Osamu Miura

BCR/ABL and IL-3 activate Rap1 to stimulate the B-Raf/MEK/Erk and Akt signaling pathways and to regulate proliferation, apoptosis, and adhesion.

The Ras family small GTPase Rap1 is activated by hematopoietic cytokines, such as interleukin (IL)-3, to induce β1 integrin-mediated cell adhesion or by the BCR/ABL fusion tyrosine kinase to stimulate the MEK/Erk signaling pathway. Here, we demonstrate that the abrogation of Rap1 activation by SPA-1, a Rap1-specific GAP, inhibits activation of B-Raf, MEK, Erk, and Akt in a murine hematopoietic cell line, Ton.B210, stimulated with IL-3 or inducibly expressing BCR/ABL. Furthermore, Rap1 inactivation had an inhibitory effects on proliferation and survival of Ton.B210 cells, which were more remarkable when cells were stimulated by BCR/ABL than by IL-3. Induction of BCR/ABL expression increased adhesion of Ton.B210 cells to fibronectin in a manner at least partly dependent on its kinase activity, and Rap1 inhibition by SPA-1 partially inhibited BCR/ABL-induced adhesion of cells. Thus, IL-3- or BCR/ABL-induced activation of Rap1 may play important roles in regulation of cell proliferation and survival through activation of the B-Raf/MEK/Erk and Akt signaling pathways and in induction of integrin-mediated cell adhesion. Furthermore, as compared with IL-3, BCR/ABL is more dependent on Rap1-mediated signaling to induce cell proliferation and survival and, thus, Rap1 may represent an attractive target for novel therapies for leukemias caused by BCR/ABL.

Novel mouse xenograft models reveal a critical role of CD4+ T cells in the proliferation of EBV-infected T and NK cells.

Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, ectopically infects T or NK cells to cause severe diseases of unknown pathogenesis, including chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). We developed xenograft models of CAEBV and EBV-HLH by transplanting patients PBMC to immunodeficient mice of the NOD/Shi-scid/IL-2Rγnull strain. In these models, EBV-infected T, NK, or B cells proliferated systemically and reproduced histological characteristics of the two diseases. Analysis of the TCR repertoire expression revealed that identical predominant EBV-infected T-cell clones proliferated in patients and corresponding mice transplanted with their PBMC. Expression of the EBV nuclear antigen 1 (EBNA1), the latent membrane protein 1 (LMP1), and LMP2, but not EBNA2, in the engrafted cells is consistent with the latency II program of EBV gene expression known in CAEBV. High levels of human cytokines, including IL-8, IFN-γ, and RANTES, were detected in the peripheral blood of the model mice, mirroring hypercytokinemia characteristic to both CAEBV and EBV-HLH. Transplantation of individual immunophenotypic subsets isolated from patients PBMC as well as that of various combinations of these subsets revealed a critical role of CD4+ T cells in the engraftment of EBV-infected T and NK cells. In accordance with this finding, in vivo depletion of CD4+ T cells by the administration of the OKT4 antibody following transplantation of PBMC prevented the engraftment of EBV-infected T and NK cells. This is the first report of animal models of CAEBV and EBV-HLH that are expected to be useful tools in the development of novel therapeutic strategies for the treatment of the diseases.

Sorafenib induces apoptosis specifically in cells expressing BCR/ABL by inhibiting its kinase activity to activate the intrinsic mitochondrial pathway.

Although the BCR/ABL tyrosine kinase inhibitor imatinib is highly effective for treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia, relapse with emerging imatinib resistance mutations in the BCR/ABL kinase domain poses a significant problem. Here, we show that the multikinase inhibitor sorafenib inhibits proliferation and induces apoptosis at much lower concentrations in Ton.B210 cells when driven by inducibly expressed BCR/ABL than when driven by interleukin-3. The increased sensitivity to sorafenib was also observed in cells inducibly expressing BCR/ABL with the imatinib-resistant E255K or T315I mutation. Sorafenib-induced apoptosis in these cells and Ph+ leukemic cells was synergistically enhanced by rottlerin, bortezomib, or ABT-737 and inhibited by the pan-caspase inhibitor BOC-d-fmk or the overexpression of Bcl-XL. It was further revealed that sorafenib activates Bax and caspase-3 and reduces mitochondrial membrane potential specifically in BCR/ABL-driven cells. Sorafenib also inhibited BCR/ABL-induced tyrosine phosphorylation of its cellular substrates and its autophosphorylation in Ton.B210. It was finally shown that sorafenib inhibits the kinase activity of BCR/ABL as well as its E255K and T315I mutants in in vitro kinase assays. These results indicate that sorafenib induces apoptosis of BCR/ABL-expressing cells, at least partly, by inhibiting BCR/ABL to activate the mitochondria-mediated apoptotic pathway. Thus, sorafenib may provide an effective therapeutic measure to treat Ph+ leukemias, particularly those expressing the T315I mutant, which is totally resistant to imatinib and the second generation BCR/ABL inhibitors.

