Myeloid Hematopoiesis Research Articles

Congenital hypoplastic bone marrow failure associated with a de novo partial deletion of the MECOM gene at 3q26.2

Congenital hypoplastic bone marrow failure is a rare condition in neonates. The genetics and mechanisms behind are largely obscure.Here we characterize a neonate presenting with congenital thrombocytopenia and anemia. During the first 2–4 weeks after birth the neonate developed severe neutropenia while the lymphoid lineages were unaffected. The neonate was without dysmorphic signs. A de novo mono-allelic constitutional microdeletion of 175.1 kb at 3q26.2 affecting exon 2 of MECOM, involving MDS1 but not EVI1, was identified as the only copy number alteration by oligo-based array-CGH analysis. Expression analysis showed profoundly reduced expression of multiple MECOM transcripts in the bone marrow cells. Whole exome sequencing detected no pathogenic mutations in genes known to be associated with inherited bone marrow failure syndromes. The patient was successfully treated with hematopoietic stem cell transplantation at 5 months of age.Interstitial deletions encompassing the 3q26.2 region are very rare. A literature search revealed two previous cases with microdeletions involving this region, and the cases were associated with congenital thrombocytopenia and anemia, but unaffected lymphopoiesis. Together these data indicate that MECOM may be important for normal myeloid hematopoiesis in humans but dispensable for lymphoid differentiation. We suggest that partial deletion in MECOM may be a primary event associated with congenital pancytopenia.

FOXM1 Transcription Factor: A New Component of Chronic Myeloid Leukemia Stem Cell Proliferation Advantage.

FOXM1 transcription factor is a central component of tumor initiation, growth, and progression due to its multiple effects on cell cycle, DNA repair, angiogenesis and invasion, chromatin, protein anabolism, and cell adhesion. Moreover, FOXM1 interacts with β-catenin promoting its nuclear import and transcriptional activation. Here, we show that FOXM1 is involved in the advantage of chronic myeloid leukemia hematopoiesis over the normal counterpart. FOXM1 hyper-activation associated with BCR-ABL1 results from phosphorylation by the fusion protein kinase-dependent activation of Polo-like kinase 1. FOXM1 phosphorylation lets its binding with β-catenin and β-catenin transcriptional activation, a key event for persistence of the leukemic stem cell compartment under tyrosine kinase inhibitor therapy. Polo-like kinase 1 inhibitor BI6727, already advanced for clinical use, breaks β-catenin interaction with FOXM1, hence hampering FOXM1 phosphorylation, β-catenin binding, nuclear import, and downstream signaling. In conclusion, our results support Polo-like kinase 1/FOXM1 axis as a complementary target to eradicate leukemic early progenitor/stem cell compartment in chronic myeloid leukemia. J. Cell. Biochem. 118: 3968-3975, 2017. © 2017 Wiley Periodicals, Inc.

Jak2a regulates erythroid and myeloid hematopoiesis during zebrafish embryogenesis.

Zebrafish (Danio rerio) are an attractive vertebrate model for the molecular dissection of disease mechanisms. Janus kinase (JAK)/signal transducer and activator of transcription (stat) has been defined through studies of cytokine signaling pathways in mammals. Here, we examined the expression level of Jak2a, which is a homolog of mammalian jak2 in zebrafish, by quantitative reverse transcriptase (RT)-PCR, and the peak of mRNA expression occurred at 3.75 hours post fertilization (hpf). The overexpression of Jak2a was proven by real-time Q-PCR and Western blot in 1-4-cell stage embryos injected with 400 ng/µl full-length jak2a mRNA as well as gfi1.1, gata1, mpo and β-embryonic hemoglobin as detected by real-time Q-PCR. Moreover, jak2a mRNA significantly increased the GFP+ population in the transgenic zebrafish lines Tg (gata1: gfp) (uninjected embryos: 17.22±1.70%; embryos injected with jak2a mRNA: 21.31±2.11%, p<0.01) and Tg (mpo: gfp) (uninjected embryos: 3.86±1.94; embryos injected with jak2a mRNA: 6.64±1.30%, p<0.01) compared with the control group. Thus, our data indicate that Jak2a plays an important role in erythropoiesis and myeloid hematopoiesis.

