Adenylate Kinase 2 deficiency causes NAD+ depletion and impaired purine metabolism during myelopoiesis

2021 ◽  
Author(s):  
Wenqing Wang ◽  
Andrew DeVilbiss ◽  
Martin Arreola ◽  
Thomas Mathews ◽  
Misty Martin-Sandoval ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Adenylate Kinase ◽  
De Novo ◽  
Bone Marrow Cells ◽  
Severe Combined Immunodeficiency ◽  
Purine Metabolism ◽  
Model Systems ◽  
Hematopoietic Stem ◽  
Mechanistic Basis ◽  
Nad Depletion

Reticular Dysgenesis is a particularly grave from of severe combined immunodeficiency (SCID) that presents with severe congenital neutropenia and a maturation arrest of most cells of the lymphoid lineage. The disease is caused by biallelic loss of function mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) as substrate for adenosine triphosphate (ATP) synthesis in the mitochondria. Accordingly, it has long been hypothesized that a decline in OXPHOS metabolism is the driver of the disease. The mechanistic basis for Reticular Dysgenesis, however, remained incompletely understood, largely due to lack of appropriate model systems to phenocopy the human disease. We have used single cell RNA-sequencing of bone marrow cells from 2 reticular dysgenesis patients to gain insight into the disease pathology. Gene set enrichment for differentially expressed genes in different subsets of myeloid and lymphoid progenitor cells pointed to processes involving RNA and ribonucleoprotein assembly and catabolism as well as cell cycle defects. To investigate these findings and precisely mimic the failure of human myelopoiesis in culture, we developed a cell-tracible model of Reticular Dysgenesis based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. In this model, we have identified that AK2-deficienct myeloid progenitor cells exhibit NAD+ depletion and high levels of reductive stress accompanied by an accumulation of AMP and IMP while ADP and ATP are only mildly decreased. Our studies further show that AK2-deficienct cells have decreased de novo purine synthesis and increased purine breakdown, accompanied by decreased RNA and ribosome subunit cellular content. These data highlight the profound impact of mitochondrial dysfunction on the cellular redox state and nucleotide pool and identify the mechanistic basis of Reticular Dysgenesis as a defect in purine metabolism.

The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal

Blood ◽  
2021 ◽  
Author(s):  
Yuqing Yang ◽  
Andrew J Kueh ◽  
Zoe Grant ◽  
Waruni Abeysekera ◽  
Alexandra L Garnham ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
High Rate ◽  
Embryonic Patterning ◽  
Self Renewal ◽  
Hematopoietic Stem

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac) and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used two complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1 null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow two to six weeks after Hbo1 deletion. Hbo1 deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors (HPCs). The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1 and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 700-704 ◽  
Author(s):  
Kimberly A. Gush ◽  
Kai-Ling Fu ◽  
Markus Grompe ◽  
Christopher E. Walsh
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Fanconi Anemia ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Hematopoietic Stem ◽  
Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

scholarly journals Bone Marrow Oxidative Stress and Acquired Lineage-Specific Genotoxicity in Hematopoietic Stem/Progenitor Cells Exposed to 1,4-Benzoquinone

2020 ◽  
Vol 17 (16) ◽  
pp. 5865
Author(s):  
Ramya Dewi Mathialagan ◽  
Zariyantey Abd Hamid ◽  
Qing Min Ng ◽  
Nor Fadilah Rajab ◽  
Salwati Shuib ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Dna Damage ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Protein Carbonyls ◽  
Mouse Bone Marrow ◽  
Tail Moment ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem

Hematopoietic stem/progenitor cells (HSPCs) are susceptible to benzene-induced genotoxicity. However, little is known about the mechanism of DNA damage response affecting lineage-committed progenitors for myeloid, erythroid, and lymphoid. Here, we investigated the genotoxicity of a benzene metabolite, 1,4-benzoquinone (1,4-BQ), in HSPCs using oxidative stress and lineage-directed approaches. Mouse bone marrow cells (BMCs) were exposed to 1,4-BQ (1.25–12 μM) for 24 h, followed by oxidative stress and genotoxicity assessments. Then, the genotoxicity of 1,4-BQ in lineage-committed progenitors was evaluated using colony forming cell assay following 7–14 days of culture. 1,4-BQ exposure causes significant decreases (p < 0.05) in glutathione level and superoxide dismutase activity, along with significant increases (p < 0.05) in levels of malondialdehyde and protein carbonyls. 1,4-BQ exposure induces DNA damage in BMCs by significantly (p < 0.05) increased percentages of DNA in tail at 7 and 12 μM and tail moment at 12 μM. We found crucial differences in genotoxic susceptibility based on percentages of DNA in tail between lineage-committed progenitors. Myeloid and pre-B lymphoid progenitors appeared to acquire significant DNA damage as compared with the control starting from a low concentration of 1,4-BQ exposure (2.5 µM). In contrast, the erythroid progenitor showed significant damage as compared with the control starting at 5 µM 1,4-BQ. Meanwhile, a significant (p < 0.05) increase in tail moment was only notable at 7 µM and 12 µM 1,4-BQ exposure for all progenitors. Benzene could mediate hematological disorders by promoting bone marrow oxidative stress and lineage-specific genotoxicity targeting HSPCs.

