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A decreased percentage of CD177 pos neutrophils is frequently present in MDS and AML and is a useful flow cytometry (FCM) marker for the identification of MDS. The underlying mechanism leading to the low percentage of CD177 pos neutrophils in MDS has not been explained. The aim of this study was to identify whether specific somatic mutations in myeloid neoplasms are associated with the low percentage of CD177 pos neutrophils. 507 myeloid neoplasms with one or more pathogenic molecular abnormality identified by NGS and in which CD177 expression was assessed were evaluated. Correlation with CD177 expression was determined for 39 variables (including genes mutated, diagnostic groups and gender) using a 40 % cutoff level for low CD177 expression. In multivariate analysis mutations involving NPM1 (OD 0.26), RUNX1 (OD 0.39), TET2 (OD 0.58), and U2AF1 S34F (OD 0.25) were associated with low percentage of CD177 pos neutrophils when all cases were evaluated. JAK2 (OD 2.5) alteration was associated with increased percentage of CD177 pos neutrophils. Differences were noted between diagnostic subgroups with no single mutation associated with decreased CD177 pos neutrophils in MDS and CCUS. The findings demonstrate an association between the percentage of CD177 pos neutrophils and somatically acquired mutations involving several genes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The Ten Eleven Translocation 1 (TET1) gene encodes an epigenetic modifying molecule that is involved in demethylation of 5-methylcytosine. In hematological malignancies, loss-of-function mutations of TET2, which is one of the TET family genes including TET1, are frequently found, while the mutations of TET1 are not. However, clinical studies have revealed that TET1 is highly expressed in some cases of the hematological malignancies including acute myeloid leukemia. Indeed, studies by mouse models using conventional Tet1 knockout mice demonstrated that Tet1 is involved in myeloid leukemogenesis by Mixed Lineage Leukemia (MLL) fusion gene or TET2 mutant. Meanwhile, the other study showed that Tet1 is highly expressed in hematopoietic stem cells (HSCs), and that deletion of Tet1 in HSCs enhances potential self-renewal capacity, which is potentially associated with myeloid leukemogenesis. To examine the role of Tet1 in myeloid leukemogenesis more precisely, we generated novel conditional Tet1-knockout mice, which were used to generate the compound mutant mice by crossing with the inducible MLL-ENL transgenic mice that we developed previously. The leukemic immortalization in vitro was not critically affected by conditional ablation of Tet1 in HSCs with the induced expression of MLL-ENL or in hematopoietic progenitor cells retrovirally transduced with MLL-ENL. In addition, the leukemic phenotypes caused by the induced expression of MLL-ENL in vivo was not also critically affected in the compound mutant mouse model by conditional ablation of Tet1, although we found that the expression of Evi1, which is one of critical target genes of MLL fusion gene, in tumor cells was remarkably low under Tet1-ablated condition. These results revealed that Tet1 was dispensable for the myeloid leukemogenesis by MLL-ENL, suggesting that the therapeutic application of Tet1 inhibition may need careful assessment., Competing Interests: Sanikai is Mie University, Faculty of Medicine, School of Medicine, Alumni Association, a non-profit organization. There are no other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Growth factor independent 1 (Gfi1) controls myeloid differentiation by regulating gene expression and limits the activation of p53 by facilitating its de-methylation at Lysine 372. In human myeloid leukemia, low GFI1 levels correlate with an inferior prognosis. Here, we show that knockdown (KD) of Gfi1 in mice causes a fatal myeloproliferative disease (MPN) that could progress to leukemia after additional mutations. Both KO and KD mice accumulate myeloid cells that show signs of metabolic stress and high levels of reactive oxygen species. However, only KO cells have elevated levels of Lysine 372 methylated p53. This suggests that in contrast to absence of GFI1, KD of GFI1 leads to the accumulation of myeloid cells because sufficient amount of GFI1 is present to impede p53-mediated cell death, leading to a fatal MPN. The combination of myeloid accumulation and the ability to counteract p53 activity under metabolic stress could explain the role of reduced GF1 expression in human myeloid leukemia.

The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

Epigenetic alteration may play a role in age-associated dysfunction of stem cells and predispose to the development of hematological cancers. We analyzed global levels of hematopoietic 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in a cross-sectional study comprising 198 unrelated individuals from four age categories (neonates, 25-30, 70-75, and >90 years old) by liquid chromatography-electrospray ionization-tandem mass spectrometry with multiple reaction monitoring. X-chromosome inactivation (XCI) ratios and telomere length (TL) were measured in all individuals by polymerase chain reaction. Sequencing of epigenetic regulator genes (including TET2, DNMT3A, ASXL1, IDH1, IDH2, and WT1) was performed in the two older subcohorts. We found that global 5hmC levels declined with age in human blood cells (27.5% reduction from birth to old age, p < 0.0005). The levels of 5mC underwent a more modest reduction (2.4% drop) between newborns and the elderly (p < 0.0005). Low 5hmC was associated with increased skewing of XCI (age-adjusted p = 0.0304) and reduced TL (age-adjusted p = 0.0354), both surrogate markers of clonal dominance. Of the 100 individuals over the age of 70, 16 had somatic mutations in TET2, 14 in DNMT3A, and none in IDH1, IDH2, or WT1. Individuals with TET2 mutations had significantly lower 5hmC (relative to unmutated individuals), whereas DNMT3A-mutated subjects did not. However, mutations in TET2 cannot account solely for the decline in 5hmC levels observed with aging because unmutated older individuals also had lower 5hmC levels compared with younger individuals. This suggests that the age-associated decline in 5hmC is multifactorial. Larger prospective studies are needed to determine whether 5hmC reduction is a biomarker of hematological cancer development., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Background: The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells., Methods: U937T&#95;EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice., Results: Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis., Conclusions: Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.

Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are characteristic of AML. Zinc finger protein 382 (ZNF382) has been suggested to be a tumor suppressor gene possibly regulated by promoter hypermethylation in various types of human cancer. However, ZNF382 expression and methylation status in pediatric AML is unknown. In the present study, ZNF382 transcription levels were evaluated by quantitative reverse-transcription PCR. Methylation status was investigated by methylation-specific (MSP) PCR and bisulfate genomic sequencing (BGS). The prognostic significance of ZNF382 expression and promoter methylation was assessed in 105 cases of pediatric AML. The array data suggested that the ZNF382 promoter was hypermethylated in the AML cases examined. MSP PCR and BGS analysis revealed that ZNF382 was hypermethylated in leukemia cell lines. Furthermore, treatment with 5-aza-2'-deoxycytidine (5-Aza) upregulated ZNF382 expression in the selected leukemia cell lines. The aberrant methylation of ZNF382 was observed in 10% (2/20) of the control samples compared with 26.7% (28/105) of the AML samples. ZNF382 expression was significantly decreased in the 105 AML patients compared with the controls. Patients with ZNF382 methylation showed lower ZNF382 transcript levels compared with patients exhibiting no methylation. There were no significant differences in clinical characteristics or cytogenetic analysis between the patients with or without ZNF382 methylation. ZNF382 methylation correlated with minimal residual disease (MRD). Kaplan-Meier survival analysis revealed similar survival times in the samples with ZNF382 methylation, and multivariate analysis revealed that ZNF382 methylation was not an independent prognostic factor in pediatric AML. The epigenetic inactivation of ZNF382 by promoter hypermethylation can be observed in AML cell lines and pediatric AML samples. Therefore, our study suggests that ZNF382 may be considered a putative tumor suppressor gene in pediatric AML. However, further studies focusing on the mechanisms responsible for ZNF382 downregulation in pediatric leukemia are required.

