Chromatin State Shaping the Gene Expression Profiling in the Inducible Differentiation of Promyelocytic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5188-5188
Author(s):  
Chunhua Song ◽  
Lisa Chung ◽  
Xiaokang Pan ◽  
Li Zhanjun ◽  
Yali Ding ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epigenetic Regulation ◽  
Conflicts Of Interest ◽  
Promyelocytic Leukemia ◽  
Epigenetic Mechanism ◽  
Chromatin State ◽  
Hematopoietic Stem ◽  
Signature Genes ◽  
Lineage Differentiation ◽  
On Chip

Abstract Background Epigenetic changes in DNA and chromatin are regarded as emerging major players for hematopoietic stem cells development and lineage differentiation. Epigenetic deregulation of gene expression leads to leukemia and reversibility of epigenetic modifications makes DNA and chromatin changes attractive targets for therapeutic intervention. The promyelocytic leukemia HL-60 cell can differentiate into microphage, granulocyte and monocyte with stimulation of phorbol myristic acid (PMA), dimethylsulfoxide (DMSO), and Vitamin D3, respectively, by affecting the gene expression on cell cycle and cell differentiation. However, how epigenetic regulation on gene expression in the inducible differentiation is still undetermined. Methods and Results We did the microarray analysis to identify the genome-wide gene expression profile of HL-60 cells in various time points(0h, 6h, 1d,2d,4d and 6d) under the treatment of PMA, DMSO and D3 and found that around 3000 genes are significantly altered commonly in the cells upon the 3 treatments. The percentage of down-regulated genes in the all commonly altered genes is significantly higher than that of up-regulated genes, and the significantly altered genes showed significantly physical clustering on chromosome loci, indicating the epigenetic regulation involved in the regulation of the gene expression. We did observed the expression changes of epigenetic enzymes in the process and further did the ChIP-on-ChIP analysis by using the customer array tiling the selected changed genes and 5 genomic loci hybridizing with ChIP’d DNA of histone markers (H3K4me3, H3K9me3 and H3K27me3). We found that the obvious decrease of H3K4me3 binding and increase of H3K9me3 and H3K27me3 are observed in the promoter of the commonly down-regulated genes. These data indicated histone H3K4, H3K9 and H3K27 marker play important roles in shaping the chromatin state and regulating gene expression. Conclusion We identified the signature genes controlling the inducible differentiation of promyelocytic leukemiacells and found the epigenetic mechanism regulating the gene expression in the process. Disclosures No relevant conflicts of interest to declare.

Epigenetic Control of C/EBPa by Noncoding RNAs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3644-3644
Author(s):  
Annalisa Di Ruscio ◽  
Alexander K Ebralidze ◽  
Francesco D'Alò ◽  
Maria Teresa Voso ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Messenger Rna ◽  
Noncoding Rna ◽  
Gene Locus ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Factor C

Abstract Abstract 3644 Poster Board III-580 Little is currently known about the role of noncoding RNA transcripts (ncRNA) in gene regulation; although most, and perhaps all, gene loci express such transcripts. Our previous results with the PU.1 gene locus showed a shared transcription factor complex and chromatin configuration requirements for biogenesis of both messenger and ncRNAs. These ncRNAs were localized within the nuclear and cytoplasmic compartments. Disrupting ncRNAs in the cytoplasmic cellular fraction results in increased PU.1 mRNA and protein. Recently, we have focused on the C/EBPa gene locus and observed extensive noncoding transcription. The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo. In this study, we characterize ncRNAs derived from the C/EBPa locus and demonstrate their functional role in regulation of C/EBPa gene expression. First, northern blot analysis and RT PCR determined a predominantly nuclear localization of the C/EBPa ncRNAs. Second, strand-specific quantitative RT PCR demonstrated a concordant expression of coding and noncoding C/EBPa transcripts. Next, we investigated the results of ablation of ncRNAs using a lentiviral vector containing ncRNA-targeting shRNAs on the expression of the C/EBPa gene. We have observed that reduced levels of ncRNAs leads to a significant downregulation of the expression of coding messenger RNA. These data strongly suggest that C/EBPa ncRNAs play an important role in maintaining optimal expression of the C/EBPa gene at different stages of hematopoiesis and makes targeting noncoding transcripts a novel and attractive tool in correcting aberrant gene expression levels. Disclosures: No relevant conflicts of interest to declare.

