scholarly journals Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing in Transgenic Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 827-827
Author(s):  
Cara Lunn Shirai ◽  
James N Ley ◽  
Brian S. White ◽  
Justin Tibbitts ◽  
Jin Shao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Transgenic Mice ◽  
Rna Sequencing ◽  
Peripheral Blood ◽  
Mrna Splicing ◽  
Splice Acceptor Site ◽  
Isoform Expression

Abstract Our group and others discovered recurrent heterozygous missense mutations in U2AF1 in 11% of patients with myelodysplastic syndromes (MDS). The U2AF1 gene encodes a splicing factor involved in intronic 3’-splice site recognition, which suggests that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. To study the effects of the most common U2AF1 mutation, U2AF1(S34F), on hematopoiesis and pre-mRNA splicing in vivo, we created site-specific, single-copy, doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type recipient mice prior to induction of transgene expression. Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice have reduced total WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (4.3 vs 7.11 and 7.13 K/µl, respectively, p≤0.01), but no significant changes in bone marrow cellularity or spleen size (n=9-11). U2AF1(S34F)-recipient mice have a perturbed mature cell lineage distribution, including reduced monocytes and B cells in both peripheral blood (p≤0.05) and bone marrow (p≤0.01) when compared to control mice (n=9-11). Reduction of bone marrow monocytes occurs as early as 5 days and is associated with increased Annexin V+ (p≤0.05) and phospho-H2AX (p≤0.05) compared to controls, suggesting loss of these cells may be due to apoptosis. In addition, U2AF1(S34F)-recipient mice have increased numbers of progenitors in both bone marrow and spleen by CFU-C methylcellulose assay and flow cytometry for c-Kit+/Lineage- cells, as well as common myeloid progenitors (CMPs), when compared to U2AF1(WT) and rtTA only controls (p≤0.05, n=5-10). U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow hematopoietic stem cells (HSCs) measured by flow cytometry for bone marrow KLS (c-Kit+/Lineage-/Sca-1+) cells (p≤0.05). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice is seen as early as 5 days and is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to U2AF1(WT) controls (p<0.05, n=8-13). Competitive repopulation studies show a disadvantage for bone marrow cells expressing mutant U2AF1(S34F) compared to U2AF1(WT) at ≥4 months post-transplant in both primary and secondary transplant recipient mice (p≤0.05, n=3-12), suggesting that the increase in KLS cell cycling following U2AF1(S34F) expression may lead to stem cell exhaustion. Collectively, these data indicate U2AF1(S34F) expression alters hematopoiesis in vivo. Next, we performed unbiased RNA sequencing on sorted bone marrow CMPs following 5 days of transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice (n=3 each). We identified 460 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) controls (FDR <5%). We observed a preference of the mutant U2AF1(S34F) to skip exons (p=1.3e-05, n=72) and alternative splice sites (p=0.014, n=45) with a T in the -3 position relative to the AG splice acceptor site of differentially-spliced genes; this effect has been previously reported in AML patient samples with U2AF1 mutations. To prioritize altered junctions for further analysis, we intersected mouse CMP junction results with RNA sequencing data from AML patient samples with and without U2AF1 mutations and primary human CD34+ cells over-expressing U2AF1(S34F) or U2AF1(WT). Across species and present in all 3 datasets, we identified homologous dysregulated junctions in 2 genes known to be involved in cancer and stem cell biology: H2AFY and MED24. We validated concordant changes in both H2AFY and MED24 isoform expression by RT-PCR using MDS patient bone marrow samples that have mutant U2AF1(S34F) versus U2AF1(WT) (p<0.001, n=5-6). We are currently testing these isoform changes for their functional contribution to mutant U2AF1-associated phenotypes. Together, these results suggest that mutant U2AF1 expression contributes to the altered hematopoiesis and pre-mRNA splicing observed in patients with U2AF1 mutations. This study also identifies changes in gene isoform expression unique to U2AF1 mutations that may have functional significance for MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

Efficient and Durable Gene Marking of Hematopoietic Progenitor Cells in Nonhuman Primates After Nonablative Conditioning

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2271-2286 ◽  
Author(s):  
M. Rosenzweig ◽  
T.J. MacVittie ◽  
D. Harper ◽  
D. Hempel ◽  
R.L. Glickman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
High Level

