The Role of Forkhead Transcription Factors FoxO1, FoxO3 and FoxO4 in Hematopoiesis and Leukemogenesis.

Abstract FoxO is a family of forkhead transcription factors that negatively regulate proliferation and survival signals in hematopoietic cells. We and others have previously shown that inhibition of the three members of this family (FoxO1, FoxO3 and FoxO4) by leukemogenic tyrosine kinase fusion genes results in enhanced proliferative and survival signaling in leukemic cells. For example, the transforming activities of the lymphoma associated NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) fusion, BCR-ABL, or FLT3-ITD, are mediated in part by inactivation of FoxO through phosphorylation and ubiquitin mediated degradation by constitutively active Akt (Gu TL, et al. Blood 2004), with subsequent induction of proliferative and survival signals. Furthermore, inhibition of FoxO is required for efficient transformation of hematopoietic cells. However, the roles of FoxO in adult hematopoiesis are unknown. We have initiated studies to examine the role of FoxO in the context of normal hematopoiesis and leukemogenesis using a triple conditional knockout mouse for each of the FoxO1, FoxO3 and FoxO4 alleles. The FoxO alleles are flanked by lox-P sites and conditional excision is mediated by Cre expression under the control of the interferon inducible Mx1 promoter. Based on the normal function of FoxO family members to repress proliferative and survival signals, we hypothesized that the deletion of FoxO subfamily members would lead to an enhanced proliferation and survival in the hematopoietic compartment, and might contribute to the development of a myeloproliferative and/or lymphoproliferative phenotype in vivo. Triple homozygous conditional FoxO knockout mice were generated in an Mx1-Cre background to allow for excision of the FoxO alleles in the hematopoietic stem cell compartment after treatment with pIpC. Complete excision of each of the three alleles in the hematopoietic compartment was confirmed. However, in contrast with our working hypothesis, we observed that loss of function of FoxO family members was associated with a relatively subtle hematopoietic phenotype with 12 months of follow-up. The phenotype includes a non-fatal mild myeloproliferative phenotype that is progressive over time and characterized by modest splenomegaly, extramedullary hematopoiesis and increased mature myeloid populations in bone marrow and spleen. In addition, there are subtle alterations in both B and T lymphoid cell populations, including a decrease in both immature and mature B cells in the spleen and bone marrow; and abnormalities of CD4+CD8+ double positive and CD4+ and CD8+ T cells in the thymus. Examination of stem and progenitor populations also revealed subtle differences in the HSC and CLP progenitor populations at 4 weeks post pIpC. Thus, these data indicate that complete loss of FoxO function in the adult hematopoietic compartment results in a relatively subtle hematopoietic phenotype. They further demonstrate that although inhibition of FoxO family members is required for efficient transformation of hematopoietic cells by leukemogenic fusion tyrosine kinases, loss of FoxO function alone is not sufficient to induce a leukemic phenotype.

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The Role of AP-1 Transcription Factors in Plasma Cell Biology and Multiple Myeloma Pathophysiology

Cancers ◽

10.3390/cancers13102326 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2326

Author(s):

Fengjuan Fan ◽

Klaus Podar

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Transcription Factors ◽

Cell Differentiation ◽

Plasma Cell ◽

Cell Biology ◽

Hematologic Malignancy ◽

Family Members ◽

Plasma Cell Differentiation

Multiple myeloma (MM) is an incurable hematologic malignancy characterized by the clonal expansion of malignant plasma cells within the bone marrow. Activator Protein-1 (AP-1) transcription factors (TFs), comprised of the JUN, FOS, ATF and MAF multigene families, are implicated in a plethora of physiologic processes and tumorigenesis including plasma cell differentiation and MM pathogenesis. Depending on the genetic background, the tumor stage, and cues of the tumor microenvironment, specific dimeric AP-1 complexes are formed. For example, AP-1 complexes containing Fra-1, Fra-2 and B-ATF play central roles in the transcriptional control of B cell development and plasma cell differentiation, while dysregulation of AP-1 family members c-Maf, c-Jun, and JunB is associated with MM cell proliferation, survival, drug resistance, bone marrow angiogenesis, and bone disease. The present review article summarizes our up-to-date knowledge on the role of AP-1 family members in plasma cell differentiation and MM pathophysiology. Moreover, it discusses novel, rationally derived approaches to therapeutically target AP-1 TFs, including protein-protein and protein-DNA binding inhibitors, epigenetic modifiers and natural products.

