Proliferation In Liquid Culture Research Articles

Tumor Associated Macrophages Promoted Fatal Patient Derived Acute Promyelocytic Leukemia In Vivo

With immune therapies on the rise, an in-depth understanding of the immunological changes in leukemic bone marrow (BM) niches becomes indispensable. Being an crucial part of the tumor microenvironment (TME) in solid tumours, tumour-associated macrophages are often associated with poor prognosis (Bruni et al. 2020). Yet, in acute myeloid leukaemia (AML) the role of macrophages has not been thoroughly studied. The expression of the M2-markers CD163 and CD206 in the AML BM cell population predicted poor clinical outcome. We identified that this expression emerges from a more mature (CD45 midSSC highHLA-DR +CD14 +CD16 +/-) myeloid cell population (hereafter called AML-associated macrophages - AAM) and not from the leukemic blasts. By employing flow cytometry analysis (FACS) we noted a decrease in the expression of the M1-marker (CD80) and an increase of the M2-markers CD163/CD206on AAM (n=70) compared to healthy donors (HD, n=10). Unsupervised clustering based on the CD163/CD206 levels detected on AAM generated 4 distinct clusters, whereby patients within the CD163 low/CD206 low cluster displayed better overall survival than the other clusters.In vitro, the co-culture of HD-derived M1 macrophages and AML primary/cell lines reduced AML growth via apoptosis induction and cell cycle arrest, while M2-macrophages promoted AML survival and phagocytosis/drug-resistance when treated with FLT3/BCL2 inhibitors. Primary AML cells were also able to repolarize M1- into M2-macrophages, suggesting that leukemic cells actively remodel their microenvironment. Next, we evaluated the impact of M2-macrophages on leukemogenesis in a patient derived xenograft (PDX) model, using the notoriously difficult to engraft primary Acute Promyelocytic Leukaemia (APL) cells (n=7 patient samples). Intra-BM injection of M2-macrophages and retro-orbital transplant of primary APL cells induced full-blown APL in NSGS mice. More strikingly, ex vivo culture of APL cells on M2-macrophages (48h) was sufficient to “train” these cells to engraft and induce fatal APL. Maintenance of self-renewal was shown in a secondary transplant and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays.To identify the biological changes acquired by leukemic blasts, we performed RNA sequencing comparing AML/APL samples at diagnosis to cells that were “trained” (48 h) on M2-macrophages or on MS5 mesenchymal BM stromal cells. Gene ontology and gene set enrichment analysis on the genes up-regulated upon M2 co-culture were significantly enriched for cell migration, cell cycle progression and oxidative phosphorylation (OXPHOS) signatures. In line with our RNAseq data, we noted improved in vivo homing of primary APL cells to the BM within 18 h post-transplant upon ex vivo M2 co-culture compared to diagnosis (n=7 APL blasts). Concurrently, we detected increased levels of surface protein expression Integrin-α4 (CD49d) and -α5 (CD49e) on APL/AML blast cells after M2 exposure. The CD49d expression remained high in primary and secondary transplants. Using seahorse measurements, we confirmed the increased respiration capacity (basal and maximum) of primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5.FACS analysis revealed that M2-macrophages were able to transfer more mitochondria than MS5 cells to primary AML cells, which could underlie the observed increase in OXPHOS mitochondrial metabolism. Treatment with Etomoxir (50 µM), prevented the gain in functional respiration when AML blast were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures, suggesting increased fatty acid oxidation to drive the OXPHO-like state. Finally, we noted that training on M2 macrophages significantly increased colony formation and endowed the cells with long term proliferation in liquid cultures for over 30 days.Overall, we reveal that the frequency of M2-macrophages is up-regulated in a subgroup of AML patients representing a group with poor prognosis. M2 macrophages can support leukemic growth and therapy-resistance, and support fatal APL in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter adhesion, homing and metabolic characteristics of leukemic blasts to allow efficient engraftment and fatal leukemogenesis. Our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions. DisclosuresSilveira: BMS/Celgene: Research Funding; Servier/Agios: Research Funding; Abbvie: Speakers Bureau; Astellas: Speakers Bureau. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding. Mota: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Technopharma: Speakers Bureau; Bristol Myer Squibb: Speakers Bureau; Bayer: Speakers Bureau; Pfizer: Speakers Bureau; AstraZeneca: Speakers Bureau; Astellas: Speakers Bureau; Ipsen: Speakers Bureau; Amgen: Speakers Bureau.

Expression of the Tumor Suppressor WT1 Is Induced By PML-Rara in Acute Promyelocytic Leukemia. Â

Inactivating mutations in Wilms Tumor 1 (WT1) are found in 5-10% of cases of acute myeloid leukemia (AML). Paradoxically, wild type WT1 is expressed highly in most cases of AML compared to normal hematopoietic progenitors, and high levels of WT1 have been associated with poor outcomes. Therefore, it is not yet clear whether loss-of-function WT1 mutations-or paradoxically, wild type WT1 overexpression-both promote disease progression in AML. We performed a genome-wide screen for mutations in acute promyelocytic leukemia (APL), a morphologic subtype of AML characterized by the PML-RARA fusion. We identified WT1 mutations in 11/42 patients, suggesting that WT1 mutations may cooperate specifically with PML-RARA in APL. Further, expression analysis demonstrated a high level of wild type WT1 expression in non-mutated cases. To test the effect of Wt1 inactivation in a mouse model of APL, we bred mice carrying a floxed Wt1 allele (give Baylor reference) to transgenic mice expressing tamoxifen-inducible Cre recombinase (ERT2-Cre). Tamoxifen treatment leads to deletion of the C-terminal zinc finger domains of Wt1, mimicking some of the mutations that are commonly found in AML/APL cases. These mice were crossed with mice expressing the PML-RARA fusion cDNA in early myeloid progenitors with Cathepsin G (Ctsg) regulatory sequences (Ctsg-PML-RARA). Bone marrow cells from Ctsg-PML-RARA x Wt1fl/fl x ERT2-Cre mice were transplanted into irradiated recipients. Tamoxifen-treated mice (i.e. Wt1 deficient) developed APL with a significantly shortened latency compared to untreated controls (198 days vs. endpoint not reached, N=8-10 each group, p=.0001), suggesting that Wt1 loss-of-function mutations can cooperate with PML-RARA to drive APL. Next, to test the effect of wild type Wt1 overexpression on hematopoietic cells, bone marrow cells from wild type or Ctsg-PML-RARA mice was transduced with retroviruses expressing full length Wt1 cDNA or an empty vector control, and subjected to serial replating. As previously reported, untransduced bone marrow cells from Ctsg-PML-RARA mice were able to form colonies in methylcellulose week after week, while cells overexpressing wild type Wt1 were selected against during serial replating, as demonstrated by a gradual reduction in GFP percentage and Wt1 expression, as measured by western blotting. Interestingly, in contrast to human AML/APL, Wt1 expression could not be detected in a panel of murine tumors with initiating mutations in DNMT3A, PML-RARA, or MLL-AF9 . Taken together, these findings suggest that Wt1 expression suppresses APL development in mice.To reconcile these findings with the high level of WT1 expression in human AML/APL, we speculated that in human progenitor cells, WT1 expression may be induced by leukemia-associated oncogenes (like PML-RARA) as a physiologic, inhibitory response to oncogenic stress. In this case, subsequent inactivating mutations in WT1 would provide an additional growth advantage. Given the observed interspecies difference between WT1/Wt1 expression levels in mouse and human leukemia cells, we used human umbilical cord blood-derived CD34+ cells transduced with retroviruses expressing PML-RARA to model early events in human APL development. CD34+ cells expressing PML-RARA share some characteristics of fully transformed APL cells, including enhanced proliferation in liquid culture, and reduced expression of the myeloid maturation markers CD11b and CD14. WT1 mRNA expression is low in freshly isolated CD34+ cells, but rapidly increases in cells expressing PML-RARA over two weeks in culture (10.5-fold increase compared to empty-vector transduced controls, N=3 separate experiments, p<0.05). Western blotting confirmed robust induction of WT1 protein in transformed cells compared to empty-vector transduced controls, suggesting that WT1 expression is a normal consequence of PML-RARA activation in human hematopoietic progenitor cells. In summary, these findings support a cooperating role for WT1 mutations in APL development, and a potential mechanism to explain the high level of wild type WT1 expression observed in APL cells. Further experiments will be required to determine the downstream mechanisms by which WT1 antagonizes APL development, and whether these observations are generalizable to other AML subtypes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mutant TET2 Allele Dosage Affects Response to 5-Azacitidine in Acute Myeloid Leukemia

