U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival

Abstract Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

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Mutant ASXL1 Disrupts Paraspeckle Formation through Aberrant Interaction with Nono in Hematopoietic Cells

Blood ◽

10.1182/blood-2019-125472 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2514-2514

Author(s):

Yamamoto Keita ◽

Susumu Goyama ◽

Shuhei Asada ◽

Takeshi Fujino ◽

Tomofusa Fukuyama ◽

...

Keyword(s):

Bone Marrow ◽

Alternative Splicing ◽

Rna Splicing ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Wild Type ◽

Truncated Form ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Marrow Cells

Paraspeckles are sub-nuclear structures found in the interchromatin space of mammalian cells. The core paraspeckle components include a lncRNA NEAT1 and members of the DBHS family of proteins: NONO, SFPQ, and PSPC1. Paraspeckles and their components play diverse roles in gene regulatory networks, including transcription, alternative RNA splicing, nuclear retention of RNA, and DNA repair. Although a previous study showed the presence of paraspeckles in hematopoietic stem and progenitor cells (HSPCs), their roles in normal and malignant hematopoiesis remain largely unknown. ASXL1 regulates gene expression through interactions with multiple epigenetic regulators. Somatic mutations in ASXL1 gene occur frequently in myeloid neoplasms. We previously generated a hematopoietic lineage-specific conditional knockin (KI) mouse of a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), and showed that ASXL1-MT inhibited repopulating capability of HSPCs. We performed deep RNA sequencing using HSPCs from ASXL1-MT-KI mice, and found aberrant alternative splicing in multiple genes involved in hematopoiesis. The altered splicing in ASXL1-MT-KI HSPCs included abnormal exon skipping or retention in Runx1, Traf6, Atm, and Dnmt3b. These findings, together with a previous report showing that ASXL1 mutations affect alternative splicing in U937 cells, strongly indicate the involvement of ASXL1 in RNA splicing machinery. Because a previous interactome analysis suggested the association between NONO and ASXL1, we hypothesized that ASXL1 may play a role in RNA maturation processes through interactions with paraspeckle proteins. To test this hypothesis, we examined physical and functional interactions between paraspeckle components and ASXL1. We found that both wild-type and mutant ASXL1 interact with NONO and SFPQ in 293T cells. Interestingly, protein and RNA immunoprecipitation (RIP) analyses revealed that coexpression of wild-type ASXL1, but not mutant ASXL1, enhanced interactions between NONO and histone H3 as well as NONO and NEAT1. These results suggest that ASXL1 acts as a scaffolding protein that assembles paraspeckle proteins and histones to promote transcription and RNA processing. Importantly, mutant ASXL1 loses this function. Next, we assessed subcellular localization of Nono in HSPCs from control and ASXL1-MT-KI mice. We observed predominant cytoplasmic expression of Nono in ASXL1-MT KI HSPCs, while Nono mainly localized in the nucleus in control cells (Figure 1). In addition, expression of NEAT1_2 isoform, which is essential for paraspeckle formation and maintenance, was substantially downregulated in ASXL1-MT-KI HSPCs. Consistent with these observations, RNA FISH against NEAT1 and immunofluorescence against NONO revealed disrupted paraspeckle formation in ASXL1-MT-KI HSPCs. These data suggest that ASXL1-MT promotes nuclear export of Nono, which results in disruption of paraspeckles in HSPCs. NONO has nuclear localization signal (NLS) at its C-terminus, and it was previously shown that a cytoplasmic C-truncated form of NONO induced senescence in fibroblasts. To assess the effect of forced expression of the cytoplasmic NONO in hematopoietic cells, we transduced vector or a NONO mutant lacking the NLS domain (NONO-ΔNLS) into c-Kit+ bone marrow cells, and transplanted these cells into recipient mice. NONO-ΔNLS induced overproduction of reactive oxygen species (ROS) and reduced engraftment of bone marrow progenitors as ASXL1-MT did. We then assessed the effect of Nono depletion in ASXL1-MT-KI HSPCs using CRISPR/Cas9 system. We crossed ASXL1-MT-KI mice with Rosa26-LSL-Cas9-KI mice, and c-Kit+ bone marrow cells derived from these mice were transduced with a non-targeting or Nono-targeting sgRNAs. This experiment revealed that Nono depletion reverted the impaired repopulation of ASXL1-MT-KI HSPCs after transplantation. Taken together, these data indicate that the cytoplasmic localization of Nono induced by ASXL1-MT has the negative impact on HSPC function. In summary, this study reveals a novel link between an epigenetic regulator ASXL1 and paraspeckle formation. The aberrant interaction between mutant ASXL1 and NONO results in NONO mislocalization, paraspeckle disruption and HSPC dysfunction. Our findings also suggest potentially important roles for paraspeckles to maintain normal hematopoiesis. Disclosures Ogawa: Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Kan Research Laboratory, Inc.: Consultancy.