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 μg/kg) and G-CSF (20 μg/kg) for 7 days and autologous CD34+ peripheral blood stem cells harvested by leukapheresis. CD34+ cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naı̈ve (CD45RA+and CD62L+) CD4+ and CD8+cells were the predominant phenotype of the marked CD3+ T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naı̈ve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

The “MarrowMiner”: A Novel, Minimally Invasive Device for the Harvest of Bone Marrow: Initial Human Experience Reveals Safety, Efficacy and Richer Cell Product Than Standard Harvest Methods

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4130-4130
Author(s):  
Daniel L. Kraft ◽  
Vartan Ghazarossian ◽  
Mike Crocker ◽  
Sergio Najar ◽  
Antonio A. Carrasco-Yalðn
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
T Cell ◽  
Minimally Invasive ◽  
Progenitor Cells ◽  
Local Anesthesia ◽  
Peripheral Blood ◽  
Single Entry

Abstract INTRODUCTION: Bone marrow (BM) contains a rich supply of adult stem and progenitor cells, including hematopoieitic and mesenchymal stem cells which are used in Bone Marrow Transplantation (BMT) and an increasing array of regenerative therapies. Traditional marrow harvest methods utilize percutaneous large bore needle aspiration, result in marrow highly diluted by peripheral blood, and are crude, tedious, labor intensive and expensive, usually requiring general anesthesia, and &gt;100 serial small volume aspirates to obtain adequate cell numbers for BMT. BM is showing increasing long-term advantages over mobilized PBSC for many alloegeneic BMTs, in terms of less cGVHD and in some cases improved survival. Improved BM harvest methods are needed. A novel device, the “MarrowMiner” (MM), was developed for the minimally invasive harvest of BM to enable the rapid, convenient, outpatient harvest of large quantities of BM under local anesthesia for use in allogeneic and autologous BMT and cell therapies utilizing autologous marrow derived cells. The MarrowMiner utilizes a single marrow entry site into the anterior or posterior iliac, through which the flexible, powered, guidable FlexShaft catheter can access the majority of the marrow space and aspirate rich marrow. Extensive testing in human cadavers and porcine models demonstrated a 10X increase in stem cells activity/ml (by CFU) compared to that of traditional needle harvests. The MM recently received both FDA and CE Mark regulatory approved, and ‘First In Human’ trials were successfully completed under local anesthesia, demonstrating safety, efficacy and higher stem cell yields compared to traditional methods. METHODS: In an ongoing prospective study, 10 patients undergoing autologous marrow derived therapy for use in regenerative medicine, had marrow harvested from their anterior or posterior ileac by the MM under local anesthesia on one hip, with direct comparison to standard needle serial marrow aspirates on the patients opposite hip (up to 350 ml per side). Cell viability, counts, CD34+, T cell, and MSC populations were assessed by flow cytometry. RESULTS: The MM successfully harvested marrow from a single entry sites and 2–3 paths under local anesthesia, without complications. Compared to standard harvest in the same patients, MM harvests had significantly number of Total Nucleated Cells ml compared to marrow harvested from the same patient by standard needle ( mean 1.98 fold greater TNC (range 0.87–3.36, p&lt;.05). Viability was equivalent at (&gt;99). In addition to higher TNC/ml, significantly higher levels (mean 3.56 fold) of Aldeflour/ALDH+ cells/ml, CD34+, and phenotypic MSC (CD45−,34−,90+,105+) and endothelial progenitor cells were obtained, as measured by flow cytometry. Mean CD3+ T-cell counts per ml were lower with MM harvests. CONCLUSIONS: The novel FDA approved MarrowMiner system demonstrated safety and efficacy in clinical use, harvesting more stem cells per unit volume in a single entry compared to standard harvest methods. These results suggests the MM may enable improved clinical stem cell harvests in a more rapid and minimally invasive manner in the outpatient setting, while harvesting a richer marrow product with less peripheral blood contamination. Such a system, facilitating convenient, on demand stem cell collection may have significant application for BMT and other marrow based cellular therapies.

