Factor-dependent Cell Line Research Articles

Effective Suicide Gene Therapy for Leukemia in a Model of Insertional Oncogenesis in Mice

The safety of gene therapy using hematopoietic stem cells may be increased by including a suicide gene in the therapeutic vector to eliminate adverse events like insertional oncogenesis while retaining the clinical benefits. We have developed a model of experimental insertional oncogenesis by transducing the murine factor-dependent leukemia cell line Ba/F3 with a bicistronic Moloney murine leukemia virus retroviral vector encoding a murine oncogene (cKit(D814V)) in addition to one of three suicide genes: Herpes simplex virus thymidine kinase (HSV-TK); SR39, an HSV-TK mutant with an increased affinity for the drug substrate Ganciclovir (GCV); or sc39, a splice-corrected version of SR39. Following intravenous challenge with transduced Ba/F3 clones and treatment with GCV, leukemia developed in mice given cells expressing HSV-TK, but not SR39 or sc39. In vitro GCV resistance was observed in heterogeneously transduced Ba/F3 pools at 2.5-14%, and single-nucleotide changes or partial loss of the suicide gene were identified as mechanisms of drug escape. However, GCV treatment resulted in 80-100% survival of mice challenged even with pools of partially resistant Ba/F3 cells expressing SR39 or sc39. Thus, in this model of vector-driven insertional oncogenesis, a suicide gene approach was effective for eliminating leukemia using modified HSV-TK variants with improved biological activity.

Mitotic Arrest Deficiency 2 (Mad2) Is Required for Proliferative Synergy of GM-CSF Plus SCF in Hematopoietic Progenitors Via Effects on Apoptosis and Is Mediated by Ligand-Dependent Association of Mad2 with SCF and GM-CSF Receptors.

Mitotic arrest deficiency 2 (Mad2) is a component of the mitotic spindle checkpoint complex and is essential for accurate chromosome segregation. We previously reported that Mad2 is involved in synergistic proliferation of hematopoietic progenitor cells in response to SCF combined with GM-CSF, but the underlying mechanism remains unclear. Recently, it was reported that Mad2 physically associates with common β chain (βc) of GM-CSF receptor. Because c-Kit constitutively binds to βc, we hypothesized that Mad2 protein physically associates with c-Kit receptor. We examined if Mad2 interacts with c-Kit using human growth factor-dependent cell line, MO7e, which expresses both c-Kit and GM-CSF receptors. Immunoprecipitation assays demonstrated interaction of c-Kit with Mad2. We then examined whether the interaction of Mad2 with c-Kit is cytokine dependent. Mad2 dissociates from c-Kit after stimulation with SCF plus GM-CSF, but not with SCF or GM-CSF alone. Additionally, because bone marrow hematopoietic progenitor cells from Mad2+/− mice lack synergy in response to SCF plus GM-CSF, it is possible that interaction of Mad2 with c-Kit mediates their synergistic proliferative effects. To address this, we examined intracellular cytokine signaling and apoptosis in MO7e cells depleted of Mad2 by RNA interference. We observed no difference between control and Mad2-depleted cells in phosphorylation of Erk1/2 at Thr202/Tyr204 and Akt at Ser473 after synergistic stimulation with SCF plus GM-CSF. In contrast, the percent of apoptosis of Mad2-depleted cells was significantly higher than that of control (26.2±0.7% vs. 20.3±0.4%, P<0.01). This was compatible with the results of apoptosis assays using c-Kit+Lineage− bone marrow cells from Mad2+/− and wild type mice. These results suggest that Mad2 may be involved in synergistic proliferation of hematopoietic progenitor cells via regulation of apoptosis rather than early cytokine signaling. These effects are likely mediated through Mad2 interaction with c-Kit and the beta chain of the GM-CSF receptor.

