Takuo Katsumoto

Regulation of Th2 cell differentiation by mel-18, a mammalian polycomb group gene.

Polycomb group (PcG) gene products regulate homeobox gene expression in Drosophila and vertebrates and also cell cycle progression of immature lymphocytes. In a gene-disrupted mouse for polycomb group gene mel-18, mature peripheral T cells exhibited normal anti-TCR-induced proliferation; however, the production of Th2 cytokines (IL-4, IL-5, and IL-13) was significantly reduced, whereas production of IFNgamma was modestly enhanced. Th2 cell differentiation was impaired, and the defect was associated with decreased levels in demethylation of the IL-4 gene. Significantly, reduced GATA3 induction was demonstrated. In vivo antigen-induced IgG1 production and Nippostrongylus brasiliensis-induced eosinophilia were significantly affected, reflecting the deficit in Th2 cell differentiation. Thus, the PcG gene products play a critical role in the control of Th2 cell differentiation and Th2-dependent immune responses.

The Hbo1-Brd1/Brpf2 complex is responsible for global acetylation of H3K14 and required for fetal liver erythropoiesis

The histone acetyltransferases (HATs) of the MYST family include TIP60, HBO1, MOZ/MORF, and MOF and function in multisubunit protein complexes. Bromodomain-containing protein 1 (BRD1), also known as BRPF2, has been considered a subunit of the MOZ/MORF H3 HAT complex based on analogy with BRPF1 and BRPF3. However, its physiologic function remains obscure. Here we show that BRD1 forms a novel HAT complex with HBO1 and regulates erythropoiesis. Brd1-deficient embryos showed severe anemia because of impaired fetal liver erythropoiesis. Biochemical analyses revealed that BRD1 bridges HBO1 and its activator protein, ING4. Genome-wide mapping in erythroblasts demonstrated that BRD1 and HBO1 largely colocalize in the genome and target key developmental regulator genes. Of note, levels of global acetylation of histone H3 at lysine 14 (H3K14) were profoundly decreased in Brd1-deficient erythroblasts and depletion of Hbo1 similarly affected H3K14 acetylation. Impaired erythropoiesis in the absence of Brd1 accompanied reduced expression of key erythroid regulator genes, including Gata1, and was partially restored by forced expression of Gata1. Our findings suggest that the Hbo1-Brd1 complex is the major H3K14 HAT required for transcriptional activation of erythroid developmental regulator genes.

PU.1-mediated upregulation of CSF1R is crucial for leukemia stem cell potential induced by MOZ-TIF2

Leukemias and other cancers possess self-renewing stem cells that help to maintain the cancer. Cancer stem cell eradication is thought to be crucial for successful anticancer therapy. Using an acute myeloid leukemia (AML) model induced by the leukemia-associated monocytic leukemia zinc finger (MOZ)-TIF2 fusion protein, we show here that AML can be cured by the ablation of leukemia stem cells. The MOZ fusion proteins MOZ-TIF2 and MOZ-CBP interacted with the transcription factor PU.1 to stimulate the expression of macrophage colony–stimulating factor receptor (CSF1R, also known as M-CSFR, c-FMS or CD115). Studies using PU.1-deficient mice showed that PU.1 is essential for the ability of MOZ-TIF2 to establish and maintain AML stem cells. Cells expressing high amounts of CSF1R (CSF1Rhigh cells), but not those expressing low amounts of CSF1R (CSF1Rlow cells), showed potent leukemia-initiating activity. Using transgenic mice expressing a drug-inducible suicide gene controlled by the CSF1R promoter, we cured AML by ablation of CSF1Rhigh cells. Moreover, induction of AML was suppressed in CSF1R-deficient mice and CSF1R inhibitors slowed the progression of MOZ-TIF2–induced leukemia. Thus, in this subtype of AML, leukemia stem cells are contained within the CSF1Rhigh cell population, and we suggest that targeting of PU.1-mediated upregulation of CSF1R expression might be a useful therapeutic approach.

Roles of the histone acetyltransferase monocytic leukemia zinc finger protein in normal and malignant hematopoiesis.

