scholarly journals Elucidating the Role of the Endocytic Protein Ap2a2 in Haematopoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1568-1568
Author(s):  
Stephen Ting ◽  
Sara Rhost ◽  
Nicole Lee ◽  
Sarah Ghotb ◽  
Ruth Meex ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fatty Acid ◽  
Cell Division ◽  
Adaptor Protein ◽  
Conditional Knockout ◽  
Metabolic Effects ◽  
Lacz Reporter ◽  
Self Renewal ◽  
Endocytic Protein

Abstract The rare subpopulation of quiescent HSCs exists in limited numbers but upon in vivo requirement have the highest proliferative potency. Self-renewal via asymmetrical cell division (ACD) is vital for maintenance of HSC numbers and perturbation of this process can lead to an impaired hematopoietic system, with subsequent blood diseases such as acute leukemia and/or bone marrow failure. How and which HSCs are programmed for ACD remain central and unanswered questions. We have demonstrated that the endocytic protein, Ap2a2 distinguishes an asymmetrical from a symmetrical cell division during HSC mitoses, and when overexpressed enhances mouse HSC activity.Ap2a2 is a component of the Adaptor protein 2 complex that functions in clathrin-dependent endocytosis of receptors and nutrient cargo. The tetracycline-induceable H2B-GFP mouse (Foudi A. et al, Nature Biotech 2009) has revealed functional heterogeneity within the CD150+48- Lin-Sca+Kit+(LSK) HSC population, such that the GFPhigh subpopulation within the CD150+48- LSK is the true Long-Term repopulating HSCs (GFPhigh LT-HSCs). Using this mouse, we have shown that Ap2a2 is not only more highly expressed in the GFPhigh LT-HSCs but that Ap2a2 overexpression increases the fraction of this specific subpopulation from 20% to 60% at plus 20 weeks post-transplantation. To identify novel genes and pathways driving LT-HSC function and self-renewal, we are currently performing comparative gene expression analyses from the subpopulation of Ap2a2-transduced against control, vector-transduced GFPhighCD150+48-LSK LT-HSCs. To further investigate Ap2a2 in haematopoiesis, we have constructed an Ap2a2 conditional knockout mouse (Ap2a2 cKO) line, which has a LacZ reporter expressed from the endogenous Ap2a2 promoter. Our initial expression analyses with tissue LacZ staining shows relative increased staining in subpopulations of bone marrow cells and using a β-galactosidase (β-gal) flow cytometry method co-stained with CD150+48-LSK, have identified β-galhigh and β-gallow subpopulations. This suggests Ap2a2 expression may be an independent marker of LT-HSCs, which we are functionally assessing via comparative LSK -β-galhigh versus -β-gallow versus -β-galnegative transplantation assays. The specific function of Ap2a2 in developmental (Vav-Cre) and adult (Mx-Cre) haematopoiesis is being investigated by respective tissue specific-Cre matings with the Ap2a2fl/flmice. Mechanistically, a recent publication (Ito K. et al, Nature Med 2012) identified the importance of PPAR-δ/β-fatty acid oxidation regulation for both maintenance and ACD ofHSCs. Ap2a2 is a target of PPAR-α (Buroker N. et al Protein J 2012). Our hypothesis states that the role of Ap2a2 in LT-HSCs involves the lipid metabolic pathway, which would uniquely link endocytosis to metabolism. To this end, using our Ap2a2-LacZ reporter mouse, we have shown intense and specific LacZ expression in adipocytes of the bone marrow and other lipid containing organs. In addition, perturbation of Ap2a2 results in altered lipid droplet formation and impaired triacylgylceride storage. We are currently investigating the oxidative phosphorylation, anaerobic glycolysis and fatty acid metabolic effects in the context of altered Ap2a2 expression. Disclosures No relevant conflicts of interest to declare.