Tribbles homolog 2 inactivates C/EBPα and causes acute myelogenous leukemia

Tribbles homolog 2 (Trib2) was identified as a downregulated transcript in leukemic cells undergoing growth arrest. To investigate the effects of Trib2 in hematopoietic progenitors, mice were reconstituted with hematopoietic stem cells retrovirally expressing Trib2. Trib2-transduced bone marrow cells exhibited a growth advantage ex vivo and readily established factor-dependent cell lines. In vivo, Trib2-reconstituted mice uniformly developed fatal transplantable acute myelogenous leukemia (AML). In mechanistic studies, we found that Trib2 associated with and inhibited C/EBPalpha. Furthermore, Trib2 expression was elevated in a subset of human AML patient samples. Together, our data identify Trib2 as an oncogene that induces AML through a mechanism involving inactivation of C/EBPalpha.

Gangliosides of the stroma layer participate in the interferon-gamma receptor-dependent controls of myelopoiesis

Stroma-mediated myelopoiesis depends upon growth-factors and an appropriate intercellular microenvironment, whose polarity is relevant for granulocyte-macrophage colony stimulating factor (GM-CSF) mediated myeloid cell proliferation. Here we have studied qualitative and quantitative aspects of ganglioside participation in controls of the microenvironment required to sustain myelopoiesis. We analysed ganglioside synthesis, expression and shedding by two primary liver stromal cell cultures isolated from wild type and interferon-gamma (IFNγ) receptor knockout mice. The latter one has a higher capacity to sustain myelopoiesis. FDC-P1 myeloid growth factor-dependent cell line was used as the reporter system, monitoring the cell survival and proliferation that reflect the bio-availability and the activity of GM-CSF. Although the two stromal cells synthesised the same gangliosides their relative content was quite different. FDC-P1 proliferation decreased in cultures in which ganglioside synthesis was inhibited in the stroma, as well as in presence of stroma cell supernatants in which GM3 was neutralised by the anti-GM3 monoclonal antibody. Addition of exogenous GM3 reverted the inhibition and sustained proliferation of FDC-P1 cells. FDC-P1 cells do not accumulate GM3, but they are able to take up the stroma-produced sphingolipids. Thus, stroma has a double role in sustaining myelopoiesis, providing both growth factor(s) and ganglioside(s) required for the optimal stimulation of the myeloid cell proliferation, and the IFNγ mediated stroma-dependent controls of myelopoiesis are determinant for this cell interaction.

A CSF‐1 receptor kinase inhibitor targets effector functions and inhibits pro‐inflammatory cytokine production from murine macrophage populations

CSF-1 regulates macrophage differentiation, survival, and function, and is an attractive therapeutic target for chronic inflammation and malignant diseases. Here we describe the effects of a potent and selective inhibitor of CSF-1R-CYC10268-on CSF-1R-dependent signaling. In in vitro kinase assays, CYC10268 was active in the low nanomolar range and showed selectivity over other kinases such as Abl and Kit. CYC10268 blocked survival mediated by CSF-1R in primary murine bone marrow-derived macrophages (BMM) and in the factor-dependent cell line Ba/F3, in which the CSF-1R was ectopically expressed. CYC10268 also inhibited CSF-1 regulated signaling (Akt, ERK-1/2), gene expression (urokinase plasminogen activator, toll-like receptor 9, and apolipoprotein E), and priming of LPS-inducible cytokine production in BMM. In thioglycollate-elicited peritoneal macrophages (TEPM), which survive in the absence of exogenous CSF-1, CYC10268 impaired LPS-induced cytokine production and regulated expression of known CSF-1 target genes. These observations support the conclusion that TEPM are CSF-1 autocrine and that CSF-1 plays a central role in macrophage effector functions during inflammation. CSF-1R inhibitors such as CYC10268 provide a powerful tool to dissect the role of the CSF-1/CSF-1R signaling system in a range of biological systems and have potential for a number of therapeutic applications.

