ALBERT

Beschreibung: Although diffuse large B-cell lymphoma (DLBCL) is potentially curable with current therapy, a significant number of DLBCL patients still die of their disease. In a broad-based screen for genes and pathways associated with poor DLBCL outcome, we previously identified a novel risk-related gene, termed BAL (B-aggressive lymphoma). Thereafter, we cloned and characterized a major BAL partner protein, BBAP (B-aggressive lymphoma and BAL binding partner), described the nuclear trafficking patterns of both proteins and demonstrated that BAL functions as a modulator of transcription. In the current study, we characterized BAL expression in normal tonsil and primary DLBCLs and defined BAL regulation and potential functions in both cell types. Immunohistochemical staining of normal tonsil revealed that BAL was expressed by small numbers of germinal center (GC) cells and interfollicular cells with the morphologic features of centroblasts (with the GC) and immunoblasts (within the interfollicular areas). In primary DLBCLs, BAL was expressed by the malignant B cells. To gain insights regarding the regulation of BAL and BBAP expression in DLBCLs, we analyzed a series of 176 primary tumor biopsies transcriptionally profiled an U133A/B Affymetrix microarrays. BAL/BBAP high-expressing DLBCLs had evidence of a brisk host immune response, including increased expression of T/NK-cell receptor and activation pathway components, complement cascade members, macrophage/dendritic cells markers and γIFN-induced transcripts, raising the possibility that BAL was induced by γIFN. Consistent with this hypothesis, γIFN treatment of DLBCL cell lines with low basal levels of BAL markedly increased BAL expression. In silico analysis revealed that the BAL and BBAP genes are located in 3q21 in head-to-head orientation and share the same CpG-related promoter. This shared promoter contains a conserved γIFN-responsive module composed of IRF and STAT binding elements. The BAL/BBAP bidirectional promoter was cloned into a pGL3 luciferase promoterless reporter vector and found to increase luciferase activity 〉 20-fold following γIFN stimulation. To gain further insights into regulation of BAL transcription, mutant versions of BAL promoter were generated and cloned. Luciferase assays demonstrated that the IRF binding site was necessary for γIFN-induced BAL transcription, whereas the STAT1 binding site had an accessory role. Taken together, these results demonstrate that BAL and BBAP genes are transcriptionally activated by γIFN in DLBCLs with features of a brisk host immune response including γIFN signaling. Preliminary studies suggest that BAL limits the efficacy of the observed host inflammatory response.

Beschreibung: Juvenile Myelomonocytic Leukemia (JMML) is a clonal hematopoietic disorder of childhood that is principally characterized by proliferation of cells of the granulocytic and monocytic lineages. Previous studies showed that Ras or Nf1 mutations contribute to ~65% cases of JMML. More recently, mutations in the protein tyrosine phosphatase Shp2 (PTPN11) have been found in ~35% of sporadic JMML, and at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and childhood B-cell acute lymphoblastic leukemia (B-ALL). Interestingly, germline mutations of Shp2 cause the autosomal dominant genetic disorder Noonan Syndrome (NS) and NS patients may have an increased incidence of JMML. Nearly all disease-associated Shp2 mutations affect residues known to control catalytic activity. NS and JMML mutations can involve the same residues, but when they do, the latter are less conservative, suggesting that they may be more activated. We have compared the biochemical and biological effects of NS and JMML mutations. When produced as recombinant proteins in bacteria, nearly all NS mutations studied showed increased PTP activity. However, the JMML mutations E76K and D61Y have the highest activity, resembling the fully active mutant E76A that we defined earlier. Retroviral-mediated expression of E76K or D61Y, but not wild type (WT) Shp2 in murine bone marrow (BM) cells results in cytokine-independent myeloid colony outgrowth, as well as hypersensitivity to both IL-3 and GM-CSF. Notably, NS- associated mutants (e.g., D61G or N308D) also could transform BM cells to factor-independence, but yielded far fewer colonies. Transformation by the leukemia-associated E76K mutant required the phosphatase activity and intact FLVRE motifs in the Shp2 SH2 domains. Gab2, a major Shp2 SH2 domain binding protein, also was required for transformation. When retrovirally transduced BM cells were transplanted into lethally irradiated recipients, E76K and D61Y but not WT Shp2 evoked fatal myeloproliferative disease (MPD) in 30–40% of recipients characterized by splenomegaly, leukocytosis, neutrophilia and monocytosis with occasional anemia and thrombocytopenia. Another ~20% showed less severe MPD at early stage and ultimately succumbed to T-ALL at later times. Histological analysis of mice dying MPD revealed BM and spleen packed with myeloid cells and livers with perivascular myeloid infiltrates. Flow cytometry on BM and spleen confirmed the presence of MPD. Moreover, BM from E76K and D61Y transplanted recipients exhibited enhanced factor-independent colony formation. Furthermore, mast cells derived from E76K and D61Y transplanted mice exhibit increased proliferation and enhanced activation of Erk, Akt and Stat5 in response to IL-3. In contrast, mice with a knock-in mutation of NS (D61G) displayed only a mild MPD. These data support a model in which leukemia-associated mutants of Shp2 are strong hypermorphs that enhance signaling via βc cytokines, whereas NS mutants are less severe gain of function alleles. Our results provide the first evidence that Shp2 mutants have a causal role in leukemogenesis and therefore, Shp2 is the first bona fide PTP proto-oncogene.

