scholarly journals Changes in Activated Bone Marrow Macrophages and Mast Cells in Patients with Myelofibrosis Following Ruxolitinib Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3184-3184 ◽  
Author(s):  
Hans Michael Kvasnicka ◽  
Juergen Thiele ◽  
Carlos E. Bueso-Ramos ◽  
Shilpa Kamalanabhaiah ◽  
Jorge E. Cortes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Research Funding ◽  
Statistical Significance ◽  
Tnf Alpha ◽  
Spleen Size ◽  
Who Grade ◽  
Expression Levels ◽  
Anti Inflammatory

Abstract Background: Macrophages (MAC) are key regulators of malignant progression in solid tumors by promoting tumor cell invasion, migration and angiogenesis. Only few studies have investigated the role of activated MAC phenotypes, such as M1 and M2, in the bone marrow (BM) microenvironment of myelofibrosis (MF). MAC are generally increased in the BM of patients (pts) with MF and recent studies have also identified mast cells (MC) as playing an important role in regulating the underlying inflammatory process in MF. With regard to the substantial improvement in the mostly cytokine-driven constitutional symptoms following JAK inhibition therapy, we have investigated the effect of ruxolitinib (RUX) on BM MAC subtypes and MC and their association with BM fibrosis. Methods: A total of 63 pts with high-risk MF were included in this analysis. All pts had both baseline (BL) and sequential BM trephine biopsy at 24 months (mo) following RUX therapy. Using specific immunohistochemical stains, we analyzed assorted MAC markers, including CD68 and CD163. CD68 is a pan-MAC marker that recognizes both M1 and anti-inflammatory M2 subtypes. CD163 is a scavenger receptor upregulated by MAC within an anti-inflammatory environment and regarded as a highly specific monocyte/macrophage marker for the M2 subtype. Anti-Mast Cell Tryptase was applied as specific antibody to identify BM MC. All sections were stained following routine procedures and quantification of positivity was performed for each marker by consensus after independent review by 3 pathologists. Cytokine expression levels (TNF-alpha, MIP-1-alpha, IgE) at week 4 and 24 mo following RUX therapy were available for a subset of 23 and 16 pts. Individual changes were categorized as increase, stable, or decrease and correlated to the degree of WHO BM fibrosis grade and hematological features. Results: At BL, 81.0% of MF pts presented with an advanced fibrotic stage of disease (WHO grade 2 or 3). Grade of BM fibrosis significantly correlated with the frequency of CD68+ and CD168+ MAC. Furthermore, advanced fibrosis was associated with a higher frequency of BM MC. Following RUX treatment, 14.3% of cases showed an improvement in BM fibrosis, while 58.7% showed stabilization. RUX induced in 48.3% of cases a significant decrease in the overall amount of BM CD68+ MAC, whereas a further increase was observed in only 6.9%. Similar results were obtained for the specific CD163+ anti-inflammatory M2 subtype. Post-RUX a significant reduction of this cell lineage was seen in 47.6%, while 12.7% of cases revealed a further increase. Improvement in BM fibrosis was highly correlated with an overall reduction of CD68+ MAC, and in particular with modulation of the CD163+ M2 subtype. RUX therapy induced a profound reduction in the expression of associated cytokines such as TNF-alpha and MIP-1-alpha, both at 4 weeks and 24 mo. Frequency of BM MC was reduced in 26.3% of pts during therapy, however, in 49.1% therapy showed no change. Pts with improvement in BM fibrosis revealed in most cases lower frequencies of MC, but this association did not reach statistical significance. In contrast, expression levels of IgE were strongly reduced in almost all pts at week 4 and 24 mo. The amount of MAC or MC did not correlate with BL hematological or clinical parameters such as BL spleen size, anemia or platelet counts. Following RUX, decreases in M1 and M2 MAC were associated with changes in hemoglobin levels and spleen size reduction. Conclusions: Our results significantly extend previous observations on the role of the BM microenvironment in MF. RUX treatment meaningfully and directionally impacts the amount of activated anti-inflammatory MAC in BM of MF patients. Overall, our data advocate the strong disease modulation capacity of anti-JAK therapy. Disclosures Kvasnicka: Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Honoraria. Thiele:Novartis: Consultancy, Honoraria; Incyte Corporation: Consultancy, Honoraria. Kantarjian:Novartis: Research Funding. Verstovsek:NS Pharma, Inc: Research Funding.

