Hematopoietic Precursor Cell Line Research Articles

Abstract 971: PRMT5 modulates splicing for genome integrity and preserves proteostasis of hematopoietic stem cells

Abstract Protein arginine methyltransferase 5 (PRMT5) has recently been identified as a promising therapeutic target in various solid and hematologic cancers, while PRMT5 inhibitors are currently in clinical development. Functionally, PRMT5 has an established role in regulating splicing, and is essential for hematopoiesis. However, the mechanisms underlying PRMT5 dependency in HSCs, particularly PRMT5-mediated splicing, remain unclear. Moreover, how pharmacological inhibition of PRMT5 affects HSCs remains undetermined. To clarify the function of PRMT5 in HSCs, we adopted a loss-of-function approach, coupled with functional and transcriptome profiling. We report that depletion of PRMT5 activity severely perturbs HSC maintenance. HSCs from Mx1-Cre Prmt5fl/fl mice (PRMT5Δ/Δ), which exhibit severe bone marrow (BM) failure, failed to reconstitute lethally irradiated wild-type recipients. Notably, this was associated with rapid loss of Prmt5Δ/Δ HSCs. Similarly, EPZ015666-mediated PRMT5 inhibition led to a dose-dependent decrease in the growth and viability of EML cells (murine BM-derived hematopoietic precursor cell line). Interestingly, this was associated with increased HSC size and PI3K/AKT/mTOR pathway activity. In addition, protein synthesis rate was also elevated in an mTOR-dependent manner. Thus, we confirm the cell-intrinsic requirement for PRMT5 activity in HSCs, and demonstrate a potential role for PRMT5 in regulating HSC proteostasis. We also analyzed the splice variant landscape of PRMT5Δ/Δ HSCs, and observed substantial upregulation of intron retention and exon skipping events; which were overrepresented for processes associated with maintenance of genomic integrity. Notably, we show that splicing of key genes involved in base excision repair, telomere maintenance and the Fanconi anemia pathway were dysregulated in PRMT5Δ/Δ HSCs, as well as EPZ015666-treated EML cells. Thus, suggesting that HSCs depleted of PRMT5 activity are potentially deficient in DNA repair. In agreement, PRMT5Δ/Δ HSCs exhibited elevated γH2A.X levels, replicative stress-associated oxidative DNA lesions and DNA strand breaks. Likewise, EPZ015666-treated EML cells exhibited dose-dependent increments in γH2A.X levels, and increased sensitivity to DNA damaging agents. Corroborating these observations, loss of PRMT5 activity in both PRMT5Δ/Δ HSCs and EPZ015666-treated EML cells led to elevated reactive oxygen species (ROS) levels, p53 activation and induction of apoptosis; which was significantly rescued by antioxidant treatment or co-depletion of p53. Therefore, depletion of PRMT5 activity renders HSCs vulnerable to DNA damage. Collectively, our findings uncover a critical role for PRMT5 in maintaining genomic integrity, and highlight a novel link between PRMT5 and proteostasis in HSCs. Citation Format: Darren Qiancheng Tan, Ying Li, Chong Yang, Jia Li, Shi Hao Tan, Desmond W. Chin, Ayako Nakamura-Ishizu, Henry Yang, Toshio Suda. PRMT5 modulates splicing for genome integrity and preserves proteostasis of hematopoietic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 971.

Engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP2) with Sickle or β-Thalassemia Mutation: An in-Vitro System for Testing Pharmacological Induction of Fetal Hemoglobin