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

Blood ◽

10.1182/blood.v118.21.859.859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 859-859 ◽

Cited By ~ 1

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.

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Protective Role of Connexin 32 in Experimental Leukemogenesis.

Blood ◽

10.1182/blood.v110.11.2239.2239 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2239-2239

Author(s):

Yoko Hirabayashi ◽

Byung-Il Yoon ◽

Isao Tsuboi ◽

Yan Huo ◽

Yukio Kodama ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Bone Marrow Cells ◽

Pcr Analysis ◽

Wild Type ◽

Hematopoietic Stem ◽

Immunomagnetic Bead ◽

Competitive Assay ◽

Marrow Cells

Abstract Connexin (Cx) functions in the organization of cell-cell communication in multicellular organisms. Gap junctions have been implicated in the homeostatic regulation of various cellular functions, including growth control and differentiation, apoptosis, and the synchronization of electrotonic and metabolic functions. Primitive hemopoietic progenitor cells form a multicellular system, but a previous report describes that Cx32 is not expressed in the bone marrow. Thus, a question arises as to why Cx molecules are not detected in the hematopoietic tissue other than stromal cells. Based on our preliminary study that suggested a potential impairment of hematopoiesis in Cx32-knockout (KO) mice, the objectives of the present study were to determine whether Cx32 functions in the bone marrow during steady-state hematopoiesis and further to examine its possible protective roles during regeneration after chemical abrasions and during leukemogenesis after the administration of a genotoxic chemical, methyl nitrosourea (MNU). As results, the Cx32 molecule functioning in the hematopoietic stem cell (HSC) compartment during steady-state hematopoiesis was observed for the first time; the expression of Cx32 at the mRNA level determined by PCR analysis and that at the protein level determined using an anti-Cx32 antibody were observed only in the lin−c-kit+ HSC fraction using a combination of immunobead-density gradient and immunomagnetic-bead separation. Hematopoiesis was impaired in the absence of Cx32; it was delayed during regeneration after chemical abrasion with 5-fluorouracil at 150 mg/kg body weight in Cx32-KO mice. Cx32-KO mice also showed increased leukemogenicity compared with wild-type mice after MNU injection; furthermore, in a competitive assay for leukemogenicity in mice that had been lethally irradiated and repopulated with a mixed population of equal amount of bone marrow cells from Cx32-KO mice and wild-type mice, the resulting leukemias were originated predominantly from Cx32-KO bone marrow cells. The present competitive assay clearly showed that Cx32-KO bone marrow cells have a higher risk of becoming leukemogenic. The above-mentioned findings in this study imply that Cxs play an essential role in maintaining the steady-state hematopoiesis and suppressing the neoplastic change. In summary, the role of Cx32 in hematopoiesis was not previously recognized and Cx32 was expressed only in HSCs and their progenitors. The results indicate that Cx32 in wild-type mice protects HSCs from chemical abrasion and leukemogenic impacts. Our results indicate that the risk of developing leukemia in patients with X-chromosome-linked Cx32 deficiency, called Charcot-Marie-Tooth syndrome, may not be incidental.

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R-Ras Regulates Hematopoietic Stem/Progenitor Cell Adhesion, Migration, and Mobilization through Rac GTPase-Mediated Signals.

