSCs home to solid tumors and their metastases and can be successfully used as gene-delivery vehicles, both in murine models and in patients (Studeny et al. JCI 2001, Andreeff et al. AACR 2018). This model is the first to conduct studies of circulating MSCs and to further analyze their role in tumor biology and therapy. Lengner, C.J., Steinman, H.A., Gagnon, J., Smith, T.W., Henderson, J.E., Kream, B.E., Stein, G.S., Lian, J.B., and Jones, S.N. (2006). Osteoblast differentiation and skeletal development are regulated by Mdm2-p53 signaling. J Cell Biol 172, 909-921. Mizoguchi, T., Pinho, S., Ahmed, J., Kunisaki, Y., Hanoun, M., Mendelson, A., Ono, N., Kronenberg, H.M., and Frenette, P.S. (2014). Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development. Dev Cell 29, 340-349. Figure Disclosures Andreeff: Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Amgen: Research Funding.

scholarly journals p53 Mediated Bone Marrow Mesenchymal Stem Cell Expansion Supports Acute Myeloid Leukemia Development

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 523-523
Author(s):  
Rasoul Pourebrahimabadi ◽  
Zoe Alaniz ◽  
Lauren B Ostermann ◽  
Hung Alex Luong ◽  
Rafael Heinz Montoya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Osteoblast Differentiation ◽  
Hematopoietic Cells ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Mdm2 Inhibitor ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a heterogeneous disease that develops within a complex microenvironment. Reciprocal interactions between the bone marrow mesenchymal stem/stromal cells (BM-MSCs) and AML cells can promote AML progression and resistance to chemotherapy (Jacamo et al., 2014). We have recently reported that BM-MSCs derived from AML patients (n=103) highly express p53 and p21 compared to their normal counterparts (n=73 p<0.0001) (Hematologica, 2018). To assess the function of p53 in BM-MSCs, we generated traceable lineage specific mouse models targeting Mdm2 or Trp53 alleles in MSCs (Osx-Cre;mTmG;p53fl/fl and Osx-Cre;mTmG;Mdm2fl/+) or hematopoietic cells (Vav-Cre;mTmG;p53fl/fl and Vav-Cre;mTmG;Mdm2fl/+). Homozygote deletion of Mdm2 (Osx-Cre;Mdm2fl/fl) resulted in death at birth and displayed skeletal defects as well as lack of intramedullary hematopoiesis. Heterozygote deletion of Mdm2 in MSCs was dispensable for normal hematopoiesis in adult mice, however, resulted in bone marrow failure and thrombocytopenia after irradiation. Homozygote deletion of Mdm2 in hematopoietic cells (Vav-Cre;Mdm2fl/fl) was embryonically lethal but the heterozygotes were radiosensitive. We next sought to examine if p53 levels in BM-MSCs change after cellular stress imposed by AML. We generated a traceable syngeneic AML model using AML-ETO leukemia cells transplanted into Osx-Cre;mTmG mice. We found that p53 was highly induced in BM-MSCs of AML mice, further confirming our findings in primary patient samples. The population of BM-MSCs was significantly increased in bone marrow Osx-Cre;mTmG transplanted with syngeneic AML cells. Tunnel staining of bone marrow samples in this traceable syngeneic AML model showed a block in apoptosis of BM-MSCs suggesting that the expansion of BM-MSCs in AML is partly due to inhibition of apoptosis. As the leukemia progressed the number of Td-Tomato positive cells which represents hematopoietic lineage and endothelial cells were significantly decreased indicating failure of normal hematopoiesis induced by leukemia. SA-β-gal activity was significantly induced in osteoblasts derived from leukemia mice in comparison to normal mice further supporting our observation in human leukemia samples that AML induces senescence of BM-MSCs. To examine the effect of p53 on the senescence associated secretory profile (SASP) of BM-MSCs, we measured fifteen SASP cytokines by qPCR and found significant decrease in Ccl4, Cxcl12, S100a8, Il6 and Il1b upon p53 deletion in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) compared to p53 wildtype mice. To functionally evaluate the effects of p53 in BM-MSCs on AML, we deleted p53 in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) and transplanted them with syngeneic AML-ETO-Turquoise AML cells. Deletion of p53 in BM-MSCs strongly inhibited the expansion of BM-MSCs in AML and resulted in osteoblast differentiation. This suggests that expansion of BM-MSCs in AML is dependent on p53 and that deletion of p53 results in osteoblast differentiation of BM-MSCs. Importantly, deletion of p53 in BM-MSCs significantly increased the survival of AML mice. We further evaluated the effect of a Mdm2 inhibitor, DS-5272, on BM-MSCs in our traceable mouse models. DS-5272 treatment of Osx-cre;Mdm2fl/+ mice resulted in complete loss of normal hematopoietic cells indicating a non-cell autonomous regulation of apoptosis of hematopoietic cells mediated by p53 in BM-MSCs. Loss of p53 in BM-MSCs (Osx-Cre;p53fl/fl) completely rescued hematopoietic failure following Mdm2 inhibitor treatment. In conclusion, we identified p53 activation as a novel mechanism by which BM-MSCs regulate proliferation and apoptosis of hematopoietic cells. This knowledge highlights a new mechanism of hematopoietic failure after AML therapy and informs new therapeutic strategies to eliminate AML. Disclosures Khoury: Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding. Bueso-Ramos:Incyte: Consultancy. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy. OffLabel Disclosure: Mdm2 inhibitor-DS 5272

Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation

2017 ◽  
Vol 60 (6) ◽  
pp. 326-334 ◽  
Author(s):  
Carla Martins Kaneto ◽  
Patrícia S. Pereira Lima ◽  
Karen Lima Prata ◽  
Jane Lima dos Santos ◽  
João Monteiro de Pina Neto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenesis Imperfecta ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Osteoblast Differentiation ◽  
Marrow Mesenchymal Stem Cells

scholarly journals Myeloma Cells Deplete Bone Marrow Glutamine and Inhibit Osteoblast Differentiation Limiting Asparagine Availability

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3267
Author(s):  
Martina Chiu ◽  
Denise Toscani ◽  
Valentina Marchica ◽  
Giuseppe Taurino ◽  
Federica Costa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Amino Acid ◽  
Stromal Cells ◽  
Osteoblast Differentiation ◽  
Essential Amino Acids ◽  
Asparagine Synthetase ◽  
Osteolytic Lesions ◽  
Glutamine Transporter

Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.

Biological Characteristics of ZUC3 Antigen Expressed on Rat Bone Marrow Mesenchymal Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4254-4254
Author(s):  
He Huang ◽  
Jing Zheng ◽  
Xiaoyu Lai ◽  
Junli Cao ◽  
Jianling Fan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Flow Cytometric Analysis ◽  
Osteoblastic Differentiation ◽  
Significant Decline ◽  
Human Mscs ◽  
Down Regulation ◽  
Flow Cytometric ◽  
Marrow Mesenchymal Stem Cells