Role of common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of murine mast cells

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2172-2180 ◽  
Author(s):  
Kotaro Suzuki ◽  
Hiroshi Nakajima ◽  
Norihiko Watanabe ◽  
Shin-ichiro Kagami ◽  
Akira Suto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Cytokine Receptor ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Wild Type ◽  
Peritoneal Mast Cells ◽  
The Common

Abstract The regulatory roles of the common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using γc-deficient (γc−) and Jak3-deficient (Jak3−) mice. Although the mast cells in γc− and Jak3− mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in γc− and Jak3− mice as compared with that in wild-type mice. Among γc-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow–derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from γc− and Jak3−mice. In addition, IL-4Rα, γc, and Jak3, but not IL-2Rβ or IL-7Rα, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Rα1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from γc− and Jak3− mice. These results indicate that γc- and Jak3-dependent signaling is essential for IL-4– and IL-9–induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a γc- and Jak3-dependent manner.

Potential Therapeutic Role of the Selective Adhesion Molecule (SAM) Inhibitor Natalizumab in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1850-1850 ◽  
Author(s):  
Klaus Podar ◽  
Alexander Zimmerhackl ◽  
Ursula Hainz ◽  
Mariateresa Fulciniti ◽  
Sonia Vallet ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Adhesion Molecule ◽  
Research Funding ◽  
Therapeutic Role ◽  
Potential Therapeutic Role

Abstract Abstract 1850 Poster Board I-876 Multiple Myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells in the bone marrow. Despite current therapeutic approach and prolongation of the median survival, new therapies are urgently needed. Integrins are cell surface receptors which mediate both cell-cell adhesion and cell-extracellular matrix (ECM) protein adhesion. beta1-integrins, including very-late antigen-4 (VLA-4;á4β1), are typically expressed on MM cells. In MM, VLA-4-mediated binding to ECMS and bone marrow stromal cells (BMSCs) confers protection against drug-induced apoptosis and triggers transcription and secretion of IL-6, the major MM growth and survival factor. In addition to up-regulation of cell surface-clustering, integrin activity can also be triggered by multiple agonists through ‘inside-out’ signaling, independent of changes in integrin expression levels. Importantly, VEGF-induced migration of MM cells on fibronectin is also associated with β1-integrin- and PI3-kinase- dependent PKC activation. Targeting VLA-4 is therefore of potential high therapeutic interest in MM. Indeed, an antibody against murine á4 induces inhibition of MM growth in a murine model. Natalizumab is a recombinant humanized IgG4 monoclonal antibody, which belongs to a new class of molecules known as selective adhesion molecule (SAM) inhibitors and binds to á4-integrin. Clinically, Natalizumab has demonstrated activity in patients with multiple sclerosis and Crohn's disease. Here we tested the potential therapeutic role of Natalizumab on MM cell survival, and migration in the BM microenvironment. VLA-4 is expressed by all MM cell lines investigated (NCIH929, RPMI8226, INA-6, MM.1S, and OPM2). Functionally, Natalizumab but not a control antibody, triggered dose-dependent inhibition of MM cell adhesion to fibronectin, BMSCs, and endothelial cells (ECs). Importantly, inhibition of adhesion to fibronectin, BMSCs, or ECs was observed in MM cells pretreated with Natalizumab. Moreover, inhibition of MM cell adhesion to fibronectin, BMSCs, or ECs was also observed when Natalizumab was added to already adherent MM cells. Taken together, Natalizumab decreases adhesion of non-adherent MM cells as well as binding of already adherent MM cells to non-cellular and cellular components of the microenvironment. Given the protective role of the microenvironment on MM cell survival, we next sought to evaluate the chemosensitizing activity of Natalizumab. Specifically, we investigated dose- and time- dependent effects of Natalizumab, alone and when combined with conventional and novel therapies, on MM cells. Our results show that Natalizumab alone did not inhibit growth or survival of MM cells when cultured without components of the microenvironment. However, Natalizumab enhanced sensitivity of tumor cells to both bortezomib and dexamethasone in MM-BMSC and, MM-EC co-cultures. These data indicate a potential role of Natalizumab in bortezomib- and dexamethasone-containing treatment regimens including MPV. Moreover, Natalizumab decreases IL-6 and VEGF secretion triggered in MM-BMSC co-cultures. Consequently, angiogenesis triggered by supernatants of Natalizumab- treated MM-BMSC co-cultures was inhibited. Moreover, Natalizumab blocked MM cell migration on fibronectin triggered by both VEGF and IGF-1. Finally, our previous results implicate an PKC signaling in MM cell migration on fibronectin, and our current results show that Natalizumab inhibits phosphorylation of á4 integrins and PKC induced by co-stimulation with VEGF/ fibronectin, IGF-1/ fibronectin, and patient serum. Taken together, our data indicate a potential therapeutic role of Natalizumab in MM. Ongoing studies evaluating the effect of Natalizumab in a SCID-hu murine model of MM will also be reported. Disclosures: Podar: Biogen Idec: Research Funding. Off Label Use: natalizumab, integrin inhibitor. Zimmerhackl:Biogen Idec: Research Funding. Olsen:Biogen Idec: Employment.