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Adoptive Transfer of Nf1+/− Bone Marrow Is Sufficient for Neurofibroma Progression in Krox20;Nf1flox/flox Mice.

Blood ◽

10.1182/blood.v106.11.3064.3064 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3064-3064

Author(s):

Fengchun Yang

Keyword(s):

Bone Marrow ◽

Mast Cells ◽

Tumor Microenvironment ◽

Schwann Cells ◽

Genetic Disorder ◽

Hematopoietic Cells ◽

Tumor Formation ◽

Conditional Knockout ◽

Axial Tomography

Abstract Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a GTPase activating protein for Ras called neurofibromin. NF1 is a genetic disorder that affects approximately 250,000 individuals in the US, Europe, and Japan alone. Neurofibromas, the hallmark of NF1, are complex tumors characterized by tumorigenic Schwann cells, neoangiogenesis, fibrosis, and degranulating mast cells. Studies in experimental models have emphasized the role of inflammatory cells in altering the microenvironment and facilitating malignant outgrowth. Similarly, Parada (Science, 2002) found that nullizygosity of Nf1 in Schwann cells of conditional knockout mice (Krox20;Nf1flox/flox) was necessary but not sufficient for neurofibroma formation and haploinsufficiency of Nf1 in lineages within the tumor microenvironment was required for neurofibroma progression. We previously provided the first genetic, cellular, and biochemical evidence that haploinsufficiency of Nf1 alters Ras activity and cell fates in mast cells (JEM, 2000, 2001) and identified a mechanism underlying the recruitment of mast cells to tumorigenic Schwann cells (JCI 2003). However, it remains unclear whether Nf1 +/− bone marrow derived hematopoietic cells can directly contribute to neurofibroma formation in vivo. To address this question, Nf1+/− or wildtype (WT) EGFP+ bone marrow (BM) was adoptively transferred into lethally irradiated Krox20;Nf1flox/flox mice and cohorts were followed prospectively for tumor formation using positron emission tomography and computerized axial tomography. Mice transplanted with Nf1+/− but not WT BM developed progressive enlargement of the trigeminal nerve, dorsal root ganglia, peripheral nerves, and motor paralysis similar to Krox20;Nf1flox/− mice that are haploinsufficient at Nf1 in all lineages of the tumor microenvironment. Postmortem analysis revealed that Krox20;Nf1flox/flox mice transplanted with Nf1+/− BM had cellular neurofibromas containing Schwann cells, fibroblasts, blood vessels and mast cells, which closely resembled the cellular architecture of human neurofibromas. Mice transplanted with WT BM did not develop neurofibromas. These studies establish that recruitment of Nf1 +/− BM derived cells to the neurofibroma microenvironment is directly linked to neurofibroma formation and progression. Given our observations, therapies which prevent both the recruitment and the tumor promoting functions of Nf1 +/− hematopoietic cells in neurofibroma formation are currently being tested in vivo as pre-clinical trials.

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Requirement of CREB in Normal and Malignant Hematopoiesis.

Blood ◽

10.1182/blood.v108.11.1168.1168 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1168-1168

Author(s):