Blood ◽

10.1182/blood.v110.11.221.221 ◽

2007 ◽

Vol 110 (11) ◽

pp. 221-221

Author(s):

Xun Shang ◽

Lina Li ◽

Jose Concelas ◽

Fukun Guo ◽

Deidre Daria ◽

...

Keyword(s):

Bone Marrow ◽

Cell Adhesion ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Wild Type ◽

Rac Gtpase ◽

Hematopoietic Stem ◽

And Migration ◽

Marrow Cells

Abstract Hematopoietic stem/progenitor cells (HSPCs) are maintained by strictly regulated signals in the bone marrow microenvironment. One challenge in understanding the complex mode of HSPC regulation is to link intracellular signal components with extracellular stimuli. R-Ras is a member of the Ras family small GTPases. Previous mouse genetic studies suggest that R-Ras mRNA is primarily expressed in endothelial cells and R-Ras is involved in vascular angiogenesis. In clonal cell lines, although dominant mutant overexpression studies suggest a possible role of R-Ras in regulating cell adhesion and spreading, proliferation and/or differentiation in a cell-type dependent manner, it remains controversial whether R-Ras activity may promote or inhibit cell adhesion and migration. Here, in a mouse knockout model, we have examined the role of R-Ras in HSPC regulation by a combined in vivo and in vitro approach. Firstly, we found that R-Ras is expressed in the Lin− low density bone marrow cells of wild-type mice, and R-Ras activity in the cells is downregulated by cytokines and chemokines such as SCF and SDF-1a (∼ 20% and 40% of unstimulated control, respectively). Secondly, R-Ras deficiency did not significantly affect peripheral blood CBC, nor alter the frequency or distribution of long-term and short-term hematopoietic stem cells (defined by IL7Ra−Lin−Sca-1+c-Kit+CD34− and IL7Ra−Lin−Sca-1+c-Kit+CD34+ genotypes, respectively) in the bone marrow, peripheral blood and spleen. Competitive repopulation experiments using the wild-type and R-Ras−/− bone marrow cells at 1:1 ratio in lethally irradiated recipient mice showed no significant difference of blood cells of the two genotypes in the recipients up to 6 months post-transplantation. R-Ras−/− bone marrow cells did not show a detectable difference in colony forming unit activities assayed in the presence of various combinations of SCF, TPO, EPO, IL3, G-CSF and serum, compared with the matching wild-type cells. Thirdly, upon challenge with G-CSF, a HSPC mobilizing agent, R-Ras−/− mice demonstrated a markedly enhanced ability to mobilize HSPCs from bone marrow to peripheral blood as revealed by genotypic and colony-forming unit analyses (WT: 150 vs. KO: 320 per 200uL blood, p=0.018), and R-Ras−/− HSPCs exhibit significantly decreased homing activity (WT: 4.3% vs. KO: 2.8%, p<0.001). Fourthly, isolated R-Ras−/− HSPCs displayed a constitutively assembled cortical actin cytoskeleton structure in the absence of cytokine or chemokine stimulation, similar to that of activated wild-type HSPCs. The R-Ras−/− HSPCs were defective in adhesion of cobblestone area-forming cells to a bone marrow-derived stroma cell line (FBMD-1) and in adhesion to fibronectin CH296 fragment, and showed a drastically increased ability to migrate toward a SDF-1a gradient (WT: 16% vs. KO: 38%, p<0.001). These data point to a HSPC-intrinsic role of R-Ras in adhesion and migration. Finally, the functional changes of R-Ras−/− cells were associated with a ∼3 fold increase in Rac-GTP species and constitutively elevated Rac downstream signals of phsopho-PAK1 and phospho-myosin light chain. Partial inhibition of Rac activity by NSC23766, a Rac GTPase-specific inhibitor, readily reversed the migration phenotype under SDF-1a stimulation. Taken together, these studies demonstrate that R-Ras is a critical signal regulator for HSPC adhesion, homing, migration, and mobilization through a mechanism involving Rac GTPase-regulated cytoskeleton and adhesion machinery.

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C-Cbl Regulates Interaction of Immature Hematopoietic Cells with the Bone Marrow Microenvironment by Rac GTPase-Mediated Cytoskeletal Signals.