The EVI1 oncogene at human chr 3q26 is rearranged and/or overexpressed in a subset of acute myeloid leukemias and myelodysplasias. The EVI1 protein is a 135 kDa transcriptional regulator with DNA-binding zinc finger domains. Here we provide a critical review of the current state of research into the molecular mechanisms by which this gene plays a role in myeloid malignancies. The major pertinent cellular effects are blocking myeloid differentiation and preventing cellular apoptosis, and several potential mechanisms for these phenomena have been identified. Evidence supports a role for EVI1 in inducing cellular quiescence, and this may contribute to the resistance to chemotherapy seen in patients with neoplasms that overexpress EVI1. Another isoform, MDS1-EVI1 (or PRDM3), encoded by the same locus as EVI1, harbors an N-terminal histone methyltransferase(HMT) domain; experimental findings indicate that this protein and its HMT activity are critical for the progression of a subset of AMLs, and this provides a potential target for therapeutic intervention., (Copyright © 2014. Published by Elsevier Inc.)

Deregulation of Ras/ERK signaling in myeloid leukemias makes this pathway an interesting target for drug development. Myeloid leukemia cell lines were screened for idarubicin-induced apoptosis, cell-cycle progression, cell-cycle-dependent MAP kinase kinase (MEK-1/2) activation, and Top2 expression. Cell-cycle-dependent activation of MEK/ERK signaling was blocked using farnesyltransferase inhibitor (FTI) BMS-214,662 and dual prenyltransferase inhibitor (DPI) L-778,123 to disrupt Ras signaling. Idarubicin caused a G2/M cell-cycle arrest characterized by elevated diphosphorylated MEK-1/2 and Top2α expression levels. The FTI/DPIs elicited distinct effects on Ras signaling, protein prenylation, cell cycling and apoptosis. Combining these FTI/DPIs with idarubicin synergistically inhibited proliferation of leukemia cell lines, but the L-778,123+idarubicin combination exhibited synergistic growth inhibition over a greater range of drug concentrations. Interestingly, combined FTI/DPI treatment synergistically inhibited cell proliferation, induced apoptosis and nearly completely blocked protein prenylation. Inhibition of K-Ras expression by RNA interference or blockade of its post-translational prenylation led to increased BMS-214,662-induced apoptosis. Our results suggest that nearly complete inhibition of protein prenylation using an FTI + DPI combination is the most effective method to induce apoptosis and to block anthracycline-induced activation of ERK signaling.

Recent studies have demonstrated the significance of the leukemia oncogene Evi1 as the regulator of hematopoietic stem cells and marker of poor clinical outcomes in myeloid malignancies. Evi1-mediated leukemogenic activities include a wide array of functions such as the induction of epigenetic modifications, transcriptional control, and regulation of signaling pathways. We have recently succeeded in comprehensively elucidating the oncogenic function of Evi1 in a model of the polycomb-Evi1-PTEN/AKT/mTOR axis. These results may provide us with novel therapeutic approaches to conquer the poor prognosis associated with Evi1-activated leukemia or other solid tumors with high Evi1 expression. Here, we review the current understanding of the role of Evi1 in controlling the development of leukemia and highlight potential modalities for targeting factors involved in Evi1-regulated signaling.

The human mixed lineage leukemia-5 (MLL5) gene is frequently deleted in myeloid malignancies. Emerging evidence suggests that MLL5 has important functions in adult hematopoiesis and the chromatin regulatory network, and it participates in regulating the cell cycle machinery. Here, we demonstrate that MLL5 is tightly regulated through phosphorylation on its central domain at the G(2)/M phase of the cell cycle. Upon entry into mitosis, the phosphorylated MLL5 delocalizes from condensed chromosomes, whereas after mitotic exit, MLL5 becomes dephosphorylated and re-associates with the relaxed chromatin. We further identify that the mitotic phosphorylation and subcellular localization of MLL5 are dependent on Cdc2 kinase activity, and Thr-912 is the Cdc2-targeting site. Overexpression of the Cdc2-targeting MLL5 fragment obstructs mitotic entry by competitive inhibition of the phosphorylation of endogenous MLL5. In addition, G(2) phase arrest caused by depletion of endogenous MLL5 can be compensated by exogenously overexpressed full-length MLL5 but not the phosphodomain deletion or MLL5-T912A mutant. Our data provide evidence that MLL5 is a novel cellular target of Cdc2, and the phosphorylation of MLL5 may have an indispensable role in the mitotic progression.

The Mre11-Rad50-Nbs1 complex and autophosphorylated Ser(1981)-ATM are involved in recognizing and repairing DNA damage, such as double-strand breaks (DSB). However, the role of these factors in response to stalled replication forks is not clear. Nucleoside analogues are agents that are incorporated into DNA during replication, which cause stalling of replication forks. The molecular mechanisms that sense these events may signal for DNA repair and contribute to survival but are poorly understood. Cellular responses to both DSBs and stalled replication forks are marked by H2AX phosphorylation on Ser(139) (gamma-H2AX), which forms nuclear foci at sites of DNA damage. Here, concentrations of the nucleoside analogues 1-beta-d-arabinofuranosylcytosine (cytarabine; ara-C), gemcitabine, and troxacitabine, which inhibited DNA synthesis by 90% within 2 hours, were determined for each agent. Using gamma-H2AX as a marker for changes in chromatin structure, we show that Mre11, Rad50, Nbs1, and phosphorylated ATM respond to nucleoside analogue-induced stalled replication forks by forming nuclear foci that colocalize with gamma-H2AX within 2 hours. Because neither DSBs nor single-strand breaks were detectable after nucleoside analogue exposure, we conclude that this molecular response is not due to the presence of DNA breaks. Deficiencies in ATM, Mre11, or Rad50 led to a 2- to 5-fold increase in clonogenic sensitization to gemcitabine, whereas Nbs1 and H2AX deficiency did not affect reproductive growth. Taken together, these results suggest that ATM, Mre11, and Rad50 are required for survival after replication fork stalling, whereas Nbs1 and H2AX are inconsequential.

Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After specific ligand binding, the intracellular part of Notch is cleaved off and translocates to the nucleus, where it targets the DNA binding protein RBP-Jkappa. In the absence of Notch, RBP-Jkappa represses Notch target genes by recruiting a corepressor complex. We and others have previously identified SHARP as one component of this complex. Here, we show that the corepressor ETO as well as the leukemogenic fusion protein AML1/ETO directly interacts with SHARP, that ETO is part of the endogenous RBP-Jkappa-containing corepressor complex, and that ETO is found at Notch target gene promoters. In functional assays, corepressor ETO, but not AML1/ETO, augments SHARP-mediated repression in an histone deacetylase-dependent manner. Furthermore, either the knockdown of ETO or the overexpression of AML1/ETO activates Notch target genes. Therefore, we propose that AML1/ETO can disturb the normal, repressive function of ETO at Notch target genes. This activating (or derepressing) effect of AML1/ETO may contribute to its oncogenic potential in myeloid leukemia.

EVI is a proto-oncogene that is activated in acute myeloid leukemia with chromosomal rearrangements that map to chromosome 3q26. We previously reported the clinicopathologic features of five cases of acute myeloid leukemia carrying t(3;8)(q26;q24). Using fluorescence in situ hybridization analysis, we demonstrate in the current study that the breakpoint on chromosome 3 is at EVI1/MDS1, and the breakpoint on chromosome 8 is just distal to the PVT1 oncogene homolog, a C-MYC activator in mice. The breakpoint on chromosome 8 was detected between the components of the LSI MYC dual-color break-apart rearrangement probe. Reverse-transcriptase polymerase chain reaction assay showed expression of EVI1 in all four cases analyzed, and DNA sequence analysis confirmed the findings. Reverse transcriptase polymerase chain reaction assay also demonstrated the expression of PVT1 and C-MYC in all four cases assessed. Western blot analysis detected EVI1 in one case analyzed. We conclude that the t(3;8)(q26;q24) results in deregulated EVI1 expression, similar to other balanced or unbalanced chromosomal translocations involving chromosome 3q26.

The EVI1 gene codes for a zinc finger transcription factor with important roles both in normal development and in leukemogenesis. Transcriptional activation of this gene through chromosome rearrangements or other, yet to be identified mechanisms leads to particularly aggressive forms of human myeloid leukemia. In vitro as well as in animal model systems, EVI1 affected cellular proliferation, differentiation, and apoptosis in cell type specific ways. Retroviral integrations into the EVI1 locus provided cells with increased abilities to engraft, survive, and proliferate in bone marrow transplantation experiments. Experimental overexpression of EVI1 by itself was insufficient to cause leukemia in animal model systems, but it cooperated with other genes in this process. This review summarizes the currently available experimental evidence for the proposed biochemical and biological functions of this important oncogene.

Nur77 (NR4A1) and Nor-1 (NR4A3) are highly homologous orphan nuclear receptors that regulate the transcription of overlapping target genes. The transcriptional activity of both proteins is regulated in a ligand-independent manner by cell- and stimulus-specific gene induction and protein phosphorylation. Nor-1 and Nur77 have been implicated in a variety of cellular processes, including the transduction of hormonal, inflammatory, mitogenic, apoptotic and differentiative signals. Cellular responses to these proteins suggest that they may function as homeostatic regulators of proliferation, apoptosis and differentiation, and thus may regulate cellular susceptibility to tumorigenesis. Their physiological functions, however, remain poorly understood. Here we describe a previously unsuspected function of Nor-1 and Nur77-as critical tumor suppressors of myeloid leukemogenesis. The abrogation of these proteins in mice led to rapidly lethal acute myeloid leukemia (AML), involving abnormal expansion of hematopoietic stem cells (HSCs) and myeloid progenitors, decreased expression of the AP-1 transcription factors JunB and c-Jun and defective extrinsic apoptotic (Fas-L and TRAIL) signaling. We found that downregulation of NR4A3 ( NOR-1 ) and NR4A1 ( NUR77 ) was a common feature in leukemic blasts from human AML patients, irrespective of karyotype. Thus Nor-1 and Nur77 may provide potential targets for therapeutic intervention in AML.

Cooperative activation of Meis1 and Hoxa9 perturbs myeloid differentiation and eventually leads myeloid progenitors to leukemia, yet it remains to be clarified what kinds of subsequent molecular processes are required for development of overt leukemia. To understand the molecular pathway in Hoxa9/Meis1-induced leukemogenesis, retroviral insertional mutagenesis was applied using retrovirus-mediated gene transfer. The mice that received Hoxa9/Meis1-transduced bone marrow cells developed acute myeloid leukemia (AML), and Trib1, Evi1, Ahi1, Raralpha, Pitpnb, and AK039950 were identified as candidate cooperative genes located near common retroviral integration sites. Trib1 and Evi1 were up-regulated due to retroviral insertions, and coexpression of these genes significantly accelerated the onset of Hoxa9/Meis1-induced AML, suggesting that Trib1 and Evi1 are the key collaborators. Furthermore, Trib1 by itself is a novel myeloid oncogene, enhancing phosphorylation of ERK, resulting in inhibition of apoptosis. These results demonstrate the importance of specific oncogene interaction in myeloid leukemogenesis.

Among the topoisomerase (topo) II isozymes (alpha and beta), topo IIbeta has been suggested to regulate differentiation. In this study, we examined the role of topo IIbeta in all-trans retinoic acid (ATRA)-induced differentiation of myeloid leukemia cell lines. Inhibition of topo IIbeta activity or downregulation of protein expression enhanced ATRA-induced differentiation/growth arrest and apoptosis. ATRA-induced apoptosis in topo IIbeta-deficient cells involved activation of the caspase cascade and was rescued by ectopic expression of topo IIbeta. Gene expression profiling led to the identification of peroxiredoxin 2 (PRDX2) as a candidate gene that was downregulated in topo IIbeta-deficient cells. Reduced expression of PRDX2 validated at the mRNA and protein level, in topo IIbeta-deficient cells correlated with increased accumulation of reactive oxygen species (ROS) following ATRA-induced differentiation. Overexpression of PRDX2 in topo IIbeta-deficient cells led to reduced accumulation of ROS and partially reversed ATRA-induced apoptosis. These results support a role for topo IIbeta in survival of ATRA-differentiated myeloid leukemia cells. Reduced expression of topo IIbeta induces apoptosis in part by impairing the anti-oxidant capacity of the cell owing to downregulation of PRDX2. Thus, suppression of topo IIbeta and/or PRDX2 levels in myeloid leukemia cells provides a novel approach for improving ATRA-based differentiation therapy.