scholarly journals Interferon gamma selectively inhibits very primitive CD342+CD38- and not more mature CD34+CD38+ human hematopoietic progenitor cells.

1994 ◽  
Vol 180 (3) ◽  
pp. 1177-1182 ◽  
Author(s):  
H W Snoeck ◽  
D R Van Bockstaele ◽  
G Nys ◽  
M Lenjou ◽  
F Lardon ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Interferon Gamma ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Ifn Gamma ◽  
Hematopoietic Stem ◽  
First Time ◽  
Dose Dependent

To assess the effects of interferon gamma (IFN-gamma) on very primitive hematopoietic progenitor cells, CD34(2+)CD38- human bone marrow cells were isolated and cultured in a two-stage culture system, consisting of a primary liquid culture phase followed by a secondary semisolid colony assay. CD34(2+)CD38- cells needed at least the presence of interleukin 3 (IL-3) and kit ligand (KL) together with either IL-1, IL-6, or granulocyte-colony-stimulating factor (G-CSF) in the primary liquid phase in order to proliferate and differentiate into secondary colony-forming cells (CFC). Addition of IFN-gamma to the primary liquid cultures inhibited cell proliferation and generation of secondary CFC in a dose-dependent way. This was a direct effect since it was also seen in primary single cell cultures of CD34(2+)CD38- cells. The proliferation of more mature CD34+CD38+ cells, however, was not inhibited by IFN-gamma, demonstrating for the first time that IFN-gamma is a specific and direct hematopoietic stem cell inhibitor. IFN-gamma, moreover, preserves the viability of CD34(2+)CD38- cells in the absence of other cytokines. IFN-gamma could, therefore, play a role in the protection of the stem cell compartment from exhaustion in situations of hematopoietic stress and may be useful as stem cell protecting agent against chemotherapy for cancer.

Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element

Blood ◽  
2003 ◽  
Vol 102 (13) ◽  
pp. 4369-4376 ◽  
Author(s):  
James C. Mulloy ◽  
Jorg Cammenga ◽  
Francisco J. Berguido ◽  
Kaida Wu ◽  
Ping Zhou ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Hematopoietic Cells ◽  
The Self ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Human Cd34

AbstractHematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo. (Blood. 2003; 102:4369-4376)

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

Hemangiogenic Role of ELF4 in Bone Marrow Post Myeloablation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1783-1783
Author(s):  
Mariela Sivina ◽  
Takeshi Yamada ◽  
Natalie Dang ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Vessels ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Huvec Cells