Epigenetic Regulation of Lipid Signalling Pathways In Low-Risk MDS Patients During Azacitidine Treatment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 233-233
Author(s):  
Matilde Y Follo ◽  
Sara Mongiorgi ◽  
Cristina Clissa ◽  
Carla Filì ◽  
Chiara Colombi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Promoter Methylation ◽  
Lower Risk ◽  
Conflicts Of Interest ◽  
Stem Cell Differentiation ◽  
Epigenetic Therapy ◽  
Signalling Pathways ◽  
Molecular Profile ◽  
Hematopoietic Stem ◽  
Lipid Signalling

Abstract Abstract 233 Azacitidine, a DNA methyltransferase inhibitor currently used for the treatment of higher-risk myelodysplastic syndromes (MDS) patients, was shown to delay the evolution into acute myeloid leukemia (AML) and prolong overall survival (Fenaux P et al, Lancet Oncol 2009). In addition, azacitidine has recently been shown to potentially be a feasible and effective treatment even for patients with lower-risk MDS (Musto P et al, Cancer 2010). Lipid signalling pathways are involved in many important biological processes, such as cell growth, differentiation and apoptosis and play a role in the progression of MDS towards AML (Follo MY et al, J Cell Biochem 2010). Moreover, we recently demonstrated that phosphoinositide-phospholipase C beta1 (PI-PLCbeta1) promoter gene is hyper-methylated in higher-risk MDS and that azacitidine treatment can induce an increase in the level of PI-PLCbeta1 splicing variants as well as a down-regulation of activated Akt (Follo MY et al, Leukemia 2008; Follo MY et al, PNAS 2009). In fact, responding patients showed an increase in PI-PLCbeta1 expression in correlation with the therapeutic response, whereas their PI-PLCbeta1 promoter methylation was reduced. Furthermore, the decrease of promoter methylation anticipated the hematologic response, since the variations in PI-PLCbeta1 gene expression were observed prior to the clinical outcome. Stemming from these data, we further investigated the role of inositide signalling pathways during the epigenetic therapy, focusing on the effect of azacitidine on lipid signal transduction pathways in lower-risk MDS patients. The study included 25 patients (IPSS risk: low or intermediate-1) treated with azacitidine (75mg/m2 subcutaneous daily for 5 consecutive days every 28 days, for a total of 8 courses). For each patient we followed the effect of azacitidine in correlation to both PI-PLCbeta1 promoter methylation and gene expression, as well as the molecular profile of key molecules involved in the regulation of methylation processes, such as histone deacetylases (HDACs), methyl-CpG binding domain proteins (MBDs), and transcription factors correlated to hematopoietic stem cell differentiation and proliferation. Our results show that 8/25 (34%) of our lower-risk MDS patients, showing hematologic improvements after azacitidine therapy, had a significant increase in PI-PLCbeta1 expression, as compared with the amount of the pre-treatment period, thus confirming the involvement of this molecule in the response to demethylating agents. As for the remaining patients, mainly showing a stable disease, we observed slight increases or almost constant levels of PI-PLCbeta1 expression. Moreover, ongoing analyses are trying to disclose whether lower-risk MDS patients responding to azacitidine show a specific molecular epigenetic profile during the regulation of methylation processes. Taken together, our data suggest a correlation between azacitidine treatment and PI-PLCbeta1 signalling even in lower-risk MDS, thus hinting at a role for PI-PLCbeta1 in the evaluation of patients likely to respond to azacitidine and paving the way for the development of innovative therapeutic strategies in lower-risk MDS patients. Disclosures: No relevant conflicts of interest to declare.

Role of Notch Signaling in Hematopoietic Stem Cell Function

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-15-SCI-15
Author(s):  
Lluis Espinosa ◽  
Anna Bigas
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Notch Pathway ◽  
Cell Types ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
On Chip ◽  
Stem Cell Populations

Abstract Abstract SCI-15 The Notch pathway controls the generation of different cell types in most tissues including blood, and dysregulation of this pathway is strongly associated with oncogenic processes. In many systems, Notch is also required for the maintenance of the stem cell populations. However, in the adult hematopoietic system this link between Notch and stemness has not been established. Instead, work of several groups, including ours, has clearly demonstrated that Notch has a prominent role in the generation of hematopoietic stem cells (HSC) during embryonic development. Although the first wave of blood cells appears in the mouse embryo around day 7.5 of development and is independent of Notch function, embryonic HSC are formed around day 10 of development from endothelial-like progenitors that reside in the embryonic aorta surrounded by the gonad and mesonephros, also called AGM region. By analyzing different Notch pathway mutant mouse embryos, we have demonstrated the involvement of the Jagged1-Notch1-GATA2 axis in this event. However, the formal demonstration that Notch regulates the GATA2 gene during HSC generation is still lacking. We have now found that GATA2 is a direct Notch target in vivo during embryonic HSC generation. However, whereas Notch positively activates GATA2 transcription in the HSC precursors, it simultaneously activates hes1 transcription, which acts a repressor of the same GATA2 gene. This finding directly implicates hes1 in the regulation of HSC development although further studies using loss-of-function mutant embryos are still needed. Altogether, our results indicate that both Notch and hes1 are required to finely regulate the levels, distribution, and likely the timing of GATA2 expression through an incoherent feed-forward loop. In parallel, we have identified other downstream targets of Notch in the AGM region by ChIP-on-chip and expression microarray analysis that we are currently characterizing. Disclosures: No relevant conflicts of interest to declare.