Jerry C. Cheng ◽

Deepa Shankar ◽

Stanley F. Nelson ◽

Kathleen M. Sakamoto

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Western Blot ◽

Blot Analysis ◽

Hematopoietic Cells ◽

Wild Type ◽

Qrt Pcr ◽

T315i Mutation

Abstract CREB is a nuclear transcription factor that plays an important role in regulating cellular proliferation, memory, and glucose homeostasis. We previously demonstrated that CREB is overexpressed in bone marrow cells from a subset of patients with acute leukemia at diagnosis. Furthermore, CREB overexpression is associated with an increased risk of relapse and decreased event-free survival in adult AML patients. Transgenic mice that overexpress CREB in myeloid cells developed myeloproliferative/myelodysplastic syndrome after one year. To further understand the role of CREB in leukemogenesis and in normal hematopoiesis, we employed RNA interference methods to inhibit CREB expression. To achieve sustained, CREB-specific gene knockdown in leukemia and normal hematopoietic cells, a lentiviral-based small hairpin (shRNA) approach was taken. Three CREB specific shRNAs were generated and tested for efficiency of gene knockdown in 293T cells. Knockdown efficiency approached 90 percent by Western blot analysis compared to vector alone and luciferase controls. Human myeloid leukemia cell lines, K562, TF1, and MV411, were then infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed using quantitative real time (qRT)-PCR, Western blot analysis, and growth and viability assays. Lentiviral CREB-shRNA achieved between 50 to 90 percent knockdown of CREB compared to control shRNAs at the protein and mRNA levels. To control for non-specific effects, we performed qRT-PCR analysis of the interferon response gene, OAS1, which was not upregulated in cells transduced with CREB shRNA constructs. Within 72 hours, cells transduced with CREB shRNA had decreased proliferation and survival. Similar results were obtained with murine leukemia cells (NFS60 and BA/F3 bcr-abl).To study the role of CREB in normal hematopoiesis, both primary murine and human hematopoietic cells were transduced with our shRNA constructs, and methylcellulose-based colony assays were performed. Primary hematopoietic cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in colony number compared to control virus-infected cells (p<0.05). Bone marrow colonies consisted of myeloid progenitor cells that were mostly Mac-1+ by FACs analysis. Interestingly, there were fewer differentiated cells in the CREB shRNA transduced cells compared to vector control or wild type cells, suggesting that CREB is critical for both myeloid cell proliferation and differentiation. To study the in vivo effects of CREB knockdown on leukemia progression, we studied mice injected with BA/F3 cells that express both bcr/abl with the T315I mutation and a luciferase reporter gene. BA/F3 cells expressing the T315I mutation have a 2-fold increase in CREB overexpression compared to wild-type cells. Disease progression was monitored using bioluminescence imaging with luciferin. CREB knockdown was 90 percent after transduction and prior to injection into SCID mice. We observed improved survival of mice injected with CREB shRNA transduced BA/F3 bcr-abl (T315I) compared to vector control cells. To understand the mechanism of growth suppression resulting from CREB downregulation, we performed microarray analysis with RNA from CREB shRNA transduced K562 and TF1 cells. Several genes were downregulated using a Human Affymetrix chip. Most notable was Beclin1, a tumor suppressor gene often deleted in prostate and breast cancer that has been implicated in autophagy. Our results demonstrate that CREB is required for normal and leukemic cell proliferation both in vitro and in vivo.

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

Blood ◽

10.1182/blood.v110.11.3365.3365 ◽

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.

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Selected Commensals Mediate GvHD Via MyD88-Dependent Signaling in Non-Hematopoietic Cells.

Blood ◽

10.1182/blood.v120.21.3005.3005 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3005-3005 ◽

Cited By ~ 1

Author(s):

Matthew Horch ◽

Nathanael Sahli ◽

Elizabeth Zale ◽

Kyle Tretina ◽

Vu H. Nguyen

Keyword(s):

