Immature Hematopoietic Cells Research Articles

Extramedullary hematopoiesis involving the central nervous system and surrounding structures

Extramedullary hematopoiesis in central nervous system is a rare condition that can present in association with hematologic conditions. In this single-center retrospective study from 1988 to 2010, we identified 8 cases of extramedullary hematopoiesis involving the central nervous system or meninges. Most were incidental autopsy findings, usually microscopic collections of cells in the meninges or around blood vessels in the brain of infants (4 cases; age, 10 days to 5 months), in 1 young child (age, 6.5 years with anemia), and 1 elderly adult (age, 81 years with end-stage idiopathic myelofibrosis). Two individuals (both with polycythemia rubra vera) presented with symptomatic intracranial or spinal canal lesions (age, 67 and 68 years). Mixed populations of immature hematopoietic cells were identified by characteristic morphology and appropriate immunohistochemistry. The precise pathogenesis of extramedullary hematopoiesis in central nervous system is unknown. In infants, extramedullary hematopoiesis is unlikely to be pathologic and most likely represents a resolving fetal state. The older children and adults with incidental or symptomatic extramedullary hematopoiesis usually have some underlying hematologic condition, which should be sought if not already recognized.

C-myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb-silenced CD34(+) cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets, which can account for c-myb knockdown effects. Indeed, chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing, whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34(+) cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.

Antibody‐directed lentiviral gene transduction in early immature hematopoietic progenitor cells

The specific and efficient transduction of retroviral particles remains problematic for in vivo and ex vivo gene therapy studies, where the targeting cell population is a heterogeneous bulk population. Pseudotyping lentiviral particles with Sindbis virus envelope (Env) proteins modified with an immunoglobulin Fc-binding domain presents a method of selecting cells within a mixed population through antibody (Ab)-mediated targeting. Conditions were tested for targeted lentiviral gene delivery to hematopoietic progenitor cells via Ab-conjugated envelopes independent of CD34. Conditions to optimize the efficiency of gene delivery were established using the ABCG2 multidrug resistance protein, associated with stem cell phenotypes, as the cell surface target. By varying the proportion of ABCG2 expressing cells in a population, ABCG2-targeted gene delivery was detectable by flow cytometry when ABCG2(+) cells comprised greater than 5% of the population. Conditions that increased the efficiency of gene transfer, including cholesterol independent Env proteins and pH, increased nonspecific gene delivery. The feasibility of this cell-Ab-virus sandwich system in targeting transduction in a mixed population was tested in cells derived from human cord blood (CB). Conjugation of viral particles with anti-CD133 and anti-ABCG2 hematopoietic stem cell-associated Ab resulted in targeted gene transfer into early immature hematopoietic progenitor cells. Enhancement was found when the hematopoietic progenitor cells were enriched from CB cells via the depletion of lineage(+) committed cells. Gene transfer to lineage(-) early immature hematopoietic progenitors from human umbilical CB was obtained using CD133, ABCG2 or HLA-1 antibodies conjugated to lentiviruses pseudotyped with modified Sindbis viral Env proteins.

C-Myb Regulates Mir-101 Expression During the Early Stages of Erythropoiesis

AbstractAbstract 3876The proto-oncogene c-myb encodes the transcription factor c-Myb, which is predominantly expressed in immature hematopoietic cells where it plays an obligate role in definitive hematopoiesis. Given the critical functions of c-Myb in lineage commitment, proliferation, and differentiation, c-Myb regulatory factors are of great interest but remain incompletely defined. In recent years, c-Myb has been shown to regulate the expression of microRNA (miRNA) molecules in hematopoietic cells. MiRNA molecules are noncoding RNA molecules that are 21–23 nucleotides in length and function to hybridize to the 3′UTR region of its target mRNA to stimulate/repress translation or induce mRNA degradation. For example, in hematopoietic cells, miR-15a and c-Myb form an autoregulatory negative feedback loop in that over-expression of miR-15a in hematopoietic cells was determined to block erythroid and myeloid colony formation. In megakaryocytes, the hormone thrombopoietin induced miR-150 expression which subsequently functioned to degrade c-myb mRNA through direct interaction with c-myb's 3′-UTR. Our studies have focused on determining the physiologic function of the neuroendocrine Neuromedin U (NmU) during the early stages of erythropoiesis, because we recently determined that silencing NmU in primary human CD34+ cells impairs burst-forming units-erythroid and colony-forming unit-erythroid formation. In subsequent studies, we determined that c-Myb directly interacts with the NmU promoter at Myb Response Elements (MREs) distal to its transcription start site. Also, the expression profiles of NmU and c-myb are similar in CD34+ cells cultured under erythroid inducing conditions for 10 days, and silencing c-myb expression in hematopoietic cells inhibits NmU expression. To gain insight into the regulatory mechanism involved in NmU expression during the early stages of erythropoiesis, we hypothesized that miRNA molecules regulated by c-Myb would inhibit NmU expression through a negative feedback loop. To address this hypothesis, we first scanned the 3′-UTR of NmU and identified 24 different miRNA molecules predicted to interact with NmU's 3′-UTR. Second, we used luciferase reporter assays to determine which of the miRNA molecules interacted with NmU's 3′-UTR. Of the three miRNA molecules we tested, miR-101 directly interacted with NmU's 3′-UTR in a dose-dependent manner. Third, we determined the expression profile of miR-101 in primary CD34+ cells cultured under erythroid inducing conditions. The gene expression of miR-101 was inversely correlated with NmU and c-myb. Finally, because miR-101 contained 6 MREs, we determined the ability of c-Myb to directly interact with the promoter of miR-101 using chromatin immunoprecipitation (ChIP) assays. Using primers that flank the MREs proximal to miR-101's transcription start site, we observed a greater than 2-fold increase in the amplification of DNA recovered from ChIP assays completed with c-Myb antibody compared to ChIP assays completed with irrelevant antibody. Studies are underway to confirm by luciferase-reporter assays that c-Myb directly binds to and transactivates the miR-101 promoter. Collectively, these data identify a regulatory loop comprised of c-Myb, NmU, and miR-101 that could be of potential importance during human erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

