Manabu Kusakabe

Transcription factor GATA-3 is essential for lens development

During vertebrate lens development, the anterior, ectoderm‐derived lens vesicle cells differentiate into a monolayer of epithelial cells that retain proliferative potential. Subsequently, they exit the cell cycle and give rise to posterior lens fiber cells that form the lens body. In the present study, we demonstrate that the transcription factor GATA‐3 is expressed in the posterior lens fiber cells during embryogenesis, and that GATA‐3 deficiency impairs lens development. Interestingly, expression of E‐cadherin, a premature lens vesicle marker, is abnormally prolonged in the posterior region of Gata3 homozygous mutant lenses. Furthermore, expression of γ‐crystallin, a differentiation marker for fiber cells, is reduced. This suppressed differentiation is accompanied by an abnormal cellular proliferation, as well as with diminished levels of the cell‐cycle inhibitors Cdkn1b/p27 and Cdkn1c/p57 and increased Ccnd2/cyclin D2 abundance. Thus, these observations suggest that GATA‐3 is essential for lens cells differentiation and proper cell cycle control. Developmental Dynamics 238:2280–2291, 2009.

c-Maf plays a crucial role for the definitive erythropoiesis that accompanies erythroblastic island formation in the fetal liver

c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the activated protein-1 super family of basic leucine zipper proteins. Despite its overexpression in hematologic malignancies, the physiologic roles c-Maf plays in normal hematopoiesis have been largely unexplored. On a C57BL/6J background, c-Maf(-/-) embryos succumbed from severe erythropenia between embryonic day (E) 15 and E18. Flow cytometric analysis of fetal liver cells showed that the mature erythroid compartments were significantly reduced in c-Maf(-/-) embryos compared with c-Maf(+/+) littermates. Interestingly, the CFU assay indicated there was no significant difference between c-Maf(+/+) and c-Maf(-/-) fetal liver cells in erythroid colony counts. This result indicated that impaired definitive erythropoiesis in c-Maf(-/-) embryos is because of a non-cell-autonomous effect, suggesting a defective erythropoietic microenvironment in the fetal liver. As expected, the number of erythroblasts surrounding the macrophages in erythroblastic islands was significantly reduced in c-Maf(-/-) embryos. Moreover, decreased expression of VCAM-1 was observed in c-Maf(-/-) fetal liver macrophages. In conclusion, these results strongly suggest that c-Maf is crucial for definitive erythropoiesis in fetal liver, playing an important role in macrophages that constitute erythroblastic islands.

A Novel Transgenic Mouse Model of the Human Multiple Myeloma Chromosomal Translocation t(14;16)(q32;q23)

Multiple myeloma (MM) is a currently incurable neoplasm of terminally differentiated B cells. The translocation and/or overexpression of c-MAF have been observed in human MM. Although c-MAF might function as an oncogene in human MM, there has been no report thus far describing the direct induction of MM by c-MAF overexpression in vivo. In this study, we have generated transgenic (TG) mice that express c-Maf specifically in the B-cell compartment. Aged c-Maf TG mice developed B-cell lymphomas with some clinical features that resembled those of MM, namely, plasma cell expansion and hyperglobulinemia. Quantitative RT-PCR analysis demonstrated that Ccnd2 and Itgb7, which are known target genes of c-Maf, were highly expressed in the lymphoma cells. This novel TG mouse model of the human MM t(14;16)(q32;q23) chromosomal translocation should serve to provide new insight into the role of c-MAF in tumorigenesis.

Overexpression of RORγt under control of the CD2 promoter induces polyclonal plasmacytosis and autoantibody production in transgenic mice.

