scholarly journals Extravesicular Mir-7977 Released from Leukemic Cells Inhibites the PCBP1-Mediated mRNA Stabilization and Hippo-YAP Signaling Pathway in Bone Marrow Mesenchymal Stromal Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1519-1519
Author(s):  
Masahiro Yoshida ◽  
Satoshi Iyama ◽  
Hiroto Horiguchi ◽  
Akari Goto ◽  
Shohei Kikuchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Signaling Pathway ◽  
Conflicts Of Interest ◽  
Target Prediction ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Leukemic Cells ◽  
Mirna Biogenesis ◽  
Mrna Stabilization ◽  
Underlying Mechanisms

Abstract [Background] We and others have revealed that various abnormalities of the bone marrow (BM) environment such as aberrant cytokine expression and impaired microRNA (miRNA) biogenesis are observed in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, underlying mechanisms to induce BM stromal dysfunction have not yet been clarified. Recently, we have found that abundant extracellular vesicles including exosomes, were detected in the BM interstitial fluid. Furthermore, we have shown that exosomal miR-7977 was highly released from AML cells (GSE64029) and was taken up by BM mesenchymal stem cells (MSCs). Based on these findings, we hypothesized that exosomal miR-7977 could be involved in the alteration of BM stromal function of MDS/AML. [Materials and Methods] To gain an insight into the function of exosomal miR-7977 in BM, we firstly performed transduction of a miR-7977 mimic into BM MSCs and compared transcriptomes between control-transduced and miR-7977-transduced MSCs (GSE108186). Subsequently, we selected the differentially expressed genes (DEGs) and conduced gene set enrichment analysis (GSEA). [Results] Regarding DEGs, we focused on poly(rC) binding protein 1 (PCBP1), since PCBP1 was predicted to be a target gene of miR-7977 by an online database for miRNA target prediction using miRDB. The reductions in PCBP1 mRNA and protein were confirmed in MSCs after miR-7977 transfer by real time PCR. Because PCBP1 is known to be involved in splicing, and the expression levels of several genes, we analyzed the expression of several hematopoietic factors and found that expression level of stem cell factor (SCF), angiopoietin-1 (ANGPT1) and Jagged-1 (JAG1) were remarkably decreased. Regarding the pathway analysis, GSEA showed that the gene sets of Yes-associated protein 1 (YAP1)_up were significantly enriched (p<0.001, q<0.25), suggesting that miR-7977 modulates the Hippo-YAP signaling pathway. Visualization of pathway and network showed that miR-7977 significantly reduced the expression of Hippo core kinase, STK4. The transfer of miR-7977 mimic inactivated the Hippo-YAP signaling pathway as proven by GFP-tagged YAP nuclear trans localization and TEAD reporter assay. The miR-7977-transduced MSCs showed elevated saturation density and enhanced entry into the cell cycle. [Conclusion] Collectively, these results indicated that miR-7977 is an important factor that inhibits normal hematopoiesis via PCBP1 reduction and up-regulate the growth of functionally-disturbed MSCs via inactivation of Hippo-YAP signaling pathway in MDS/AML. Disclosures No relevant conflicts of interest to declare.

G-CSF Treatment Induces Toll-Like Receptor Signaling and Regulates Hematopoietic Stem Cell Function

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1181-1181 ◽  
Author(s):  
Laura G. Schuettpelz ◽  
Joshua N. Borgerding ◽  
Priya Gopalan ◽  
Matt Christopher ◽  
Molly Romine ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Intestinal Flora ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Tlr Signaling ◽  
Hematopoietic Stem

