Yoko Okitsu

Accelerated lymphangiogenesis in malignant lymphoma : possible role of VEGF-A and VEGF-C

There is little information regarding the lymphangiogenesis of malignant lymphoma. In this study, we evaluated the lymphangiogenesis and angiogenesis in 44 lymph nodes of 39 malignant lymphomas and five non‐reactive normal lymph nodes, based on the lymphatic vessel density (LVD) and microvessel density (MVD) calculated by the computer‐assisted assessment of vessel density. The LVD of malignant lymphomas was significantly higher than that of non‐reactive normal lymph nodes, irrespective of subtypes (P = 0·00077). On the contrary, there was no difference in MVD between malignant lymphomas and non‐reactive normal lymph nodes, except for diffuse large B cell lymphomas, which had a significantly low value of MVD, in comparison with non‐reactive normal lymph nodes (P = 0·009). We further examined the expression of vascular endothelial growth factor (VEGF)‐C and VEGF‐A, which function on lymphangiogenesis in lymph node samples. VEGF‐C was expressed in 36 of 39 malignant lymphomas. All 39 of the malignant lymphoma samples expressed VEGF‐A. Furthermore, the level of LVD and VEGF‐A or VEGF‐C was positively correlated. These findings suggest that lymphangiogenesis is actively developed in lymph nodes of malignant lymphomas and it may be induced by both VEGF‐A and VEGF‐C secreted from lymphoma cells.

3-Deazaneplanocin A (DZNep), an inhibitor of S-adenosylmethionine-dependent methyltransferase, promotes erythroid differentiation.

Background: S-adenosylmethionine-dependent methyltransferase inhibitor, DZNep, targets the degradation of histone methyltransferase EZH2 that catalyzes H3K27 trimethylation. Results: DZNep induced erythroid-related genes, which may not be directly related to EZH2 inhibition but may be partly associated with reduced protein level of hematopoietic corepressor ETO2. Conclusion: DZNep has the capacity to induce erythroid differentiation. Significance: Our data may be exploited for therapeutic applications for hematological diseases, including anemia. EZH2, a core component of polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through histone H3 Lys-27 trimethylation and is involved in various biological processes, including hematopoiesis. It is well known that 3-deazaneplanocin A (DZNep), an inhibitor of S-adenosylmethionine-dependent methyltransferase that targets the degradation of EZH2, preferentially induces apoptosis in various hematological malignancies, suggesting that EZH2 may be a new target for epigenetic treatment. Because PRC2 participates in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation, inhibition of EZH2 may influence erythropoiesis. To explore this possibility, we evaluated the impact of DZNep on erythropoiesis. DZNep treatment significantly induced erythroid differentiation of K562 cells, as assessed by benzidine staining and quantitative RT-PCR analysis for representative erythroid-related genes, including globins. When we evaluated the effects of DZNep in human primary erythroblasts derived from cord blood CD34-positive cells, the treatment significantly induced erythroid-related genes, as observed in K562 cells, suggesting that DZNep induces erythroid differentiation. Unexpectedly, siRNA-mediated EZH2 knockdown had no significant effect on the expression of erythroid-related genes. Transcriptional profiling of DZNep-treated K562 cells revealed marked up-regulation of SLC4A1 and EPB42, previously reported as representative targets of the transcriptional corepressor ETO2. In addition, DZNep treatment reduced the protein level of ETO2. These data suggest that erythroid differentiation by DZNep may not be directly related to EZH2 inhibition but may be partly associated with reduced protein level of hematopoietic corepressor ETO2. These data provide a better understanding of the mechanism of action of DZNep, which may be exploited for therapeutic applications for hematological diseases, including anemia.

Successful treatment with bortezomib and thalidomide for POEMS syndrome.