Genome Wide DNA Methylation Profiling In Patients with Multiple Myeloma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3622-3622
Author(s):  
Yang Liu ◽  
Shenghua Duan ◽  
Xavier Leleu ◽  
Yong Zhang ◽  
Abdel Kareem A. Azab ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Cell Lines ◽  
Promoter Region ◽  
Genomic Dna ◽  
Research Funding ◽  
Plasma Cells ◽  
Genome Wide

Abstract Abstract 3622 Introduction: Epigenetic factors such as DNA methylation have been shown to play a crucial role in the pathogenesis and progression of multiple myeloma (MM), yet studies of DNA methylation in MM are still limited. Therefore, in order to better understand the role of DNA methylation and identify specific genes that may be affected by differential methylation in MM patients, we conducted genome-wide DNA methylation profiling in cd138+ plasma cells purified from bone marrow of the patients with MM and normal donors. Methods: Genomic DNA of CD138+ Plasma cell selected from both MM patients and normal primary bone marrow was extracted using QIAGEN genome isolation kit. Following extraction, methylated DNA was isolated by Chip and hybridized to Affymetrix Human 2.0 tiling arrays. Chip assay and array hybridization was performed by Genepathway Inc. CEL files were processed and normalized using the MAT program, and methylation peaks were called from the resulting MAT scores using a custom segmentation method. Peak annotation and characterization of different genomic regions was done with custom tools and using genome annotation files from the UCSC genome database. All peaks were visualized by IGB online software. Medip-PCR was done in human MM cell lines to validate the methylation status. Methylated gene expression was determined by both Semi-quantitative PCR and real-time PCR. 5′aza was used for demethylation in human MM cell lines. Methylated gene expression with or without 5′aza treatment was determined by both Semi-quantitative PCR and real-time PCR. Results: Genomic DNA from CD138+ plasma cells from bone marrow of MM patients showed a significant increase in methylation levels compared to normal controls. We demonstrated that the hypermethylated sites were distributed across the genome in the following proportions: 3.2% in the promoter region; 45.6% in the intragenic region; 5.4 % in the 3′ end region; and 46.8 % in the intergenic region. Furthermore, around 9 % promoter CpG islands (CGIs); 11% intragenic CGIs; 15 % CGIs in 3′end region; and 14.3 % intergenic CGIs of patients genomic DNA were methylated. Moreover 2.1% promoter CGIs; 2.3 % intragenic CGIs; 2.5% CGIs in 3′end region; and 4.7% intergenic CGIs were methylated for the normal control. Medip-PCR showed that the identified methylation pattern in MM patients showed similar results in MM cell lines. Expectedly, we also observed that suppressor of cytokine signaling 1 (SOCS1) was hypermethylated at the promoter region (MAT score=19.986) as has been reported in human cell lines. Importantly, another member of SOCS family SOCS3 showed much stronger signal in the promoter region with CpG island (MAT score=31.707) in MM patients compared to normal control. Notably, the expression of two members of TNFR superfamily TNFRSF18 and TNFRSF4 which play an important role in development and programmed cell death of lymphocyte significantly have increased 283 and 141-fold after treatment with 5′aza in MM cell lines. Conclusion: These findings enhance our understanding of the role of DNA methylation in MM, as one of the epigenetic changes that may contribute to the pathogenesis of this disease. The identification and functional characterization of novel key molecules affected by DNA methylation will provide deeper insight into the molecular basis of MM disease. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2424-2424
Author(s):  
Yang Liu ◽  
Yong Zhang ◽  
Phong Quang ◽  
Hai T Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Advisory Committees ◽  
Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Minimal Residual Disease Monitoring During Maintenance In Multiple Myeloma Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3126-3126 ◽  
Author(s):  
Alberto Rocci ◽  
Manuela Gambella ◽  
Paola Omedè ◽  
Daniela Drandi ◽  
Francesca Gay ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Maintenance Therapy ◽  
Research Funding ◽  
Plasma Cells ◽  
Residual Disease ◽  
Clinical Relapse ◽  
Advisory Committees ◽  
Minimal Residual