Abstract Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are unclear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect. RNA profiling and flow cytometry studies of HSCs from G-CSF treated mice show that multiple toll- like receptors (TLRs) are upregulated in HSCs upon G-CSF treatment, and gene set enrichment analysis shows enhancement of TLR signaling in G-CSF-treated HSCs. G-CSF-induced expansion of phenotypic HSCs is reduced in mice lacking the TLR signaling adaptors MyD88 or Trif, and the induction of quiescence is abrogated in mice lacking these adaptors. Furthermore, loss of TLR4 mitigates the G-CSF-mediated HSC repopulating defect. Interestingly, baseline HSC function is also dependent on TLR signaling. We show that HSC long-term repopulating activity is enhanced in Tlr4-/- and MyD88-/- mice, but not Trif-/- mice. One potential source of TLR ligands affecting HSC function in the bone marrow is the gut microbiota. Indeed, we show that in mice treated with antibiotics to suppress intestinal flora, G-CSF induced HSC quiescence and hematopoietic progenitor mobilization are attenuated. Moreover, in germ free mice, HSC long-term repopulating activity is enhanced. Collectively these data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling. Our finding of enhanced TLR signaling upon G-CSF treatment, and the mitigation of G-CSF’s effects in mice deficient for TLR signaling or commensal organisms, suggest that TLR antagonists and/or agonists may ultimately be used clinically to enhance engraftment following bone marrow transplantation or applied toward the treatment of patients with bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Adhesion Signaling States in AML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2386-2386 ◽  
Author(s):  
Chenyue W Hu ◽  
Amina A Qutub ◽  
Yihua Qiu ◽  
Suk Young Yoo ◽  
Nianxiang Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Principal Component ◽  
Leukemic Cells ◽  
Holistic View ◽  
Npm1 Mutation ◽  
Expression Levels

Abstract Background Stromal contact in the bone marrow microenvironment is known to affect the resistance of leukemic cells to therapy, in particular the homing and engraftment of leukemic stem cells. This stromal interaction is mediated by the adhesion signaling pathway including extracellular matrix proteins (e.g. FN1, SPP1), cell surface and transmembrane proteins (e.g. CD44, integrin, CAV1), as well as intracellular binding proteins and enzymes (e.g. IGFBP2, PTK2, TGM2). Previous studies mostly examined these proteins in isolation, and hence they were unable to capture the coordination among subpathways and within patient subpopulations. Therefore, it is of key interest to study these proteins in their ensemble and obtain a holistic view of how adhesion signaling pathway gives rise to and affects different AML subpopulations. Methods To profile protein expressions in AML, we made a reverse phase protein array (RPPA) with proteins from leukemia enriched cells from 511 new AML patients. Both bone marrow (n=387) and peripheral blood (n=283) samples were used, with 140 cases having both. The RPPA was probed with 231 strictly validated antibodies, including antibodies against ITGA2, ITGB3, FN1, ITGAL, PTK2, IGFBP2, CD44, SPP1, CAV1 and TGM2. The normal bone marrow derived CD34+ cells were used for comparison. The protein expression data generated from this RPPA was then analyzed by the Standard Proteomic Analysis (SPA), a combination of computational methods including clustering, principal component analysis, network reconstruction (glasso), survival analysis, correlation tests and data mining from public databases. Results Based on the expression levels of ten proteins in the adhesion pathway, we first built a heatmap (Figure A) using “Prototype Clustering” that grouped all patients into six distinct clusters featured by C1) pan low, C2) high SPP1-CD44; C3) high CAV1; C4) high PTK2-ITGA2-ITGB3-FN1-IGFBP2-TGM2; C5) high TGM2; C6) high ITGAL and pan intermediate high expression levels. The adhesion pathway in AML showed literature-consistent patterns, e.g. the coupling between CD44 and SPP1 and the co-expression among integrin subunits, FN1 and PTK2, but also demonstrated new patterns, e.g. independent regulation of CAV1, as well as decoupled expression of TGM2 from the integrins. Each patient cluster represents an adhesion signaling state that can be seen in AML. As shown in this transition map (Figure B), there is an OFF state (C1), two isolated activation states of either CAV1 (C3) or CD44-SPP1 (C2), two intermediate activation states of either TGM2 (C5) or ITGAL (C6), and a combined activation state (C4) from the two intermediate states. By combining both connections inferred from the data and interactions collected from public databases (e.g. String, KEGG), we were able to expand the protein network beyond adhesion pathway and examine their expression levels in each adhesion signaling state. We observed positive co-regulation of SRC and PRKCA with the integrin subunits, connections between IGFBP2 and metabolism/synthesis proteins (e.g. GADPH, EIF4E, GSK), as well as the association of CD44 with histone modification (H3K4Me2, H3K4Me3), most of which have not been reported before. The adhesion activation states are not associated with most clinical correlates, including FLT3 and NPM1 mutation, gender and response status, with the exception of the CAV1 activation state (C3). A significant amount of patients with high CAV1 expression levels are in the favorable cytogenetics group (35% vs. 8% in general, p=0.00001), thus have fewer relapses (relapse rate of 26% vs. 64% in general, p=0.001) and superior overall survival (Figure C) and remission duration (Figure D). Conclusions We have discovered previously unrecognized protein expression patterns and activation states that control stromal contact and adhesion in AML. This includes independent activation of SPP1-CD44 and CAV1, intermediate activation of TGM2 and ITGAL and combined activation of integrin-FN1-PTK2, indicating diverse stromal interaction states in the bone marrow. In particular, the activation of CAV1 is prognostically favorable, suggesting a potential target for future therapeutics in AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Transmembrane p24 Trafficking Protein 2 Regulates Inflammation Through the TLR4/NF-κB Signaling Pathway in Lung Adenocarcinoma