Introduction: Sickle cell disease (SCD) and β-thalassemia are inherited blood disorders caused by mutations in the β-globin gene (HBB). Elucidation of the multiple pathophysiologic mechanisms in SCD and β-thalassemia has resulted in an increasing efforts to identify new treatment modalities to ameliorate the consequences of the disease. However, no consistent in vitro system exists for studies of pharmacological therapies for the diseases. Human umbilical cord-derived erythroid progenitor cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell-derived erythroid precursor cell line that can differentiate into red blood cells. Here, we engineered sickle HUDEP2 and β-thalassemia HUDEP2 clonal lines through CRISPR/Cas9-mediated editing of the human HBB. We sought to establish if these engineered cell lines exhibit disease phenotypes, and if upon in vitro erythroid differentiation they produce fetal hemoglobin (HbF) in response to hydroxyurea, the only FDA-approved drug for HbF induction. Our goal is to create an in vitro system to test new HbF inducers for treating SCD or β-thalassemia.Materials and Methods: We delivered Hi-Fidelity Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex in conjunction with a single-stranded DNA donor (ssODN) template to introduce the sickle or K17X (A<T) or codon 6 [-G] β-thalassemia mutation into the HBB locus of HUDEP2 cells. Edited HUDEP2 cells were single-cell sorted into multiple 96-well plates and expanded. The genotype of the clones was determined using a probe-based droplet digital PCR assay and confirmed through Sanger sequencing. Native polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC) were used to confirm the hemoglobin phenotype. Normal parental cell line, sickle clone, and two individual β-thalassemia clones were used to test the pharmacological induction of HbF. We initiated drug treatment in the expansion phase with 30 µM hydroxyurea. Trypan Blue staining and CD71/CD233/CD235 staining determined the effect of the drugs on the viability, growth rate and erythroid development of HUDEP2 lines. After 10 days of drug treatment, differentiated HUDEP2 were analyzed for globin expression through RT-qPCR and HPLC, and HbF positive cells (F-cells) were quantified via flow cytometry. Cells were placed at 2% O2 for four hours, fixed in glutaraldehyde, stained, and viewed under magnification to assess sickling potential.Results and Discussion: We generated multiple clones with biallelic sickle or β-thalassemia mutations. Sickle HUDEP2 clones almost exclusively expressed sickle hemoglobin with low level of HbF and hemoglobin A2 (HbA2), and β-thalassemia HUDEP2 clones produced no normal adult hemoglobin, 8-10% HbF, and 26-28% HbA2. On HPLC analysis, β-Thalassemia HUDEP2 clones had an unknown tall peak (39-45%) between HbF and HbA consistent with an α-globin homotetramer (α4). When subjected to hypoxic conditions for 4 hours, sickle HUDEP2 produced sickle cells. HUDEP2 parent cells did not sickle under hypoxic conditions. Hydroxyurea induced 3.8-fold, 1.8-fold, and 1.6-fold increases in γ-globin gene (HBG) expression; 2.9-fold, 1.4-fold, and 1.4-fold increases in the percentages of F-cells; 1.4-fold, 1.2-fold, and 1.6-fold increase in the percentages of HbF in sickle, K17X(A<T) and codon 6[-G] β-thalassemia HUDEP2 clones, respectively. No change was observed in CD71/CD235 positive HUDEP2 cells in the presence hydroxyurea. This finding demonstrated that hydroxyurea treatment induces HBG expression as well as HbF and F-cells in engineered sickle and β-thalassemia HUDEP2 clones. Future work will include screening other pharmacological compounds as well as studying the mechanism of HbF induction by using HUDEP2 clones.Conclusions: Our engineered sickle and β-thalassemia HUDEP2 cell lines have properties similar to those of patient erythroid cells and respond to the known HbF inducer hydroxyurea. This in vitro model system may facilitate the drug-discovery process by enabling multimodal drug screening on a large scale with consistent and reproducible results.Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G.B.) and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) DisclosuresNo relevant conflicts of interest to declare.

Genetic Ablation or Pharmacological Inhibition of PRMT5 Affects Maintenance of Genomic Integrity in Hematopoietic Stem Cells

Protein arginine methyltransferase 5 (PRMT5) has been implicated in diverse processes, including spliceosome assembly. In addition, the therapeutic potential of PRMT5-selective inhibitors has recently been demonstrated in several hematological malignancies. PRMT5 was also recently reported to be essential for hematopoiesis, and required for splicing in hematopoietic stem/progenitor cells. However, the role of PRMT5, particularly its importance in splicing regulation, in hematopoietic stem cells (HSCs) has not been extensively studied. Moreover, how pharmacological inhibition of PRMT5 affects the HSC compartment remains undetermined. To do so, we employed a loss-of-function approach, coupled with functional and transcriptome profiling.We report that PRMT5 is essential for HSCs. Mx1-Cre Prmt5fl/fl mice (PRMT5Δ/Δ) exhibited bone marrow (BM) failure; characterized by rapid reduction in BM cellularity, severe pancytopenia and lethality within 17.5 days. Importantly, PRMT5Δ/Δ HSCs failed to reconstitute lethally irradiated wild-type recipients. This was associated with transient expansion of the PRMT5Δ/Δ HSC compartment, which was less quiescent and had higher reactive oxygen species (ROS) levels, followed by significant loss of HSCs a week after. Correspondingly, pharmacological inhibition (using EPZ015666) of PRMT5 in EML cells (murine BM-derived hematopoietic precursor cell line) lead to a dose-dependent increase in the proportion of lineage-negative EML cells, increased ROS levels and decreased cell viability. Thus, confirming the cell-intrinsic requirement for PRMT5 in HSCs.We also analyzed the splice variant landscape in PRMT5Δ/Δ HSCs, and identified differential splicing events (DSEs) across several splicing classes; of which intron retention and exon skipping events were substantially elevated. Interestingly, DSEs were overrepresented for processes associated with maintenance of genome integrity. Notably, we show that splicing of key genes involved in base excision repair, telomere maintenance and the Fanconi anemia pathway were dysregulated in PRMT5Δ/Δ HSCs. We also detected dose-dependent dysregulated splicing of these genes in EPZ015666-treated EML cells. Further analysis in-silico determined that these dysregulated splicing events generated premature termination codons. Thereby suggesting that loss of PRMT5 activity in HSCs potentially leads to deficient DNA repair.In agreement, PRMT5Δ/Δ HSCs exhibited elevated γH2A.X levels, replicative stress-associated oxidative DNA lesions and DNA strand breaks. Likewise, EPZ015666-treated EML cells exhibited dose-dependent increments in γH2A.X levels. Corroborating these observations, apoptosis was increased among PRMT5Δ/Δ HSCs and EPZ015666-treated EML cells. This was associated with aberrant splicing of MDM4 (negative regulator of p53) and p53 activation; which involves upregulation of several pro-apoptotic genes and CDKN1A . Therefore, loss of PRMT5 activity renders HSCs vulnerable to DNA damage. Collectively, our study highlights a novel link between PRMT5 and maintenance of genome integrity in HSCs. DisclosuresNo relevant conflicts of interest to declare.

