Regulation of AML1/RUNX1 Function by Protein Arginine Methyltransferase 4 (PRMT4) in Myeloid Differentiation

Abstract Abstract 549 RUNX1 (also known as AML1) is the DNA binding component of the Core Binding Factor (CBF)-transcriptional regulatory complex, which plays an important role in hematopoiesis. Upon binding to the common binding sequence -PyGpyGGTPy (Py = pyrimidine) in the regulatory regions of promoters and enhancers of its target genes, RUNX1 acts either as an activator or a repressor, depending on promoter context and its interacting partners. Thus, modulation of the network of RUNX1 interactions can influence hematopoiesis. However, how RUNX1 selects one set of partners over another to assemble a functional complex is largely unknown. Posttranslational modifications, including ubiquitination, phosphorylation, acetylation and methylation, present a viable mean to fine-tune its functions. Here we shown that RUNX1 is arginine methylated at a specific residue, R223, by PRMT4, a type I arginine methyltransferase generally thought of as a co-activator molecule. We hypothesized that arginine methylation of RUNX1 by PRMT4 affects its protein-protein interactions, therefore, to identify proteins that specifically interact with unmethylated and/or methylated-R223 RUNX1, in an unbiased manner, we performed a peptide pull-down experiment, using a methyl-R223 RUNX1 peptide and an unmodified RUNX1 peptide as bait, following by mass spectrometry analysis. We identified several proteins that preferentially interacted with the R223 methyl peptide, but focused on a novel interacting protein, DPF2 (double PhD Finger 2), which is a widely expressed member of the d4 protein family, characterized by the presence of a tandem plant-homodomain (PHD domain). We confirmed the specific interaction between methylated-RUNX1 with DPF2 in vivo by immunoprecipitation. We generated an antibody specific for the R223 methylated-RUNX1 protein, and found that RUNX1 methylation decreases during the myeloid differentiation of human CD34+ haematopoietic stem/progenitor cells (HSPCs), without a change in the total level of RUNX1 protein, and this occurred co-incident with a downregulation of PRMT4 protein expression. Having determined that PRMT4 expression declines during myeloid differentiation, we examined the role of PRMT4 in this process, using short hairpin RNAs to knockdown PRMT4 expression in CD34+ cells. Knockdown of PRMT4 accelerates the myeloid differentiation of the cells, whereas overexpression of PRMT4 in human CD34+ cells blocked their myeloid differentiation. When analyzing the expression of several “master” regulators of myeloid differentiation, we identified microRNA-223, a myeloid specific microRNA, as a common target gene of PRMT4 and RUNX1. Furthermore, we have found that by promoting the assembly of a functional complex containing R223 methylRUNX1 and DPF2 at the transcriptional regulatory region of the microRNA-223 promoter, PRMT4 can control miR-223 expression and myeloid differentiation. We have verified the role of DPF2 in this process, as DPF2 represses miR-223 expression and loss of DPF2 promotes myeloid differentiation. Thus, DPF2 acts in a common pathway with PRMT4 to regulate myeloid differentiation. In conclusion, our study elucidates a novel mechanism, where the arginine methylation of RUNX1 regulates its recruitment of interacting partner(s). In addition to demonstrating that PRMT4 can trigger repression of gene expression, we have identified a novel role for PRMT4 (aka CARM1) in myeloid differentiation. Disclosures: No relevant conflicts of interest to declare.

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Downregulation of Protein Arginine Methyltransferase – 4 (PRMT4) Promotes MiR-223 Expression and Myeloid Differentiation.

Blood ◽

10.1182/blood.v116.21.3632.3632 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3632-3632

Author(s):

Ly P Vu ◽

Xinyang Zhao ◽

Fabiana Perna ◽

Stephen D Nimer

Keyword(s):