Enhancement of imatinib-induced apoptosis of BCR/ABL-expressing cells by nutlin-3 through synergistic activation of the mitochondrial apoptotic pathway

The BCR/ABL tyrosine kinase inhibitor imatinib is highly effective for treatment of chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). However, relapses with emerging imatinib-resistance mutations in the BCR/ABL kinase domain pose a significant problem. Here, we demonstrate that nutlin-3, an inhibitor of Mdm2, inhibits proliferation and induces apoptosis more effectively in BCR/ABL-driven Ton.B210 cells than in those driven by IL-3. Moreover, nutlin-3 drastically enhanced imatinib-induced apoptosis in a p53-dependent manner in various BCR/ABL-expressing cells, which included primary leukemic cells from patients with CML blast crisis or Ph+xa0ALL and cells expressing the imatinib-resistant E255K BCR/ABL mutant. Nutlin-3 and imatinib synergistically induced Bax activation, mitochondrial membrane depolarization, and caspase-3 cleavage leading to caspase-dependent apoptosis, which was inhibited by overexpression of Bcl-XL. Imatinib did not significantly affect the nutlin-3-induced expression of p53 but abrogated that of p21. Furthermore, activation of Bax as well as caspase-3 induced by combined treatment with imatinib and nutlin-3 was observed preferentially in cells expressing p21 at reduced levels. The present study indicates that combined treatment with nutlin-3 and imatinib activates p53 without inducing p21 and synergistically activates Bax-mediated intrinsic mitochondrial pathway to induce apoptosis in BCR/ABL-expressing cells.

Clinical features of adult-onset chronic active Epstein–Barr virus infection: a retrospective analysis

We performed a retrospective analysis of patients with adult-onset chronic active Epstein–Barr virus infection (CAEBV). First, we analyzed five patients (aged 28–72) diagnosed at our hospitals with EBV-infected clonally proliferating T cells. Four patients were administered cyclophosphamide/doxorubicin/vincristine/prednisone (CHOP) chemotherapy, but no remarkable decrease of viral load was observed in three of the patients. The other patient died 19 days after initiation of CHOP treatment due to disease progression. Addition of high-dose cytarabine to the regimens of two of the patients was discontinued shortly after administration, due to the development of grade 4 pericardial effusion. Together, these regimens may be insufficient for treating adult-onset CAEBV. We next reviewed 23 adult-onset CAEBV patients, adding 18 previously reported patients to the five patients described in the present study. T cells were frequently infected (87%), whereas NK- and T-cell types are known to be almost equally prevalent in childhood-onset cases. The time duration from the onset of disease to initiation of treatment averaged 20 months. Reports showed that 12 patients died; seven patients died at an average of 8 months after initiation of treatment. Patients’ disease courses seemed to be rapidly progressive and more aggressive than those of childhood-onset cases. More cases must be studied to clarify clinical features and establish an optimal treatment strategy.

Role for protein geranylgeranylation in adult T-cell leukemia cell survival

Adult T-cell leukemia (ATL) is a fatal lymphoproliferative disease that develops in human T-cell leukemia virus type I (HTLV-I)-infected individuals. Despite the accumulating knowledge of the molecular biology of HTLV-I-infected cells, effective therapeutic strategies remain to be established. Recent reports showed that the hydroxyl-3-methylglutaryl (HMG)-CoA reductase inhibitor statins have anti-proliferative and apoptotic effects on certain tumor cells through inhibition of protein prenylation. Here, we report that statins hinder the survival of ATL cells and induce apoptotic cell death. Inhibition of protein geranylgeranylation is responsible for these effects, since simultaneous treatment with isoprenoid precursors, geranylgeranyl pyrophosphate or farnesyl pyrophosphate, but not a cholesterol precursor squalene, restored the viability of ATL cells. Simvastatin inhibited geranylgeranylation of small GTPases Rab5B and Rac1 in ATL cells, and a geranylgeranyl transferase inhibitor GGTI-298 reduced ATL cell viability more efficiently than a farnesyl transferase inhibitor FTI-277. These results not only unveil an important role for protein geranylgeranylation in ATL cell survival, but also implicate therapeutic potentials of statins in the treatment of ATL.