Beschreibung: The design and evaluation of therapies for the sickle cell and β-thalassemia syndromes rely on our understanding of hemoglobin accumulation during human erythropoiesis. Here we report direct measurements of hemoglobin composition and messenger RNA (mRNA) levels in cultured CD34+ cells and correlate those measurements with studies of freshly obtained bone marrow samples. Hemoglobin levels in differentiating cells were also compared with morphologic, immunophenotypic, and cell cycle assessments. A population of large preproerythroblasts was first identified within 24 hours and became the dominant population by day 5. The transition from proerythroblast to basophilic normoblast occurred later, from days 7 to 9, and correlated with a peak of 74.1% ± 3.9% of the cells in the S phase of cell cycle. Orthochromatic normoblasts were the dominant and final cell type by day 13. High-performance liquid chromatography–based quantitation of fetal (HbF) and adult (HbA) hemoglobin and real-time polymerase chain reaction globin mRNA quantitation demonstrated a coordinate rise in the accumulation of both proteins and mRNA among these developmentally staged populations. Quantitative analyses on freshly sorted bone marrow populations demonstrated a similar rising pattern with β-globin and HbA as the dominant species at both early and late stages of differentiation. We found no evidence for HbF dominant populations or switching during differentiation in adult cells. Instead, rapid increases in both HbF (heterocellular) and HbA (pancellular) content were observed, which coincided with the apex in cell cycling and the proerythroblast-basophilic normoblast transition. Based on these measurements, we conclude that HbF and HbA content are regulated with the rate of proliferation during adult erythropoiesis.

Beschreibung: PU.1 is a member of the Ets family of transcription factors and plays an essential role in the development of both myeloid and lymphoid cells. To examine zebrafish pu.1 (zpu.1) expression in subpopulations of blood cells during zebrafish development, we linked a 9-kb zebrafish genomic fragment upstream of the zpu.1 initiator codon to green fluorescent protein (GFP) and microinjected this construct to generate stable transgenic lines. GFP-positive fluorescent myeloid precursors were observed migrating from the anterolateral mesoderm in living embryos from 16 to 28 hours after fertilization (hpf) in a pattern that overlaps the expression pattern of endogenous zpu.1 mRNA. Analysis of larval histologic sections revealed GFP-expressing hematopoietic cells in the developing zebrafish kidney. Flow cytometric analysis of cells from adult whole kidney marrow revealed 2 discrete subpopulations of GFP-positive cells, which after cell sorting exhibited either myeloid or early lymphoid morphology. Thus, the zebrafish zpu.1 promoter fragment used here is capable of driving reporter gene expression in subsets of embryonic and adult hematopoietic cells. These transgenic lines will be useful to dissect the cellular and molecular control of myeloid cell differentiation, and this promoter fragment may prove useful in the development of zebrafish models of acute myeloid leukemia.