Numerous strategies have been proposed to specifically inhibit telomerase (human telomerase reverse transcriptase (hTERT)) but to date only a few are clinically relevant in anticancer therapy. Recently, we have shown that long-term treatment with all-trans retinoic acid (ATRA), a compound clinically approved for differentiation therapy of acute promyelocytic leukemia (APL), represses hTERT in differentiation-resistant APL cell lines leading to telomere shortening and death. This signaling requires the co-activation of the retinoic acid receptor alpha (RARalpha) and the retinoic X receptor (RXR). In contrast to differentiation-therapy, which is only successful in this subtype of leukemia, the telomerase-targeted pathway could also be of use in non-APL. Here, we demonstrate that repression of hTERT occurs in fresh blasts cells from patients with myeloid leukemias of various subtypes exposed ex vivo to ATRA or synthetic retinoids. These results support the idea that, by hTERT targeting, retinoids can induce telomere shortening and cell death and their integration in therapy protocols for myeloid leukemias refractory to maturation should be considered.

Acute myeloid leukemia (AML) is a heterogeneous disease with multiple different cytogenetic and molecular aberrations contributing to leukemic transformation. We compared gene expression profiles of 4608 genes using cDNA-arrays from 20 AML patients (nine with -7/del7q and 11 with normal karyotype) with 23 CD34+ preparations from healthy bone marrow donors. SKI, a nuclear oncogene, was highly up regulated. In a second set of 183 AML patients analyzed with real-time PCR, the highest expression level of SKI in AML with -7/del7q could be confirmed. As previously described, Ski associates with the retinoic acid receptor (RAR) complex and can repress transcription. We wanted to investigate the interference of Ski with RARalpha signaling in AML. Ski was co-immunoprecipitated and colocalized with RARalpha. We also found that overexpression of wild-type Ski inhibited the prodifferentiating effects of retinoic acid in U937 leukemia cells. Mutant Ski, lacking the N-CoR binding, was no more capable of repressing RARalpha signaling. The inhibition by wild-type Ski could partially be reverted by the histone deacetylase blocking agent valproic acid. In conclusion, Ski seems to be involved in the blocking of differentiation in AML via inhibition of RARalpha signaling.

Myeloid leukemias in AKXD23 mice contain proviral insertions at Evi1, resulting in transcriptional activation. Although Evi1 is clearly involved in leukemia, gene transfer studies in mice with Evi1 fail to cause leukemia, arguing that cooperating events are necessary. We reanalyzed AKXD-23 tumors for cooperating proviral insertion and found that each tumor had a proviral insertion in Sox4, which encodes an HMG-box transcription factor. RNA analysis revealed these insertions cause increased Sox4 expression. Overexpression of Sox4 in 32Dcl3 cells markedly inhibited cytokine-induced granulocyte maturation, as documented by morphologic and mRNA analysis. Sox4-expressing cells had higher levels of transcripts associated with proliferation, including Evi1. Conversely, in leukemic cells that express Sox4 and bear provirally activated Evi1, suppression of Sox4 with short hairpin RNAs resulted in down-regulation of both Sox4 and Evi1. By cotransfection studies, Sox4 is able to transactivate the AKV long terminal repeat, which likely explains how Sox4 transcriptionally up-regulates provirally activated Evi1; however, Sox4 does not appear to regulate the native Evi1 promoter. We propose that Sox4 proviral activation is selected for in the setting of prior proviral activation of Evi1, because it transactivates the relatively weak LTR of AKV leading to higher Evi1 expression and consequent block to differentiation.

Chromosomal translocations are primary events in tumorigenesis. Those involving the mixed lineage leukaemia (MLL) gene are found in various guises and it is unclear whether MLL fusions can affect haematopoietic differentiation. We have used a model in which chromosomal translocations are generated in mice de novo by Cre-loxP-mediated recombination (translocator mice) to compare the functionally relevant haematopoietic cell contexts for Mll fusions, namely pluripotent stem cells, semicommitted progenitors or committed cells. Translocations between Mll and Enl or Af9 cause myeloid neoplasias, initiating in pluripotent stem cells or multipotent myeloid progenitors. However, while Mll-Enl translocations can also cause leukaemia from T-cell progenitors, no tumours arose with Mll-Af9 translocations in the T-cell compartment. Furthermore, Mll-Enl translocations in T-cell progenitors can cause lineage reassignment into myeloid tumours. Therefore, a permissive cellular environment is required for oncogenicity of Mll-associated translocations and Mll fusions can influence haematopoietic lineage commitment.

The JAK-STAT signal transduction cascade participates in various cellular processes, including immune response, cell replication, differentiation and oncogenesis. Here, we report that this cascade is induced in two human myeloid HL-60 leukemia cell variants by the granulocyte differentiation inducer dimethyl sulfoxide (DMSO) and macrophage differentiation inducer phorbol 12-myristate 13-acetate (PMA). DMSO and PMA also induced the expression and catalytic activity of 2'-5' oligoadenylate synthetase (2-5A synthetase), a known interferon (IFN) inducible enzyme. The HL-60 cell variants included HL-205, which is susceptible to DMSO- and PMA-induced differentiation, and HL-525, which is susceptible to DMSO- but not to PMA-induced differentiation. Treatment of HL-205 and HL-525 cells with DMSO and HL-205 cells with PMA-induced JAK1 phosphorylation, JAK1/STAT1 association, formation of STAT1-STAT2 heterodimers, and the binding of the active IFN stimulating growth factor 3 (ISGF3) to the IFN-stimulated response element (ISRE) fragment isolated from the 2-5A synthetase promoter. These events were either reduced or absent in the resistant HL-525 cells treated with PMA. Taken together, our data implicate the above signaling cascade in DMSO- and PMA-induced 2-5A synthetase expression and catalytic activity in the HL-60 cell system.

CTCF is a transcription factor and a candidate tumor suppressor that contains a DNA-binding domain composed of 11 zinc fingers. We reported previously that CTCF is differentially regulated during differentiation of human myeloid leukemia cells. In this study we aimed to investigate the role of CTCF in myeloid cell differentiation. A human cell line, K562, that can be chemically induced to differentiate into various hematopoietic lineages was chosen as a model system for this study. Several K562 cell lines with constitutive and conditional expression of CTCF have been generated. By using these model systems we demonstrated that: (i) ectopic expression of CTCF in K562 cells led to growth retardation and promotion of differentiation into the erythroid lineage; (ii) CTCF knock-down significantly inhibited differentiation of K562 cells into erythroid lineage; (iii) differentiation of K562 into the megakaryocytic lineage was not significantly affected; and (iv) down-regulation of MYC has been identified as one of the mechanisms by which CTCF promotes erythroid differentiation. Taken together our results demonstrate that CTCF is involved in the control of myeloid cell growth and differentiation.

The AML1/ETO and PML/RARalpha leukemia fusion proteins induce acute myeloid leukemia by acting as transcriptional repressors. They interact with corepressors, such as N-CoR and SMRT, that recruit a multiprotein complex containing histone deacetylases on crucial myeloid differentiation genes. This leads to gene repression contributing to generate a differentiation block. We expressed in leukemia cells containing PML/RARalpha and AML1/ETO N-CoR protein fragments derived from fusion protein/corepressor interaction surfaces. This blocks N-CoR/SMRT binding by these fusion proteins, and disrupts the repressor protein complex. In consequence, the expression of genes repressed by these fusion proteins increases and differentiation response to vitamin D3 and retinoic acid is restored in previously resistant cells. The alteration of PML/RARalpha-N-CoR/SMRT connections triggers proteasomal degradation of the fusion protein. The N-CoR fragments are biologically effective also when directly transduced by virtue of a protein transduction domain. Our data indicate that fusion protein activity is permanently required to maintain the leukemia phenotype and show the route to developing a novel therapeutic approach for leukemia, based on its molecular pathogenesis.

