BCL2A1 Is A Survival and Immortalization Factor for Primitive Myeloid Hematopoietic Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3365-3365
Author(s):  
Jean-Yves Metais ◽  
Ashley E. Dunfee ◽  
Rodrigo T. Calado ◽  
Cynthia E. Dunbar
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Gene Product ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Marker Genes ◽  
Empty Vector ◽  
Myeloid Progenitor ◽  
The Impact

Abstract We recently reported development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2005). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene in the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on myeloid cell behavior. To investigate the impact of the BCL2A1 gene product on hematopoietic cells, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentivirus vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins on hematopoiesis. We confirmed overexpression of the proteins in the producer cell line as well as in transduced cells by western blot using an anti-HA monoclonal antibody. BaF3 cell proliferation and survival are dependant on IL-3, and under IL-3 replete conditions overexpression of murine or human BCL2A1 did alter proliferation compared with untransduced cells or cells transduced with an empty vector. Removal of IL-3 from the cell culture media leads to rapid apoptosis of BaF3 cells, with cell cycle arrest in the G1 and an apoptotic subpopulation appearing within 24 hours of IL-3 removal. 45% untransduced or empty vector cells were apoptotic, and this fraction decreased to 30% and 15% respectively for BaF3 cells expressing murine or human BCL2A1. These results were confirmed by direct analysis of apoptosis. Only BaF3 cells over-expressing human BCL2A1 were still alive and arrested in G1 after 3 days of culture without IL-3. The murine BCL2A1 had similar but less striking effects. Gene expression analyses on the BaF3 cell populations are ongoing, to identify potential downstream targets of the BCL2A1 protein. The BCL2A1 and empty vectors were also utilized in murine bone marrow cell immortalization assay, previously utilized to identify genes impacting on the survival and expansion of primary myeloid progenitor cells (Du, 2005). In an initial set of experiments, clonal clonal expansion was obtained with marrow cells expressing murine (4 clones) and human (5 clones) BCL2A1 but not for empty vector or untransduced murine marrow. Mice have also been transplanted with primary bone marrow cells transduced with the BCL2A1 and control vectors, and are being followed for in vivo expansion of transduced clones and development of leukemia. In conclusion, we have confirmed the role of BCL2A1 as an anti-apoptotic protein, now in myeloid hematopoietic cells, and will continue to investigate the role of this gene product in hematopoiesis and leukemogenesis.

G-CSF and GM-CSF Have Opposite Effects on the Growth and Survival of Cells Containing Abnormal RUNX1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2760-2760
Author(s):  
Fariborz Mortazavi ◽  
Arun Sharma ◽  
Marie Baraoidan ◽  
Vinzon Ibanez ◽  
SiJun Yang ◽  
...  
Keyword(s):  
Myelodysplastic Syndrome ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Leukemia Cells ◽  
Growth And Survival ◽  
Gm Csf ◽  
Molecular Abnormalities ◽  
Empty Vector ◽  
Human Cd34

Abstract Primary leukemia cells can respond very differently to cytokines such as G-CSF and GM-CSF, demonstrating marked increases in colony-forming ability to one but not the other (Park et al, Blood1989; 74: 56–65). To examine if such differences might correlate with underlying molecular abnormalities, we examined the growth and survival response of hematopoietic cells containing abnormal RUNX1 variants (a frequent occurrence in leukemia and myelodysplastic syndrome) to treatment with G-CSF and GM-CSF. In the RUNX1-ETO containing cell-line Kasumi-1, GM-CSF decreased colony forming ability whereas G-CSF preserved or increased it. The 32Dcl3 murine myelomonocytic cell-line terminally differentiates in response to G-CSF or mGM-CSF. We transduced 32Dcl3 with RUNX1-ETO or Empty Vector: G-CSF promoted the growth and survival of 32D RUNX1-ETO but caused 32D Empty Vector to terminally differentiate and die. In contrast, 32D RUNX1-ETO cultured in mGM-CSF demonstrated accelerated terminal differentiation and death compared to 32D Empty Vector. Many of the RUNX1 variants seen in leukemia and myelodysplastic syndrome, including RUNX1-ETO, lack the RUNX1 C-terminal transcription regulating domain (TRD). In human CD34+ hematopoietic cells transduced with RUNX1 with TRD deleted (CD34 RUNX1noTRD), colony forming ability was retained even after >40 days of culture in IMDM 10% FBS, SCF 10ng/ml, IL-3 30ng/ml, G-CSF 100ng/ml (CD34 Empty Vector lost colony forming ability after 14 days). However, culture in media supplemented with 100ng/ml GM-CSF instead of G-CSF significantly impaired colony forming ability of CD34 RUNX1noTRD compared to CD34 Empty Vector. In conclusion, RUNX1 variants seen in acute myeloid leukemia and myelodysplastic syndrome promote growth and survival in G-CSF but accelerate differentiation and death in response to GM-CSF. The molecular basis for this effect appears to relate to the presence or absence of the RUNX1 C-terminal transcription regulating domain. These findings may have clinical relevance.