Transcription Factors ◽

Binding Site ◽

Rna Binding ◽

Myeloid Differentiation ◽

Type I ◽

Protein Arginine Methyltransferase ◽

Arginine Methyltransferase ◽

Cd34 Cells ◽

Wide Range ◽

Arginine Residues

Abstract Abstract 3632 Protein arginine methyltransferase 4 is a Type I member of PRMT family, that catalyses the addition of a methyl-group to arginine residues of a wide range of proteins, including histones, transcription factors, and RNA binding proteins. PRMT4 has been shown to regulate gene expression through its interaction with various transcription factors and via methylation of numerous substrates. Although PRMT4 has been reported to play an important role in T cell development, lung development and adipocyte differentiation in mouse models, the function of PRMT4 during hematopoiesis has not been studied. To investigate the function of PRMT4 in the hematopoietic system, we utilized human CD34+ haematopoietic stem/progenitor cells (HSPCs). We observed that PRMT4 protein level is markedly downregulated during the myeloid differentiation of CD34+ cells without a significant change in the mRNA level. We then utilized a loss of function approach, using short hairpin RNAs, and found that knockdown of PRMT4 leads to an acceleration of myeloid differentiation, with a concomitant loss of the clonogenic potential of the cells. Interestingly, knocking down PRMT4 results in upregulation of miR-223, a myeloid specific microRNA. We also found that, during the myeloid differentiation of CD34+ cells, miR-223 expression steadily increased. Using a microRNA target prediction program, we identified a binding site for miR-223 in the 3′-UTR region of PRMT4 and found that when we over-expressed miR-223 in CD34+ cells, PRMT4 protein expression decreased. To determine the importance of PRMT4 downregulation in myeloid differentiation, we expressed the PRMT4-ORF (that should not be regulated by microRNAs) in CD34+ cells. The forced expression of PRMT4, that lacks the 3′-UTR region, leads to a block in myeloid differentiation and the inability of cells to up-regulate miR-223 during differentiation. Taken together, these data indicate a regulatory loop between PRMT4 and miR-223 that controls the differentiation of CD34+ toward the myeloid lineage. To examine how PRMT4 regulates transcription of miR-223, we examined the miR-223 locus and found a RUNX1 binding site in the promoter of pri-miR-223. We discovered that PRMT4 interacts with RUNX1 and methylates RUNX1 at a specific arginine residue. This results in the recruitment of several novel interacting partners, which appear to control the expression of miR-223. Thus our results indicate that PRMT4 regulate the transcription of miR-223 transcription via its effects on RUNX1. Our study demonstrates a novel function of PRMT4 in myeloid differentiation, through regulation of RUNX1 function and miR-223 expression. Disclosures: No relevant conflicts of interest to declare.

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An evolutionary recent IFN/IL-6/CEBP axis is linked to monocyte expansion and tuberculosis severity in humans

eLife ◽

10.7554/elife.47013 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 6

Author(s):

Murilo Delgobo ◽

Daniel AGB Mendes ◽

Edgar Kozlova ◽

Edroaldo Lummertz Rocha ◽

Gabriela F Rodrigues-Luiz ◽

...

Keyword(s):

Large Data ◽

Myeloid Differentiation ◽

Data Sets ◽

Type I ◽

Gene Module ◽

Evolutionary Selection ◽

Human Cd34 ◽

Cd34 Cells ◽

Mycobacterial Growth

Monocyte counts are increased during human tuberculosis (TB) but it has not been determined whether Mycobacterium tuberculosis (Mtb) directly regulates myeloid commitment. We demonstrated that exposure to Mtb directs primary human CD34+ cells to differentiate into monocytes/macrophages. In vitro myeloid conversion did not require type I or type II IFN signaling. In contrast, Mtb enhanced IL-6 responses by CD34+ cell cultures and IL-6R neutralization inhibited myeloid differentiation and decreased mycobacterial growth in vitro. Integrated systems biology analysis of transcriptomic, proteomic and genomic data of large data sets of healthy controls and TB patients established the existence of a myeloid IL-6/IL6R/CEBP gene module associated with disease severity. Furthermore, genetic and functional analysis revealed the IL6/IL6R/CEBP gene module has undergone recent evolutionary selection, including Neanderthal introgression and human pathogen adaptation, connected to systemic monocyte counts. These results suggest Mtb co-opts an evolutionary recent IFN-IL6-CEBP feed-forward loop, increasing myeloid differentiation linked to severe TB in humans.

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Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4

Biochemical Journal ◽

10.1042/bj20031176 ◽

2004 ◽

Vol 379 (2) ◽

pp. 283-289 ◽

Cited By ~ 47

Author(s):

Marie-Chloé BOULANGER ◽

Tina Branscombe MIRANDA ◽

Steven CLARKE ◽

Marco di FRUSCIO ◽

Beat SUTER ◽

...