The translocation t(8;21)(q22;q22) in acute myeloid leukemia (AML) results in the expression of the fusion protein RUNX1/MTG8, which in turn recruits histone deacetylases (HDAC) to silence RUNX1 target genes [e.g., interleukin-3 (IL-3)]. We previously reported that expression of the RUNX1/MTG8 target gene IL-3 is synergistically restored by the combination of inhibitors of HDACs (i.e., depsipeptide) and DNA methyltransferases (DNMT; i.e., decitabine) in RUNX1/MTG8-positive Kasumi-1 cells. Thus, we hypothesized that DNMT1 is also part of the transcriptional repressor complex recruited by RUNX1/MTG8. By a chromatin immunoprecipitation assay, we identified a RUNX1/MTG8-DNMT1 complex on the IL-3 promoter in Kasumi-1 cells and in primary RUNX1/MTG8-positive AML blasts. The physical association of RUNX1/MTG8 with DNMT1 was shown by coimmunoprecipitation experiments. Furthermore, RUNX1/MTG8 and DNMT1 were concurrently released from the IL-3 promoter by exposure to depsipeptide or stabilized on the promoter by decitabine treatment. Finally, we proved that RUNX1/MTG8 and DNMT1 were functionally interrelated by showing an enhanced repression of IL-3 after coexpression in 293T cells. These results suggest a novel mechanism for gene silencing mediated by RUNX1/MTG8 and support the combination of HDAC and DNMT inhibitors as a novel therapeutic approach for t(8;21) AML.

Exogenous expression of the transcription factor Scl (Tal1) in WEHI-3B D+ myelomonocytic leukemia cells interferes with their capacity to respond to all-trans retinoic acid (ATRA) induced differentiation; combination of ATRA with LiCl, however, circumvents the inhibition of differentiation produced by Scl. To gain information on the possible involvement of this transcription factor in the non-responsiveness of acute myelocytic leukemia (AML) patients to ATRA, we compared the endogenous expression levels of Scl and its transcription complex partners [i.e., Rbtn1 (LMO1), Rbtn2 (LMO2), Ldb1, and GATA family proteins] in leukemic blast cells from patients with AML and acute promyelocytic leukemia (APL), and determined the effects of lithium chloride alone or in combination with ATRA on the capacity of blast cells to differentiate during short-term ex vivo culture. Levels of Scl, Rbtn2, GATA1, and Ldb1 expression were comparable in AML and APL blasts, while the levels of expression of Rbtn1, GATA2, and GATA3 were absent or markedly lower in APL cells. Differentiation markers (cell surface myeloid antigens CD11b, CD15, CD14, and CD33) were also analyzed in blast cells. ATRA produced changes in at least one surface antigen differentiation marker in 89% of patient blasts, while LiCl caused such changes in 72% of the leukemic cells of patients. The combination of LiCl and ATRA induced the differentiation of leukemic blasts from 94% of patients. Although the expression of the transcription factors did not act as individual predictors of responsiveness or non-responsiveness to the inducers of differentiation, ATRA or ATRA plus LiCl, the addition of LiCl to ATRA increased the differentiation response over that of ATRA alone in a number of leukemic samples. These findings suggest that the combination of LiCl and ATRA may produce some clinical benefit in the treatment of the myeloid leukemias.

p73, the homologue of p53, is a nuclear protein whose ectopic expression, in p53+/+ and p53-/- cells, recapitulates the most well-characterized p53 effects, such as growth arrest, apoptosis and differentiation. Unlike p53, which is mutated in half of human cancers, p73 is rarely mutated. However, altered expression of the p73 gene has been reported in neuroblastoma, lung cancer, prostate cancer and renal cell carcinoma. To investigate the potential involvement of p73 in acute myeloid leukemias (AMLs), we analyzed 71 samples from AML patients for the expression pattern of N-terminal transactivation-p73alpha (TA-p73alpha), its spliced isoforms and N-terminal-deleted-p73 transcripts (DeltaN-p73). We detected p73 gene expression in AML irrespective of FAB (French-American-British) subtypes. Notably, the analysis of DeltaN-p73 expression, which has been reported to inactivate both p53 and p73 antitumor effects, revealed a rather peculiar pattern. In fact, DeltaN-p73 transcript and protein were detectable in 27/28 (96.4%) cases of M0, M1, M2, M4, M5 and M6 AML and in 13/41 (31.7%) cases of PML-RARalpha-positive M3 AML (P<0.01). Thus, the distinct gene expression profile of p73 further supports the notion that acute promyelocytic leukemia is a biologically different subset of AML.

TEL2 is a member of the ETS family of transcription factors, which is highly similar to TEL1/ETV6. It binds to DNA via the ETS domain and interacts with itself or TEL1 via the pointed domain. The expression of TEL2 in normal and leukemic hematopoietic cells suggests a role in hematopoietic development. In this article, we describe the role of TEL2 in hematopoietic differentiation and cellular transformation. Quantitative reverse transcription-PCR showed that the expression of TEL2 mRNA was down-regulated during monocytic differentiation of U937 and HL60 induced by 1,25-(OH)2 vitamin D3 and 12-O-tetradecanoylphorbol 13-acetate, respectively. Overexpression of TEL2 in U937 cells inhibited differentiation induced by vitamin D3. In contrast, overexpression of a TEL2 mutant lacking either the pointed domain or a functional ETS domain induced both differentiation of U937 cells and inhibited their growth in vitro and in vivo. In addition, these mutants blocked TEL2-mediated transcriptional repression of a synthetic promoter containing TEL2 binding sites. These data suggest that dominant-negative inhibition of TEL2 might cause differentiation. Quantitative reverse transcription-PCR demonstrated that TEL2 is expressed at higher level in some primary human leukemia samples than in normal bone marrow. Furthermore, overexpression of TEL2 in NIH3T3-UCLA cells blocked the inhibitory effect of TEL1 on Ras-induced cellular transformation. These results suggest that TEL2 may play an important role in hematopoiesis and oncogenesis.

The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 15% of acute myeloid leukemia (AML) cases. This study shows that AML1-ETO, as well as ETO, inhibits transcriptional activation by E proteins through stable interactions that preclude recruitment of p300/CREB-binding protein (CBP) coactivators. These interactions are mediated by a conserved ETO TAF4 homology domain and a 17-amino acid p300/CBP and ETO target motif within AD1 activation domains of E proteins. In t(8;21) leukemic cells, very stable interactions between AML1-ETO and E proteins underlie a t(8;21) translocation-specific silencing of E protein function through an aberrant cofactor exchange mechanism. These studies identify E proteins as AML1-ETO targets whose dysregulation may be important for t(8;21) leukemogenesis, as well as an E protein silencing mechanism that is distinct from that associated with differentiation-inhibitory proteins.