Metallothionein in human immunomagnetically selected CD34+haematopoietic progenitor cells

Human umbilical CD34(+) immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34(+) was cultured in serum-free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34(+) cells. CD34(+) cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34(+) cells by 5.12 ± 2.47-fold (mean ± S.D., n = 3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81 ± 1.07%, whereas it was 69.39 ± 10.37% in none-expanded CD34(+) cells (mean ± S.D., n = 3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine-rich metal-binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34(+) cells and compared it with more mature CD34(-) cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34(-) cells was higher than in the immature CD34(+) cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34(-) cells, greater than in CD34(+) cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.

Chronic RPS19 Deficiency Leads to Bone Marrow Failure In a Mouse Model for Diamond-Blackfan Anemia

AbstractAbstract 193Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al, 2009 ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the Collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells, and flow cytometric analysis of bone marrow after a short-term induction reveals increased frequencies of hematopoietic stem and progenitor cells reflecting the onset of stress hematopoiesis. In the current study we have analyzed the long-term effect of RPS19 deficiency in bone marrow. In contrast to a short-term induction, flow cytometric analysis of bone marrow after 51 days revealed decreased frequencies of hematopoietic stem and progenitor cells that correlate with a severe peripheral blood phenotype. In addition, we observed a 3–6 fold increase in apoptosis in RPS19-deficient bone marrow compared to controls based on TUNEL assay. Furthermore, transplantation of whole bone marrow cells from transgenic donors into wild type lethally irradiated recipients confirms that the observed phenotype is autonomous to the blood system. To study whether long-term RPS19 deficiency functionally impairs hematopoietic stem cells, we pre-induced mice for 30 days followed by 15 days without doxycycline to restore the RPS19 expression. Mice were sacrificed and total bone marrow cells were transplanted together with wild-type competitor cells (1:1) into wild type lethally irradiated recipients without doxycycline. This experimental setting allows us to assess the functionality of pre-induced hematopoietic stem cells in absence of ribosomal stress. Flow cytometric analysis of peripheral blood one month after transplantation clearly demonstrates decreased reconstitution from pre-induced donors compared to the wild-type competitor. While this time point reflects mainly the function of transplanted progenitors, long-term analysis of hematopoietic stem cell function in these recipients is ongoing. To study the molecular mechanisms underlying the hematopoietic defect we performed comparative microarray analysis. We chose to analyze preCFU-E/CFU-E erythroid progenitors since we have previously located the erythroid defect at the CFU-E – proerythroblast transition based on flow cytometry and clonogenic proliferation cultures of prospectively isolated erythroid progenitors. Microarray analysis of preCFU-E/CFU-E progenitors reveals deregulation of several genetic pathways, including a robust upregulation of p53 pathway genes, and these targets have been confirmed by real-time PCR. Furthermore, many of p53 target genes are also upregulated in the Lineage− Sca-1+ c-Kit+ (LSK) population that contains immature hematopoietic progenitors and stem cells suggesting that the activation of p53 is not restricted to the erythroid lineage. To ask whether increased activity of p53 can solely explain the hematopoietic phenotype, we have crossed our mouse model into a p53-null background. In summary, our data suggest that RPS19-deficient mice fail to uphold stress hematopoiesis for extended periods of time, with chronic RPS19 deficiency causing bone marrow failure. Disclosures:No relevant conflicts of interest to declare.