A Novel Role For Histone Deacetylase 11 (HDAC11) As a Regulator Of Neutrophil Function and Differentiation In Normal and Malignant Hematopoesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2267-2267
Author(s):  
Eva Sahakian ◽  
John Powers ◽  
Jie Chen ◽  
Allison Distler ◽  
Jennifer Rock-Klotz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Cytokine Production ◽  
Hematopoietic Cells ◽  
Negative Regulator ◽  
Over Expression

Abstract Histone Deacetylase 11 (HDAC11) is the newest member of the HDAC family of enzymes, which we have previously reported to function as a negative regulator of IL-10 expression in macrophages and dendritic cells. Thus far, its role in other hematopoietic cells has not been completely elucidated. We hereby report for the first time a lineage-restricted over-expression of HDAC11 in neutrophils, committed neutrophil precursors and myeloid leukemias exhibiting neutrophilic differentiation demonstrating a novel physiological role of HDAC11 as a negative regulator of neutrophil cytokine production. Leukocyte subpopulations from murine bone marrow and spleen were flow-sorted and analyzed by qRT-PCR for HDAC11 mRNA, revealing a higher level of mRNA expression on neutrophils and promyelocytes, as compared to monocytes and lymphoid subsets. Similarly, sorted human peripheral blood leukocytes from normal donors, showed higher levels of HDAC11 mRNA in neutrophils, as compared to monocytes. To further investigate the transcriptional activity of HDAC11 in myeloid and lymphoid cells, we utilized a HDAC11 promoter-driven eGFP reporter mice, where eGFP expression indicates HDAC11 transcription (Heintz, N Nat. Rev. Neuroscience 2001). Using multiparametric flow cytometry with lineage-specific markers on this mouse model, we confirmed a marked over-expression of HDAC11 on neutrophils, compared to other subpopulations including monocytes, B-cell, T-cells, NK cells and plasma cells. Furthermore, analysis of bone marrow hematopoietic cells revealed a swift over-expression of HDAC11 at the promyelocyte stage of neutrophil differentiation, with low to undetectable expression in upstream uncommitted common myeloid progenitors and lineage-unrelated monocytic precursors. To study whether this lineage-specific overexpression applies to malignant processes, we studied human cell lines and found overt overexpression of HDAC11 in the acute promyelocytic leukemia cell line NB4, as compared to the myeloblastic cell line Kasumi and two monocyte/macrophage cell lines U937 and THP1. Moreover, in-vitro maturation of the differentiation-inducible myeloid cell line HL60 demonstrated a marked increase in HDAC11 mRNA, paralleling the acquisition of nuclear segmentation characteristic of neutrophil maturation. In order to investigate the physiologic role of HDAC11 overexpression on neutrophils, we utilized a model of germline-HDAC11KO mice. Surprisingly, highly purified neutrophils lacking HDAC11 showed an overt overproduction of TNF-alpha and IL-6 upon stimulation with LPS, as compared to their wild type counterparts. We hereby report a previously un-described lineage-specific over-expression of HDAC11 in neutrophils and its precursors, which actively functions as a physiological repressor of cytokine production and possibly involved in their regulation. Given the predominance of neutrophils which account for 70% of leukocytes in the peripheral blood, and their pivotal role in the first line of defense, results highlight a novel mechanism for HDAC11, as a key regulator and modulator of neutrophil cytokine production with potential implications for autoimmunity, inflammation, and infection. Disclosures: No relevant conflicts of interest to declare.