Germline Calr Mutation and Thrombocytosis Presenting with Concomitant BCR-ABL1+ CML

CALR mutations are present in 70-84% of JAK2 wild-type myeloproliferative neoplasms (MPN) and 67% and 88% of essential thrombocytopenia (ET) and primary myelofibrosis (PMF) respectively. Most cases of MPN are apparently sporadic, but 7-11% have evidence of familial predisposition. While germline mutations in ET-associated genes, MPL and JAK2, have been described in hereditary thrombocytosis, germline mutations in CALR have not been described in any setting. Two types of CALR mutations are common in MPN: a 52-base pair deletion (bp) and a 5 bp insertion, both in exon 9. With rare exceptions, CALR mutations are generally mutually exclusive with JAK2 or MPL mutations and have very rarely been reported in conjunction with the BCR-ABL1translocation.Here, we report a patient with a germline CALR mutation, thrombocytosis, and subsequent development of BCR-ABL+ CML. A 67-year-old female with no significant medical history presented with severe abdominal pain and nausea. Peripheral blood analysis revealed a marked leukocytosis composed of 66% neutrophils, 16% myelocytes, 6.5% monocytes, 3.5% basophils, 2.5% promyelocytes, 2.5% metamyelocytes, 1.5% lymphocytes, 1.5% blasts, and no eosinophils. The patient was non-anemic and had a normal platelet count (340,000/mm3). Bone marrow biopsy revealed a hypercellular marrow with myeloid predominant trilineage hematopoiesis and 1-2% blasts with morphology consistent with chronic myelogenous leukemia (CML). Fluorescence in-situ hybridization analysis of peripheral blood identified a BCR-ABL1fusion in 98.5% of interphase cells.After 3 months of standard imatinib therapy, quantitative RT-PCR showed a reduction of BCR-ABL1/ABL1 in the peripheral blood, however platelet count was elevated at 539,000/mm3. Thrombocytosis persisted over 2 years with a maximal platelet count of 584,000/mm3. Given the patient's thrombocytosis, her peripheral blood was subjected to a next generation sequencing of JAK2, MPL, and CALR genes. A 52-bp out-of-frame deletion in exon 9 of the CALR gene was detected (52% allele frequency) in peripheral blood. In addition, the same 52-bp CALR deletion (63% allele frequency) was present at the time of diagnosis and within a buccal specimen (47% allele frequency) when the BCR-ABL1 transcript was 1% in the peripheral blood. Immunostain of the buccal sample was strongly positive for cytokeratin (CK) AE1/AE3 but CD45 was not detected indicating no leukocyte contamination. This case reports the first instance of a germline CALR mutation associated with thrombocytosis and is the fourth report of the co-occurrence of BCR-ABL1 and CALR mutation in a single patient. Evolution to BCR-ABL1+ CML suppressed the CALR-mutant thrombocytosis phenotype, emphasizing the effect of these genes on lineage determination in abnormal myeloid proliferation. DisclosuresNo relevant conflicts of interest to declare.

GSK3β Inhibition Promotes Efficient Myeloid and Lymphoid Hematopoiesis from Non-human Primate-Induced Pluripotent Stem Cells.

SummaryAdvances in the scalable production of blood cells from induced pluripotent stem cells (iPSCs) open prospects for the clinical translation of de novo generated blood products, and evoke the need for preclinical evaluation of their efficacy, safety, and immunogenicity in large animal models. Due to substantial similarities with humans, the outcomes of cellular therapies in non-human primate (NHP) models can be readily extrapolated to a clinical setting. However, the use of this model is hampered by relatively low efficiency of blood generation and lack of lymphoid potential in NHP-iPSC differentiation cultures. Here, we generated transgene-free iPSCs from different NHP species and showed the efficient induction of mesoderm, myeloid, and lymphoid cells from these iPSCs using a GSK3β inhibitor. Overall, our studies enable scalable production of hematopoietic progenitors from NHP-iPSCs, and lay the foundation for preclinical testing of iPSC-based therapies for blood and immune system diseases in an NHP model.

SRSF2 Is Essential for Hematopoiesis, and Its Myelodysplastic Syndrome-Related Mutations Dysregulate Alternative Pre-mRNA Splicing.