Several reports have described various strategies of dendritic cell (DC) vaccination to induce specific T-cell responses in patients with acute myeloid leukaemia (AML). About 50-60% of AML cases blasts have chromosomal abnormalities, such as inv(16) or t(8,21), which could encode for leukaemia-specific antigenic peptide sequences, possibly presented in the context of self-major histocompatibility complex (MHC) molecules. As the co-culture of AML blasts with T lymphocytes seldom resulted in T-cell stimulation, we fused AML blasts with autologous DC to enhance this effect. The fusion cells expressed MHC class I and II, CD40, B7-1, B7-2, CD209 and several adhesion molecules. In a mixed lymphocyte hybrid reaction, the fusion cells induced the proliferation of autologous T cells. Moreover, in the special case of fusion cells established from AML blasts with the chromosomal abnormality inv(16), the autologous T lymphocytes could be primed to induce cytotoxicity against up to 70% autologous AML blasts in a effector:target ratio of 20:1. Blocking assays demonstrated that the lysis was chiefly mediated by CD8(+), CCR7(-) T lymphocytes, which could be further expanded in the form of effector memory CD8(+) T cells by repeated co-cultures with the autologous fusion cells.

Inv(16)(p13q22) is associated with acute myeloid leukemia subtype M4Eo that is characterized by the presence of myelomonocytic blasts and atypical eosinophils. This chromosomal rearrangement results in the fusion of CBFB and MYH11 genes. CBF beta normally interacts with RUNX1 to form a transcriptionally active nuclear complex. The MYH11 gene encodes the smooth muscle myosin heavy chain. The CBF beta-SMMHC fusion protein is capable of binding to RUNX1 and form dimers and multimers through its myosin tail. Previous results from transgenic mouse models show that Cbfb-MYH11 is able to inhibit dominantly Runx1 function in hematopoiesis, and is a key player in the pathogenesis of leukemia. In recent years, molecular and cellular biological studies have led to the proposal of several models to explain the function of CBF beta-SMMHC. In this review, we will first focus our attention on the molecular mechanisms proposed in the recent publications. We will next examine recent gene expression profiling studies on inv(16) leukemia cells. Finally, we will describe a recent study from one of our labs on the identification of cooperating genes for leukemogenesis with CBFB-MYH11.

The Runx genes present a challenge to the simple binary classification of cancer genes as oncogenes or tumor suppressors. There is evidence that loss of function of two of the three mammalian Runx genes promotes cancer, but in a highly lineage-restricted manner. In human leukemias, the RUNX1 gene is involved in various chromosomal translocation events that create oncogenic fusion proteins, at least some of which appear to function as dominant-negative inhibitors of the normal gene product. Paradoxically, evidence is mounting that structurally intact Runx genes are also oncogenic when overexpressed. All the three murine genes act as targets for transcriptional activation by retroviral insertional mutagenesis, and the oncogenic potential of Runx2 has been confirmed in transgenic mice. Moreover, the RUNX1 gene is often amplified or overexpressed in cases of acute leukemia. The state of progress in elucidating the oncogenic roles of the Runx genes is the subject of this review, and we draw together recent observations in a tentative model for the effects of Runx deregulation on hematopoietic cell differentiation. We suggest that lineage-specific factors determine the sensitivity to the oncogenic effects of loss or overexpression of Runx factors.

Molecular mechanisms of leukemogenesis have been successfully unraveled by studying genes involved in simple rearrangements including balanced translocations and inversions. In contrast, little is known about genes altered in complex karyotypic abnormalities. We studied acute myeloid leukemia (AML) patients with complex karyotypes and abnormal chromosome 21. High-resolution bacterial artificial chromosome (BAC) array-based comparative genomic hybridization disclosed amplification predominantly in the 25- to 30-megabase (MB) region that harbors the APP gene (26.3 MB) and at position 38.7-39.1 MB that harbors the transcription factors ERG and ETS2. Using oligonucleotide arrays, APP was by far the most overexpressed gene (mean fold change 19.74, P = 0.0003) compared to a control group of AML with normal cytogenetics; ERG and ETS2 also ranked among the most highly expressed chromosome 21 genes. Overexpression of APP and ETS2 correlated with genomic amplification, but high APP expression occurred even in a subset of AML patients with normal cytogenetics (10 of 64, 16%). APP encodes a glycoprotein of unknown function previously implicated in Alzheimer's disease, but not in AML. We hypothesize that APP and the transcription factors ERG and ETS2 are altered by yet unknown molecular mechanisms involved in leukemogenesis. Our results highlight the value of molecularly dissecting leukemic cells with complex karyotypes.

Fanconi anemia (FA) is an autosomal recessive chromosomal instability disorder caused by mutations in one of seven known genes (FANCA,C,D2,E,F,G and BRCA2). Mutations in the FANCA gene are the most prevalent, accounting for two-thirds of FA cases. Affected individuals have greatly increased risks of acute myeloid leukemia (AML). This raises the question as to whether inherited or acquired mutations in FA genes might be involved in the development of sporadic AML. Quantitative fluorescent PCR was used to screen archival DNA from sporadic AML cases for FANCA deletions, which account for 40% of FANCA mutations in FA homozygotes. Four heterozygous deletions were found in 101 samples screened, which is 35-fold higher than the expected population frequency for germline FANCA deletions (P<0.0001). Sequencing FANCA in the AML samples with FANCA deletions did not detect mutations in the second allele and there was no evidence of epigenetic silencing by hypermethylation. However, real-time quantitative PCR analysis in these samples showed reduced expression of FANCA compared to nondeleted AML samples and to controls. These findings suggest that gene deletions and reduced expression of FANCA may be involved in the promotion of genetic instability in a subset of cases of sporadic AML.

The EVI1 proto-oncogene encodes a nuclear zinc finger protein that acts as a transcription repressor factor. In myeloid leukemia it is often activated by chromosomal rearrangements involving band 3q26, where the gene has been mapped. Here we report two leukemia cases [a chronic myeloid leukemia blast crisis (CML-BC) and an acute myeloid leukemia (AML) M4] showing a t(3;7)(q26;q21) translocation in a balanced and unbalanced form, respectively. Fluorescent in situ hybridization (FISH) analysis revealed that both patients showed a breakpoint on chromosome 3 inside the clone RP11-33A1 containing the EVI1 oncogene and, on chromosome 7, inside the clone RP11-322M5, partially containing the CDK6 oncogene which is a D cyclin-dependent kinase gene, observed to be overexpressed and disrupted in many hematological malignancies. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed overexpression of EVI1 in both cases, but excluded the presence of any CDK6/ EVI1 fusion transcript. CDK6 expression was also detected. Together, these data indicate that EVI1 activation is likely due not to the generation of a novel fusion gene with CDK6 but to a position effect dysregulating its transcriptional pattern.