Bone Marrow ◽

Bacterial Translocation ◽

Broad Spectrum ◽

Hematopoietic Cells ◽

Host Cells ◽

Allogeneic Bone ◽

Transplant Recipients ◽

High Exposure ◽

Myd88 Signaling

Abstract Abstract 3005 Graft-versus-host disease (GVHD) primarily affects the epithelial compartments of skin, liver, and gut and is thought to display tissue tropism for two main reasons: the conditioning regimen can selectively damage these epithelial cell compartments due to rapid cell turnover; and these epithelial organs have high exposure to host commensals and their products that promote tissue-specific inflammation. To understand how commensals mediate GVHD, we first tested the hypothesis that the disruption of the MyD88 signaling pathway, which is critical for host-microbe interaction, would alter GVHD outcomes. We created bone marrow chimeras that received a second allogeneic bone marrow transplant (BMT) and showed that MyD88 deficiency in recipient hematopoietic cells reduced BMT mortality, while deficiency in the non-hematopoietic cells (NHC) increased BMT mortality (p<0.05). These findings indicate the protective role of MyD88 expression in the latter compartment. Furthermore, deficiency of TLR6, but not TLR1,2,4, and 5, in NHC recapitulated similar BMT outcomes, suggesting regulation of MyD88 by TLR6 in our BMT model. Based on a hypothesis that MyD88 signaling on NHC promotes epithelial healing and barrier function, we first determined whether the level of bacterial translocation in MyD88KO BMT recipients are different. 16s DNA, a measurement of bacteria content, was significantly increased in livers of MyD88 knock-out (KO) transplant recipients compared to WT counterparts. We next decontaminated MyD88KO transplant recipients with a cocktail of broad spectrum antibiotics (Abx) prior to BMT and maintained these mice in sterile isolators. MyD88KO mice treated with Abx and untreated WT BMT recipients had similar survival; in contrast, untreated MyD88KO recipients died rapidly within the first 2 weeks following BMT (p<0.05). 16S DNA level was reduced in MyD88KO mice treated with Abx prior to BMT compared to their untreated MyD88KO counterpart. Furthermore, MyD88KO radiation controls treated with Abx had improved survival, similar to WT radiation controls, compared to untreated MyD88KO controls. These series of findings suggest a critical role of MyD88 in promoting barrier protection and healing via the NHC following BMT. Interestingly, similar Abx decontamination in WT BMT recipients did not change survival, suggesting that broad microbial decontamination in normal recipients without significant risk for bacterial translocation is not beneficial. Based on these findings, we next hypothesized that selective versus broad decontamination of commensal populations would improve GVHD outcomes following allogeneic BMT in WT mice. In MHC-matched and mismatched BMT murine models, we used different antibiotic cocktails to selectively remove subsets of commensals from recipients pre- and peri-BMT. Transplant recipients which received vancomycin (V) alone had significantly improved GVHD measures and survival compared to those that were administered more broad spectrum antibiotic cocktails (p <0.05). Flow cytometric analysis of the colon lamina propria and intraepithelial layer showed a significant increase of Foxp3-expressing lymphocytes in mice given V vs other groups, suggesting that the reduced GVHD in the former may be due to a higher regulatory cell population that suppresses local inflammation in the gut. In sum, our studies suggest a critical interaction between commensals and host cells via MyD88 signaling in mediating GVHD and BMT outcomes, and support consideration of selective versus broad microbial decontamination with BMT. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v122.21.2267.2267 ◽

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.

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Investigating The Role Of ARAP3 In The Regulation Of Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v122.21.2409.2409 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2409-2409

Author(s):

Yiwen Song ◽

Sonja Vermeren ◽

Wei Tong

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

Conditional Knockout ◽

Ph Domain ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Stem And Progenitor Cells

