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T-LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of constitutive activation of the STAT3 and ERK pathway. Notably, in 40% of patients, constitutive STAT3 activation is due to STAT3activating mutations, whereas in 60% this is unknown. As miRNAs are amongst the most potent regulators in health and disease, we hypothesized that aberrant miRNA expression could contribute to dysregulation of these pathways. miRNA sequencing in T-LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miR-181a. Furthermore, geneset enrichment analysis (GSEA) of downregulated targets of miR-181a implicated involvement in regulating STAT3 and ERK1/2 pathways. Flow cytometric analyses showed increased SOCS3+ and DUSP6+ T-LGL cells upon miR-181a inhibition. In addition, miR-181a-transfected human CD8+ T cells showed increased basal STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line, we could show that miR-181a is an actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition and ERK1/2 phosphorylation by DUSP6 inhibition and verified this mechanism in an independent cell line. In addition, miR-181a inhibition resulted in a higher sensitivity to FAS-mediated apoptosis. Collectively, our data show that miR-181a could be the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

MIR139is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139targets that mediate the tumor-suppressing activities of miR-139in MLL-AF9 AML. We revealed that two enhancer regions control MIR139expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139and reveal a role for POLR2Min the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML.

Genome-wide association studies (GWAS) have identified many susceptibility loci for cardiometabolic disorders. Most of the associated variants reside in non-coding regions of the genome including long non-coding RNAs (lncRNAs), which are thought to play critical roles in diverse biological processes. Here, we leveraged data from the available GWAS meta-analyses on lipid and obesity-related traits, blood pressure, type 2 diabetes, and coronary artery disease and identified 179 associated single-nucleotide polymorphisms (SNPs) in 102 lncRNAs (p-value < 2.3 × 10−7). Of these, 55 SNPs, either the lead SNP or in strong linkage disequilibrium with the lead SNP in the related loci, were selected for further investigations. Our in silico predictions and functional annotations of the SNPs as well as expression and DNA methylation analysis of their lncRNAs demonstrated several lncRNAs that fulfilled predefined criteria for being potential functional targets. In particular, we found evidence suggesting that LOC157273(at 8p23.1) is involved in regulating serum lipid-cholesterol. Our results showed that rs4841132 in the second exon and cg17371580 in the promoter region of LOC157273are associated with lipids; the lncRNA is expressed in liver and associates with the expression of its nearby coding gene, PPP1R3B. Collectively, we highlight a number of loci associated with cardiometabolic disorders for which the association may act through lncRNAs.

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.

Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only.

Neutrophilic differentiation is dependent on CCAATenhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPAis located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPAis active and engages with the CEBPApromoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpalevels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPAexpression in myeloid cells only.

Interstrand crosslinks (ICLs) are toxic DNA lesions that cause severe genomic damage during replication, especially in Fanconi anemia pathway-deficient cells. This results in progressive bone marrow failure and predisposes to acute myeloid leukemia (AML). The molecular mechanisms responsible for these defects are largely unknown. Using Ercc1-deficient mice, we show that Trp53 is responsible for ICL-induced bone marrow failure and that loss of Trp53 is leukemogenic in this model. In addition, Ercc1-deficient myeloid progenitors gain elevated levels of miR-139-3p and miR-199a-3p with age. These microRNAs exert opposite effects on hematopoiesis. Ectopic expression of miR-139-3p strongly inhibited proliferation of myeloid progenitors, whereas inhibition of miR-139-3p activity restored defective proliferation of Ercc1-deficient progenitors. Conversely, the inhibition of miR-199a-3p functions aggravated the myeloid proliferation defect in the Ercc1-deficient model, whereas its enforced expression enhanced proliferation of progenitors. Importantly, miR-199a-3p caused AML in a pre-leukemic mouse model, supporting its role as an onco-microRNA. Target genes include HuR for miR-139-3p and Prdx6, Runx1, and Suz12 for miR-199a-3p. The latter genes have previously been implicated as tumor suppressors in de novo and secondary AML. These findings show that, in addition to TRP53-controlled mechanisms, miR-139-3p and miR-199a-3p are involved in the defective hematopoietic function of ICL-repair deficient myeloid progenitors.

Interstrand crosslinks (ICLs) are toxic DNA lesions that cause severe genomic damage during replication, especially in Fanconi anemia pathway-deficient cells. This results in progressive bone marrow failure and predisposes to acute myeloid leukemia (AML). The molecular mechanisms responsible for these defects are largely unknown. Using Ercc1-deficient mice, we show that Trp53is responsible for ICL-induced bone marrow failure and that loss of Trp53is leukemogenic in this model. In addition, Ercc1-deficient myeloid progenitors gain elevated levels of miR-139-3pand miR-199a-3pwith age. These microRNAs exert opposite effects on hematopoiesis. Ectopic expression of miR-139-3pstrongly inhibited proliferation of myeloid progenitors, whereas inhibition of miR-139-3pactivity restored defective proliferation of Ercc1-deficient progenitors. Conversely, the inhibition of miR-199a-3pfunctions aggravated the myeloid proliferation defect in the Ercc1-deficient model, whereas its enforced expression enhanced proliferation of progenitors. Importantly, miR-199a-3pcaused AML in a pre-leukemic mouse model, supporting its role as an onco-microRNA. Target genes include HuRfor miR-139-3pand Prdx6, Runx1, and Suz12for miR-199a-3p.The latter genes have previously been implicated as tumor suppressors in de novo and secondary AML. These findings show that, in addition to TRP53-controlled mechanisms, miR-139-3pand miR-199a-3pare involved in the defective hematopoietic function of ICL-repair deficient myeloid progenitors.