MN1-TEL, the product of the t(12;22) in human myeloid leukemia, immortalizes murine myeloid cells and causes myeloid malignancy in mice

MN1-TEL is the product of the recurrent t(12;22)(p12;q11) associated with human myeloid malignancies. MN1-TEL functions as an activated transcription factor, exhibiting weak transforming activity in NIH3T3 fibroblasts that depends on the presence of a functional TEL DNA-binding domain, the N-terminal transactivating sequences of MN1 and C-terminal sequences of MN1. We determined the transforming activity of MN1-TEL in mouse bone marrow (BM) by using retroviral transfer. MN1-TEL-transduced BM showed increased self-renewal capacity of primitive progenitors in vitro, and prolonged in vitro culture of MN1-TEL-expressing BM produced immortalized myeloid, interleukin (IL)-3/stem cell factor-dependent cell lines with a primitive morphology. Transplantation of such cell lines into lethally irradiated mice rescued them from irradiation-induced death and resulted in the contribution of MN1-TEL-expressing cells to all hematopoietic lineages, underscoring the primitive nature of these cells and their capacity to differentiate in vivo. Three months after transplantation, all mice succumbed to promonocytic leukemia. Transplantation of freshly MN1-TEL-transduced BM into lethally irradiated mice also caused acute myeloid leukemia within 3 months of transplantation. We infer that MN1-TEL is a hematopoietic oncogene that stimulates the growth of hematopoietic cells, but depends on secondary mutations to cause leukemia in mice.

The JAK2V617F tyrosine kinase mutation in myeloproliferative disorders: Summary of published literature and a perspective

A common somatic point mutation has recently been identified in the Janus kinase 2 (JAK2) gene in virtually all cases of polycythemia vera and in a majority of patients with essential thrombocythemia and idiopathic myelofibrosis. This common mutation in the pseudokinase autoinhibitory domain of the enzyme results in constitutive tyrosine kinase activation, which in turn leads to cytokine hypersensitivity and factor independence in factor-dependent cell lines, and causes polycythemia in mice. This discovery has led to greater understanding of the molecular pathogenesis of the chronic myeloproliferative disorders, which may translate into targeted therapy.

Role of the monomeric GTPase Rho in hematopoietic progenitor cell migration and transplantation

To investigate the role of the monomeric guanosine triphosphatase (GTPase) Rho on migration of hematopoietic progenitor cells (HPC), we employed different clostridial toxins which inhibit the Rho family of GTPases. Pretreatment with C2I-C3, a cell-accessible C3 transferase fusion protein that targets Rho, increased chemokinetic migration of the factor-dependent multipotent cell line Factor Dependent Cell Paterson with mixed lineage differentiation potential (FDCP-mix) and of primary lineage marker-depleted HPC in vitro. In contrast, treatment with lethal toxin (LT) from Clostridium sordellii, which predominantly inactivates Rac, and with toxin B from C. difficile, which inactivates Rho, Rac and Cdc42, decreased in vitro migration. When HPC pretreated with LT or toxin B were transplanted into mice, homing to the bone marrow was impaired, whereas C2I-C3 treatment did not alter HPC homing. However, in a competitive hematopoietic repopulation experiment in C57BL/6 mice, pretreatment of bone marrow cells with any of the inhibitors, including the Rho inhibitor C2I-C3, resulted in suppressed donor-type hematopoiesis. Our data indicate that whereas Rac supports HPC cell cycling, migration, short-term homing and hematopoietic regeneration, Rho coordinates down-regulation of HPC migration and is required for hematopoietic regeneration.

Distinct Characteristics of t(8;21) Acute Myeloid Leukemia with Active KIT Mutation.

The t(8;21) translocation, which generates a chimeric gene, AML1/ETO, is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML) and is associated with a good clinical outcome. However, the clinical features of t(8;21)AML with additional genetic aberrations have not been well elucidated. In this study, we screened for KIT mutations in 44 patients with t(8;21)AML. While 2 cases had a silent type mutation at codon 546 in the juxtamembrane domain, Lyn546Lyn, 8 cases (17.8%) had Asp816 mutations in the tyrosine kinase domain, Asp>Asn in 2 cases, Asp>Tyr in 4 cases, Asp>Val in 1 case and Asp>His in 1 case. Patients with Asp816 mutations showed leukocytosis (P = 0.001) and poor overall survival (P < 0.0001), when compared to those without Asp816 mutations. When the Asp816 mutation was transfected into a stem cell factor-dependent cell line, Mo7e, the cells grew rapidly and displayed stem cell factor-independent proliferation. These results suggest that t(8;21)AML with Asp816 mutations in KIT may be a distinctive subtype of t(8;21)AML and that this genetic aberration has an adverse effect on the clinical outcome for t(8;21)AML.

