Dexamethasone and lenalidomide have distinct functional effects on erythropoiesis
ABSTRACT: We explored the effects of dexamethasone and lenalidomide, individually and in combination, on the differentiation of primary human bone marrow progenitor cells in vitro. Both agents promote erythropoiesis, increasing the absolute number of erythroid cells produced from normal CD34+ cells and from CD34+ cells with the types of ribosome dysfunction found in DBA and del(5q) MDS. However, the drugs had distinct effects on the production of erythroid progenitor colonies; dexamethasone selectively increased the number burst-forming units-erythroid (BFU-E), while lenalidomide specifically increased colony-forming units-erythroid (CFU-E). Use of the drugs in combination demonstrates that their effects are not redundant. Human CD34+ cells were cultured for 48 hours in the erythroid differentiation media described above with the addition of 40 ng/mL of FMS-like tyrosine kinase 3 (flt-3; Miltenyi Biotech) and 15 ng/mL of Granulocyte colony-stimulating factor (G-CSF; Amgen). Cells were cultured in the presence of the drugs of interest for 24 hours. RNA was purified using Trizol (Invitrogen). RNA was amplified and labeled by in vitro transcription and hybridized to the Affymetrix HT Human Genome U133A Array. Three replicates per condition.
Project description:Burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45+GPA-IL-3R-CD34+CD36-CD71low and CD45+GPA-IL-3R-CD34-CD36+CD71high phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity ~90%. The BFU-E colony forming ability of CD45+GPA-IL-3R-CD34+CD36-CD71low cells required SCF and erythropoietin, while the CFU-E colony forming ability of CD45+GPA-IL-3R-CD34-CD36+CD71high cells required only erythropoietin. Bioinformatic analysis of the RNA-seq data revealed unique transcriptomes in each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematological disease. Our data provide important resource for future studies. Transcription profiles of Human erythroid progenitors at distinct developmental stages were generated by deep sequencing, in triplicate, using IlluminaHiSeq 2000. The complete dataset comprises 4 sample types: CD34, BFU, CFU, and Pro (reanalysis of GSM1304777-GSM1304779).
Project description:The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their anti-tumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the anti-myeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to and putatively resistant to lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity, and a possible biomarker for the clinical assessment of anti-myeloma efficacy. We included 15 samples from multiple myeloma cell lines.
Project description:The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their anti-tumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the anti-myeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to and putatively resistant to lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity, and a possible biomarker for the clinical assessment of anti-myeloma efficacy. We included two isogenic MM1.S cell lines, which differ in the sensibiligy to lenalidomide. We included MM1.S and MM1.S res, which were sensitive and resistant to lenalidomide, respectively.
Project description:Expression of P190 and P210 BCR/ABL1 in normal human CD34(+) cells induces similar gene expression profiles and results in a STAT5-dependent expansion of the erythroid lineage The P190 and P210 BCR/ABL1 fusion genes are mainly associated with different types of hematologic malignancies, but it is presently unclear whether they are functionally different following expression in primitive human hematopoietic cells. We investigated and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood. Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under in vitro conditions favoring B-cell differentiation, neither P190 nor P210 BCR/ABL1-expressing cells formed detectable levels of CD19-positive cells. Gene expression profiling revealed that P190 BCR/ABL1 and P210 BCR/ABL1 induced almost identical gene expression profiles, and we identified a common set of 222 differentially expressed genes. Our data suggest that the early cellular and transcriptional effects of P190 BCR/ABL1 and P210 BCR/ABL1 expression are very similar when they are expressed in the same human progenitor cell population, and that STAT5 is an important regulator of BCR/ABL1-induced erythroid cell expansion. Keywords: global gene expression profiling, BCR/ABL1, CD34+ cord blood cells, CML, Ph+ ALL Overall design: The transcriptional effects of retroviral expression of P190 and P210 BCR/ABL1 in human cord blood (CB) CD34+ cells were compared to the expression profiles of CD34+ control cells transduced with an empty vector. Total RNA was isolated from sorted GFP+ cells isolated at day 2, 3, and 4 posttransduction in two independent experiments. RNA extraction, labeling, hybridization, washing, scanning, and feature analysis were performed as described (Järås et al., 2009, Exp Hematol).
