Restoration of MYC-repressed targets mediates the negative effects of GM-CSF on RUNX1-ETO leukemogenicity.

Granulocyte macrophage-colony-stimulating factor (GM-CSF) signaling regulates hematopoiesis and immune responses. CSF2RA, the gene encoding the α-subunit for GM-CSF, is significantly downregulated in t(8;21) (RUNX1-ETO or RE) leukemia patients, suggesting that it may serve as a tumor suppressor. We previously reported that GM-CSF signaling is inhibitory to RE leukemogenesis. Here we conducted gene expression profiling of primary RE hematopoietic stem/progenitor cells (HSPCs) treated with GM-CSF to elucidate the mechanisms mediating the negative effects of GM on RE leukemogenicity. We observed that GM treatment of RE HSPCs resulted in a unique gene expression profile that resembles primary human cells undergoing myelopoiesis, which was not observed in control HSPCs. Additionally, we discovered that GM-CSF signaling attenuates MYC-associated gene signatures in RE HSPCs. In agreement with this, a functional screen of a subset of GM-CSF-responsive genes demonstrated that a MYC inhibitor, MXI1 (Max interactor 1), reduced the leukemic potential of RE HSPCs and t(8;21) acute myeloid leukemia (AML) cells. Furthermore, MYC knockdown and treatment with the BET (bromodomain and extra terminal domain) inhibitor JQ1 reduced the leukemic potential of t(8;21) cell lines. Altogether, we discovered a novel molecular mechanism mediating the GM-CSF-induced reduction in leukemic potential of RE cells, and our findings support MYC inhibition as an effective strategy for reducing the leukemogenicity of t(8;21) AML.

Fatty acid-binding protein FABP4 mechanistically links obesity with aggressive AML by enhancing aberrant DNA methylation in AML cells.

Obesity is becoming more prevalent worldwide and is a major risk factor for cancer development. Acute myeloid leukemia (AML), the most common acute leukemia in adults, remains a frequently fatal disease. Here we investigated the molecular mechanisms by which obesity favors AML growth and uncovered the fatty acid-binding protein 4 (FABP4) and DNA methyltransferase 1 (DNMT1) regulatory axis that mediates aggressive AML in obesity. We showed that leukemia burden was much higher in high-fat diet-induced obese mice, which had higher levels of FABP4 and interleukin (IL)-6 in the sera. Upregulation of environmental and cellular FABP4 accelerated AML cell growth in both a cell-autonomous and cell-non-autonomous manner. Genetic disruption of FABP4 in AML cells or in mice blocked cell proliferation in vitro and induced leukemia regression in vivo. Mechanistic investigations showed that FABP4 upregulation increased IL-6 expression and signal transducer and activator of transcription factor 3 phosphorylation leading to DNMT1 overexpression and further silencing of the p15INK4B tumor-suppressor gene in AML cells. Conversely, FABP4 ablation reduced DNMT1-dependent DNA methylation and restored p15INK4B expression, thus conferring substantial protection against AML growth. Our findings reveal the FABP4/DNMT1 axis in the control of AML cell fate in obesity and suggest that interference with the FABP4/DNMT1 axis might be a new strategy to treat leukemia.

AML1/ETO cooperates with HIF1α to promote leukemogenesis through DNMT3a transactivation.

The mechanisms by which AML1/ETO (A/E) fusion protein induces leukemogenesis in acute myeloid leukemia (AML) without mutagenic events remain elusive. Here we show that interactions between A/E and hypoxia-inducible factor 1α (HIF1α) are sufficient to prime leukemia cells for subsequent aggressive growth. In agreement with this, HIF1α is highly expressed in A/E-positive AML patients and strongly predicts inferior outcomes, regardless of gene mutations. Co-expression of A/E and HIF1α in leukemia cells causes a higher cell proliferation rate in vitro and more serious leukemic status in mice. Mechanistically, A/E and HIF1α form a positive regulatory circuit and cooperate to transactivate DNMT3a gene leading to DNA hypermethylation. Pharmacological or genetic interventions in the A/E-HIF1α loop results in DNA hypomethylation, a re-expression of hypermethylated tumor-suppressor p15(INK4b) and the blockage of leukemia growth. Thus high HIF1α expression serves as a reliable marker, which identifies patients with a poor prognosis in an otherwise prognostically favorable AML group and represents an innovative therapeutic target in high-risk A/E-driven leukemia.