Efficiency of Leukemic Stem Cell Separation From Patients with Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4997-4997
Author(s):  
Lu Zhang ◽  
Susanne Hofmann ◽  
Lars Bullinger ◽  
Marlies Goetz ◽  
Markus Wiesneth ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cell Sorting ◽  
Peripheral Blood ◽  
Recovery Rate ◽  
Mononuclear Cells ◽  
Separation Efficiency ◽  
Conflicts Of Interest ◽  
Cell Number ◽  
Hematopoietic Stem

Abstract Abstract 4997 Leukemic stem cells (LSC) are the source for leukemic disease self-renewal and account for disease relapse after treatment. Therefore LSCs probably represent a critical target for therapeutic options. Xenograft models confirmed repeatedly that LSCs from AML patients reside mainly in CD34+CD38- compartment of leukemic blasts which makes the pure and efficient separation of this population mandatory to identify new therapeutic drugs to target LSC in different AML subtypes. We separated this subpopulation out of primary AML peripheral blood mononuclear cells (PBMC) samples with fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) and compared the efficiency of both methods. In order to profile gene expression of LSCs and hematopoietic stem cells (HSC) MicroArrays were performed using GeneChip Human Genome U133 Plus 2.0 from Affymetrix. The CD34+CD38- subpopulation was separated from PBMCs of 12 AML patients and 5 healthy volunteers using FACS. Concerning the 12 primary AML samples, the ratio of CD34+CD38- cells ranges between 0.79% and 86.2% using 1–5×107 PBMC for separation. After sorting, the purity of those AML samples increased to 88.4–98.4% while 2×104-3.6×106 cells were obtained. MACS was used to separate 2 representative samples, in which the CD34+CD38- subpopulation was rather small (sample1: 0.78%) or large (sample2: 86.1%). Those sorted subpopulations were compared to the samples sorted via FACS. In order to evaluate separation efficiency in a standardized manner, we defined the recovery rate: (CD34+CD38- cell number obtained /total CD34+CD38- cell number) × 100%. The total CD34+CD38- cell number was calculated through a pre-sorting FACS analysis. For sample 1, MACS resulted in a recovery rate of 4.2–6.4% with a purity of 86.6–90.3%, which is inferior to the recovery rate of 17% and the purity of 92.1% using FACS. For Sample 2, MACS resulted in a recovery rate of 0.4% with a purity of 98.8%, compared to the recovery rate of 11.6% with a purity of 98.1% by FACS. Comparing both methods it is obvious that the purity doesn't differ a lot, but the yield is much higher using FACS. This could represent a powerful tool, when managing rare samples. Finally, by comparing purity and yield, we showed that FACS is the adequate separation method. At the moment MicroArrays are being performed in order to investigate the gene expression profile for 12–15 AML patients and 5 HVs. Taken together, we showed a widely efficient method to routinely separate LSCs from patients with different subtypes of AML. Microarrays, that have been performed, represent a method that allows the comparison of the characteristics of LSCs in different AML subtypes and also of LSCs from bone-marrow with LSCs from peripheral blood and with HVs. These array data analyses are ongoing and will be presented. Disclosures: No relevant conflicts of interest to declare.

Disease-Relevant Expression Of TRAF6 In Mouse HSC Results In An MDS-Like Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 101-101
Author(s):  
Jing Fang ◽  
Xiaona Liu ◽  
Brenden Barker ◽  
Lyndsey Bolanos ◽  
Yue Wei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Bone Marrow Failure ◽  
Gene Signature ◽  
Sublethal Dose ◽  
Hematopoietic Tissue ◽  
Hematopoietic Stem ◽  
Disease Evolution ◽  
Cd34 Cells