Inhibition of the PI3K/Akt/GSK3 Pathway Downstream of BCR/ABL, Jak2-V617F, or FLT3-ITD Downregulates DNA Damage-Induced Chk1 Activation as Well as G2/M Arrest and Prominently Enhances Induction of Apoptosis

Constitutively-activated tyrosine kinase mutants, such as BCR/ABL, FLT3-ITD, and Jak2-V617F, play important roles in pathogenesis of hematopoietic malignancies and in acquisition of therapy resistance. We previously found that hematopoietic cytokines enhance activation of the checkpoint kinase Chk1 in DNA-damaged hematopoietic cells by inactivating GSK3 through the PI3K/Akt signaling pathway to inhibit apoptosis. Here we examine the possibility that the kinase mutants may also protect DNA-damaged cells by enhancing Chk1 activation. In cells expressing BCR/ABL, FLT3-ITD, or Jak2-V617F, etoposide induced a sustained activation of Chk1, thus leading to the G2/M arrest of cells. Inhibition of these kinases by their inhibitors, imatinib, sorafenib, or JakI-1, significantly abbreviated Chk1 activation, and drastically enhanced apoptosis induced by etoposide. The PI3K inhibitor GD-0941 or the Akt inhibitor MK-2206 showed similar effects with imatinib on etoposide-treated BCR/ABL-expressing cells, including those expressing the imatinib-resistant T315I mutant, while expression of the constitutively activated Akt1-myr mutant conferred resistance to the combined treatment of etoposide and imatinib. GSK3 inhibitors, including LiCl and SB216763, restored the sustained Chk1 activation and mitigated apoptosis in cells treated with etoposide and the inhibitors for aberrant kinases, PI3K, or Akt. These observations raise a possilibity that the aberrant kinases BCR/ABL, FLT3-ITD, and Jak2-V617F may prevent apoptosis induced by DNA-damaging chemotherapeutics, at least partly through enhancement of the Chk1-mediated G2/M checkpoint activation, by inactivating GSK3 through the PI3K/Akt signaling pathway. These results shed light on the molecular mechanisms for chemoresistance of hematological malignancies and provide a rationale for the combined treatment with chemotherapy and the tyrosine kinase or PI3K/Akt pathway inhibitors against these diseases.

Clonal evolution with double Ph followed by tetraploidy in imatinib-treated chronic myeloid leukemia with e19a2 transcript in transformation

Chronic myeloid leukemia (CML) with the e19a2 transcript coding for p230 is typically associated with a benign clinical course unless accompanied at presentation with additional chromosomal abnormalities. We report here a case of CML with e19a2 who did not show additional chromosomal abnormalities at diagnosis, but progressed to the fatal advanced stage in approximately 2 years. The patient was initially treated with imatinib, which, however, could be administered only intermittently at reduced doses because of recurrent thrombocytopenia and fluid retention. Nine months after starting imatinib, fluorescence in situ hybridization (FISH) with the BCR/ABL-ES fusion probe revealed 96% and 3% of bone marrow cells with one and two BCR/ABL1 fusion signals, respectively. Two years after starting therapy, leukocytosis recurred and the bone marrow contained 8.2% large and bizarre myeloblasts. Cytogenetic analysis revealed double Ph clones as well as tetraploid cells with four to five Ph chromosomes. FISH analysis confirmed the presence of cells with two to five BCR/ABL1 fusion signals. The patient died of disease progression in 2 months. No point mutation was detected in the region coding for the BCR/ABL tyrosine kinase domain by sequence analysis. It is speculated that the amplification of the BCR/ABL1 fusion gene by duplication of Ph and tetraploidy led to the progression of CML with the e19a2 transcript.

Proliferation and survival signaling from both Jak2-V617F and Lyn involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 cell line newly established from acute myeloid leukemia transformed from polycythemia vera.

The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),−16,+21,−22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.

Concurrent administration of intravenous systemic and intravitreal methotrexate for intraocular lymphoma with central nervous system involvement.

Intraocular lymphoma (IOL) is rare lymphoma that frequently infiltrates the central nervous system (CNS). An optimal treatment has not been established, and its prognosis is quite poor. We treated three IOL patients with CNS involvement by concurrent administration of intravenous and intravitreal methotrexate (MTX) injection. The intraocular lesion responded in all patients. One patient achieved complete response (CR), whereas the other 2 patients were in partial response for CNS lesion, added whole brain radiation and achieved CR. In 3 eyes of 2 patients, an intravitreal MTX injection (vMTX) was administered 2xa0h after a systemic MTX injection (sMTX) and the intravitreal MTX concentration was measured twice: 2xa0h after sMTX and 24xa0h after vMTX. The half-life of MTX in the vitreous fluid was estimated to be 12.4–21.5xa0h by assuming the first-order elimination kinetics. Although the concentration was still high 24xa0h after vMTX (69.94–82.89xa0μM), there were no ocular complications. The serum MTX concentration was not influenced by adding vMTX to sMTX. Grade 3 adverse event, leukocytopenia, was observed in only 1 patient. No grade 4 event was observed. Although further evaluation is required, concurrent sMTX and vMTX may be effective for IOL with CNS involvement.