GATA-2 Regulates Dendritic Cell Differentiation

(Background) Dendritic cells (DCs) are critical regulators of the immune response, but their differentiation mechanism remains unclear. Heterozygous germline GATA-2 mutations in humans cause MonoMAC syndrome, characterized by monocytopenia and predisposition to myelodysplasia/acute myeloid leukemia. In this syndrome, DC count decreases profoundly, with an increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. In the present study, we analyzed the role of GATA-2 in DC differentiation and the underlying molecular mechanisms.(Method)Gata2 haploinsufficient mice (Gata2+/−: Tsai et al. Nature 1994) and tamoxifen-inducible Gata2-knockout mice (Gata2flox/flox/ER-Cre: Charles et al. Molecular Endocrinology 2006) were used. To generate conditional Gata2 knockouts in vivo, Gata2flox/flox/ER-Cre mice were intraperitoneally injected with 1-μg tamoxifen on days 1-3 and 8-10 and evaluated on days 20-22. Isolation of splenic DCs and bone marrow (BM) precursors, including LSK (Lin- Sca1+ Kit+ cell), CMP (common myeloid-restricted progenitor), GMP (granulocyte-macrophage progenitor), CLP (common lymphoid-restricted progenitor), and CDP (common dendritic cell precursor), were separated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For the in vitro analysis of Gata2-knockout, BM cells were cultured with CD45.1+ BM feeder cells from SJL mice (The Jackson Laboratory) with FLT3L (200 ng/mL) and 4-hydroxytamoxifen (Sigma). For transcription profiling, SurePrint G3 mouse GE microarray (Agilent) was used, and the data was subsequently analyzed with ImmGen database (http://www.immgen.org). Promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using CMP fraction and erythroid-myeloid-lymphoid (EML) hematopoietic precursor cell line (ATCC) with antibodies to GATA-2 (sc-9008, Santa Cruz Biotechnology).(Results) Quantitative RT-PCR analysis showed abundant Gata2 expression in LSK and CMP fractions, with detectable expression in GMP, CLP, and CDP fractions and in vitro differentiated DCs. Although the DC count did not change in Gata2 haploinsufficient mice, it significantly and profoundly decreased in Gata2 conditional knockout mice. To examine the role of GATA-2 during DC differentiation, we knocked out Gata2 during in vitro DC differentiation, starting from LSK, CMP, GMP, CLP, and CDP fractions obtained from Gata2flox/flox/ER-Cre mice. Gata2 knockout significantly decreased CD11c+ DC counts from LSK, CMP, and CDP fractions, while those from CLP and GMP were unaffected, implying the importance of GATA-2 during DC differentiation in the pathway from LSK to CDP via CMP, not via CLP nor GMP.To elucidate the underlying molecular mechanisms, we performed expression profiling with control and Gata2 -knockout DC progenitors from CMP of Gata2flox/flox/ER-Cre mice. Gata2 knockout caused >5-fold upregulation and downregulation of 67 and 63 genes, respectively. Although genes critical for the DC differentiation, e.g., Spi1, Ikzf1, and Gfi1, were not detected among the GATA-2-regulated gene ensemble, we found significant enrichment of myeloid-related and T lymphocyte-related genes among the downregulated and upregulated gene ensembles, respectively. We focused on Gata3 upregulation (7.33-fold) as a potential key mechanism contributing to Gata2 knockout-related impaired DC differentiation. Quantitative ChIP analysis with both CMP fraction and EML cell line demonstrated obvious GATA-2 chromatin occupancy at the consensus GATA-binding motif within Gata3+190 kb, which was conserved with human. Furthermore, addition of Gata3 +190 kb region to the Gata3 promoter (~0.5 kb) significantly decreased luciferase activity, which was significantly recovered by the deletion of GATA sequence within Gata3 +190 kb, in EML cells.(Conclusion) GATA-2 seems to play an important role for cell fate specification toward myeloid versus T lymphocytes, and thus contributing to the DC differentiation. Our data offer a better understanding of the pathophysiology of MonoMAC syndrome. DisclosuresFujiwara:Chugai Pharmaceuticals. Co., Ltd.: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Ishizawa:GSK: Research Funding; Takeda: Research Funding; Celgin: Speakers Bureau; Kyowa Kirin: Research Funding; Celgin: Research Funding; Janssen: Research Funding; Takeda: Speakers Bureau; Kyowa Kirin: Speakers Bureau; Pfizer: Speakers Bureau.

Complex interactions in EML cell stimulation by stem cell factor and IL-3

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34- cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone.

