Yasuyoshi Morita

Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients

Somatic mutation of RUNX1 is implicated in various hematological malignancies, including myelodysplastic syndrome and acute myeloid leukemia (AML), and previous studies using mouse models disclosed its critical roles in hematopoiesis. However, the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder (FPD)/AML is an autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. By in vitro differentiation assays, FPD-iPSCs were clearly defective in the emergence of hematopoietic progenitors and differentiation of megakaryocytes, and overexpression of wild-type (WT)-RUNX1 reversed most of these phenotypes. We further demonstrated that overexpression of mutant-RUNX1 in WT-iPSCs did not recapitulate the phenotype of FPD-iPSCs, showing that the mutations were of loss-of-function type. Taken together, this study demonstrated that haploinsufficient RUNX1 allele imposed cell-intrinsic defects on hematopoietic differentiation in human experimental settings and revealed differential impacts of RUNX1 dosage on human and murine megakaryopoiesis. FPD-iPSCs will be a useful tool to investigate mutant RUNX1-mediated molecular processes in hematopoiesis and leukemogenesis.

Extramedullary blast crisis derived from 2 different clones in the central nervous system and neck during complete cytogenetic remission of chronic myelogenous leukemia treated with imatinib mesylate.

We describe a patient with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) who developed an extramedullary blast crisis in the central nervous system (CNS) and then a subcutaneous tumor of the neck during treatment with imatinib mesylate. Administered 400 mg of imatinib mesylate after the diagnosis of chronic-phase CML, the patient achieved a complete cytogenetic remission 4 months later. However, he developed a mixed myeloid/B-cell blast crisis with additional karyotype abnormalities only in the CNS during a complete cytogenetic remission in the bone marrow. Several doses of intrathecal chemotherapy and whole-brain irradiation were effective in treating the blast crisis in the CNS. After 7 months of complete cytogenetic remission, the patient experienced a subcutaneous tumor in the right neck. A biopsy of the tumor revealed a mixed myeloid/T-cell blast crisis. The cytogenetic analysis showed that the blast crisis clone in the neck tumor was different from that of the CNS. An increased dose of imatinib mesylate was ineffective in treating the neck tumor. Irradiation to the right neck was therefore undertaken. This case suggests that the development of a clone resistant to imatinib mesylate is not always detected in the bone marrow and that multiple Ph-positive clones have the potential to become transformed into a blast crisis.

CD34+ progenitors from MDS patients are unresponsive to SDF-1, despite high levels of SDF-1 in bone marrow plasma.

CD34 + progenitors from MDS patients are unresponsive to SDF-1, despite high levels of SDF-1 in bone marrow plasma

Clathrin Assembly Protein CALM Plays a Critical Role in KIT Signaling by Regulating Its Cellular Transport from Early to Late Endosomes in Hematopoietic Cells

CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage−Sca-1+KIT+ (LSK) cells decreased in the fetal liver of CALM −/− mice. Also, colony forming activity was impaired in CALM−/− LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM−/− LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM −/− murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM −/− MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM −/− MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM −/− MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM −/− MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM −/− KIT+ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.

Clinical evaluation of WT1 mRNA expression levels in peripheral blood and bone marrow in patients with myelodysplastic syndromes.

Abstract A study to evaluate WT1 mRNA expression levels in peripheral blood (PB) and bone marrow aspirate (BM) was conducted in 172 patients, including 115 with myelodysplastic syndromes (MDS), in Japan. The level of WT1 mRNA expression was evaluated according to the French–American–British (FAB) and World Health Organization (WHO) classifications (2001, 2008) and using the International Prognostic Scoring System and the WHO Prognostic Scoring System scales. WT1 mRNA expression levels in PB and BM were well correlated (r = 0.85), and they tended to increase with disease stage progression and in those at higher risk of leukemic transformation. WT1 mRNA expression can be a useful marker for the diagnosis and risk evaluation of MDS.

All-trans retinoic acid attacks reverse transcriptase resulting in inhibition of HIV-l replication

Abstract We previously reported that all-trans retinoic acid (ATRA) inhibited growth in HTLV-l-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. Interestingly, ATRA significantly inhibited reverse transcriptase (RT) activity similar to azidothimidine (AZT) in HTLV-l-positive T-cell lines. To clarify whether ATRA has an inhibitory effect on the replication of HIV, we examined HIV proviral DNA in a HIV-l-positive cell line (8E5) using real time PCR. ATRA as well as AZT reduced the proviral DNA load of 8E5 in a dose-dependent manner. These results suggest that there is a common element of ATRA signaling in both HTLV-l and HIV. Furthermore, we examined the effects of ATRA on viral replication in primary lymphocytes of three individuals infected with HIV. ATRA reduced viral replication significantly similar to AZT. These findings suggested that ATRA acts as a RT inhibitor, reducing the HIV-l proviral DNA load. Finally, we conclude that ATRA may be a potential therapeutic agent for HIV infection.