Dear Editor, A 60-year-old female patient was admitted to our hospital because of progressive pleural effusion. Physical examination revealed lymphadenopathy in the neck, axillary, and inguinal regions, edema of the lower extremities, skin changes such as hyperpigmentation and hypertrichosis, and distal weakness and paresthesias in extremities. She was unable to walk. A computed tomography scan showed multiple lymphadenopathy, osteosclerotic lesions on ribs, thoracic vertebrae, ilium and sacrum, massive pleural effusion, and mild splenomegaly with ascites. A positron-emission tomography (PET) scan revealed increased accumulation of [F] fluorodeoxyglucose in the osteosclerotic lesions. Immunoglobulin levels were normal, but serum monoclonal IgG λ protein was detected by immunoelectrophoresis. Bone marrow aspiration showed no increase of plasma cells. Biopsy of an osteosclerotic lesion in the rib was compatible with plasma cell neoplasm with λ light-chain restriction. Her serum vascular endothelial growth factor (VEGF) level was elevated at 378 pg/ml (normal range, <38 pg/ml). Endocrinological tests showed hypothyroidism and hyperprolactinemia. Nerve conduction studies demonstrated severe sensorimotor polyneuropathy. The diagnosis of polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome was made [1]. The autologous transplant was not planned because of poor performance status and progressive pleural effusion and ascites. The patient was initially treated with high dose dexamethasone, and then, MP regimen (melphalan and prednisolone), but her conditions rapidly deteriorated. We decided to use bortezomib because this agent has been reported to inhibit the secretion of VEGFwhich is the dominant-driving cytokine in POEMS syndrome [2], and there have been three case reports of POEMS syndrome successfully treated with bortezomib-based regimen (Table 1) [3–5]. Bortezomib was started at a dosage of 1.3 mg/m on days 1, 4, 8, and 11 of a 21-day cycle. After the second cycle, her extravascular volume overload dramatically improved, and the serum VEGF level decreased to 54 pg/ml. On a PET scan after 3 cycles of bortezomib, no abnormal uptake of tracer was seen. Bortezomib was reduced to 1.3 mg/m on days 1 and 8 for grade 3 diarrhea from the fourth cycle, which became manageable with this dose reduction. Although her conditions significantly improved and the serum VEGF level further decreased to 41 pg/ml, fluid retention and serum monoclonal protein was still present. We then added thalidomide at 100 mg/day to bortezomib from the sixth cycle, because several reports have suggested that thalidomide is effective for POEMS syndrome [6–9]. After the addition of thalidomide, pleural effusion and ascites completely disappeared, and serum monoclonal protein did not become detectable by immunofixation. Peripheral neuropathy is a common side effect of bortezomib and thalidomide, but symptoms of polyneuropathy improved gradually, and she was able to walk with equipment, suggesting the benefit of these drugs exceed the risk of those neurotoxicities, consistent with previous reports [3–9]. Administration of bortezomib was H. Ohguchi (*) : R. Ohba :Y. Onishi :N. Fukuhara :Y. Okitsu : J. Yamamoto :K. Ishizawa :H. Harigae Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan e-mail: ohguchi@med.tohoku.ac.jp

Allogeneic hematopoietic stem cell transplant following chemotherapy containing L-asparaginase as a promising treatment for patients with relapsed or refractory extranodal natural killer/T cell lymphoma, nasal type

The prognosis of advanced extranodal NK/T cell lymphoma (ENKTL) is poor. Allogeneic hematopoietic stem cell transplant (allo-HSCT) has been suggested to be a promising treatment for this disease, but its utility has yet to be established. Here we retrospectively analyzed five cases of ENKTL treated with allo-HSCT in our institute. After induction chemotherapy, disease status at allo-HSCT was second CR in three patients and refractory in two patients. All patients received a myeloablative conditioning regimen, and GVHD prophylaxis consisted of tacrolimus or cyclosporine with short-term methotrexate. Only one patient who received conventional induction chemotherapy developed severe complications, which needed long-term treatment, while others who received chemotherapy containing l-asparaginase did not have severe complications. There were no cases of treatment-related mortality, and all patients survived without disease for a median follow-up period of 1911 days. These results suggested that allo-HSCT following l-asparaginase-containing induction chemotherapy might improve the outcome of advanced ENKTL.

Acquired hemophilia A with sigmoid colon cancer : successful treatment with rituximab followed by sigmoidectomy

Acquired hemophilia A is a rare and potentially fatal condition of coagulopathy caused by autoantibodies against clotting factor VIII (factor VIII inhibitor). We report a case of a 63-year-old woman, who presented with a sudden onset of severe hemorrhagic tendency with exclusively prolonged activated partial thromboplastin time (APTT). She was diagnosed with acquired hemophilia A due to a decrease in factor VIII activity and a high titer of factor VIII inhibitor. Hemorrhage was well controlled by recombinant activated factor VII. Although the level of factor VIII inhibitor did not decline with prednisolone and cyclophosphamide, it became undetectable with rituximab. In parallel with controlling hemorrhage, malignancy, which may cause acquired hemophilia A, was searched for and sigmoid colon cancer was found. After the eradication of factor VIII inhibitor, surgical resection was performed uneventfully. Thereafter, acquired hemophilia A has been in complete remission without any additional therapy. The present case suggests the efficacy of rituximab for refractory acquired hemophilia A and the importance of the identification of underlying diseases that can cause acquired hemophilia A.