Myelodysplastic syndromes (MDS) are a group of neoplasms characterized by ineffective myeloid hematopoiesis and various risks for leukemia. SRSF2, a member of the serine/arginine-rich (SR) family of splicing factors, is one of the mutation targets associated with poor survival in patients suffering from myelodysplastic syndromes. Here we report the biological function of SRSF2 in hematopoiesis by using conditional knockout mouse models. Ablation of SRSF2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased levels of hematopoietic stem/progenitor cells. Induced ablation of SRSF2 in adult Mx1-Cre Srsf2(flox/flox) mice upon poly(I):poly(C) injection demonstrated a significant decrease in lineage(-) Sca(+) c-Kit(+) cells in bone marrow. To reveal the functional impact of myelodysplastic syndromes-associated mutations in SRSF2, we analyzed splicing responses on the MSD-L cell line and found that the missense mutation of proline 95 to histidine (P95H) and a P95-to-R102 in-frame 8-amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact SRSF2 is essential for the functional integrity of the hematopoietic system and that its mutations likely contribute to development of myelodysplastic syndromes.

About hematopoietic properties of peripheral blood lymphocytes RNA from patients with polycythemia vera and healthy donors

Total RNA isolated from peripheral blood lymphocytes of donor and patient with polycythemia, stimulates hematopoiesis in rats with toxic aplastic anemia due to benzene administration. Total RNA of lymphocytes from polycythemia patient has a more pronounced effect on the erythroid, myeloid and megakaryocytic hematopoiesis comparing to total RNA from donor lymphocytes. The greatest stimulatory effect of RNA observed after 21 days from the start of experiment. Total RNA of lymphocytes from polycythemia patient largely activates erythropoiesis promoting restoration of reticulocyte count in animals with aplastic anemia.

Prognostic significance of morbidly hematopoietic characteristics in 69 patients with myelodysplastic syndrome

This study was to investigate the influence of morbidly hematopoietic characteristics on the prognosis of patients with myelodysplastic syndrome (MDS). A total of 69 cases of MDS were analyzed retrospectively on ralatienship between sex, age, MDS types, WBC count, hemoglobin (Hb) level, platelet (Plt) count at diagnosis, morbidly cytologic features of bone marrow and survival time of MDS patients. The median survival time of 69 cases of MDS was 29.90 months. The patients of different sexes and Plt level at diagnosis did not display statistically significant difference in median survival time (P > 0.05); the patients with different ages, WBC count and Hb level showed statistically significant difference in median survival time (P < 0.05); the median survival time of patients with different MDS types was significant different (P < 0.01); the MDS patients with myeloid lineage containing nuclear plasma development imbalance, micronuclei, abnormal mitotic figures, with erythroid lineage containing megaloblastic degeneration, cell size disparity, nuclcar budding and muclear fragmentation, and with megakaryocyte lineage containing micromegaryocytes, excessive muclear leaves, displayed significant difference in median survival time (P < 0.05). The MDS patients with ALIP positive, fibrosis in bone marrow blopsy showed significant difference in median survival time. The age, MDS types, Hb level and WBC count at diagnosis are indicators influencing the prognosis. The unbalanced development of muclear plasma, micronuclei, abnormal mitotic figures in myeloid morbid hematopoiesis, the megaloblastic degeneration, cell size disperity, muclear budding, nuclear fragmentation in erythroid morbid hematopoiesis, the micro-megakaryocytes, excessive nuclear leaves in megakaryocytic morbid hematopoiesis, and existance of ALIP posstive and fibrosis in bone marrow biopsy indicate important values for evaluation of MDS prognosis.

Pyruvate Kinase Deficiency Children with New Types of PK-LR Gene Mutation Were Cured by Allogeneic Hematopoietic Stem Cell Transplantation