Supplemental Digital Content is available in the text.

MicroRNAs (miRNAs) belong to an abundant class of highly conserved small (22nt) non-coding RNAs. MiRNA profiling studies indicate that their expression is highly cell type-dependent. DICER1 is an essential RNase III endoribonuclease for miRNA processing. Hematopoietic cell type- and developmental stage-specific Dicer1deletion models show that miRNAs are essential regulators of cellular survival, differentiation and function. For instance, miRNA deficiency in hematopoietic stem cells and progenitors of different origins results in decreased cell survival, dramatic developmental aberrations or dysfunctions in mice. We recently found that homozygous Dicer1deletion in myeloid-committed progenitors results in an aberrant expression of stem cell genes and induces a regained self-renewal capacity. Moreover, Dicer1deletion causes a block in macrophage development and myeloid dysplasia, a cellular condition that may be considered as a preleukemic state. However, Dicer1-null cells do not develop leukemia in mice, indicating that depletion of miRNAs is not enough for tumorigenesis. Surprisingly, we found that heterozygous Dicer1deletion in myeloid-committed progenitors, but not Dicer1knockout, collaborates with p53deletion in leukemic progression and results in various types of leukemia. Our data indicate that Dicer1is a haploinsufficient tumorsuppressor in hematopoietic neoplasms, which is consistent with the observed downregulation of miRNA expression in human leukemia samples. Here, we review the various hematopoietic specific Dicer1deletion mouse models and the phenotypes observed within the different hematopoietic lineages and cell developmental stages. Finally, we discuss the role for DICER1 in mouse and human malignant hematopoiesis.

Recent data show that microRNAs play critical roles in the regulation of the developmental process of hematopoietic stem and progenitor cells toward mature myeloid cells. The main focus of the article is the function of some evolutionary conserved microRNAs that are abundantly expressed and tightly regulated during myelopoiesis.

MicroRNAs (miRNAs) have the potential to regulate cellular differentiation programs; however, miRNA deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions un-answered. To address this issue, we deleted Dicer1, which encodes an essential RNase III enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein α (C/EBPA)–positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA–positive progenitor cells. Analysis of gene-expression profiles from wild-type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multipotent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating the regained self-renewal potential of these cells. In mice, Dicer1deletion blocked monocytic differentiation, depleted macrophages, and caused myeloid dysplasia with morphologic features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion toward myeloid differentiation in GMPs.

MicroRNAs (miRNAs) have the potential to regulate cellular differentiation programs; however, miRNA deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions un-answered. To address this issue, we deleted Dicer1, which encodes an essential RNase III enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein α (C/EBPA)–positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA–positive progenitor cells. Analysis of gene-expression profiles from wild-type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multipotent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating the regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages, and caused myeloid dysplasia with morphologic features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion toward myeloid differentiation in GMPs.

MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93and -106, all AAAGUGC seed-containing miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of Sqstm1transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106expression in AML samples. ShRNA-mediated silencing of Sqstm1phenocopied the effects of ectopic miR-17/20/93/106expression in hematopoietic progenitors in vitro and in mice. Further, SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment, but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion, replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways.

MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93 and -106, all AAAGUGC seed-containing miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further, SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment, but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion, replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways.

The granulocyte colony-stimulating factor receptor (G-CSF-R) transmits signals for proliferation and differentiation of myeloid progenitor cells. Here we report on the identification of a rare single nucleotide polymorphism within its intracellular domain (G-CSF-R_Glu785Lys). Screening a cohort of 116 patients with primary myelodysplastic syndromes (MDS), de novo acute myeloid leukemia (AML) (84 patients), as well as 232 age- and sex-matched controls revealed a highly significant association of the G-CSF-R_785Lys allele with the development of high-risk MDS as defined by more than 5% bone marrow blasts (9.7% versus 0.9% in controls; P = .001; odds ratio [OR], 12.5; 95% confidence interval [CI], 2.4-58.9) or an International Prognostic Scoring System score of intermediate-2 or high (13.0% versus 0.9%; P < .001; OR, 14.0; 95% CI, 3.4-85.0). Functional analysis by retroviral transfer of G-CSF-R_785Lys into myeloid progenitor cells of G-CSF-R–deficient mice showed a significantly diminished colony-formation capacity after G-CSF stimulation as compared with cells transduced with the wild-type receptor. These results suggest that lifelong altered G-CSF response by the G-CSF-R_785Lys may render individuals susceptible to development of high-risk MDS.