Dual Role Of MERIT40 In Hematopoietic Stem and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 797-797
Author(s):  
Krasimira Rozenova ◽  
Jing Jiang ◽  
Chao Wu ◽  
Junmin Wu ◽  
Bernadette Aressy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Adaptor Protein ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is maintained by cell intrinsic and extrinsic mechanisms, including tight regulation of signaling pathways such as Tpo-Mpl and SCF-ckit. Posttranslational modifications, such as phosphorylation and ubiquitination, regulate these pathways. While the role of protein phosphorylation is well established, the importance of ubiquitination in HSC self-renewal has not been well addressed. It is known that of the seven different lysines on ubiquitin, Lys48 polyubiquitination is a marker for protein degradation, and Lys63 polyubiquitination is associated with regulation of kinase activity, protein trafficking, and localization. In this study, we provide evidence that the adaptor protein MERIT40 has multiple roles in hematopoietic stem/progenitor cells (HSPCs). MERIT40 is a scaffolding protein shared by two distinct complexes with Lys63 deubiquitinase (DUB) activities: the nuclear RAP80 complex with a known role in DNA damage repair in breast/ovarian cancer cells, whereas the functions of the cytoplasmic BRISC remains less characterized. MERIT40 is important for integrity of both complexes, and its deficiency leads to their destabilization and a >90% reduction in deubiquitinase activity. By using MERIT40 knockout (M40-/-) mice, we found that lack of MERIT40 leads to a two-fold increase in phenotypic and functional HSCs determined by FACS and limiting dilution bone marrow transplantation (BMT), respectively. More importantly, M40-/- HSCs have increased regenerative capability demonstrated by increased chimerism in the peripheral blood after BMT of purified HSCs. The higher self-renewal potential of these HSCs provides a survival advantage to M40-/- mice and HSCs after repetitive administration of the cytotoxic agent 5-flurouracil (5FU). MERIT40 deficiency also preserves HSC stemness in culture as judged by an increase in peripheral blood chimerism in recipient mice transplanted with M40-/- Lin-Sca1+Kit+ (LSK) cells cultured in cytokines for nine days compared to recipient mice receiving cultured wildtype (WT) LSK cells. In contrast to the increased HSC homeostasis and superior stem cell activity due to MERIT40 deficiency, M40-/- mice are hypersensitive to DNA damaging agents caused by inter-cross linking (ICL), such as Mitomycin C (MMC) and acetaldehydes that are generated as side products of intracellular metabolism. MMC injection caused increased mortality in M40-/- mice compared to WT controls attributable to DNA damage-induced bone marrow failure. MMC-treated M40-/- mice showed marked reduction in LSK progenitor numbers accompanied by increased DNA damage, in comparison to WT mice. Consistent with the in vivo studies, M40-/- progenitor cells are hypersensitive to MMC and acetaldehyde treatment in a cell-autonomous manner in colony forming assays. ICL repair is known to require Fanconi Anemia (FA) proteins, an ICL repair network of which mutations in at least 15 different genes in humans cause bone marrow failure and cancer predisposition. Thus, M40-/- mice represent a novel mouse model to study ICL repair in HSPCs with potential relevance to bone marrow failure syndromes. Taken together, our data establishes a complex role of MERIT40 in HSPCs, warranting future investigation to decipher functional events downstream of two distinct deubiquitinating complexes associated with MERIT40 that may regulate distinct aspects of HSPC function. Furthermore, our findings reveal novel regulatory pathways involving a previously unappreciated role of K63-DUB in stem cell biology, DNA repair regulation and possibly bone marrow failure. DUBs are specialized proteases and have emerged as potential “druggable” targets for a variety of diseases. Hence, our work may provide insights into novel therapies for the treatment of bone marrow failure and associated malignancies that occur in dysregulated HSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Adult Hematopoietic Stem and Progenitor Cell Maintenance Requires Histone Variant H3.3 Regulation By Hira

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2473-2473
Author(s):  
Rebecca Murdaugh ◽  
Kevin Hoegenauer ◽  
Xiangguo Shi ◽  
Ayumi Kitano ◽  
Richard Chapple ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Division ◽  
S Phase ◽  
Histone Variant ◽  
Conditional Knockout ◽  
Epigenetic Mechanism ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System