Objective To investigate PK-LR genetic diagnosis and hematopoietic stem cell transplantation (allo-HSCT) of pyruvate kinase deficiency (PKD) through clinical symptoms of two children with PKD and new types of PK-LR gene mutation report.Methods In February 2009 and August 2013,two children with PKD in Shanghai Children's Medical Center,Affiliated to Shanghai Jiao Tong University School of Medicine were collected into this study.The study protocol was approved by the Ethical Review Board of Investigation in Human at Shanghai Children's Medical Center Affiliated to Shanghai Jiao Tong University School of Medicine.Informed consent was obtained from all participants' parents.The blood specimens from the two PKD children were collected.PK-LR gene analysis of two children were detected by Sanger sequencing and whole exon capture sepuencing.Two PKD children were treated by allo-HSCT,and their clinical symptoms,diagnosis and treatment were retrospectively analyzed.Results ① Pyruvate kinase (PK) activation of two PKD children were reduced to 8.00 IU/gHb and 7.61 IU/gHb respectively.② Four types of PK-LR gene missense mutation were found in gene detection of two children.PK-LR gene c.T941T＞C(p.Ile314Thr) mutation was reported formerly,and c.119G＞A (p.Arg40Gln),c.1015G＞A(p.Asp339Asn) and c.848T＞ C (p.Va1283Ala) mutations were new.SIFT function prediction results of the 4 mutations were 0.20,0.37,0 and 0.23 respectively.③ Two PKD children were treated by alloHSCT.After transplantation,myeloid and erythroid hematopoiesis of children 1 were recovered on the 14th day.Platelet (PLT) count was ＞5× 1010/L on the 19th day.Hematopoietic stem cell (HSC) was complete chimerism on the 22th day,and the blood type of children 1 was converted to donor's.After transplantation,myeloid and erythroid hematopoiesis of children 2 were recovered on the 12th day.PLT count was ＞3× 1010/L on the 19th day.HSC was complete chimerism on the 18th day,and the blood type of children 2 was converted to donor's.Conclusions The whole exon capture sequencing can be used in the clinical diagnosis of PKD and searching for new types of PK-LR gene mutation.PK-LR gene c.119 G＞A (p.Arg40Gln),c.1015G＞A(p.Asp339Asn) and c.848T＞C(p.Va1283Ala) missense mutations could also lead to PKD.allo-HSCT could be an effective treatment for PKD. Key words: Pyruvate kinase deficiency of red cells; Mutation,missense; Hematopoietic stem cell transplantation

Graft-Versus-Host-Disease Recruits Both Monocyte and Pre-Conventional Dendritic Cell-Derived Tissue Antigen Presenting Cells Independent Of CCR2 and CCR6

Graft-versus-host disease (GVHD) limits the broader application of allogeneic hematopoietic stem cell transplantation. In prior studies we defined roles for both host and donor-derived antigen presenting cells (APCs) in the activation of alloreactive donor T cells and in promotion of GVHD. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs, we have consistently observed MHCII+ donor-derived APCs, including dendritic cells (DCs), in histopathologic GVHD lesions, frequently adjacent to infiltrating T cells, suggesting they have a role in local GVHD reactions. Donor-derived tissue APCs (t-APCs), including tissue-DCs (t-DCs) could activate donor T cells through indirect or cross-presentation of host antigens, produce chemokines that recruit other effectors, and elaborate inflammatory mediators or suppressors of inflammation. We first characterized t-DC subsets in the skin and bowel of GVHD-affected mice. 129 (H-2b) hosts were irradiated and reconstituted with B6 (H-2b) BM with or without CD4+ and CD8+ T cells to induce GVHD and analyzed mononuclear cells from skin and bowel approximately 4 weeks post transplant. In skin, both main dermal DC populations (CD11b+ and CD103+) were significantly increased in GVHD mice as compared to BM alone controls, though the ratios of CD11b+: CD103+ DCs were similar. In the bowel lamina propria, the ratios of CD11b+CD103- to CD11b+CD103+ were increased in GVHD mice in the colon but were similar to that in BM alone controls in the small bowel. We next studied the roles of CCR6 and CCR2 in the recruitment of donor-derived APCs to skin and bowel. We transplanted mice with CCR6-/- BM in competition with wild type (wt) BM and found that the contribution of each to skin and bowel APCs matched their contributions to myeloid hematopoiesis in BM, spleen and blood, indicating that CCR6 is not required. To study the role of CCR2 we first compared mice transplanted with either wt or CCR2-/- BM with wt T cells. Despite having a profound reduction in blood monocytes, all skin and bowel t-APC subsets were present in CCR2-/- recipients, indicating that CCR2 is not required for t-APC recruitment in contrast to its role in many other models of inflammation. However, CD103+ DCs were more prevalent relative to CD11b+ DCs, consistent with a pre-cDC origin. Despite monocytopenia, recipients of CCR2-/- BM developed clinical GVHD; histology data is being analyzed and will be presented. To better define the contributions of CCR2 to t-APC recruitment and to determine monocyte versus pre-cDC origin of t-DCs, we transplanted mice with CCR2-/- BM in competition with wt BM and compared ratios of BM and blood precursors (pre-cDCs and monocytes) to t-DC ratios. For CD103+ DCs, wt/KO ratios matched the ratios of general myeloid hematopoiesis and pre-cDCs, indicating a pre-cDC origin. For CD11b+CD103- DCs, the ratio of wt/KO matched that in blood monocytes. We further subsetted CD11b+ t-DCs based on the expression of Ly6C, MAR1, CD64 and CD24, used to differentiate pre-cDC from mono-derived DCs in other organs, and did not identify any population with wt/KO ratios that did not match that of the general CD11b+ DC population, suggesting that most if not all CD11b+ t-DCs are of monocyte origin. Experiments are underway examining the role of CX3CR1 in t-APC recruitment and these data will be presented. Disclosures: No relevant conflicts of interest to declare.