Keyword(s):

Rna Binding ◽

Developmental Stages ◽

Rna Binding Proteins ◽

Genetic System ◽

Arginine Methylation ◽

Type I ◽

Protein Arginine Methyltransferases ◽

Arginine Residues ◽

Drosophila Protein

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 (Drosophilaarginine methyltransferases 1–9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

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The Role of Protein Arginine Methyltransferase 1 in Gastrointestinal Cancers

10.5772/intechopen.96197 ◽

2021 ◽

Author(s):

Jin Zou ◽

Wei Shen ◽

Yu Zhang ◽

Shibo Ying

Keyword(s):

Tumor Cells ◽

Cancer Progression ◽

Cancer Metastasis ◽

Arginine Methylation ◽

Gastrointestinal Tumors ◽

Migration And Invasion ◽

Arginine Methyltransferase ◽

Tumorigenic Mechanism ◽

Methyltransferase 1

Mammals can produce nine kinds of arginine methylation enzymes that can be divided into three types (I, II, and III) according to their catalytic activity. Arginine methyltransferase 1 (PRMT1), as the first discovered arginine methyltransferase type I, has been reported to be involved in cell signal transduction, DNA damage repair, RNA transcription and other processes. Its imbalance or abnormal expression is also involved in cancer metastasis. PRMT1 is highly expressed in gastrointestinal tumors and promotes tumor biomarkers expression, chemotherapy resistance and tumorigenicity to promote cancer progression, while downregulation of PRMT1 expression can inhibit the migration and invasion of related tumor cells or promote tumor cells apoptosis and inhibit the progression of cancer. Therefore, PRMT1 may be a cancer therapeutic target. In this paper, arginine methylase 1 expression in various types of gastrointestinal tumors, the tumorigenic mechanism and the role of PRMT1 in tumorigenesis and development were reviewed.

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MicroRNA hsa-mir-16 Contributes to Regulation of Myeloid Differentiation of Human CD34+ Cells.

Blood ◽

10.1182/blood.v108.11.1343.1343 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1343-1343

Author(s):

Richard Hildreth ◽

Robert W. Georgantas ◽

Roshan Patel ◽

Sebastien Morisot ◽

Jonathan Alder ◽

...

Keyword(s):

Protein Expression ◽

K562 Cells ◽

Negative Regulator ◽

Luciferase Reporter ◽

Myeloid Differentiation ◽

Expression Data ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Human Cd34 ◽

Cd34 Cells

Abstract In a large microRNA-array and bioinformatics study, we determined all of the microRNAs (miRs) expressed by human CD34+ hematopoietic stem-progenitor cells (HSPCs) from bone marrow and G-CSF mobilized blood. When we combined miR expression data, mRNA expression data fro a previous study (Georgantas et al, Cancer Research 64:4434), and data from various published mir-target prediction algorithms, we were able to bioinformaticly predict the actions of miRs within the hematopoietic system. MicroRNA hsa-mir-16 was highly expressed in CD34+ HSPCs, and was predicted to target several HSPC-expressed mRNAs (CXCR4, HoxB7, Runx-1, ETS-1, and Myb) that encode proteins known to be critically involved specifically in myelopoiesis within the hematopoietic system. We first confirmed that protein expression from each of these putative target mRNAs was in fact regulated by mir-16. The 3′UTR sequence from each of these mRNAs was cloned behind a luciferase reporter. Each reporter construct was transfected into K562 cells, which strongly express mir-16. In all cases, protein expression from the predicted target mRNA was greatly reduced in K562 cells, as compared to controls. As a first determination of mir-16’s function in hematopoietic cells, HL60 and K562 cells were transduced with hsa-mir-16 lentivirus, then treated with various chemical differentiation inducers. As was predicted by bioinformatics, hsa-mir-16 halted myeloid differentiation of HL60 cells, but did not affect megakaryocytic differentiation or erythroid differentiation of K562 cells. These initial findings suggest that mir-16 is a specific negative regulator of myelopoiesis. We are currently evaluating the effects of mir-16 on normal human CD34+ cells by in vitro CFC and suspension culture assays, as well as in vivo by transplantation of hsa-mir-16 lentivirus transduced cells in immunodeficient mice.

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Reduced HSPA9B Expression, a 5q31.2 Candidate Gene, in Primary Human CD34+ Cells Recapitulates Features of Ineffective Hematopoiesis Observed in MDS.