2021 ◽  
Author(s):  
Longhua Feng ◽  
Pengjiang Cheng ◽  
Zhengyun Feng ◽  
Xiaoyu Zhang
Keyword(s):  
Lung Adenocarcinoma ◽  
Signaling Pathway ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Inflammatory Factors ◽  
Normal Tissues ◽  
Kaplan Meier ◽  
New Strategy

Abstract Background: To investigate the role of transmembrane p24 trafficking protein 2 (TMED2) in lung adenocarcinoma (LUAD) and determine whether TMED2 knockdown could inhibit LUAD in vitro and in vivo.Methods: TIMER2.0, Kaplan-Meier plotter, gene set enrichment analysis (GSEA), Target Gene, and pan-cancer systems were used to predict the potential function of TMED2. Western blotting and immunohistochemistry were performed to analyze TMED2 expression in different tissues or cell lines. The proliferation, development, and apoptosis of LUAD were observed using a lentivirus-mediated TMED2 knockdown. Bioinformatics and western blot analysis of TMED2 against inflammation via the TLR4/NF-κB signaling pathway were conducted. Results: TMED2 expression in LUAD tumor tissues was higher than that in normal tissues and positively correlated with poor survival in lung cancer and negatively correlated with apoptosis in LUAD. The expression of TMED2 was higher in tumors or HCC827 cells. TMED2 knockdown inhibited LUAD development in vitro and in vivo and increased the levels of inflammatory factors via the TLR4/NF-κB signaling pathway. TMED2 was correlated with TME, immune score, TME-associated immune cells, their target markers, and some mechanisms and pathways, as determined using the TIMER2.0, GO, and KEGG assays.Conclusions: TMED2 may regulate inflammation in LUAD through the TLR4/NF-κB signaling pathway, and enhance the proliferation, development, and prognosis of LUAD by regulating inflammation, which provide a new strategy for treating LUAD by regulating inflammation.

CENPF/CDK1 Signaling Pathway Enhances the Progression of Adrenocortical Carcinoma by Regulating the G2/M-phase Cell Cycle

2021 ◽  
Author(s):  
Yugang Huang ◽  
Dan Li ◽  
Li Wang ◽  
Xiaomin Su ◽  
Xian-bin Tang
Keyword(s):  
Cell Cycle ◽  
Signaling Pathway ◽  
Adrenocortical Carcinoma ◽  
Immune Cell ◽  
Interaction Network ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Phase Cell ◽  
Aberrant Expression ◽  
M Phase

