Akihiko Gotoh

The function of BCR/ABL and related proto-oncogenes

The BCR/ABL oncogene product is one of the genes associated with and possibly responsible for human leukemias. Chronic myelogenous leukemia and Philadelphia chromosome‐positive acute lymphoblastic leukemia are associated with p210BCR/ABL and p185BCR/ABL, respectively. Abelson murine leukemia virus encodes the related oncogene product, v‐Abl, and also causes pre‐B‐cell leukemia. In this article, recent advances in understanding the function of these oncogenes as well as the function of normal counterparts, c‐Abl and Bcr, are discussed. Intracellular signaling events initiated from these oncogene products are emphasized. The possibilities are also discussed that inhibition of apoptosis and altered adhesive properties to the bone marrow microenvironment by BCR/ABL might contribute to disease initiation.

Can eradication therapy for Helicobacter pylori really improve the thrombocytopenia in idiopathic thrombocytopenic purpura? Our experience and a literature review

Helicobacter pylori has recently been postulated to play a role in the pathogenesis of autoimmune diseases, including idiopathic thrombocytopenic purpura (ITP). We investigated the prevalence ofH pylori infection and the effects of its eradication in 61 patients with ITP.H pylori infection was found in 50 patients (83%), an incidence significantly higher than not only healthy volunteers in Japan (60%) but also subjects in other reported ITP series (approximately 43%–71%). In our study, the mean age ofH pylori-positive ITP patients (58.0 years) was significantly higher than that ofH pylori-negative ITP patients (40.5 years). Bacterium eradication efforts were performed in 29 infected ITP patients and succeeded in 27 patients (93%). The 29 patients with eradicatedH pylori infections showed significant increases in platelet counts compared with patients with uneradicated infections or who wereH pylori-negative. During the follow-up period (median, 11.0 months), 16 (55%) of 29 patients achieved a major or a minor response. The patients who achieved a major response had not received previous prednisolone therapy, suggesting a relationship between prednisolone therapy and the response to eradication efforts.The assessment ofH pylori infection and its eradication should be attempted in cases of ITP, because this approach may be a good new strategy for treating some ITP patients, especially elderly Japanese patients. Some regional factors have been suggested as causes ofH pylori-associated ITP.

Macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress-mediated CHOP induction in myeloma cells

The specific 26S proteasome inhibitor bortezomib (BZ) potently induces autophagy, endoplasmic reticulum (ER) stress and apoptosis in multiple myeloma (MM) cell lines (U266, IM-9 and RPMI8226). The macrolide antibiotics including concanamycin A, erythromycin (EM), clarithromycin (CAM) and azithromycin (AZM) all blocked autophagy flux, as assessed by intracellular accumulation of LC3B-II and p62. Combined treatment of BZ and CAM or AZM enhanced cytotoxicity in MM cell lines, although treatment with either CAM or AZM alone exhibited almost no cytotoxicity. This combination also substantially enhanced aggresome formation, intracellular ubiquitinated proteins and induced the proapoptotic transcription factor CHOP (CADD153). Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (BIM, BAX, DR5 and TRB3) were all enhanced by combined treatment with BZ plus CAM, compared with treatment with each reagent alone. Like the MM cell lines, the CHOP+/+ murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and upregulation of CHOP and its transcriptional targets with a combination of BZ and one of the macrolides. In contrast, CHOP−/− MEF cells exhibited resistance against BZ and almost completely canceled enhanced cytotoxicity with a combination of BZ and a macrolide. These data suggest that ER stress-mediated CHOP induction is involved in pronounced cytotoxicity. Simultaneously targeting two major intracellular protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances ER stress-mediated apoptosis in MM cells. This result suggests the therapeutic possibility of using a macrolide antibiotic with a proteasome inhibitor for MM therapy.