Sequential Acquisition of Somatic Mutations In Progenitors with Differential Proliferative Potential In Human Acute Myeloid Leukemia

AbstractAbstract 755 Background:Evidence suggests that leukemic transformation is a multistep process in acute myeloid leukemia (AML). However, the cell of origin for human leukemogenesis remains ill defined. Experimental systems with murine hematopoetic cells indicate that not only immature multipotent hematopoietic stem cells but also more mature, committed myeloid precursors can be a target of murine AML transformation. These findings suggest that requisite mutations for leukemogenesis may be acquired sequentially in myeloid progenitors with differential proliferative potential. To investigate this possibility in human AML, we studied primary specimens from pediatric patients who had previously been identified as having 2 distinct, disease-relevant somatic mutations. Methods:Diagnostic bone marrow or peripheral blood specimens from pediatric AML patients with two distinct genomic alterations were obtained from Children's Oncology Group (COG) AML Reference Laboratory. Early myeloid CD34+/CD33- progenitors and more differentiated CD34+/CD33+ progenitors were isolated using fluorescence-activated cell sorting (FACS). Twenty-eight of 43 patient specimens had FACS-isolated CD34+/CD33- progenitor cells and molecular data available for both somatic alterations, and were included in this study. The genomic alterations included those with core-binding factor (CBF) translocations and FLT3/ITD (n=5), CBF translocations and KIT abnormalities (n=19), or nucleophosmin (NPM1) mutations and FLT3/ITD (n=4). Aliquots of sorted subpopulations were subjected to direct colony forming cell (CFC) assays. Freshly sorted cells and resulting colony forming units-granulocyte and/or monocyte (CFU-GM) were evaluated for the presence of the patient-specific genomic alterations. Results:In 20/28 specimens (71.4%), both mutations were detected in CD34+/CD33- cells. By comparison, in 6 (21.4%) patient specimens, the receptor tyrosine kinase (FLT3/ITD or KIT) alteration was not detected in CD34+/CD33- cells, whereas in 2 (7.1%) specimens, CD34+/CD33- cells did not harbor either of the two patient-specific alterations. In contrast to these less mature CD34+/CD33- progenitor cells, analysis of more differentiated CD34+/CD33+ progenitors revealed both genomic alterations in 26/28 samples. For the remaining 2 samples, only the CBF mutation was detected. FACS-isolated CD34+/CD33- cells from 22 specimens were subjected to CFC assay. There was sufficient colony growth and evaluable molecular data for the paired mutations for 7/22 samples. Within these specimens, the majority (43 of 60) of the evaluable CFU-GM demonstrated the CBF or NPM1 abnormality but lacked the RTK mutation; in the remaining colonies, both somatic alterations were detected (10 of 60), or neither mutation was detected (7 of 60). This finding suggests that the majority of CFU-GM analyzed were derived from an abnormal cell that had not yet acquired the requisite RTK mutation necessary for frank leukemic transformation. Conclusion:These findings suggest the potential for sequential acquisition of RTK mutations in some cases of pediatric CBF or NPM1+ AML. Specifically, the presumptive class I mutation confering a proliferative advantage (e.g. the receptor tyrosine kinase mutation) appears to arise, in some instances, in a more committed myeloid progenitor with limited proliferative potential. Disclosures:No relevant conflicts of interest to declare.

Microenvironmental Changes In An In Vivo Model of Myeloid Leukemia Negatively Regulate Osteoblastic Cells.