Personalised medicine is predicted to significantly improve outcomes for cancer patients, but implementation requires comprehensive genetic characterisation of malignant cells to identify therapeutically exploitable vulnerabilities. Using an isogenic cell model system with CRISPR-inactivated TET2 in HEL acute myeloid leukemia (AML) cells and an orthotopic mouse xenograft model we demonstrate that mutant TET2 allele dosage significantly affects sensitivity to 5-azacitidine hypomethylating therapy in AML, with biallelic mutation conferring hypersensitivity relative to monoallelic mutation. In the presence of 5-azacitidine, cell clones with biallelic TET2 mutation had significantly lower cloning efficiency (P = 3 x 10-3) and proliferation in liquid culture (P &amp;lt; 1 x 10-4) compared to isogenic clones with monoallelic TET2 mutation. Mixed populations of monoallelic and biallelicTET2 mutated HEL AML cells were transplanted via intrafemoral injection into Rag2−/−Il2rg−/−129×Balb/c mice, and treatment with 5-azacitidine resulted in significant negative in vivo selection against TET2 null cells relative to cells with monoallelic TET2 mutation (P = 4 x 10-4). Methylation analysis revealed the acquisition of an overall hypermethylation phenotype in TET2 null cells and RNA sequencing identified significant down-regulation of ABCB1 transcript, resulting in concomitant pronounced down-regulation of the MDR1 drug efflux transporter at the protein level. RNA sequencing pathway analysis also identified a global effect on ribosome pathway (KEGG pathway ko03010) transcript levels (Padjusted = 0.002), evidenced by down-regulation of numerous RNA polymerase II components in cells with bi-allelic TET2 mutation compared to cells with monoallelic TET2 mutation. Consistent with our isogenic model data, we characterise biallelic somatic TET2 mutation in a patient with AML that was chemoresistant to anthracycline/cytarabine-based chemotherapy but acutely sensitive to 5-azacitidine, resulting in durable cytomorphological remission. Integration of next generation sequencing, interphase FISH and SNP array analysis of bone marrow at AML presentation, relapse and during remission was used to infer tumour phylogeny which indicated that disease pathogenesis was initiated by a TET2 nonsense mutation (c.2815C&amp;gt;T, Q939*) with subsequent deletion of the second TET2 allele and a NPM1 mutation (c.863_864ins, TCTG) that arose after the acquisition of bi-allelic TET2 mutation. Furthermore, our data demonstrate that 5-azacitidine treatment almost completely eliminated the TET2/NPM1-mutated clone. 5-azacitidine also induced a modest reduction in ancestral pre-leukemic cells carrying bi-allelic TET2 mutation but negative for the NPM1 mutation, although the majority retained viability and re-acquired the ability to differentiate and recapitulate normal haematopoiesis rendering a cytomorphological remission. These observations suggest that bi-allelic TET2 mutation confers sensitivity to the cytotoxic effects of 5-azacitidine, but that the major effect of 5-azacitidine is the induction of phenotypic re-programming. The frequency of TET2 mutation in primary AML is estimated at 10-20%, with the majority of these being monoallelic. We determined the frequency of TET2 alterations in AML patients presenting with a chromosome 4 abnormality discernible cytogenetically. TET2 copy number and mutational status were determined using high density SNP arrays and gene sequencing, respectively. In a panel of 30 AML cases with a chromosome 4 abnormality, four patients were heterozygous for TET2 mutation (all deletions resulting in reduced copy number) and three patients were homozygous for TET2 mutation (deletion plus base substitution in two cases and homozygous base substitution resulting from uniparental disomy in one case). Furthermore, all seven cases with TET2 mutation were characterised by cytogenetics that included loss or gain of material on chromosome 4. In contrast, only 1 case with a TET2 mutation had a translocation affecting chromosome 4. In summary, our data argue in favour of using 5-azacitidine in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of personalised medicine for cancer patients. Disclosures Stoelzel: JAZZ Pharmaceuticals: Consultancy; Neovii: Other: Travel funding; Shire: Consultancy, Other: Travel funding. Jackson:Celgene, Amgen, Roche, Janssen, Sanofi: Honoraria. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Tumor Suppressor FOXO1 Serves As a Critical Oncogenic Mediator in AML1-ETO Leukemia