Abstract Adrenocortical carcinoma (ACC) is an aggressive and rare malignant tumor and prone to local invasion and metastasis. While, overexpressed Centromere Protein F (CENPF) is closely related to oncogenesis of various neoplasms, including ACC. However, the prognosis and exact biological function of CENPF in ACC remains largely unclear. In present essay, the expression of CENPF in human ACC samples, GEO and TCGA databases depicted that CENPF were overtly hyper-expressed in ACC patients and positively correlated with tumor stage. The aberrant expression of CENPF was significantly correlated with unfavorable overall survival (OS) in ACC patients. Then, the application of gene-set enrichment analysis (GSEA) declared that CENPF was mainly involved in the G2/M-phase mediated cell cycle and p53 signaling pathway. Further, a small RNA interference experiment was conducted to demonstrate that the interaction between CENPF and CDK1 enhanced the G2/M-phase transition of mitosis, cell proliferation and might induce p53 mediated anti-tumor effect in human ACC cell line, SW13 cells. Lastly, two available therapeutic strategies, including immunotherapy and chemotherapy, have been further probed. Immune infiltration analysis highlighted that ACC patients with high CENPF expression harbored significantly different immune cell populations, and high TMB/MSI score. Then, the gene-drug interaction network stated that CENPF inhibitors, such as Cisplatin, Sunitinib, and Etoposide, might serve as potential drugs for the therapy of ACC. Briefly, CENPF and related genes might be served as a novel prognostic biomarker or latent therapeutic target for ACC patients.

scholarly journals Analysis of Ferroptosis-Mediated Modification Patterns and Tumor Immune Microenvironment Characterization in Uveal Melanoma

2021 ◽  
Vol 9 ◽  
Author(s):  
Yi Jin ◽  
Zhanwang Wang ◽  
Dong He ◽  
Yuxing Zhu ◽  
Lian Gong ◽  
...  
Keyword(s):  
Uveal Melanoma ◽  
Enrichment Analysis ◽  
Progression Free Survival ◽  
Gene Set Enrichment Analysis ◽  
Consensus Clustering ◽  
Training Set ◽  
Poor Overall Survival ◽  
Competing Endogenous Rna Network ◽  
Underlying Mechanisms ◽  
Cox Analysis

Uveal melanoma (UVM) is an intraocular malignancy in adults in which approximately 50% of patients develop metastatic disease and have a poor prognosis. The need for immunotherapies has rapidly emerged, and recent research has yielded impressive results. Emerging evidence has implicated ferroptosis as a novel type of cell death that may mediate tumor-infiltrating immune cells to influence anticancer immunity. In this study, we first selected 11 ferroptosis regulators in UVM samples from the training set (TCGA and GSE84976 databases) by Cox analysis. We then divided these molecules into modules A and B based on the STRING database and used consensus clustering analysis to classify genes in both modules. According to the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA), the results revealed that the clusters in module A were remarkably related to immune-related pathways. Next, we applied the ESTIMATE and CIBERSORT algorithms and found that these ferroptosis-related patterns may affect a proportion of TME infiltrating cells, thereby mediating the tumor immune environment. Additionally, to further develop the prognostic signatures based on the immune landscape, we established a six-gene-regulator prognostic model in the training set and successfully verified it in the validation set (GSE44295 and GSE27831). Subsequently, we identified the key molecules, including ABCC1, CHAC1, and GSS, which were associated with poor overall survival, progression-free survival, disease-specific survival, and progression-free interval. We constructed a competing endogenous RNA network to further elucidate the mechanisms, which consisted of 29 lncRNAs, 12 miRNAs, and 25 ferroptosis-related mRNAs. Our findings indicate that the ferroptosis-related genes may be suitable potential biomarkers to provide novel insights into UVM prognosis and decipher the underlying mechanisms in tumor microenvironment characterization.

scholarly journals The Role of Circular RNA CDR1as/ciRS-7 in Regulating Tumor Microenvironment: A Pan-Cancer Analysis

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 429 ◽  
Author(s):  
Zou ◽  
Zheng ◽  
Deng ◽  
Yang ◽  
Xie ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Signaling Pathway ◽  
Malignant Tumors ◽  
Enrichment Analysis ◽  
Functional Enrichment Analysis ◽  
Circular Rna ◽  
Gene Set Enrichment Analysis ◽  
Functional Enrichment ◽  
Receptor Interaction