AbstractAbstract 1219Patients with myelogenous leukemias can present with symptoms of bone pain and pathologic fractures, however little is known about the interactions between malignant cells and the bone marrow microenvironment. Additionally leukemia is known to severely interfere with normal hematopoiesis. To further characterize interactions between leukemic cells and the microenvironment, we used a model of blast crisis CML (bcCML) in which immature murine hematopoietic cells are engineered to express the BCR/ABL and Nup98/HoxA9 translocation products. Injection of these cells into naïve mice results in rapid accumulation of leukemic cells in the bone marrow (Dash et al. PNAS, 2002). We investigated the effect of leukemia on the bone marrow microenvironment by first performing immunohistochemical analyses. Leukemic cells were observed to preferentially localize in close contact with bony trabeculae. In addition, leukemic mice also exhibited a dramatic loss of trabecular bone as measured by micro-CT scanning (22.8 ± 1.5% vs 13.6 ± 1.5%, BV/TV n=5 in each group p=0.0048), prompting us to examine bone resorption and the abundance of osteoclasts. Histologic sections from leukemic mice showed an increase in mature osteoclasts (TRAP+, multinucleated cells) at the endosteal surface of the long bones (51 ± 4 OC/section vs 64 ± 3 OC/section p=0.0229). Additionally leukemic mice had a 50% increase in serum C-telopeptide (CTX), a well-established marker of global bone resorption (15.5 ± 0.3 ng/ml vs 21.8 ± 1.0 ng/ml, p=0.0003). Therefore, the presence of leukemic cells appears to strongly stimulate osteoclastogenesis and bone resorption. In addition to increased osteoclasts, a rapid and severe reduction in bone formation was identified in leukemic mice by decreased serum osteocalcin, a well-established marker of bone formation (70.8 ± 6.9 ng/ml vs 39.9 ± 3.2 ng/ml, p=0.0036). To determine effects of leukemia on bone-forming cells, we analyzed osteoblastic cells from the long bones of leukemic animals. Marrow was flushed from the bone and minced bone fragments were digested in collagenase. Cells isolated in this fashion have strong osteoblastic activity and can support hematopoietic stem cells (HSCs) (Chitteti et al, Blood 2010). Leukemic cells were present in the isolated fraction confirming that leukemic cells were closely associated with the bone and not entirely removed when the marrow was flushed. Total cells isolated by collagenase digestion from the long bones of leukemic mice were cultured with leukemic cells and evaluated for osteoblastic colony-forming ability. These cultures demonstrated a reduced ability to form osteoblastic colonies compared to controls (26 ± 2 colonies vs 13 ± 2 colonies p=0.0014). Freshly isolated cells were CD45 depleted to remove leukemic cells and again evaluated for colony-forming ability. CD45 negative cells from leukemic mice also demonstrated reduced ability to form mineralizing osteoblastic colonies in vitro compared to controls when an identical number of cells were cultured (10 ± 2 colonies vs 0 colonies) suggesting that their previous exposure to leukemic cells in vivo was sufficient to decrease their osteoblastic activity. Further in vivo osteoblastic evaluation in leukemic mice showed reduced immunohistochemical staining for the osteoblastic marker osteopontin at the endosteal surface, supporting a leukemic-induced reduction of the mature osteoblastic population. These data demonstrate a severe reduction in both number and function of osteoblastic cells in a leukemic environment. Together these data show severe effects of leukemia on both osteoblastic and osteoclastic cells, which could contribute to the bone-specific problems associated with leukemic disease. Moreover, we propose that osteoblastic defects observed in this model may contribute to the leukemia-induced inhibitory effects on normal hematopoiesis. Studies are ongoing to assess the microenvironmental support for normal hematopoiesis in the leukemic setting and to identify the leukemic signals that modify the bone marrow microenvironment. Disclosures:No relevant conflicts of interest to declare.

Clinico-Pathological Profile and Outcome of Acute Myeloid Leukemia: An Experience of Al-Amal Hematology/Oncology Center In Qatar

AbstractAbstract 4384AML is a group of neoplastic disorders resulting from clonal expansion of immature hematopoietic cells in the bone marrow, blood and other tissues. AML represents approximately 90% of all acute leukemias in adults. The median age at diagnosis is 65 years. Geographic variations in the incidence and subtype of AML have been reported in different parts of the world. The classification of AML has evolved from the standard morphological FAB classification to the most recent 2008 World Health Organization (WHO) classification that consider the morphological, immunological, cytogenetic, genetic, and clinical features. In this study we report and for the first time from Qatar the clinico-pathological features and the outcome of adult patients diagnosed as AML between January 2006 and January 2010 at Al-Amal Oncology/Hematology Center. Diagnosis was based on combined morphologic, immunophenotypic & cytogenetic studies. Seventy three patients were found to have AML accounting for 73% of all adult acute leukemias diagnosed in our center. Age was between 17 and 100 years with a median age of 40 years. Fifty five patients were males and 18 were females with a male to female ratio of 3:1. Most of the patients were non-Qatari (83.5%). The most common FAB subtype was AML –M2 (27.3%). The second most prevalent is AML-M3 (23.2%). Within this group (17.7%) were of the classical hyper granular type, while (82.3%) showed different morphological variants. Cytogenetic analysis was attempted in 64 cases. Intermediate was the most common cytogenetic risk group (64%). The most common clinical presentations were Bleeding tendency (55 patients), symptomatic anemia (52 patients) and fever (29 patients). Fifty patients were treated in our center in Qatar. Pre-treatment death occurred in 14 patients (8 patients presented with septic shock and 6 with intracranial haemorrhage). Three patients were initially palliative, 33 patients received treatment in forms of 2 inductions with Doxorubicin + Cytosine Arabinoside (DA 3+7) and 3 consolidations with Cytosine Arabinoside, pre-treatment required in 13 patients, 2 of them required leukopharesis and 11 underwent cyto-reduction by hydroxy-carbamide. Complete remission was achieved in 28 patients (85%), and 5 patients were resistant disease. Patients were followed from 60 to 1620 days with average of 429 days and Median of 330 days. As of June 2010, 24 (73%) patients are still alive, and 9 (27%) patients died. Infection was the main cause of death. In conclusion AML in Qatar have a lower median age, and an increase incidence of AML-M3 (Acute Promyelocytic) subtype that exhibit significant morphological heterogenecity (variant APL). Doxorubicin is still in use for induction in our center due to unavailability of other anthracyclines. Despite this, cardiac toxicity was not seen in our cases so far, and our complete remission and disease free survival are comparable to international data. Disclosures:No relevant conflicts of interest to declare.