Retinoic acid related orphan receptor gamma‐t (RORγt) is known to be a master regulator of Th17‐cell development. In this study, we generated RORγt‐overexpressing transgenic (RORγt Tg) mice in which transgene expression was driven by the CD2 promoter, and found that these mice developed polyclonal plasmacytosis and autoantibody production. RORγt Tg mice were generated on a C57BL/6 background, and also were intercrossed with BALB/c mice. BALB/c F1 (BALB/F1) RORγt Tg mice developed massive polyclonal plasma‐cytosis, and had shorter life spans. Splenomegaly and infiltration of plasma cells into the lung were observed. Hyperglobulinemia, anti‐double‐stranded DNA antibodies, anti‐erythrocyte antibodies, and anti‐platelet antibodies were detected in BALB/F1 RORγt Tg mice. In the present study, polyclonal plasmacytosis in BALB/F1 RORγt Tg mice appeared to be due to the induction of excessive IL‐6 production by IL‐17. We detected increased numbers of CD11b+ cells that produced IL‐6. We also generatedIL‐6‐deficient RORγt Tg BALB/F1 background mice, which displayed high levels of serum IL‐17, but did not develop severe hyperglobulinemia. Excessive IL‐6 production by several cell types, including macrophages, in BALB/F1 RORγt Tg mice, might effect the development of plasma‐cytosis. These results suggest that RORγt plays important roles in the development of plasmacytosis and autoantibody production.

Detection of the STAT5B–RARA fusion transcript in acute promyelocytic leukemia with the normal chromosome 17 on G-banding

Acute promyelocytic leukemia (APL) is characterized by chromosomal rearrangements of 17q21, leading to fusion of the gene‐encoding retinoic acid receptor alpha (RARA) with a number of alternative partner genes. Signal transducer and activator of transcription 5 beta (STAT5B) is one of the alternative partners. We report a rare case of APL with STAT5B–RARA fusion transcript and the normal chromosome 17 on G‐banding. Administration of all trans‐retinoic acid improved disseminated intravascular coagulation without decrease of the leukemia cells in his peripheral blood and bone marrow. The molecular mechanism of fusion between STAT5B and RARA by chromosomal rearrangement is discussed based on the data from genome database. Clinical characteristics of APL with STAT5B–RARA are also discussed.

c-Maf is essential for the F4/80 expression in macrophages in vivo

c-Maf, which is one of the large Maf transcription factors, can bind to Maf recognition element (MARE) and activates transcription of target genes. Although c-Maf is expressed in macrophages and directly regulates the expression of interleukin-10, detailed information regarding its function in the null mutant phenotype of tissue macrophages remain unknown. In this study, we demonstrated that c-Maf is specifically expressed in the F4/80 positive fetal liver and adult macrophages. The expression of F4/80, which is a tissue macrophage-specific seven trans-membrane receptor, was dramatically suppressed in the c-Maf-deficient macrophage, whereas the expression of Mac-1 was not affected, suggesting that c-Maf is not necessary for the lineage commitment of macrophages. Luciferase reporter and EMSA showed that c-Maf directly regulates the expression of F4/80 by interacting with the half-MARE site of the F4/80 promoter. These results suggest that c-Maf is required for the F4/80 expression in macrophages in vivo.

Blastic plasmacytoid dendritic cell neoplasm arising from clonal hematopoiesis

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of myeloid neoplasm. Clonal evolution in the development of BPDCN remains to be elucidated. In the present study, we examined clonal evolution in a case of BPDCN by analyzing the distribution of gene mutations in tumor cells and non-tumor blood cells. The p.D1129fs and p.K1005fs TET2 mutations, p.P95H SRSF2 mutation, and p.L287fs NPM1 mutation were identified in a skin tumor at diagnosis and peripheral blood mononuclear cells at relapse. Notably, the p.D1129fs TET2 and p.L287fs NPM1 mutations were observed only in tumor cells, while the p.K1005fs TET2 and p.P95H SRSF2 mutations were found in both tumor cells and non-tumor blood cells. Recent genetic studies have suggested that some blood cancers may originate from clonal hematopoiesis, harboring somatic mutations. In the present case, the data suggest that BPDCN originated from clonal hematopoiesis with the p.K1005fs TET2 and p.P95H SRSF2 mutations via acquisition of the additional p.D1129fs TET2 and p.L287fs NPM1 mutations.