AML1-ETO (AE) fusion protein, produced by the chromosome translocation (8;21), accounts for 10%-15% of acute myeloid leukemia (AML). We use CD34+ human umbilical cord blood (CB) cells retrovirally transduced with AE to model AE leukemia (AE cells). This model represents the pre-leukemic stage of AE leukemia since AE by itself does not lead to overt disease but largely enhances the self-renewal and impedes the differentiation of CB cells. However the molecular mechanism by which AE deregulates the hematopoietic program is still largely unknown.The FOXO subfamily of forkhead transcription factors, which in mammals includes FOXO1, FOXO3, FOXO4, and FOXO6, are well known tumor suppressor proteins due to their ability to arrest cell cycle and induce apoptosis. Although FOXO1 is silenced in many types of solid tumors, unexpectedly, we found expression of FOXO1 (but not other family members) significantly upregulated in AE AML patient samples compared to other AML subtypes. Increased protein expression was confirmed by western blot in a cohort of patient samples and our AE cells. AE bound to a region within intron 1 of the FOXO1 gene as shown by chromatin immunoprecipitation, the sequence of which includes several AML1 binding motifs. The expression of FOXO1 was dependent on the presence of AE as shown by overexpression and knockdown studies. These findings suggest that FOXO1 could be a direct target of AE. Knockdown of FOXO1 by shRNA resulted in decreased proliferation of AE cells, and conditional deletion of Foxo1 in murine AE cells led to decreased CFU replating in methylcellulose compared to non-deleted control. In contrast, conditional deletion of Foxo3 using the same approach did not significantly influence the clonogenicity of AE cells. These data indicate FOXO1 is critical for AE cell growth.Interestingly, overexpression of FOXO1 in CD34+ CB cells promoted their long-term proliferation in liquid culture, inhibited erythroid differentiation while promoting myeloid lineage development and enhanced their replating ability in colony forming assays. FOXO1-expressing cells also achieved a higher engraftment level when transplanted into immunodeficient mice. These effects partially phenocopy the outcomes observed upon AE expression in CB cells. These effects require the transcriptional activity of FOXO1, as shown with a FOXO1 mutant lacking DNA binding capacity, and are not seen upon FOXO3 expression. Although inhibition of reactive oxygen species (ROS) production is regarded as a general function of FOXO family proteins, ROS regulation is unlikely to be the major downstream mechanism, since ROS levels were only transiently and slightly decreased upon FOXO1 expression. In addition, treatment of CB cells with the ROS scavenger N-acetyl-cysteine could not recapitulate FOXO1’s phenotype.To gain molecular insight into FOXO1’s function in CB cells, RNA-Seq experiments were performed in triplicate. CD34+ CB cells were transduced with empty vector (MIT), FOXO1 DNA-binding-deficient mutant (FOXO1 DB), wildtype FOXO1 (FOXO1 WT) or AE. The MIT and FOXO1 DB groups showed very similar gene expression profiles. 1349 genes were upregulated and 1113 genes downregulated in the AE group while FOXO WT showed 608 genes increased and 490 genes decreased (P<=0.01, fold change>=1.5). To our surprise, 282 upregulated and 231 downregulated genes were common targets of AE and FOXO WT, suggesting that about 20% of AE downstream targets are potentially regulated via FOXO1. More strikingly, pathway enrichment analysis showed that published AE gene signature datasets were enriched in the FOXO1-WT gene list. Among genes upregulated by FOXO1, several have been reported as AE transcriptional targets, such as POU4F1 and JUP. In addition, some FOXO1 target genes, SOX4 and HLF for example, have been implicated in leukemogenesis.In summary, we find FOXO1 is required for AE cell growth, and overexpression of FOXO1 in CB cells can partially recapitulate the AE phenotype at both the morphological and molecular level. Taken together, these data suggest that instead of acting as a tumor suppressor, FOXO1 is a critical oncogenic mediator of the AE leukemia program. It is possible that FOXO1 serves as one signaling hub of the AE molecular network, similar to the role MEIS1-HOXA9 play in MLL-fusion AML. Fully dissecting the AE-FOXO1 pathway will enhance our understanding of AE leukemogenesis and provide potential therapeutic targets. DisclosuresMulloy:Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

Trisomy 21-Associated Abnormalities in IGF Signalling and the Fetal Microenvironment Both Contribute to Disruption of Fetal Hematopoiesis in Down Syndrome

In Down syndrome (DS), trisomy 21 (T21) causes perturbation of fetal liver (FL) hematopoiesis leading to expansion of megakaryocyte-erythroid (MK-E) progenitors. On this background, FL hematopoietic stem/progenitor cells (HSPC) may acquire GATA1 gene mutations which usually present as a neonatal preleukemic condition, Transient Myeloproliferative Disorder (TMD). TMD develops only in fetal life and most cases spontaneously regress after birth, suggesting unique features of the T21 fetal microenvironment may be important in driving both abnormal DS FL hematopoiesis and mutant GATA1 clones in TMD. Previous work showed activation of insulin-like growth factor (IGF) signalling in TMD and DS-AMKL and fetal-specific IGF dependence of murine MK development implicating T21 in altered fetal IGF signalling.To investigate the role of IGF signalling in fetal MK-E development in DS FL without GATA1 mutations, we performed gene expression profiling (GEP) of DS FL (n=5) and normal FL CD34+ cells (n=3). GSEA showed significant enrichment of IGF targets. We therefore measured expression of the principal IGF receptor, IGF1R, on DS FL HSPC (n=6). Surface IGF1R was uniformly expressed on all DS FL HSPC subpopulations. However, there was no difference in IGF1R surface or gene expression between DS and normal FL HSPC (n=8) suggesting that increased IGF1R expression on TMD and DS-AMKL cells is an early event and may reflect their fetal rather than T21 origin. To determine if the enrichment of IGF targets in DS FL HSPC might reflect increased IGF production in the FL microenvironment, we compared IGF expression in DS FL mesenchymal stromal cells (MSC)(n=4) and hepatocytes with normal FL MSC (n=4). Both DS and normal FL MSC expressed large amounts of IGF2, but little or no IGF1 with no difference between DS and normal MSC. Similarly, IGF2 was expressed by IHC at similar levels in both DS and normal fetal hepatocytes and MSC whereas IGF1 was barely detectable. Normal and DS fetal bone marrow (BM) MSC also expressed high levels of IGF2, but not IGF1 in contrast to adult BM MSC. Thus, IGF2 is the main IGF produced in the human fetal hematopoietic environment. Consistent with a specific role in DS FL hematopoiesis, IGF2 caused marked proliferation of DS FL clonogenic MK and MK-E cells (n=3) and stimulated MK proliferation in liquid culture while in normal FL, IGF2 stimulated EPO-independent BFU-E and Pre-BFU-E but had no effect on MK cells. Since differential effects of IGF2 on DS/normal FL HSPC might be due to altered expression of the Insulin Receptor (InsR), which binds IGF2 with low affinity, we compared InsR surface and gene expression in DS and normal FL HSPC but found no significant difference. IGF2 also binds to IGF2R, a negative regulator of IGF2, however there was no difference in IGF2R expression between DS and normal FL HSPC. Co-culture of normal FL CD34+ cells with DS or normal FL MSC (n=3) promoted MK and erythroid cell proliferation and differentiation. Since the effect of DS FL MSC on normal FL CD34+ cells was significantly greater (~4-fold) than that of normal FL MSC, the data above suggest this is unlikely to be due to IGF2 alone. To investigate whether T21 alters the MSC secretome, we performed GEP of DS FL MSC (n=3) and compared this with normal FL MSC (n=5). Both normal FL and DS FL expressed known hematopoietic cytokine genes, including SCF, CXCL12, G-CSF, M-CSF, Flt3 and IL-6, as well as IGF2 but not IGF1, EPO or TPO. Of the top 80 differentially expressed genes (Fold Change>2), ~40% encode secreted proteins, including several which affect IGF availability.Conclusion: T21 perturbs FL hematopoiesis through both HSPC-intrinsic mechanisms, including changes in IGF responsiveness, and through alterations to the FL microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3015: Identification of a novel gene target involved in tumor growth and metastasis of non-small cell lung cancer.