Abstract Objective: Bone marrow mesenchymal stem cells (MSCs) are widely studied in recent years. As an important part of cell identification, specific surface markers of MSCs have been paid a lot of attention to for long, but no breakthrough as yet. Monoclonal antibodies (McAb) against surface of certain cells have been used to characterize cell lineages. ZUC3, a novel murine McAb was produced by hybridoma technology previously, which was specifically reactive with both human MSCs and rat MSCs. Studying the expression of ZUC3 antigen on rat MSCs after passage and differentiation, it was to define whether ZUC3 antigen would be available for the identification of rat MSCs or their differentiation lineages. Methods: Rat MSCs isolated by a single step of adhesion to cell culture plastic, and purified via replacement of medium and a serial of passage, then the cells were identified by surface molecules CD90, CD44 and CD45 by flow cytometry. Enzyme immunocytochemistry and indirect immunofluorescence were used to evaluate the availability of ZUC3 expression by rat MSCs as a surface marker. Then further exploratory researches were carried out concerning ZUC3 expression by rat MSCs during passages (P1 to P5) and multiple differentiation (neuron, osteoblasts and adipocytes) in the certain condition. Results: Homogeneous rat MSCs could be obtained in vitro, which were uniformly positive for adhesion molecules CD90, CD44, and negative for CD45. The McAb was specifically reactive with rat MSCs as the positive cells were more than 99% by immunohistochemistry and immunofluorescence staining, and ZUC3 antigen located on the membrane of rat MSCs. The flow cytometric analysis show ZUC3 antigen expression by rat MSCs from P1 to P5 were all more than 85%. Analysis by multiple comparison, it was found some differences between P2 and P1 (93.95±2.44% v.s. 86.90±1.80%, P<0.01). The maximal expression was reached at P3 (97.10±1.25%), and the flow cytometric analysis showed a single symmetrical peak. Data of P4 (94.50±2.23%) population were slightly lower than P3 (P>0.05). By contrast, P5 (88.35±2.99%) showed a significant decline comparing with the former passages (P<0.01). Furthermore, rat MSCs could be successfully induced to differentiate into neuron-like cells, osteoblasts, and adipocytes and there was to some extent a downward trend of ZUC3 expression after differentiation (P<0.01). More than 90% rat MSCs could transform to an neuron-like appearance which were positive for NeuN, NF-M after treated with alpha-thioglycerol, and there was some downward degree of ZUC3 expression (97.77±1.03% to 80.07±2.70%, P<0.01). During the osteoblastic differentiation, it was observed an obvious down-regulation of ZUC3 expression from the 10th day (96.63±1.03% to 90.07±2.40%, P<0.01 ) and percentage on the 10th (90.07±2.40%), 15th (84.43±2.80%), 20th (64.53±7.63%) and 25th (53.40±10.02%) day were significantly lower than their anterior time respectively (P<0.05). The results of adipogenic differentiation after MSCs incubated with proper medium were similar to what observed during osteoblastic differentiation and ZUC3 expression were down-regulation on the 7th (84.33±2.70%), 14th (75.90±2.00%) and 21st (70.57±0.47%) day compared with their anterior dots respectively (P<0.01). Conclusion: ZUC3 antigen could be used for identification of rat MSCs. Significant decline of ZUC3 expression had be observed after rat MSCs were induced to differentiate along neuronal, osteoblastic and adipogenic pathways, which indicated that ZUC3 antigen would be a marker of progenitor.

The Serine/Threonine Kinase Pim-2 Is a Novel Anti-Apoptotic Mediator in Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 243-243
Author(s):  
Jin Asano ◽  
Masahiro Abe ◽  
Shiro Fujii ◽  
Osamu Tanaka ◽  
Ai Mihara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Stromal Cells ◽  
Osteoblast Differentiation ◽  
Family Members ◽  
Bone Marrow Microenvironment ◽  
Serine Threonine Kinase ◽  
Growth And Survival ◽  
Threonine Kinase ◽  
Cell Growth And Survival

Abstract Myeloma (MM) cells stimulate bone resorption by enhancing osteoclast (OC) formation and suppress bone formation by inhibiting osteoblast differentiation, leading to destructive bone lesions. In these lesions, OCs and stromal cells with defective osteoblast differentiation create a microenvironment suitable for myeloma cell growth and survival (a MM niche) to protect MM cells from various apoptotic insults. IL-6 and the TNF family members BAFF and APRIL have been demonstrated to be among predominant anti-apoptotic cytokines for MM cells elaborated by the bone marrow microenvironment in MM. The serine/threonine kinase Pim-2 is a novel apoptotic inhibitor which is transcriptionally up-regulated to promote survival of hematopoietic cells in response to environmental growth factors and cytokines. Up-regulation of Pim-2 expression has also been observed in various malignancies including MM. However, the roles for Pim-2 in growth and survival of MM cells are largely unknown. In the present study we therefore investigated the regulatory mechanism for Pim-2 expression in MM cells and the impact of Pim-2 on MM cell growth and survival with special reference to the interaction between MM cells and bone marrow components. Pim-2 protein is constitutively overexpressed in the absence of IL-6 in IL-6-dependent INA-6 as well as IL-6-independent RPMI8226 and U266 MM cell lines. Addition of IL-6, BAFF and TNFalpha up-regulated Pim-2 protein expression in INA-6 and RPMI8226 cells. A JAK/STAT3 inhibitor, cucurbitacin I, suppresses Pim-2 expression induced by IL-6, indicating Pim-2 as a downstream target of a JAK/STAT3 pathway. Stromal cells and OCs are regarded as a predominant cell type in MM bone marrow microenvironment to produce IL-6 and the TNF family members BAFF and APRIL, respectively. Co-cultures with stromal cells as well as OCs enhanced Pim-2 expression in INA-6 cells, suggesting up-regulation of Pim-2 in MM cells by surrounding cells in the bone marrow. In order to clarify the roles for Pim-2 in growth and survival of MM cells we next looked at the effects of Pim-2 siRNA. Suppression of Pim-2 expression by Pim-2 siRNA partly reduced the proliferation of INA-6 cells stimulated by IL-6 as well as the co-cultures with stromal cells or OCs. Pim-2 silencing also enhanced the cytotoxic effects of dexamethason on MM cells. Interestingly, further addition of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces cell death in concert with Pim-2 silencing in INA-6 cells, suggesting a cooperative roles for PI3K/Akt and Pim-2-mediated pathways in growth and survival of MM cells. Furthermore, Pim-2 silencing induced the cleavage of caspase9 but not caspase8; enforced expression of Pim-2 phosphorylated the BH3 only protein Bad; Pim-2 silencing suppressed phosphorylation of Bad by IL-6. Thus, Pim-2 appears to activate the intrinsic pathway of apoptotic machinery involving Bad phosphorylation. Taken together, our results suggest that Pim-2 is an important prosurvival mediator in MM cells, and that up-regulation of its expression in MM cells by bone marrow components may at least in part contribute to resistance to spontaneous and drug-induced apoptosis in MM cells. Therefore, Pim-2 may become a target for novel therapeutic strategies against MM.