The adult hematopoietic system is sustained by a balance of self-renewal and differentiation in a small pool of stem and progenitor cells. This balance must be maintained to ensure a continuous supply of blood cells throughout life and prevent malignancy from arising. There are many facets of epigenetic regulation that are well known to be key components of healthy and diseased hematopoiesis, such as DNA methylation and histone post-translational modifications. However, the role of histone variant incorporation in hematopoiesis remains relatively unexplored. In this study, we explore the role of histone variant H3.3 regulation in the hematopoietic system by assessing the function of the histone H3.3 chaperone, Hira. Toward this goal, we use inducible and early developmental conditional knockout (cKO) mouse models to assess the role of Hira within the hematopoietic system. Following Hira cKO early in hematopoietic development (Vav-iCre; Hirafl/fl), we find that HSPCs are unaffected in the fetal liver but deplete quickly after homing to the bone marrow. Using polyinosinic-polycytidylic (pIpC) inducible Hira cKO mice (Mx1-Cre; Hirafl/fl), we find a similarly severe depletion of HSPCs in adult mice within 1 month after Hira loss. In contrast, differentiated cells remain largely unaffected following Hira cKO, demonstrating that Hira is especially important in the hematopoietic stem and progenitor compartment. Since Hira is known to incorporate H3.3 throughout the cell cycle and not just during S-phase like H3.1/2, we hypothesized that adult HSPCs are more dependent upon Hira to regulate histone H3 dynamics since they are slowly dividing. The loss of Hira-mediated H3.3 deposition would also be particularly detrimental to the function of these cells given its association with actively transcribed and bivalent genes. To test the role of Hira in maintaining gene expression patterns, we performed bulk RNA-seq on adult HSPCs and found that hematopoietic differentiation genes are dysregulated after Hira cKO with increased erythroid lineage and decreased lymphoid lineage gene expression. We then assessed gene expression changes in Hira cKO HSPCs in a doxycycline-inducible H2B-GFP background (Mx1-Cre; Hirafl/fl; R26-M2rtTa; TetOP-H2B-GFP) to distinguish between the gene expression changes caused by Hira loss before and after cell division. In the absence of Hira-mediated H3.3 incorporation, we expect some highly expressed genes in slowly dividing adult HSPCs to be affected by Hira loss prior to cell division due to nucleosome turnover in the wake of RNA Polymerase II. At other loci, like bivalent promoters, H3.3 would be diluted after cell division by H3.1/2 during S-phase in Hira cKO HSPCs. In support of this hypothesis, we found that increased expression of the erythroid differentiation gene Klf1 in Hira cKO MPPs after cell division (H2B-GFPLow) relative to Hira cKO MPPs before division (H2B-GFPHigh) and WT MPPs that have divided (H2B-GFPLow). The findings from both of these transcriptome analyses point toward a role of Hira in regulating HSPC differentiation genes and are supported by our in vitro and in vivo data showing increased differentiation of Hira cKO HSPCs and decreased self-renewal. In order to more fully understand the H3.3-dependent gene expression changes after Hira cKO in HSPCs, we correlated H3.3 enrichment patterns from chromatin-immunoprecipitation and sequencing (ChIP-seq) with our data from assay for transposase-accessible chromatin and sequencing (ATAC-seq). Our results demonstrate that Hira cKO HSPCs have more open chromatin and fewer H3.3 peaks, suggesting that loss of Hira-mediated H3.3 deposition increases DNA accessibility. This study identifies a novel epigenetic mechanism required for adult HSPC maintenance and elucidates a previously unappreciated regulator of normal hematopoietic homeostasis. Further understanding how Hira-mediated H3.3 regulation maintains adult HSPCs will provide greater depth to our current understanding of the epigenetic regulators essential for hematopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of Kdm6a in Normal Hematopoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1467-1467
Author(s):  
Ling Tian ◽  
Lukas D. Wartman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Young Female ◽  
Conditional Knockout ◽  
Transplant Experiment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Aged Mice