BCL2A1 Is a Survival and Immortalization Factor for Primitive Hematopoietic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2957-2957
Author(s):  
Jean-Yves Metais ◽  
Thomas Winkler ◽  
Rodrigo T. Calado ◽  
Cynthia E. Dunbar
Keyword(s):  
Vector Control ◽  
Myeloid Leukemia ◽  
Chromosome 9 ◽  
Marker Genes ◽  
Control Groups ◽  
Cell Counts ◽  
The Impact ◽  
Marrow Cells

Abstract Abstract 2957 Poster Board II-933 We recently reported the development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2003). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene belonging to the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on hematopoiesis. To further investigate the impact of the BCL2A1 gene product on normal and malignant hematopoiesis, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentiviral vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins in vitro. We confirmed the role of BCL2A1 as an anti-apoptotic protein. The proliferation and survival of the BaF3 cells is dependant on IL-3. Upon removal of IL-3 from the media, BaF3 cells underwent an arrest in the G1 phase of the cycle. Untransduced cells or cells transduced with the empty lentiviral vector were 45% apoptotic, but this fraction decreased to 30 and 15% respectively with the cells transduced with murine and human BCL2A1. Similar results were obtained in murine 32Dcl3 cells and human UT7/Epo-S1 cells, two cells line that are respectively dependant on IL-3 and erythropoietin. In order to study the in vivo impact of BCL2A1 on hematopoiesis, C57/bl6 (Ly5.2) mice have been transplanted with primary bone marrow cells (from C57/bl6 (Ly5.1)) transduced with the BCL2A1 and control vectors, and have been followed for in vivo expansion of transduced clones and development of leukemia. The mice cohort consisted of 5 MOCK controls, 15 control vector expressing GFP only, and 15 murine BCL2A1. We frequently checked the blood counts and analyzed the lineage of blood by FACS. Mice transplanted with marrow cells transduced with the BCL2A1 vector had higher overall marking levels in the blood compared to the vector control (80% vs. 10% cells GFP+ respectively). Interestingly, preliminary histopathology revealed that 7 mice from the BCL2A1 group, but none of the control groups developed a further to be classified very poorly differentiated hematologic malignancy consisting of circulating blasts, splenomegaly, and lymphadenopathy. Cells responsible for this fatal disease were not stained by markers used for FACS analysis or by immunohistochemistry. The median overall survival was significantly different for mice treated with the vector (420 days) and mice treated with BCL2A1 (328 days). The median disease free survival was more striking as the median could not be defined for the vector while it was of 397 days for BCL2A1. Two hundred and fifty three days after the transplantation we selected 3 primary mice from BCL2A1 and vector control groups to perform secondary transplant. Less than a month after reinfusion all BCL2A1 mice developed a disease characterized by high white blood cell counts with a majority of undifferenciated cells, as well as splenomegaly, and hepatomegaly. These results were repeated in a second set of secondary transplant carried out 289 days after transplantation confirming that the disease is transplantable. The preliminary histopathology findings showed a similar phenotype to the lymphatic malignancy seen in the primary mice. In conclusion, we have confirmed the anti-apoptotic role of BCL2A1, and we are now investigating its role in hematopoiesis and leukemogenesis. We are investigating the exact phenotype of the disease seen in primary and secondary mice in order to clarify the role of BCL2A1 as a survival factor. Disclosures: Dunbar: ASH: Honoraria.

scholarly journals SETDB1 Mediated H3K9 Methylation Suppresses MLL-Fusion Target Expression and Leukemic Transformation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 110-110
Author(s):  
Andrew G. Muntean ◽  
James Ropa ◽  
Nirmalya SAHA ◽  
Hsiang-Yu Hu ◽  
Justin Serio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
High Expression ◽  
Leukemic Transformation ◽  
Acute Myeloid