TEL-AML1 Transgenic Zebrafish Model of Acute Lymphoblastic Leukemia (ALL).

TEL-AML1 (ETV6-RUNX1) fusion is the product of the t(12;21) chromosomal translocation, the most common chromosomal rearrangement in childhood cancer. The translocation fuses two highly conserved transcription factors, TEL and AML1 that have essential roles in hematopoiesis. Genetic studies of identical twins with concordant leukemia, the detection of leukemia-specific fusion genes in neonatal blood spots, and the existence of multiple leukemic subclones at ALL diagnosis point to prenatal origin of the fusion and long latency before leukemia development. Additionally, a study in growth factor-dependent cell lines and transgenic mice, and several mouse transplant models suggest that TEL-AML1 is insufficient by itself for leukemic transformation. To determine whether TEL-AML1 has selective transforming impact on a particular stem cell lineage, we established stable zebrafish transgenic lines expressing the TEL-AML1 fusion protein both constitutively, and within the lymphoid progenitors. In these lines, either the zebrafish β-actin (ZBA) promoter or the xenopus elongation factor-1 (XEF1) promoter drives TEL-AML1 expression constitutively, while the zebrafish RAG-2 promoter (RAG2) selectively drives TEL-AML1 expression in committed lymphocyte precursors, but not in earlier multilineage hematopoietic precursors. TEL-AML1 expression, alone or fused to EGFP, was detected at 24-hour post fertilization (hpf) with fluorescent microscopy or RT-PCR and confirmed with western blot analysis. In-Situ Hybridization and confocal microscopy revealed that transgenic zebrafish maintained TEL-AML1 expression throughout adulthood. The expression of TEL-AML1 was associated with accumulation of immature hematopoietic progenitor cells, mostly in the kidney and spleen of several transgenic zebrafish lines, within a few weeks of development, indicating an expansion of the progenitor cell population. To date, a small number (5 out of 391 transgenic fish; 1.3%) of founders and progeny of zebrafish transgenic for ZBA- or XEF1-TEL-AML1 fusion developed an infiltrating lymphoid neoplasm, most likely acute lymphoblastic leukemia with a latency of 8–12 month. The lymphoblasts were negative for myeloperoxidase and PAS staining, with abundant expression of both EGFP in lymphoblasts from EGFP-TEL-AML1 line, and pre-B-cell leukemia transcription factor-1 (PBX1; also called cALLa) indicating that Leukemias originated in a TEL-AML1 expressing cell(s), and mimic the human CD10-positive precursor-B cell ALL. None of 350 wild-type, 125 control EGFP fish or 353 RAG2-TEL-AML1 transgenic zebrafish had any leukemia or hematopoietic changes. Therefore, the expression of TEL-AML1 fusion in our ZBA- and XEF1-TEL-AML1 transgenic lines generates a premalignant state, which appears to require additional genetic events for acquisition of the leukemic phenotype. The fact that none of the RAG2 zebrafish expressing TEL-AML1 in committed lymphoid progenitors developed leukemia or progenitor expansion indicates that the leukemic stem cell associated with TEL-AML1 leukemia is an immature progenitor earlier than the committed lymphoid progenitors where RAG2 expression is detected. The TEL-AML1 transgenic zebrafish lines provide a basis for large scale and targeted mutagenesis screens aimed at identifying mutations and cooperating events that are required for TEL-AML1 induced leukemias.