Abstract Overexpression of immune-related genes is widely reported in Myelodysplastic Syndrome (MDS), and chronic immune stimulation increases the risk for developing MDS. We find that TNF receptor associated factor 6 (TRAF6), an innate immune protein, is overexpressed approximately 2-fold in CD34+ cells from 40% of MDS patients, and may explain immune pathway activation in the MDS-initiating hematopoietic stem/progenitor cell (HSPC). In support of these observations and our hypothesis that TRAF6 is important in the pathophysiology of MDS, a gene expression analysis revealed that TRAF6 controls an MDS gene signature in human cells. We, and others, have previously shown that retroviral overexpression of TRAF6 in mouse HSPC results in MDS and Acute Myeloid Leukemia (AML). However, interpretations of these findings are hampered by supra-physiological levels of TRAF6 (>10-fold overexpression) and the stress associated with HSPC transduction/transplantation. To investigate the consequences of TRAF6 overexpression to MDS, we generated a transgenic mouse model overexpressing TRAF6 from a hematopoietic-specific Vav promoter. Expression of TRAF6 in HSPC was approximately 2-fold higher as compared to endogenous TRAF6 and in line with MDS patient CD34+ cells. By 15 months of age, half of Vav-TRAF6 mice succumbed to a hematologic disease resembling MDS associated with bone marrow failure (BMF). In contrast to the retroviral overexpression approach, Vav-TRAF6 mice did not develop AML. Examination of sick mice revealed stage-specific disease evolution. Initially, all Vav-TRAF6 mice exhibit an inversion of myeloid/lymphoid proportions. For Vav-TRAF6 mice that develop a fatal disease, they present with a hypocellular marrow, dysplasic myeloid cells, and neutropenia. A subset of mice also display anemia with nucleated red blood cells, poikilocytosis, and extramedullular erythropoiesis. In support of a BMF phenotype, HSPC from Vav-TRAF6 mice form fewer colonies in methylcellulose. To investigate the consequences of an acute exposure to pathogen, early-stage Vav-TRAF6 mice were treated with a single sublethal dose of lipopolysaccharide (LPS). Unlike wild-type (WT) mice, Vav-TRAF6 mice developed a rapid and reversible anemia, suggesting environmental factors can influence the severity of the disease. To gain insight into the mechanism contributing to BMF, gene expression profiling was performed in WT and Vav-TRAF6 HSPC. One of the enriched pathways consisted of AKT activation and FOXO downregulation. Consistent with the microarray analysis, AKT is constitutively phosphorylated at Thr308 in hematopoietic tissue from Vav-TRAF6 mice. SOD2, a transcriptional target of FoxO3a that is suppressed by activated AKT, is decreased in Vav-TRAF6 HSPC. Given that AKT/FOXO regulate reactive oxygen species (ROS) in cells, we investigated ROS levels in HPSC from Vav-TRAF6 and WT mice. Intracellular ROS is significantly elevated in BM cells from Vav-TRAF6 mice, and restored to normal levels when AKT was inhibited. In conclusion, we propose the potential role of TRAF6 in the development of MDS-associated BMF, partly due to constitutive activation of AKT and subsequent ROS elevation in HSPC cells. Disclosures: No relevant conflicts of interest to declare.

Large Conserved Domains Of Low DNA Methylation Maintained By 5-Hydroxymethycytosine and Dnmt3a

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2406-2406
Author(s):  
Mira Jeong ◽  
Deqiang Sun ◽  
Min Luo ◽  
Yun Huang ◽  
Myunggon Ko ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Hox Genes ◽  
Conflicts Of Interest ◽  
P Value ◽  
Genomic Feature ◽  
Conserved Domains ◽  
Hematopoietic Stem ◽  
Genome Wide ◽  
The Impact