Abstract Bone marrow suppression is an important cause of death in patients exposed to radiation or in cancer patients treated with conventional chemotherapeutic agents. Myeloablative treatments (i.e. 5-fluorouracil administration) lead to apoptosis of blood forming cells and to regression of blood vessels in bone marrow. It is well known that hematological recovery post-bone marrow insult depends on the capacity of hematopoietic stem cells to regenerate the entire hematopoietic system, however, the transcriptional machinery involved in the regeneration of sinusoidal blood vessels in bone marrow from endothelial progenitor cells is largely unknown. Endothelial cells express the Tie2 receptor tyrosine kinase (a.k.a. Tek), which is involved in the angiogenic remodeling and vessel stabilization. Gene targeting of Tie2 showed that it is not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo although Tie2 is needed during postnatal bone marrow hematopoiesis. ELF is a subgroup of the ETS family of transcription factors composed by ELF1, ELF2 (a.k.a. NERF), ELF3, ELF4 (a.k.a. MEF) and ELF5. ELF1 and ELF2 have been shown to regulate Tie2 expression in vitro. Recently we showed that ELF4 modulates the exit of hematopoietic stem cells (HSC) from quiescence (Lacorazza et al., Cancer Cell2006, 9:175–187). Given the high homology between ELF1 and ELF4 and the same origin of HSC and endothelial progenitor cells, we hypothesize that ELF4 regulates proliferation and Tie2 expression of endothelial cells. We used a luciferase gene reporter system in COS-7 and HEK cells to examine the capacity of ELF proteins to activate Tie2. ELF4 is the strongest activator of Tie2 expression following the hierarchy ELF4>ELF1>ELF2 variant 1>ELF2 variant 2. Site directed mutagenesis of each of the five ETS-binding sites (EBS) present in the Tie2 promoter shows that ELF4 binds preferentially to EBS 1, 3 and 5. Binding of ELF4 to the Tie2 promoter was confirmed by chromatin immunoprecipitation and EMSA. Although Elf1 gene expression is essentially normal in Elf4−/− bone marrow cells collected after 5-FU treatment, we detected diminished Tie2 expression compared to Elf4+/+ bone marrow cells. The association of this effect to human endothelial cells derived from umbilical cord (HUVEC cells) was investigated. All-trans retinoic acid (ATRA) and vascular-endothelial growth factor (VEGF) induced ELF4 expression in HUVEC cells in a dose and time dependent manner which was followed by increased Tie2 expression, suggesting that expression of ELF4 is modulated by angiogenic signals. Moreover, endothelial cells treated with ATRA showed rapid wound colonization in a wound assay. Expression of the pan-endothelial marker MECA-32 was determined by immunohistochemistry to correlate Tie2 with the regeneration of blood vessels: myeloablated Elf4−/− femurs exhibited a reduction of MECA-32 positive arterioles. Finally, temporal and spatial expression of Tie2 during hematological recovery post ablation was measured in bone marrow using transgenic Tie2-LacZ mice crossed to Elf4−/− mice. Collectively, our data suggests that ELF4 regulates Tie2 expression in endothelial cells but most importantly their proliferative capacity in response to angiogenic signals.

Requirement for p85α Regulatory Subunit of Class IA PI3Kinase and Rac2 GTPase in Myeloproliferative Disease Driven by Activation Loop Mutant of KIT.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 89-89
Author(s):  
Veerendra Munugalavadla ◽  
Emily C. Sims ◽  
Stephen D. Lenz ◽  
Reuben Kapur
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Regulatory Subunit ◽  
Activation Loop ◽  
Myeloproliferative Disease ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Oncogenic activation-loop mutants of KIT, the receptor for stem cell factor (SCF), are commonly observed in acute myeloid leukemia (AML) and systemic mastocytosis (SM); however, unlike the KIT juxtamembrane mutants (found in patients with gastrointestinal stromal tumors [GISTs]), the activation-loop mutants are commonly insensitive to inhibition by tyrosine kinase inhibitors. Furthermore, little is known about the signaling pathways that contribute to oncogenic KIT-induced transformation in SM or AML. We demonstrate that expression of KITD814V (KIT activation-loop mutant) in primary hematopoietic stem and progenitor cells induces constitutive KIT autophosphorylation, promotes ligand-independent hyperproliferation, skews myeloid differentiation towards the granulocytic lineage, and promotes promiscuous cooperation with multiple cytokines, including G-CSF, M-CSF and IL-3. KITD814V expressing primary mast cells also demonstrated hyperproliferation in response to SCF, IL-3, IL-4 and IL-10. Biochemical analyses of KITD814V expressing cells revealed constitutively elevated levels of phosphatidylinositol-3-kinase (PI3K) and its downstream substrate, the Rho family GTPase Rac. Genetic disruption of p85a, the regulatory subunit of class IA PI-3Kinase, but not of p85β, or genetic disruption of the hematopoietic cell-specific Rho GTPase, Rac2, normalized KITD814V-induced ligand independent hyperproliferation in vitro. Additionally, deficiency of p85α or Rac2 corrected the promiscuous hyperproliferation observed in response to multiple cytokines in both KITD814V expressing stem/progenitor cells as well as mast cells in vitro. Although p85α is hyperphosphorylated and constitutively bound to KITD814V in bone marrow cells in vitro; its physiologic role in transformation in vivo is not known. To address this, we generated a new mouse model to study KITD814V induced transformation in myeloid cells as opposed to previously described models that primarily result in the generation of phenotypes resembling acute lymphocytic leukemia via this mutation. Our results show that transplantation of KITD814V expressing bone marrow cells from C57/BL6 strain of mice into syngeneic recipients results in a fatal myeloproliferative disease (MPD) characterized by leukocytosis, splenomegaly, disruption of the splenic architecture as well as myeloid cell infiltration in the lung and liver. Importantly, in this model, transplantation of KITD814V expressing p85α deficient bone marrow cells rescued the MPD phenotype, including splenomegaly, peripheral blood leukocytosis and the reduced life span associated with the transplantation of KITD814V expressing wildtype bone marrow cells. Treatment of KITD814V-expressing hematopoietic progenitors with either a Rac inhibitor (NC23766) or rapamycin showed a dose-dependent suppression in KITD814V induced growth. Taken together, our results describe the generation of a new murine transplant model to study KITD814V induced transformation and identify p85a and Rac2 as potential novel therapeutic target for the treatment of KITD814V-bearing diseases including SM and AML.