Blood ◽

10.1182/blood.v110.11.116.116 ◽

2007 ◽

Vol 110 (11) ◽

pp. 116-116

Author(s):

Tim H. Chen ◽

Mark Walshauser ◽

Amal Kambal ◽

Gagan Raju ◽

Matthew J. Walter

Keyword(s):

Culture Medium ◽

S Phase ◽

Myeloid Differentiation ◽

Intrinsic Defect ◽

Genetic Event ◽

Human Cd34 ◽

Control Shrna ◽

Cd34 Cells ◽

Key Features ◽

Number Of Cells

Abstract Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis. Deletions spanning chromosome 5q31.2 are among the most common karyotypic abnormalities in MDS, and evidence suggests that del(5q) maybe an early genetic event. We hypothesize that haploinsufficiency of one or more of the 28 candidate genes in the 5q31.2 commonly deleted segment (CDS) are important for MDS pathogenesis. Many genes in the CDS, including HSPA9B, have dose reduced mRNA levels in CD34+ cells from MDS patients with del(5q), and zebrafish carrying a heterozygous mutation in the orthologue of the human HSPA9B gene display increased apoptosis in blood cells. Therefore, we asked whether reduced HSPA9B expression in primary human hematopoietic cells can recapitulate key features of ineffective hematopoiesis (i.e. abnormal proliferation, apoptosis, and differentiation). To address this question, we purified human CD34+ hematopoietic progenitors from cord blood samples (>90% purity) and infected them with a shRNA expressing lentivirus that also carries the puromycin resistance gene. Cell culture densities were normalized after 4 days in selection medium, and 3 days later apoptosis (AnnexinV/7-AAD), cell cycle status (BrdU/7-AAD), and erythroid or myeloid differentiation was measured using flow cytometry. We performed 4–5 independent experiments using 5 individual shRNAs targeting HSPA9B (30% to >90% knockdown) and 2 control shRNAs. Cells transduced with control shRNAs expand 18.2 fold from days 4–7 in RBC unilineage differentiation culture medium (25ng/ml SCF, 10ng/ml IL-6, 10ng/ml IL-3 and 0.5U/ml Epo), vs. only a 0.8–5.2 fold expansion when HSPA9B is knocked down (N=5 for each shRNA, p≤0.007). The reduced cell numbers observed in HSPA9B knockdown cultures is associated with an increase in apoptosis and a decrease in the number of cells entering S-phase compared to control shRNA expressing cells. 8.2% of cells in control cultures were AnnexinV+/7AAD+ vs. 20–62% of cells in HSPA9B knockdown cultures (N=5 for each shRNA, p≤0.0004). Following 1 hour of BrdU exposure, 60% of cells in control cultures were in S-phase vs. only 22–48% of HSPA9B knockdown cultures (N=5 for each shRNA, p≤0.008). In addition, after 7 days in RBC unilineage differentiation culture medium, 58% of control shRNA expressing cells were CD71+/Gycophorin A+ vs. 7.1–32% of cells in the HSPA9B knockdown cultures (N=4 for each shRNA, p≤0.004). The decreased number of GPA+ cells in HSPA9B knockdown cultures was concomitant with retention of CD34+ expression on cells. Similar results were observed using myeloid unilineage culture conditions (10ng/ml SCF, 100ng/ml G-CSF, 20% FCS), where the number of cells expressing CD15+ was reduced from 47% in control cultures to 28–37% in HSPA9B knockdown cultures. A delay in erythroid and myeloid differentiation was confirmed by cell morphology. Lentiviral knockdown of the murine orthologue of HSPA9B (Hspa9a) in 2 independent transduction/transplantation experiments resulted in a loss of transduced cells over 2 months (2 shRNAs each) compared to a control shRNA, implicating a cell intrinsic defect when Hspa9a levels are reduced in vivo. Collectively, these results implicate that reduced HSPA9B expression in human CD34+ progenitor cells results in abnormal proliferation, increased apoptosis, and altered differentiation, key features of ineffective hematopoiesis, in a dose dependent manner.

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MKL1 Enhances Megakaryocytic Differentiation of Primary CD34+ Cells.

Blood ◽

10.1182/blood.v110.11.2218.2218 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2218-2218

Author(s):

Matthew J. Renda ◽

James A. Troy ◽

Ee-Chun Cheng ◽

Lin Wang ◽

Diane S. Krause

Keyword(s):