Abstract Background The quality of response and the residual disease after treatment are important prognostic factors in several hematological diseases including multiple myeloma (MM). Several papers demonstrated that the deeper the response after treatment, the longer the survival. However few data are available on the monitoring of minimal residual disease (MRD) during the maintenance therapy in transplant eligible MM patients. Aims to evaluate the role of maintenance therapy in reducing MRD and the role of monitoring the response to predict clinical relapse. Patients and Methods newly diagnosed MM patients enrolled in the RV-MM-EMN-441 trial (NCT01091831) and achieving at least a very good partial response (VGPR) after consolidation were included in the study. Patients received 4 Lenalidomide-Dexamethasone (RD) courses as induction, Cyclophosphamide to mobilize bone marrow stem cells (BMSC) and then were randomized to receive 6 cycles of Cyclophosphamide-Lenalidomide-Dexamethasone (CRD) or Autologous Stem Cell Transplantation (ASCT) with Melphalan 200 mg/m2. All patients received maintenance therapy with Lenalidomide (R) or Lenalidomide-Dexamethasone (RD) until relapse. MRD analysis was performed in a single laboratory (University of Turin, Italy) using flow cytometry according to European Myeloma Network guideline (Rawstron AC, Haematologica 2008). Samples of bone marrow (BM) were collected at diagnosis, after consolidation, after 3 and 6 courses of maintenance and then every 6 months until clinical relapse. The samples were considered MRD +ve if ≥ 0.01% of PC were detected. Immunophenotypic (IF) relapse was defined as an increase of ≥ 25% in the amount of malignant plasma cells in BM compared to the previous determination. Results Fifty patients (27 female/23 male) with a median age of 57 yrs (40-65) entered the study. According to ISS, 27 patients were stage I, 15 stage II and 8 stage III. Fish risk profile was standard in 31 patients, high in 11 and not available in 8. Twenty-five patients received CRD as consolidation and 25 underwent ASCT. The median follow-up was 28.6 months. After consolidation 16 (32%) patients achieve a complete response (CR) and 34 (68%) a VGPR. MRD was negative in 19/48 (40%) patients, of which 12 received ASCT (out of 23, 52%) and 7 received CRD (out of 25, 28%). Patients receiving ASCT showed a lower value of residual cells (median 0.08%, range 0 – 1.00) compared to patients receiving CRD (median 0.5%, range 0 – 2.9%, p=0.0134). The lower MRD value was achieved after consolidation in 31 patients (62%), after 3 courses of maintenance in 6 patients (12%) and after 6 or more courses of maintenance in 13 patients (26%). The increase in quality of response was observed primarily in patients receiving CRD: the average amount of residual plasma cells in bone marrow was 71/uL after induction, lowering to 51/uL after 6 and 12 courses of maintenance therapy. Nine patients clinically relapsed after an average time of 25.6 months from the beginning of the therapy and in all patients this was anticipated by immunophenotypic relapse. Conclusion 1) consolidation therapy with ASCT determines a deeper response compared to CRD; 2) maintenance therapy can improve the quality of response, in particular in patients not receiving ASCT; 3) Immunophenotypic relapse anticipate the clinical relapse. These results suggest the possible role of MRD monitoring to better assess the response to therapy also during maintenance and as marker of early relapse. Disclosures: Ladetto: Celgene: Research Funding, Speakers Bureau; Janssen Cilag: Research Funding, Speakers Bureau; Mundipharma: Research Funding, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Amgen: Research Funding, Speakers Bureau. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Palumbo:Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Consultancy, Honoraria.