Molecular Mechanism Regulating Foxo In Leukemia Initiating Cells of Chronic Myeloid Leukemia.

AbstractAbstract 3391Chronic myeloid leukemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. Although the development of imatinib, a small molecule inhibitor of ABL, represents a breakthrough in the treatment of CML, major part of patients treated in chronic phase CML are not off therapy due to resistance or intolerance. Recent studies have suggested that imatinib is a potent inhibitor against differentiated leukemia cells, but does not deplete leukemia-initiating cells (LICs) responsible for recurrence of CML. To date, therapeutics that can eradicate CML LICs, however, have remained under investigation. To overcome these clinical problems, here we studied the molecular mechanisms regulating maintenance of imatinib-resistant CML LICs by forkhead transcription factor Foxo3a.We first generated a mouse CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature hematopoietic cells, and the cells were subsequently transplanted into irradiated recipient mice. These experiments showed that CML LICs were highly enriched in c-Kit+Lin−Sca-1+ (KLS+) population in BCR-ABL+ CML cells. Serial transplantation experiments for CML LICs originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency reduced lethality of recipient mice at third transplantation. Although recipients that transplanted with wild-type LICs developed CML and acute lymphocytic leukemia (ALL) at third transplantation, we did not observe development of ALL or CML in recipients of Foxo3a deficient LICs after 45 days post-third transplantation, suggesting that the Foxo3a deficient LICs lose their potential to generate malignancies. In addition, a combination of Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo, indicating that Foxo3a plays an essential role for the maintenance of imatinib-resistant CML LICs (Naka et al., Nature 463, 676–680, 2010).Interestingly, when we examined sub-cellular localization of Foxo3a transcription factor in the CML LICs, we found two CML LIC populations; one population was the cells with nuclear localization of Foxo3a (Foxo3a transcription factor is active) and the other population was the cells with cytoplasmic localization of Foxo3a (Foxo3a is inactive). To understand the molecular mechanisms regulating Foxo3a in CML LICs, we next evaluated the activity of upstream BCR-ABL, PI3K, PDK1, and Akt signaling pathway by fluorescence immunohistochemistry. BCR-ABL activity that was determined by phosphorylation levels of CrkL, a down-stream target of BCR-ABL, was detected in almost all of the CML LICs. However, unexpectedly, phosphorylation levels of Akt in the CML LICs with nuclear localization of Foxo3a appeared to be lower than that in the CML LICs with cytoplasmic localization of Foxo3a, despite it is widely believed that BCR-ABL induces activation of Akt signal. Consistent with Akt phosphorylation status, we detected low levels phosphorylation of PDK1 and PI3K p85, upstream regulators for Akt, in the CML LICs with nuclear localization of Foxo3a. Interestingly, expression levels of the cell proliferation antigen Ki67 were lower in the CML LICs with nuclear Foxo3a than that in the CML LICs with cytoplasmic Foxo3a. These results suggest that Foxo3a responsible for maintenance of imatinib-resistant CML LICs may be regulated by molecular mechanisms that are involved in dormancy in CML LICs. Disclosures:No relevant conflicts of interest to declare.

The Neurexin I Alpha/Neurexophilin Axis as An Anti-Proliferative Factor In Human Cord Blood and Murine Bone Marrow Hematopoiesis.