Blood ◽

10.1182/blood.v114.22.3643.3643 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3643-3643

Author(s):

Eisuke Uehara ◽

Takahiro Suzuki ◽

Hiroshi Okabe ◽

Masuzu Ueda ◽

Tadashi Nagai ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Bone Marrow Microenvironment ◽

Negative Regulator ◽

Wild Type ◽

Rac Gtpase ◽

Hematopoietic Stem ◽

Deficient Mice ◽

Marrow Cells

Abstract Abstract 3643 Poster Board III-579 <Background and purpose> c-Cbl is a ubiquitin E3 ligase and functions as a negative regulator for signals induced by various activated tyrosine kinases, by promoting ubiquitination and proteasomal degradation of these kinases. This inhibitory action is mainly mediated by the tyrosine kinase binding (TKB) and RING finger domains located in the NH3-terminal part of the molecule and these domains are evolutionally well-conserved from nematodes. But mammalian c-Cbl has a stretched structure toward the COOH-terminal end with putative tyrosine residues that can interact with p85 subunit of PI3-kinase, and several reports have denoted that this interaction may be important for cytoskeletal regulation. Recently, mutations of the c-cbl gene have been reported in hematopoietic malignancies, and regulation of hematopoietic stem/progenitor cells (HSPCs) by Cbl is attracting attention. Since it is well-known that cytoskeletal dysregulation is often associated with malignant transformation, in this study, we investigated cytoskeletal regulatory mechanisms mediated by Cbl in hematopoietic cells, using Cbl deficient mice. <Methods and results> To examine the migratory capacity of the HSPCs, lineage negative (Lin(-)) bone marrow cells were set in the Boyden Chamber assay for SDF-1 and fibronectin (FN) were performed. We found that Cbl deficient Lin(-) cells showed significantly decreased migration to these chemoattractants; the migration capacity of the Cbl deficient cells was one eighth for SDF-1 (p = 0.01) and one third for FN (p = 0.007), respectively, compared with the wild-type counterparts. Then, to evaluate in vivo homing ability to the bone marrow microenvironment, transplantation assays were performed. We transplanted 2.5 × 10e6 of Lin(-) HSPCs from Cbl deficient or wild-type Ly5.2 mice into sublethally irradiated wild-type Ly5.1 mice. Three hours after transplantation, chimerism of the transplanted Ly5.2 cells in the bone marrow was examined, and we found that the number of transplanted cells was significantly smaller in Cbl deficient cells, showing that homing capacity of HSPCs in Cbl deficient mice was impaired. Moreover, when we administered G-CSF to Cbl deficient mice, a significantly larger number of Lin(-) Sca1(+) c-Kit(+) cells were mobilized from the bone marrow (p = 0.01), indicating that in Cbl deficient mice, mobilization of HSPCs by G-CSF was also affected. In bone marrow cells or Lin(-) HPSCs of Cbl deficient mice, activity of Rac, a member of small G-protein GTPases, was significantly decreased (p = 0.002). These data ware supported by the experiments with embryonic fibroblast; Rac activities induced by FN stimulation were weaker in Cbl deficient fibroblasts than wild-type ones, and Cbl deficient fibroblasts showed impaired actin rearrangement. <Discussion> We found that Cbl deficient HSPCs showed impaired migration activities to chemoattractants and altered homing and mobilization to and from the bone marrow. Furthermore, Cbl deficient cells had impaired activation of Rac. Currently, little is known about the relationship between Cbl-mediated signals and cytoskeletal regulator Rac, and in this study, we found that Cbl is a positive regulator of Rac activity in the bone marrow hematopoietic cells. Rac activity is reported to be necessary for homing and retention of HSPCs in the bone marrow microenvironment, and our results indicate that interaction of HSPCs with the bone marrow microenvironment, e.g. trafficking of these cells, is regulated by Cbl via Rac GTPase signals. Disclosures: No relevant conflicts of interest to declare.