Abstract We have discovered a potential oncogenic role of a gene in non-small cell lung cancer (NSCLC) using RNAi technology. Silencing of the gene significantly inhibits soft agar colony formation of NSCLC cell lines, whereas its inhibition on cell proliferation in liquid culture is modest. This effect was observed in multiple murine and human NSCLC cell lines with varied Kras and p53 status. The inhibition on soft agar colony formation is reversed with ectopic overexpression of a cDNA encoding the full-length gene. Knocking down the expression of the gene also significantly impairs migration and enhances detachment-induced anoikis of NSCLC cells. An investigation of the molecular mechanisms reveals that silencing of the gene cytoskeleton signaling network. In xenograft studies using stable luciferase-expressing inducible shRNA cell lines, silencing of the gene upon doxycycline treatment leads to a modest reduction in tumor volumes while significantly decreases bioluminescent signals at metastatic sites including lung and lymph nodes. Furthermore, gene expression analyses in KrasG12D/p53 deletion-driven mouse tumors and clinical tumor samples show significantly increased expression of the gene in lung cancer as well as multiple other cancer types. Taken together, we have uncovered a novel oncogenic function of a gene in NSCLC, which could provide a potential new therapeutic target for cancer intervention. Citation Format: Honglin Chen, James Lee, Noelyn M. Kljavin, Leisa Johnson, Yuxin Liang. Identification of a novel gene target involved in tumor growth and metastasis of non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3015. doi:10.1158/1538-7445.AM2013-3015 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Reduced C/EBPα Expression Favors Monopoiesis Over Granulopoiesis

Abstract 1230C/EBPα is required for the formation of the granulocyte-monocyte progenitor; however, its role in subsequent lineage specification remains uncertain. We previously compared G-CSF with M-CSF signaling in murine marrow, finding that G-CSF more potently induces phosphorylation of SHP2 whereas M-CSF more potently activates ERK. Knockdown (KD) of SHP2 impaired granulopoiesis relative to monopoiesis and unexpectedly led to reduced C/EBPα RNA. A. Cantor and colleagues recently reported that SHP2 dephosphorylates Src kinase-modified RUNX1 and increases the ability of RUNX1 to induce megakaryopoiesis, and we demonstrated that in myeloid cells RUNX1 binds and activates the CEBPA gene, via its promoter and via a conserved 450 bp enhancer located at +37 kb containing 4 RUNX1-binding sites. Thus, G-CSF or other cytokines potentially direct SHP2 to activate RUNX1 which then increases C/EBPα transcription in myeloid progenitors to favor granulopoiesis. As a test of this model, we have now transduced lineage-depleted murine marrow cells, cultured in SCF, TPO, and Flt3L to minimize myeloid differentiation, with shRNAs targeting CEBPA or with a control lentiviral vector. Western blotting indicated 2-fold KD by shRNAs B9 or B11 after 2 days of transduction followed by 2 days of puromycin selection. As this analysis was done on a mixed population, greater KD may occur in rare progenitors. Upon transfer to IL3, IL6, and SCF to favor myeloid maturation, vector-transduced cells produced 4-fold more CFU-G than CFU-M in methylcellulose, whereas CEBPA KD resulted in 4- or 5-fold more CFU-M than CFU-G. FACS analysis of pooled CFUs demonstrated significant reduction of Mac-1+Gr-1+ granulocytes and an increase in Mac-1+Gr-1− monocytes, and examination of cell morphology after Wright’s-Giemsa staining further confirmed a shift towards monopoiesis in response to reduced C/EBPα. Similar findings were obtained for cells placed in liquid culture. In addition, on day 3 of liquid culture Q-RTPCR analysis showed that CEBPA shRNAs induced 2- or 3-fold CEBPA RNA KD, reduced granulocytic MPO, NE, PR3, and GCSFR RNAs, increased monocytic MCSFR and CD14 RNAs, and increased expression of EGR1, IRF8, and c-Jun RNAs, encoding transcription factors that help direct monopoiesis. RUNX1 RNA was unchanged, and PU.1 RNA was reduced <2-fold. To determine the effect of CEBPA KD in vivo, transduced, puromycin-selected CD45.2+ marrow cells were transplanted into lethally irradiated, syngeneic CD45.1+ recipients together with CD45.1+ carrier cells. At day 28, FACS analysis indicated a shift towards monopoiesis amongst shRNA B9- or B11-transduced marrow cells, and CFU assay of sorted CD45.2+ cells demonstrated significantly reduced CFU-G with increased CFU-M. Besides affecting myeloid lineage determination, CEBPA KD increased the rate of cell proliferation in liquid culture, associated with blasts evident upon morphologic analysis and an increase in Mac-1−Gr-1− cells, which mainly consisted of c-Kit+Sca-1− myeloid progenitors. In addition, CEBPA KD by shRNA B9, which impaired granulopoiesis more effectively than shRNA B11, increased CFU replating to 8 generations, compared with 4 generations for control cells. Analysis on day 3 of liquid culture did not reveal an alteration in the proportion of cells in G1, S, or G2/M or increased apoptosis, and c-Myc and Bcl-2 RNA levels were unaffected. We propose that CEBPA KD not only strongly impairs granulopoiesis, but also modestly impedes monopoiesis to produce a transient accumulation of cells with blast morphology and the ability to proliferate. Overall, our findings suggest that relative to monopoiesis, granulopoiesis requires increased level or increased activity of C/EBPα. Offering a potential mechanism, we previously found that C/EBPα :AP-1 leucine zipper heterodimers are more abundant than C/EBPα homodimers during monopoiesis compared with granulopoiesis, in part due to c-Fos, c-Jun, and JunB induction, and that C/EBPα :AP-1 heterodimers can direct marrow monopoiesis. Perhaps higher levels of C/EBPα favor homodimer formation, with reduced levels still able to heterodimerize with AP-1 proteins. ERK modification of C/EBPα on S21, as occurs downstream of M-CSF, reduces its ability to direct granulopoiesis; phospho-C/EBPα (S21) potentially retains the ability to direct AP-1 proteins to hybrid DNA elements recently identified in numerous monocytic enhancers. Disclosures:No relevant conflicts of interest to declare.

Effects of Sea Salts on Induction of Cell Proliferation in Liquid Cultures of Mangrove Plants, Sonneratia caseolaris and S. alba

The effects of five salt ingredients of sea water, KCl, NaCl, CaCl2, MgCl2 and MgSO4, on induction of cell prolif-eration in Sonneratia caseolaris were investigated. Proliferation was examined in tissue explants derived from such as leaves, cotyledons, and hypocotyls using a small-scale liquid culture method. Addition of 12.5-25 mM of MgCl2 was unique in stimulating cell proliferation in all tissues of S. caseolaris. Otherwise, different effects of salts were observed among the three tissues. In hypocotyl culture, 25-50 mM of NaCl and CaCl2 stimulated cell divisions. Tolerance to 100 mM of MgSO4 was observed in leaves. Three osmotically active compounds commonly used in tissue culture, sorbitol, mannitol and glycinebetaine, were also tested to assess the importance of osmotic effects on cell proliferation. No significant stimulation by these was observed over a wide range of concentrations. Data were compared with those of cotyledon cultures of another mangrove, S. alba, which exhibits no stimulation by MgCl2, stimulation by KCl and tolerance to NaCl. Mechanisms for adaptation of mangrove plants to various and high salts were discussed by comparing the differences in reaction to salts in cultures of two Sonneratia mangrove species of the same genera growing different salt environment.