A novel application of furazolidone: anti-leukemic activity in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common malignant myeloid disorder of progenitor cells in myeloid hematopoiesis and exemplifies a genetically heterogeneous disease. The patients with AML also show a heterogeneous response to therapy. Although all-trans retinoic acid (ATRA) has been successfully introduced to treat acute promyelocytic leukemia (APL), it is rather ineffective in non-APL AML. In our present study, 1200 off-patent marketed drugs and natural compounds that have been approved by the Food and Drug Administration (FDA) were screened for anti-leukemia activity using the retrovirus transduction/transformation assay (RTTA). Furazolidone (FZD) was shown to inhibit bone marrow transformation mediated by several leukemia fusion proteins, including AML1-ETO. Furazolidone has been used in the treatment of certain bacterial and protozoan infections in human and animals for more than sixty years. We investigated the anti-leukemic activity of FZD in a series of AML cells. FZD displayed potent antiproliferative properties at submicromolar concentrations and induced apoptosis in AML cell lines. Importantly, FZD treatment of certain AML cells induced myeloid cell differentiation by morphology and flow cytometry for CD11b expression. Furthermore, FZD treatment resulted in increased stability of tumor suppressor p53 protein in AML cells. Our in vitro results suggest furazolidone as a novel therapeutic strategy in AML patients.

Live-attenuated Listeria-based immunotherapy

For decades Listeria monocytogenes has been used as a model of host–disease immunology, and a considerable body of knowledge has been amassed regarding the complex immune response to L. monocytogenes. Attenuated strains of L. monocytogenes are currently being assessed as therapeutic bacterial vectors to present tumor-associated antigens to the immune system for the clinical treatment of cancer. L. monocytogenes immunotherapy utilizes many synchronous and disparate action mechanisms that stimulate innate and cell-mediated adaptive immunity while reducing immunosuppressive influences in the tumor microenvironment. Other effects not typically associated with immunotherapy include the stimulation of myeloid hematopoiesis and vascular changes that enable chemotaxis. Preliminary clinical results using L. monocytogenes bearing the HPV oncogene E7 indicate good tolerability and a strong efficacy signal, warranting further development. This article reviews the current status of L. monocytogenes as a cancer immunotherapeutic and the complex immune responses that underlie L. monocytogenes immunotherapy.

Combination therapy with dendritic cell-based vaccine and chemotherapy is active against acute myeloid leukemia in a mouse model

Acute myeloid leukemia (AML) is a malignant disorder of progenitor cells in myeloid hematopoiesis. The majority of AML patients achieve complete remission with the current therapeutic treatment but relapse frequently occurs which shortens survival of AML patients. The dismal cure rate of current therapeutic strategies stresses the urgent need of novel strategy to prevent relapse and lengthen the survival of AML patients. Therefore, DC based cancer immunotherapy presents as an attractive adjunct for the treatment of AML. We hypothesized that AML dendritic cell (DC)-based vaccine plus chemotherapy using cytosine arabinoside (AraC) induces better antitumor effect in AML mouse model. Therapeutic efficacy of syngeneic DC-based vaccine alone or in combination with chemotherapy in antitumor immunity was evaluated in vivo. Female C57BL/6 mice were challenged subcutaneously with murine C1498 AML cells and tumor bearing mice were treated as follows: 1) PBS injection; 2) cytosine arabinoside (AraC, 100 mg/kg) alone; 3) DC vaccine (2.0×106 cells/mouse) alone; 4) combination of AraC followed by DC vaccine (AraC-DC) treatment; and 5) DC vaccine followed by AraC and repeat DC injection (DC-AraC-DC). We found that individual treatments (DC vaccine or AraC treatment alone) induced only transient tumor regression with minimal survival advantage as compared to PBS control. The combinatorial therapy, both AraC-DC (P = 0.093) and DC-AraC-DC (P < 0.001) treatment, conferred greater tumor suppression effects and strongly prolonged median survival time of AML bearing mice (median survival 15 days & 20 days vs 10 days, respectively). The DC-AraC-DC treatment approach appears to be superior in increasing the tumor eradication as compared to AraC-DC treatment (P < 0.05). These findings raise the possibility of utilizing this approach to treat AML patients clinically.