The granulocyte colony-stimulating factor receptor (G-CSF-R) transmits signals for proliferation and differentiation of myeloid progenitor cells. Here we report on the identification of a rare single nucleotide polymorphism within its intracellular domain (G-CSF-R_Glu785Lys). Screening a cohort of 116 patients with primary myelodysplastic syndromes (MDS), de novo acute myeloid leukemia (AML) (84 patients), as well as 232 age- and sex-matched controls revealed a highly significant association of the G-CSF-R_785Lys allele with the development of high-risk MDS as defined by more than 5% bone marrow blasts (9.7% versus 0.9% in controls; P= .001; odds ratio [OR], 12.5; 95% confidence interval [CI], 2.4-58.9) or an International Prognostic Scoring System score of intermediate-2 or high (13.0% versus 0.9%; P< .001; OR, 14.0; 95% CI, 3.4-85.0). Functional analysis by retroviral transfer of G-CSF-R_785Lys into myeloid progenitor cells of G-CSF-R–deficient mice showed a significantly diminished colony-formation capacity after G-CSF stimulation as compared with cells transduced with the wild-type receptor. These results suggest that lifelong altered G-CSF response by the G-CSF-R_785Lys may render individuals susceptible to development of high-risk MDS.

The genetic defects underlying the pathogenesis of acute myeloid leukemia (AML) are still largely unknown. Retroviral insertion mutagenesis in mice has become a powerful tool to identify candidate genes involved in the development of leukemia and lymphoma. We have used this strategy with the 1.4 strain of Graffi murine leukemia virus (MuLV), which predominantly causes myeloid leukemias. Here, we report that Graffi-1.4–induced AML frequently harbors virus integrations in the gene encoding the transcription factor Yin Yang 1 (YY1). These integrations occurred in both orientations, and all were located in the 5′ promoter region of the gene, 0.5 to 1.5 kb upstream of the major transcriptional start site. Luciferase reporter assays showed that virus integration in this region increases promoter activity and renders it independent of a functional binding site for Sp1, a major transcriptional regulator of YY1. We used the murine 32D model to study the consequence of perturbed YY1 expression for myelopoiesis. YY1 protein levels were high in 32D parental cells maintained in interleukin-3–containing medium, but they dropped when the cells were induced to differentiate by granulocyte–colony-stimulating factor (G-CSF). Strikingly, G-CSF–induced neutrophilic differentiation was reduced in 32D cell transfectants ectopically expressing YY1. In similar experiments on primary bone marrow cells, enforced YY1 expression blocked the outgrowth of CFU-GM colonies. Increased YY1 expression was seen in some cases of human AML. Collectively, these data imply a possible role of perturbed expression of YY1 in the development of AML through interference with the myeloid differentiation program in the leukemic progenitor cells.

The genetic defects underlying the pathogenesis of acute myeloid leukemia (AML) are still largely unknown. Retroviral insertion mutagenesis in mice has become a powerful tool to identify candidate genes involved in the development of leukemia and lymphoma. We have used this strategy with the 1.4 strain of Graffi murine leukemia virus (MuLV), which predominantly causes myeloid leukemias. Here, we report that Graffi-1.4–induced AML frequently harbors virus integrations in the gene encoding the transcription factor Yin Yang 1 (YY1). These integrations occurred in both orientations, and all were located in the 5′ promoter region of the gene, 0.5 to 1.5 kb upstream of the major transcriptional start site. Luciferase reporter assays showed that virus integration in this region increases promoter activity and renders it independent of a functional binding site for Sp1, a major transcriptional regulator of YY1. We used the murine 32D model to study the consequence of perturbed YY1 expression for myelopoiesis. YY1 protein levels were high in 32D parental cells maintained in interleukin-3–containing medium, but they dropped when the cells were induced to differentiate by granulocyte–colony-stimulating factor (G-CSF). Strikingly, G-CSF–induced neutrophilic differentiation was reduced in 32D cell transfectants ectopically expressing YY1. In similar experiments on primary bone marrow cells, enforced YY1 expression blocked the outgrowth of CFU-GM colonies. Increased YY1 expression was seen in some cases of human AML. Collectively, these data imply a possible role of perturbed expression of YY1 in the development of AML through interference with the myeloid differentiation program in the leukemic progenitor cells.

No relevant conflicts of interest to declare.

Background:C/EBPa is a lineage determining transcription factor, critical for terminal cell differentiation in different tissues including the bone marrow (BM), lung, liver, and adipocytes. Adequate CEBPAlevels are needed to maintain the haematopoietic stem cell (HSC) pool and to promote neutrophilic differentiation. This knowledge points towards the importance of CEBPAdosage at different stages of differentiation.

No relevant conflicts of interest to declare.

Abstract 2542This icon denotes a clinically relevant abstract

No relevant conflicts of interest to declare.