SDF‐1 suppresses cytokine‐induced adhesion of human haemopoietic progenitor cells to immobilized fibronectin

The process of haemopoietic cell homing to the microenviroment includes migration and adhesion. SDF‐1 is a C‐X‐C chemokine that acts as a chemoattractant for haemopoietic progenitors. Adhesion of haemopoietic progenitors to immobilized fibronectin is up‐regulated by stimulation with cytokines. We assessed the effects of SDF‐1 on cytokine‐induced adhesion of progenitor cells to fibronectin. In factor‐dependent human MO7e cells and primary CD34+ cord blood cells, treatment with SDF‐1 dose‐dependently suppressed cytokine‐induced adhesion. Pretreatment with pertussis toxin reversed adhesion‐inhibition, suggesting that activation of G‐coupled proteins are involved in the intracellular signalling of this process. These data suggest that SDF‐1 not only acts as a chemoattractant but also participates in modulating adhesiveness of haemopoietic progenitors to extracellular matrix components.

Targeting the integrated networks of aggresome formation, proteasome, and autophagy potentiates ER stress-mediated cell death in multiple myeloma cells

The inhibitory effects of macrolide antibiotics including clarithromycin (CAM) on autophagy flux have been reported. Although a macrolide antibiotic exhibits no cytotoxicity, its combination with bortezomib (BZ), a proteasome inhibitor, for the simultaneous blocking of the ubiquitin (Ub)-proteasome and autophagy-lysosome pathways leads to enhanced multiple myeloma (MM) cell apoptosis induction via stress overloading of the endoplasmic reticulum (ER). As misfolded protein cargo is recruited by histone deacetylase 6 (HDAC6) to dynein motors for aggresome transport, serving to sequester misfolded proteins, we further investigated the cellular effects of targeting proteolytic pathways and aggresome formation concomitantly in MM cells. Pronounced apoptosis was induced by the combination of vorinostat [suberoylanilide hydroxamic acid (SAHA); potently inhibits HDAC6] with CAM and BZ compared with each reagent or a 2-reagent combination. CAM/BZ treatment induced vimentin positive-aggresome formation along with the accumulation of autolysosomes in the perinuclear region, whereas they were inhibited in the presence of SAHA. The SAHA/CAM/BZ combination treatment maximally upregulated genes related to ER stress including C/EBP homologous protein (CHOP). Similarly to MM cell lines, enhanced cytotoxicity with CHOP upregulation following SAHA/CAM/BZ treatment was shown by a wild-type murine embryonic fibroblast (MEF) cell line; however, a CHOP-deficient MEF cell line almost completely canceled this pronounced cytotoxicity. Knockdown of HDAC6 with siRNA exhibited further enhanced CAM/BZ-induced cytotoxicity and CHOP induction along with the cancellation of aggresome formation. Targeting the integrated networks of aggresome, proteasome, and autophagy is suggested to induce efficient ER stress-mediated apoptosis in MM cells.

Thrombocytopenia induced by imatinib mesylate (Glivec) in patients with chronic myelogenous leukemia: is 400 mg daily of imatinib mesylate an optimal starting dose for Japanese patients?

Imatinib mesylate (Glivec) is a potent and selective inhibitor of the tyrosine kinase activity of BCR-ABL. Because the mechanism of action of imatinib mesylate is inhibition of BCR-ABL tyrosine kinase, it seems likely that to achieve maximum therapeutic benefit for chronic myelogenous leukemia (CML), imatinib mesylate must be administered at a dose that maximally inhibits BCR-ABL kinase activity or, alternatively, that inhibits sufficient BCR-ABL kinase activity to induce apoptosis. According to in vitro study results, 1 M levels of imatinib mesylate are optimal for inducing cell death, and 1 M trough levels are achieved in patients using imatinib mesylate at a daily dose of more than 300 mg [1]. In a phase I/II study in which the dose-response curve in chronic-phase CML patients was examined, the therapeutic effect was reported to plateau at a dose at or slightly above 300 mg. In addition, there was no convincing evidence of dose-limiting toxicity in patients receiving a maximum dose of 1000 mg [2]. These data were used to set the standard therapeutic dose of imatinib mesylate for chronicphase patients at 400 mg daily, and thereafter, the therapeutic benefits for CML patients of imatinib mesylate at this dose were demonstrated in clinical trials [2,3]. This standard therapeutic dose has been used in administering imatinib mesylate to Japanese patients as well as patients in the United States and Europe. At our institute, however, a series of CML patients treated with 400 mg of imatinib mesylate showed thrombocytopenia, and imatinib mesylate adminisThrombocytopenia Induced by Imatinib Mesylate (Glivec) in Patients with Chronic Myelogenous Leukemia: Is 400 mg Daily of Imatinib Mesylate an Optimal Starting Dose for Japanese Patients?