AbstractAbstract 2627The membrane-bound receptors Neurexin I alpha (NRXN1α) and Dystroglycan (DAG1) serve as ligands for each other, and the Neurexophilins (NXPHs) are thought to function as natural antagonists for this interaction. This signaling axis centered on NRXN1α, DAG1 and the three highly homologous NXPHs, plays a well-defined role in neuronal development and synapse development. The failure of DAG1 knock-out mice to develop a mesoderm led us to hypothesize that this axis plays a role in hematopoiesis. Consistent with previous research, DAG1 is heavily (>95%) expressed in human (hu) umbilical cord blood (CB) CD34+ cells, including the primitive CD38- subpopulation. NRXN1α is expressed at similar levels in these populations as well. Hematopoietic cell populations are more precisely defined in the murine (mu) system, and adult mu bone marrow (BM) exhibits a similar trend to hu CB with NRXN1α expression being higher in the more primitive cells and decreasing as the cells differentiate. A high concentration of Neurexophilins is present in the hu CB plasma (900ng/ml), though it is absent in adult mu blood plasma and fetal bovine serum. Neither blocking DAG1 with a well-described blocking antibody, nor stimulation with recombinant NRXN1α has any observable effect on stimulation of hematopoietic progenitor cell (HPC) proliferation in vitro. This led us to investigate the other receptor present in the axis, NRXN1α. We first used alpha-latrotoxin, an excitatory toxin for NRXN1α. While no stimulatory or inhibitory proliferation effect was observed under normal growth conditions, alpha-latrotoxin did promote HPC survival under growth factor starved conditions as effectively as SDF1α/CXCL12 (a known HPC survival enhancing factor), suggesting a possible protective role for the DAG1-NRXN1α axis during stress. In contrast, we identified the Neurexophilins as anti-proliferative agents for immature hematopoietic cells. When injected in vivo, recombinant NXPH1 acts in a myelo- and lympho-suppressive manner. Absolute numbers of mu HPCs were suppressed in a dose- and time-dependent manner, with the maximal dosage decreasing HPC numbers by over 50%. The decrease in absolute colony number was mirrored by a decrease in the cycling population of HPCs in mice treated with NXPH1, as measured by the high specificity activated tritiated thymidine kill assay. Peripheral blood neutrophils, lymphocytes, monocytes and platelets, were also decreased in a time-dependant fashion after in vivo exposure to NXPH1. Intriguingly, the most primitive LTR-HSCs behave in the opposite manner, with a three-fold increase in cycling status induced via the Rapamycin sensitive mTOR pathway. During in vitro culture NXPH1 is only able to suppress colony forming ability of unseparated HPCs treated with both GM-CSF and SCF in combination in comparison to stimulation by other cytokines. This response is most likely due to increased NRXN1α expression in response to stimulation by GM-CSF and SCF. Given the high level of DAG1 expression on colony forming hematopoietic cells, we hypothesized that endogenous DAG1 may be competing with NXPH1 for NRXN1α binding. We thus exposed cells to a well studied DAG1 blocking antibody or plated CD34+ cells in single cell culture. The in vitro results were then able to recapitulate the results observed in vivo and a 50% decrease in colony forming ability is observed in combination with the following cytokines: GM-CSF, IL-3, GM-CSF+SCF, and IL-3+SCF as well as under maximally stimulatory conditions (IL-3, GM-CSF, SCF, and Epo for hu cells and SCF, PWMSCM, and Epo for mu cell). The ability of NXPH1 to inhibit proliferation of CD34+ cells in single cell culture suggests that NXPH1 acts directly on HPCs. The anti-proliferative function of the NXPHs are consistent with previous literature correlating NXPH expression in cancers with negative prognoses and these data may provide a mechanism for the immune system evasion of cancer. Additionally, the high concentration of NXPH in CB plasma may help explain the relatively long time to engraftment of transplanted CB cells. More broadly, these results demonstrate the presence and function of a tightly regulated signaling axis centered on NRXN1α in hematopoiesis. Disclosures:Broxmeyer:CordUse: Honoraria, Membership on an entity's Board of Directors or advisory committees.

A Child With Myelodysplastic Syndrome With Hypocellular Fibrosis

A 9-year-old girl with intractable anemia, rare mucocutaneous bleeding, and pallor was presented. Hemoglobin was 49 g/L; reticulocyte 0.79%, mean corpuscular volume 81 fL, platelet 37×10⁹/L; white blood cell count 3.2×10⁹/L with dysmorphic cells in peripheric blood. Further evaluation revealed 10% cellularity with grade IV reticulin fibrosis, immature, and/or dysplastic hematopoietic cells without sideroblasts, or blast increase in biopsy, Monosomy 8 was found in bone marrow aspiration material using FISH. Vitamin B12, folic acid, hemoglobin electrophoresis, immunoglobulin levels, CD55, CD59, complement 3, 4, abdominal ultrasonography, chest x-ray were normal; diepoxybutane, acid ham, sucrose lysis tests, viral serologies, antinuclear antibody, anti DNA were negative. On diagnosis of "Myelodysplastic Syndrome-refractory cytopenia with hypocellular fibrosis," she received a successful allogeneic BM transplantation from her full matched sibling.

In vitro reconstruction of a three-dimensional mouse hematopoietic microenvironment in the pore of polyurethane foam

Hematopoietic stem cells exist in specific niches in the bone marrow, and generate either more stem cells or differentiated hematopoietic progeny. In such microenvironments, cell-cell and cell-matrix interactions are as important as soluble factors such as cytokines. To provide a similar environment for in vitro studies, a three-dimensional culture technique is necessary. In this manuscript, we report the development of a three-dimensional culture system for murine bone marrow mononuclear cells (mBMMNCs) using polyurethane foam (PUF) as a scaffold. The mBMMNCs were inoculated into two kinds of PUF disks with different surface properties, and cultured without exogenous growth factors. After seeding the inside of the PUF pores with mBMMNCs, PUF disks were capable of supporting adherent cell growth and continuous cell production for up to 90days. On days 21-24, most nonadherent cells were CD45 positive, and some of the cells were of the erythroid type. From comparisons of the cell growth in each PUF material, the mBMMNC culture in PUF-W1 produced more cells than the PUF-R4 culture. However, the mBMMNC culture in PUF-W1 had no advantages over PUF-R4 with regard to the maintenance of immature hematopoietic cells. The results of scanning electron microscopy and colony-forming assays confirmed the value of the different three dimensional cultures.