Dutch Scientific Organisation (NWO-VENI) and Dutch Cancer Society (KWF)No relevant conflicts of interest to declare.

Abstract 43

Abstract 746

No relevant conflicts of interest to declare.

Abstract 1463

G-CSF is the major regulator of neutrophil development and controls the proliferation, differentiation and survival of myeloid progenitors in both steady state and “emergency” granulopoiesis. More recently however, novel activities of the G-CSF/G-CSF receptor (CSF3R) axis have been suggested, especially after severe forms of tissue damage. For instance, upon myocardial infarction, G-CSF was shown to protect cardiomyocytes from apoptosis, reducing the extent of myocardial damage. Likewise, in ischemic stroke models, G-CSF treatment reduced infarct volumes by promoting neuronal survival and differentiation of neural stem cells in the brain. Finally, G-CSF has been suggested to promote hepatic regeneration after severe liver damage by directly stimulating the proliferation of oval cells. To study whether and to what extent CSF3R expression in damaged tissues reflects reactivation of a normal development program, we generated a csf3r-Cre knock-in model. To this end, the csf3r locus was targeted with a construct containing the cre gene and a puromycin selection cassette flanked by a 2.7 kb fragment of the csf3r 5′UTR and a 4 kb fragment directly downstream of the initiation codon of csf3r. Correct insertion of the cre gene was confirmed by Southern blotting and sequence analysis and subsequent crossing with FLP deleter mice resulted in successful germ line transmission and deletion of the puromycin cassette. These mice were crossed with the R26R LacZ reporter mouse and β-galactosidase (β-gal) activity was examined by FACS and histochemical analysis. At E9.5, no β-gal activity was detected, indicating that csf3r is not expressed at that stage of development. In contrast, in E12.5 embryos, β-gal staining was observed in fetal liver, heart, kidney, brain and intestine. Analysis of adult tissues showed that cardiomyocytes were β-gal positive in a mosaic pattern. A similar mosaicism was observed in liver, kidney and the intestines. In liver both hepatocytes and vascular endothelial cells stained positive, whereas in the kidney β-gal staining was detected in vascular endothelium, glomeruli and afferent and efferent vessels. In ileum and colon, β-gal activity was mainly seen in the stem and progenitor cell-containing crypts and the developing progeny migrating upwards along the villus. In bone marrow, high levels (>40%) of β-gal activity were detected in Gr1+ and CD11b+ myeloid cells, CD19+ (B) and CD3ε+ (T) lymphoid cells, NK cells and CD11c+ dendritic cells, indicating that G-CSFR was expressed in a common progenitor of these cell types. In agreement with this, β-gal activity was also detected in the primitive LSK population. In conclusion, our data indicate that CSF3R is expressed in both hematopoietic and multiple non-hematopoietic organs from embryonic development stage E12.5 onward. This may have important ramifications for a more wide-spread application of G-CSF in tissue regeneration.

G-CSF is the major regulator of neutrophil development and controls the proliferation, differentiation and survival of myeloid progenitors in both steady state and “emergency” granulopoiesis. More recently however, novel activities of the G-CSF/G-CSF receptor (CSF3R) axis have been suggested, especially after severe forms of tissue damage. For instance, upon myocardial infarction, G-CSF was shown to protect cardiomyocytes from apoptosis, reducing the extent of myocardial damage. Likewise, in ischemic stroke models, G-CSF treatment reduced infarct volumes by promoting neuronal survival and differentiation of neural stem cells in the brain. Finally, G-CSF has been suggested to promote hepatic regeneration after severe liver damage by directly stimulating the proliferation of oval cells. To study whether and to what extent CSF3Rexpression in damaged tissues reflects reactivation of a normal development program, we generated a csf3r-Cre knock-in model. To this end, the csf3rlocus was targeted with a construct containing the cregene and a puromycin selection cassette flanked by a 2.7 kb fragment of the csf3r5′UTR and a 4 kb fragment directly downstream of the initiation codon of csf3r. Correct insertion of the cregene was confirmed by Southern blotting and sequence analysis and subsequent crossing with FLP deleter mice resulted in successful germ line transmission and deletion of the puromycin cassette. These mice were crossed with the R26R LacZreporter mouse and β-galactosidase (β-gal) activity was examined by FACS and histochemical analysis. At E9.5, no β-gal activity was detected, indicating that csf3ris not expressed at that stage of development. In contrast, in E12.5 embryos, β-gal staining was observed in fetal liver, heart, kidney, brain and intestine. Analysis of adult tissues showed that cardiomyocytes were β-gal positive in a mosaic pattern. A similar mosaicism was observed in liver, kidney and the intestines. In liver both hepatocytes and vascular endothelial cells stained positive, whereas in the kidney β-gal staining was detected in vascular endothelium, glomeruli and afferent and efferent vessels. In ileum and colon, β-gal activity was mainly seen in the stem and progenitor cell-containing crypts and the developing progeny migrating upwards along the villus. In bone marrow, high levels (>40%) of β-gal activity were detected in Gr1+and CD11b+myeloid cells, CD19+(B) and CD3ε+(T) lymphoid cells, NK cells and CD11c+dendritic cells, indicating that G-CSFR was expressed in a common progenitor of these cell types. In agreement with this, β-gal activity was also detected in the primitive LSK population. In conclusion, our data indicate that CSF3Ris expressed in both hematopoietic and multiple non-hematopoietic organs from embryonic development stage E12.5 onward. This may have important ramifications for a more wide-spread application of G-CSF in tissue regeneration.