Circular RNA CDR1as/ciRS-7 functions as an oncogenic regulator in various cancers. However, there has been a lack of systematic and comprehensive analysis to further elucidate its underlying role in cancer. In the current study, we firstly performed a bioinformatics analysis of CDR1as among 868 cancer samples by using RNA-seq datasets of the MiOncoCirc database. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), CIBERSORT, Estimating the Proportion of Immune and Cancer cells (EPIC), and the MAlignant Tumors using Expression data (ESTIMATE) algorithm were applied to investigate the underlying functions and pathways. Functional enrichment analysis suggested that CDR1as has roles associated with angiogenesis, extracellular matrix (ECM) organization, integrin binding, and collagen binding. Moreover, pathway analysis indicated that it may regulate the TGF-β signaling pathway and ECM-receptor interaction. Therefore, we used CIBERSORT, EPIC, and the ESTIMATE algorithm to investigate the association between CDR1as expression and the tumor microenvironment. Our data strongly suggest that CDR1as may play a specific role in immune and stromal cell infiltration in tumor tissue, especially those of CD8+ T cells, activated NK cells, M2 macrophages, cancer-associated fibroblasts (CAFs) and endothelial cells. Generally, systematic and comprehensive analyses of CDR1as were conducted to shed light on its underlying pro-cancerous mechanism. CDR1as regulates the TGF-β signaling pathway and ECM-receptor interaction to serve as a mediator in alteration of the tumor microenvironment.

scholarly journals Ganghuo Kanggan Decoction in Influenza: Integrating Network Pharmacology and In Vivo Pharmacological Evaluation

2020 ◽  
Vol 11 ◽  
Author(s):  
Yanni Lai ◽  
Qiong Zhang ◽  
Haishan Long ◽  
Tiantian Han ◽  
Geng Li ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Influenza A ◽  
Target Prediction ◽  
Enrichment Analysis ◽  
New Drugs ◽  
Network Pharmacology ◽  
Pathway Enrichment Analysis ◽  
Active Components ◽  
Compound Screening

Background: Ganghuo Kanggan decoction (GHKGD) is a clinical experience prescription used for the treatment of viral pneumonia in the Lingnan area of China, and its clinical effect is remarkable. However, the mechanism of GHKGD in influenza is still unclear.Objective: To predict the active components and signaling pathway of GHKGD and to explore its therapeutic mechanism in influenza and to verified it in vivo using network pharmacology.Methods: The potential active components and therapeutic targets of GHKGD in the treatment of influenza were hypothesized through a series of network pharmacological strategies, including compound screening, target prediction and pathway enrichment analysis. Based on the target network and enrichment results, a mouse model of influenza A virus (IAV) infection was established to evaluate the therapeutic effect of GHKGD on influenza and to verify the possible molecular mechanism predicted by network pharmacology.Results: A total of 116 candidate active compounds and 17 potential targets were identified. The results of the potential target enrichment analysis suggested GHKGD may involve the RLR signaling pathway to reduce inflammation in the lungs. In vivo experiments showed that GHKGD had a protective effect on pneumonia caused by IAV-infected mice. Compared with the untreated group, the weight loss in the GHKGD group in the BALB/c mice decreased, and the inflammatory pathological changes in lung tissue were reduced (p &lt; 0.05). The expression of NP protein and the virus titers in lung were significantly decreased (p &lt; 0.05). The protein expression of RIG-I, NF-kB, and STAT1 and the level of MAVS and IRF3/7 mRNA were remarkably inhibited in GHKGD group (p &lt; 0.05). After the treatment with GHKGD, the level of Th1 cytokines (IFN-γ, TNF-α, IL-2) was increased, while the expression of Th2 (IL-5, IL4) cytokines was reduced (p &lt; 0.05).Conclusion: Through a network pharmacology strategy and in vivo experiments, the multi-target and multi-component pharmacological characteristics of GHKGD in the treatment of influenza were revealed, and regulation of the RLR signaling pathway during the anti-influenza process was confirmed. This study provides a theoretical basis for the research and development of new drugs from GHKGD.

p53 Activation of Mesenchymal Stromal Cells (MSC) Partially Abrogates Microenvironment-Mediated Resistance to FLT3 Inhibition In FLT3/ITD AML