Impaired Lentiviral Transgene Expression In Vivo Caused by Massive Methylation of SFFV Promoter Sequences.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3760-3760 ◽  
Author(s):  
Friederike Herbst ◽  
Claudia R Ball ◽  
Francesca Tuorto ◽  
Wei Wang ◽  
Ulrich Kloz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Es Cells ◽  
Methylation Status ◽  
Lentiviral Vectors ◽  
Control Vector ◽  
Lentiviral Integration

Abstract Abstract 3760 Lentiviral vectors (LV) assure stable transgene expression in vivo, allowing to investigate genes and their functions. In recent years, lentiviral gene transfer was considered to facilitate the generation of transgenic mice with a higher yield of transgenic offspring as compared to commonly used DNA microinjection. We applied LV to generate a mouse model transgenic for SETBP1 and eGFP. Murine zygotes were infected at dE0.5 with lentiviral particles directly injected into the perivitelline space. Specific PCRs for either the SETBP1 transgene or for the WPRE element of the lentiviral construct verified complete lentiviral integration in newborn pups (F0). Lentiviral integration sites were detected using highly sensitive LAM-PCR in 65% of 31 analyzed F0 mice. Germline transmission was shown in a total of 33% vector positive offspring from 5 out of 9 F0 mice. However, no ectopic transcription and overexpression of neither SETBP1 nor eGFP could be detected in transgenic mice. We therefore analyzed the methylation status of the internal SFFV promoter (SFFVp) by bisulfite sequencing. Extensive methylation (around 90%) could be assessed in 18 of 18 analyzed CpGs within the promoter region in F0 animals and in all progeny determined (n=12). We transduced mES cells with LV.SFFV.Setbp1.IRES.eGFP or the corresponding eGFP-expressing control vector to exclude transgene effects on epigenetic silencing of SFFVp sequences in self-inactivating LVs. Differentiation of ES cells infected with the transgene vector and SFFV driven control vector led to a 1.8 – 3.5 fold decrease of eGFP expression. To analyze whether methylation of SFFVp sequences is a common event even in adult tissues, we analyzed the methylation status of peripheral blood in mice transplanted with bone marrow cells transduced with either gammaretroviral vectors (RV) or LV 3 months after transplantation (n=7). Interestingly, SFFVp sequences in peripheral blood of mice transplanted with LV transduced bone marrow were stronger methylated than CpGs of SFFVp in RV transplants. Our data demonstrate that the commonly used SFFV promotor is highly methylated with remarkable strength and frequency during development in vivo and differentiation in vitro. We conclude that lentiviral vectors using an internal SFFV promoter are not suitable for the generation of transgenic mice or constitutive expression studies in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2476-2476
Author(s):  
Kasia Mierzejewska ◽  
Ewa Suszynska ◽  
Sylwia Borkowska ◽  
Malwina Suszynska ◽  
Maja Maj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Sex Hormones ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vivo administration of stem cell factor to mice increases the absolute number of pluripotent hematopoietic stem cells

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 445-455 ◽  
Author(s):  
DM Bodine ◽  
NE Seidel ◽  
KM Zsebo ◽  
D Orlic
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Stem Cell Factor ◽  
Absolute Number ◽  
Hematopoietic Stem ◽  
The Absolute ◽  
In Vivo Administration

We have examined the effects of administration of stem cell-factor (SCF) on the number and distribution of pluripotent hematopoietic stem cells (PHSC) in normal mice. Using the competitive repopulation assay we found that in vivo administration of SCF increases the absolute number of PHSC per mouse threefold. The increased numbers of PHSC are found in the peripheral blood and spleen of the SCF-treated animals. The spleen and peripheral blood stem cells completely repopulated the erythroid, myeloid, and lymphoid lineages of irradiated or W/Wv hosts, similar to bone marrow PHSC. PHSC from the peripheral blood of SCF- treated mice have a lineage marker-negative, c-kit-positive phenotype that is indistinguishable from that of bone marrow PHSC. The increase in the absolute number of spleen PHSC is associated with efficient gene transfer to these cells without prior treatment with 5-fluorouracil. This is a US government work. There are no restrictions on its use.

scholarly journals Case Report: Graft Versus Tumor Effect After Non-Myeloablative Allogeneic Stem-Cell Transplantation in a Patient With Brentuximab-Vedotin Refractory Sezary Syndrome

2021 ◽  
Vol 11 ◽  
Author(s):  
Georg-Nikolaus Franke ◽  
Konstantin Dumann ◽  
Madlen Jentzsch ◽  
Astrid Monecke ◽  
Christine Doehring ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Conditioning Regimen ◽  
Brentuximab Vedotin ◽  
Sézary Syndrome ◽  
Sezary Syndrome