The granulocyte colony-stimulating factor receptor (G-CSF-R) induces proliferation, survival and differentiation of myeloid progenitor cells in a tightly controlled temporal fashion. These responses depend on multiple signaling mechanisms that are activated via distinct regions in the cytoplasmic domain of wild type (WT) G-CSF-R. About 20% of severe congenital neutropenia patients acquire mutations that truncate the C-terminus of G-CSF-R, which is often associated with disease progression to acute myeloid leukemia. Myeloid cells expressing these truncated G-CSF-R hyperproliferate and are hampered in differentiation in response to G-CSF. Multiple mechanisms have been linked to perturbed signaling of truncated G-CSF-R. Specifically, defective internalization has been associated with a prolonged activation status of the truncated G-CSF-R, which is e.g. reflected by strongly increased and sustained activation of STAT5. We found that internalized WT G-CSF-R are rapidly targeted to lysosomes, suggesting that lysosomal degradation is a major mechanism for signal attenuation. Lysine (K) residues in the cytoplasmic tail of transmembrane receptors are often determinants for ubiquitin-mediated sorting into multi-vesicular bodies and lysosomes. We observed that G-CSF-R is ubiquitinated and subsequently assessed the contribution of K residues in G-CSF-R routing and signaling. To this end, we generated mutant G-CSF-R-K5R, in which all five conserved cytoplasmic K residues were replaced by arginine (R). To study differential localization of internalized K5R and WT G-CSF-R, we introduced constitutively active Rab5 (Rab5-Q79L) to enlarge early endosomes. Under these conditions, internalized WT G-CSF-R colocalized extensively with endosomal microdomains containing the endosome-to-lysosome sorting protein Hrs. In contrast, internalized G-CSF-R-K5R was mostly found outside these domains. Consequently, lysosomal routing of G-CSF-R-K5R was severely impaired, which resulted in strongly increased receptor protein levels. Upon removal of G-CSF, downregulation of STAT5 activity in 32D/K5R cells was significantly delayed compared to 32D/WT cells, establishing that the lysine residues are not only crucial for receptor stability, but also for duration of signaling. Moreover, in the continuous presence of G-CSF, 32D/K5R cells showed strongly increased STAT5 activity and proliferation and reduced granulocytic differentiation compared to 32D/WT. Similar results were obtained in colony assays with g-csfr deficient primary bone marrow progenitors transduced with K5R or WT G-CSF-R. To substantiate a relationship between signal duration and receptor degradation, we inhibited lysosomal degradation by pharmacological inhibitors and by knockdown of Hrs and the related protein Tsg101 by RNA interference. As expected, receptor stability was significantly increased by these treatments. Surprisingly however, this did not influence STAT5 activity, suggesting that signal attenuation predominantly occurred in a pre-lysosomal compartment. We conclude from these data that ubiquitinated lysine residues of G-CSF-R are required to target internalized receptors for lysosomal degradation. However, they also appear to be involved in contracting an as yet unidentified inhibitor, perhaps a phosphatase, to a specific Hrs-positive subendosomal compartment, thereby switching off G-CSF-R already prior to its degradation in lysosomes.