Abstract Identification of recurrent leukemia-associated mutations in genes encoding regulators of DNA methylation such as DNMT3A and TET2 have underscored the critical importance of DNA methylation in maintenance of normal physiology. To gain insight into how DNA methylation exerts the central role, we sought to determine the genome-wide pattern of DNA methylation in the normal precursors of leukemia cells: the hematopoietic stem cell (HSC), and investigate the factors that affect alterations in DNA methylation and gene expression. We performed whole genome bisulfite sequencing (WGBS) on purified murine HSCs achieving a total of 1,121M reads, resulting in a combined average of 40X coverage. Using Hidden Markov Model we identified 32,325 under-methylated regions (UMRs) with average proportion of methylation ≤ 10% and by inspecting the UMR size distribution, we discovered exceptionally large “methylation Canyons” which span highly conserved domains frequently containing transcription factors and are quite distinct from CpG islands and shores. Methylation Canyons are a distinct genomic feature that is stable, albeit with subtle differences, across cell-types and species. Canyon-associated genes showed a striking pattern of enrichment for genes involved in transcriptional regulation (318 genes, P=6.2 x 10-123), as well as genes containing a homeobox domain (111 genes, P=3.9 x 10-85). We compared Canyons with TF binding sites as identified from more than 150 ChIP-seq data sets across a variety of blood lineages (>10)19 and found that TF binding peaks for 10 HSC pluripotency TFs are significantly enriched in entirety of Canyons compared with their surrounding regions. Low DNA methylation is usually associated with active gene expression. However, half of Canyon genes associated with H3K27me3 showed low or no expression regardless of their H3K4me3 association while H3K4me3-only Canyon genes were highly expressed. Because DNMT3A is mutated in a high frequency of human leukemias24, we examined the impact of loss of Dnmt3a on Canyon size. Upon knockout of Dnmt3a, the edges of the Canyons are hotspots of differential methylation while regions inside of Canyon are relatively resistant. The methylation loss in Dnmt3a KO HSCs led Canyon edge erosion, Canyon size expansion and addition of 861 new Canyons for a total of 1787 Canyons. Canyons marked with H3K4me3 only were most likely to expand after Dnmt3a KO and the canyons marked only with H3K27me3 or with both marks were more likely to contract. This suggests Dnmt3a specifically is acting to restrain Canyon size where active histone marks (and active transcription) are already present. WGBS cannot distinguish between 5mC and 5hmC, so we determined the genome-wide distribution of 5hmC in WT and Dnmt3a KO HSCs using the cytosine-5-methylenesulphonate (CMS)-Seq method in which sodium bisulfate treatment convert 5hmC to CMS; CMS-containing DNA fragments are then immunoprecipitated using a CMS specific antiserum. Strikingly, 5hmC peaks were enriched specifically at the borders of Canyons. In particular, expanding Canyons, typically associated with highest H3K4me3 marking, were highly enriched at the edges for the 5hmC signal suggesting a model in which Tet proteins and Dnmt3a act concomitantly on Canyon borders opposing each other in alternately effacing and restoring methylation at the edges, particularly at sites of active chromatin marks. Using Oncomine data, we tested whether Canyon-associated genes were likely to be associated with hematologic malignancy development and found Canyon genes were highly enriched in seven signatures of genes over-expressed in Leukemia patients compared to normal bone marrow; in contrast, four sets of control genes were not similarly enriched. Further using TCGA data, we found that expressed canyon genes are significantly enriched for differentially expressed genes between patients with and without DNMT3A mutation (p value<0.05) Overall, 76 expressed canyon genes, including multiple HOX genes, are significantly changed in patients with DNMT3A mutation (p=0.0031). Methylation Canyons, the novel epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development. Disclosures: No relevant conflicts of interest to declare.

MiR-150 Inhibits MLL-AF9 Associated Leukemia By Suppressing Leukemic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3764-3764
Author(s):  
Patali S Cheruku ◽  
Marina Bousquet ◽  
Guoqing Zhang ◽  
Guangtao Ge ◽  
Wei Ying ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Gene Profiling ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Signature Genes

Abstract Leukemic stem cells (LSCs) are derived from hematopoietic stem or progenitor cells and often share gene expression patterns and specific pathways. Characterization and mechanistic studies of LSCs are critical as they are responsible for the initiation and potential relapse of leukemias, however the overall framework, including epigenetic regulation, is not yet clear. We previously identified microRNA-150 (miR-150) as a critical regulator of mixed lineage leukemia (MLL) -associated leukemias by targeting oncogenes. Our additional results suggest that miR-150 can inhibit LSC survival and disease initiating capacity by suppressing more than 30% of “stem cell signature genes,” hence altering multiple cancer pathways and/or stem cell identities. MLL-AF9 cells derived from miR-150 deficient hematopoietic stem/progenitor cells displayed significant proliferating advantage and enhanced leukemic colony formation. Whereas, with ectopic miR-150 expression, the MLL-AF9 associated LSC population (defined as Lin-ckit+sca1- cells) was significantly decreased in culture. This is further confirmed by decreased blast leukemic colony formation in vitro. Furthermore, restoration of miR-150 levels in transformed MLL-AF9 cells, which often display loss of miR-150 expression in AML patients with MLL-fusion protein expressing, completely blocked the myeloid leukemia development in a transplantation mouse model. Gene profiling analysis demonstrated that an increased level of miR-150 expression down regulates 30 of 114 stem cell signature genes by more than 1.5 fold, partially mediated by the suppressive effects of miR-150 on CBL, c-Myb and Egr2 oncogenes. In conclusion, our results suggest that miR-150 is a potent MLL-AF9 leukemic inhibitor that may act by suppressing the survival and leukemic initiating potency of MLL-AF9 LSCs. Disclosures: No relevant conflicts of interest to declare.