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.

The Transcription Repressor Gfi1 Directly Interacts With and Is Phosphorylated By Aurora A Kinase, Which Abrogates Myeloma-Induced Gfi1 Repression Of The Runx2 Promoter In Pre-Osteoblasts

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1844-1844
Author(s):  
Jixin Ding ◽  
Fengming Wang ◽  
ShunQian Jin ◽  
Judy Anderson ◽  
Deborah L. Galson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Binding ◽  
Bone Disease ◽  
Stromal Cells ◽  
Protein Turnover ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells ◽  
Aurora A Kinase

Abstract Multiple myeloma (MM) is a plasma cell malignancy that is the most frequent cancer to involve the skeleton. MM bone disease is characterized by the formation of lytic bone lesions adjacent to MM cells that rarely heal even when patients are in long-term remission. This is due to the persistent suppression of bone marrow stromal cell (BMSC) differentiation into osteoblasts. We previously reported that MM cells induce long-lasting suppression of osteoblast differentiation by repression of the Runx2 gene through elevated expression of the transcriptional repressor Gfi1. However, how Gfi1 activity in BMSC is regulated by MM cells remains unclear. Using bioinformatics analysis, we found that there are three putative phosphorylation sites in the Gfi1 protein for Aurora A kinase (AurA) at S216, S326, and T418. We confirmed that Gfi1 was phosphorylated by AurA at multiple sites using an in vitro kinase assay. Co-immunoprecipitation assays revealed that AurA physically interacted with Gfi1 and phosphorylated Gfi1 protein. The interaction with AurA stabilized Gfi1 protein by blocking Gfi1 protein turnover, thereby extending the Gfi1 half-life from 2 hrs to 6 hrs. Further, co-transfection studies using wildtype and mutant AurA and Gfi1 showed that AurA inhibition of Gfi1 protein turnover was dependent on AurA kinase activity and phosphorylation of the S326 and T418 amino acid residues of Gfi1. Studies with co-transfected Myc-ubiquitin, FLAG-Gfi1, and HA-AurA revealed that AurA decreased Gfi1 ubiquitination, thereby leading to increased Gfi1 protein stability. Amino acids S326 and T418 are in Gfi1 zinc fingers (ZF) 3 and 6, respectively. It is known that Gfi1 ZF3, 4, and 5 are required for DNA binding, and that the K403R mutation in ZF6 interferes with DNA binding. Therefore we investigated if AurA phosphorylation of Gfi1 interferes with DNA binding. Chromatin immunoprecipitation and mRunx2 promoter oligo-pull down assays demonstrated that phosphorylated Gfi1 can still bind the Runx2 promoter. However, co-transfection studies with AurA and Gfi1 expression vectors with mRunx2-promoter luciferase reporters demonstrated that AurA phosphorylation of Gfi1 blocked repression of the Runx2 promoter. These data indicate that although AurA increased the amount of Gfi1 protein present on Runx2, AurA phosphorylation of Gfi1 appeared to lock Gfi1 in an “Off” (inactive) status and abrogated Gfi1 repression of Runx2 expression in osteoblast precursor cells. Since AurA phosphorylation of Gfi1 is not blocking DNA binding, the difference between Gfi1 “OFF” and “ON” status probably involves altered protein-protein interactions between Gfi1 and other factors that regulate Runx2 transcription. TNFa treatment, which we showed also represses Runx2 via Gfi1 activity, decreased the AurA protein level in MC-4 osteoblast precursor cells. Importantly, we found that AurA mRNA was decreased in both MC-4 cells treated with MM cells in vitro, and in bone marrow stromal cells isolated from MM patients. In conclusion, these data indicate that MM cells lower the levels of AurA in bone marrow stromal cells, thereby decreasing AurA phosphorylation of Gfi1. This helps to maintain Gfi1 in the “ON” status and allows Gfi1 repression of the Runx2 gene, thereby preventing osteoblast differentiation. These data suggest that AurA is an important regulator of Gfi1 function in MM bone disease. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Eli Lilly: Research Funding.