Prostaglandin E2 Inhibits Apoptosis in Hematopoietic Stem and Progenitor Cells and Enhances Their Survival Following Sub-Lethal Radiation Injury,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3401-3401
Author(s):  
Rebecca L Porter ◽  
Mary A Georger ◽  
Laura M Calvi
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Radiation Injury ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Cox 2 ◽  
Apoptotic Genes

Abstract Abstract 3401 Hematopoietic stem and progenitor cells (HSPCs) are responsible for the continual production of all mature blood cells during homeostasis and times of stress. These cells are known to be regulated in part by the bone marrow microenvironment in which they reside. We have previously reported that the microenvironmentally-produced factor Prostaglandin E2 (PGE2) expands HSPCs when administered systemically in naïve mice (Porter, Frisch et. al., Blood, 2009). However, the mechanism mediating this expansion remains unclear. Here, we demonstrate that in vivo PGE2 treatment inhibits apoptosis of HSPCs in naïve mice, as measured by Annexin V staining (p=0.0083, n=6–7 mice/group) and detection of active-Caspase 3 (p=0.01, n=6–7 mice/group). These data suggest that inhibition of apoptosis is at least one mechanism by which PGE2 expands HSPCs. Since PGE2 is a local mediator of injury and is known to play a protective role in other cell types, we hypothesized that it could be an important microenvironmental regulator of HSPCs during times of injury. Thus, these studies explored the role of PGE2 signaling in the bone marrow following myelosuppressive injury using a radiation injury model. Endogenous PGE2 levels in the bone marrow increased 2.9-fold in response to a sub-lethal dose of 6.5 Gy total body irradiation (TBI)(p=0.0004, n=3–11 mice/group). This increase in PGE2 correlated with up-regulation of microenvironmental Cyclooxygenase-2 (Cox-2) mRNA (p=0.0048) and protein levels at 24 and 72 hr post-TBI, respectively. Further augmentation of prostaglandin signaling following 6.5 Gy TBI by administration of exogenous 16,16-dimethyl-PGE2 (dmPGE2) enhanced the survival of functional HSPCs acutely after injury. At 24 hr post-TBI, the bone marrow of dmPGE2-treated animals contained significantly more LSK cells (p=0.0037, n=13 mice/group) and colony forming unit-spleen cells (p=0.037, n=5 mice/group). Competitive transplantation assays at 72 hr post-TBI demonstrated that bone marrow cells from irradiated dmPGE2-treated mice exhibited increased repopulating activity compared with cells from vehicle-treated mice. Taken together, these results indicate that dmPGE2 treatment post-TBI increases survival of functional HSPCs. Since PGE2 can inhibit apoptosis of HSPCs in naïve mice, the effect of dmPGE2 post-TBI on apoptosis was also investigated. HSPCs isolated from mice 24 hr post-TBI demonstrated statistically significant down-regulation of several pro-apoptotic genes and up-regulation of anti-apoptotic genes in dmPGE2-treated animals (3 separate experiments with n=4–8 mice/group in each), suggesting that dmPGE2 initiates an anti-apoptotic program in HSPCs following injury. Notably, there was no significant change in expression of the anti-apoptotic gene Survivin, which has previously been reported to increase in response to ex vivo dmPGE2 treatment of bone marrow cells (Hoggatt et. al., Blood, 2009), suggesting differential effects of dmPGE2 in vivo and/or in an injury setting. Additionally, to ensure that this inhibition of apoptosis was not merely increasing survival of damaged and non-functional HSPCs, the effect of early treatment with dmPGE2 post-TBI on hematopoietic recovery was assayed by monitoring peripheral blood counts. Interestingly, dmPGE2 treatment in the first 72 hr post-TBI significantly accelerated recovery of platelet levels and hematocrit compared with injured vehicle-treated mice (n=12 mice/group). Immunohistochemical analysis of the bone marrow of dmPGE2-treated mice also exhibited a dramatic activation of Cox-2 in the bone marrow microenvironment. This suggests that the beneficial effect of dmPGE2 treatment following injury may occur, both through direct stimulation of hematopoietic cells and also via activation of the HSC niche. In summary, these data indicate that PGE2 is a critical microenvironmental regulator of hematopoietic cells in response to injury. Exploitation of the dmPGE2-induced initiation of an anti-apoptotic program in HSPCs may represent a useful method to increase survival of these cells after sub-lethal radiation injury. Further, amplification of prostaglandin signaling by treatment with PGE2 agonists may also represent a novel approach to meaningfully accelerate recovery of peripheral blood counts in patients with hematopoietic system injury during a vulnerable time when few therapeutic options are currently available. Disclosures: No relevant conflicts of interest to declare.