Sequence Motifs in IL-4Rα Mediating Cell-Cycle Progression of Primary Lymphocytes

IL-4 signaling through the IL-4Ralpha chain regulates the development and proliferation of the Th2 lineage of effector CD4(+) T cells. Analyses of the IL-4R in factor-dependent cell lines led to the development of two apparently conflicting models of the primary structural determinants of IL-4R-mediated proliferative signaling. In one model, proliferation was dependent on the first conserved tyrosine in the cytoplasmic tail (Y1), while in the second, proliferation was independent of cytoplasmic tyrosines. We found that in activated primary T cells, mutation of only the Y1 residue resulted in a modest decrease in IL-4-induced S phase entry, a further decrease in cell-cycle completion, and a complete failure of IL-4 to induce p70S6 kinase phosphorylation. Consistent with a role for the PI3K/mammalian target of rapamycin pathway in mediating cytokine acceleration of G(2)/M transit, pretreatment of activated T cells with rapamycin resulted in only a modest decrease in IL-4-induced S phase entry, but a total block of cell-cycle completion. Strikingly, IL-4Ralpha chains that lacked all cytoplasmic tyrosines were competent to signal for STAT5 phosphorylation, mediated efficient S phase entry, and promoted cell-cycle progression. The ability of tyrosine-deficient IL-4Rs to mediate proliferative signaling and STAT phosphorylation was absolutely dependent on the presence of an intact ID-1 region. These findings show that IL-4Ralpha lacking cytoplasmic tyrosine residues is competent to induce ID-1-dependent proliferation, and indicate that IL-4 can promote G(2)/M progression via activation of the mammalian target of rapamycin pathway initiated at the Y1 residue.

The polycomb group protein enhancer of zeste homolog 2 (EZH2) is an oncogene that influences myeloma cell growth and the mutant ras phenotype

Three distinct proliferative signals for multiple myeloma (MM) cell lines induce enhancer of zeste homolog 2 (ezh 2) transcript expression. EZH 2 is a polycomb group protein that mediates repression of gene transcription at the chromatin level through its methyltransferase activity. Normal bone marrow plasma cells do not express ezh2; however, gene expression is induced and correlates with tumor burden during progression of this disease. We therefore investigated how EZH 2 expression is deregulated in MM cell lines and determined the consequence of this activity on proliferation and transformation. We found that EZH 2 protein expression is induced by interleukin 6 (IL-6) in growth factor-dependent cell lines and is constitutive in IL-6-independent cell lines. Furthermore, EZH 2 expression correlates with proliferation and B-cell terminal differentiation. Significantly, EZH 2 protein inhibition by short interference RNA treatment results in MM cell growth arrest. Conversely, EZH 2 ectopic overexpression induces growth factor independence. We found that the growth factor-independent proliferative phenotype in MM cell lines harboring a mutant N- or K-ras gene requires EZH 2 activity. Finally, this is the first report to demonstrate that EZH 2 has oncogenic activity in vivo, and that cell transformation and tumor formation require histone methyltransferase activity.

Role of c-KIT expression level and phosphatidylinositol 3-kinase activation in survival and proliferative responses of early myeloid cells

The receptor tyrosine kinase c-KIT and its ligand Stem Cell Factor (SCF) are critical in haemopoiesis but pathways linking receptor activation to specific responses in progenitor cells are still unclear. We have investigated the role of c-KIT expression level and the phosphatidylinositol 3-kinase (PI3-K) pathway in survival and cell division of early myeloid cells in response to SCF. Two factor-dependent murine early myeloid cell lines, FDC-P1 and Myb-immortalised haemopoietic cells (MIHC), were transduced to express wild-type c-KIT or a mutant form of the receptor (Y721F) that lacks the major recruitment site for the p85 regulatory subunit of PI3-K. Several clones expressing different receptor levels were analysed in each case. Growth of cells expressing either the wild-type or Y721F mutant KIT was strongly dependent on receptor level within the physiological range. Using an assay that allows quantitative measurement of the contributions of cell survival and cell division, diminished cell growth in response to SCF under limiting conditions of receptor copy number or PI3-K recruitment was shown to be almost entirely due to decreased cell survival. Further studies with the PI3-K inhibitor LY294002 indicated that PI3-K activation was also required for cell division. Alternate binding and/or indirect activation of PI3-K could support cell division mediated by Y721F mutant KIT, but was insufficient for the survival response.