Beschreibung: The highly variable levels of fetal hemoglobin (HbF) reactivation among patients with sickle cell disease is not well understood. We previously determined that pancellular reversal of gamma-globin gene silencing is achievable through the activation of specific signal transduction pathways using stem cell factor (SCF) and transforming growth factor beta (TGF-B). Based upon the reliability of this culture assay for HbF reactivation, the culture system is additionally being utilized to screen for inhibitors of HbF reactivation. As an initial screening strategy, published reports, high-throughput sequencing efforts, and array-based transcription profiles were examined to identify erythroblast-expressed receptors with known ligands that may serve a signaling role in the regulation of globin expression. A dose-escalation strategy was used to screen seventeen ligands that bind to a subset of G-protein coupled receptors (GPCR). Among those ligands, oleoyl-lysophosphatidic acid (OLPA) and neurokinin A (NKA) were identified as potential inhibitors of HbF reactivation. Lysophosphatidic acid is a lipid metabolite that is released after tissue injury and may play a role in vascular remodeling. Neurokinin A is a small neuropeptide mainly involved in pain signaling. OLPA inhibited HbF reactivation in a dose-dependent manner (HbF/HbF+HbA ratios: EPO: 0.7±0.01%; EPO+SCF+TGF-B (EST): 36.3±0.1%; EST+10nM OLPA: 36.5±0.1%; EST+100nM OLPA: 31.6±0.1%; EST+1uM OLPA: 12.7±0.1%, p=1.9E-03). NKA also inhibited HbF reactivation, but the magnitude of the NKA effect was donor-specific. Cells from one donor demonstrated a robust response (HbF/HbF+HbA ratios: EPO: 1.7%; EST: 26.7%; EST+10nM NKA: 24.5%; EST+100nM NKA: 8.8%; EST+1uM NKA: 7.8%). Since OLPA and NKA may both act through adenylate cyclase, the role of this enzyme was examined further using the activator, forskolin (0.8 uM-20uM) and inhibitor, SQ22536 (40uM – 600uM). While we were unable to demonstrate a significant increase in HbF among cultures grown in forskolin, the addition of SQ22536 resulted in a profound, dose-dependent inhibitory effect on the expression of HbF (EPO: 1.3±1.2%; EST: 36.7±11.3%; EST+40uM SQ: 30.3±10.2%; EST+200 uM SQ : 14.5±4.5%, p=1.9E-02; EST+600uM SQ: 2.3±2.7%, p=4.1E-03). Flow cytometry revealed that the inhibitory effects of SQ22536 on HbF were pancellular. Importantly, inhibition of the main downstream target of adenylate cyclase (protein kinase A) with H89 (10nM-1.0 uM) did not result in a significant reduction in HbF. However, phosphorylation studies demonstrated that SQ22536 altered the kinetics of MEK activation. MEK is a kinase that helps mediate the HbF-activating effect of SCF [Blood 2004, 1;103(5):1929–33]. These data suggest that inhibitory effects upon HbF reactivation in adult erythroblasts can be mediated by endogenous signaling molecules. Based upon these results, we propose that patient-specific responses to tissue injury and pain may play a negative role in HbF reactivation in sickle-cell disease.