Transfusion-transmitted hepatitis E in a patient with myelodysplastic syndromes

Patients with haematological diseases occasionally exhibit liver dysfunction during treatment. This liver dysfunction can have various causes such as therapy-related drugs and hepatitis B and C infections, although the cause is unclear in some cases. It was recently reported that some patients initially diagnosed with drug-induced liver dysfunction actually had hepatitis E1. Several cases of transfusion-transmitted hepatitis E infections have also been reported1,2. In Japan, screening for hepatitis E does not appear to be performed at the initial examination of patients with acute hepatitis. This might be because hepatitis E is believed to be orally transmitted and to occur mainly in developing countries and rarely in developed countries. However, hepatitis E is a zoonotic infectious disease. Cases of regional endemic hepatitis E virus (HEV) infection have been increasing in Europe, the United States, and Japan1. Although HEV usually causes self-limited acute hepatitis, it sometimes progresses to a chronic infection. Most cases of chronic infection occur in patients undergoing solid organ or haematopoietic stem cell transplantation, in those receiving anti-cancer or immunosuppressant drugs, and in patients with human immunodeficiency virus infection, in whom the condition may progress to liver cirrhosis3. HEV RNA persisted for a long period during treatment in a patient with T-cell lymphoma4. Reactivation of HEV hepatitis was reported after an allogeneic haematopoietic stem cell transplant in a patient with Philadelphia chromosome-positive acute lymphoblastic leukaemia5. On the other hand, a low risk of HEV reactivation after haematopoietic stem cell transplantation was also reported6. More studies on the risk of HEV reactivation are, therefore, required. Here, we report the case of a patient with a myelodysplastic syndrome (MDS) who developed acute hepatitis due to transfusion-transmitted HEV infection. We also review the literature on the topic.

Leukocytosis is linked to thrombosis at diagnosis, while JAK2 V617F mutation is associated with thrombosis during the course of essential thrombocythemia