Lgr4-Mediated Potentiation Of Wnt/β-Catenin Signaling Promotes MLL Leukemogenesis Via An Rspo3/Wnt3a-Gnaq Pathway In Leukemic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 887-887 ◽  
Author(s):  
Hangyu Yi ◽  
Jianlong Wang ◽  
Maria Kavallaris ◽  
Jenny Yingzi Wang
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
G Protein ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Active Form ◽  
Leukemic Stem Cells ◽  
Western Blots ◽  
Hematopoietic Stem

Abstract Although the clinical importance of aberrant Wnt/β-catenin signaling has been recognized in various cancers, including MLL-rearranged acute myeloid leukemia (MLL AML), its key tractable pathway components have not yet been discovered in leukemic stem cells (LSC). Our studies have identified an Rspo3/Wnt3a-Lgr4-Gnaq pathway, which significantly potentiates β-catenin signaling in MLL LSC. Genetic and pharmacological targeting of this pathway impairs LSC self-renewal and survival, inhibiting MLL-AF9-induced leukemia progression in vivo. Gene expression analysis of AML patient samples (Nucleic Acids Res, 41:D1034-9, 2013) revealed an approximately 3-fold increase (p=0.00002) in expression of leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) in leukemic cells from patients with MLL AML compared to normal human hematopoietic stem cells (HSC). As recent studies have highlighted a critical link between R-spondin (Rspo)/Lgr4 and Wnt/β-catenin signaling pathways, we hypothesized that up-regulation of Lgr4 is associated with aberrant activation of β-catenin signaling in MLL LSC. We have previously demonstrated that β-catenin is highly expressed in HSC transformed by MLL-AF9 and is lower in HSC transduced with leukemic oncogenes such as Hoxa9/Meis1, while increased β-catenin expression is correlated with a poor survival rate in mice. In this study, western blots confirmed high levels of Lgr4 expression in HSC expressing MLL-AF9 compared to Hoxa9/Meis1. ShRNA-mediated stable knockdown of Lgr4 markedly reduced colony formation of HSC expressing MLL-AF9 by 55-65% (p=0.0001) and significantly prolonged mouse survival (p=0.0019) through its inhibition of endogenous β-catenin expression. This deficient phenotype could be rescued by expression of a constitutively active form of β-catenin. Furthermore, ectopic expression of Lgr4 alone was not sufficient for triggering the leukemic transformation of HSC but conferred a growth advantage in vivo to HSC expressing Hoxa9/Meis1 and significantly accelerated the onset of Hoxa9/Meis1-induced AML in mice (p=0.0011). These data support an oncogenic role of Lgr4 in promoting tumor formation through activation of β-catenin signaling. As Lgr4 has recently been identified as a receptor for the Rspo family of secreted proteins (Rspo1–Rspo4), we sought to determine if Rspo is a positive regulator of β-catenin signaling in MLL AML. We found that only the combination of Rspo3 and Wnt3a potently enhanced β-catenin signaling in HSC expressing MLL-AF9 whereas Rspo and Wnt3a alone or the combination of Wnt3a with other Rspo had no effects on β-catenin activity. Depletion of Lgr4 completely abolished Rspo3/Wnt3a-induced β-catenin signaling, suggesting Rspo3/Wnt3a potentiating β-catenin signaling through Lgr4. Next, we assessed if Lgr4 signals through G protein pathways. By testing G protein alpha inhibitors in MLL LSC, we demonstrated that G protein alpha-q (Gnaq) was required for maintenance of stem cell properties by chemical suppression of the Gnaq-activated β-catenin pathway with a Gnaq selective inhibitor, which exhibited a 3-fold decrease in colony formation (p=0.0001) and a 4-fold reduction in cell number (p=0.0009), and was sufficient to induce substantial cell differentiation and apoptosis. Treatment with Gnaq inhibitor abolished the effect of Lgr4 on β-catenin transactivation, implicating an Lgr4-Gnaq-β-catenin signaling pathway in MLL LSC. Microarray analysis of gene expression confirmed enrichment of genes related to cancer cell proliferation, migration and growth, as well as enrichment of Wnt target genes in LSC expressing Lgr4. Taken together, we report here an Rspo3/Wnt3a-Lgr4-Gnaq-β-catenin signaling circuit in MLL leukemogenesis. Interference with components of the circuit can block β-catenin signaling and perturb leukemia development. Thus, our findings provide potential therapeutic targets in treating LSC-based hematological malignancy driven by Wnt/β-catenin signaling. Disclosures: No relevant conflicts of interest to declare.