Contribution of increased ISG15, ISGylation and deregulated type I IFN signaling in Usp18 mutant mice during the course of bacterial infections.

Host genetics has a key role in susceptibility to Salmonella Typhimurium infection. We previously used N-ethyl-N-nitrosourea (ENU) mutagenesis to identify a loss-of-function mutation within the gene ubiquitin-specific peptidase 18 (Usp18(Ity9)), which confers increased susceptibility to Salmonella Typhimurium. USP18 functions to regulate type I interferon (IFN) signaling and as a protease to remove ISG15 from substrate proteins. Usp18(Ity9) mice are susceptible to infection with Salmonella Typhimurium and have increased expression and function of ISG15, but Usp18(Ity9) mice lacking Isg15 do not show improved survival with Salmonella challenge. Type I IFN signaling is increased in Usp18(Ity9) mice and inhibition of type I IFN signaling is associated with improved survival in mutant mice. Hyperactivation of type I IFN signaling leads to increased IL-10, deregulated expression of autophagy markers and elevated interleukin (IL)-1ß and IL-17. Furthermore, Usp18(Ity9) mice are more susceptible to infection with Mycobacterium tuberculosis, have increased bacterial load in the lung and spleen, elevated inflammatory cytokines and more severe lung pathology. These findings demonstrate that regulation of type I IFN signaling is the predominant mechanism affecting the susceptibility of Usp18(Ity9) mice to Salmonella infection and that hyperactivation of signaling leads to increased IL-10, deregulation of autophagic markers and increased proinflammatory cytokine production.

JAK inhibitors suppress t(8;21) fusion protein-induced leukemia.

Oncogenic mutations in components of the JAK/STAT pathway, including those in cytokine receptors and JAKs, lead to increased activity of downstream signaling and are frequently found in leukemia and other hematological disorders. Thus, small-molecule inhibitors of this pathway have been the focus of targeted therapy in these hematological diseases. We previously showed that t(8;21) fusion protein acute myeloid leukemia (AML)1-ETO and its alternatively spliced variant AML1-ETO9a (AE9a) enhance the JAK/STAT pathway via downregulation of CD45, a negative regulator of this pathway. To investigate the therapeutic potential of targeting JAK/STAT in t(8;21) leukemia, we examined the effects of a JAK2-selective inhibitor TG101209 and a JAK1/2-selective inhibitor INCB18424 on t(8;21) leukemia cells. TG101209 and INCB18424 inhibited proliferation and promoted apoptosis of these cells. Furthermore, TG101209 treatment in AE9a leukemia mice reduced tumor burden and significantly prolonged survival. TG101209 also significantly impaired the leukemia-initiating potential of AE9a leukemia cells in secondary recipient mice. These results demonstrate the potential therapeutic efficacy of JAK inhibitors in treating t(8;21) AML.

Measuring population differentiation using GST or D? A simulation study with microsatellite DNA markers under a finite island model and nonequilibrium conditions.