A microscale assay for the identification of TGF-β antagonists based on functional coupling of the heterodimeric TGF-β receptor to STAT6-driven promoter activation

Inadequate function of Transforming Growth Factor- (TGF-) β is involved in numerous disease states including immune disorders and cancer, rendering this polypeptide and its receptor an attractive target for pharmaceutical interference. We have developed a novel microscale functional test system suited for the investigation of ligand-induced receptor activation and the automatable evaluation of potential agonists and antagonists. Hybrid receptors were constructed from the cytoplasmic domain of the human interleukin-4 (IL-4) receptor α-chain and extracellular domains of a TGF-β type I and type II receptor, respectively. These chimeras were stably introduced into the factor-dependent murine cell line Ba/F3 along with an IL-4-inducible luciferase reporter gene construct, yielding a reporter cell line which responds to productive ligand?receptor interactions by specific luciferase activity in a dose-dependent fashion. A model experiment employing inhibitory peptides demonstrates that the devised reporter cell provides a rational readout for TGF-β activity on target cells and its impairment by specific antagonists.

Role for the Nuclear Factor κB Pathway in Transforming Growth Factor-β1 Production in Idiopathic Myelofibrosis: Possible Relationship with FK506 Binding Protein 51 Overexpression

The release of transforming growth factor-beta1 (TGF-beta1) in the bone marrow microenvironment is one of the main mechanisms leading to myelofibrosis in murine models and probably in the human idiopathic myelofibrosis (IMF). The regulation of TGF-beta1 synthesis is poorly known but seems regulated by nuclear factor kappaB (NF-kappaB). We previously described the overexpression of an immunophilin, FK506 binding protein 51 (FKBP51), in IMF megakaryocytes. Gel shift and gene assays show that FKBP51's overexpression in a factor-dependent hematopoietic cell line, induces a sustained NF-kappaB activation after cytokine deprivation. This activation correlates with a low level of IkappaBalpha. A spontaneous activation of NF-kappaB was also detected in proliferating megakaryocytes and in circulating CD34(+) patient cells. In normal cells, NF-kappaB activation was only detected after cytokine treatment. The expression of an NF-kappaB superrepressor in FKBP51 overexpressing cells and in derived megakaryocytes from CD34(+) of IMF patients revealed that NF-kappaB activation was not involved in the resistance to apoptosis after cytokine deprivation of these cells but in TGF-beta1 secretion. These results highlight the importance of NF-kappaB's activation in the fibrosis development of this disease. They also suggest that FKBP51's overexpression in IMF cells could play an important role in the pathogenesis of this myeloproliferative disorder.

FLT3/ITD Mutation Signaling Includes Suppression of SHP-1

Mutations in the FLT3 gene are the most common genetic alteration found in AML patients. FLT3 internal tandem duplication (ITD) mutations result in constitutive activation of FLT3 tyrosine kinase activity. The consequences of this activation are an increase in total phosphotyrosine content, persistent downstream signaling, and ultimately transformation of hematopoietic cells to factor-independent growth. The Src homology (SH)2 domain-containing protein-tyrosine phosphatase (SHP)-1 is involved in the down-regulation of a broad range of growth factor and cytokine-driven signaling cascades. Loss-of-function or deficiency of SHP-1 activity results in a hyperproliferative response of myelomonocytic cell populations to growth factor stimulation. In this study, we examined the possible role of SHP-1 in regulating FLT3 signaling. We found that transformation of TF-1 cells with FLT3/ITD mutations suppressed the activity of SHP-1 by approximately 3-fold. Suppression was caused by decreased SHP-1 protein expression, as analyzed at both the protein and RNA levels. In contrast, protein levels of SHP-2, a phosphatase that plays a stimulatory role in signaling through a variety of receptors, did not change significantly in FLT3 mutant cells. Suppressed SHP-1 protein levels in TF-1/ITD cells were partially overcome after cells were exposed to CEP-701, a selective FLT3 inhibitor. SHP-1 protein levels also increased in naturally occurring FLT3/ITD expressing AML cell lines and in primary FLT3/ITD AML samples after CEP-701 treatment. Furthermore, a small but reproducible growth/survival advantage was observed in both TF-1 and TF-1/ITD cells when SHP-1 expression was knocked down by RNAi. Taken together, these data provide the first evidence that suppression of SHP-1 by FLT3/ITD signaling may be another mechanism contributing to the transformation by FLT3/ITD mutations.