Project description:CD34+ hematopoietic stem/progenitor cells (HSC) reside in the bone marrow in close vicinity to the endosteal bone surface, surrounded by osteoblasts, stromal cells and various extracellular matrix molecules. We utilized a bioartificial matrix containing fibrillar collagen I, the major matrix component of bone, as scaffold for ex vivo expansion of HSCs. CD34+ HSCs were isolated from umbilical cord blood and cultivated within reconstituted collagen I fibrils in presence of FLT3-ligand, SCF and IL-3. After seven days of culture cell number, colony-forming units and gene expression profile of the cultured cells were assessed. Although the total expansion factor of CD34+ cells was slightly lower when cells were cultivated in the collagen I gel, the frequency of colony-forming units (CFU-C) increased compared to control suspension cultures. Gene expression analysis with microarray chip technology revealed the upregulation of more than 50 genes in presence of collagen I. Among them, genes for several growth factors, cytokines and chemokines (e.g. interleukin 8, MIP1-α) could be confirmed by quantitative PCR. Furthermore, increased expression of the negative cell-cycle regulator BTG2/TIS21 and an inhibitor of MAP kinase pathway, DUSP2, underline the regulatory role of the extracellular matrix. Together, these data show that the expansion of CD34+ cord blood cells in a culture system containing a three-dimensional collagen I matrix induces a qualitative change in the gene expression profile of cultivated HSCs. Experiment Overall Design: Gene expression profiling of CD34+ hematopoietic cells expanded in a collagen I matrix
Project description:The serine threonine kinase Stk40 has been shown to involve in mouse embryonic stem cell differentiation, pulmonary maturation and adipocyte differentiation. Here we report that targeted deletion of Stk40 leads to fetal liver hypoplasia and anemia in the mouse embryos. The reduction of erythrocytes in the fetal liver is accompanied by increased apoptosis and compromised erythroid maturation. Stk40-/- fetal liver cells have significantly reduced colony forming units (CFUs) capable of erythroid differentiation, including burst forming unit-erythroid (BFU-E), colony forming unit-erythroid (CFU-E), and CFU-granulocyte, erythrocyte, megakaryocyte and macrophage (CFU-GEMM), but not CFU-granulocyte/macrophages (CFU-GM). Purified Stk40-/- megakaryocyte-erythrocyte progenitors (MEPs) produced substantially fewer CFU-E colonies compared to control cells. Moreover, Stk40-/- fetal liver erythroblasts failed to form normal erythroblastic islands in association with wild type or Stk40-/- macrophages, indicating an intrinsic defect of Stk40-/- erythroblasts. Furthermore, the hematopoietic stem and progenitor cell pool is reduced in Stk40-/- fetal livers but still retains the multi-lineage reconstitution capacity. Finally, analysis of microarray data of E14.5 fetal liver cells suggests a potential role of aberrantly activated TNF-α signaling in Stk40 depletion induced dyserythropoiesis with a concomitant increase in cleaved Caspase-3 and decrease in Gata1 proteins. Altogether, the identification of Stk40 as a regulator for fetal erythroid differentiation, maturation and survival provides new clues to the molecular regulation of erythropoiesis and related diseases. Overall design: Six E14.5 mouse fetal liver cells of same genotype were pooled together for each sample. There are two replicates for each genetype. mRNA extraction and hybridization on Affymetrix microarrays were performed by Shanghai Biochip Company.
Project description:Schilling2009 - ERK distributive
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Theoretical and experimental
analysis links isoform-specific ERK signalling to cell fate
Schilling M, Maiwald T, Hengl S,
Winter D, Kreutz C, Kolch W, Lehmann WD, Timmer J,
Mol. Syst. Biol. 2009; 5: 334
Cell fate decisions are regulated by the coordinated
activation of signalling pathways such as the extracellular
signal-regulated kinase (ERK) cascade, but contributions of
individual kinase isoforms are mostly unknown. By combining
quantitative data from erythropoietin-induced pathway
activation in primary erythroid progenitor (colony-forming unit
erythroid stage, CFU-E) cells with mathematical modelling, we
predicted and experimentally confirmed a distributive ERK
phosphorylation mechanism in CFU-E cells. Model analysis showed
bow-tie-shaped signal processing and inherently transient
signalling for cytokine-induced ERK signalling. Sensitivity
analysis predicted that, through a feedback-mediated process,
increasing one ERK isoform reduces activation of the other
isoform, which was verified by protein over-expression. We
calculated ERK activation for biochemically not addressable but
physiologically relevant ligand concentrations showing that
double-phosphorylated ERK1 attenuates proliferation beyond a
certain activation level, whereas activated ERK2 enhances
proliferation with saturation kinetics. Thus, we provide a
quantitative link between earlier unobservable signalling
dynamics and cell fate decisions.
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Project description:Forced expression of Gata1, Tal1, Lmo2 and c-Myc reprograms murine adult fibroblasts into erythroid progenitor cells. The resulting cells, called induced erythroid progenitors/precursors (iEPs), resemble bona fide erythroid cells in terms of morphology, colony forming capacity, and gene expression. We used microarrays to characterize the reprogrammed cells (iEP_red and iEP_non-red) at the molecular level and compare their global gene expression profile with bona fide erythroid cells (FL and BM BFU-Es) and starting fibroblasts (Fibs). Overall design: To obtain mRNA from pure cell populations, BFU-E colony-forming assays were performed on day 5-iEPs as well as on mouse E14.5 fetal liver cells and bone marrow cells. Eight days after, colonies were picked and processed for RNA extraction. Cultured fibroblasts were collected as controls.
Project description:Derivation and expansion of human umbilical cord blood-derived endothelial colony forming cells under serum-free conditions - a transcriptome analysis. Endothelial colony forming cells (ECFCs) were isolated from term umbilical cord blood units. ECFCs were expanded under standard, fetal bovine serum (FBS) containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture. Comparison of the expression patterns of ECFCs that were either cultured in FBS-containing medium or in serum-free medium (five replicates each).
Project description:In vivo changes of gene expression profiles (GEP) of tumor cells 48hr after single agent therapy may vary by treatment and provide added predictive power over baseline GEP information. In newly diagnosed patients with multiple myeloma (MM), GEP data were obtained on tumor cells prior to and 48hr after dexamethasone (n=45) or thalidomide treatment (n=42); in case of relapsed MM, GEP data were obtained prior to (n=36) and after (n=19) lenalidomide administration. Dexamethasone and thalidomide induced both common and unique GEP changes. Combined baseline and 48hr changes of GEP in a subset of genes that were discovered in newly diagnosed MM also predicted event-free and overall survival in relapsed patients receiving lenalidomide. Combined with baseline molecular features, changes in GEP following short-term single agent treatment may help guide treatment decisions for patients with MM. The genes whose altered expression is related to eventual survival may also point to mechanisms of action and resistance to different classes of drugs. Keywords: drug response Overall design: See above (Series_summary)