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2151-2151
Author(s):  
Kensuke Kojima ◽  
Teresa McQueen ◽  
Elizabeth J. Shpall ◽  
Rui-Yu Wang ◽  
Michael Andreeff
Keyword(s):  
Bone Marrow ◽  
Protective Effect ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Protein Levels ◽  
Dismal Prognosis ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
P53 Activation

Abstract Abstract 2151 Fms-like tyrosine kinase-3 (FLT3) inhibitors have recently been introduced to overcome the dismal prognosis of acute myeloid leukemia (AML) with FLT3/ITD mutations. However, while ciculating blasts are rapidly eliminated, bone marrow (BM) responses are in general less impressive (Zhang et al., JCI 2009). One potential explanation for the reduced bone marrow response compared to the striking activity against circulating blast cells may be microenvironmental resistance to FLT3 inhibitors, including protection of FLT3/ITD+ blasts through the SDF-1 (CXCL12)/CXCR4 axis (Zeng et al., BLOOD 2009) and novel strategies to overcome microenvironmental resistance may further enhance the clinical benefit of FLT3 inhibitors. Here we investigated the role of p53 in bone marrow stromal cells in stromal cell-mediated resistance to FLT3 inhibition in FLT3/ITD AML. We confirmed that Mdm2-p53 interaction and mutant FLT3 signaling in leukemic blasts were inhibited by the Mdm2 inhibitor Nutlin-3a and the selective FLT3 inhibitor FI-700, respectively, as previously reported (Kojima et al., BLOOD 2004; Kojima et al., LEUKEMIA 2009). BM samples were obtained from normal individuals or from FLT3/ITD AML patients with more than 70% leukemia cells after informed consent. BM-derived stromal cells were seeded in 12-well plates in MEM-alpha medium and exposed to Nutlin-3a for 24 hours, gamma-irradiated (2 Gy), or treated with 100 nM doxorubicin. The wells were then washed three times and AML cells were added. Cell cycle distribution was determined by propidium iodide staining and apoptosis by Annexin V binding. In FLT3/ITD AML cell lines and primary cells, apoptosis was induced by FI-700, but apoptosis induction was diminished under stromal coculture conditions (43.2 ± 0.5% versus 17.0 ± 1.2% in MOLM-13; p < .01). FI-700–induced reduction of Mcl-1 protein levels and activation of Bax were essentially abrogated when FLT3/ITD AML cells were co-cultured with MSC (20.9 ± 1.6% versus 8.3 ± 0.3% cells with activated Bax levels in MOLM-13;p < .01). Separation of MOLM-13 cells from stroma by 0.4 μM culture filters did not affect the protective effect of stromal cells (21.2 ± 2.3% versus 21.6 ± 1.6%). The protection appeared to be mediated partially by SDF-1/CXCR4 signaling because the addition of recombinant SDF-1 to control medium protected FLT3/ITD AML cells from FI-700–induced apoptosis while the addition of anti-CXCR4 antibody (R&D Systems) partially abrogated the protection conferred by stromal cells. The protective effect of stromal cells on MOLM-13 was significantly reduced when MSC were exposed to p53 inducers including Nutlin-3a (17.0 ± 1.2% versus 26.0 ± 1.8%; p < .01), gamma-irradiation (13.5 ± 3.0% versus 18.6 ± 3.1%; p < .05) or doxorubicin (14.2 ± 3.0% versus 19.7 ± 0.9%; p < .05), although p53 activation was not itself cytotoxic to stromal cells. However, SDF-1 mRNA and protein levels were reduced in p53-activated stroma cells. Our findings confirm that selective FLT3 inhibition may not eradicate FLT3/ITD AML cells protected by stroma and show, for the first time, that p53 activation in stroma cells blunts stroma cell-mediated resistance to a kinase, here a FLT3 inhibitor, in part through regulation of SDF-1. Results suggest that combinations of HDM2/p53 (R7112/Nutlin-3a, Andreeff et al. ASH 2010) and/or CXCR4 inhibitors (Plerixafor, Konopleva et al. ASH 2010) with FLT3 inhibitors should be investigated in clinical trials targeting FLT3 mutant leukemias, with the dual goal of inducing apoptosis in leukemic cells and, concomittantly, reduce the protective effects exerted by the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Establishment of a Novel CNS Infiltrated Xenograft Model through Engraftment of Patient-Derived Acute Lymphoblastic Leukemic Cells Into NOD/SCID/γc Null Mouse