Abstract The role of the X-linked H3K27 demethylase KDM6A in normal hematopoiesis remains unclear. We generated Kdm6a conditional knockout mice (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre mice to inactivate Kdm6a in hematopoietic stem/progenitor cells. We characterized normal hematopoiesis from young (6 to 8 week old) and aged (50 to 55 week old) Kdm6a conditional KO mice. We included female and male animals. The inactivation of Kdm6a alone, both in male and female mice, results in a myeloid replating phenotype indicative of aberrant self-renewal. This is present in both young and aged mice with an increased number of colonies at week 2 in the female KO mice as compared to the hemizygous male KO mice. Interestingly, heterozygous female KO mice only acquire this abnormal replating phenotype with age. Next, we found that the genetic inactivation of Kdm6a has age- and gender-dependent effects on primitive hematopoietic cell populations and progenitors (Figure 1). The phenotype outlined in Figure 1 is specific to female Kdm6a-null mice and is not present in male hemizygous or female heterozygous mice at any time point. We went on to show that the decrease in the KLS compartment seen in the young female Kdm6a-null mice is associated with an increase in the fraction of these cells in G0/G1 along with increased apoptosis. The deletion of Kdm6a in young female homozygous mice causes mild thrombocytopenia but in aged mice is associated with anemia and thrombocytopenia. There is also a myeloid skewing with an increased number of neutrophils and a B-cell lymphopenia in the bone marrow, spleen and peripheral blood that becomes more pronounced with age in these mice. The female homozygous KO mice also have mild splenomegaly and an increased number of red blood cell precursors in the spleen. We went on to perform a competitive transplant experiment mixing donor marrow from all cohorts of young Kdm6a conditional KO x Vav1-Cre mice (CD 45.2) with WT competitor marrow (CD 45.1 x 45.2) in a 1:1 ratio, which was then transplanted into lethally-irradiated CD45.1 recipients so that we could easily follow donor versus recipient chimerism. It has been shown that hematopoietic stem cells (HSCs) from female Kdm6a-null mice had a cell migration defect (Thieme S et al., Blood, 2013). We have now shown a decreased repopulation potential for all three cohorts of Kdm6a KO mice (homozygous and heterozygous females and hemizygous males). The finding is most pronounced in the female homozygous Kdm6a KO mice. Strikingly, and in keeping with results noted in the non-transplanted aged mice, there is a selective preservation of the donor SLAM compartment from all cohorts of the Kdm6a conditional KO mice, which is most pronounced in the female Kdm6a-null mice. The competitive repopulation disadvantage was observed when the bone marrow from primary recipients was transplanted into secondary mice. Again, however, relative preservation of the SLAM compartment was sustained. To circumvent the effects of a migration/engraftment defect of female Kdm6a-null stem cells, we repeated a competitive transplant experiment using young, female homozygous Kdm6a conditional KO x ERT2-Cre mice (and appropriate controls). After engraftment (6-weeks post-transplant), these mice were given tamoxifen to activate the Cre locus and to inactivate Kdm6a. In the bone marrow, we achieved approximately 50% floxing of the Kdm6a conditional allele 2-weeks after the last dose of drug. Again, we observed decreased repopulation potential in all lineages with the exception of the relative preservation of the SLAM compartment. We also repeated serial replating assays and showed that the floxed Kdm6a allele increased with serial replating as expected. Finally, we performed gene expression profiling via exon arrays on flow-sorted SLAM cells from aged female Kdm6a-null mice and aged controls. Hierarchical clustering revealed a clear distinction between cohorts. In sum, our data shows that female Kdm6a KO mice have a gender-specific phenotype that emerges with aging and is similar to human myelodysplastic syndrome (MDS). The female KO aged mice have an expansion of their HSCs with aberrant self-renewal, but these HSCs do not differentiate into downstream progeny as in normal hematopoiesis. As such, these mice become anemic and thrombocytopenic-but do not develop overt leukemia or die of these abnormalities. Disclosures No relevant conflicts of interest to declare.