Characteristics of Myeloid Differentiation and Maturation Pathway Derived from Human Hematopoietic Stem Cells Exposed to Different Linear Energy Transfer Radiation Types

Exposure of hematopoietic stem/progenitor cells (HSPCs) to ionizing radiation causes a marked suppression of mature functional blood cell production in a linear energy transfer (LET)- and/or dose-dependent manner. However, little information about LET effects on the proliferation and differentiation of HSPCs has been reported. With the aim of characterizing the effects of different types of LET radiations on human myeloid hematopoiesis, in vitro hematopoiesis in Human CD34+ cells exposed to carbon-ion beams or X-rays was compared. Highly purified CD34+ cells exposed to each form of radiation were plated onto semi-solid culture for a myeloid progenitor assay. The surviving fractions of total myeloid progenitors, colony-forming cells (CFC), exposed to carbon-ion beams were significantly lower than of those exposed to X-rays, indicating that CFCs are more sensitive to carbon-ion beams (D 0 = 0.65) than to X-rays (D 0 = 1.07). Similar sensitivities were observed in granulocyte-macrophage and erythroid progenitors, respectively. However, the sensitivities of mixed-type progenitors to both radiation types were similar.In liquid culture for 14 days, no significant difference in total numbers of mononuclear cells was observed between non-irradiated control culture and cells exposed to 0.5 Gy X-rays, whereas 0.5 Gy carbon-ion beams suppressed cell proliferation to 4.9% of the control, a level similar to that for cells exposed to 1.5 Gy X-rays. Cell surface antigens associated with terminal maturation, such as CD13, CD14, and CD15, on harvest from the culture of X-ray-exposed cells were almost the same as those from the non-irradiated control culture. X-rays increased the CD235a+ erythroid-related fraction, whereas carbon-ion beams increased the CD34+CD38− primitive cell fraction and the CD13+CD14+/−CD15− fraction. These results suggest that carbon-ion beams inflict severe damage on the clonal growth of myeloid HSPCs, although the intensity of cell surface antigen expression by mature myeloid cells derived from HSPCs exposed to each type of radiation was similar to that by controls.

Altered hematopoiesis in trisomy 21 as revealed through in vitro differentiation of isogenic human pluripotent cells

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver, a preleukemic condition termed transient myeloproliferative disorder, and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins, human embryonic stem (hES), and induced pluripotent stem (iPS) cells, were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation, we isolated disomic and trisomic subclones from the same parental iPS line, thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like, γ-globin expressing, definitive hematopoiesis, we found that trisomic cells of hES, iPS, or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells, accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage.

Abstract 1349: Xenograft models of human acute myeloid leukemia (AML) with biomarker strategies are valuable in development of new drugs targeting AML

Abstract Acute myeloid leukemia (AML) is a malignant disease caused by disorder of progenitor cells in myeloid hematopoiesis. The rapid progress and high fatality make it difficult to cure the patients suffering from AML. Choice of treatment is limited, consisting primarily of chemotherapy and hematopoietic stem cell transplantation. Pharmaceutical companies are focused on designing new drugs that may be efficacious in treating the disease and prolong the survival of the patients. However, the lack of AML model and specific biomarkers hinder the progress of research on this cancer type. Here we successfully validated subcutaneous and systemic models derived from a number of AML cell lines, such as MV4-11, HL-60, Molm-16, and ML-2, in NOD/SCID mice to evaluate the antitumor activities of new compounds. Biomarkers, such as CD45, CD38 and CD15 in PBMC, spleen and bone marrow from the systemic models, are used in detecting and monitoring the disease progress. These biomarkers correlated with tumor take rate, disease progression and response to standard of care treatments. In conclusion, AML xenograft models with biomarker strategies provide valuable platforms for the development of new compounds targeting this deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1349. doi:1538-7445.AM2012-1349