GATA2 regulates dendritic cell differentiation

Dendritic cells (DCs) are critical immune response regulators; however, the mechanism of DC differentiation is not fully understood. Heterozygous germ line GATA2 mutations induce GATA2-deficiency syndrome, characterized by monocytopenia, a predisposition to myelodysplasia/acute myeloid leukemia, and a profoundly reduced DC population, which is associated with increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. To define the role of GATA2 in DC differentiation and function, we studied Gata2 conditional knockout and haploinsufficient mice. Gata2 conditional deficiency significantly reduced the DC count, whereas Gata2 haploinsufficiency did not affect this population. GATA2 was required for the in vitro generation of DCs from Lin(-)Sca-1(+)Kit(+) cells, common myeloid-restricted progenitors, and common dendritic cell precursors, but not common lymphoid-restricted progenitors or granulocyte-macrophage progenitors, suggesting that GATA2 functions in the myeloid pathway of DC differentiation. Moreover, expression profiling demonstrated reduced expression of myeloid-related genes, including mafb, and increased expression of T-lymphocyte-related genes, including Gata3 and Tcf7, in Gata2-deficient DC progenitors. In addition, GATA2 was found to bind an enhancer element 190-kb downstream region of Gata3, and a reporter assay exhibited significantly reduced luciferase activity after adding this enhancer region to the Gata3 promoter, which was recovered by GATA sequence deletion within Gata3 +190. These results suggest that GATA2 plays an important role in cell-fate specification toward the myeloid vs T-lymphocyte lineage by regulating lineage-specific transcription factors in DC progenitors, thereby contributing to DC differentiation.

GATA2 regulates differentiation of bone marrow-derived mesenchymal stem cells.

The bone marrow microenvironment comprises multiple cell niches derived from bone marrow mesenchymal stem cells. However, the molecular mechanism of bone marrow mesenchymal stem cell differentiation is poorly understood. The transcription factor GATA2 is indispensable for hematopoietic stem cell function as well as other hematopoietic lineages, suggesting that it may maintain bone marrow mesenchymal stem cells in an immature state and also contribute to their differentiation. To explore this possibility, we established bone marrow mesenchymal stem cells from GATA2 conditional knockout mice. Differentiation of GATA2-deficient bone marrow mesenchymal stem cells into adipocytes induced accelerated oil-drop formation. Further, GATA2 loss- and gain-of-function analyses based on human bone marrow mesenchymal stem cells confirmed that decreased and increased GATA2 expression accelerated and suppressed bone marrow mesenchymal stem cell differentiation to adipocytes, respectively. Microarray analysis of GATA2 knockdowned human bone marrow mesenchymal stem cells revealed that 90 and 189 genes were upregulated or downregulated by a factor of 2, respectively. Moreover, gene ontology analysis revealed significant enrichment of genes involved in cell cycle regulation, and the number of G1/G0 cells increased after GATA2 knockdown. Concomitantly, cell proliferation was decreased by GATA2 knockdown. When GATA2 knockdowned bone marrow mesenchymal stem cells as well as adipocytes were cocultured with CD34-positive cells, hematopoietic stem cell frequency and colony formation decreased. We confirmed the existence of pathological signals that decrease and increase hematopoietic cell and adipocyte numbers, respectively, characteristic of aplastic anemia, and that suppress GATA2 expression in hematopoietic stem cells and bone marrow mesenchymal stem cells.