The granulocyte colony-stimulating factor receptor (G-CSF-R) induces proliferation, survival and differentiation of myeloid progenitor cells in a tightly controlled temporal fashion. These responses depend on multiple signaling mechanisms that are activated via distinct regions in the cytoplasmic domain of wild type (WT) G-CSF-R. About 20% of severe congenital neutropenia patients acquire mutations that truncate the C-terminus of G-CSF-R, which is often associated with disease progression to acute myeloid leukemia. Myeloid cells expressing these truncated G-CSF-R hyperproliferate and are hampered in differentiation in response to G-CSF. Multiple mechanisms have been linked to perturbed signaling of truncated G-CSF-R. Specifically, defective internalization has been associated with a prolonged activation status of the truncated G-CSF-R, which is e.g. reflected by strongly increased and sustained activation of STAT5. We found that internalized WT G-CSF-R are rapidly targeted to lysosomes, suggesting that lysosomal degradation is a major mechanism for signal attenuation. Lysine (K) residues in the cytoplasmic tail of transmembrane receptors are often determinants for ubiquitin-mediated sorting into multi-vesicular bodies and lysosomes. We observed that G-CSF-R is ubiquitinated and subsequently assessed the contribution of K residues in G-CSF-R routing and signaling. To this end, we generated mutant G-CSF-R-K5R, in which all five conserved cytoplasmic K residues were replaced by arginine (R). To study differential localization of internalized K5R and WT G-CSF-R, we introduced constitutively active Rab5 (Rab5-Q79L) to enlarge early endosomes. Under these conditions, internalized WT G-CSF-R colocalized extensively with endosomal microdomains containing the endosome-to-lysosome sorting protein Hrs. In contrast, internalized G-CSF-R-K5R was mostly found outside these domains. Consequently, lysosomal routing of G-CSF-R-K5R was severely impaired, which resulted in strongly increased receptor protein levels. Upon removal of G-CSF, downregulation of STAT5 activity in 32D/K5R cells was significantly delayed compared to 32D/WT cells, establishing that the lysine residues are not only crucial for receptor stability, but also for duration of signaling. Moreover, in the continuous presence of G-CSF, 32D/K5R cells showed strongly increased STAT5 activity and proliferation and reduced granulocytic differentiation compared to 32D/WT. Similar results were obtained in colony assays with g-csfr deficient primary bone marrow progenitors transduced with K5R or WT G-CSF-R. To substantiate a relationship between signal duration and receptor degradation, we inhibited lysosomal degradation by pharmacological inhibitors and by knockdown of Hrs and the related protein Tsg101 by RNA interference. As expected, receptor stability was significantly increased by these treatments. Surprisingly however, this did not influence STAT5 activity, suggesting that signal attenuation predominantly occurred in a pre-lysosomal compartment. We conclude from these data that ubiquitinated lysine residues of G-CSF-R are required to target internalized receptors for lysosomal degradation. However, they also appear to be involved in contracting an as yet unidentified inhibitor, perhaps a phosphatase, to a specific Hrs-positive subendosomal compartment, thereby switching off G-CSF-R already prior to its degradation in lysosomes.

Acute myeloid leukemia (AML) is a heterogeneous disease with variable responses to treatment, characterized by specific chromosomal breakpoint products such as AML-ETO, PML-RAR or mutations in e.g., c/EBPα or FLT3 genes. These abnormalities are not sufficient to cause AML and approximately 40% of AML cases present without these defects or have more complex abnormalities. To find genes involved in AML, we recently isolated common integration sites (CIS) from Graffi-1.4 MuLV (Gr1.4)-induced mouse myeloid leukemia and identified the genes affected by these integrations (Erkeland S.J., et al, J. Virol., 2004). To assess the significance of these genes for human AML, we determined their contribution to 16 distinct classes of patients recently defined based on gene expression profiling of 285 AML samples using Affymetrix U133A microarrays (Valk P.J., et al, NEJM, 2004) plus 11 additional groups based on known chromosomal aberrations or gene mutations. Using the significance analysis of microarrays (SAM) methods we could link specific gene sets to these 27 patient classes. Genes were considered to contribute to the signature of the class when the following criteria were met: an RNA expression Fold Change over 1.5 or under 0.67, a score over 4 or less than −4 and a q-value less then 5%. Three gene lists derived from the Gr1.4-induced leukemia samples were generated: (I) genes (n=51, represented by 116 probe sets), within or most proximal to CIS; (II) genes (n=53, 81 probe sets) located next to the CIS flanking genes; (III) genes (n=279, 468 probe sets) located more distantly from the CIS (up to 1 megabases), with a maximum of 5 genes in each direction. Each of these gene lists was compared to the 27 outcomes of the SAM analyses to count the number of cases where a probe set of one of the three lists was significantly deregulated. To establish whether the fraction of deregulated genes in one of the three lists was significantly higher than the expected fraction of the total list, a chi-square test was performed. The list of CIS shows a significant overrepresentation of deregulated genes (Ψ2 =13.4 and p=0.0002). In contrast, the list of two closest flanking genes does not show overrepresentation (Ψ2= 0.4 and p=0.54), and neither does the list of 10 neighboring genes (Ψ2= 1.7 and p= 0.19). We repeated the analysis on a profiling dataset from 130 childhood AML samples (Ross ME, et al, Blood 2004). Using this dataset, we performed 5 SAM analyses based on known chromosomal aberrations or specific FAB-type. Again, this showed an overrepresentation of deregulated genes in case of the CIS (Ψ2= 6.3, p=0.012) but not for the more distant genes (2 closest genes: Ψ2= 0.006, p=0.936, 10 neighboring genes: Ψ2= 1.67, p=0.196). We conclude from these analyses that the closest CIS flanking genes have the highest probability to be involved in human AML. We next performed a similar analysis using the list of virus integrations from the BXH2 myeloid leukemia model (http://genome2.ncifcrf.gov/RTCGD/). This list, consisting of 53 genes represented by 111 probe sets also showed a higher representation of deregulated genes of adult AML (Ψ2= 112.2, p=0.000) as well as of pediatric AML samples (Ψ2= 14.0 and p=0.0001). Examples of CIS genes that are found associated with specific subgroups of clinical AML are CTNNA1, IRS-2, VDUP1, DUSP10 and PRDXII. These results demonstrate the power of combining retroviral tagging of cancer genes with expression array analysis of clinical leukemia samples for the identification of pathogenetic mechanisms in human AML.