Lentiviral Vectors ◽

Absolute Number ◽

High Concentration ◽

Megakaryoblastic Leukemia ◽

Human Cd34 ◽

Healthy Donors ◽

Cd34 Cells ◽

Megakaryocyte Differentiation ◽

In Cells

Abstract Acute Megakaryoblastic Leukemia (AMKL or AML variant M7), which occurs most often in infants and young children, is characterized by a failure of megakaryocyte (MK) differentiation, bone marrow fibrosis, cytogenetic abnormalities, and a poor prognosis. We are particularly interested in AMKL that is associated with the translocation t(1;22)(p13;q13), which yields an in-frame fusion of RBM15 (OTT) and MKL1 (MAL) on chromosomes 1 and 22, respectively. The resultant fusion, RBM15-MKL1 is believed to include all of the functional domains of each component. In order to better understand the role of RBM15-MKL1 in AMKL, it is necessary to understand the roles of the constituent genes, RBM15 and MKL1, in hematopoiesis. We have studied the role of human MKL1 in megakaryopoiesis using primary human CD34+ cells purified from G-CSF mobilized PBMC from healthy donors (n=4). To optimize the CD34+ model, we tested the ability of TPO vs. TPO+SCF vs. TPO+SCF+IL–3 to induce megakaryocytopoiesis. TPO and TPO+SCF gave the highest percentages of MK (12% and 7%, respectively) on day 9. However, due to enhanced cell proliferation with TPO+SCF, the absolute number of MK was highest using this cytokine combination. To test the effect of MKL1 overexpression on megakaryopoiesis, we generated VSVG-pseudotyped lentiviral vectors containing human MKL1 and tested the effect of retronectin on viral transduction of CD34 cells. Surprisingly, retronectin decreased the level of transduction when compared to no retronectin (12% vs. 15% transduction respectively). We also found that polybrene enhanced transduction compared to lipofectamine 2000 (20% vs. 6% transduction, respectively). Using our optimized protocols, we examined the effect of MKL1 overexpression on megakaryocytopoiesis. One million CD34+ cells were thawed, infected the following two days with either empty lentivirus (pCCL) or lentivirus containing human MKL1 (pCCL-MKL), and cultured in TPO+SCF for 9 days. Since both lentiviral vectors included GFP driven by the PGK promoter, we measured the levels of CD41a, CD42d, and CD61 in GFP+ cells at day 9. In a representative experiment (of 4), CD41a levels increased in cells containing pCCL-MKL1 vs. pCCL (50% vs. 40%). Moreover, CD42d levels (22% vs. 7%) and CD61 levels (53% vs. 44%) were increased in cells containing pCCL-MKL1 virus when compared to cells containing pCCL virus. We also tested the ability of MKL1 to increase megakaryocyte differentiation using a semisolid Megacult assay from Stem Cell Technologies. CD34+ cells were cultured and infected as described above with either pCCL or pCCL-MKL1 virus. Two days post infection, GFP+ cells were FACS sorted and plated at two different concentrations in semisolid Megacult medium containing collagen, TPO, IL-6, and IL-3. Eleven days post plating, cells were stained for CD41/CD61. Cells infected with pCCL-MKL1 cells gave approximately 2 fold more MK colonies than pCCL infected cells at both low cell concentration plating (395 vs. 182 colonies, respectively) and high concentration plating (900 vs. 389 colonies, respectively). These data suggest that overexpression of human MKL1 enhances megakaryocyte differentiation of primary human CD34+ cells. A further understanding of the normal roles of RBM15 and MKL1 in megakaryopoiesis will allow us to better understand the role of the RBM15-MKL1 fusion in AMKL, and aid in the development of treatments for this disease.

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PRMT1 Mediated FLT3 Methylation As a Therapeutic Vulnerability in FLT3-ITD+ AML

Blood ◽

10.1182/blood-2018-99-112766 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 760-760

Author(s):

Xin He ◽

Yinghui Zhu ◽

Haojie Dong ◽

Sierra Min Li ◽

Zonghui Ding ◽

...