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Kras Plays An Important Role In Generating Differentiated Blood Cells

Blood ◽

10.1182/blood.v122.21.2451.2451 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2451-2451 ◽

Cited By ~ 1

Author(s):

Alisa Damnernsawad ◽

Guangyao Kong ◽

Yangang Liu ◽

Yuan-I Chang ◽

Jingfang Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Blood Cells ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Conditional Knockout ◽

Wild Type ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract Background Kras is a small GTPase essential for mouse embryonic development. Although Kras-/- fetal liver cells reconstitute recipient mice indistinguishably from wild-type cells, chimeric mice generated from injection of Kras-/- embryonic stem cells into wild-type blastocysts show little contribution of knockout cells to hematopoietic tissues even when these cells contribute to all the other tissues to a high degree. These results suggest that Kras plays an important role in adult hematopoiesis. However, early embryonic lethality of Kras-/- mice prevents further investigation of Kras functions in adulthood. To overcome this problem, we generated Kras conditional knockout mice (Krasfl/fl), which allow the deletion of Kras by the Cre recombinase in desired tissues and at desired developmental stages. Method We used two transgenic Cre lines, Mx1-Cre and Vav-Cre, to knockout Kras in adult hematopoietic system. The Mx1 promoter is induced by interferon signaling, which can be triggered by injections of polyinosinic-polycytidylic acid (pI-pC). The Vav promoter drives Cre expression specifically in fetal liver hematopoietic cells since E11.5 as well as in adult hematopoietic tissues. Both Cre lines efficiently deleted Kras expression in above 95% of hematopoietic cells as judged by single hematopoietic stem cell (HSC) genotyping. Results obtained from these two different Cre lines were essentially same. Results We found that the frequency and absolute number of Kras-/- HSCs, multipotent progenitors (MPPs), LSK (Lin- Sca1+ cKit+) cells, myeloid progenitors and common lymphoid progenitors are comparable to wild-type control cells. Consistent with this observation, cytokine signaling in Kras-/- hematopoietic stem/progenitor cells (HSPCs, Lin- cKit+) is indistinguishable from control HSPCs. In contrast, the percentage of CD19+ B-cells is moderately but significantly reduced in Kras-/- spleens and concomitantly cytokine-evoked ERK1/2 activation is greatly reduced in differentiated blood cells. To determine whether Kras plays an important role in regulating HSC functions, we performed a competitive bone marrow reconstitution assay using CD45.2+ control or Kras-/- bone marrow cells mixed together at ratios 1:1 and 3:1 with congeneic competitor cells (CD45.1+ bone marrow cells). Kras-/- bone marrow cells show significantly reduced long-term reconstitution in recipient mice compared to control cells (10% vs 45%). The reduced reconstitution is persistent in the secondary and tertiary recipients. However, detailed analysis in primary and secondary recipients revealed that the frequency of Kras-/- HSCs and MPPs is comparable to that of control cells and Kras-/- progenitor cells are also largely normal, indicating that Kras is dispensable for adult HSC functions but might play an important role in generating differentiated blood cells. The reduced generation of myeloid cells is further validated in an in vitro culture assay, in which we quantitatively measured the myeloid cell production from Lin- progenitor cells. Conclusions Our results indicate that loss-of-Kras could be compensated by other Ras isoforms in adult HSCs. However, in mature blood cells, Kras deficiency results in greatly reduced cytokine-evoked ERK1/2 activation. Under a stressed condition (e.g. competitive bone marrow transplantation), the generation of Kras-/- blood cells is defective. Taken together, our study reveals a novel and unique function of Kras in regulating adult hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.2504.2504 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2504-2504

Author(s):

Russell Garrett ◽

Gerd Bungartz ◽

Alevtina Domashenko ◽

Stephen G. Emerson

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Poly I:C ◽

Hematopoietic Stem ◽

Marrow Cells ◽

Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

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Understanding the Impact of Inflammation on Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.2629.2629 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2629-2629

Author(s):