Abstract ARAP3 is a member of the dual Arf-and-Rho GTPase-activating proteins (GAP) family, functioning specifically to inactivate its substrates Arf6 and RhoA GTPases. ARAP3 is translocated to the plasma membrane after PIP3 binding to the first two of its five PH domains, facilitating its GAP activity in a PI3K-mediated manner. Rho family GTPases are found to play critical roles in many aspects of hematopoietic stem and progenitor cells (HSPCs), such as engraftment and migration, while a role for Arf family GTPases in hematopoiesis is less defined. Previous studies found that either exogenous ARAP3 expression in epithelial cells or RNAi-mediated ARAP3 depletion in endothelial cells disrupts F-actin or lamellipodia formation, respectively, resulting in a cell rounding phenotype and failure to spread. This implies that ARAP3 control of Arf6 and RhoA is tightly regulated, and maintaining precise regulation of ARAP3 levels is crucial to actin organization in the cell. Although ARAP3 was first identified in porcine leukocytes, its function in the hematopoietic system is incompletely understood. Germline deletion of Arap3 results in embryonic lethality due to angiogenic defects. Since endothelial cells are important for the emergence of HSCs during embryonic development, early lethality precludes further studying the role of ARAP3 in definitive hematopoiesis. Therefore, we generated several transgenic mouse models to manipulate ARAP3 in the hematopoietic compartment: (1) Arap3fl/fl;Vav-Cretg conditional knockout mice (CKO) deletes ARAP3 specifically in hematopoietic cells, (2) Arap3fl/fl;VE-Cadherin -Cretg CKO mice selectively deletes ARAP3 in embryonic endothelial cells and thereby hematopoietic cells, and (3) Arap3R302,3A/R302,3A germline knock-in mice (KI/KI) mutates the first PH domain to ablate PI3K-mediated ARAP3 activity in all tissues. We found an almost 100% and 90% excision efficiency in the Vav-Cretg- and VEC-Cretg- mediated deletion of ARAP3 in the bone marrow (BM), respectively. However, the CKO mice appear normal in steady-state hematopoiesis, showing normal peripheral blood (PB) counts and normal distributions of all lineages in the BM. Interestingly, we observed an expansion of the Lin-Scal+cKit+ (LSK) stem and progenitor compartment in the CKO mice. This is due to an increase in the multi-potent progenitor (MPP) fraction, but not the long-term or short-term HSC (LT- or ST-HSC) fractions. Although loss of ARAP3 does not alter the frequency of phenotypically-characterized HSCs, we performed competitive BM transplantation (BMT) studies to investigate the functional impact of ARAP3 deficiency. 500 LSK cells from Arap3 CKO (Arap3fl/fl;Vav-Cretg and Arap3fl/fl;VEC-Cretg) or Arap3fl/fl control littermate donors were transplanted with competitor BM cells into irradiated recipients. We observed similar donor-derived reconstitution and lineage repopulation in the mice transplanted with Arap3fl/fl and Arap3 CKO HSCs. Moreover, Arap3 CKO HSCs show normal reconstitution in secondary transplants. Arap3 KI/KI mice are also grossly normal and exhibit an expanded MPP compartment. Importantly, Arap3KI/KI LSKs show impaired reconstitution compared to controls in the competitive BMT assays. Upon secondary and tertiary transplantation, reconstitution in both PB and BM diminished in the Arap3KI/KI groups, in contrast to sustained reconstitution in the control group. Additionally, we observed a marked skewing towards the myeloid lineage in Arap3KI/KI transplanted secondary and tertiary recipients. These data suggest a defect in HSC function in Arap3KI/KI mice. Myeloid-skewed reconstitution also points to the possibility of selection for “myeloid-primed” HSCs and against “balanced” HSCs, as HSCs exhaust during aging or upon serial transplantation. Taken together, our data suggest that ARAP3 plays a non-cell-autonomous role in HSCs by regulating HSC niche cells. Alternatively, the ARAP3 PH domain mutant that is incapable of locating to the plasma membrane in response to PI3K may exert a novel dominant negative function in HSCs. We are investigating mechanistically how ARAP3 controls HSC engraftment and self-renewal to elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in HSCs. In summary, our studies identify a previously unappreciated role of ARAP3 as a regulator of hematopoiesis and hematopoietic stem and progenitor cell function. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v126.23.1158.1158 ◽

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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Role of Forkhead Transcription Factors in Diabetes-Induced Oxidative Stress

Experimental Diabetes Research ◽

10.1155/2012/939751 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 100

Author(s):

Bhaskar Ponugoti ◽

Guangyu Dong ◽

Dana T. Graves

Keyword(s):

Oxidative Stress ◽

Transcription Factors ◽

Cellular Response ◽

Cell Damage ◽

Cell Types ◽

Biological Processes ◽

Oxygen Species ◽

Forkhead Transcription Factors ◽

Foxo Transcription Factors

Diabetes is a chronic metabolic disorder, characterized by hyperglycemia resulting from insulin deficiency and/or insulin resistance. Recent evidence suggests that high levels of reactive oxygen species (ROS) and subsequent oxidative stress are key contributors in the development of diabetic complications. The FOXO family of forkhead transcription factors including FOXO1, FOXO3, FOXO4, and FOXO6 play important roles in the regulation of many cellular and biological processes and are critical regulators of cellular oxidative stress response pathways. FOXO1 transcription factors can affect a number of different tissues including liver, retina, bone, and cell types ranging from hepatocytes to microvascular endothelial cells and pericytes to osteoblasts. They are induced by oxidative stress and contribute to ROS-induced cell damage and apoptosis. In this paper, we discuss the role of FOXO transcription factors in mediating oxidative stress-induced cellular response.

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SCL Assembles a Multifactorial Complex That Determines Glycophorin A Expression

Molecular and Cellular Biology ◽

10.1128/mcb.24.4.1439-1452.2004 ◽

2004 ◽

Vol 24 (4) ◽

pp. 1439-1452 ◽

Cited By ~ 112

Author(s):

Rachid Lahlil ◽

Eric Lécuyer ◽

Sabine Herblot ◽

Trang Hoang

Keyword(s):

Transcription Factors ◽

Hematopoietic Cells ◽

Inhibitory Effect ◽

Erythroid Cell ◽

Glycophorin A ◽

Protein Activity ◽

Protein Levels ◽

Helix Loop Helix

ABSTRACT SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression.

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