scholarly journals Multiple Myeloma Cells Induce Lipolysis in Adipocytes and Uptake Fatty Acids through Fatty Acid Transporter Proteins

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 41-42
Author(s):  
Cristina Panaroni ◽  
Keertik Fulzele ◽  
Tomoaki Mori ◽  
Chukwuamaka Onyewadume ◽  
Noopur S. Raje
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Fatty Acid ◽  
Board Of Directors ◽  
Metabolic Reprogramming ◽  
Glycerol Production ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Long Chain

Multiple myeloma (MM) originates in the bone marrow where adipocytes occupy 65% of the cellular volume in a typical myeloma patient. Cancer associated adipocytes support the initiation, progression, and survival of solid tumors via mechanisms including adipokine secretion, modulation of the tumor microenvironment, and metabolic reprogramming of cancer cells. Although MM cells are surrounded by abundant bone marrow adipocytes (BMAd), the nature of their interaction remains unclear. Recent studies have elucidated the role of BMAds in supporting the survival of MM cells, in part, through secreted adiponectin. Increased fatty acid (FA) metabolism may result in metabolic reprogramming of cancer cells impacting their growth and survival. Here, we hypothesize that MM cells extract FA from adipocytes for their growth. We first characterized mesenchymal stem cells (MSCs) from MGUS, smoldering MM (SMM), and newly diagnosed MM (NDMM) patients by flow cytometry analysis. MSCs showed significant increase in Pref1, leptin receptor and perilipin A, suggesting increased adipogenic commitment. MSCs from healthy donors (HD), MGUS, SMM, and NDMM patients were induced to differentiate into adipocytes and then co-cultured with human MM MM.1S cells. After 72 hr of co-culture, CyQUANT assay demonstrated significant increase in proliferation of MM.1S cells in the presence of BMAd from HD; this was further increased in the presence of BMAd from MGUS/SMM and NDMM. These data suggest that the BMAd support the growth of MM cells and this effect is more pronounced in patient derived BMAd. A PCR-array targeting lipid metabolism on BM fat aspirates showed significant deregulation of genes involved in FA synthesis and lipolysis. Taken together, our data suggest that BMAd in MM patients are altered to further support the aggressive expansion of MM cells. The proliferative-supportive role of adipocytes was further validated in co-culture of OP9 murine BM stromal preadipocytes with 5TGM1 murine MM cells. To study the bidirectional interaction of MM/ BMAd, mature OP9 adipocytes were co-cultured with 5TGM1 or human OPM2 MM cells for 24 hr. Intracellular lipid droplets were labelled with Deep Red LipidTox stain. The lipid droplet sizes were significantly decreased in the presence of both 5TGM1 and OPM2 cells compared to OP9 alone. The decrease in lipid size suggested that MM cells may induce lipolysis in adipocytes. Indeed, 24hr co-culture of 5TGM1 cells with OP9 mature adipocytes significantly increased lipolysis 3-fold as measured by glycerol secretion in conditioned media. Co-culture of OP9 adipocytes with other MM cell lines of human origin, MM.1S, INA6, KMS-12 PE, and OPM2 also significantly increased the glycerol production as much as 4-fold. Taken together these data indicate that MM cells induce lipolysis in adipocytes. In contrast, treatment of 5TGM1 cells with synthetic catecholamine isoproterenol did not induce lipolysis, or glycerol production, indicating lack of triglyceride storage. Next, we hypothesized that the free FAs released from adipocytes are taken up by MM cells for various biological processes. To test this, 5TGM1, MM.1S and OPM2 cells were incubated with BODIPY-C12 and BODIPY-C16, the BODIPY-fluorophore labelled 12-carbon and 16-carbon long chain FA. All MM cells showed saturated uptake of the FA within 10 minutes suggesting that MM cells have efficient FA transporters. To confirm this uptake, unstained 5TGM1, OPM2 and KMS12 PE cells were co-cultured with the LipidTox-labelled OP9 mature adipocytes. After 24 hours, flow cytometric analysis showed LipidTox signal in MM cells. These data demonstrate that FAs released by MM induced adipocyte lipolysis are taken up by MM cells. Long-chain FAs such as BODIPY-C12 and BODIPY-C16 are transported into cells through FA transporter protein (FATP) family of lipid transporters. We therefore analyzed patient samples which showed that CD138+ plasmacells and myeloma cells expressed high levels of FATP1 and FATP4 whereas, their expression was absent in lineage-sibling T-cells. Moreover, pretreatment with Lipofermata, a FATP inhibitor, was able to decrease the uptake of BODIPY-C12 and -C16 in 5TGM1 cells. Taken together, our data show that myeloma cells induce lipolysis in adipocytes and the released free FAs are then uptaken by myeloma cells through FATPs. Inhibiting myeloma cell induced lipolysis or uptake of FA through FATPs may be a potential anti-tumor strategy. Disclosures Fulzele: FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Raje:Amgen: Consultancy; bluebird bio: Consultancy, Research Funding; Caribou: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immuneel: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Immuneel: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

scholarly journals GIRK currents in VTA dopamine neurons control the sensitivity of mice to cocaine-induced locomotor sensitization

2018 ◽  
Vol 115 (40) ◽  
pp. E9479-E9488 ◽  
Author(s):  
Robert A. Rifkin ◽  
Deborah Huyghe ◽  
Xiaofan Li ◽  
Manasa Parakala ◽  
Erin Aisenberg ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Conditional Knockout ◽  
Dopamine Neurons ◽  
Substantia Nigra Pars Compacta ◽  
Locomotor Sensitization ◽  
Girk Channels ◽  
Sorting Nexin ◽  
Inwardly Rectifying Potassium Channels ◽  
Dependent Inhibition

GABABR-dependent activation of G protein-gated inwardly rectifying potassium channels (GIRK or KIR3) provides a well-known source of inhibition in the brain, but the details on how this important inhibitory pathway affects neural circuits are lacking. We used sorting nexin 27 (SNX27), an endosomal adaptor protein that associates with GIRK2c and GIRK3 subunits, to probe the role of GIRK channels in reward circuits. A conditional knockout of SNX27 in both substantia nigra pars compacta and ventral tegmental area (VTA) dopamine neurons leads to markedly smaller GABABR- and dopamine D2R-activated GIRK currents, as well as to suprasensitivity to cocaine-induced locomotor sensitization. Expression of the SNX27-insensitive GIRK2a subunit in SNX27-deficient VTA dopamine neurons restored GIRK currents and GABABR-dependent inhibition of spike firing, while also resetting the mouse’s sensitivity to cocaine-dependent sensitization. These results establish a link between slow inhibition mediated by GIRK channels in VTA dopamine neurons and cocaine addiction, revealing a therapeutic target for treating addiction.