Beschreibung: Adenosine signal transduction in human cells is achieved through four distinct G-protein-coupled receptors named A1, A2A, A2B and A3. Adenosine signaling events affect several physiological processes including growth regulation and cellular responses to hypoxia. Despite extensive studies of native and pharmacologically-targeted adenosine signal transduction, the potential for adenosine to regulate erythropoiesis remains largely unexplored. Here we systematically studied adenosine receptor expression, activation, and inhibition among CD34+ human erythroid progenitor cells cultured in the presence of erythropoietin. This 14 day culture system has been studied extensively in the context of erythroblast commitment and terminal differentiation (BLOOD 99(8):3005–13). Using real-time RT-PCR, we demonstrated that the A2A, A2B and A3 adenosine receptors are expressed in highly regulated patterns. The expression of A2A and A2B were highest early in the culture period and later declined as the cells underwent terminal differentiation. Receptor A3 gene expression gradually increased during the terminal differentiation. Expression of the A1 receptor gene was not detected above background levels at any stage of differentiation (compared with the positive control). Based upon those receptor expression patterns, several adenosine receptor signaling agonists and antagonists were screened for possible effects on erythroblast growth and differentiation. Among those screened molecules, the selective A3 adenosine receptor agonist, CI-IB-MECA, had effects on the proliferation and differentiation of the cells without overt toxicity in the dose range of 10–100uM. CI-IB-MECA significantly inhibited the proliferation of the cells with resulting cell counts being only 10% of those in matched controls after 14 days. Cell cycle analyses demonstrated that the growth inhibitory effects of CI-IB-MECA were largely due to a significant reduction in the percentage of S-phase proerythroblasts (S-phase: 16 ± 5% in CI-IB-MECA versus 51 ± 2% in matched controls; p80%) in the matched controls. Consistent with the morphological examination, HPLC analyses revealed barely detectable levels of hemoglobin in the presence of CI-IB-MECA after 14 days. These finding suggest that selective activation of adenosine A3 receptors permits erythroid commitment, but profoundly inhibits the proliferation of those committed cells and retards their terminal differentiation. As a novel regulator of erythropoiesis, adenosine A3 receptor signaling should be explored in patients with growth-related erythroid diseases.

Beschreibung: Peripheral destruction of sickled erythrocytes is recognized as the central pathologic mechanism of SCD. Less well established is the contribution of ineffective erythropoiesis to the pathophysiology of SCD. Patients with SCD frequently develop mixed hematopoietic chimerism after nonmyeloablative stem cell transplantation (NMA-SCT). This provides an opportunity to directly compare the differentiation and survival of normal donor and SCD erythropoiesis in vivo. To this end, total genomic DNA (gDNA) and erythroid-lineage specific chimerism in peripheral blood (PB) and marrow (BM) were analyzed in 5 patients (3 with SCD, 2 with non-SCD) following NMA-SCT with HLA-matched donors. RBC chimerism was measured by beta-globin RNA pyrosequencing, which directly and quantitatively sequences coding region polymorphisms in beta-globin RNA that distinguish recipient and donor-derived erythroid precursors. The clinical characteristics of the 5 patients are shown in the table below. All patients developed mixed gDNA chimerism, ranging from 18–50%, between days 60–180, with no differences between PB and BM levels. BM erythroid precursor chimerism, determined by Y chromosome FISH and ABO staining in 4 of 5 patients, were identical to total BM gDNA chimerism. PB from all 3 SCD patients had a ~2 fold greater level of donor-derived beta-globin RNA compared with total gDNA. These levels were similar for the 2 non-SCD patients. The presence of ineffective SS erythropoiesis was directly revealed by analysis of BM, which similarly revealed higher expression of donor-derived beta-globin RNA relative to the degree of donor erythroid progenitor engraftment in Pts 1–3. These findings were not present in Pts 4–5. Chimerism was determined in purified marrow populations of immature (pro-and basophilic –– glycophorin [GYPA+, CD71+ hi]) versus mature (polychromatophilic, orthochromatophilic [GYPA+, CD71+dim] ) erythroblasts to determine the stage of maturation at which SS erythroblasts are lost. While non-SCD patients demonstrated similar rates of donor and host erythropoiesis, all 3 SCD patients demonstrated progressive intramedullary loss of SS erythroblasts with maturation, with enrichment of donor erythroid precursor chimerism appearing from the earliest stages of hemoglobinization. The presence of ineffective erythropoiesis in SCD explains the maturation advantage of AA or SA donor erythroid precursor cells over SS cells that allows for greater donor contribution to overall erythropoiesis following SCT. This is the first definitive in vivo demonstration of ineffective erythropoiesis in SCD, and supports the notion that NMA-SCT can be curative for SCD in the setting of stable donor engraftment. Further studies examining the underlying mechanism of ineffective erythropoiesis in SCD are under investigation. Patient clinical characteristics Pt 1 Pt 2 Pt 3 Pt 4 Pt 5 Pt age/sex 34/F 52/M 5/M 45/M 45/F Pt dx SCD SCD+ myeloma SCD CLL HD Dnr sickle genotype/sex SA/M SA/M SA/M ––/F ––/M stem cell source PBSC PBSC BM PBSC CB % donor at d60–180     PB gDNA 25 45 50 54 10     BM gDNA 26 52 50 49 20     Cells: erythroid progenitors 23 49 –– 50 22     PB beta-globin RNA 66 100 100 50 18     BM beta-globin RNA 55 100 100 54 15