The genetic differentiation of populations is a key parameter in population genetic investigations. Wright's F(ST) (and its relatives such as G(ST) ) has been a standard measure of differentiation. However, the deficiencies of these indexes have been increasingly realized in recent years, leading to some new measures being proposed, such as Jost's D (Molecular Ecology, 2008; 17, 4015). The existence of these new metrics has stimulated considerable debate and induced some confusion on which statistics should be used for estimating population differentiation. Here, we report a simulation study with neutral microsatellite DNA loci under a finite island model to compare the performance of G(ST) and D, particularly under nonequilibrium conditions. Our results suggest that there exist fundamental differences between the two statistics, and neither G(ST) nor D operates satisfactorily in all situations for quantifying differentiation. D is very sensitive to mutation models but G(ST) noticeably less so, which limits D's utility in population parameter estimation and comparisons across genetic markers. Also, the initial heterozygosity of the starting populations has some important effects on both the individual behaviours of G(ST) and D and their relative behaviours in early differentiation, and this effect is much greater for D than G(ST) . In the early stages of differentiation, when initial heterozygosity is relatively low (<0.5, if the number of subpopulations is large), G(ST) increases faster than D; the opposite is true when initial heterozygosity is high. Therefore, the state of the ancestral population appears to have some lasting impacts on population differentiation. In general, G(ST) can measure differentiation fairly well when heterozygosity is low whatever the causes; however, when heterozygosity is high (e.g. as a result of either high mutation rate or high initial heterozygosity) and gene flow is moderate to strong, G(ST) fails to measure differentiation. Interestingly, when population size is not very small (e.g. N ≥ 1000), G(ST) measures differentiation quite linearly with time over a long duration when gene flow is absent or very weak even if mutation rate is not low (e.g. µ = 0.001). In contrast, D, as a differentiation measure, performs rather robustly in all these situations. In practice, both indexes should be calculated and the relative levels of heterozygosities (especially H(S) ) and gene flow taken into account. We suggest that a comparison of the two indexes can generate useful insights into the evolutionary processes that influence population differentiation.

Overexpression of an isoform of AML1 in acute leukemia and its potential role in leukemogenesis.

AML1/RUNX1 is a critical transcription factor in hematopoietic cell differentiation and proliferation. From the AML1 gene, at least three isoforms, AML1a, AML1b and AML1c, are produced through alternative splicing. AML1a interferes with the function of AML1b/1c, which are often called AML1. In this study, we found a higher expression level of AML1a in acute lymphoblastic leukemia and acute myeloid leukemia (AML)-M2 patients in comparison to the controls. Additionally, AML1a represses transcription of promoter of macrophage colony-stimulating factor receptor mediated by AML1b, indicating that AML1a antagonized the effect of AML1b. To investigate the role of AML1a in hematopoiesis and leukemogenesis in vivo, murine bone marrow mononuclear cells were transduced with AML1a and then transplanted into lethally irradiated mice, which developed lymphoblastic leukemia after transplantation. Taken together, these results indicate that overexpression of AML1a may be an important contributing factor to leukemogenesis.

The multi-functional cellular adhesion molecule CD44 is regulated by the 8;21 chromosomal translocation.

The 8;21 translocation is a common chromosomal abnormality in acute myeloid leukemia (AML). We recently identified a naturally occurring leukemogenic splice variant, AML1-ETO9a (acute myeloid leukemia-1 transcription factor and the eight-twenty-one corepressor-9a), of t(8;21). To understand the leukemic potential of AML1-ETO9a, we performed microarray analysis with the murine multipotential hematopoietic FDCP-mix A4 cell line. We identified changes in expression of various genes including CD44. CD44 is a type I transmembrane protein and functions as the major cellular adhesion molecule for hyaluronic acid, a component of the extracellular matrix. CD44 is expressed in most human cell types and is implicated in myeloid leukemia pathogenesis. We show that the presence of AML1-ETO9a significantly increased the expression of CD44 at both RNA and protein levels. Furthermore, the CD44 promoter is bound by AML1-ETO9a and AML1-ETO at the chromatin level. In addition, in the AML1-ETO9a leukemia mouse model CD44 is regulated in a cell context-dependent manner. Thus, our observations suggest that AML1-ETO and its splice variant AML1-ETO9a are able to regulate the expression of the CD44 gene, linking the 8;21 translocation to the regulation of a cell adhesion molecule that is involved in the growth and maintenance of the AML blast/stem cells.

AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations.