A Unique Activating Mutation in JAK2 (V617F) Is at the Origin of Polycythemia Vera and Allows a New Classification of Myeloproliferative Diseases

Myeloproliferative disorders (MPDs) are heterogeneous diseases that occur at the level of a multipotent hematopoietic stem cell. They are characterized by increased blood cell production related to cytokine hypersensitivity and virtually normal cell maturation. The molecular pathogenesis of the MPDs has been poorly understood, except for chronic myeloid leukemia (CML), where the Bcr-Abl fusion protein exhibits constitutive kinase activity. Since some rare MPDs are also related to a dysregulated kinase activity, a similar mechanism was thought to be likely responsible for the more frequent MPDs. We investigated the mechanisms of endogenous erythroid colony formation (EEC) by polycythemia vera (PV) erythroid progenitor cells and found that EEC formation was abolished by a pharmacological inhibitor of JAK2 as well as an siRNA against JAK2. JAK2 sequencing revealed a unique mutation in the JH2 domain leading to a V617F substitution in more than 80% of the PV samples. This mutation in the pseudokinase autoinhibitory domain results in constitutive kinase activity and induces cytokine hypersensitivity or independence of factor-dependent cell lines. Retroviral transduction of the mutant JAK2 into murine HSC leads to the development of an MPD with polycythemia. The same mutation was found in about 50% of patients with idiopathic myelofibrosis (IMF) and 30% of patients with essential thrombocythemia (ET). Using different approaches, four other teams have obtained similar results. The identification of the JAK2 mutation represents a major advance in our understanding of the molecular pathogenesis of MPDs that will likely permit a new classification of these diseases and the development of novel therapeutic approaches.

NF-κB Family Proteins Participate in Multiple Steps of Hematopoiesis through Elimination of Reactive Oxygen Species

To examine the roles for NF-kappaB family proteins in hematopoiesis, we first expressed dominant negative Rel/NF-kappaB(IkappaBSR) in a factor-dependent cell line, Ba/F3. Although IkappaBSR neither affected thrombopoietin-dependent nor gp130-mediated growth, it suppressed interleukin-3- and erythropoietin-dependent growth at low concentrations. In addition, IkappaBSR enhanced factor-deprived apoptosis through the accumulation of reactive oxygen species (ROS). When expressed in normal hematopoietic stem/progenitor cells, IkappaBSR induced apoptosis even in the presence of appropriate cytokines by accumulating ROS. We also expressed IkappaBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from embryonic stem cells. When IkappaBSR was expressed at the stage of Flk-1(+) cells (putative hemangioblasts), IkappaBSR inhibited the development of primitive hematopoietic progenitor cells by inducing apoptosis through the ROS accumulation. Furthermore, when IkappaBSR was expressed after the development of hematopoietic progenitor cells, it inhibited their terminal differentiation toward erythrocytes, megakaryocytes, and granulocytes by inducing apoptosis through the ROS accumulation. These results indicate that NF-kappaB is required for preventing apoptosis at multiple steps of hematopoiesis by eliminating ROS.

Pim Kinases Mediate Viability Signals Downstream of the Tyrosine Kinase Oncogenes BCR-ABL and FLT3-ITD.