Akt Requires Glucose Metabolism to Suppress Puma Expression and Prevent Apoptosis of Leukemic T Cells

The PI3K/Akt pathway is activated in stimulated cells and in many cancers to promote glucose metabolism and prevent cell death. Although inhibition of Akt-mediated cell survival may provide a means to eliminate cancer cells, this survival pathway remains incompletely understood. In particular, unlike anti-apoptotic Bcl-2 family proteins that prevent apoptosis independent of glucose, Akt requires glucose metabolism to inhibit cell death. This glucose dependence may occur in part through metabolic regulation of pro-apoptotic Bcl-2 family proteins. Here, we show that activated Akt relies on glycolysis to inhibit induction of Puma, which was uniquely sensitive to metabolic status among pro-apoptotic Bcl-2 family members and was rapidly up-regulated in glucose-deficient conditions. Importantly, preventing Puma expression was critical for Akt-mediated cell survival, as Puma deficiency protected cells from glucose deprivation and Akt could not readily block Puma-mediated apoptosis. In contrast, the pro-apoptotic Bcl-2 family protein Bim was induced normally even when constitutively active Akt was expressed, yet Akt could provide protection from Bim cytotoxicity. Up-regulation of Puma appeared mediated by decreased availability of mitochondrial metabolites rather than glycolysis itself, as alternative mitochondrial fuels could suppress Puma induction and apoptosis upon glucose deprivation. Metabolic regulation of Puma was mediated through combined p53-dependent transcriptional induction and control of Puma protein stability, with Puma degraded in nutrient-replete conditions and long lived in nutrient deficiency. Together, these data identify a key role for Bcl-2 family proteins in Akt-mediated cell survival that may be critical in normal immunity and in cancer through Akt-dependent stimulation of glycolysis to suppress Puma expression.

Up- or downregulation of tescalcin in HL-60 cells is associated with their differentiation to either granulocytic or macrophage-like lineage

Tescalcin is a 25-kDa EF-hand Ca2+-binding protein that is differentially expressed in several mammalian tissues. Previous studies demonstrated that expression of this protein is essential for differentiation of hematopoietic precursor cell lines and primary stem cells into megakaryocytes. Here we show that tescalcin is expressed in primary human granulocytes and is upregulated in human promyelocytic leukemia HL-60 cells that have been induced to differentiate along the granulocytic lineage. However, during induced macrophage-like differentiation of HL-60 cells the expression of tescalcin is downregulated. The decrease in expression is associated with a rapid drop in tescalcin mRNA level, whereas upregulation occurs via a post-transcriptional mechanism. Tescalcin is necessary for HL-60 differentiation into granulocytes as its knockdown by shRNA impairs the ability of HL-60 cells to acquire the characteristic phenotypes such as phagocytic activity and generation of reactive oxygen species measured by respiratory burst assay. Both up- and downregulation of tescalcin require activation of the MEK/ERK cascade. It appears that commitment of HL-60 cells toward granulocytic versus macrophage-like lineage correlates with expression of tescalcin and kinetics of ERK activation. In retinoic acid-induced granulocytic differentiation, the activation of ERK and upregulation of tescalcin occurs slowly (16–48 h). In contrast, in PMA-induced macrophage-like differentiation the activation of ERK is rapid (15–30 min) and tescalcin is downregulated. These studies indicate that tescalcin is one of the key gene products that is involved in switching differentiation program in some cell types.

Glucose Metabolism Attenuates p53 and Puma-dependent Cell Death upon Growth Factor Deprivation

Growth factor stimulation and oncogenic transformation lead to increased glucose metabolism that may provide resistance to cell death. We have previously demonstrated that elevated glucose metabolism characteristic of stimulated or cancerous cells can stabilize the anti-apoptotic Bcl-2 family protein Mcl-1 through inhibition of GSK-3. Here we show that the pro-apoptotic Bcl-2 family protein, Puma, is also metabolically regulated. Growth factor deprivation led to the loss of glucose uptake and induction of Puma. Maintenance of glucose uptake after growth factor withdrawal by expression of the glucose transporter, Glut1, however, suppressed Puma up-regulation and attenuated growth factor withdrawal-induced activation of Bax, DNA fragmentation, and cell death. Conversely, glucose deprivation led to Puma induction even in the presence of growth factor. This regulation of Puma expression was a central component in cell death as a consequence of growth factor or glucose deprivation because Puma deficiency suppressed both of these cell death pathways. Puma induction in growth factor or glucose withdrawal was dependent on p53 in cell lines and in activated primary T lymphocytes because p53 deficiency suppressed Puma induction and delayed Bax and caspase activation, DNA fragmentation, and loss of clonogenic survival. Importantly, although p53 levels did not change or were slightly reduced, p53 activity was suppressed by elevated glucose metabolism to inhibit Puma induction after growth factor withdrawal. These data show that p53 is metabolically regulated and that glucose metabolism initiates a signaling mechanism to inhibit p53 activation and suppress Puma induction, thus promoting an anti-apoptotic balance to Bcl-2 family protein expression that supports cell survival.

Roles of Raf/MEK/ERK and PI3K/Akt/mTOR Signaling and p53 Pathways on Apoptosis, Drug Resistance and Therapeutic Sensitivity of Early Hematopoietic Precursor Cells