Sezary Syndrome (SS) is a rare leukemic variant of primary cutaneous T-cell lymphoma. Relapsed or refractory disease is generally considered incurable by conventional therapeutic approaches, although durable responses can be achieved with novel monoclonal antibodies. Allogeneic hematopoietic stem cell transplantation (alloHSCT) may have potential value by inducing graft vs-lymphoma (GvL) effects, but there is currently no consensus regarding the timing of alloHSCT or type of conditioning regimen. Here we present the case of a male patient who achieved a complete remission (CR) of primary refractory SS after non-myeloablative alloHSCT. Patient: Two years prior to HSCT, the patient had been refractory to CHOEP-based chemotherapy, interferon, extracorporeal photopheresis (ECP), and bexarotene. Directly prior to alloHSCT brentuximab-vedotin (BV) was applied resulting in a partial remission of the skin compartment and overall in a stable disease. Prior to HSCT, flow cytometry of the bone marrow and peripheral blood showed an infiltration with T-cells positive for CD5, CD4, low CD3, low CD2 and negative for CD7, CD38, HLA-DR and CD8. The trephine biopsy showed a 7% infiltration of SS cells. The CD4:CD8 ratio in peripheral blood (pb) was massively increased at 76.67, with 63.5% of white blood cells expressing a SS immune phenotype. The conditioning regimen included 30 mg/m2 fludarabine on days -5, -4 and -3 and total body irradiation with 2 Gy on day -1. Immunosuppression consisted of cyclosporine A from day-1 and mycophenolate mofetil from day 0. The patient received 6.55x106 CD34+ cells and 1.11x108 CD3+ cells/kg body weight. Bone marrow evaluation on day 28 still showed persistent SS cells by flow cytometry. After tapering immunosuppression until day 169, the CD4:CD8 ratio in pb normalized. CR was documented on day 169 after alloHSCT and is now ongoing for almost 3 years after alloHSCT. Conclusions: We confirm that an alloHSCT can be a curative option for refractory patients with SS. The achievement of a CR after tapering the immunosuppressive therapy indicates a significant role of the GvL effect. In present treatment algorithms for patients with SS, the timing of an alloHSCT and the intensity of conditioning should be further explored.

Bone Morphogenetic Protein 4 Regulates Hematopoietic Stem Cell Maintenance in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1399-1399
Author(s):  
Devorah C. Goldman ◽  
Alexis S. Bailey ◽  
Dana L. Pfaffle ◽  
Jan L. Christian ◽  
William H. Fleming
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Morphogenetic Protein ◽  
Peripheral Blood ◽  
Bone Morphogenetic Protein 4 ◽  
Hematopoietic Stem ◽  
Morphogenetic Protein ◽  
Fold Reduction ◽  
Donor Cells

Abstract The secreted signaling molecule Bone Morphogenetic Protein 4 (BMP4) is expressed by osteoblasts and other cell types that comprise the hematopoietic microenvironment. Therefore, we hypothesized that BMP4 may play an important regulatory role in the maintenance and function of hematopoietic stem cells (HSCs). As the deletion of BMP4 is lethal during early embryogenesis, we exploited a viable BMP4 hypomorph in which a point mutation in BMP4 reduces, but does not abolish the amount of active BMP4 ligand. In these mutants, peripheral blood cell lineages and bone marrow cellularity are normal during steady state conditions. However, consistent with our hypothesis, 40% fewer c-kit+, Sca-1+, lineage- (KSL) cells were present in the femurs of mutants (7.4 x103 ± 0.4 x103 SEM) than in age-and sex-matched wild-type (WT) controls (12.4 x103 ± 1.3 x103, p&lt;0.005). Transplantation of mutant KSL cells produced levels of hematopoietic engraftment indistinguishable from transplanted WT KSL cells, consistent with a stem cell extrinsic effect of BMP4. To functionally assess the mutant hematopoietic microenvironment, unfractionated WT bone marrow cells (BM) were transplanted into lethally irradiated mutants or WT controls. Although WT cells could engraft mutant primary recipients to the same degree as WT recipients, serial transplantation of these WT cells into secondary WT hosts revealed a marked depletion of hematopoietic reconstitution activity. Specifically, nearly a 4-fold reduction of donor cells was found in the peripheral blood of secondary recipients that received BM from reconstituted mutant hosts (p&lt;0.0005). To determine whether such defects in the mutant microenvironment exist in the absence of myeloablative conditioning and transplantation, a parabiotic mouse model was employed. CD45.1 WT mice were joined to CD45.2 BMP4 hypomorphs for 8 weeks and then separated. As early as one month following separation, a greater than 2- fold reduction in circulating WT donor cells was detected in mutant hosts relative to the number of mutant donor cells detected in the WT hosts (p&lt;0.005). Analysis of BM at 24 weeks following separation revealed a striking 23- fold reduction in WT donor hematopoietic cells in the mutant host BM compared to the number of mutant hematopoietic cells detected in WT host BM. Together, our findings reveal a novel, critical role for BMP4 in maintaining HSCs in vivo.