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3248-3248
Author(s):  
Itaru Kato ◽  
Akira Niwa ◽  
Megumu Saito ◽  
Hisanori Fujino ◽  
Satoshi Saida ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Hematologic Malignancy ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Leukemic Cells ◽  
Null Mouse ◽  
Immunodeficient Mice ◽  
Mouse Xenograft Model ◽  
Nog Mice

Abstract Abstract 3248 Background and Purpose: Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although improvements in treatment regimen have raised the 5-year survival rate as high as 80% for pediatric ALL patients, a minority of patients with various risk factors, including central nervous system (CNS) infiltration continue to have poor prognosis. Recently, bone marrow (BM) microenvironments which support leukemic stem cells have become noticed as an important element which can influence treatment response and relapse of the disease. Although leukemic cells appear to be completely eradicated through treatment, they are thought to survive within bone marrow and/or extramedullary microenvironments, such as CNS, causing disease recurrence. However, little is known about the CNS microenvironment for leukemic cells because of the lack of appropriate animal model. Even though several investigators have tried to establish a CNS infiltrated model of leukemia, major limitation with these studies are the use of leukemic cell lines and the preconditioning of recipient mice, which did not represent CNS leukemia observed in patients. Here we report the establishment of a novel xenograft model for primary human ALL using NOD/SCID/γc null (NOG) mouse. Without irradiation, this model recapitulates CNS as well as extramedullary leukemic infiltration (hereby referred to as the h-leukemic NOG model). Result: Primary bone marrow samples were collected from 9 children with ALL at the time of diagnosis with informed consent. The leukemic cells (1×106cells) were injected into the tail veins of non-irradiated 8- to 10-week old NOG mice. Primary samples from 8 out of 9 patients were successfully engrafted. Engrafted leukemic cells could be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as 95% BM chimerism and showed that blast phenotypes were conserved through serial transplantations. Of note, extramedullary organs including the CNS, liver, spleen, and kidneys showed the leukemic invasion consistent with those of the donor ALL patients. Liver pathology in the h-leukemic NOG model is identical to that seen in the ALL patients. We also showed the existence of a functional niche in the liver mediated by SDF-1/CXCR4 axis. In terms of the CNS involvement, we observed the progressive infiltration of leukemic cells into the Virchow-Robin space that is consistent with the pathology of human ALL patients. Using this model, we examined the mechanism of dissemination and harboring of leukemic cells in the CNS niche. Discussion: NOG mice model for engraftment of human leukemic cells provides useful insights into the biology of ALL and allows us to answer various questions concerning the mechanism of extramedullary invasion and expansion. We have reported that NOG mice have significantly better human hematopoietic cell engraftment in the BM and extramedullary organs than other immunodeficient mice (Hiramatsu H. Blood. 2003), and is capable of supporting the growth of human neoplastic cells (Kato M. Nature. 2009). Here we report that this non-preconditioned mouse xenograft model reproduces leukemic extramedullary involvement, including the CNS, in sustaining leukemic cells. This approach provides a more sophisticated and physiological model suitable for the evaluation of molecular interactions between patient leukemic cells and host niche. Our h-leukemic NOG model will provide a powerful tool to analyze the CNS niche that harbors leukemia initiating cells. Moreover, this model would be a useful platform for developing novel anti-leukemic therapies that target CNS extramedullary niche. Disclosures: No relevant conflicts of interest to declare.