The t(8;21) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). The translocation, which involves the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generates an AML1-ETO fusion transcription factor. To examine the effect of the AML1-ETO fusion protein on leukemogenesis, we made transgenic mice in which expression of AML1-ETO is under the control of the human MRP8 promoter (hMRP8-AML1-ETO). AML1-ETO is specifically expressed in myeloid cells, including common myeloid progenitors of hMRP8-AML1-ETO transgenic mice. The transgenic mice were healthy during their life spans, suggesting that AML1-ETO alone is not sufficient for leukemogenesis. However, after treatment of newborn hMRP8-AML1-ETO transgenic mice and their wild-type littermates with a strong DNA-alkylating mutagen, N-ethyl-N-nitrosourea, 55% of transgenic mice developed AML and the other 45% of transgenic mice and all of the wild-type littermates developed acute T lymphoblastic leukemia. Our results provide direct evidence that AML1-ETO is critical for causing myeloid leukemia, but one or more additional mutations are required for leukemogenesis. The hMRP8-AML1-ETO-transgenic mice provide an excellent model that can be used to isolate additional genetic events and to further understand the molecular pathogenesis of AML1-ETO-related leukemia.

Dichotomy of AML1-ETO functions: growth arrest versus block of differentiation.

The fusion gene AML1-ETO is the product of t(8;21)(q22;q22), one of the most common chromosomal translocations associated with acute myeloid leukemia. To investigate the impact of AML1-ETO on hematopoiesis, tetracycline-inducible AML1-ETO-expressing cell lines were generated using myeloid cells. AML1-ETO is tightly and strongly induced upon tetracycline withdrawal. The proliferation of AML1-ETO(+) cells was markedly reduced, and most of the cells eventually underwent apoptosis. RNase protection assays revealed that the amount of Bcl-2 mRNA was decreased after AML1-ETO induction. Enforced expression of Bcl-2 was able to significantly delay, but not completely overcome, AML1-ETO-induced apoptosis. Prior to the onset of apoptosis, we also studied the ability of AML1-ETO to modulate differentiation. AML1-ETO expression altered granulocytic differentiation of U937T-A/E cells. More significantly, this change of differentiation was associated with the down-regulation of CCAAT/enhancer binding protein alpha (C/EBPalpha), a key regulator of granulocytic differentiation. These observations suggest a dichotomy in the functions of AML1-ETO: (i) reduction of granulocytic differentiation correlated with decreased expression of C/EBPalpha and (ii) growth arrest leading to apoptosis with decreased expression of CDK4, c-myc, and Bcl-2. We predict that the preleukemic AML1-ETO(+) cells must overcome AML1-ETO-induced growth arrest and apoptosis prior to fulfilling their leukemogenic potential.

AML1-ETO downregulates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia.

The transcription factor CCAAT/enhancer binding protein alpha, or C/EBPalpha, encoded by the CEBPA gene, is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and Cebpa knockout mice exhibit an early block in maturation. Dominant-negative mutations of CEBPA have been found in some patients with acute myeloid leukemia (AML), but not in AML with the t(8;21) translocation which gives rise to the fusion gene RUNX1-CBF2T1 (also known as AML1-ETO) encoding the AML1-ETO fusion protein. RUNX1-CBF2T1 positive-AML blasts had eight-fold lower CEBPA RNA levels and undetectable C/EBPalpha protein levels compared with other subgroups of AML patients. Conditional expression of RUNX1-CBF2T1 in U937 cells downregulated CEBPA mRNA, protein and DNA binding activity. AML1-ETO appears to suppress C/EBPalpha expression indirectly by inhibiting positive autoregulation of the CEBPA promoter. Conditional expression of C/EBPalpha in AML1-ETO-positive Kasumi-1 cells results in neutrophilic differentiation. We suggest that restoring C/EBPalpha expression will have therapeutic implications in RUNX1-CBF2T1-positive leukemias.

Fusion AML1 transcript in a radiation-associated leukemia results in a truncated inhibitory AML1 protein.

AML1 is a transcription factor that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. Recently, fluorescence in situ hybridization was used to identify a novel syndrome of radiation-associated secondary acute myelogenous leukemia that had AML1 translocations. Using polymerase chain reaction, the AML1 fusion transcript was isolated from the patient who had a t(19;21) radiation-associated leukemia. The AML1 gene is fused out of frame to chromosome 19 sequences, resulting in a truncated AML protein bearing the DNA binding domain but not the transcriptional activation domain. This fusion AML1 protein functions as an inhibitor of the normal AML1 protein. (Blood. 2001;97:2168-2170)

Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model.