Gene Therapy Corrects the Lethal Bone Marrow Failure in a Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 513-513
Author(s):  
Pekka Jaako ◽  
Shubhranshu Debnath ◽  
Karin Olsson ◽  
Axel Schambach ◽  
Christopher Baum ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Stem And Progenitor Cells

Abstract Abstract 513 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical abnormalities and predisposition to cancer. Mutations in genes that encode ribosomal proteins have been identified in approximately 60–70 % of the patients. Among these genes, ribosomal protein S19 (RPS19) is the most common DBA gene (25 % of the cases). Current DBA therapies involve risks for serious side effects and a high proportion of deaths are treatment-related underscoring the need for novel therapies. We have previously demonstrated that enforced expression of RPS19 improves the proliferation, erythroid colony-forming potential and differentiation of patient derived RPS19-deficient hematopoietic progenitor cells in vitro (Hamaguchi, Blood 2002; Hamaguchi, Mol Ther 2003). Furthermore, RPS19 overexpression enhances the engraftment and erythroid differentiation of patient-derived hematopoietic stem and progenitor cells when transplanted into immunocompromised mice (Flygare, Exp Hematol 2008). Collectively these studies suggest the feasibility of gene therapy in the treatment of RPS19-deficient DBA. In the current project we have assessed the therapeutic efficacy of gene therapy using a mouse model for RPS19-deficient DBA (Jaako, Blood 2011; Jaako, Blood 2012). This model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of Collagen A1 gene. Transgenic animals were bred either heterozygous or homozygous for the shRNA-D in order to generate two models with intermediate or severe Rps19 deficiency, respectively. Indeed, following transplantation, the administration of doxycycline to the recipients with homozygous shRNA-D bone marrow results in an acute and lethal bone marrow failure, while the heterozygous shRNA-D recipients develop a mild and chronic phenotype. We employed lentiviral vectors harboring a codon-optimized human RPS19 cDNA driven by the SFFV promoter, followed by IRES and GFP (SFFV-RPS19). A similar vector without the RPS19 cDNA was used as a control (SFFV-GFP). To assess the therapeutic potential of the SFFV-RPS19 vector in vivo, transduced c-Kit enriched bone marrow cells from control and homozygous shRNA-D mice were injected into lethally irradiated wild-type mice. Based on the percentage of GFP-positive cells, transduction efficiencies varied between 40 % and 60 %. Three months after transplantation, recipient mice were administered doxycycline in order to induce Rps19 deficiency. After two weeks of doxycycline administration, the recipients transplanted with SFFV-RPS19 or SFFV-GFP control cells showed no differences in blood cellularity. Remarkably, at the same time-point the recipients with SFFV-GFP homozygous shRNA-D bone marrow showed a dramatic decrease in blood cellularity that led to death, while the recipients with SFFV-RPS19 shRNA-D bone marrow showed nearly normal blood cellularity. These results demonstrate the potential of enforced expression of RPS19 to reverse the severe anemia and bone marrow failure in DBA. To assess the reconstitution advantage of transduced hematopoietic stem and progenitor cells with time, we performed similar experiments with heterozygous shRNA-D bone marrow cells. We monitored the percentage of GFP-positive myeloid cells in the peripheral blood, which provides a dynamic read-out for bone marrow activity. After four months of doxycycline administration, the mean percentage of GFP-positive cells in the recipients with SFFV-RPS19 heterozygous shRNA-D bone marrow increased to 97 %, while no similar advantage was observed in the recipients with SFFV-RPS19 or SFFV-GFP control bone marrow, or SFFV-GFP heterozygous shRNA-D bone marrow. Consistently, SFFV-RPS19 conferred a reconstitution advantage over the non-transduced cells in the bone marrow. Furthermore, SFFV-RPS19 reversed the hypocellular bone marrow observed in the SFFV-GFP heterozygous shRNA-D recipients. Taken together, using mouse models for RPS19-deficient DBA, we demonstrate that the enforced expression of RPS19 rescues the lethal bone marrow failure and confers a strong reconstitution advantage in vivo. These results provide a proof-of-principle for gene therapy in the treatment of RPS19-deficient DBA. Disclosures: No relevant conflicts of interest to declare.

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