Absence of the Rho GTPase Activating Protein p190-B Enhances Long Term Hematopoietic Stem Cell Engraftment

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 614-614 ◽  
Author(s):  
Haiming Xu ◽  
Hartmut Geiger ◽  
Kathleen Szczur ◽  
Deidra Deira ◽  
Yi Zheng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Ex Vivo ◽  
Gtpase Activating Protein ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation ◽  
Serial Transplantation

Abstract Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow (BM), self-renewal, proliferation and differentiation to mature blood cells. However, the molecular regulation of HSC engraftment is still poorly defined. Small Rho GTPases are critical regulator of cell migration, proliferation and differentiation in multiple cell types. While their role in HSC functions has begun to be understood, the role of their regulator in vivo has been understudied. P190-B GTPase Activating Protein (GAP), a negative regulator of Rho activity, has been implicated in regulating cell size and adipogenesis-myogenesis cell fate determination during fetal development (Sordella, Dev Cell, 2002; Cell 2003). Here, we investigated the role of p190-B in HSC/P engraftment. Since mice lacking p190-B die before birth, serial competitive repopulation assay was performed using fetal liver (FL) tissues from day E14.5 WT and p190-B−/− embryos. WT and p190-B−/− FL cells exhibited similar levels of engraftment in primary recipients. However, the level of contribution of p190-B−/− cells to peripheral blood and bone marrow was maintained between the primary and secondary recipients and still easily detectable in tertiary recipients, while the level of contribution of FL WT cells dramatically decreased with successive serial transplantion and was barely detectable in tertiary recipients. The contribution to T cell, B cell and myeloid cell reconstitution was similar between the genotypes. A pool of HSC was maintained in serially transplanted p190-B−/− animals, since LinnegScaposKitpos (LSK) cells were still present in the BM of p190-B−/− secondary engrafted mice while this population disappeared in WT controls. Importantly, this enhanced long term engraftment was due to a difference in the functional capacity of p190-B−/− HSC compared to WT HSC since highly enriched p190-B−/− HSC (LSK) demonstrated similar enhanced serial transplantation potential. Because previous studies have suggested that the loss of long term function of HSC during serial transplantation can depend, at least in part, on the upregulation of the cyclin dependent kinase inhibitor p16Ink4a (Ito et al, Nat Med 2006), the expression of p16Ink4a was examined during serial transplantation. While expression of p16Ink4a increased in WT HSC in primary and secondary recipients, p16Ink4a remained low in p190-B−/− HSC, which indicated that p190-B-deficiency represses the upregulation of p16Ink4a in HSC in primary and secondary transplant recipients. This provides a possible mechanism of p190-B-mediated HSC functions. We next examined whether p190-B-deficiency may preserve the repopulating capacity of HSC/P during ex vivo cytokine-induced culture. While freshly isolated LSK cells from WT and p190-B−/− mice exhibited comparable intrinsic clonogenic capacity, the frequency of colony-forming unit after 7 days in culture was 2 fold-higher in p190-B−/− compared with WT cultures, resulting in a net CFU expansion. Furthermore, competitive repopulation assays showed significantly higher repopulating activity in mice that received p190-B−/− cultured cells compared with WT cells equivalent to a 4.4-fold increase in the estimated frequency of repopulating units. Interestingly, p190-deficiency did not alter cell cycling rate or survival both in vivo and in vitro. Therefore, p190-B-deficiency maintains key HSC functions either in vivo or in ex vivo culture without altering cycling rate and survival of these cells. These findings define p190-B as a critical regulator of HSC functions regulating self renewal activity while maintaining a balance between proliferation and differentiation.

Functional Role of BAALC In Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4194-4194
Author(s):  
Tobias Berg ◽  
Michael Heuser ◽  
Florian Kuchenbauer ◽  
Gyeongsin Park ◽  
Stephen Fung ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Myeloid Differentiation ◽  
Self Renewal ◽  
Murine Bone Marrow ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Marrow Cells

Abstract Abstract 4194 Cytogenetically normal acute myeloid leukemia (CN-AML) patients with high BAALC or MN1 expression have a poor prognosis. Whereas the oncogenic function of MN1 is well established, the functional role of BAALC in hematopoiesis is not known. We therefore compared the expression of BAALC and MN1 in 140 CN-AML patients by quantitative PCR. To further assess the impact of BAALC on leukemogenesis we used retroviral gene transfer into primary murine bone marrow cells and cells immortalized with NUP98-HOXD13 (ND13) and HOXA9. Transduced cells were assessed in vitro by colony forming assays and for their sensitivity to treatment with all-trans retinoic acid (ATRA). They were also evaluated by in vivo transplantation into lethally-irradiated mice. In the 140 CN-AML patients analyzed, the expression of BAALC and MN1 was highly correlated (R=0.71). Retroviral overexpression of MN1 or BAALC in the Hox gene-immortalized bone marrow cells did not cause upregulation of the other gene, suggesting that these genes do not regulate each other. In murine bone marrow cells BAALC did not immortalize the cells in vitro as assessed by serial replating of transduced cells in methylcellulose assays. Transplantation of transduced cells resulted in negligible engraftment of approximately 1 percent at 4 weeks after transplantation. However, co-transduction of BAALC into NUP98-HOXD13 cells (which are very sensitive to the treatment with all-trans retinoic acid) increased the 50 percent inhibitory concentration (IC50) of ATRA by 4.3-fold, suggesting a negative impact of BAALC on myeloid differentiation. We next evaluated whether the differentiation inhibiting effects of BAALC may cooperate with the self renewal-promoting effects of HOXA9 to induce leukemia in mice. Mice receiving transplants of murine bone marrow cells transduced with BAALC and HOXA9 developed myeloid leukemias with a median latency of 139.5 days that were characterized by leukocytosis, massively enlarged spleens (up to 1.02 g), anemia and thrombocytopenia. Infiltrations of myeloid cells were also found in liver, spleen, and kidney. The disease was transplantable into secondary animals. By Southern blot analysis we found one to two BAALC viral integrations per mouse, suggesting that clonal disease had developed from BAALC-transduced cells. We demonstrate for the first time that BAALC blocks myeloid differentiation and promotes leukemogenesis when combined with the self-renewal promoting oncogene HOXA9. Due to its prognostic and functional effects BAALC may become a valuable therapeutic target in leukemia patients. Disclosures: No relevant conflicts of interest to declare.