As reported previously, AML1-ETO knock-in mice were generated to investigate the role of AML1-ETO in leukemogenesis and to mimic the progression of t(8;21) leukemia. These knock-in mice died in midgestation because of hemorrhaging in the central nervous system and a block of definitive hematopoiesis during embryogenesis. Therefore, they are not a good model system for the development of acute myeloid leukemia. Therefore, mice were generated in which the expression of AML1-ETO is under the control of a tetracycline-inducible system. Multiple lines of transgenic mice have been produced with the AML1-ETO complementary DNA controlled by a tetracycline-responsive element. In the absence of the antibiotic tetracycline, AML1-ETO is strongly expressed in the bone marrow of AML1-ETO and tet-controlled transcriptional activator double-positive transgenic mice. Furthermore, the addition of tetracycline reduces AML1-ETO expression in double-positive mice to nondetectable levels. Throughout the normal murine lifespan of 24 months, mice expressing AML1-ETO have not developed leukemia. In spite of this, abnormal maturation and proliferation of progenitor cells have been observed from these animals. These results demonstrate that AML1-ETO has a very restricted capacity to transform cells. Either the introduction of additional genetic changes or the expression of AML1-ETO at a particular stage of hematopoietic cell differentiation will be necessary to develop a model for studying the pathogenesis of t(8;21).

Hepatocytes contribute to soluble CD14 production, and CD14 expression is differentially regulated in hepatocytes and monocytes.

CD14 presents as a glycosylphosphatidylinositol-linked membrane protein on the surface of monocytes/macrophages and as a soluble protein in the serum. Our previous studies have shown that an 80-kilobase pair (kb) genomic DNA fragment containing the human CD14 gene is sufficient to direct CD14 expression in a monocyte-specific manner in transgenic mice. In addition, we discovered that human CD14 is highly expressed in hepatocytes. Here, we report the generation of transgenic mice with either a 24- or 33-kb human CD14 genomic DNA fragment. Data from multiple transgenic lines show that neither the 24- nor the 33-kb transgenic mice express human CD14 in monocytes/macrophages. However, human CD14 is highly expressed in the liver of the 33-kb transgenic mice. These results demonstrate that human CD14 expression is regulated differently in monocytes and hepatocytes. Furthermore, we identified an upstream regulatory element beyond the 24-kb region, but within the 33-kb region of the human CD14 gene, which is critical for CD14 expression in hepatocytes, but not in monocytes/macrophages. Most importantly, the data demonstrate that the liver is one of the major organs for the production of soluble CD14. These transgenic mice provide an excellent system to further explore the functions of soluble CD14.

Cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine UBP43 (Usp18).

The ubiquitin-specific proteases (UBP) are a family of enzymes that cleave ubiquitin from ubiquitinated protein substrates. We have recently cloned UBP43, a novel member of this family from AML1-ETO knock-in mice. To analyze the role of UBP43 in hematopoiesis and leukemogenesis, we have cloned a full-length human UBP43 cDNA by screening a human monocytic cDNA library as well as by 5'- and 3'-rapid amplification of cDNA ends analyses. This cDNA encodes a polypeptide of 372 amino acids with all of the structural motifs of a deubiquitinating enzyme. The human UBP43 mRNA is strongly expressed in human liver and thymus. Transfection analysis has demonstrated that UBP43 is a nuclear protein. Interestingly, the gene encoding human UBP43 maps to chromosome 22q11.2. This region, known as DiGeorge syndrome critical region, contains a minimal area of 2 Mb and is consistently deleted in DiGeorge syndrome and related disorders. The syndrome is marked by thymic aplasia or hypoplasia, parathyroid hypoplasia, or congenital cardiac abnormalities. Taken together, our results broaden the understanding of a new human ubiquitin-specific protease, UBP43, and suggest that this gene may also be related to DiGeorge syndrome.

CCAAT/enhancer-binding protein activates the CD14 promoter and mediates transforming growth factor beta signaling in monocyte development.