SHP2 Tyrosine Phosphatase Enhances CEBPA Gene Expression, STAT3 Activation, and Cytokine-Dependent Granulopoiesis.

AbstractAbstract 1550Culture of individual murine granulocyte-monocyte progenitors (GMP) with myeloid cytokines demonstrates that M-CSF or G-CSF help direct monocyte versus granulocyte lineage specification (T. Schroeder and colleagues, Science, 2009). We evaluated signaling pathways induced in murine, lineage-negative marrow cells to demonstrate that M-CSF more potently induces ERK activation via PLCγ and PKC whereas G-CSF more potently activates STAT3 and induces SHP2 phosphorylation on tyrosines 542 and 580, potentially increasing SHP2 activity or altering its substrate specificity (Jack et al 2009). In the same study, we found that chemical inhibition of ERK reduces CFU-M but not CFU-G, whereas exposure of marrow cells to the SHP1/SHP2 inhibitor NSC-87877 reduces CFU-G but not CFU-M. SHP2 indirectly activates ERK, and NSC-87877 prevented ERK phosphorylation induced by G-CSF but not M-CSF, underscoring the specificity of G-CSF for SHP2 activation. We postulate that constitutively active SHP2 found in JMML cases excessively activates ERK to enable a monocytic phenotype. To further investigate the role of SHP2 in normal granulopoiesis, we screened a panel of lentiviral vectors expressing SHP2 shRNAs for their ability to reduce SHP2 protein expression in the murine 32Dcl3 granulocyte progenitor cell line. shRNA77 reduced SHP2 expression 5-fold in pooled transductants, whereas the other shRNAs produced at most 3-fold inhibition. Five 32Dcl3-shRNA77 subclones were obtained, two with 90%, one with 80%, and two with approximately 65% reduction in SHP2 levels. Proliferation in IL-3 correlated inversely with the degree in SHP2 knockdown. Expression of myeloperoxidase and lactoferrin RNAs were reduced 24 hrs after transfer from IL-3 to G-CSF in each subclone, in comparison to cells transduced with the empty pLKO.1 lentiviral vector, again correlated with the degree of SHP2 knockdown. C/EBPα RNA and protein levels were markedly reduced in all five subclones, whereas PU.1 RNA or protein or RUNX1 protein levels were unaffected. As expected, knockdown of SHP2 reduced ERK activation, and STAT3 activation was also reduced in the two shRNA77 sublcones evaluated. We also developed two 32Dcl3 subclones containing >90% knockdown of both ERK1 and ERK2, using separate shRNAs, and demonstrated that these lines each retain normal levels of C/EBPα protein, normal STAT3 activation, and normal induction of MPO, PR3, and LF by G-CSF, despite have markedly reduced proliferation. Thus, reduced C/EBPα RNA and protein expression or STAT3 activation as a consequence of SHP2 knockdown is not due to loss of ERK activation or to reduced growth rate. Lineage-negative murine marrow cells cultured in TPO, Flt3 ligand, and SCF were transduced with shRNA77 or empty vector. Cell numbers expanded approximately 7-fold in each group during this period. Upon transfer to IL3, IL6, and SCF, control cells continued to expand whereas shRNA77-transduced cells showed little proliferation in liquid culture. SHP2 protein levels were reduced 3-fold. Quantitative RT-PCR analysis after 48 hrs in IL3/IL6/SCF demonstrated that the granulocytic markers MPO, PR3, LF, NE, and C/EBPε were significantly reduced (p < 0.05) and the monocytic marker CD68 was significantly increased (p < 0.05) in cells transduced with shRNA77 compared to empty vector, and there was a significant increase in Mac-1+Gr1- monocytes compared with Mac-1+Gr1+ granulocytes, each in three independent experiments. SHP2 shRNA77 reduced CFU-G from 75% to 50% of total CFU-G + CFU-M, again in three experiments (p = 0.005). Knockdown of SHP2 in marrow cells with shRNA75 also reduced C/EBPα protein levels and expression of granulocytic markers. In summary, SHP2 RNA knockdown extends prior results obtained using a chemical inhibitor to support the conclusion that SHP2 is required for granulocyte lineage commitment and maturation. Reduced C/EBPα expression and/or STAT3 activation provides a mechanistic basis for this finding, with a novel SHP2 substrate rather than ERK activation likely responsible. Disclosures:No relevant conflicts of interest to declare.

Mir-9 Is Aberrantly Expressed In MPN Patients and Accelerates Erythropoietic Cell Growth and Differentiation

AbstractAbstract 1983The myeloproliferative neoplasms (MPNs), PV, ET and IMF, harbor a common gain-of-function mutation of JAK2V617F but in the past several years other molecular lesions have been noted as well such as mutation of TET2, chromsomal gains and losses, aberrant mRNA expression and aberrant microRNA expression. Since miRNAs are important regulators of hematopoiesis, we measured miRNA expression levels in CD34+ cells isolated from 8 patient samples (4 PV with JAK2V617F, 3 ET with wild-type JAK2 and 1 IMF with unknown JAK2 status) and 4 healthy controls using a Taqman Low Density Array (TLDA) representing 667 known miRNAs. We identified 28 miRNAs that were significantly deregulated in MPN patients compared to controls (p<0.05). Many of these miRNAs (18/24; 75%) were also deregulated in an independent miRNA array study using MPN patient granulocytes (n=39, 25 PV, 14 ET). Among these miRNAs, miR-9 expression was significantly upregulated in MPN patient CD34+ cells as well as in mature granulocytes. Furthermore, miR-9 expression was responsive to modulation of JAK2 activity in both gain of function (overexpression of JAK2V617F in TF-1 cells) and loss of function (inhibition of JAK2 activity in HEL cells) systems, suggesting that miR-9 may be dysregulated by aberrant JAK2V617F signaling. Analysis of miR-9 expression in ex-vivo, uni-lineage human CD34+ cultures, revealed that miR-9 is expressed at low levels during erythropoiesis, while levels gradually increase during myeloid differentiation. To determine how deregulated expression of miR-9 affected erythropoiesis, we transduced human bone marrow CD34+ cells (AllCells) with empty vector or miR-9 lentivirus, and examined erythroid differentiation and proliferation in liquid culture over 12 days. miR-9 expressing cells displayed a consistent growth advantage (∼30%), and accelerated erythroid differentiation (CD71+, GlyA+) at days 7 and 9. HITS-CLIP is a high throughput miRNA target prediction method developed in the Darnell lab based on crosslinking Argonaute protein to miRNA and target mRNA, providing highly specific identification of direct miRNA targets. We previously identified genes differentially expressed in PV patient CD34+ cells compared to normal CD34+ cells using Affymetrix HG-U133A arrays (117 genes, p<0.05). A comparison of predicted miR-9 mRNA targets identified using HITS-CLIP in P13 mouse brain, with genes downregulated in PV patient microarray studies, identified KLF4 which encodes a zinc finger protein involved in pluripotency and YWHAZ a 14-3-3 protein involved in signaling pathways regulating proliferation and survival as potential miR-9 targets relevant to aberrant hematopoiesis in MPN. Dysregulated cell growth in MPN may result in part from aberrant miRNA expression. Disclosures:Carroll:Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.