The interleukin-3 (IL-3) dependent FL5.12 hematopoietic precursor cell line was isolated from murine fetal liver, an important site of early hematopoiesis, by panning with an anti-AA4 antibody and culturing the cells in medium containing IL-3. The AA4 cell antigen (human analogue, Clq Receptor) is a key cell surface marker expressed on early hematopoietic stem cells along with the IL-3R (CD123) and others. Upon Hoechst-33342 staining and flow cytometric analysis, FL5.12 cells normally have approximately 0.4% of the cells present in side population (SP) which are proposed to possess “stem cell like characteristics”. Some leukemic stem cells often display drug resistance properties. FL5.12 cells are normally sensitive to chemotherapeutic drugs such as doxorubicin, however, drug resistant cells (FL/Doxo) were isolated by subcloning the cells in doxorubicin. These drug resistant cells contain approximately 2-fold more SP cells than drug sensitive FL5.12 cells. Drug resistance was associated with 6-fold increased ERK activation and 11-fold decreased p53 induction after doxorubicin treatment. Furthermore the drug resistant cells displayed decreased apoptosis (1.8 fold) and caspase 3, 8 and 10 activation (4.2, 2.3, 2 fold respectively) upon culture with doxorubicin than the parental cells. A 3-fold higher level of proteasomal degradation of p53 was observed in the drug resistant cells which was due to 8-fold higher levels of MDM2. Drug resistant FL/Doxo cells were more sensitive to proteosome inhibitors (IC50 of 75 nM compared to 125 nM for parental cells). Treatment with proteosome inhibitors resulted in 12-fold and 2-fold more p53 detection in FL/Doxo and FL5.12 cells respectively. Synergistic responses to doxorubicin and proteosome inhibitor treatment were observed as the IC50 for doxorubicin was reduced 16- and 5-fold respectively with FL/Doxo and FL5.12 respectively. FL/Doxo cells were also highly sensitive to mTOR inhibitors and synergistic effects were observed upon combining doxorubicin and rapamycin as the IC50s decreased 40- and 6-fold for FL/Doxo and FL5.12 cells respectively, documenting the importance of the mTOR pathway in their drug resistance. Introduction of dominant negative (DN) p53 or activated MEK1 (MEK1 CA) further increased the resistance of the FL/Doxo cells to doxorubicin approximately 18- and 34-fold respectively. The DN p53 or MEK1 CA transduced cells contained 3.3- and 5.4-fold more SP positive cells than parental FL5.12 cells respectively. While both DN p53 and MEK1 CA increased drug resistance and the frequency of SP cells, this occurred by different mechanisms. DN p53 suppressed Bax induction after doxorubicin treatment and decreased the sensitivity of the cells to proteosome (1.6-fold), MEK (1.2-fold), MDM- 2 (5-fold) and mTOR (3-fold) inhibitors compared to the parental FL5.12 cells. MEK1 CA also decreased the sensitivity to proteosome inhibitors 3-fold, yet the cells remained sensitive to MEK, mTOR and MDM2 inhibitors. Thus while DN p53 or MEK1 CA both increased the frequency of SP hematopoietic “leukemia stem” like cells, they do this by different mechanisms which also alters the sensitivities of small molecule inhibitors. Mutation of p53 or activation of the Raf/MEK/ERK pathway in leukemia stem cells may render them resistant to chemotherapy as well as certain targeted therapeutic approaches.

Regulation of Myoblast Motility and Fusion by the CXCR4-associated Sialomucin, CD164

Myoblast fusion is fundamental to the development and regeneration of skeletal muscle. To fuse, myoblasts undergo cell-cell recognition and adhesion and merger of membranes between apposing cells. Cell migration must occur in advance of these events to bring myoblasts into proximity, but the factors that regulate myoblast motility are not fully understood. CD164 is a cell surface sialomucin that is targeted to endosomes and lysosomes via its intracellular region. In hematopoietic progenitor cells, CD164 forms complexes with the motility-stimulating chemokine receptor, CXCR4, in response to the CXCR4 ligand, CXCL12/SDF-1 (Forde, S., Tye, B. J., Newey, S. E., Roubelakis, M., Smythe, J., McGuckin, C. P., Pettengell, R., and Watt, S. M. (2007) Blood 109, 1825-1833). We have previously shown that CD164 stimulates myotube formation in vitro. We report here that CD164 is associated with CXCR4 in C2C12 myoblasts. Cells in which CD164 levels are increased or decreased via overexpression or RNA interference-mediated knockdown, respectively, show enhanced or reduced myotube formation and cell migration, the latter both basally and in response to CXCL12/SDF-1. Furthermore, expression of CD164 cytoplasmic tail mutants that alter the endosome/lysosome targeting sequence and, consequently, the subcellular localization in myoblasts, reveals a similar correlation between cell motility and myotube formation. Finally, Cd164 mRNA is expressed in the dorsal somite (the early myogenic compartment of the mouse embryo) and in premuscle masses. Taken together, these results suggest that CD164 is a regulator of myoblast motility and that this property contributes to its ability to promote myoblast fusion into myotubes.

Activating PP2A Tumor Suppressor Activity as a Potential Therapy for Treating Jak2 V617F-Driven Myeloproliferative Disorders.