Transcription factors from the CCAAT/enhancer-binding protein (C/EBP) family play important roles in myeloid cell differentiation. CD14 is a monocyte/macrophage differentiation marker and is strongly up-regulated during monocytic cell differentiation. Here, we report the direct binding of C/EBP to the monocyte-specific promoter of CD14. Transactivation analyses demonstrate that C/EBP family members significantly activate the CD14 promoter. These data indicate that C/EBP is directly involved in the regulation of CD14 gene expression. When myelomonoblastic U937 cells are treated with vitamin D(3) and TGF-beta, they differentiate toward monocytic cells. Using specific antibodies against different C/EBP family members in electrophoretic mobility shift assays and Western blot assays, we have identified a specific increase in the DNA binding and the expression of C/EBPalpha and C/EBPbeta during U937 monocytic cell differentiation, and we found C/EBPalpha and C/EBPbeta bind to the promoter in heterodimer. Furthermore, with stably transfected cell lines, we demonstrate that the C/EBP binding site in the CD14 promoter plays a critical role for mediating TGF-beta signaling in the synergistic activation of CD14 expression by vitamin D(3) and TGF-beta during U937 differentiation. This may indicate that C/EBPs have important functions in the process of TGF-beta signal transduction during monocyte differentiation.

AML1 (CBFalpha2) cooperates with B cell-specific activating protein (BSAP/PAX5) in activation of the B cell-specific BLK gene promoter.

AML1 plays a critical role during hematopoiesis and chromosomal translocations involving AML1 are commonly associated with different forms of leukemia, including pre-B acute lymphoblastic leukemia. To understand the function of AML1 during B cell differentiation, we analyzed regulatory regions of B cell-specific genes for potential AML1-binding sites and have identified a putative AML1-binding site in the promoter of the B cell-specific tyrosine kinase gene, blk. Gel mobility shift assays and transient transfection assays demonstrate that AML1 binds specifically to this site in the blk promoter and this binding site is important for blk promoter activity. Furthermore, in vitro binding analysis revealed that the AML1 runt DNA-binding domain physically interacts with the paired DNA-binding domain of BSAP, a B cell-specific transcription factor. BSAP has been shown previously to be important for B cell-specific regulation of the blk gene. Physical interaction of AML1 with BSAP correlates with functional cooperativity in transfection studies where AML1 and BSAP synergistically activate blk promoter transcription by more than 50-fold. These results demonstrate physical and functional interactions between AML1 and BSAP and suggest that AML1 is an important factor for regulating a critical B cell-specific gene, blk.

A novel ubiquitin-specific protease, UBP43, cloned from leukemia fusion protein AML1-ETO-expressing mice, functions in hematopoietic cell differentiation.

Using PCR-coupled subtractive screening-representational difference analysis, we have cloned a novel gene from AML1-ETO knockin mice. This gene is highly expressed in the yolk sac and fetal liver of the knockin mice. Nucleotide sequence analysis indicates that its cDNA contains an 1,107-bp open reading frame encoding a 368-amino-acid polypeptide. Further protein sequence and protein translation analysis shows that it belongs to a family of ubiquitin-specific proteases (UBP), and its molecular mass is 43 kDa. Therefore, we have named this gene UBP43. Like other ubiquitin proteases, the UBP43 protein has deubiquitinating enzyme activity. Protein ubiquitination has been implicated in many important cellular events. In wild-type adult mice, UBP43 is highly expressed in the thymus and in peritoneal macrophages. Among nine different murine hematopoietic cell lines analyzed, UBP43 expression is detectable only in cell lines related to the monocytic lineage. Furthermore, its expression is regulated during cytokine-induced monocytic cell differentiation. We have investigated its function in the hematopoietic myeloid cell line M1. UBP43 was introduced into M1 cells by retroviral gene transfer, and several high-expressing UBP43 clones were obtained for further study. Morphologic and cell surface marker examination of UBP43/M1 cells reveals that overexpression of UBP43 blocks cytokine-induced terminal differentiation of monocytic cells. These data suggest that UBP43 plays an important role in hematopoiesis by modulating either the ubiquitin-dependent proteolytic pathway or the ubiquitination state of another regulatory factor(s) during myeloid cell differentiation.