MLL5 is a mammalian trithorax group (trx-G) gene identified within chromosome band 7q22, a frequently deleted element found in cytogenetic aberrations of acute myeloid malignancies. MLL5 cDNA was linked with the FLAG and V5 tags at the N and C terminus, respectively, and transfected into 293T cells. Immunofluoresence staining of the expressed tagged MLL5 protein showed localization to the nucleus and exclusion from nucleoli, and no surface staining was detected. Both ectopically introduced and endogenous MLL5 protein displayed a speckled nuclear distribution. By using a series of MLL5-truncated mutants fused with enhanced GFP, a domain (residues 945-1,156) required for foci accumulation was identified, and regions containing functional nuclear localization signals were mapped. Ectopic overexpression of GFP-MLL5 induced cell cycle arrest in G(1) phase. This inhibition of cell cycle progression was indicated by delayed progression into nocodazole-induced mitotic arrest and was confirmed by a lack of BrdUrd incorporation. These findings suggest that MLL5 forms intranuclear protein complexes that may play an important role in chromatin remodeling and cellular growth suppression.

Background: Inactivation of genes that suppress neoplasia by aberrant DNA methylation is a key event that occurs during the development of leukemia. The inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (5AZA), which can re-activate these genes, is under clinical investigation for therapy of leukemia. The objective of this study was to determine the concentrations of 5AZA that will re-activate target silent genes in human leukemic cell lines., Materials and Methods: RT-PCR was used to evaluate the effect of concentrations of 1 to 100 ng/ml of 5AZA on the re-activation of p15 and p73 in KG1a myeloid leukemic cells and E-cadherin in HL-60 myeloid leukemic cells. The effect of 5AZA on inhibition of growth, DNA synthesis and colony formation in these cell lines was also investigated., Results: The extent of activation of the target genes was dependent on the concentration of 5AZA. For p15, pronounced activation was observed at 10 ng/ml or greater. For p73 and E-cadherin significant activation was observed at 100 ng/ml of 5AZA. Maximal inhibition of growth, DNA synthesis and colony formation occurred at 100 ng/ml., Conclusion: The in vitro antineoplastic and gene re-activation activity of 5AZA is dependent on the concentration of this analog. These data may be helpful in the design of the optimal dose-schedule of 5AZA for the clinical therapy of leukemia.

The transcription factor FKHR, which is controlled by Akt-PKB signaling, is involved in regulating cell cycle progression and cell death. In this study, the phosphorylation of FKHR was observed in 45 (73.8%) of 61 patients with acute myeloid leukemia (AML). The phosphorylation of Akt-PKB was found to be significantly associated with phospho-FKHR (P<0.001). Patients with phospho-FKHR had a significantly shorter overall survival than those without (P<0.05). In conclusion, the constitutive phosphorylation of FKHR was observed in the majority of AML, and the detection of phospho-FKHR might provide a new tool for identifying AML patients with an unfavorable outcome.

Transcriptional deregulation through the production of dominant-acting chimeric transcription factors derived from chromosomal translocations is a common theme in the pathogenesis of acute leukemias; however, the essential target genes for acute leukemogenesis are unknown. We demonstrate here that primary myeloid progenitors immortalized by various MLL oncoproteins exhibit a characteristic Hoxa gene cluster expression profile, which reflects that preferentially expressed in the myeloid clonogenic progenitor fraction of normal bone marrow. Continued maintenance of this MLL-dependent Hoxa gene expression profile is associated with conditional MLL-associated myeloid immortalization. Moreover, Hoxa7 and Hoxa9 were specifically required for efficient in vitro myeloid immortalization by an MLL fusion protein but not other leukemogenic fusion proteins. Finally, in a bone marrow transduction/transplantation model, Hoxa9 is essential for MLL-dependent leukemogenesis in vivo, a primary requirement detected at the earliest stages of disease initiation. Thus, a genetic reliance on Hoxa7 and Hoxa9 in MLL-mediated transformation demonstrates a gain-of-function mechanism for MLL oncoproteins as upstream constitutive activators that promote myeloid transformation via a Hox-dependent mechanism.

STAT5 phosphorylation has been noted in 69-95% of AML cases by Western blotting. We used flow cytometry to measure phosphorylated STAT5 on a semi-quantitative scale. The method was validated on K562 cells, which constitutively express phosphorylated STAT5, but lose this when BCR-abl tyrosine kinase activity is blocked by STI571. Phosphorylated STAT5 was found to measure 2.22+/-0.09 relative fluorescence units (RFU) falling to 0.925+/-0.005RFU in the presence of STI571. Phosphorylated STAT5 expression was 0.99 to 2.09RFU in 28 primary AML samples. There was no logical cut-off point between positive and negative fluorescence. FLT3 internal tandem duplications, found in 11/28 samples, were not significantly associated with the level of phosphorylated STAT5 expression. We conclude that STAT5 phosphorylation can be measured sensitively by flow cytometry in AML and that its expression should not be dichotomised as present or absent.

ETS proteins (such as PU.1, Fli-1 and ETS-1) have been shown to play important roles in normal and abnormal hematopoiesis. We examined the expression of the ELF subfamily of ETS genes (ELF-1, MEF and NERF) in acute myeloid leukemia (AML) cells using Northern blot analysis. ELF-1 and MEF were expressed in all samples, whereas NERF was not. The relative expression (RE) of MEF, but not ELF-1, was significantly lower (P<0.0001) in AML with t(8;21) and t(15;17) compared with AML with normal karyotype. The pattern of MEF expression was not uniform among cells with CD34(+)/CD33(+). It is suggested that the low RE of MEF might be part of a gene expression profile characterizing AML with a good prognosis.

Double strand breaks (DSBs) are considered the most lethal form of DNA damage for eukaryotic cells, and misrepair of DSB can cause cell death, chromosome instability, and cancer. Nonhomologous end-joining (NHEJ) is a major mechanism for the repair of DSBs. We previously reported that the cancer predisposition Bloom's syndrome and myeloid leukemias demonstrate increased NHEJ activity and consequent misrepair. In this study, we link this increased NHEJ activity and infidelity to ongoing or induced DNA damage at sites that recruit key NHEJ proteins. We show here that in myeloid leukemia cells and normal hemopoietic cells, agents that induce DSBs produce an up to 2-fold increase in this DSB misrepair activity, whereas an alkylating agent produces little or no increases. Furthermore, NHEJ overactivity after induction of DSBs is dependent on the presence of Ku70/Ku86. We also present data to explain the constitutively activated NHEJ in myeloid leukemias. Using an immunofluorescence-based assay for DNA damage, myeloid leukemias demonstrate constitutive DNA damage in the absence of treatment with DSB-inducing agents compared with normal hemopoietic cells. Importantly, damaged foci from myeloid leukemia and normal cells colocalize with NHEJ proteins Ku70 and Ku86. These data suggest that the generation of increased constitutive DNA damage may be a common pathway for the creation of NHEJ-dependent genomic instability.