Signaling from Fibroblast Growth Factor Receptor 2 in Immature Hematopoietic Cells Facilitates Donor Hematopoiesis After Intra-Bone Marrow–Bone Marrow Transplantation

Fibroblast growth factor (FGF) and FGF receptor (FGFR) are expressed in various cells including endothelial progenitor cells and hematopoietic cells. The interaction between FGF and FGFR is associated with the proliferation, migration, and survival of these cells. In this report, we examined the effects of FGFR2 signaling on hematopoiesis in immature hematopoietic cells, using mutant mice in which a constitutively active form of FGFR2 mutant was caused to be overexpressed by the Tie2 promoter (FGFR2 Tg mice). Under normal conditions, hematopoiesis of FGFR2 Tg mice and wild type (Wt) mice do not differ significantly, except for the weight and cell numbers of the thymus. However, the c-kit(+)Sca-1(+)lineage⁻ bone marrow cells (BMCs) of FGFR2 Tg mice facilitate the formation of colony-forming units of culture. When these BMCs were transplanted into the recipient bone marrow (intra-bone marrow-bone marrow transplantation), there was better reconstitution of donor hematopoietic cells. In the in vitro experiment, the c-kit(+)Sca-1(+)lineage⁻ BMCs from FGFR2 Tg mice showed fewer apoptotic cells than those from Wt mice. These results suggest that the antiapoptotic effect of FGFR2 signaling facilitates the hematopoiesis of FGFR2 Tg mice.

Natural sesquiterpene lactones as inhibitiors of leukaemia-associated transcription factor c-Myb

The transcription factor c-Myb is essential for immature hematopoietic cell proliferation. It has been discovered that changes in the c-myb gene are responsible for development of some kinds of acute lymphatic leukaemia. Inhibition of this target might be of interest as a novel therapeutic strategy [1]. Up to present, however, no inhibitor is known that might be used in therapy. Our main interest is directed towards sesquiterpene lactones (STLs). Such compounds show a wide variety of biological activities and some are known to inhibit transcription factors, e.g. NF-κB. We have recently discovered that some STLs inhibit transcription of a c-Myb regulated gene. Especially the helenanolide Mexicanin-I (structure as depicted) showed high potency as a c-Myb inhibitor [2]. Based on a bioassay that reflects a direct connection between fluorescence and c-Myb activity, we found that Mexicanin-I at concentrations of 1, 3 and 10µM inhibits the c-Myb induced fluorescence activity to 66, 42 and 16%, respectively, compared to an untreated control. It has been verified by MTT-assay, that these results are not solely based on cytotoxic effects. A selection of further STLs of different structural types was shown to cause effects of varying magnitude in this assay. Furthermore, several crude extracts of Asteraceae were tested and the active compounds obtained by bioactivity-guided isolation. Based on the chemical structures of the investigated compounds, it is possible to draw first conclusions about structure-activity-relationships.

In Silico Analyses of Proteomic Data Suggest a Role for Heat Shock Proteins in Umbilical Cord Blood Hematopoietic Stem Cells

Umbilical-cord blood (UCB) has growingly become an accepted alternative source of hematopoietic stem cells for transplantation purposes. However, the low cell dose limit within a single unit is still an obstacle hindering the way of a broader diffusion. The real deal is the lack of knowledge about the molecular processes governing the events of expansion and differentiation of these cells. In order to fill this void, several studies were focused on the identification of the peculiar whole protein profile of UCB-derived hematopoietic stem cells. In this review article we provide a referenced list of overall proteins from UCB-derived hematopoietic stem and progenitor cells. This list has been elaborated for pathway and network analyses, along with GO term enrichment for biological and molecular functions, in order to individuate main classes of proteins governing functioning of these cells. From these analyses it seems to emerge a central role for heat shock proteins in immature hematopoietic stem cells. Their role might be relevant in protecting crucial transcription factors which drive proliferation and differentiation towards a specific lineage (e.g. erythroid, myeloid). Hereby we also stress the helpfulness of interactomics elaboration in providing a unified overview of independent proteomics data. It appears that maturation, other than representing a bottleneck to protein expression, could sculpt interaction maps via reducing complexity of immature interactomics profiles.