Polycythemia vera (PV) is a myeloproliferative disorder (MPD) caused by a valine to phenylalanine mutation in the inhibitory JH2 “pseudokinase” domain of the signaling protein Janus Kinase 2 (Jak2). While Jak2 has several known regulators (e.g. SOCS1, SOCS3, PP2A, and SHP-1), the role played by these signaling molecules in the development of PV is still largely unclear. One of these regulators, the tumor suppressor phosphatase PP2A, has been found functionally inactivated in different hematologic myeloid and lymphoid malignancies characterized by the expression of constitutively activated oncogenic tyrosine kinases (e.g. BCR/ABL). To investigate the role of PP2A in the pathogenesis of Jak2 V617F+ MPDs and the potential therapeutic relevance of PP2A activating drugs (e.g. FTY720), we determined the effects of wild type and V617F Jak2 expression on PP2A activity and assessed the molecular and biological effects of FTY720 in hematopoietic precursor cell lines and/or primary lineage-negative bone marrow cells engineered to expressed either wild type or V617F Jak2 protein. Herein we report that PP2A activity is significantly reduced by about 82% and 78% (P< 0.01) in mJak2 V617F-transduced growth factor-dependent and erythropoietin receptor-expressing 32Dcl3 (32D-EPO) and Ba/F3 cells, respectively, compared to MigR1-transduced controls. Furthermore, addition of the PP2A activator FTY720 (2.5μM) for 10 hours restored PP2A activity to 66% and 75% respectively compared to that of MigR1 controls. Mechanistically, we demonstrated that inactivation of PP2A was due to constitutive Jak2 activity. In fact, treatment of V617F Jak2-expressing cells with Jak inhibitor I (1μM; 10 hours) restored PP2A activity to 80% of controls in 32Dcl3 cells, while 600nM was sufficient to restore activity to 108% of controls in Ba/f3 cells. Likewise, transduction of murine lineage-negative bone marrow cells with wild-type Jak2 produced a 62% reduction in PP2A activity (P<0.01), while expression of the Jak2 V617F mutant produced a 97% reduction (P< 0.01). Moreover, as we previously reported for BCR/ABL, pharmacologic restoration of PP2A activity by treatment of Ba/F3 or 32D-EPO cells with the PP2A activator FTY720 (2.5μM for 10 hours) led to reduced expression and dephosphorylation of wild type and V617F Jak2. This, in turn, resulted in a 60% reduction in the colony forming ability of IL-3 cultured cells overexpressing wild type Jak2, while a 94% suppression of colony formation was evident in Jak2 V617-expressing cells maintained in the absence of cytokines. Moreover, addition of IL-3 to the FTY720-containing semisolid medium restored viability to levels similar to those of FTY720-treated wild type Jak2-expressing cells (70% suppression of colony formation upon treatment), indicating that restoration of PP2A activity counteracts cytokine-independent pathways triggered by the V617F Jak2 mutated tyrosine kinase. Altogether, these results suggest that inactivation of PP2A is essential for Jak2 (V617F mutant included) -driven cell/proliferation and survival and that pharmacologic activation of PP2A might represent a potential avenue for treatment of PV and, perhaps, of other MPDs characterized by the expression of a mutated Jak2 kinase.

Serum-induced expression of metallothionein isoforms in K-562 cells

The metallothionein (MT) expression was studied in the hematopoietic precursor cell line K-562, after serum deprivation and reconstitution of the cells in medium with 10% (v/v) FCS. Serum deprivation for 72 h markedly downregulated the MT mRNA expression, only the isoforms most abundant in normal K-562 cells were clearly detectable. Within 1–1.5 h after serum supplementation however, a definite induction of MT mRNA was noticed, and all isoforms were induced. Forty-eight hours after serum stimulation, the MT mRNA expression of all isoforms decreased again. Also MT protein levels increased twofold 24 h after serum stimulation. These results suggest that MT has a function in the re-entry of resting cells into the cell cycle, this function however could not be assigned to a specific MT isoform. The induction of MT after serum stimulation was independent of protein synthesis, but dependent on phosphorylation.

Sodium caseinate induces expression and secretion of murine multipotent myeloid cell line 32D macrophage colony-stimulating factor

Background Evidence that sodium caseinate (CasNa) is capable of inhibiting proliferation of hematopoietic precursor cell line 32D and inducing its differentiation into macrophage cells has recently been published. Taking into consideration that hematopoiesis is regulated by growth factors and that macrophage colony-stimulating factor (M-CSF) is a well-known growth factor that induces differentiation of macrophages, in this work we evaluated whether CasNa is capable of inducing expression and secretion of M-CSF in 32D cells. Methods We cultured 32D cells in presence and absence of CasNa and compared their proliferation and viability. RNA was extracted from cell lysates to evaluate expression of the gene for M-CSF and its receptor. Cultured conditioned media was used to evaluate presence of M-CSF. Results Our results showed that CasNa inhibited proliferation of 32D cells and that conditioned media (CM) of these cultures contained M-CSF-like activity. Presence of M-CSF in CM was detected by inhibiting M-CSF activity with anti-M-CSF and presence of this growth factor was confirmed by ELISA assay. We also provided evidence that CasNa induced expression of mRNA for M-CSF in 32D cells as well as increased expression of mRNA for its receptor. Conclusions CasNa inhibits proliferation of 32D cells and induces expression of the gene for M-CSF and that of its receptor. It also induces secretion of the bioactive form of M-CSF.

The leukemia-associated transcription repressor AML1/MDS1/EVI1 requires CtBP to induce abnormal growth and differentiation of murine hematopoietic cells.