Role of hERG1 K+ Channels as a Positive Regulator In SDF-1alpha-Mediated Proliferation of Leukemia Cells and Leukemia Stem/Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4786-4786
Author(s):  
Fang Zheng ◽  
Huiyu Li ◽  
Fang Liu ◽  
Wen Du ◽  
Shiang Huang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Leukemic Cell ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
K Channels

Abstract Abstract 4786 Background: Mounting evidence that leukemia stem cells (LSCs) occupy and receive important signals from specialized areas (“niches”) that alter the stromal microenvironment and disrupt normal hematopoiesis. The innovative therapeutic strategies focus on targeting of microenvironmental interactions in leukemia. Therefore, it is important to fully elaborate the mechanisms of microenvironment- mediated leukemogenesis. Stromal-cell derived factor-1alpha (SDF-1à) is the main cytokine produced by bone marrow stromal cells. The SDF-1à/CXCR4 axis specifically mediates homing and migration of leukemic blasts. While our previous work has shown that SDF-1à significantly increases hERG1 K+ tail current and a specific hERG1 K+ channels inhibitor significantly blocks SDF-1à- induced migration of leukemic cells. In fact, recent studies suggested that the human ether à-go-go-related gene (HERG) K+ channels are constitutively expressed in AML stem/progenitor cells, and regulate cell proliferation as well as clinical prognosis. Here we investigate the hypothesis that a new leukemic blast–stromal interaction is mediate by hERG1 K+ channels and SDF-1à. Methods: Proliferation assay, apoptosis and cell cycle analysis were used to analyze effects of E-4031(a specific hERG1 K+ channels inhibitor) in the presence of SDF-1à on leukemia cell lines HL-60. RT–PCR and western blot analysis were used to determine changes in herg1 expression and Wnt/β-catenin signaling pathway in response to SDF-1à in the presence and absence of E-4031. Primary leukemias obtained from the bone marrow of de novo AML patients (n=6) at diagnosis. Mononuclear cells were isolated from the samples using Ficoll-Paque density gradient separation, and cultured with SDF-1à in the presence and absence of E-4031. AML colony-forming cell (CFC) assays and flow cytometry were performed to assess the effects of E-4031 in the presence of SDF-1à on LSCs. Results: SDF-1a enhanced cell proliferation in a dose-dependent manner. The maximal increase by 1.6 times was obtained for 100ng/ml. While this effect was impaired by E-4031, which significantly impaired cell proliferation induced by SDF-1a with a concentration of 100ng/mL by (40.3±8.4)%. In addition, E-4031 inhibited SDF-1a-stimulated leukemic cell proliferation by inducing G0/G1 arrest. Cell apoptosis analysis revealed that either E-4031 or SDF-1a has direct effect on HL-60 cell apoptosis. Unexpected, there was no significant synergistic effect upon apoptosis. After exposures to 100ng/ml SDF-1à, hERG1 mRNA and protein levels increased significantly, by approximately 1.5-fold above control levels. Moreover, SDF-1a increased the expression of Wnt/β-catenin target genes, including β-catenin, cyclin-D1, and c-myc. Interestingly, this manner was abolished by E-4031. The presence of progenitor cells was evaluated by plating suspension cells cultured with SDF-1a in CFC assays. E-4031 decreased numbers of CFC in suspension to 77.3%. Upon expansion with SDF-1a, E-4031 resulted in a significant reduction in the number of progenitors to 31.8%. The effects on LSCs were determined on phenotypically described stem cells from AML. Treatment with 1μ M E-4031 for 48 hours inhibited the proliferation of LCSs compared with untreated controls, a mean viability of 11.8% for CD34+CD38- and 10.4% for CD34+CD38+. In contrast, a significant decrease in the viability of stem cells after E-4031 in the present of SDF-1a treatment, with only 9.6% for CD34+CD38- and 9.5% for CD34+CD38+. Conclusions: Initial studies provided evidence that the hERG1 K+ channels and SDF-1 emerged as mediators of stromal/leukemic cell interactions, which largely contribute to the proliferation mediated by the microenvironment. Likewise, other components of bone marrow microenvironment, such as Wnt/β-catenin signaling pathway, may modulate hERG1 K+ channels in leukemic cells. Taken together, these results provided rationale for studies of new molecular events involved in bone marrow microenvironment and leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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