Circulating Blasts in a Crohn’s Patient Treated With Natalizumab

### Patient: 34-year-old Caucasian male. ### Chief Complaint: Pathologist’s review of peripheral smear is requested secondary to presence of circulating blasts. ### History of Present Illness: The patient has a 7-year history of Crohn’s disease. He has been treated with Natalizumab (Tysabri) since April 2008, after infliximab and high-dose steroids failed to control his symptoms. He stated he has an average of 5–7 bowel movements daily. He denies changes in the number of bowel movements, hematochezia, abdominal pain, fatigue, night sweats, fever/chills, weight loss, or neurologic symptoms, including visual changes, changes in thought process, or headaches. He also denies recent travel or recent illness. ### Past Medical History: Crohn’s disease diagnosed in 2003, status post partial small bowel resection and multiple stricturoplasties. Well documented iron deficiency anemia. ### Medications: Natalizumab; 300 mg IV every 4 weeks and iron infusions; monthly. ### Family History: Non-contributory. ### Physical Exam: Vital signs: blood pressure, 143/92 mmHg; temperature, 36.1°C; pulse, 81 bpm; respirations, 14. HEENT: Conjunctivae are clear. Sclerae are anicteric. Oropharynx is clear. There is no lymphadenopathy. Abdomen: Active bowel sounds, soft and nontender, no hepatosplenomegaly or masses. ### Principal Laboratory Finding: The laboratory findings at presentation are listed in Table 1. ### Hematopathology Review of Peripheral Blood Smear: Occasional blasts (Image 1) and rare myelocytes present. Adequate platelets; erythrocytes with mild microcytosis, moderate anisocytosis, and polychromasia. 1. What are this patient’s most striking clinical and laboratory findings? 2. What is the differential diagnosis of these findings? 3. What is Natalizumab and what is its therapeutic effect? 4. How does Natalizumab contribute to this patient’s peripheral blood findings? 5. What are Natalizumab’s additional risks? 6. What are Natalizumab’s possible additional applications? 1. The most striking finding in this patient is the presence of circulating immature hematopoietic cells (blasts) in his peripheral smear. The hematology analyzer used in our institution defines the leukocyte differential by flow cytometric principles and staining with myeloperoxidase. In addition to the white cell fractions, the instrument also reports a large unstained cell (LUC) value, relating to populations …

Pure Erythropoiesis in Clear Cell Renal Cell Carcinoma

Extramedullary hematopoiesis (EMH) is a phenomenon in which benign immature hematopoietic cells are found in sites other than the bone marrow. Although usually associated with an underlying hematologic abnormality such as myelofibrosis, it can be found in some physiologic states or occasionally within solid tumors. In most instances, foci of EMH contain elements of all cell lineages. Presented here is the second reported case of EMH occurring in a low-grade clear cell renal cell carcinoma in a patient without evidence of hematologic disease. As previously, it manifested as pure erythropoiesis as confirmed by morphology and immunohistochemistry. It is possible that this finding is related to erythropoietin secretion by the tumor and not hematologic malignancy.

Second hideout for HIV-1

Immature multipotent haematopoietic progenitor cells are identified as another reservoir for latent HIV-1 infection.

Abstract 4237: Foxo3a is essential for survival of leukemia-initiating cells in chronic myeloid leukemia

Abstract Chronic myelogenous leukemia (CML) is caused by a defined genetic abnormality, BCR-ABL, a constitutively active protein tyrosine kinase. Although the introduction of imatinib, a small molecule inhibitor of ABL, represented a breakthrough in the treatment of CML, major part of patients treated in chronic-phase CML are not off therapy due to resistance or intolerance. Recent studies have suggested that imatinib is a potent inhibitor of the production of differentiated leukemic cells, but does not deplete leukemia-initiating cells (LICs). To date, therapeutics that can eradicate CML LICs, however, have remained under investigation. To overcome these clinical problems, we here evaluated molecular mechanisms on survival of the CML LICs. We first generated a mouse CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature hematopoietic cells, and the cells were subsequently transplanted into irradiated recipient mice. These experiments showed that CML LICs were highly enriched in c-Kit+Lin-Sca-1+ (KLS+) population in BCR-ABL+ CML cells, as previously reported. Unexpectedly, phosphorylation levels of Akt in CML LICs appeared to be lower than that in non-LICs, KLS- cells, despite it is widely believed that BCR-ABL induces activation of Akt signal. Since Forkhead O (Foxo) transcription factors, which are important downstream targets of PI3K-Akt signaling, are essential for the maintenance of self-renewal capacity of normal HSCs, we focused on analysis of Foxo3a in CML. Consistent with Akt phosphorylation status, we found that cells with nuclear localization of Foxo3a were enriched in the CML LICs, whereas non-LICs showed the cytoplasmic localization, suggesting that Foxo3a is activated via Akt inactivation in the CML LICs, but not in majority of CML cells. Serial transplantation experiments for CML LICs originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency reduced lethality of recipient mice at 3rd transplantation. Although recipients that transplanted with wild-type LICs developed CML and acute lymphocytic leukemia (ALL) at 3rd transplantation, we did not observe development of ALL or CML in recipients of Foxo3a deficient LICs after 45 days post-3rd transplantation, suggesting that the Foxo3a deficient LICs lose their potential to generate malignancies. Furthermore, deficiency of Foxo3a led to enhanced efficiency in elimination of CML LICs in combination with imatinib treatment. These results demonstrate a critical role of Foxo pathway in survival of CML LICs, and provide a novel therapeutics approach for CML patients by suppression of FOXO signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4237.