Graffi-1.4 (Gr-1.4) and CasBrM murine leukemia viruses induce myeloid leukemias due to deregulation of genes by proviral integration. Some of these genes, such as peroxiredoxin (PRDX-2), vitamin D upregulated protein 1 (VDUP1), and dual specific phosphatase 10 (DUSP10), are involved in the regulation of reactive oxygen species (ROS) induced stress pathways. ROS are known to influence signal transduction by modulating the activity of protein and lipid phosphatases and cell differentiation at relatively low levels, whereas high ROS levels induce apoptosis. Perturbations of the cellular redox state have a high impact on these processes. VDUP1 is a recently identified oxidative stress-responsive gene that suppresses thioredoxin activity in cardiomyocytes, thereby controlling cell survival. Integrations in the gene encoding VDUP1 all occurred at the 5′ and 3′ region with a frequency of 100% of the Gr-1.4 and 62% of the CasBrM induced leukemias. All cases of CasBrM leukemias with 3′ integrations (33% of leukemias) have additional integrations at the 5′end of VDUP1, suggesting that multiple virus integration sites cooperate in gene deregulation. A hotspot of integrations was found around 900 base pairs upstream of the ATG, near two newly identified heat shock elements. The presence of Gr-1.4 LTR sequences in the VDUP1 promoter and in the 3′ untranslated region results in a 2–2.5 times enhanced luciferase signal when compared to normal promoter activity. This effect was even greater (up to 6-fold) under stress conditions, suggesting that normal VDUP1 regulation is disrupted by viral integration. In human AML, we found that expression of VDUP1 transcripts is different in distinct patient clusters recently identified by gene expression profiling (Valk et al NEJM 2004, 350:1617-28), suggesting a specific involvement of this gene in certain subgroups of AML. For instance, AML samples exhibiting t(8;21) have significantly higher VDUP1 transcript levels compared to other AML patients. Furthermore, high VDUP1 protein levels significantly correlated with FAB classifications M4 and M5 and with younger age (<35 yrs), whereas low VDUP1 expression were found in FAB-M1 and M2 and in patients older than 50 years. To study the consequences of VDUP1 overexpression in normal myelopoiesis, we overexpressed the gene in murine hematopoietic progenitors by retroviral gene transfer and performed in vitro colony assays with G-CSF and liquid culture assay with different cytokine cocktails. Irrespective of the cytokine used, ectopic expression of VDUP1 resulted in accelerated apoptosis and inhibited proliferation, indicating that deregulation of VDUP1 as a single event does not confer a growth advantage and implying that additional events are needed for full leukemic transformation of myeloid precursors. DUSP10 or other members of the DUSP family might be candidates, as we observed that overexpression of DUSP10 in myeloid 32D cells decreased oxidative stress-induced activation of JNK and p38MAPK and inhibited apoptosis. We are currently generating 32D models in which VDUP1 and DUSP10 can be inducibly expressed to further address this hypothesis. In conclusion, we found that VDUP1 expression is frequently enhanced in mouse leukemia models due to viral integrations and also in distinct subgroups of human AML. Our data thus identify disruption of VDUP1-controlled oxidative stress responses as a novel mechanism involved in the pathogenesis of AML.