Histone‐modified enzymes are involved in various cell functions, including proliferation, differentiation, cell death and carcinogenesis. The protein MOZ (monocytic leukemia zinc finger protein) is a Myst (MOZ, Ybf2 (Sas3), Sas2, Tip60)‐type histone acetyltranseferase (HAT) that generates fusion genes, such as MOZ–TIF2, MOZ–CBP and MOZ–p300, in acute myeloid leukemia (AML) by chromosomal translocation. MOZ associates with AML1 (RUNX1), PU.1, and p53, and cooperatively activates target gene transcription. Gene targeting in mice has revealed that MOZ is essential for the generation and maintenance of hematopoietic stem cells (HSC) and for the appropriate development of myeloid, erythroid and B‐lineage cell progenitors. In AML, MOZ fusion genes lead to repressed differentiation, hyper‐proliferation, and self‐renewal of myeloid progenitors through deregulation of MOZ‐regulated target gene expression. It is therefore necessary to analyze the roles of MOZ and MOZ fusion genes in normal and malignant hematopoiesis to elucidate the mechanisms underlying and develop therapies for MOZ‐related AML. (Cancer Sci 2008; 99: 1523–1527)

PML-retinoic acid receptor alpha inhibits PML IV enhancement of PU.1-induced C/EBPepsilon expression in myeloid differentiation.

ABSTRACT PML and PU.1 play important roles in myeloid differentiation. PML-deficient mice have an impaired capacity for terminal maturation of their myeloid precursor cells. This finding has been explained, at least in part, by the lack of PML action to modulate retinoic acid-differentiating activities. In this study, we found that C/EBPε expression is reduced in PML-deficient mice. We showed that PU.1 directly activates the transcription of the C/EBPε gene that is essential for granulocytic differentiation. The type IV isoform of PML interacted with PU.1, promoted its association with p300, and then enhanced PU.1-induced transcription and granulocytic differentiation. In contrast to PML IV, the leukemia-associated PML-retinoic acid receptor α fusion protein dissociated the PU.1/PML IV/p300 complex and inhibited PU.1-induced transcription. These results suggest a novel pathogenic mechanism of the PML-retinoic acid receptor α fusion protein in acute promyelocytic leukemia.

Characterization of spheres derived from canine mammary gland adenocarcinoma cell lines.

There is increasing evidence for the presence of cancer stem cells in several solid tumors, and these cancer stem cells have a potential role in tumor initiation, aggression, and recurrence. The stem cell-like properties of spheres derived from canine mammary tumors remain largely elusive. We attempted to induce sphere formation using four cell lines of canine mammary adenocarcinoma, and characterized the spheres derived from a CHMp line in vitro and in vivo. The CHMp-derived spheres showed predominantly CD44+CD24- population, higher expression of stem cell-related genes, such as CD133, Notch3 and MDR, and higher resistance to doxorubicin compared with the CHMp-derived adherent cells. Xenograft transplantations in nude mice demonstrated that only 1 × 10(4)sphere cells were sufficient for tumor formation. Use of the sphere assay on these sphere-derived tumors showed that sphere-forming cells were present in the tumors, and were maintained in serial transplantation. We propose that spheres derived from canine mammary adenocarcinoma cell lines possess a potential characteristic of cancer stem cells. Spheres derived from canine mammary tumors could be a powerful tool with which to investigate novel therapeutic drugs and to elucidate the molecular and cellular mechanisms that underlie tumorigenesis.

Brpf1, a subunit of the MOZ histone acetyl transferase complex, maintains expression of anterior and posterior Hox genes for proper patterning of craniofacial and caudal skeletons.