Pathogenesis of Acute Myeloid Leukemia: In Vitro Studies Using Primary Human Cells.

Many cases of acute myeloid leukemia (AML) are associated with non-random chromosomal rearrangements and most of these result in fusions involving retinoic acid receptor α (RARα), CBF transcription factors, MLL, and nucleoporins. Here, we report the effects of key members of these four major groups of AML-associated chimeric fusion proteins on differentiation, proliferation, self-renewal and gene expression in primary human CD34+ hematopoietic cells. We expressed the PML-RARα, AML1-ETO, MLL-AF9 and NUP98-HOXA9 fusion genes in human peripheral blood CD34+ cells by retroviral transduction and compared them to cells transduced with empty vector. By colony forming assays, morphological examination and flow cytometric immunophenotyping we found that PML-RARα causes some degree of myeloid differentiation block. AML1-ETO had no obvious effect on differentiation, while MLL-AF9 and NUP98-HOXA9 caused a block in both myeloid and erythroid differentiation. All fusion oncoproteins, except PML-RARα, induced dramatic long-term proliferation in liquid culture and a marked increase in the numbers of primitive long-term culture-initiating cells (LTC-ICs). In order to understand the molecular basis of these effects, gene expression profiling was performed for each fusion gene by oligonucleotide microarray analysis at 3 different time points (6 h, 3 days, and 8 days post transduction). At the 6 h time point both AML1-ETO and PML-RARα caused extensive changes in gene expression with a predominance of downregulated genes. The number of genes dysregulated by AML1-ETO and PML-RARα decreased dramatically at the 3-day time point. These data are consistent with the fact that these two oncoproteins are DNA-binding transcription factors that are known to repress transcription. In contrast, MLL-AF9 and NUP98-HOXA9 altered the expression of fewer genes at the 6 h time point with a preponderance of upregulated genes; at the 3 day time point, the number of genes dysregulated by NUP98-HOXA9 and MLL-AF9 increased. These findings suggest that NUP98-HOXA9 and MLL-AF9 may have delayed effects that are not due to direct transcriptional regulation. Homeobox transcription factors were upregulated by both MLL-AF9 and NUP98-HOXA9, but not by either AML1-ETO or PML-RARα. The results of the biological assays and gene profiling show marked similarities between NUP98-HOXA9 and MLL-AF9, and suggest that they transform cells by similar pathways that are different from those used by AML1-ETO and PML-RARα.

DNMT3b's Role in Hematopoietic Stem Cell Self-Renewal.

DNA methylation is an epigenetic means of gene regulation and is carried out by a family of methyltransferases of which DNMT1 acts to maintain methylation marks following DNA replication and DNMT3a and DNMT3b methylate DNA de novo. DNMT3b has been shown to be essential for mammalian development and necessary for differentiation of germline and neural progenitor cells. Mutations of DNMT3b in humans lead to a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability, and facial abnormalities. We have shown by real-time, RT-PCR that DNMT3b mRNA is uniquely over-expressed by approximately 30-fold in immunophenotypically-defined longterm repopulating hematopoietic stem cells (HSCs) that are CD34−lineage−c-kit+Sca-1+ as compared to progenitor and differentiated cell types within the bone marrow and with respect to the other members of the DNMT family, namely DNMT1 and DNMT3a. To determine DNMT3b's function in HSCs competitive bone marrow transplantation was undertaken. Isolated lineage− enriched bone marrow cells were transduced with a retroviral backbone based on the Murine Stem Cell Virus (MSCV) carrying either GFP and a short, hairpin RNA (shRNA) targeting DNMT3b or GFP alone. Following transduction 1×105 GFP+ cells along with 1×105 competitor cells were transplanted into 9.5 Gray irradiated congenic recipients. Two months following transplantation mice receiving bone marrow cells transduced with DNMT3b shRNA showed a significantly lower engraftment of donor cells as a percentage of total competitor cell engraftment in the peripheral blood as compared to those receiving cells transduced with GFP alone (24.8 vs 3.7, p<0.05) which persisted at 3 months (22.8 vs 1.5, p<0.05). Similarly, within the donor derviced cells in the peripheral blood there was a lower percentage of myeloid (CD11b+) cells at 2 and 3 months in the recipients of DNMT3b shRNA transduced cells as compared to controls. However there was no observed difference in the percentage of peripheral B (CD45R+) or T (CD3+) cells within the donor-derived cells. To determine the mechanism behind the observed engraftment defect with DNMT3b knockdown we cultured GFP+ transduced bone marrow cells in vitro with minimal cytokine support. As a control for our targeting methodology we also transduced bone marrow cells from mice harboring two floxed DNMT3b alleles with a MSCV carrying Cre recombinase and GFP. While lineage− bone marrow cells transduced with GFP alone increased 10-fold in number over two weeks of culture, cells in which DNMT3b was down regulated by shRNA or Cre-mediated recombination only doubled. Culture of lineage− bone marrow cells in methylcellulose medium by the colony-forming cell (CFC) assay revealed increases in the granulocytic and total number of colonies with DNMT3b knockdown or Cre-mediated recombination of DNMT3b similar to the increased myeloid engraftment of DNMT3b shRNA transduced cells observed 1 month following competitive bone marrow transplantation. However when 5,000 of these cells from the first CFC assay were sub-cultured there was a significant loss of colony forming ability within all lineages when DNMT3b was targeted by shRNA or Cre-mediated recombination. Taken together with the decreased engraftment of DNMT3b shRNA cells following competitive bone marrow transplantation, the observed limited proliferation in liquid culture and loss of colony forming ability during serial CFC assays is suggestive of a self-renewal defect of HSCs in the absence of DNMT3b, that was previously only reported in the absence of both DNMT3a and DNMT3b. Further elucidation of this proposed self-renewal defect is being undertaken and results of ongoing studies including long-term culture initiating cell (LTC-IC) assays and identification of genomic sites of DNA methylation within different hematopoietic subsets will also be presented.

The MLL PHD Fingers of MLL Block MLL Fusion Protein Mediated Transformation.

