Akihito Matsuoka

Lenalidomide induces cell death in an MDS-derived cell line with deletion of chromosome 5q by inhibition of cytokinesis.

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by refractory cytopenias and susceptibility to leukemic transformation. On a subset of MDS patients with deletion of the long arm of chromosome5 (del(5q)), lenalidomide exerts hematological and cytogenetic effects, but the underlying pharmacological mechanisms are not fully understood. In this study, we have investigated the in vitro effects of lenalidomide on an MDS-derived cell line, MDS-L, which carries del(5q) and complex chromosome abnormalities. We found that the growth of MDS-L cells was specifically suppressed mainly by apoptosis, and in addition, multinucleated cells were frequently formed and finally died out in the presence of lenalidomide. Time-lapse microscopic observation and the DNA ploidy analysis revealed that lenalidomide does not affect DNA synthesis but inhibits cytokinesis of MDS-L cells. The gene expression profile showed decreased expression of M phase-related genes such as non-muscle myosin heavy-chain 10, polo-like kinase 1, aurora kinase B, citron kinase and kinesin family member 20A(KIF20A). Interestingly, KIF20A is located at 5q31. These data contribute to the understanding of action mechanisms of lenalidomide on MDS with del(5q) and complex abnormalities.

Potentiated activation of VLA-4 and VLA-5 accelerates proplatelet-like formation

Fibronectin (FN) plays important roles in the proliferation, differentiation, and maintenance of megakaryocytic-lineage cells through FN receptors. However, substantial role of FN receptors and their functional assignment in proplatelet-like formation (PPF) of megakaryocytes are not yet fully understood. Herein, we investigated the effects of FN receptors on PPF using the CHRF-288 human megakaryoblastic cell line, which expresses VLA-4 and VLA-5 as FN receptors. FN and phorbol 12-myristate 13-acetate (PMA) were essential for inducing PPF in CHRF-288 cells. Blocking experiments using anti-β1-integrin monoclonal antibodies indicated that the adhesive interaction with FN via VLA-4 and VLA-5 were required for PPF. PPF induced by FN plus PMA was accelerated when CHRF-288 cells were enforced adhering to FN by TNIIIA2, a peptide derived from tenascin-C, which we recently found to induce β1-integrin activation. Adhesion to FN enhanced PMA-stimulated activation of extracellular signal-regulated protein kinase 1 (ERK1)/2 and enforced adhesion to FN via VLA-4 and VLA-5 by TNIIIA2-accelerated activation of ERK1/2 with FN plus PMA. However, c-Jun amino-terminal kinase 1 (JNK1), p38, and phosphoinositide-3 kinase (PI3K)/Akt were not stimulated by FN plus PMA, even with TNIIIA2. Thus, the enhanced activation of ERK1/2 by FN, PMA plus TNIIIA2 was responsible for acceleration of PPF with FN plus PMA.

Validation of the revised 2008 WHO diagnostic criteria in 75 suspected cases of myeloproliferative neoplasm

The objective of this study was to validate the recently revised 2008 WHO diagnostic criteria of myeloproliferative neoplasms (MPN) together with the analysis of correlation of JAK2 (Janus kinase 2)-V617F mutant allele burden with clinical/laboratory findings on each patient. We made a diagnosis of 75 suspected MPN patients based on both diagnostic criteria of the 2001 WHO classification and the revised 2008 WHO classification, and found that both criteria show a quite similar diagnostic power except for two patients (idiopathic erythrocytosis (IE) and thrombocytosis) who were diagnosed as essential thrombocythemia by the 2008 WHO criteria. From JAK2-V617F analysis, hemoglobin and hematocrit values were significantly higher and platelet count was lower in JAK2-V617F high allele burden group than JAK2-V617F middle allele burden group. Mutant allele burden of polycythemia vera (PV) group was higher than that of essential thrombocythemia group. Therefore, the amount of mutant allele seemed to define the disease phenotypes. We further found a PV case presenting a rare type of JAK2-exon12 mutation. In contrast, IE presented a good prognosis unlike MPN. Hereafter, the 2008 WHO criteria with JAK2 gene analysis are useful for precise diagnosis of MPN and the patients with erythrocytosis.

Calmodulin antagonists induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in vivo in human multiple myeloma

BackgroundHuman multiple myeloma (MM) is an incurable hematological malignancy for which novel therapeutic agents are needed. Calmodulin (CaM) antagonists have been reported to induce apoptosis and inhibit tumor cell invasion and metastasis in various tumor models. However, the antitumor effects of CaM antagonists on MM are poorly understood. In this study, we investigated the antitumor effects of naphthalenesulfonamide derivative selective CaM antagonists W-7 and W-13 on MM cell lines both in vitro and in vivo.MethodsThe proliferative ability was analyzed by the WST-8 assay. Cell cycle was evaluated by flow cytometry after staining of cells with PI. Apoptosis was quantified by flow cytometry after double-staining of cells by Annexin-V/PI. Molecular changes of cell cycle and apoptosis were determined by Western blot. Intracellular calcium levels and mitochondrial membrane potentials were determined using Fluo-4/AM dye and JC-10 dye, respectively. Moreover, we examined the in vivo anti-MM effects of CaM antagonists using a murine xenograft model of the human MM cell line.ResultsTreatment with W-7 and W-13 resulted in the dose-dependent inhibition of cell proliferation in various MM cell lines. W-7 and W-13 induced G1 phase cell cycle arrest by downregulating cyclins and upregulating p21cip1. In addition, W-7 and W-13 induced apoptosis via caspase activation; this occurred partly through the elevation of intracellular calcium levels and mitochondrial membrane potential depolarization and through inhibition of the STAT3 phosphorylation and subsequent downregulation of Mcl-1 protein. In tumor xenograft mouse models, tumor growth rates in CaM antagonist-treated groups were significantly reduced compared with those in the vehicle-treated groups.ConclusionsOur results demonstrate that CaM antagonists induce cell cycle arrest, induce apoptosis via caspase activation, and inhibit tumor growth in a murine MM model and raise the possibility that inhibition of CaM might be a useful therapeutic strategy for the treatment of MM.