The proto-oncogene EVI1 encodes a DNA binding protein and is located on chromosome 3q26. The gene is aberrantly expressed in acute myeloid leukemia (AML) patients carrying 3q26 abnormalities. Two mRNAs are transcribed from this locus: EVI1 and a fusion of EVI1 with MDS1 (MDS1-EVI1), a gene located 5' of EVI1. The purpose of this study was to investigate which of the 2 gene products is involved in transformation in human AML. To discriminate between EVI1 and MDS1-EVI1 transcripts, distinct real-time quantitative polymerase chain reaction (PCR) assays were developed. Patients with 3q26 abnormalities often showed high EVI1 and MDS1-EVI1 expression. In a cohort of 319 AML patients, 4 subgroups could be distinguished: EVI1(+) and MDS1-EVI1(-) (6 patients; group I), EVI1(+) and MDS1-EVI1(+) (26 patients; group II), EVI1(-) and MDS1-EVI1(+) (12 patients; group III), and EVI1(-) and MDS1-EVI1(-) (275 patients; group IV). The only 4 patients with a 3q26 aberration belonged to groups I and II. Interestingly, high EVI1 and not MDS1-EVI1 expression was associated with unfavorable karyotypes (eg, -7/7q-) or complex karyotypes. Moreover, a significant correlation was observed between EVI1 expression and 11q23 aberrations (mixed lineage leukemia [MLL] gene involvement). Patients from groups I and II had significantly shorter overall and event-free survival than patients in groups III and IV. Our data demonstrate that high EVI1 expression is an independent poor prognostic marker within the intermediate- risk karyotypic group.

The EVI1 gene in chromosome band 3q26 exhibits a number of properties consistent with a role as an oncogene, and its expression is activated in most myeloid leukemia patients with, as well as in a minority of patients without, 3q26 rearrangements. A splice variant of this gene, MDS1/EVI1, acts as its antagonist at least in some tissue culture assays. We established real-time quantitative reverse transcriptase polymerase chain reaction (RTQ-RT-PCR) assays for these mRNA variants to compare their expression levels in a quantitatively reliable manner. EVI1 was overexpressed to highly variable extents in all patients with, as well as in 14% of patients without, 3q26 rearrangements. In some of these samples, MDS1/EVI1 was also transcribed at elevated levels compared to those of healthy controls. However, although the induction of MDS1/EVI1 was comparable to, or higher than, that of EVI1 in three of five samples with a normal EVI1 locus, this was true for only two of 13 patients with a 3q26 aberration. We further provide preliminary evidence that the RTQ-RT-PCR assay may be useful for disease monitoring in patients overexpressing EVI1., (Copyright 2002 Wiley-Liss, Inc.)

Loss of transforming growth factor (TGF) beta signaling has been implicated in malignant transformation of various tissues. To investigate a potential role of Smad4 in acute myeloid leukemia (AML), the expression of Smad4 was determined in blast cells from AML patients. Western analysis of nuclear extracts of nine AML samples indicated the absence of Smad4 protein in two cases. Smad4 RT-PCR analysis of these cases indicated normal Smad4 mRNA expression, and sequencing of one of these cases revealed no mutations as compared to wild type Smad4. Next, it was investigated whether Smad4 protein from these AML cases was subject to proteolytic degradation by incubating cell extracts of these Smad4-negative AML cells with extracts from COS-7 cells in which a tagged Smad4 was overexpressed. Inhibitor studies indicated that the extracts of AML blasts lacking Smad4 possessed a serine-dependent proteolytic activity, capable of degrading Smad4. Transfection studies using an SBE containing reporter construct as well as RT-PCR analysis of endogenous TGFbeta1 responsive genes indicated that the AML blasts were still able to respond to TGFbeta1, despite the observed degradation of Smad4. It was, therefore, concluded that the degradation of Smad4 was possibly AML subtype-dependent, in vitro phenomenon, occurring during the preparation of nuclear and cellular extracts despite the addition of a protease inhibitor cocktail. The results indicate that care should be taken when interpreting data obtained from protein expression studies using AML blast cells.

In CBFbeta-SMMHC, core binding factor beta (CBFbeta) is fused to the alpha-helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba/F3 hematopoietic cells expressing a CBFbeta-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBFbeta-SMMHC(DeltaACD) multimerized less effectively than either wild-type protein or a variant lacking a different 28-residue segment. In contrast to the control proteins, the DeltaACD mutant did not inhibit CBF DNA binding, AML1-mediated reporter activation, or G(1) to S cell cycle progression, the last being dependent upon activation of CBF-regulated genes. We also linked the CBFbeta domain to 149 or 83 C-terminal CBFbeta-SMMHC residues, retaining 86 or 20 amino acids N-terminal to the ACD. CBFbeta-SMMHC(149C) multimerized and slowed Ba/F3 proliferation, whereas CBFbeta-SMMHC(83C) did not. The majority of CBFbeta-SMMHC and CBFbeta-SMMHC(149C) was detected in the nucleus, whereas the DeltaACD and 83C variants were predominantly cytoplasmic, indicating that multimerization facilitates nuclear retention of CBFbeta-SMMHC. When linked to the simian virus 40 nuclear localization signal (NLS), a significant fraction of CBFbeta-SMMHC(DeltaACD) entered the nucleus but only mildly inhibited CBF activities. As NLS-CBFbeta-SMMHC(83C) remained cytoplasmic, we directed the ACD to CBF target genes by linking it to the AML1 DNA binding domain or to full-length AML1. These AML1-ACD fusion proteins did not affect Ba/F3 proliferation, in contrast to AML1-ETO, which markedly slowed G(1) to S progression dependent upon the integrity of its DNA-binding domain. Thus, the ACD facilitates inhibition of CBF by mediating multimerization of CBFbeta-SMMHC in the nucleus. Therapeutics targeting the ACD may be effective in acute myeloid leukemia cases associated with CBFbeta-SMMHC expression.

Signal transducer and activator of transcription 5 (STAT5) is constitutively activated by BCR/ABL, the oncogenic tyrosine kinase responsible for chronic myelogenous leukemia. The mechanism of BCR/ABL-mediated STAT5 activation is unknown. We show here that the BCR/ABL SH3 and SH2 domains interact with hematopoietic cell kinase (Hck), leading to the stimulation of Hck catalytic activity. Active Hck phosphorylated STAT5B on Tyr699, which represents an essential step in STAT5B stimulation. Moreover, a kinase-dead Hck mutant and Hck inhibitor PP2 abrogated BCR/ABL-dependent activation of STAT5 and elevation of expression of STAT5 downstream effectors A1 and pim-1. These data identify a novel BCR/ABL-Hck-STAT5 signaling pathway, which plays an important role in BCR/ABL-mediated transformation of myeloid cells.