The leukemia-associated fusion gene AML1/MDS1/EVI1 (AME) encodes a chimeric transcription factor that results from the (3;21)(q26;q22) translocation. This translocation is observed in patients with therapy-related myelodysplastic syndrome (MDS), with chronic myelogenous leukemia during the blast crisis (CML-BC), and with de novo or therapy-related acute myeloid leukemia (AML). AME is obtained by in-frame fusion of the AML1 and MDS1/EVI1 genes. We have previously shown that AME is a transcriptional repressor that induces leukemia in mice. In order to elucidate the role of AME in leukemic transformation, we investigated the interaction of AME with the transcription co-regulator CtBP1 and with members of the histone deacetylase (HDAC) family. In this report, we show that AME physically interacts in vivo with CtBP1 and HDAC1 and that these co-repressors require distinct regions of AME for interaction. By using reporter gene assays, we demonstrate that AME represses gene transcription by CtBP1-dependent and CtBP1-independent mechanisms. Finally, we show that the interaction between AME and CtBP1 is biologically important and is necessary for growth upregulation and abnormal differentiation of the murine hematopoietic precursor cell line 32Dc13 and of murine bone marrow progenitors.

Multipotent hematopoietic progenitor cells immortalized by Lhx2 self-renew by a cell nonautonomous mechanism.

Direct molecular and cellular studies of hematopoietic stem cells (HSCs) are hampered by the low levels of HSCs in hematopoietic tissues. To address these issues, we generated immortalized multipotent hematopoietic precursor cell (HPC) lines by expressing the LIM-homeobox gene Lhx2 (previously LH2) in hematopoietic progenitors derived from embryonic stem cells differentiated in vitro. To validate further the relevance of the HPC lines as a model for normal HSCs, we analyzed in detail the growth requirements of HPC lines in vitro. Lhx2 immortalized the HPC lines by a putatively novel and cell nonautonomous mechanism. Self-renewal of the HPC lines is dependent on functional Lhx2 expression. Most early-acting hematopoiesis-related growth factors show synergistic effects on the HPC lines, whereas late-acting factors do not induce differentiation by themselves. Transforming growth factor-beta(1) is a potent inhibitor of proliferation of the HPC lines. HPC lines form cobblestone areas with high efficiency when seeded onto stromal cell lines, and the cobblestone area-forming cell can be maintained in these cultures for several months. Our data show that, in many respects, HPC lines are similar to normal hematopoietic progenitor/stem cells on the cellular level, in contrast to most previously described multipotent hematopoietic cell lines. The cell nonautonomous mechanism for immortalization of the HPC lines suggests that Lhx2 regulates, directly or indirectly, soluble mediators involved in self-renewal of the HPC lines.

Real-time kinetics of ligand/cell surface receptor interactions in living cells: binding of epidermal growth factor to the epidermal growth factor receptor.

We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing using flow rates as great as 4.0 mL/s, allowing rapid processes to be quantitated with dead times as short as 10 ms. 32D cells do not express any endogenous epidermal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Association of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-expressing 32D cells was observed by measuring time-dependent changes in fluorescence anisotropy following rapid mixing. Dissociation of F-EGF from EGF-receptor-expressing 32D cells was measured both by chase experiments using unlabeled mEGF and by experiments in which equilibrium was perturbed by dilution. Comparison of these dissociation experiments showed that little, if any, ligand-induced dissociation occurs in the chase dissociation experiments. Data from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cell densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations having association rate constants of k(on1) = 8.6 x 10(6) M(-1) s(-1) and k(on2) = 2.4 x 10(6) M(-1) s(-1) and dissociation rate constants of k(off1) = 0.17 x 10(-2) s(-1) and k(off2) = 0.21 x 10(-2) s(-1). The magnitudes of these parameters suggest that under physiological conditions, in which cells are transiently exposed to nanomolar concentrations of ligand, ligand capture and release may function as the first line of regulation of the EGF receptor-induced signal transduction cascade.

A Thrombopoietin Receptor Mutant Deficient in Jak-STAT Activation Mediates Proliferation But Not Differentiation in UT-7 Cells

Thrombopoietin (TPO) stimulates proliferation and differentiation of cells of the megakaryocytic lineage. It exerts its function by binding and activating c-mpl, a member of the hematopoietic receptor superfamily. Upon binding of TPO to its receptor, numerous signaling events are triggered. These include activation of the Jak-STAT (signal transducers and activators of transcription) pathway, mitogen-activated protein kinase (MAPK), Tec, and phospatidylinositol (PI) 3-kinase and phosphorylation of Shc and Vav. The contribution of different signaling pathways to the induction of specific cellular processes such as proliferation and differentiation is incompletely understood. We have previously described a mutant of c-mpl that fails to activate the Jak-STAT pathway but nevertheless retains its ability to mediate proliferation and activation of most signaling events in the murine hematopoietic precursor cell lines BAF/3 and 32D. We confirm here the ability of this mutant to mediate proliferation in the absence of Jak-STAT activation in the human cell line UT-7 and further show that this mutant fails to mediate TPO-induced megakaryocytic differentiation. Comparison of the signaling capacity of this mutant in UT-7 and BAF/3 cells shows considerable cell-type-specific differences. Whereas in BAF/3 cells the mutant still mediates activation of Shc, MAPK, Vav, and PI 3-kinase at levels comparable to the wild-type receptor, these events are strongly diminished in UT-7 cells expressing the mutant. Furthermore, we show that the C-terminal 25 amino acid residues of the receptor mutant are crucial for the mitogenic response in UT-7 cells.

MDS1/EVI1 enhances TGF-beta1 signaling and strengthens its growth-inhibitory effect but the leukemia-associated fusion protein AML1/MDS1/EVI1, product of the t(3;21), abrogates growth-inhibition in response to TGF-beta1.