NKT cell-infiltrating aseptic meningitis on the central nervous system in Philadelphia chromosome-positive acute lymphoblastic leukemia treated with dasatinib.

Dear Editor, Among the TKIs, dasatinib is an ABL kinase inhibitor characterized by strong binding affinity to the ABL kinase domain [1]. Dasatinib is different from the other resting TKIs as a unique immunological activator. It is thought that dasatinib exerts an immunomodulatory effect to enhance the innate immune system involving natural killer (NK), natural killer T (NKT) cells, and large granular lymphocytes (LGL) under certain circumstances [2, 3]. A 53-year-old female was transferred to our institute due to febrile leukocytosis. She was diagnosed as having Ph ALL and received induction chemotherapy consisting of cyclophosphamide, daunorubicin, vincristine, and prednisolone, followed by imatinib 600 mg/day (days 8–63). She achieved complete remission after the induction chemotherapy, as her bone marrow aspiration showed molecular remission confirmed by the evaluation of BCR-ABL messenger RNA (mRNA) (<1.0×10). We performed a first consolidation chemotherapy consisting of high-dose methotrexate 1 g/m and cytarabine 2 g/m/day for 2 days, followed by dasatinib 140 mg/day (days 29–56). The patient maintained complete remission with undetectablemolecular evidence of BCR-ABL oncogene product. We initiated a second consolidation, with the same regimen as the first consolidation, and on day 34, she felt hypersensitivity on both palms. The patient’s neurological examination showed no abnormal findings included. A cranial MRI revealed no intracranial lesion explaining her neurological symptoms, while a spinal tap revealed elevatedmononuclear cell counts. The infiltrating cells consisted of granulocytes (CD3433) 15 %, B cells (CD19) 0.6 %, T cells (CD3) 98.2 %, NK cells (CD356) 1.21 %, and NKT cells (CD356) 3.0 % (Fig. 1a). Some infiltrating cells revealed LGL morphology (Fig. 1b). Cultured cerebrospinal fluid (CSF) showed no bacterial organisms or acid-fast bacillus. Molecular biological methods using CSF, a fluorescence in situ hybridization (FISH) analysis, and mRNA for BCR-ABL did not uncover findings of leukemic involvement of the CNS and bone marrow. Soon after the discontinuation of dasatinib, the patient’s neurological symptoms were ameliorated, and the infiltrating mononuclear cells disappeared completely. A high proportion of NK cells (LGLs) and NKT cells is thought to be an immunological reaction involved in dasatinib-induced immunomodulation, which has been observed several times in various clinical situations [3, 4]. The cell surface antigen analysis we performed by a flow cytometry technique revealed the NK and NKT cell infiltration, and notably, the NKT cell composition was concentrated in the CSF more than in the peripheral blood (at most 0.01 %). Almost all of the infiltrating cell population was T cells, but a minor population of NK and NKT cells was present, which was similar to the case presenting pleural effusion during dasatinib therapy.We did not determine the antigen specificity of Tcells. Even so, an accumulation of NKTcells in the CSF is an unusual event, and this case appeared to be “immunogenic” aseptic meningitis. Although the mechanisms by which activated NKTcells confer disease protection remain to be examined in future studies, those immunomodulatory properties of NKT cells suggested in the present patient may be useful for the development of immunotherapies against hematological malignancies other than Ph ALL that involve the CNS. O. Imataki (*) :A. Matsuoka :M. Uemura Division of Hematology, Department of Internal Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan e-mail: oima@med.kagawa-u.ac.jp

Approach to new therapeutics: investigation by the use of MDS-derived cell lines.

Myelodysplastic syndromes (MDS) are a group of aquired hematopoietic disorders characterized by ineffective hematopoiesis, and increased risk of progression of acute myeloid leukemia. For a long period of time, the standard therapy for MDS was hematopoietic stem cell transplantation, however DNA methyltransferase inhibitors (DNMT inhibitors) including decitabine (DAC) and azacitidine (AZA), and lenalidomide, a derivative of thalidomide have been highlighted as new chemotherapeutic agents for MDS. However, the underlying mechanisms of action of these drugs have not been fully defined yet. Therefore, we investigated the in vitro effects of DNMT inhibitors and lenalidomide on an MDS-derived cell line, MDS92 and its blastic subline MDS-L, both of which carry del(5q). MDS-L cells were found to be quite sensitive to DAC, which induced to cell death through DNA damage-mediated G2 arrest via p53- independent pathways. Gene expression profiling suggested that DAC affects biogroups representing hematological systems, cellular development, cell death and apoptosis. Next, we examined the effects of lenalidomide on MDS-L. Cell growth was inhibited and multinucleated cells were frequently formed prior to cell death by lenalidomide treatment. Time-lapse microscopic observation and DNA ploidy analysis revealed that lenalidomide does not affect DNA synthesis itself but inhibits cytokinesis of MDS-L cells. The gene expression profiling showed decreased expression of M phase-related genes. These data contribute to the understanding of action mechanisms of lenalidomide on MDS with del(5q).