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Peripheral Blood ◽

Mass Spectrometry Analysis ◽

Current View ◽

Clinical Activity ◽

Type I ◽

Peripheral Blood Stem Cells ◽

Normal Peripheral Blood ◽

Cd34 Cells

Abstract The current view is that treatment failures of AML patients are due to persistence of leukemia stem cells (LSCs). The presence of FMS-like tyrosine kinase-3 (FLT3) Internal tandem duplication (ITD) is associated with poor prognosis. But, FLT3 tyrosine kinase inhibitors (TKI) demonstrate transient clinical activity in FLT3-ITD+ AML patients. Persistent FLT3-ITD+ AML LSC represent a source of relapse. There is a pressing need to target LSC and improve outcomes for FLT3-ITD+ AML patients. PRMT1, the predominant arginine methyltransferase, has been implicated in pathogenesis of AML rare subtype (i.e., acute megakaryoblastic leukemia). Herein, we show that PRMT1 protein expression is significantly increased in LSC-enriched AML CD34+CD38- cells relative to the counterparts of normal peripheral blood stem cells (PBSC) (AML n=9, normal n=8, p=0.0004,). Following PRMT1-knockdown (KD), AML CD34+ cells (n=15) demonstrated varying degrees of apoptosis while the survival of normal cells were not affected. Interestingly, we observed significant apoptosis-induction in a subset of samples bearing FLT3-ITD mutation (6 out of total 15) upon PRMT1-KD (ShCtrl 13.9±3.6%, ShPRMT1 32.5±4.6%, p<0.001). PRMT1-KD induced apoptosis was also more evident in cord blood (CB) CD34+ cells expressing FLT3-ITD (ShCtrl 19.4±1.3%, ShPRMT1 45.2±2.5%, p<0.001) relative to that of FLT3-WT (ShCtrl 27.47±1.8%, ShPRMT1 36.9±1.6%, p=0.0167). In 293T cells ectopically overexpressing FLT3-WT or FLT3-ITD, co-immunoprecipitation (co-IP) indicated greater interaction between PRMT1 and FLT3-ITD. Through RNA-Seq profiling two AML lines (MV4-11, OCI-AML3) plus one FLT3-ITD transduced CB CD34+ cells with PRMT1-KD, we obtained a differentially-regulated gene set as a "PRMT1 signature". This signature was enriched in FLT3-ITD+ AML relative to FLT3-WT AML according to ssGSEA analysis using two AML datasets (GSE14468, GSE10358), indicating PRMT1 may cooperate with FLT3-ITD regulating AML maintenance. Given that PRMT1 directly interacts with FLT3-ITD, we next asked whether PRMT1 catalyzes FLT3-ITD protein methylation. Through mass-spectrometry analysis of a FLT3-ITD+ AML specimen and in vitro methylation assay, we identified that PRMT1 catalyzes FLT3-ITD arginine (R) methylation (Me) at two conserved residues, 972 and 973. Using in-house R972/973 Me antibody, we validated the expression of FLT3 R-Me in FLT3-ITD AML speciemens (7 out of 7). To test R-Me function, we transduced MLL-AF9 (MA9) overexpressing murine c-Kit+ cells with methylation-deficient FLT3-ITD (R972/973K, arginine [R] to lysine [K]) construct, and found that MA9 cells expressing R972/973K underwent more apoptosis than that of WT FLT3-ITD (WT FLT3-ITD 9.7±1.1%, R972/973K 23.7±2.1%, p=0.003). The double transformed cells were further transplanted into recipients for leukemia development. Mice receiving MA9 cells expressing R972/973K exhibited longer survival (median survival: WT FLT3-ITD 36 days, R972/973K 50 days, p=0.002, n=6). Mechanistically, expression of R972/973K did not affect the total tyrosine phosphorylation level of FLT3-ITD. Additionally, FLT3-ITD R-Me expression persisted after a TKI (AC220) treatment. These facts indicated that FLT3-ITD R-Me function is independent of FLT3-ITD kinase activity. We then used a FLT3-ITD+ patient derived xenograft (PDX) model to assess the effects of TKI and PRMT1 inhibition. Following engraftment >1% in peripheral blood, we divided mice (n=24) into 4 groups and treated each with vehicle, MS023 (a type I PRMT inhibitor, ACS Chem Biol. 2016;11:772-781) (160 mg/kg/i.p), AC220 (10 mg/kg/i.g) or combination for 4 weeks. MS023 treatment downregulating FLT3 R-Me levels enhanced elimination of FLT3-ITD AML cells by AC220 treatment (AC220 24.6±13.4% vs combination 7.6±6.5%, p=0.02, n=6). At 16 weeks post-secondary BMT, significant AML burden in single drug treated transplants was observed, but less AML cells were detected in combination-treated transplants (AC220 52.3%, vs combination 25.4%, p<0.001, n=6). MS023 had little effect on long-term in vivo engraftment of CD34+ from a human CB specimen (vehicle 76.3±5.9%, MS023 72.2±3.4%, p=0.21, n=5). In summary, our study demonstrated PRMT1 overexpression contributes to AML stem/progenitor cell survival possibly through FLT3-ITD methylation, supporting further exploration into how PRMT1-mediated FLT3 methylation governs LSC survival. Disclosures Khaled: Alexion: Consultancy, Speakers Bureau; Daiichi: Consultancy; Juno: Other: Travel Funding.