MDS1/EVI1, located on chromosome 3 band q26, encodes a zinc-finger DNA-binding transcription activator not detected in normal hematopoietic cells but expressed in several normal tissues. MDS1/EVI1 is inappropriately activated in myeloid leukemias following chromosomal rearrangements involving band 3q26. The rearrangements lead either to gene truncation, and to expression of the transcription repressor EVI1, as seen in the t(3;3)(q21;q26) and inv(3)(q21q26), or to gene fusion, as seen in the t(3;21)(q26;q22) which results in the fusion protein AML1/MDS1/EVI1. This fusion protein contains the DNA-binding domain of the transcription factor AML1 fused in-frame to the entire MDS1/EVI1 with the exclusion of its first 12 amino acids. In this report, we have analyzed the response of the hematopoietic precursor cell line 32Dcl3, expressing either the normal protein MDS1/EVI1 or the fusion protein AML1/MDS1/EVI1, to factors that control cell differentiation or cell replication. The 32Dcl3 cells are IL-3-dependent for growth and they differentiate into granulocytes when exposed to G-CSF. They are growth-inhibited by TGF-beta1. We show that whereas the expression of MDS1/EVI1 has no effect on granulocytic differentiation induced by G-CSF, expression of AML1/MDS1/EVI1 blocks differentiation resulting in cell death. This effect is similar to that previously described by others for 32Dcl3 cells that express transgenic Evil. Furthermore, we show that whereas the expression of the fusion protein AML1/MDS1/EVI1 completely abrogates the growth-inhibitory effect of TGF-beta1 and allows 32Dcl3 cells to proliferate, expression of the normal protein MDS1/EVI1 has the opposite effect, and it strengthens the response of cells to the growth-inhibitory effect of TGF-beta1. By using the yeast two-hybrid system, we also show that EVI1 (contained in its entirety in MDS1/EVI1 and AML1/MDS1/EVI1) physically interacts with SMAD3, which is an intracellular mediator of TGF-beta1 signaling. Finally, we have correlated the response of the cells to G-CSF or TGF-beta1 with the ability of the normal and fusion proteins to activate or repress promoters which they can directly regulate by binding to the promoter site. We propose that mutations of MDS1/EVI1 either by gene truncation resulting in the transcription repressor EVI1 or by gene fusion to AML1 lead to an altered cellular response to growth and differentiation factors that could result in leukemic transformation. The different response of myeloid cells ectopically expressing the normal or the fusion protein to G-CSF and TGF-beta1 could depend on the different transactivation properties of these proteins resulting in divergent expression of downstream genes regulated by the two proteins.

Early biochemical events in insulin-stimulated fluid phase endocytosis.

We examined the initial molecular mechanisms by which cells nonselectively internalize extracellular solutes in response to insulin. Insulin-stimulated fluid phase endocytosis (FPE) was examined in responsive cells, and the roles of the insulin receptor, insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3'-kinase (PI 3'-kinase), Ras, and mitogen-activated protein kinase kinase (MEK) were assessed. Active insulin receptors were essential, as demonstrated by the stimulation of FPE by insulin in HIRc-B cells (Rat-1 cells expressing 1.2 x 10(6) normal insulin receptors/cell) but not in untransfected Rat-1 cells or in Rat-1 cells expressing the inactive A/K1018 receptor. IRS-1 expression augmented insulin-stimulated FPE, as assessed in 32D cells, a hematopoietic precursor cell line lacking endogenous IRS-1. Insulin-stimulated FPE was inhibited in mouse brown adipose tissue (BAT) cells expressing the 17N dominant negative mutant Ras and was augmented in cells expressing wild-type Ras. The MEK inhibitor PD-98059 had little effect on insulin-stimulated FPE in BAT cells. In 32D cells, but not in HIRc-B and BAT cells, insulin-stimulated FPE was inhibited by 10 nM wortmannin, an inhibitor of PI 3'-kinase. The results indicate that the insulin receptor, IRS-1, Ras, and, perhaps in certain cell types, PI 3'-kinase are involved in mediating insulin-stimulated FPE.

Expression of the LIM-homeobox gene LH2 generates immortalized steel factor-dependent multipotent hematopoietic precursors.

The genes controlling self-renewal and differentiation in the hematopoietic system are largely unknown. The LIM-homeobox genes are known to be important for asymmetric cell divisions and differentiation of specific cell types and organs. One member of this family, LH2, is expressed in fetal liver at the time of active hematopoiesis. Therefore, we have assessed the function of LH2 during the formation and initial expansion of the hematopoietic system by differentiating LH2-transduced embryonic stem (ES) cells in vitro. This procedure generated multipotent hematopoietic precursor cell (HPC) lines that required Steel factor for growth. HPC lines have been maintained in an undifferentiated state in culture for >7 months. Other growth factors tested efficiently induce terminal differentiation of HPCs into various mature myeloid lineages. Steel factor is also required and acts synergistically with the other growth factors to generate multilineage colonies from the HPCs. These HPC lines express transcription factors that are consistent with an immature progenitor, and the pattern of cell surface marker expression is similar to that of early fetal multipotent hematopoietic progenitors. Collectively, these data suggest that the HPC lines represent an early fetal multipotent hematopoietic progenitor, and suggest a role for LH2 in the control of cell fate decision and/or proliferation in the hematopoietic system.