Pim1 and Pim2 belong to a family of serine/threonine kinases that are overexpressed in many leukemias. Pim1 has previously been shown to cooperate with the v-myc oncogene to transform hematopoietic cells with murine transforming viruses and overexpression of Pim2 has been shown to induce growth factor independence in a factor dependent murine BaF3 cell line. Protein expression of both Pim1 and Pim2 is low or absent in non-transformed hematopoietic cells but was found to be rapidly increased upon transformation by BCR-ABL or FLT3 with an internal tandem duplication (ITD). The exact contribution of the Pim kinases to transformation in these cells is unknown. BaF3 cell lines were created that stably expressed either BCR-ABL or FLT3-ITD tyrosine kinases, and in which either Pim1 or Pim2 could be induced with doxycycline. Treatment of BCR-ABL or FLT3-ITD expressing cells with small molecule kinase inhibitors specific for either ABL or FLT3 led to inhibition of cell growth, increased apoptosis, and downregulation of Pim expression as expected. However, if Pim2, and to a lesser extent, Pim1 expression was maintained by doxycycline, there was a substantial increase in both viability and cell growth. The molecular mechanisms by which Pim proteins exhibit their effects on target cells are not known. Using parental growth factor dependent BaF3 cell lines with doxycycline inducible Pim1 and Pim2, we show that expression of either of the Pim proteins is sufficient, by itself, to reduce apoptosis and induce modest cell growth. The effects of Pim overexpression on several pathways known to be associated with viability were studied. We found that while Pim over-expression does not activate Akt, it does result in the phosphorylation of a known Akt target, GSK-3β, a regulator of cell cycle progression by targeting the stability of cyclin D proteins. This suggests that Pim proteins may mediate their biological effects through regulation of components in the phosphatidylinositol-3′ kinase signaling cascade, independent of Akt activation. These results further suggest that upregulation of Pim kinases significantly contributes to transformation. Inhibition of Pim kinases could have beneficial therapeutic effects in leukemias.

Expression of Cyclooxygenase-2 (COX-2) in Human Leukemias and Hematopoietic Cells.

COX-2 has been implicated in the development of many epithelial cancers, as well as in tumor angiogenesis. COX-2 inhibitors have been shown to have anti-tumor activity in experimental cancer. Little information exists, however, on the expression or role of COX-2 in hematologic malignancies. We have use a variety of immunochemical assays to document expression of COX-2 in human and murine leukemias and hematopoietic cells. The factor-dependent murine cell lines FDCP1 and 32D expressed COX-2 when growing continuously in the presence of IL-3; expression declined markedly when growth factor was removed. FDCP1 cells constitutively expressing bcl-2, pim-1, or bcr-abl had markedly elevated levels of COX-2, and continued to express this enzyme even after removal of growth factor. To assess COX-2 expression in human hematopoietic cells we developed a flow cytometry assay using a FITC-labelled anti-COX-2 MoAb (Cayman). Cells were washed once in serum-free medium, fixed briefly in 1% paraformaldehyde, permeabilized with PBS/0.2% Triton X100, then stained with antibody. Negative control samples were processed similarly but stained with antibody that had been preincubated with immunizing peptide. Specific COX-2 staining was interpreted as the difference between the histograms from blocked versus unblocked anti-COX-2 antibody, as determined by Kolmogorov-Smirnoff analysis. In buffy coat preparations from normal donors, we found constitutive expression of COX-2 in lymphocytes (both B-cells and T-cells). In contrast little or no COX-2 was detected in unstimulated neutrophils or monocytes. In human acute myelogenous leukemia (AML) cell lines we found COX-2 expression to be universal and easily detected. In several cell lines we confirmed the results of our flow cytometry assay with immunoblotting. We further examined 25 cryopreserved samples of human acute leukemia blasts obtained from peripheral blood. COX-2 expression was variable, but universal. Levels generally were less than those seen in immortalized cell lines, and did not correlate with blasts morphology (AML, ALL, APL, AMoL, CML-BT). To determine if COX-2 inhibitors could play a role in the treatment of acute leukemias, we performed cytotoxicity assays using the COX-2 specific inhibitors, celecoxib and NS398. Survival and growth of human AML cell lines were inhibited by both agents. These data demonstrate that 1) a variety of oncogenes can induce expression of COX-2 in hematopoietic cells, 2) clinical human acute leukemias uniformly express COX-2 in circulating blasts, and 3) COX-2 inhibitors are cytotoxic for human leukemia cells. Combination therapies for acute leukemias may evaluate the incorporation of COX-2 inhibitors for added cytotoxic effects or angiogenesis inhibition.