Dichotomy of all-trans retinoic acid inducing signals for adult T-cell leukemia

We previously reported that all-trans retinoic acid (ATRA) inhibits growth in human T-cell leukemia virus type 1 (HTLV-1)-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. However, the mechanism of this inhibition is not clear. In the present study, we observed that NF-κB transcriptional activity as well as cell growth decreased significantly in HTLV-1-positive T-cell lines in the presence of ATRA. Furthermore, we observed that ATRA reduced HTLV-1 proviral DNA, HTLV-1 genes (gag, tax, or pol mRNA) using the real-time quantitative polymerase chain reaction. SIL-2R was reduced by ATRA in both protein level (culture supernantant) and mRNA level in HTLV-1-positive T-cell lines. Interestingly, ATRA significantly inhibited RT activity similar to azidothimidine (AZT) in HTLV-1-positive T-cell lines. Moreover, AZT inhibited proviral DNA but not NF-κB transcriptional activity, and sIL-2R on HTLV-1; however, ATRA inhibited of NF-κB, proviral DNA and sIL-2R on HTLV-1. These results suggested that the decrease in sIL-2R induced by ATRA may be caused by the actions of a NF-κB inhibitor acting on the NF-κB/sIL-2R signal pathway. These results suggested that ATRA could have two roles, as a NF-κB inhibitor and as an RT inhibitor.

Influence of Epstein–Barr virus infection in adult T-cell leukemia

Abstract The involvement of adult T-cell leukemia (ATL) cells in organs such as the skin and lymph nodes is observed in about 50% of cases of ATL. Epstein–Barr virus (EBV) infection has often been observed in the clinical course of ATL. In this study, we established two B-cell lines from peripheral blood of patients with ATL. EBV DNA, proviral DNA for HTLV-1 and Tax mRNA were detected in both lines. As part of the characterization of these cells, an enhanced expression of intercellular adhesion molecule-1 (ICAM-1) (CD54) or ICAM-3 (ICAM-3) (CD50), lymphocyte function-1 (LFA-1) (CD11a/CD18), and Mac-1 (CD11b/CD18) was observed. To investigate the role of the interaction of these viruses, we transfected EBV and/or HTLV-1 into a healthy donors lymphocytes, an EBV-infected B cell line, Raji, and a HTLV-1 negative T-cell line, Jurkat. Enhanced expression of adhesion molecules was also observed in double transfectants (EBV and HTLV-1). In the clinical course of ATL, LMP-1, EBNA-2, CD50 and CD54 were detected in lymph nodes and skin specimens by immunohistochemical staining. Furthermore, high levels of interleukin-4 (IL-4) were detected in these cell lines and transfectants. The results indicated that coinfection with HTLV-1 and EBV may induce aggressive organ involvement through the enhanced expression of adhesion molecules via IL-4 signaling. A new mechanism of ATL involvement is discussed.

Successful anticoagulant therapy for two pregnant PNH patients, and prospects for the eculizumab era

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem cell disorder characterized by intravascular hemolysis and thrombosis. The most serious complication is thrombosis, the risk of which is augmented by the hyper-coagulable state that occurs during pregnancy; despite this risk, however, young female PNH patients often desire to have a baby. We recently experienced two successful deliveries in PNH patients, who were treated with anticoagulant therapy during their pregnancies. Meanwhile, given the potential benefit of eculizumab (Soliris), a humanized monoclonal antibody against C5, in reducing thrombosis and hemolysis, it represents a promising therapeutic option for the treatment of pregnant PNH patients in combination with, or in replacement of, anticoagulant therapy.

Senescence induction therapy for the treatment of adult T-cell leukemia.

Retinoids (all-trans retinoic acid [ATRA] and tamibarotene [Am-80]) have been reported to inhibit the in vitro growth of human T-lymphotrophic virus type 1 (HTLV-I) (þ) T-cell lines and that of fresh cells obtained from patients with adult T-cell leukemia (ATL) [1,2]. The clinical efficacy of retinoid therapy in treating ATL has also been evaluated [3]. In this study, we focused on the role of retinoids in inducing cellular senescence during the treatment of ATL. Cellular senescence was detected by staining for senescence-associated bgalactosidase (SA b-Gal) [4]. SA b-Gal-positive cells were observed during spontaneous culture without retinoids (ATRA or Am-80) in HTLV-I (þ) T-cell lines (HUT102, MT-2, MT-4, ED40515, and ATL-2), but not in HTLV-I (7) T-cell lines (Jurkat and MOLT-4) [Figure 1(A)]. On treatment with ATRA or Am-80, the number of SA b-Gal-positive cells significantly increased in the HTLV-I (þ) T-cell lines, but not in the HTLV-I (7) ones [Figure 1(A)]. The same trend was observed in fresh ATL cells obtained from three patients with acute ATL [Figure 1(B)]. p16 expression was enhanced in all HTLV-I (þ) T-cell lines, but not in the HTLV-I (7) T-cell lines (Figure 2). A telomeric repeat amplification protocol (TRAP) assay (TRAPEZE Telomerase detection kit; Chemicon, Temecula, CA) revealed that telomerase activity was not inhibited in retinoid-treated HTLV-I (þ) T-cell lines; this indicated premature senescence (data not shown). We observed cellular senescence in HTLV-I (þ) T-cell lines and in fresh primary cells obtained from patients with acute ATL. The grade of cellular senescence was greater for HUT102, MT-2, MT-4, and ATL-2 cells than for ED40515 cells, which do not express Tax mRNA because of a nonsense mutation. This is an additional report pointing to Tax as an oncogene, and oncogene induced senescence (OIS) was possibly initiated. It has been reported that OIS does indeed occur in human and mouse tumor cells in vivo [5,6]. These cells cannot re-enter the cell cycle or undergo tumorigenesis once senescence is triggered [5,7]. OIS is caused by the accumulation of DNA damage. This DNA damage is, in turn, caused by oncogene-driven accumulation of reactive oxygen species (ROS) [8]. Chemotherapy using antineoplastic agents that decrease OIS and reduce cellular senescence may rejuvenate these cells and finally induce chemotherapy resistance. In conclusion, retinoids may be a reasonable agent for ATL by facilitating cellular senescence.