Tet2 Is a Novel Regulator of Murine Macrophage Differentiation and Polarization

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 646-646
Author(s):  
Alyssa Cull ◽  
Brooke Snetsinger ◽  
Michael J. Rauh
Keyword(s):  
Gene Expression ◽  
Blood Plasma ◽  
Mrna Expression ◽  
Conflicts Of Interest ◽  
Environmental Influence ◽  
Macrophage Polarization ◽  
M2 Macrophage ◽  
Mrna Levels ◽  
Hematopoietic Stem ◽  
Arginase 1

Abstract Introduction: The epigenetic regulator, TET2, catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. Inactivating TET2 mutations are common in myeloid cancers such as chronic myelomonocytic leukemia (CMML). Although TET2 has been characterized in hematopoietic stem and progenitor cells, little is known about its role in disease-relevant monocytes/macrophages (MΦ). Previously, we found increased expression of M2 MΦ-associated arginase 1 (Arg1) in TET2 -mutant CMML and Tet2 -deficient MΦ. Therefore, our goals were to (1) characterize Tet family expression during normal murine MΦ differentiation and polarization, (2) determine the effect of Tet2 -deficiency on broader M1-M2 MΦ spectrum gene signatures. Methods: Hematopoietic-specific Tet2+/- and Tet2-/- knockout mice were generated by breeding floxed Tet2(f/f) with Vav-Cre mice (JAX), in accordance with Queen's University's Animal Care protocols. MΦs obtained by peritoneal lavage (PMΦ) and bone marrow differentiation (BMMΦ) from 9-13 week old Tet2-/- and 20-40 week old Tet2+/- mice were treated with an M1 stimulus (100ng/mL LPS) or an M2 stimulus (10ng/mL Il-4). Comparative gene expression analysis was conducted using a 591 candidate gene Mouse Immunology Gene Expression CodeSet (NanoString). Blood plasma samples collected from Tet2f/f and Tet2-/- mice were sent for cytokine/chemokine array analysis (Eve Technologies). Results: A survey of Tet mRNA expression in wild-type C57BL/6 mouse whole BM showed that Tet1 was most abundantly expressed, with Tet2 and Tet3 having relative abundances of 0.56±0.05 and 0.09±0.01 respectively. In contrast, Tet2 expression peaked, while Tet1 expression diminished during BMMΦ differentiation. Suggesting a functional role, loss of murine Tet2 is associated with skewed myelomonocytic differentiation (i.e. CMML phenotype). In terminally-differentiated MΦ, Tet2 was the most abundantly expressed Tet gene, suggesting MΦ-specific functions. Consistent with this, following a 3-hour LPS stimulation, Tet2 mRNA levels increased 2- to 4-fold, whereas Il-4 failed to induce a similar increase in expression. Overall, our results suggested that Tet2 plays a role in M1 but not M2 macrophage polarization. Based on these findings, we hypothesized that loss of Tet2 would lead to M1 program dysregulation. PMΦs were obtained from Tet2f/f and Tet2-/- mice (n=2/ genotype) and RNA was harvested from untreated and LPS- or Il-4-treated cells. Pools of these RNA samples were then screened using Nanostring. Overall, M1-associated markers such as Stat1, Socs1, Nfkbiz, Il-6, Il-27, Il-12, Il-1 and Ccl2 were markedly increased by 2- to 50-fold in resting Tet2-/- PMΦs compared to matched Tet2f/f samples. These same M1 genes demonstrated a reduced ability to be induced by LPS treatment. We also found that while the expression of most M2 genes was similar in controls versus knockouts, Il-1rn and Arg1 were overexpressed, and Marco was decreased. This suggested that Tet2 -deficient MΦs possess a complex phenotype with a potential homeostatic response to M1 gene dysregulation. We have previously seen variable upregulation of Arg1 in mouse BMMΦs and PMΦs. Approximately 60% of Tet2-deficient mice (+/- and -/-) (n=20) tested for MΦ Arg1 mRNA expression demonstrated 2- to 90-fold increases in Arg1 compared to pooled Tet2f/f controls (n=5). We were interested in investigating the underlying mechanisms contributing to this dramatic increase in expression. Using Nanostring on pooled Tet2-deficient PMΦs with low (n=7) or high (n=8) Arg1 mRNA expression, we were able to identify genes whose expression significantly correlated with Arg1 overexpression: Cxcl3 (p=0.0329), Ppbp (p=0.0015), Cxcl1 (p=0.0104) and Ccl6 (p=0.0185). Of note, Ppbp was the most divergently expressed gene (46-fold difference) in Arg1 low vs Arg1 high macrophages, followed by Arg1 itself (14-fold difference). Suggesting a further environmental influence, blood plasma levels of TNF-alpha, Il-1b, Il-4, Il-10, Il-12 and Il-13 were significantly elevated in mice with high PMΦ Arg1 mRNA expression (n=5) compared to those with low expression (n=10). Conclusions: Tet2 is a novel regulator of murine MΦ, induced during MΦ differentiation and M1-polarization. Tet2 loss leads to complex disruption of the M1-M2 spectrum. We are currently exploring whether human TET2 mutations contribute to the abnormal immune environment of myeloid cancers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of Lysine-Specific Demethylase 1A (LSD1) Supports Human HSCs in Culture By Preserving Their Immature State