Addition of the Vascular Niche Component to the Human Bone Marrow-like Scaffold Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2402-2402
Author(s):  
Willy A Noort ◽  
Regina de Jong-Korlaar ◽  
Linda Lubbers-Aalders ◽  
Huipin Yuan ◽  
Joost D de Bruijn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Vessels ◽  
Human Blood ◽  
Stromal Cells ◽  
Research Funding ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Successful Implementation ◽  
Functional Evaluation ◽  
Essential Components

Abstract Previously, we have reported that our human bone marrow (BM)-like scaffold xenograft model allows the engraftment and outgrowth of normal and malignant hematopoiesis (e.g. multiple myeloma (MM), acute myelocytic/lymphocytic leukemia (AML/ALL) and MDS (Groen et al. Blood 2012; Gutierrez et al. JCI 2014 and data not shown). Whereas the presence of osteoblasts and bone of human origin mimics a human BM-like niche more closely than the murine BM in standard xenotransplant models (e.g. NOD-SCID/NSG mice), still some essential components of the human BM niche, i.e. human blood vessels, are missing. To this end, in addition to human mesenchymal stromal cells we now incorporated cord blood-derived endothelial progenitor cells (CB-EPCs) in the hybrid scaffold production process, to create a multi-tissue compartment that "maximally humanizes" the BM-like niche of our scaffolds. Towards successful implementation of a human vascular system we compared: i) scaffold material composition (biphasic calcium phosphate (BCP) vs. tricalcium phosphate (TCP)); ii) scaffold shape (particles vs. tubes); iii) different types of matrigel for CB-EPC embedding. Histological analysis of the humanized scaffolds, eight weeks after implantation in mice, showed a large number of functional human blood vessels, as indicated by hCD31+ staining and the presence of erythrocytes within. Comparison of the composition and the shapes of the scaffolds indicated superiority of TCP and tube-shaped scaffolds in supporting the formation of vessels. Further analysis of scaffolds for CD44, CD146, LEPR and nestin-positive cells, revealed the presence of other stromal niche cells besides human osteoblasts and endothelial cells. Irradiation of mice carrying these humanized implants did not have a significant deleterious effect on the established human vessels, allowing their further functional evaluation in xenotransplantation. Additionally, mice carrying tubes with and without human CB-EPC derived vessels (on either flank) were subsequently inoculated with adult BM-derived CD34-positive cells by intracardiac injection. Upon analysis 12 weeks later, all tubes showed multi-lineage hematopoietic outgrowth. Interestingly, CB-EPC embedment resulted in increased numbers of CD45+ (2-fold), CD13+ (4-fold) and CD7+ (2-fold), while CD19+ cell numbers were equal. In contrast, in mouse BM almost only CD19+ cells could be detected. Moreover, we observed that the use of CB-EPCs in our scaffolds provides faster kinetics of in vivo engraftment and growth of both patient-derived MM or AML cells. With the addition of both human CB-EPCs and human BM stromal cells, our scaffold systems now simulate both human endosteal and vascular niches of the BM, thereby more closely recapitulating the human hematopoietic niche. Disclosures Yuan: Xpand Biotechnology BV: Employment. de Bruijn:Xpand Biotechnology BV: Employment. Mitsiades:TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Novartis: Research Funding. Martens:Johnson & Johnson: Research Funding. Groen:Johnson & Johnson: Research Funding.

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