Calcineurin/NFAT signalling inhibits myeloid haematopoiesis

Nuclear factor of activated T cells (NFAT) comprises a family of transcription factors that regulate T cell development, activation and differentiation. NFAT signalling can also mediate granulocyte and dendritic cell (DC) activation, but it is unknown whether NFAT influences their development from progenitors. Here, we report a novel role for calcineurin/NFAT signalling as a negative regulator of myeloid haematopoiesis. Reconstituting lethally irradiated mice with haematopoietic stem cells expressing an NFAT-inhibitory peptide resulted in enhanced development of the myeloid compartment. Culturing bone marrow cells in media supplemented with Flt3-L in the presence of the calcineurin/NFAT inhibitor Cyclosporin A increased numbers of differentiated DC. Global gene expression analysis of untreated DC and NFAT-inhibited DC revealed differential expression of transcripts that regulate cell cycle and apoptosis. In conclusion, these results provide evidence that calcineurin/NFAT signalling negatively regulates myeloid lineage development. The finding that inhibition of NFAT enhances myeloid development provides a novel insight into understanding how the treatment with drugs targeting calcineurin/NFAT signalling influence the homeostasis of the innate immune system.

Myeloid metaplasia in canine mixed mammary tumors: occurrence and characterization

Background: Mixed tumors are among the most frequent mammary neoplasms in female dogs. Some of these tumors present bone marrow associated with the newly formed osseous tissue, characteristic of myeloid metaplasia. Objective: To evaluate the occurrence of these lesions in a series of mixed tumors, and determine its histomorphological characteristics. Animals and methods: In total, 384 canine mammary mixed tumors from 289 animals have been reviewed. The lesions were classified according to the presence of osseous metaplasia associated with myeloid metaplasia or extramedullary hematopoiesis. Myeloid metaplasia characterization was determined from the morphological characteristics and organization of the cells and adjacent tissues. Cytoplasmic staining for CD31 and Factor VIII were used as a criterion to confirm the presence of blood vessels and megakaryocytes, respectively. Results: The 384 cases included 206 benign and 178 carcinomas in mixed tumors. Osseous metaplasia was present in 16.1% and calcified areas exclusively in 3.1% lesions. Among all osseous metaplasia, 33.9% presented some type of extramedullary hematopoiesis, of which 71.4% were classified as myeloid metaplasia and 28.6% as extramedullary hematopoiesis. Myeloid metaplasia cases consisted of 67% benign mixed tumors and 33% carcinomas in mixed tumors. CD31 and Factor VIII expression occurred in all myeloid metaplasia, confirming the presence of blood capillaries and megakaryocytes. Myeloid metaplasia was observed in 24% of mixed tumors containing osseous metaplasia and in 4% of all mixed tumors analyzed. Conclusion: Despite the low frequency of this lesion, additional studies are needed to understand the implications of myeloid metaplasia in canine mammary mixed tumors.

A Novel Role of Pim Serine/Threonine Kinases in Hematopoiesis: Pim 1 Kinase Increases Hematopoietic Stem Cell Population