Genes encoding epigenetic proteins are mutated in about 70% of Acute Myeloid Leukemia patients underscoring their role in leukemic transformation. Epigenetic deregulation of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia (AML), such as those harboring MLL rearrangements, NPM mutations and others. Indeed, expression of Hoxa9 and its co-factor Meis1 is sufficient to transform bone marrow cells into a lethal AML. We have shown that epigenetic regulation of Hoxa9 and Meis1 is mediated through the Polymerase Associated Factor 1 complex (PAF1c). The PAF1c binds to RNA polymerase II and recruits enzymes that alter epigenetic landscapes and influence gene transcription. We and others have shown that the PAF1c directly binds and recruits MLL and MLL fusion proteins to target genes such as Hoxa9 and Meis1 to deliver H3K4 methylation or H3K79 methylation activity respectively. Importantly, the PAF1c-MLL1 interaction is necessary for the growth of MLL-fusion leukemia cells but appears dispensable for normal hematopoietic stem and progenitor cells (HSPCs) function. This suggests the PAF1c is differentially required in leukemic cells compared to its normal counterparts. Thus, we aimed to 1) define the role of the PAF1c in normal hematopoiesis, and 2) identify mechanisms regulating PAF1c function that may be deregulated in leukemia. To understand the role of the PAF1c in hematopoiesis, we conditionally deleted the PAF1c subunit Cdc73 in HSPCs in hematopoietic cells. Conditional hematopoietic deletion of Cdc73 leads to lethality within a week due to a depletion of c-kit+ cells. Characterization revealed a cell autonomous requirement for Cdc73 to prevent apoptosis in HSPCs. Gene expression profiling on both c-kit+ hematopoietic cells and AML cells revealed that Hoxa9/Meis1 gene programs are differentially responsive to loss of Cdc73. This work revealed an essential role for the PAF1c subunit in maintaining viability of HSPCs and points to AML specific functions for the PAF1c. Thus, we utilized immunoprecipitation followed by mass spectroscopy to identify novel PAF1c interactions in AML cells. We found a novel interaction with several H3K9 methyltransferases including SETDB1. We demonstrate that the PAF1c-SETDB1 interaction reduces expression of Hoxa9 and Meis1 by promoting deposition of H3K9me3. These findings mirrored human AML patient samples that showed SETDB1 expression was inversely correlated with HOXA9 and MEIS1 expression. Further, SETDB1 expression is correlated to disease status and overall survival in AML patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of AML cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While AML cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of HSPCs. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of AML cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with AML, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in AML that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML. Disclosures Maillard: Genentech: Consultancy; Regeneron: Consultancy.

scholarly journals Leukemia stem cell-bone marrow microenvironment interplay in acute myeloid leukemia development

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Yiyi Yao ◽  
Fenglin Li ◽  
Jiansong Huang ◽  
Jie Jin ◽  
Huafeng Wang
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chemotherapy Resistance ◽  
Bone Marrow Microenvironment ◽  
Cytotoxic Effects ◽  
High Relapse Rate ◽  
Underlying Mechanisms ◽  
Acute Myeloid

AbstractDespite the advances in intensive chemotherapy regimens and targeted therapies, overall survival (OS) of acute myeloid leukemia (AML) remains unfavorable due to inevitable chemotherapy resistance and high relapse rate, which mainly caused by the persistence existence of leukemia stem cells (LSCs). Bone marrow microenvironment (BMM), the home of hematopoiesis, has been considered to play a crucial role in both hematopoiesis and leukemogenesis. When interrupted by the AML cells, a malignant BMM formed and thus provided a refuge for LSCs and protecting them from the cytotoxic effects of chemotherapy. In this review, we summarized the alterations in the bidirectional interplay between hematopoietic cells and BMM in the normal/AML hematopoietic environment, and pointed out the key role of these alterations in pathogenesis and chemotherapy resistance of AML. Finally, we focused on the current potential BMM-targeted strategies together with future prospects and challenges. Accordingly, while further research is necessary to elucidate the underlying mechanisms behind LSC–BMM interaction, targeting the interaction is perceived as a potential therapeutic strategy to eradicate LSCs and ultimately improve the outcome of AML.

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

scholarly journals Gpr44 Mediates the Selenium-Dependent Anti-Leukemic Effect in Acute Myeloid Leukemia

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1835-1835
Author(s):  
Fenghua Qian ◽  
Fenghua Qian ◽  
Diwakar Tukaramrao ◽  
Jiayan Zhou ◽  
Nicole Palmiero ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Murine Model ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Pparγ Agonist ◽  
Acute Myeloid