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IL-3 Specifies Early Hematopoietic Development from Human iPSCs and Synergizes with M-CSF and G-CSF on Myeloid Differentiation

Blood ◽

10.1182/blood.v124.21.4308.4308 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4308-4308

Author(s):

Nico Lachmann ◽

Mania Ackermann ◽

Eileen Frenzel ◽

Christine Happle ◽

Olga Klimenkova ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Terminal Differentiation ◽

Myeloid Differentiation ◽

Hematopoietic Differentiation ◽

In Vitro Differentiation ◽

Hematopoietic Development ◽

Cd34 Cells

Abstract Hematopoietic in-vitro-differentiation of pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) holds great promise for disease modeling, drug testing, as well as cell- and gene-therapy strategies. Although hematopoietic differentiation of PSC has been shown to be feasible, the majority of current protocols apply a large number of different cytokines to direct differentiation. In this line, priming the differentiation process by a multitude of cytokines may alter the endogenous hematopoietic differentiation program of PSCs, thus hampering the usefulness of such protocols to gain insight into physiologic human hematopoietic development. To overcome this problem we have investigated the hematopoietic differentiation potential of human PSC, based on minimal cytokine application. Given the emerging role of IL3 as a critical factor in adult hematopoiesis and the pivotal role of M-CSF and G-CSF for terminal myeloid differentiation, we here employed IL3 in combination with either M-CSF or G-CSF on hematopoietic development. To prove our concept, human CD34+ cell-derived iPSC clones were subjected to an embryoid body (EB)-based myeloid differentiation protocol employing cytokines from day 5 onwards and yielding so-called “myeloid cell forming complexes” (MCFCs) within 7-10 days. Analysis of MCFC within 10 days of differentiation revealed expression of MIXL1, KDR1, GATA2, and RUNX1, as well as an early CD34+/CD45- population undergoing transition to a CD34+/CD45+ and thereafter CD34-/CD45+ phenotype. The hypothesis of a primitive hematopoietic cell arising from a population with dual (hematopoietic and vascular epithelial) potential was supported by co-staining of these populations with VE-cadherin (CD144). Here primarily the CD34+/CD45+/CD144- cells were capable of colony formation in vitro. Differentiation of PSC for more than 15-days resulted in the continuous shedding of hematopoietic cells from MCFCs and further differentiation along the IL3/M-CSF let to the generation of >99% pure monocytes/ macrophages (iPSC-MΦ), while IL3/G-CSF promoted granulopoiesis (iPSC-gra, purity >95%). Of note, hardly any CD34+ cells were detected among MCFC-shedded cells for the IL3/M-CSF as well as the IL3/G-CSF combination. In contrast, differentiation in IL3 only resulted in 10% MCFC-derived CD34+ cells, an observation further confirmed by a 10-times increased clonogenicity for cells shedded from MCFC exposed to IL3 only when compared to IL3/G-CSF or IL3/M-CSF cultures. Furthermore, cells cultured in IL3 maintained the capacity of subsequent M-CSF-driven terminal differentiation, whereas no suspension cells were observed following differentiation of PSC with G-CSF alone. Most strikingly, IL3/M-CSF or IL3/G-CSF cultures generated iPSC-MΦ or iPSC-gra from day 14-15 onwards over a period of 3-5 months at a quantity of 0.4-2.0 x 106 cells/week (cumulative 0.8-4.0 x 107 cells) per 3.5 cm well. For IL3/M-CSF cultures detailed characterization of mature myeloid cells demonstrated a typical MΦ-morphology of iPSC-MΦ by cytospins and a surface-marker profile of CD45, CD11b, CD14, CD163, and CD68. In addition, iPSC-MΦ had the ability to phagocytose latex-coated beads similar to peripheral blood (PB)-MΦ polarized to M2 and upon LPS stimulation secreted MCP1, IL6, IL8, and IL10, whereas IFNy, IL1b, IL4, IL5, and IL12 were absent. iPSC-gra showed surface expression of CD45, CD11b, CD16, CD15, CD66b and a differential count containing pro-myelocyte (3%), myelocyte (5%), meta-myelocyte (30%), bands (22%), eosinophils (2%), basophils (1%), and segmented-neutrophils (37%) . Moreover, iPSC-gra were able to migrate towards an IL8 or fMLP gradient, formed neutrophil extracellular traps, and up-regulated NADPH activity and ROS production upon PMA stimulation to a similar degree as PB granulocytes. In summary, we here present an in vitro differentiation protocols for human iPSC requiring minimal cytokine stimulation, which appears highly suited to model human hematopoietic development or generate cells for gene and cell-replacement strategies. We further provide evidence that IL3 constitutes a key cytokine driving the early hematopoietic specification of human PSC, whereas M-CSF and G-CSF function primarily as downstream “supporter” cytokines regulating the terminal differentiation towards macrophages and granulocytes, respectively. Disclosures No relevant conflicts of interest to declare.