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 194-194
Author(s):  
Agatheeswaran Subramaniam ◽  
Mehrnaz Safaee Talkhoncheh ◽  
Kristijonas Zemaitis ◽  
Shubhranshu Debnath ◽  
Jun Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Division ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Enrichment Score ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Human Hscs

Abstract The molecular mechanisms that govern hematopoietic stem cell (HSC) fate decisions remain incompletely defined. It has been a long-standing goal in the field to gain a better understanding of the genes and pathways that regulate the self-renewal ability of HSCs in order to develop optimal culture conditions in which HSCs can be expanded for clinical benefit. Lysine-specific histone demethylase 1A (LSD1), also known as lysine (K)-specific demethylase 1A (KDM1A), regulates gene expression by specifically eliminating di- and mono-methyl groups on H3 lysine K4 and K9 residues. Studies in mice have shown that, conditional knockdown of LSD1 results in an expansion of bone marrow hematopoietic stem and progenitor cells (HSPCs). However, a complete knockout of LSD1 results in pancytopenia and a dramatic reduction of HSPCs. In this study, we asked whether inhibition of LSD1 would improve the maintenance or expansion of cultured human HSCs derived from umbilical cord blood (UCB). To evaluate the effect of LSD1 inhibition we treated UCB CD34+ cells with three different LSD1 inhibitors (2-PCPA, GSK-LSD1 and RN1) at their respective IC50 values (20µM, 16nM and 70nM) and expanded the cultures for 6 days in serum free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L). Since we (Subramaniam et. al. Haematologica 2018) and others recently have shown that EPCR is a reliable cell surface marker to track UCB derived HSCs during in vitro culture, we quantified the numbers of CD34+EPCR+ cells using flow cytometry and compared to DMSO treated control cultures. Remarkably, treatment with either 2-PCPA or GSK-LSD1 resulted in a more than 10-fold increase of CD34+EPCR+ cells, compared to controls. Further, from dose response experiments we found that 2-PCPA at 1.25 µM expanded the total CD34+ cell population more efficiently than GSK-LSD1, and we therefore used 2-PCPA at this concentration for the subsequent experiments. Using carboxyfluorescein succinimidyl ester (CFSE) labeling to monitor cell division, we found that 2-PCPA did not significantly alter the cell division rate of the cultured CD34+ cells compared to DMSO controls, suggesting that the expansion of CD34+EPCR+ cells is not due to increased proliferation, and that LSD1 inhibition rather may prevent differentiation of the immature HSPCs. To further explore this, we mapped the early transcriptional changes triggered by 2-PCPA in HSCs using gene expression profiling of CD34+CD38-CD45RA-CD90+ cells following 24 hours of culture with or without 2-PCPA treatment. We found that gene sets corresponding to UCB and fetal liver HSCs were significantly enriched upon 2-PCPA treatment compared to DMSO control (Normalized Enrichment Score (NES)=1.49, q=0.05). This suggest that 2-PCPA indeed restricts differentiation and preserves the HSC state upon ex vivo culture. Strikingly, the gene signature induced by LSD1 inhibition was highly similar to that induced by the known HSC expanding compound UM171 (NES=1.43, q=0.11). UM171 is a molecule with unknown target and has also been shown to dramatically expand the EPCR+ population in culture. Finally, the frequency of functional HSCs in DMSO and 2-PCPA treated cultures were measured using limiting dilution analysis (LDA). LDA was performed by transplanting 4 doses (day 0 equivalents of 20000, 1000, 300 and 100 CD34+ cells) of DMSO and 2-PCPA treated cultures into sub lethally irradiated (300cGy) NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human CD45+ cell engraftment in the bone marrow was analyzed 18 weeks' post transplantation. Cultures treated with 2-PCPA showed a 5-fold higher content of long-term repopulating cells per day 0 CD34+ cell equivalent compared to the DMSO control (1 in 615 vs 1 in 3041, p=0.03). Thus, the 2-PCPA treated cultures had significantly enhanced HSCs numbers. To determine the absolute expansion rate, we are currently performing LDA using uncultured cells as well. Altogether our data suggest that LSD1 inhibition supports both phenotypic and functional HSCs in culture by preserving the immature state. Currently we are exploring the possibilities of using LSD1 inhibitors in combination with other known modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

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