Ying Zhao ◽

Flora Ling ◽

Hong-Cheng Wang ◽

Xiao-Hong Sun

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Chronic Inflammation ◽

Bone Marrow Cells ◽

Wild Type ◽

Hematopoietic Stem ◽

Inflammatory Conditions ◽

The Impact ◽

Lps Treatment ◽

Marrow Cells

Abstract Abstract 2629 The overall objectives of this study are to investigate the impact of inflammatory conditions on hematopoietic stem cell (HSC) maintenance and to elucidate the underlying mechanisms. HSCs are exposed to a variety of inflammatory conditions through life. How these conditions influence the integrity of HSCs is a fundamental issue of clinical importance but it is poorly understood. Equally unknown is the molecular regulation of HSC maintenance during inflammatory. In this context, our focus is on the role of basic helix-loop-helix (bHLH) proteins, which include transcription activators such as E2A proteins and their inhibitors including Id proteins. We and others have shown that these regulators are involved in normal hematopoiesis such as stem cell function and lineage specific differentiation. Recently, we have obtained evidence to suggest that signaling through Toll-like receptors (TLRs), which is closely linked to inflammation, causes down-regulation of E2A function by stimulating Id1 expression. Therefore, we hypothesize that inflammatory conditions causes down-regulation of E protein function, which disturbs the quiescence of long-term (LT)-HSC, leading to stem cell exhaustion over time. To test this hypothesis, we induced chronic inflammation in wild type and Id1-/- mice by daily injection of 1 mg of LPS, i.p. for 30 days. Peripheral blood was collected on days 15 and 30 and levels of a panel of inflammatory cytokines were assayed using a Luminex multiplex kit. On day 15, dramatic increases were found in the levels of IL-10, IL-6, KC and TNFα but not IFN-γ, IL12-p70 and IL-1β. Interestingly, levels of IL-6 and TNFα were significantly lower in Id1-/- mice compared to wild type mice. By day 30 of LPS treatment, levels of these cytokines returned to the levels in animals without LPS injection. These results suggest that this chronic LPS treatment indeed elicited an inflammatory response that included transient elevation of inflammatory cytokines. Whether secretion of these cytokines has any direct effects on HSCs remains to be determined. To measure HSC activity in these LPS-treated mice, we performed serial bone marrow transplant assays. Lin−Sca-1+c-kit+ (LSK) stem/progenitor cells were isolated from wild type or Id1-/- mice treated with or without LPS. These cells were transplanted into lethally irradiated CD45.1+ recipients along with equal numbers of YFP-expressing LSK as competitors. Six weeks later, cohorts of mice were sacrificed and bone marrow cells were collected. Pooled whole bone marrow cells within each cohort were injected into lethally irradiated secondary recipients. Secondary recipients were sacrificed 8 and 16 weeks post transplant. For assessment of primary and secondary engraftment, bone marrow cells were examined for expression of donor and lineage specific markers. Robust engraftment was observed in primary or secondary recipients. Donor derived cells were then gated for YFP− and YFP+ cells, which separate cells originated from tester and competitor LSK, respectively. While YFP− and YFP+ cells engrafted equivalently in primary recipients transplanted with cells treated with or without LPS, LPS treatment of wild type mice caused a great disparity in secondary recipients. In contrast, HSC in Id1-/- mice did not appear to be affected by the same treatment even though HSCs in Id1 deficient mice are normally lower in numbers and activities as we previously reported. These results suggest that chronic inflammation diminishes the LT-stem cell activity and this may involve the up-regulation of Id1 expression. To investigate the underlying mechanism, we performed label retaining assays to examine the quiescence of LT-HSCs. We found that BrdU-labeling in HSCs was 2-fold lower in mice treated with LPS compared to the untreated controls, suggesting that treatment with LPS promoted the cycling of HSCs, thus impairing their stem cell function. Taken together, our study illustrates that chronic inflammation has a detrimental effect on LT-stem cell activity. Although HSCs have an enormous capability to repopulate the bone marrow by compensatory proliferation, pro-longed inflammation could eventually lead to stem cell exhaustion and seriously compromise hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs

Blood ◽

10.1182/blood.v122.21.2430.2430 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2430-2430

Author(s):

Zhong-Fa Yang ◽

Wang Junling ◽

Alan G. Rosmarin

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Protein Kinase ◽

Bone Marrow Cells ◽

Specific Cell ◽

Wild Type ◽

Serine Threonine Kinase ◽

Hematopoietic Stem ◽

Threonine Kinase ◽

Marrow Cells

Abstract Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v122.21.469.469 ◽

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.

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