Leukemia-Associated Mutations Of DNMT3A Inhibit Differentiation Of Hematopoietic Stem Cells and Promote Leukemic Transformation Through Abberant Recruitment Of Bmi1

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 469-469
Author(s):  
Junji Koya ◽  
Keisuke Kataoka ◽  
Takako Tsuruta-Kishino ◽  
Hiroshi Kobayashi ◽  
Kensuke Narukawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Cpg Island ◽  
Hematopoietic Stem ◽  
Cell Accumulation ◽  
Exogenous Expression ◽  
Marrow Cells

Whole genome sequencing has revealed DNMT3A mutation is present in over 20% of cytogenetically normal acute myeloid leukemia (CN-AML) and R882 is the most frequent and recurrent mutated site. Cumulating clinical data have emphasized the importance of the mutation as a poor prognostic factor of AML. Since the functional role of DNMT3A mutation in leukemogenesis remains largely unknown, we aimed to elucidate the impact of DNMT3A mutation on the development and maintenance of AML. To investigate the effect of exogenous expression of DNMT3A R882 mutant (Mut) in hematopoiesis, we transplanted 5-FU primed mouse bone marrow cells transduced with empty vector (EV), DNMT3A wild type (WT), or DNMT3A Mut to lethally irradiated mice. Recipients transplanted with DNMT3A Mut-transduced cells exhibited hematopoietic stem cell (CD150+CD48-Lin-Sca1+c-Kit+) accumulation and enhanced repopulating capacity compared with EV and DNMT3A WT recipients. To identify the downstream target genes of DNMT3A Mut that evoked hematopoietic stem cell accumulation, we sorted vector-transduced LSK cells from transplanted mice and conducted quantitative PCR (Q-PCR) of various hematopoiesis-related genes. Q-PCR revealed that Hoxb cluster expression was up-regulated and differentiation-associated genes, such as PU.1 and C/ebpa, were down-regulated in DNMT3A Mut-transduced LSK cells. Targeted bisulfite sequencing showed hypomethylation of the Hoxb2 promoter-associated CpG island in DNMT3A Mut-transduced cells compared with EV-transduced cells, which suggests dominant-negative effect of DNMT3A R882 mutation. DNMT3A Mut caused no change in methylation status of PU.1 promoter-associated CpG island, indicating that DNA methylation-independent mechanism underlies PU.1 downregulation. Given that DNMT3A interacts with several histone modifiers to regulate target gene transcription, we performed co-immunoprecipitation to investigate whether these interactions are altered by DNMT3A mutation. We found that DNMT3A Mut has the emhanced capacity to interact with polycomb repressive complex 1 (PRC1), which is thought to be a potential mechanism of the DNMT3A Mut-induced differentiation defect. Co-immunoprecipitation experiments showed that DNMT3A R882H and R882C mutant exhibited augmented interaction with BMI1 and MEL18, respectively. In addition, RING1B, an essential component of PRC1, co-localized with DNMT3A Mut more frequently than WT, irrespective of the type of amino acid substitution. Furthermore, heterozygosity of Bmi1 restored the PU.1 mRNA to the normal level and canceled the effect of stem cell accumulation in mice transplanted with DNMT3A Mut bone marrow cells. Chromatin immunoprecipitation in AML cell lines showed that BMI1 and RING1B were more efficiently recruited to the upstream regulatory element of PU.1 upon expression of DNMT3A Mut than WT, while the amount of DNMT3A recruited were comparable between DNMT3A WT and Mut. In the murine transplantation model, we found that exogenous PU.1 expression impaired repopulating capacity in both EV and R882H-transduced cells to the similar level. Exogenous expression of DNMT3A WT inhibited proliferation and induced terminal myeloid differentiation, whereas DNMT3A Mut-transduced cells remained immature in AML cell lines. DNMT3A Mut-transduced cells were resistant to ATRA-induced differentiation compared to EV-transduced cells. Furthermore, R882 mutation promoted blastic transformation of murine c-Kit+ bone marrow cells in vitro in combination with HOXA9 which is highly expressed in clinical cases harboring DNMT3A mutation. Morphological and surface marker analysis revealed these cells were F4/80+ monocytic blasts, consistent with clinical observation that DNMT3A mutation is found frequently in FAB M4/M5 leukemia. These results indicate a distinct role for DNMT3A Mut as well as a potential collaboration between DNMT3A Mut and HOXA9 in malignant transformation of hematopoietic cells. Interestingly, Bmi1 heterozygosity impaired this monoblastic transformation of R882H and HOXA9 co-transduced progenitors. Taken together, our results highlight the functional role of DNMT3A mutation in differentiation block of hematopoietic stem cells and in promoting leukemic transformation via aberrant recruitment of Bmi1 and other PRC1 components. Disclosures: Kurokawa: Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding.