Terminal Differentiation of FLT3/ITD AML Blasts In Patients Treated with the FLT3 Inhibitor AC220

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 660-660
Author(s):  
Mark J. Levis ◽  
Amy Sexauer ◽  
Trivikram Rajkhowa ◽  
Donald Small ◽  
Michael J. Borowitz
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Peripheral Blood ◽  
Terminal Differentiation ◽  
Flt3 Inhibitor ◽  
Microscopic Evaluation ◽  
Pre Treatment

Abstract Abstract 660 AML is characterized by abnormal proliferation of myeloid cells that have a block in differentiation. FLT3/ITD mutations are relatively common in AML, and previous in vitro studies have demonstrated that signaling from ITD-mutated FLT3 blocks myeloid differentiation through repression of CEBP/a. As part of an ongoing phase 2 trial, we treated 6 patients with FLT3/ITD AML who were refractory to either primary induction therapy or salvage therapy after relapse with the highly potent and selective FLT3 inhibitor AC220. At the start of therapy, all 6 patients had circulating blasts (mean 9864 blasts/mm3; median 2970) and the median blast percentage in the bone marrow was 71.5%. Western blotting revealed a high level of sustained in vivo FLT3 inhibition in all patients. By Day 8, no patient had detectable blasts in the peripheral blood. After 14 days of treatment with AC220, all 6 patients displayed striking differentiation to the myelocyte stage within the bone marrow. By light microscopic evaluation of bone marrow aspirates, myelocytes (promyelocytes, myelocytes, and metamyelocytes) increased from a median of 10.5% pre-treatment to 52% after 2 weeks. Most patients were neutropenic on Day 1 of treatment (mean 574, median 560 neutrophils/mm3), but rose to a mean of 3275 neutrophils/mm3 after 4–8 weeks of treatment (median time to peak 34 days). By Day 28 of treatment, marrows were most often still hypercellular, but consisted primarily of fully differentiated neutrophils. Marrow blasts were markedly reduced or absent by Day 28 in all 6 cases (mean 2.3%, median 1.5%). In all 6 patients the FLT3/ITD mutation originally detected at the beginning of treatment was present in the marrow and peripheral blood despite the absence of circulating blasts after the first week of therapy. The FLT3 mutant allelic ratio did not change between pre-therapy and Day 28. Neutrophils were isolated to homogeneity (confirmed by cytospin) from peripheral blood by double ficoll density centrifugation. Using genomic DNA obtained from these purified neutrophils, we confirmed by PCR that the FLT3/ITD mutation was present, at a similar ratio as compared with the pre-treatment blasts. However, there was no detectable expression of FLT3 either by RNA (quantitative PCR) or protein (western blotting and flow cytometry) in these neutrophils. The isolated neutrophils morphologically resemble normal neutrophils by light microscopy, and by flow cytometry they express the differentiation antigen CD15 and CD11b, and have lost expression of immature markers such as cKIT and CD34. Stimulation of these neutrophils by endotoxin results in normal respiratory burst activity, as measured by reduction of nitroblue tetrazolium. They also express lactoferrin and MMP-9, proteins typically expressed in mature neutrophils. Clinically, lung nodules and fever occurred in 3 of the 6 patients within 14 days of the peak neutrophil count. They were not treated with steroids, but rather with antibiotics, and in all cases resolved. Other patients on this trial have developed Sweet's syndrome during the neutrophil surge. CEBPa transcript levels in Molm14 cells (an AML cell line with a FLT3/ITD mutation) rose 3–5-fold over baseline following treatment with AC220. This is consistent with our previously published findings, and suggests at least one mechanism for the observed release of the differentiation block observed in the AC220-treated patients. These clinical and correlative laboratory results suggest that effective, sustained in vivo FLT3 inhibition in AML patients with FLT3/ITD mutations induces terminal differentiation in blasts in many ways similar to that seen with all trans retinoic acid in acute promyelocytic leukemia. Furthermore, these findings demonstrate the direct link between the growth factor receptor pathway and control of differentiation, and provide new insight into mechanisms of leukemogenesis. Disclosures: Levis: Ambit Biosciences, Inc: Consultancy.

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