The epigenetic mechanism involving chromatin modification plays a critical role in the maintenance of the expression of Hox genes. Here, we characterize a mutant of the medaka fish, named biaxial symmetries (bis), in which brpf1, a subunit of the MOZ histone acetyl transferase (HAT) complex, is mutated. The bis mutant displayed patterning defects both in the anterior-posterior axis of the craniofacial skeleton and the dorsal-ventral axis of the caudal one. In the anterior region, the bis mutant exhibited craniofacial cartilage homeosis. The expression of Hox genes was decreased in the pharyngeal arches, suggesting that the pharyngeal segmental identities were altered in the bis mutant. In the posterior region, the bis mutant exhibited abnormal patterning of the caudal skeleton, which ectopically formed at the dorsal side of the caudal fin. The expression of Zic genes was decreased at the posterior region, suggesting that the dorsal-ventral axis formation of the posterior trunk was disrupted in the bis mutant. We also found that the MOZ-deficient mice exhibited an abnormal patterning of their craniofacial and cervical skeletons and a decrease of Hox transcripts. We propose a common role of the MOZ HAT complex in vertebrates, a complex which is required for the proper patterning for skeletal development.

IDH2 and NPM1 Mutations Cooperate to Activate Hoxa9/Meis1 and Hypoxia Pathways in Acute Myeloid Leukemia.

IDH1 and IDH2 mutations occur frequently in acute myeloid leukemia (AML) and other cancers. The mutant isocitrate dehydrogenase (IDH) enzymes convert α-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of AML in which mice were transplanted with nucleophosmin1 (NPM)(+/-) hematopoietic stem/progenitor cells cotransduced with four mutant genes (NPMc, IDH2/R140Q, DNMT3A/R882H, and FLT3/ITD), which often occur simultaneously in human AML patients. Conditional deletion of IDH2/R140Q blocked 2-HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. IDH2/R140Q was necessary for the engraftment or survival of NPMc(+) cells in vivo. Gene expression analysis indicated that NPMc increased expression of Hoxa9. IDH2/R140Q also increased the level of Meis1 and activated the hypoxia pathway in AML cells. IDH2/R140Q decreased the 5hmC modification and expression of some differentiation-inducing genes (Ebf1 and Spib). Taken together, our results indicated that IDH2 mutation is critical for the development and maintenance of AML stem-like cells, and they provided a preclinical justification for targeting mutant IDH enzymes as a strategy for anticancer therapy.

Essential role of PU.1 in maintenance of mixed lineage leukemia- associated leukemic stem cells

Acute myeloid leukemia is a clonal malignant disorder derived from a small number of leukemic stem cells (LSCs). Rearrangements of the mixed lineage leukemia (MLL) gene are found in acute myeloid leukemia associated with poor prognosis. The upregulation of Hox genes is critical for LSC induction and maintenance, but is unlikely to support malignancy and the high LSC frequency observed in MLL leukemias. The present study shows that MLL fusion proteins interact with the transcription factor PU.1 to activate the transcription of CSF‐1R, which is critical for LSC activity. Acute myeloid leukemia is cured by either deletion of PU.1 or ablation of cells expressing CSF‐1R. Kinase inhibitors specific for CSF‐1R prolong survival time. These findings indicate that PU.1‐mediated upregulation of CSF‐1R is a critical effector of MLL leukemogenesis.

Dual inhibition of EZH1/2 breaks the quiescence of leukemia stem cells in acute myeloid leukemia

Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer originating from rare populations of leukemia stem cells (LSCs). AML relapse after conventional chemotherapy is caused by a remaining population of drug-resistant LSCs. Selective targeting of the chemoresistant population is a promising strategy for preventing and treating AML relapse. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27 to maintain the stemness of LSCs. Here, we show that quiescent LSCs expressed the highest levels of enhancer of zeste (EZH) 1 and EZH2, the PRC2 catalytic subunits, in the AML hierarchy, and that dual inactivation of EZH1/2 eradicated quiescent LSCs to cure AML. Genetic deletion of Ezh1/2 in a mouse AML model induced cell cycle progression of quiescent LSCs and differentiation to LSCs, eventually eradicating AML LSCs. Quiescent LSCs showed PRC2-mediated suppression of Cyclin D, and Cyclin D-overexpressing AML was more sensitive to chemotherapy. We have developed a novel EZH1/2 dual inhibitor with potent inhibitory activity against both EZH1/2. In AML mouse models and patient-derived xenograft models, the inhibitor reduced the number of LSCs, impaired leukemia progression, and prolonged survival. Taken together, these results show that dual inhibition of EZH1/2 is an effective strategy for eliminating AML LSCs.