Abstract Objectives The relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia stem cells (LSCs) that are not targeted by existing therapies. LSCs show sensitivity to endogenous cyclopentenone prostaglandin J (CyPG) metabolites that are increased by dietary trace element selenium (Se), which is significantly decreased in AML patients. We investigated the anti-leukemic effect of Se supplementation in AML via mechanisms involving the activation of the membrane-bound G-protein coupled receptor 44 (Gpr44) and the intracellular receptor, peroxisome proliferator-activated receptor gamma (PPARγ), by endogenous CyPGs. Methods A murine model of AML generated by transplantation of hematopoietic stem cells (HSCs- WT or Gpr44−/−) expressing human MLL-AF9 fusion oncoprotein, in the following experiments: To investigate the effect of Se supplementation on the outcome of AML, donor mice were maintained on either Se-adequate (Se-A; 0.08–0.1 ppm Se) or Se-supplemented (Se-S; 0.4 ppm Se) diets. Complete cell counts in peripheral blood were analyzed by hemavet. LSCs in bone marrow and spleen were analyzed by flow cytometry. To determine the role of Gpr44 activation in AML, mice were treated with Gpr44 agonists, CyPGs. LSCs in bone marrow and spleen were analyzed. Mice transplanted with Gpr44−/- AML cells were compared with mice transplanted with wild type AML cells and the progression of the disease was followed as above. To determine the role of PPARγ activation in AML, PPARγ agonist (Rosiglitazone, 6 mg/kg, i.p, 14 d) and antagonist (GW9662, 1 mg/kg, i.p. once every other day, 7 injections) were applied to Se-S mice transplanted with Gpr44−/- AML cells and disease progression was followed. Results Se supplementation at supraphysiological levels alleviated the disease via the elimination of LSCs in a murine model of AML. CyPGs induced by Se supplementation mediate the apoptosis in LSCs via the activation of Gpr44 and PPARγ. Conclusions Endogenous CyPGs produced upon supplementation with Se at supraphysiological levels improved the outcome of AML by targeting LSCs to apoptosis via the activation of two receptors, Gpr44 and PPARg. Funding Sources NIH DK 07,7152; CA 175,576; CA 162,665. Office of Dietary Supplements, USDA Hatch funds PEN04605, Accession # 1,010,021 (KSP, RFP).

scholarly journals Silencing long non-coding RNA XIST suppresses drug resistance in acute myeloid leukemia through down-regulation of MYC by elevating microRNA-29a expression

2020 ◽  
Vol 26 (1) ◽  
Author(s):  
Chong Wang ◽  
Lingling Li ◽  
Mengya Li ◽  
Weiqiong Wang ◽  
Yanfang Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Cellular Viability ◽  
Down Regulation ◽  
Acute Myeloid

Abstract Background Long non-coding RNAs (lncRNAs) are biomarkers participating in multiple disease development including acute myeloid leukemia (AML). Here, we investigated molecular mechanism of X Inactive-Specific Transcript (XIST) in regulating cellular viability, apoptosis and drug resistance in AML. Methods XIST, miR-29a and myelocytomatosis oncogene (MYC) expression in AML bone marrow cells collected from 62 patients was evaluated by RT-qPCR and Western blot analysis. Besides, the relationship among XIST, miR-29a and MYC was analyzed by dual luciferase reporter assay, RIP, and RNA pull down assays. AML KG-1 cells were treated with anti-tumor drug Adriamycin. The role of XIST/miR-29a/MYC in cellular viability, apoptosis and drug resistance in AML was accessed via gain- and loss-of-function approaches. At last, we evaluated role of XIST/miR-29a/MYC on tumorigenesis in vivo. Results XIST and MYC were up-regulated, and miR-29a was down-regulated in AML bone marrow cells. Silencing XIST inhibited cellular activity and drug resistance but promoted cellular apoptosis of KG-1 cells by down-regulating MYC. XIST inhibited miR-29a expression to up-regulate MYC. Moreover, silencing XIST inhibited tumorigenesis of AML cells in vivo. Conclusions Overall, down-regulation of XIST decreased MYC expression through releasing the inhibition on miR-29a, thereby reducing drug resistance, inhibiting viability and promoting apoptosis of AML cells.

scholarly journals In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Hong Kiat Lim ◽  
Pravin Periasamy ◽  
Helen C. O’Neill
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Model Systems ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Stromal Cell Line ◽  
Reticular Cells

There are very few model systems which demonstrate hematopoiesis in vitro. Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro. Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic cells and to resemble perivascular reticular cells in the bone marrow through gene expression. In particular, 5G3 resembles CXCL12-abundant reticular cells or perivascular reticular cells, which are important niche elements for hematopoiesis in the bone marrow. To analyse the hematopoietic support function of 5G3, specific signaling pathway inhibitors were tested for the ability to regulate cell production in vitro in cocultures of stroma overlaid with bone marrow-derived hematopoietic stem/progenitor cells. These studies identified an important role for Wnt and Notch pathways as well as tyrosine kinase receptors like c-KIT and PDGFR. Cell production in stromal cocultures constitutes hematopoiesis, since signaling pathways provided by splenic stroma reflect those which support hematopoiesis in the bone marrow.

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