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CXCR7 Functions Together with CXCR4 in SDF-1/CXCL12 Induced CD34+ Hematopoietic Progenitor Cell Cycling Through β-Arrestin 2-Dependent Akt Activation

Blood ◽

10.1182/blood.v120.21.3448.3448 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3448-3448

Author(s):

Frederic Torossian ◽

Aurélie Chabanon ◽

Denis Clay ◽

Bernadette Guerton ◽

Adrienne Anginot ◽

...

Keyword(s):

Flow Cytometry ◽

Plasma Membrane ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Stem ◽

Akt Activation ◽

Respective Role ◽

Human Cd34 ◽

Cd34 Cells ◽

Cell Cycling

Abstract Abstract 3448 Introduction SDF-1/CXCL12 chemokine exhibits a well-known effect on retention, migration and homing of hematopoietic stem/progenitor cells (HSC/HP). We have previously demonstrated that it is also a key regulator of hematopoiesis homeostasis, acting, at low concentrations, as a survival and cell cycle promoting factor for human CD34+ HP. It has long been considered that CXCR4 was responsible for SDF-1 induced biological effects until the recent discovery of its second receptor, CXCR7. In the present study, we explored the respective role of CXCR4 and CXCR7 in the cell cycling and survival promoting effect of SDF-1/CXCL12 on human CD34+HP. Material and Methods We used CD34+ HP purified from the peripheral blood (PB) of healthy un-mobilized donors since they are mainly in G0. This allows to study the role of CXCR4 and CXCR7 receptors in 0.5ng/ml SDF-1/CXCL12 induced G0-G1 transition in synchronized quiescent cells. Gene expression was detected by RT-QPCR. Protein expression was detected and quantified using confocal microscopy, flow cytometry, immunoblotting and immunoprecipitation. Cell cycling experiments were performed using a Ki67 antibody and CXCR7 binding assay was performed using SDF-1/CXCL12AF647. Neutralization experiments were performed using a specific CXCR4 antibody or CXCR7 chemical inhibitors, a kind gift from ChemoCentryx, Inc (CCX771 and CCX733) and their respective controls (IgG and CCX704). Results Flow cytometry and confocal analysis showed that CXCR7 and CXCR4 are differentially distributed in PB CD34+ cells. In contrast to CXCR4 that is present at both the plasma membrane and intracellular level, CXCR7 expression is mainly restricted to the intracellular compartment. Confocal analysis suggested the presence of CXCR4/CXCR7 heterodimers on these cells the presence of which were confirmed by immunoprecipitation in a HP cell line. Despite its very low expression at the surface of CD34+ cells, we found that CXCR7 is capable of binding to exogenous SDF-1/CXCL12. Indeed, pretreatment with CXCR7 antibody or a chemical inhibitor reduces the mean fluorescence of bound fluorescent SDF-1/CXCL12AF647, a fully functional and specific chemokine with similar effects compared to unlabeled SDF-1/CXCL12. Neutralizing either CXCR4 or CXCR7 in PB CD34+ cells strongly reduced Akt activation induced by SDF-1/CXCL12 (0.5 ng/ml) as well as the percentage of cells in cycle (G1 and S + G2/M), colony formation and cell survival. This demonstrates that both receptors cooperate in SDF-1/CXCL12 induced functional effects. We further analyzed the respective role of CXCR4 and CXCR7 in SDF-1/CXCL12 signalization. In contrast to CXCR4, CXCR7 is reported not to activate G protein signaling pathways in response to SDF-1/CXCL12. However, it can transduce cell signaling through the β-arrestin pathway. In the present study, we showed that CXCR7 and β-arrestin 2 colocalize near the plasma membrane in freshly purified PB CD34+ cells, suggesting that CXCR7 is constitutively activated. After SDF-1/CXCL12 treatment, the majority of β-arrestin 2 was translocated to the nucleus and only a partial colocalization persisted in the cytoplasm. Using neutralizing antibodies and specific inhibitors, we showed that β-arrestin 2 nuclear translocation was dependent on both CXCR7 and CXCR4 receptors. Reducing β-arrestin 2 expression using siRNA decreased SDF-1/CXCL12 induced Akt activation in PB CD34+cells indicating the involvement of β-arrestin 2 in this process. Conclusion Altogether, our results demonstrate for the first time the role of CXCR7 together with CXCR4 in SDF-1/CXCL12-induced CD34+ cell cycling/proliferation. They also suggest the involvement of β-arrestin 2 as signalling hubs, downstream of both receptors. Disclosures: No relevant conflicts of interest to declare.

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