Beschreibung: Transcriptional profiles of cultured primary human erythroid cells were examined to identify those genes involved in the control of erythroid growth during the terminal phase of maturation. Our in silico screening strategy indicated that a hypoxia-inducible proapoptotic member of the Bcl-2 gene family called Nix is expressed during erythropoiesis. We next performed Northern blot analyses and determined that the 1.4-kb Nix transcript is expressed at lower levels in erythroleukemia cells than reticulocytes. Polymerase chain reaction (PCR)–based transcriptional patterning confirmed the increased expression of Nix during human erythropoiesis with a pattern similar to that of Bcl-xL and glycophorin A and opposite that of Bcl-2. Western blot analyses revealed Nix protein levels that were lower than expected due to increased proteosomal degradation. The expression of Nix and Bcl-xL proteins decreased relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control on the removal of erythropoietin (EPO) from the culture medium. Immunocytochemical analyses demonstrated a similar perinuclear mitochondrial expression pattern for both proteins in hemoglobinized precursors. On the basis of these data, we propose that the proapoptotic factor Nix is a highly regulated effector of growth during terminal erythroid maturation.

Beschreibung: Murine dendritic cells (DCs) can be classified into at least 2 subsets, “myeloid-related” (CD11bbright, CD8α−) and “lymphoid-related” (CD11bdull, CD8α+), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)–dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c+ population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro–derived DCs, when treated with interferon-α or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8α, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti–interleukin-6 (IL-6) antibody, but not anti–GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.

Beschreibung: Human stem cell leukemia-lymphoma syndrome usually presents as a myeloproliferative disease (MPD) that evolves to acute myeloid leukemia and/or lymphoma. The syndrome associated with t(8;13)(p11;q12) results in expression of the ZNF198-FGFR1 fusion tyrosine kinase. Current empirically-derived cytotoxic chemotherapy is inadequate treatment of this disease. We hypothesized that small molecule inhibitors of the ZNF198-FGFR1 fusion would have therapeutic efficacy. We characterized the transforming activity of ZNF198-FGFR1 in hematopoietic cells in vitro and in vivo. Expression of ZNF198-FGFR1 in primary murine hematopoietic cells caused a myeloproliferative syndrome in mice that recapitulated the human MPD phenotype. Transformation in these assays, and activation of the downstream effector molecules PLCγ, STAT5 and PI3K/AKT, required the proline-rich, but not the zinc-finger domains of ZNF198. A small molecule tyrosine kinase inhibitor, PKC412 (N-benzoyl-staurosporine) effectively inhibited ZNF198-FGFR1 tyrosine kinase activity and activation of downstream effector pathways, and inhibited proliferation of ZNF198-FGFR1 transformed Ba/F3 cells. Furthermore, treatment with PKC412 resulted in statistically significant prolongation of survival in the murine model of ZNF198-FGFR1 induced myeloproliferative disease. Based in part on these data, PKC412 was administered to a patient with t(8;13)(p11;q12) and was efficacious in treatment of progressive myeloproliferative disease with organomegaly. Therefore, PKC412 may be a useful therapy for treatment of human stem cell leukemia-lymphoma syndrome.