Graffi-1.4 (Gr-1.4) and CasBrM murine leukemia viruses induce myeloid leukemias due to deregulation of genes by proviral integration. Some of these genes, such as peroxiredoxin (PRDX-2), vitamin D upregulated protein 1 (VDUP1), and dual specific phosphatase 10 (DUSP10), are involved in the regulation of reactive oxygen species (ROS) induced stress pathways. ROS are known to influence signal transduction by modulating the activity of protein and lipid phosphatases and cell differentiation at relatively low levels, whereas high ROS levels induce apoptosis. Perturbations of the cellular redox state have a high impact on these processes. VDUP1 is a recently identified oxidative stress-responsive gene that suppresses thioredoxin activity in cardiomyocytes, thereby controlling cell survival. Integrations in the gene encoding VDUP1 all occurred at the 5′ and 3′ region with a frequency of 100% of the Gr-1.4 and 62% of the CasBrM induced leukemias. All cases of CasBrM leukemias with 3′ integrations (33% of leukemias) have additional integrations at the 5′end of VDUP1, suggesting that multiple virus integration sites cooperate in gene deregulation. A hotspot of integrations was found around 900 base pairs upstream of the ATG, near two newly identified heat shock elements. The presence of Gr-1.4 LTR sequences in the VDUP1 promoter and in the 3′ untranslated region results in a 2–2.5 times enhanced luciferase signal when compared to normal promoter activity. This effect was even greater (up to 6-fold) under stress conditions, suggesting that normal VDUP1 regulation is disrupted by viral integration. In human AML, we found that expression of VDUP1 transcripts is different in distinct patient clusters recently identified by gene expression profiling (Valk et al NEJM 2004, 350:1617-28), suggesting a specific involvement of this gene in certain subgroups of AML. For instance, AML samples exhibiting t(8;21) have significantly higher VDUP1 transcript levels compared to other AML patients. Furthermore, high VDUP1 protein levels significantly correlated with FAB classifications M4 and M5 and with younger age (<35 yrs), whereas low VDUP1 expression were found in FAB-M1 and M2 and in patients older than 50 years. To study the consequences of VDUP1 overexpression in normal myelopoiesis, we overexpressed the gene in murine hematopoietic progenitors by retroviral gene transfer and performed in vitro colony assays with G-CSF and liquid culture assay with different cytokine cocktails. Irrespective of the cytokine used, ectopic expression of VDUP1 resulted in accelerated apoptosis and inhibited proliferation, indicating that deregulation of VDUP1 as a single event does not confer a growth advantage and implying that additional events are needed for full leukemic transformation of myeloid precursors. DUSP10 or other members of the DUSP family might be candidates, as we observed that overexpression of DUSP10 in myeloid 32D cells decreased oxidative stress-induced activation of JNK and p38MAPK and inhibited apoptosis. We are currently generating 32D models in which VDUP1 and DUSP10 can be inducibly expressed to further address this hypothesis. In conclusion, we found that VDUP1 expression is frequently enhanced in mouse leukemia models due to viral integrations and also in distinct subgroups of human AML. Our data thus identify disruption of VDUP1-controlled oxidative stress responses as a novel mechanism involved in the pathogenesis of AML.

Acute myeloid leukemia (AML) is a heterogeneous disease with variable responses to treatment, characterized by specific chromosomal breakpoint products such as AML-ETO, PML-RAR or mutations in e.g., c/EBPα or FLT3 genes. These abnormalities are not sufficient to cause AML and approximately 40% of AML cases present without these defects or have more complex abnormalities. To find genes involved in AML, we recently isolated common integration sites (CIS) from Graffi-1.4 MuLV (Gr1.4)-induced mouse myeloid leukemia and identified the genes affected by these integrations (Erkeland S.J., et al, J. Virol., 2004). To assess the significance of these genes for human AML, we determined their contribution to 16 distinct classes of patients recently defined based on gene expression profiling of 285 AML samples using Affymetrix U133A microarrays (Valk P.J., et al, NEJM, 2004) plus 11 additional groups based on known chromosomal aberrations or gene mutations. Using the significance analysis of microarrays (SAM) methods we could link specific gene sets to these 27 patient classes. Genes were considered to contribute to the signature of the class when the following criteria were met: an RNA expression Fold Change over 1.5 or under 0.67, a score over 4 or less than −4 and a q-value less then 5%. Three gene lists derived from the Gr1.4-induced leukemia samples were generated: (I) genes (n=51, represented by 116 probe sets), within or most proximal to CIS; (II) genes (n=53, 81 probe sets) located next to the CIS flanking genes; (III) genes (n=279, 468 probe sets) located more distantly from the CIS (up to 1 megabases), with a maximum of 5 genes in each direction. Each of these gene lists was compared to the 27 outcomes of the SAM analyses to count the number of cases where a probe set of one of the three lists was significantly deregulated. To establish whether the fraction of deregulated genes in one of the three lists was significantly higher than the expected fraction of the total list, a chi-square test was performed. The list of CIS shows a significant overrepresentation of deregulated genes (Ψ2=13.4 and p=0.0002). In contrast, the list of two closest flanking genes does not show overrepresentation (Ψ2= 0.4 and p=0.54), and neither does the list of 10 neighboring genes (Ψ2= 1.7 and p= 0.19). We repeated the analysis on a profiling dataset from 130 childhood AML samples (Ross ME, et al, Blood 2004). Using this dataset, we performed 5 SAM analyses based on known chromosomal aberrations or specific FAB-type. Again, this showed an overrepresentation of deregulated genes in case of the CIS (Ψ2= 6.3, p=0.012) but not for the more distant genes (2 closest genes: Ψ2= 0.006, p=0.936, 10 neighboring genes: Ψ2= 1.67, p=0.196). We conclude from these analyses that the closest CIS flanking genes have the highest probability to be involved in human AML. We next performed a similar analysis using the list of virus integrations from the BXH2 myeloid leukemia model (http://genome2.ncifcrf.gov/RTCGD/). This list, consisting of 53 genes represented by 111 probe sets also showed a higher representation of deregulated genes of adult AML (Ψ2= 112.2, p=0.000) as well as of pediatric AML samples (Ψ2= 14.0 and p=0.0001). Examples of CIS genes that are found associated with specific subgroups of clinical AML are CTNNA1, IRS-2, VDUP1, DUSP10 and PRDXII. These results demonstrate the power of combining retroviral tagging of cancer genes with expression array analysis of clinical leukemia samples for the identification of pathogenetic mechanisms in human AML.