Mixed Lineage Leukemia (MLL) is a histone H3K4 methyltransferase that is rearranged in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). MLL is required for the maintenance of Hox gene expression. Deregulation of Hox genes by MLL fusion proteins, which fuse MLL in frame to one of over 50 different translocation partners, is critical for transformation. In these translocations, the DNMT homology (CXXC) domain is always included, but the set of adjacent plant homeodomains (PHD), which includes four PHD fingers and a bromodomain, is invariably excluded. PHD fingers have recently been described to bind tri-methylated histone H3K4 and others report PHD domains binding transcriptional co-repressors, such as Mi-2a of the NuRD complex. However, the role of the PHD fingers in MLL is not well understood. To determine the function of the PHD fingers in MLL, we performed bone marrow transduction and colony assays with the MLL fusion protein MLL-AF9, engineered to contain the PHD domain region (MLL-PHD-AF9). These experiments showed that inclusion of the PHD fingers inhibited immortalization as shown by the absence of compact colonies in methylcellulose replating assays and inhibition of proliferation in liquid cultures. Initial experiments with PHD finger deletions to map the inhibiting activity suggest inclusion of any PHD fingers beyond the first PHD finger, results in inhibition of transformation. To monitor the transcriptional activity of the retrovirally infected bone marrow cells, total RNA was isolated from cells harvested after the second replating, when significant differences were seen in colony morphology and size. Consistent with the transformation inhibition, Hoxa9 gene expression was found to be significantly repressed with respect to expression detected in transformed MLL-AF9 cells as determined by qPCR. To confirm this effect is directly due to the MLL fusion proteins, we performed luciferase assays with an MLL responsive myc E-box luciferase construct in MLL −/− MEFs. We found a specific and robust activation of the reporter in the presence of MLL-AF9, which was severely compromised by the inclusion of the PHD fingers. Together, these results suggest the PHD fingers act as transcriptional repressors that inhibit transformation. Our results provide an explanation for the finding that translocations including the coding region for C terminal PHD fingers do not occur in human leukemias and suggest that this region is also involved in the regulation of wild type MLL. We are currently studying the mechanisms of transcriptional repression mediated by the PHD fingers by isolating interacting proteins and assessing their effect on transcription and transformation.

Accurate, precise modeling of cell proliferation kinetics from time-lapse imaging and automated image analysis of agar yeast culture arrays

BackgroundGenome-wide mutant strain collections have increased demand for high throughput cellular phenotyping (HTCP). For example, investigators use HTCP to investigate interactions between gene deletion mutations and additional chemical or genetic perturbations by assessing differences in cell proliferation among the collection of 5000 S. cerevisiae gene deletion strains. Such studies have thus far been predominantly qualitative, using agar cell arrays to subjectively score growth differences. Quantitative systems level analysis of gene interactions would be enabled by more precise HTCP methods, such as kinetic analysis of cell proliferation in liquid culture by optical density. However, requirements for processing liquid cultures make them relatively cumbersome and low throughput compared to agar. To improve HTCP performance and advance capabilities for quantifying interactions, YeastXtract software was developed for automated analysis of cell array images.ResultsYeastXtract software was developed for kinetic growth curve analysis of spotted agar cultures. The accuracy and precision for image analysis of agar culture arrays was comparable to OD measurements of liquid cultures. Using YeastXtract, image intensity vs. biomass of spot cultures was linearly correlated over two orders of magnitude. Thus cell proliferation could be measured over about seven generations, including four to five generations of relatively constant exponential phase growth. Spot area normalization reduced the variation in measurements of total growth efficiency. A growth model, based on the logistic function, increased precision and accuracy of maximum specific rate measurements, compared to empirical methods. The logistic function model was also more robust against data sparseness, meaning that less data was required to obtain accurate, precise, quantitative growth phenotypes.ConclusionMicrobial cultures spotted onto agar media are widely used for genotype-phenotype analysis, however quantitative HTCP methods capable of measuring kinetic growth rates have not been available previously. YeastXtract provides objective, automated, quantitative, image analysis of agar cell culture arrays. Fitting the resulting data to a logistic equation-based growth model yields robust, accurate growth rate information. These methods allow the incorporation of imaging and automated image analysis of cell arrays, grown on solid agar media, into HTCP-driven experimental approaches, such as global, quantitative analysis of gene interaction networks.

RAPID ENLARGEMENT OF LILY BULBLET BY BIOREACTOR CULTURE

This study was conducted to investigate the optimal culture condition of lily bulblets in a bioreactor. Asiatic hybrid ‘Mirr’ was used to analyze the effects of growth regulator, sucrose concentration, and charcoal on the endogenous contents of ABA, t-zeatin, starch, and starch accumulation in bulblets. Treatment with growth regulator, BA 0.1 mg L -1 and IAA 0.1 mg L -1 , resulted in the highest bulblet proliferation in liquid culture with 5.8 bulblets per scale. Addition of 6% sucrose with 0.1% charcoal to the MS medium showed three times higher bulblet enlargement with 8.4 g per bulblet, compared to 3% sucrose alone at 2.7 g/bulblet. Starch content was higher with 9% sucrose with 0.1% charcoal, and accumulation of starch in the cell showed subsequent increases with sucrose concentration in the medium. The starch accumulation was higher in solid agar medium than in the liquid medium. ABA content was lower at 3% sucrose, than other concentrations. This was probably due to a relationship between the bulblet enlargement and endogenous ABA content. The t-zeatin content was highest in the 6% sucrose and 0.1% charcoal treatment. A liquid culture method using an Ebb & flow system resulted in better bulblet enlargement, especially in 6% sucrose treatments, than the ordinary liquid culture method.

Cholinergic Stress Responses Facilitate the Molecular Mechanisms Underlying Granulocytosis.

Glucocorticoid-initiated granulocytosis following trauma persists long after cortisol levels decrease, through yet unknown modulators of hematopoiesis. An intriguing candidate for such a modulatory role is the AChE-R splice variant of acetylcholinesterase. In brain and muscle a functional glucocorticoid response element (GRE) in the distal promoter of the ACHE gene induces overexpression of AChE-R following trauma. To explore the possibility that circulating and/or blood cells-associated AChE-R regulates sustained granulocytosis following stress, we employed labor and childbirth as the paradigm for acute, transient stress. In intra-partum women, increased AChE-R expression in granulocytes was observed by flow cytometry, which correlated significantly with their high cortisol levels and elevated AChE activity in plasma. In post-partum women, leukocyte counts, AChE-R positive granulocytes and AChE activity in plasma remained high for several days after cortisol levels declined. Furthermore, exposure to ARP, a synthetic peptide with the C-terminal sequence of AChE-R, promoted AChE gene expression in human CD34+ progenitors and facilitated their efficient proliferation in liquid cultures (1) in an antisense suppressible manner, supporting auto-regulatory prolongation of AChE-R expression. The intracellular interaction of the C-terminal domain of AChE-R with RACK1 and protein kinase Cε (2) supports such a mechanism. In 2 weeks cultures of CD34+ HPC, 2nM ARP induced granulocyte expansion and maturation. Additionally, exposure to ARP for 24 hours facilitated the production of pro-inflammatory cytokines interleukin (IL)-6, IL-10 and TNFα in adult PB MNC and enhanced the engraftment of CD34+ cells in NOD/SCID mice. We conclude that stress-induced AChE-R accumulation and consequent inflammatory reactions facilitate prolonged granulocytosis.