Role of Glutamine in Metabolic and Epigenetic Reprogramming in AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2559-2559
Author(s):  
Juliana Vélez Luján ◽  
Niki Zacharias ◽  
Dinesh Rakheja ◽  
Tushar D Bhagat ◽  
Jaehyuk Lee ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Epigenetic Reprogramming ◽  
Cellular Growth ◽  
Hypoxic Conditions ◽  
Enhanced Production ◽  
Enzymatic Reduction ◽  
Isocitrate Dehydrogenase 2

Abstract Glutamine (Gln) was shown to play a role in generation of oncometabolite 2-hydroxyglutarate (2-HG) in tumors with high GLS (glutaminase) expression, whereby IDH2 (Isocitrate Dehydrogenase 2) enzyme catalyzes carboxylating reduction of glutamine-derived α-ketoglutarate (α-KG) to isocitrate and noncarboxylating reduction to 2-HG (Wise PNAS 2011). 2-HG in turn is known to inhibit α-KG dependent dioxygenases that mediate epigenetic events, including DNA and histone demethylation (Licht Cancer Cell 2010). A recent report demonstrated that hypoxia induces production of the L-enantiomer of 2-HG (L-2HG), through enzymatic reduction of α-KG by lactate dehydrogenase A (LDHA) (Intlekofer Cell Metabolism 2015). We have previously demonstrated that leukemic bone marrow microenvironment is highly hypoxic (Benito PLoS One 2011). Further, our unpublished data indicate upregulation of GLS protein and increase production of total 2-HG in AML (acute myeloid leukemia) cells cultured under hypoxia. We therefore propose a link between hypoxia, Gln metabolism, and epigenetic regulation in AML. Since increased methylation (and decreased hydroxymethylation) is seen in AML, we hypothesize that GLS inhibition can abrogate these changes via reduction of 2-HG levels. First, we examined effects of hypoxia and selective GLS inhibitor CB-839 (Calithera Biosciences) on cellular growth of AML cells with wild type IDH (OCI-AML3 and HL-60), cultured alone or co-cultured with bone marrow derived stromal cells (MSC). The culture of untreated OCI-AML3 alone in normoxic and hypoxic conditions caused a decrease in viability from 96 ± 2.5% to 84 ± 4.1% respectively, while the treatment with CB-839 (1 mM) for 6 days decreased viability in OCI-AML3 cells from 94 ± 0.23% to 71 ± 2.3% respectively (P=0.015). While MSC co-cultures improved survival of floating AML cells, the attached cells that were in direct contact with MSC were more affected under hypoxic conditions, having a viability of 64 ± 8.7% at the end of the experiment. These data indicate that GLS inhibitor is more effective under hypoxic conditions mimicking leukemic BM microenvironment. Hypoxia selectively induced the production of L-2HG (measured by liquid chromatography-tandem mass spectrometry) under hypoxic conditions (>40 fold) in OCI-AML3 cells, both with and without MSC co-culture. This increase in L-2HG was partially inhibited by co-treating OCI-AML3 cells with GLS inhibitor CB-839 (reduction of 1.7-fold in media only and 1.3-fold in MSC co-culture). Determination of hydroxymethylation (hmc) levels using HELP-GT assay demonstrated a significant increase in hmc in cells treated with CB-839. Of importance, genes that were differentially hydroxymethylated after CB-839 treatment belonged to important functional categories with cancer being the dominant pathway affected by these changes. Under hypoxia, glucose metabolism is known to be directed towards anaerobic glycolysis, with increased pyruvate-lactate enzymatic conversion by LDHA. To characterize the role of Gln and GLS on these processes within leukemia microenvironment, we performed nuclear magnetic resonance imaging with hyperpolarized pyruvate in NSG (NOD scid gamma) mice engrafted with GFP/luc-labeled OCI-AML3 cells. Inhibition of GLS in vivo following exposure of mice with 200 mg/kg dose of CB-839 showed a decrease in lactate conversion rate within leukemic bone marrow (femur area) (0.31 + 0.03 (pre) to 0.20 + 0.04 (post) P < 0.05), possibly due to the reduction of the level of NADH from decreased flux of Gln in the TCA cycle. In summary, our results indicate that Gln and GLS contribute towards hypoxia-induced production of L-2HG and critical epigenetic changes in AML; as well as playing a role in enhanced production of lactate from pyruvate. These findings suggest a major importance of Gln in metabolic and epigenetic reprogramming of microenvironment. Disclosures Off Label Use: CB-839 is a potent, selective, reversible and orally bioavailable glutaminase (mitochondrial enzyme able to convert glutamine in glutamate) inhibitor that has shown to reduce cell growth and/or induce cell death in solid tumors and hematological malignancies.. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