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 859-859 ◽  
Author(s):  
Chen Zhao ◽  
Yan Xiu ◽  
John M Ashton ◽  
Lianping Xing ◽  
Yoshikazu Morita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Physiological Processes ◽  
Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

PRMT1 Activates Leukemic Stem Cell Program in MLL-Rearranged Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3493-3493
Author(s):  
Wing Chi Lui ◽  
Yuen Fan Chan ◽  
Ray Ng
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Leukemic Cells ◽  
Leukemic Stem Cell ◽  
Self Renewal ◽  
Mll Gene ◽  
Acute Myeloid

Abstract In MLL-rearranged leukemia, the Mixed Lineage Leukemia (MLL) gene undergoes chromosomal translocation that results in the loss of C-terminal histone methyltransferase SET domain, whereas the N-terminal of MLL gene fuses in-frame with one of the 60 identified partner genes. The resultant MLL fusion proteins lead to a characteristic aberrant gene expression pattern in human acute myeloid and lymphoblastic leukemia. Epigenetic dysregulation mediated by MLL fusion proteins has been suggested to be a key event in MLL-rearranged leukemia. It has been demonstrated that MLL-EEN/PRMT1 oncogenic complex induces transformation of primary myeloid progenitors via introduction of aberrant H4R3me2 at target Hoxloci. PRMT1 is the predominant protein arginine methyltransferase in mammals and is responsible for over 85% of arginine methylation activity in mammalian cells. Dysregulation of PRMT1 has been implicated in different cancers such as leukemia, suggesting the expression of PRMT1 is positively correlated with cancer progression and clinical parameters. Nevertheless, the leukemogenic role of PRMT1 in the establishment of leukemic stem cell (LSC) remains unclear. Previously we have demonstrated that a MLL fusion protein, MLL-EEN, can strongly enhance the self-renewal ability of murine primary hematopoietic cells through multiple rounds of replating assays. We have created a conditional Mll-Een invertor mouse model (MllEen/+) in which the expression of fusion protein is restricted to hematopoietic progenitors. Immunophenotypic analysis demonstrated a significant increase in the immature myeloid cell population (c-kit+Mac-1+) in bone marrow of MllEen/+ mice, suggesting that the expression of Mll-Een induces the development of acute myeloid leukemia. We have also established an Mll-Een expressing cell line from the bone marrow of MllEen/+ mouse. These leukemic cells can persistently form colonies and they also demonstrated deregulation of Hox genes, which is frequently observed in human leukemia cases. The leukemogenicity of Mll-Een is closely associated with Prmt1, which was demonstrated through knockdown of Prmt1. Strikingly, we discovered a subpopulation of CD41+Mll-Een expressing cells, which showed enhanced self-renewal ability in the serial colony forming assays. The percentage of CD41+ leukemic cells is reduced once Prmt1 was knocked down, suggesting that Prmt1 is crucial in the maintenance of this subpopulation of cells. In addition, the CD41+ cells showed enhanced expression of genes associated with hematopoietic stem cell (HSC) activities (Bmi-1, Runx1, Tal-1 and Lmo2), implying that part of the HSC transcriptional program has been re-activated in these cells. We therefore speculate that the CD41+ cells may represent a group of MLL leukemic cells that harbors strong stem cell features, and presumably functions as LSCs. The CD41+ leukemic cells will be further characterized by their LSC functions and CD41 can potentially serve as a novel LSC marker in MLL-rearranged leukemia. Taken together, studies on the role of PRMT1 can provide novel insights on the establishment of LSC and the development of effective clinical treatment for MLL-rearranged leukemia. Disclosures No relevant conflicts of interest to declare.