Induction of erythroid-specific genes by overexpression of GATA-2 in K562 cells

GATA transcription factors have been shown to play important roles in hematopoiesis. GATA-2 is expressed in stem and progenitor cells, and has been speculated to control the proliferation and maintain the immaturity of these cells. To examine whether the function of GATA-2 is changeable according to the differentiation stage, we established GATA-2 overexpress-ing subclones of K562, which is a leukemic cell line committed to the erythroid lineage. Via an increase in the GATA-2 expression level, the expression levels of erythroid-specific genes including a-, β-, and γ-globin were increased compared to control cells, while the expression level of GATA-1 was unchanged. Expression of the transferrin receptor was also increased in GATA-2 overexpressing K562 cells when examined by flow cytometry. In addition, the heme content of GATA-2 over-expressing K562 cells was more than 2 times higher than control cells. Chromatin immunoprecipitation analysis showed that GATA-2 protein binding to the GATA element in α-globin LCR was increased in GATA-2 overexpressing K562 cells. These findings suggest that GATA-2 could induce erythroid-specific genes without competition with GATA-1 when expressed in erythroid-committed cells, and thus further suggest that temporal and spatial regulation may be important for displaying specific functions of GATA-2.

A Lysophosphatidic Acid Receptors 1 and 3 Axis Governs Cellular Senescence of Mesenchymal Stromal Cells and Promotes Growth and Vascularization of Multiple Myeloma

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells and there is much interest in how MSCs contribute to the regulation of the tumor microenvironment. Whether MSCs exert a supportive or suppressive effect on tumor progression is still controversial, but is likely dependent on a variety of factors that are tumor‐type dependent. Multiple myeloma (MM) is characterized by growth of malignant plasma cells in the bone marrow. It has been shown that the progression of MM is governed by MSCs, which act as a stroma of the myeloma cells. Although stroma is created via mutual communication between myeloma cells and MSCs, the mechanism is poorly understood. Here we explored the role of lysophosphatidic acid (LPA) signaling in cellular events where MSCs were converted into either MM‐supportive or MM‐suppressive stroma. We found that myeloma cells stimulate MSCs to produce autotaxin, an indispensable enzyme for the biosynthesis of LPA, and LPA receptor 1 (LPA1) and 3 (LPA3) transduce opposite signals to MSCs to determine the fate of MSCs. LPA3‐silenced MSCs (siLPA3‐MSCs) exhibited cellular senescence‐related phenotypes in vitro, and significantly promoted progression of MM and tumor‐related angiogenesis in vivo. In contrast, siLPA1‐MSCs showed resistance to cellular senescence in vitro, and efficiently delayed progression of MM and tumor‐related angiogenesis in vivo. Consistently, anti‐MM effects obtained by LPA1‐silencing in MSCs were completely reproduced by systemic administration of Ki6425, an LPA1 antagonist. Collectively, our results indicate that LPA signaling determines the fate of MSCs and has potential as a therapeutic target in MM. Stem Cells 2017;35:739–753

A Low-Molecular-Weight Compound K7174 Represses Hepcidin: Possible Therapeutic Strategy against Anemia of Chronic Disease

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. The expression of the hepcidin gene, HAMP, is increased in patients with anemia of chronic disease. Previously, the synthetic compound K7174 was identified through chemical screening as a novel inhibitor of the adhesion of monocytes to cytokine-stimulated endothelial cells. K7174 also ameliorated anemia induced by inflammatory cytokines in mice, which suggests a possible involvement of hepcidin regulation. The present study was performed to assess the impact of K7174 on hepcidin expression in a human hematoma cell line and in mice in vivo. We first demonstrated that K7174 treatment in HepG2 cells significantly decreased HAMP expression. Then, we conducted microarray analysis to determine the molecular mechanism by which K7174 inhibits HAMP expression. Transcriptional profiling confirmed the downregulation of HAMP. Surprisingly, we found that K7174 strongly induced GDF15, known as a negative regulator of HAMP expression. Western blotting analysis as well as ELISA confirmed the induction of GDF15 by K7174 treatment. Furthermore, K7174-mediated HAMP suppression was rescued by the silencing of GDF15 expression. Interestingly, we found that K7174 also upregulates CEBPB. Promoter analysis and chromatin immunoprecipitation analysis revealed that CEBPB could contribute to K7174-mediated transcriptional activation of GDF15. Subsequently, we also examined whether K7174 inhibits hepcidin expression in mice. Quantitative RT-PCR analysis with liver samples from K7174-treated mice demonstrated significant upregulation of Gdf15 and downregulation of Hamp expression, as compared to control mice. Furthermore, serum hepcidin concentration was also significantly decreased in K7174-treated mice. In conclusion, K7174 inhibits hepcidin expression partly by inducing GDF15. K-7174 may be a potential therapeutic option to treat anemia of chronic disease.