scholarly journals Mast cells are responsible for the lack of anti-inflammatory effects of morphine in CBA mice

2004 ◽  
Vol 13 (5-6) ◽  
pp. 365-368 ◽  
Author(s):  
Elzbieta Stankiewicz ◽  
Ewa Wypasek ◽  
Barbara Plytycz
Keyword(s):  
Mast Cells ◽  
Histamine Release ◽  
Swiss Mice ◽  
Cba Mice ◽  
Peritoneal Mast Cells ◽  
Anti Inflammatory ◽  
Zymosan Peritonitis

BACKGROUND and aim: Morphine co-injection has anti-inflammatory effects on zymosan-induced peritonitis in several strains of mice except that of CBA. As peritoneal mast cells (pMCs) are much more numerous in CBA mice than in SWISS mice, the role of pMCs in morphine-modulated zymosan peritonitis is compared in CBA and SWISS males.Methods: pMCs were treatedin vitrowith morphine or C48/80 for comparison of histamine release.In vivoaccumulation of leukocytes and histamine in peritoneal exudate were recorded after intraperitoneal injection with morphine, zymosan, or zymosan plus morphine.Results and conclusion: Morphine induces histamine release by pMCs from CBA mice but not SWISS mice.In vivomorphine-induced peritonitis is stronger in CBA mice than SWISS mice. Corollary, morphine anti-inflammatory effects on zymosan peritonitis are reversed in CBA mice by its pro-inflammatory action through CBA pMCs.

Anti-Inflammatory and Anti-Allergic Effect of Agaricus blazei Extract in Bone Marrow-Derived Mast Cells

2012 ◽  
Vol 40 (05) ◽  
pp. 1073-1084 ◽  
Author(s):  
Hyuk-Hwan Song ◽  
Hee-Sung Chae ◽  
Sei-Ryang Oh ◽  
Hyeong-Kyu Lee ◽  
Young-Won Chin
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Mouse Bone Marrow ◽  
Agaricus Blazei ◽  
Soluble Extract ◽  
Anti Inflammatory ◽  
Allergic Effect

In this study, the anti-inflammatory and anti-allergic effects of the chloroform-soluble extract of Agaricus blazei in mouse bone marrow-derived mast cells (BMMCs) were investigated. The chloroform-soluble extract inhibited IL-6 production in PMA plus A23187-stimulated BMMCs, and down-regulated the phosphorylation of Akt. In addition, this extract demonstrated inhibition of the degranulation of β-hexosaminidase and the production of IL-6, prostaglandin D2 and leukotriene C4 in PMA plus A23187-induced BMMCs. In conclusion, the chloroform-soluble extract of Agaricus blazei exerted anti-inflammatory and anti-allergic activities mediated by influencing IL-6, prostaglandin D2, leukotriene C4, and the phosphorylation of Akt.

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