The Targeted Disruption of Kdm6a/Utx in Mice Has Gender-Dependent Effects on Primitive Hematopoietic Cell Populations and Myeloid Progenitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1158-1158
Author(s):  
Ling Tian ◽  
Lukas D. Wartman
Keyword(s):  
Bone Marrow ◽  
Young Female ◽  
Conditional Knockout ◽  
Cell Populations ◽  
Male Mice ◽  
Male And Female ◽  
Myeloid Progenitor ◽  
Cell Counts ◽  
Histone H3k27

Abstract Putative inactivating mutations of EZH2 (the histone H3K27 methylase) and KDM6A (a histone H3K27 demethylase) both occur in myeloid malignancies, including acute myeloid leukemia (AML). The mechanism(s) by which genetic inactivation of KDM6A contributes to leukemogenesis is not clear, and the role of KDM6A in normal hematopoiesis is largely undefined. To address the role of KDM6A in hematopoiesis, we generated a conditional knockout mouse of the Kdm6a gene on the X chromosome (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to inactivate Kdm6a in hematopoietic stem/progenitor cells. Mice were born in expected Mendelian ratios with no aberrant phenotypic abnormalities. We characterized normal hematopoiesis from young (6 to 8 week old) male and female Kdm6a conditional KO mice crossed with Vav1-Cre mice. We included both male and female (both homozygous and heterozygous Kdm6a KO mice) animals, since Kdm6a can have gender dependent effects, and human UTY (the KDM6A homologue on the Y chromosome) does have H3K27 demethylase activity (Thieme S et al., Blood, 2012 and Walport, L.J. et al., J Biol Chem, 2014). Young female Kdm6a null mice had a mild thrombocytopenia relative to all other cohorts with an average platelet count of 423 K/uL +/- 48 (n=10) vs. 794 K/ul +/- 76 for the WT littermates (n=10), p=0.006. We also observed mild splenomegaly in both the male and female Kdm6a null mice. The splenomegaly was not associated with extramedullary hematopoiesis or a shift in progenitor or mature lineage cell populations within the spleen. There was no difference in other blood cell counts, bone marrow cellularity, body weight or thymus weight between cohorts of young mice. We did not detect significant differences in the global levels of 21 histone H3 or 10 histone H4 modifications, using a multiplex colorimetric assay from lysates of whole bone marrow obtained from these mice. However, using western blotting, we did observe a decrease in H3K27 acetylation in both male and female Kdm6a null mice. We detected an aberrant self-renewal phenotype that may be relevant for leukemogenesis, which is in contrast to a previously reported impaired colony-forming ability using a knockdown approach of Kdm6a (Liu J et al., Exp Hematol, 2012). Using a serial replating assay with myeloid progenitor conditions, we found that both male and female Kdm6a null mice produced significantly more colonies in the second round of replating relative to control mice. Moreover, the homozygous female Kdm6a null mice had a significantly increased number of colonies at week 2, compared to hemizygous male mice (note that both are deficient for Kdm6a, but the male mice may compensate for it because of Uty). Flow cytometry revealed a slight myeloid skewing in the bone marrow of young female and male Kdm6a null mice with increased numbers of Gr-1+ and Cd11b+ cells. We did not detect differences in other lineages except for a slight decrease in erythroid precursors (as determined by Ter119 staining) in Kdm6a null mice. We also quantified the primitive hematopoietic and myeloid progenitor subpopulations from the bone marrow of these mice. Young female Kdm6a null mice had a significant decrease in the KLS population, which contained a lower frequency of short-term HSCs and multipotent progenitors. We also detected a significant decrease in MEPs (consistent with the observed thrombocytopenia). In young male Kdm6a null mice, the KLS population is not altered. A competitive transplant experiment validated the known engraftment defect in female Kdm6a null donor mice (Thieme S et al., Blood, 2012). Finally, we established a tumor watch consisting of female and male Kdm6a conditional KO mice and their littermate controls. After 18 months of follow-up, we did not observe the development of leukemia or other overt hematologic disease in either male or female mice deficient for Kdm6a, compared to a previous report that suggested that the rapid development of myelodysplasia (Thieme S et al., Blood, 2012). The difference in phenotype may be explained by cell-autonomous vs. non-autonomous effects (inactivation of Kdm6a in our model is essentially limited to the hematopoietic compartment). In sum, our data suggest that Kdm6a has a relatively subtle role in normal hematopoiesis, but the perturbations associated with its inactivation reveal insights into its role as a potential tumor suppressor in myeloid leukemogenesis. Disclosures No relevant conflicts of interest to declare.

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