Mutation of JAK2 V617F results in constitutive activation of the kinase that is found in approximately half of patients with essential thrombocythemia (ET) [1, 2]. It is still controversial whether JAK2 V617F mutation is linked to thrombosis in ET patients. We and others reported the positive correlation between the presence of JAK2 V617F mutation and thrombosis [3, 4], while another report indicated that a high number of leukocytes in JAK2 V617Fpositive ET might be linked to thrombosis and that the JAK2 V617F mutational status is independently related to thrombosis [5, 6]. The vast majority of patients with polycythemia vera (PV) carry the JAK2 V617F mutation [2], however the presence of ‘‘idiopathic erythrocytosis’’ suggested that either JAK2 V617F or other JAK2 mutations may be important molecular changes in PV [7]. This prompted us to evaluate the clinical implications of ET with isolated thrombocytosis. Since JAK2 V617F-negative ET patients show significantly low leukocyte and hemoglobin levels [1, 3], there is no significant difference in platelet counts, indicating that ET patients might also constitute hematologically and biologically heterogeneous groups. We studied JAK2 V617F in 54 patients with essential ET, including 49 patients reported previously [3]. For the diagnosis of ET, we used the criteria proposed by the PV study group (PVSG) for ET (platelet count of[600 9 10/ L). We collected frozen marrow cells or peripheral blood (100 lL) from patients after obtaining their informed consent, and determined JAK2 V617F status using the sequence-specific primer-single molecule fluorescence detection (SSP-SMFD) assay [3, 4]. All treated patients, except 2 patients treated with alpha-interferon, received hydroxyurea. We first separated ET patients according to whether they had JAK2 V617F mutation or not. Patients with JAK2 V617F (n = 33) showed a significantly higher leukocyte count (P = 0.0054), hemoglobin level (P = 0.0037), and hematocrit (P = 0.002), than those without JAK2 V617F (Table 1). They tended to have high frequencies of thrombosis (P = 0.0734): of note is that all ET patients developing thrombosis during their follow-up period had JAK2 V617F (6/33 vs. 0/21: P = 0.0382), while there was no significant difference in thrombotic episodes at ET diagnosis between patients with JAK2 V617F and those without JAK2 V617F (P = 0.7672). Evolution of PV was seen only in patients with JAK2 V617F (4/33 vs. 0/21: P = 0.0973). We next separated ET patients into those with a leukocyte count of 12,000 9 10/L or more and those with less (Table 2). ET patients with hyper-leukocytosis had a significantly high incidence of JAK2 V617F (P = 0.0224). They were older (P = 0.0214) and had higher platelet counts compared to those with \12,000 9 10/L leukocytes at the ET diagnosis. ET with hyper-leukocytosis had a significantly higher frequency of thrombosis throughout their courses (7/15 vs. 5/39: P = 0.0074) or at the time of ET diagnosis (4/15 vs. 2/39: P = 0.0241), and development of PV (P = 0.0008) (Table 2). These data may suggest that hyper-leukocytosis is linked to thrombosis at ET diagnosis and the presence of JAK2 V617F may influence the development of thrombosis during their follow-up period. K. Ohyashiki (&) T. Kiguchi Y. Ito H. Fujimoto A. Gotoh T. Tauchi K. Miyazawa Y. Kimura Hematology Division, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo 169-0023, Japan e-mail: ohyashik@rr.iij4u.or.jp

Hematopoietic Colony Formation from Human Growth Factor-Dependent TF1 Cells and Human Cord Blood Myeloid Progenitor Cells Depends on SHP2 Phosphatase Function

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity.

Clinical Efficacy and Safety of First-Line Dasatinib Therapy and the Relevance of Velocity of BCR-ABL1 Transcript Decline for Achievement of Molecular Responses in Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia: Report from the Juntendo Yamanashi Cooperative Study Group

Objective: The use of tyrosine kinase inhibitors led to an improvement in the prognoses of patients with chronic myeloid leukemia (CML). The aims of this study were to investigate the efficacy and safety of dasatinib in Japanese patients and to explore the factors that affect the achievement of molecular responses. Methods: The primary endpoint was a major molecular response (MMR) by 12 months. The halving time for BCR-ABL1 transcripts was calculated using transcript levels. Results: Thirty-two patients with chronic-phase CML (CML-CP) were enrolled and 30 received 100 mg dasatinib once daily. At 24 months of follow-up, 21 (72%) and 24 (83%) patients achieved an MMR by 12 and 24 months, respectively; the rates of a deep molecular response (DMR) by 12 and 24 months were 48 and 59%, respectively. A shorter halving time of BCR-ABL1 transcripts (≤10.6 days) accurately predicted both an MMR and a DMR. The incidence of pleural effusion was 50%. Our study reconfirmed the efficacy and safety of dasatinib treatment in Japanese patients with newly diagnosed CML-CP. In addition, the usefulness of the halving time of BCR-ABL1 transcripts was validated. Conclusion: These data emphasize the significance of an early treatment response in achieving a DMR during dasatinib therapy.