An Unprecedented Case of p190 BCR-ABL Chronic Myeloid Leukemia Diagnosed during Treatment for Multiple Myeloma: A Case Report and Review of the Literature

We report the case of a 76-year-old man who was diagnosed as having chronic myeloid leukemia (CML) with p190 BCR-ABL while receiving treatment for symptomatic multiple myeloma (MM). The diagnosis of MM was based on the presence of serum M-protein, abnormal plasma cells in the bone marrow, and lytic bone lesions. The patient achieved a partial response to lenalidomide and dexamethasone treatment. However, 2 years after the diagnosis of MM, the patient developed leukocytosis with granulocytosis, anemia, and thrombocytopenia. Bone marrow examination revealed Philadelphia chromosomes and chimeric p190 BCR-ABL mRNA. Fluorescence in situ hybridization also revealed BCR-ABL-positive neutrophils in the peripheral blood, which suggested the emergence of CML with p190 BCR-ABL. The codevelopment of MM and CML is very rare, and this is the first report describing p190 BCR-ABL-type CML coexisting with MM. Moreover, we have reviewed the literature regarding the coexistence of these diseases.

Genetic evidence implies that the primary and relapsed tumors arise from common precursor cells in primary CNS lymphoma

Primary central nervous system lymphoma (PCNSL) is a rare subtype of lymphoma that arises within the brain or the eyes. PCNSL recurs within the central nervous system (CNS) in most relapsed cases, whereas extra‐CNS relapse is experienced in rare cases. The present study aimed at identifying the presence of common precursor cells (CPC) for primary intra‐ and relapsed extra‐CNS tumors, and further assessing the initiating events in bone marrow (BM). Targeted deep sequencing was carried out for five paired primary intra‐ and relapsed extra‐CNS tumors of PCNSL. Two to five mutations were shared by each pair of intra‐ and extra‐CNS tumors. In particular, MYD88 mutations, L265P in three and P258L in one, were shared by four pairs. Unique somatic mutations were observed in all five intra‐CNS tumors and in four out of five extra‐CNS tumors. Remarkably, IgH clones in the intra‐ and the extra‐CNS tumors in two pairs were distinct from each other, whereas one pair of tumors shared identical monoclonal IgH rearrangement. In a cohort of 23 PCNSL patients, L265P MYD88 mutations were examined in tumor‐free BM mononuclear cells (MNC) in which the PCNSL tumors had L265P MYD88 mutations. L265P MYD88 mutations were detected by a droplet digital PCR method in nine out of 23 bone marrow mononuclear cells. These results suggest that intra‐ and extra‐tumors are derived from CPC with MYD88 mutations in most PCNSL, arising either before or after IgH rearrangement. The initiating MYD88 mutations may occur during B‐cell differentiation in BM.

Droplet digital polymerase chain reaction assay and peptide nucleic acid-locked nucleic acid clamp method for RHOA mutation detection in angioimmunoblastic T-cell lymphoma

Angioimmunoblastic T‐cell lymphoma (AITL) is a subtype of nodal peripheral T‐cell lymphoma (PTCL). Somatic RHOA mutations, most frequently found at the hotspot site c.50G > T, p.Gly17Val (G17V RHOA mutation) are a genetic hallmark of AITL. Detection of the G17V RHOA mutations assists prompt and appropriate diagnosis of AITL. However, an optimal detection method for the G17V RHOA mutation remains to be elucidated. We compared the sensitivity and concordance of next‐generation sequencing (NGS), droplet digital PCR (ddPCR) and peptide nucleic acid‐locked nucleic acid (PNA‐LNA) clamp method for detecting the G17V RHOA mutation. G17V RHOA mutations were identified in 27 of 67 (40.3%) PTCL samples using NGS. ddPCR and PNA‐LNA clamp method both detected G17V mutations in 4 samples in addition to those detected with NGS (31 of 67, 46.3%). Additionally, variant allele frequencies with ddPCR and those with NGS showed high concordance (P < .001). Three other RHOA mutations involving the p.Gly17 position (c.[49G > T;50G > T], p.Gly17Leu in PTCL198; c.[50G > T;51A > C], p.Gly17Val in PTCL216; and c.50G > A, p.Gly17Glu in PTCL223) were detected using NGS. These sequence changes could not appropriately be detected using the ddPCR assay and the PNA‐LNA clamp method although both indicated that the samples might have mutations. In total, 34 out of 67 PTCL samples (50.7%) had RHOA mutations at the p.Gly17 position. In conclusion, our results suggested that a combination of ddPCR/PNA‐LNA clamp methods and NGS are best method to assist the diagnosis of AITL by detecting RHOA mutations at the p.Gly17 position.