Exogenous C/EBPα Increases the Monocytic Maturation of Normal Murine Myeloid Progenitors.

Within hematopoiesis C/EBPα is expressed specifically in both the granulocytic and monocytic myeloid lineages. Mice lacking C/EBPα have reduced neutrophil and monocyte production, due in part to diminished expression of the G-CSF, M-CSF, and GM-CSF receptor mRNAs. C/EBPα-ER directs the granulocytic differentiation of 32Dcl3 cells, and induction of late markers of granulopoiesis by C/EBPα-ER in 32Dcl3 cells was dependent upon its ability to slow G1 to S progression. We have now assessed the effect of C/EBPα-ER on the myeloid maturation of murine marrow progenitors. Regulation of the transgene using estradiol avoids biasing cell populations during transduction. Mononuclear cells isolated from 5-FU-treated mice were transduced with pBabePuro-C/EBPαER and selected for 2 days with puromycin. Surviving cells were isolated using a density gradient, subjected to lineage-depletion, which removed 98% of Mac1+ and Gr1+ cells, and placed in liquid or methylcellulose culture with IL3/IL6/SCF +/− estradiol. By 48 hrs, 80% of the cells had acquired myeloid surface markers. Mac1+/Gr1+ cells were granulocytes and Mac1+/Gr1− cells were monocytes, as confirmed by Wright’s-Giemsa staining of cytospins. Estradiol increased the proportion of monocytes 1.7-fold and reduced the proportion of granulocytes 2.5-fold in 5 independent assessments. Real-time PCR analysis indicated a 5-fold increase in CD14 and CD68/macrosialin RNA, a 2-fold increase in M-CSF receptor and PU.1 RNA, and a 4-fold decrease in NE RNA by 48 hrs. In contrast, estradiol had little effect on the development of monocytes or granulocytes from vector, pBabePuro-transduced cells. Consistent with these findings, in an initial experiment we observed increased granulocytes when progenitors from floxxed C/EBPα mice (kindly provided by P. Johnson) were transduced with pBabeNeo-CRE, compare with pBabeNeo, subjected to lineage-depletion, and cultured in IL3/IL6/SCF. Loss of C/EBPα was demonstrated by PCR and Western blotting. During the first 48 hrs after transfer of transduced wild-type cells to estradiol, C/EBPα-ER slowed the proliferation of myeloid progenitors 2.6-fold, increased their G1/S ratio 2.8-fold, and reduced myeloid CFUs 5.1-fold. CFU-M, CFU-G, and CFU-GM were affected equally. Transduction with C/EBPαL12V-ER, harboring a defective leucine zipper, or with C/EBPαBR3-ER, mutant in 4 basic region residues, slowed proliferation in liquid culture 2-fold, reduced myeloid CFUs 1.5-fold, and had little effect on the G1/S ratio, perhaps indicating a role for DNA-binding in the regulation of the cell cycle by C/EBPα in normal myeloid progenitors (CFUs) and precursors. The L12V variant did not affect lineage output, whereas C/EBPαBR3 reduced monocytic maturation 1.4-fold and increased granulocyte formation 1.4-fold, in 4 experiments. C/EBPαp30, expressed in a subset of AML cases and lacking an N-terminal transactivation domain, increased the G1/S ratio and reduced myeloid CFUs 1.6-fold and had no effect on myeloid maturation. In summary, exogenous C/EBPα increases the monocytic and reduces the granulocytic maturation of transduced myeloid progenitors cultured in cytokines whose receptors are not regulated by C/EBPα. Cell cycle inhibition may play a role in this lineage bias, as the the L12V and p30 variants had both a reduced ability to slow G1 progression and to induce monocytes. Induction of PU.1 may also favor monocytic maturation. Finally, results with BR3 compared with L12V, neither of which bind DNA, suggest that alteration of a protein interaction with the non-DNA contact surface of the C/EBPα basic region favors granulocyte formation.

Proliferation deficiency of multipotent hematopoietic progenitors in ribosomal protein S19 (RPS19)-deficient diamond–Blackfan anemia improves following RPS19 gene transfer

Diamond–Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Since some patients with DBA develop a reduction in thrombocytes and granulocytes with age, we asked whether multipotent hematopoietic progenitors from DBA patients had normal proliferative capacity in liquid expansion cultures. CD34+ cells derived from DBA patients showed deficient proliferation in liquid culture containing IL-3, IL-6, and SCF. Single CD34+ CD38− cells from DBA patients exhibited deficient proliferation recruitment in a limiting dilution assay containing IL-3, IL-6, SCF, Tpo, FL, and G-CSF or containing IL-3, IL-6, and SCF. Our findings suggest that the underlying hematopoietic defect in DBA may not be limited to the erythroid lineage. Since a fraction of DBA patients have a deficiency in ribosomal protein S19 (RPS19), we constructed lentiviral vectors containing the RPS19 gene for overexpression in hematopoietic progenitors from RPS19-deficient DBA patients. Enforced expression of the RPS19 transgene improved the proliferation of CD34+ cells from DBA patients with RPS19 mutation. Similarly, enforced expression of RPS19 improved erythroid development of RPS19-deficient hematopoietic progenitors as determined by colony assays and erythroid differentiation cultures. These findings suggest that gene therapy for RPS19-deficient DBA is feasible.

Effect of Simvastatin Alone and in Combination With Cytosine Arabinoside on the Proliferation of Myeloid Leukemia Cell Lines

BackgroundCholesterol biosynthesis is regulated by the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Cholesterol and its derivatives are required in high concentrations by neoplastic proliferating cells for both DNA synthesis...

Purification, amplification and characterization of a population of human erythroid progenitors.

In humans, studies of the erythroid cell lineage are hampered by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, these progenitors in bone marrow or peripheral blood are scarce and no specific antibodies are available. We describe a new method which allows proliferation in liquid culture of large numbers of pure normal human erythroid progenitors. CD34+ cells were cultured for 7 d in serum-free conditions with the cytokine mixture interleukin (IL)-3/IL-6/stem cell factor (SCF). This resulted in cell expansion and the appearance of a high proportion of CD36+ cells which were purified on day 7. Methylcellulose clones from these cells were composed of 96.6% late BFU-E and 3.4% CFU-GM. These CD36+ cells could be recultured with the same cytokine mixture plus or minus erythropoietin (Epo) for a further 2-7 d. In both conditions further amplification of CD36+ cells was observed, but Epo induced a more dramatic cell expansion. Glycophorin-positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 d of secondary culture. Cells obtained from adult CD34+ progenitors mostly contained adult haemoglobin, whereas cord blood-derived cells contained equal proportions of adult and fetal haemoglobin. Activation of STAT5 and tyrosine phosphorylation of the Epo receptor and JAK2 were observed after Epo stimulation of these cells. This new method represents a straightforward alternative to the procedures previously described for the purification of normal erythroid progenitors and is useful in the study of erythropoietic regulation.