Abstract 561 BACKGROUND:Pim kinases are a small family of constitutively active, highly conserved serine/threonine kinases, and have 3 members (Pim1, 2 and 3). Pim kinase is considered to be oncogenic because ∼5-10% of murine Pim1 transgenic (Tg) mice driven by the Eμ immunoglobulin promoter developed lymphoid malignancies (van Lohuizen, M. et al, 1989, Cell 56:673-682). However, the roles of Pim kinases in the development of myeloid malignancies and in the regulation of hematopoietic stem cells (HSCs) are not completely understood. In the current study, we used Tg and knockout (KO) mouse models to characterize the roles of Pim1 kinase in myeloid oncogenesis and/or hematopoiesis. The regulation and the downstream events of Pim1 kinase in HSCs were also investigated. We used the vav hematopoietic regulatory elements to drive human PIM1 gene expression in our Tg mouse model. Vav regulatory elements have been shown to promote hematopoietic- specific expression and induce myeloid malignancies (Lin, Y.W. et al, 2005. Blood 106:287-295). METHODS:We generated PIM1 Tg mice bearing human PIM1 kinase under the vav-regulatory elements (vav-hPIM1). Pim1, 2, 3 single, double and triple KO mice were also available to us and were used in this study. The percentage and the absolute number of c-Kit+Sca-1+Lin− (LSK) hematopoietic stem/progenitor cells (HSPCs) in bone marrow and in spleen were measured by flow cytometry. The frequency of hematopoietic progenitor cells was measured by colony-forming unit assays (CFU-GM, BFU-E and CFU-GEMM). The capacity of HSPCs to protect and reconstitute lethally irradiated recipient mice was determined using a bone marrow transplantation model. The regulation of PIM1 expression in HSPCs was determined by quantitative RT-PCR following exposure to various cytokines including IL-3, IL-6, stem cell factor, TPO, and Flt3 ligand. Finally, HSPCs were transduced with lentiviral vector expressing PIM1 and its downstream gene targets were measured by quantitative RT-PCR. RESULTS:1). Vav-hPIM1 Tg mice developed lymphoid, but not myeloid, malignancies. In our vav-hPIM 1 Tg mice, human PIM1 kinase was expressed in all hematopoietic tissues including bone marrow, spleen and thymus, but not in non-hematopoietic tissues. Approximately10% of vav-hPIM 1 Tg mice developed lymphoid leukemia/lymphoma. Interestingly, no myeloid malignancies were observed in vav-hPIM 1 Tg mice. 2). Vav-hPIM1Tg mice had increased HSPC population. In those vav-hPIM1 Tg mice that were free of leukemia/lymphoma, a > 2 fold increase in the number of LSK HSPCs were noted when compared to age-matched, wild-type littermates (p <0.05). Vav-hPIM 1 Tg mice also exhibited enlarged spleen, higher CXCR4 surface expression and increased frequencies of CFUs, compared to littermate controls. Additionally, lethally irradiated mice transplanted with splenocytes from vav-hPIM1 Tg mice had better survival compared to those transplanted with splenocytes from wild-type control mice. 3). Pim1/2 KO mice had decreased HSPC population. When compared to wild-type littermate controls, Pim1/2 KO mice exhibited lower number of LSK HSPCs, lower CXCR4 surface expression, and reduced frequencies of CFUs. Furthermore, lethally irradiated mice transplanted with marrow cells from Pim1/2 KO mice had much slower hematopoietic recovery, compared to those receiving marrow from normal control mice. 4). The expression of Pim1 kinase in HSCs was up-regulated by cytokine stimulation, in particular, IL-3, IL-6, and stem cell factor, suggesting that cytokines in marrow niche microenvironment could affect Pim 1 expression in HSCs. 5). Finally, we identified a gene family that was up-regulated when Pim1 kinase was over-expressed in HSCs, illustrating the mechanism through which Pim1 enhances hematopoiesis. CONCLUSION:Our current studies demonstrate a novel role of Pim serine/threonine kinase in hematopoiesis. Our data also provide a novel molecular mechanism in the regulation of self-renewal, proliferation, and expansion of HSCs. Acknowledgment:We thank Dr. Jerry M. Adams, Walter and Eliza Hall Institute of Medical Research, Australia for the kind gift of vav-hCD4 vector, and Dr. Peter D. Aplan, NCI, Bethesda MD for advice on developing the construct for generating Tg mouse. This work is partly supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award. Disclosures:No relevant conflicts of interest to declare.

Characteristics of tumors developed in mdx mice after transplantation of GFP-positive mesenchymal stem cells isolated from bone marrow of transgenic C57BL/6 mice

The purpose of the work was morphological and histochemical examination of tissue differentiation in tumors developed in mdx mice after the intramuscular transplantation of GFP-positive mesenchymal bone-marrow stem cells (MSC-GFP) derived from C576BL/6 transgenic mice and cultivated for 43–45 passages. These cells did not generate tumors in syngeneic adult C57BL/6 mice. The tumors were classified as mesenhymomas, fibrosarcomas, and sarcoma. Adipocyte and chondrocyte clusters, as well as bone areas with erythroid, myeloid, and thrombocyte hematopoiesis and neural tissue with glia cells were observed inside of tumors. Types of tissue tumor differentiation were similar to those described in the literature for MSCs induced to differente in vitro by specific treatment. However, the differentiation spectrum in MSC-GFP-produced tumors was broader than the differentiation of tumors derived from adult mouse MSCs spontaneously transformed or transfected in vitro. The results presented here, along with our previous data, demonstrate that the transfection of stem cells, including totipotent stem cells, with genetic constructs is accompanied by the destabilization of the cell genome, even if the activity of inserted gene (GFP) does not affect general cell functioning.