Mio Yamamoto

Galectin-9 exhibits anti-myeloma activity through JNK and p38 MAP kinase pathways

Galectins constitute a family of lectins that specifically exhibit the affinity for β-galactosides and modulate various biological events. Galectin-9 is a tandem-repeat type galectin with two carbohydrate recognition domains and has recently been shown to have an anti-proliferative effect on cancer cells. We investigated the effect of recombinant protease-resistant galectin-9 (hGal9) on multiple myeloma (MM). In vitro, hGal9 inhibited the cell proliferation of five myeloma cell lines examined, including a bortezomib-resistant subcell line, with IC50 between 75.1 and 280.0 nM, and this effect was mediated by the induction of apoptosis with the activation of caspase-8, -9, and -3. hGal9-activated Jun NH2-terminal kinase (JNK) and p38 MAPK signaling pathways followed by H2AX phosphorylation. Importantly, the inhibition of either JNK or p38 MAPK partly inhibited the anti-proliferative effect of hGal9, indicating the crucial role of these pathways in the anti-MM effect of hGal9. hGal9 also induced cell death in patient-derived myeloma cells, some with poor-risk factors, such as chromosomal deletion of 13q or translocation t(4;14)(p16;q32). Finally, hGal9 potently inhibited the growth of human myeloma cells xenografted in nude mice. These suggest that hGal9 is a new therapeutic target for MM that may overcome resistance to conventional chemotherapy.

Targeting Activating Transcription Factor 3 by Galectin-9 Induces Apoptosis and Overcomes Various Types of Treatment Resistance in Chronic Myelogenous Leukemia

Tyrosine kinase inhibitors (TKI) against Bcr-Abl are the first-line therapeutics for chronic myelogenous leukemia (CML). However, the resistance to Bcr-Abl TKIs is induced in leukemic cells not only by loss of sensitivity to TKIs through Bcr-Abl–related molecular mechanisms but also by loss of addiction to Bcr-Abl TK activity by acquiring Bcr-Abl–unrelated additional oncogenic mutations. Therefore, the identification of an additional therapeutic target has been anticipated for achievement of a complete cure and to overcome resistance to treatment. We here showed that modified human Galectin-9 (hGal9), a lectin that show specific affinity for β-galactosides, inhibits the proliferation of five CML-derived cell lines by inducing apoptosis at their IC50s from 17.5 to 164.9 nmol/L. Our study revealed that activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding protein family transcription factors, is the critical mediator for cell killing by hGal9, and that Noxa is one of the downstream effector molecules of ATF3. Bim, on the other hand, the BH3-only protein essential for apoptosis by Bcr-Abl TKIs, was not associated with hGal9-induced cell death. ATF3-mediated cell death by hGal9 was not hampered by the absence of p53, the presence of mutant AblT315I, or by P-glycoprotein overexpression. In addition, hGal9 showed the additive growth-inhibitory effect with imatinib on CML cell lines. Collectively, hGal9 is a candidate agent that may overcome various kinds of resistance to treatment for CML and may suggest that ATF3 may be a new target molecule for the development of new treatment modalities that can overcome resistance to currently available chemotherapeutics. Mol Cancer Res; 8(7); 994–1001. ©2010 AACR.

Bcl-2 is a better therapeutic target than c-Myc, but attacking both could be a more effective treatment strategy for B-cell lymphoma with concurrent Bcl-2 and c-Myc overexpression

OBJECTIVE The prognosis for diffuse large B-cell lymphomas with concomitant overexpression of c-Myc and Bcl-2 remains dismal; there is an urgent need to clarify the significance of these two oncogenes as therapeutic targets for a more effective treatment strategy. MATERIALS AND METHODS We established two novel cell lines, KPUM-MS3 and KPUM-UH1, from two chemoresistant patients with diffuse large B-cell lymphomas with concomitant overexpression of c-Myc and Bcl-2, and investigated the significance of c-Myc and Bcl-2 as therapeutic targets. RESULTS KPUM-MS3 possesses t(14;18)(q32;q21) chromosomal translocation and KPUM-UH1 bcl-2 gene amplification, both of which account for Bcl-2 overexpression. Chromosomal translocation t(8;14)(q24;q34) was found to coexist only in KPUM-UH1, overexpression of pvt-1 messenger RNA was detected only in KPUM-MS3, and reduced expression of miR-143 and miR-145 was identified in both. Working together, these abnormalities can contribute to c-Myc overexpression. Using ABT-263, an inhibitor for Bcl-2, and 10058-F4, an inhibitor for c-Myc, we found that both cell lines were more highly sensitive to cell death as a result of Bcl-2 inhibition than of c-Myc inhibition. When combined with genotoxic agents, ABT-263 exerted additive and/or synergistic cell-killing effects, while 10058-F4 showed, at most, a modest combinatory effect. Importantly, the combination of ABT-263 and 10058-F4 had a synergistic cell-killing effect on both cell lines. CONCLUSIONS Our data suggest that Bcl-2 is a better therapeutic target than c-Myc, but attacking both Bcl-2 and c-Myc would be an even more effective treatment strategy for diffuse large B-cell lymphomas with concurrent Bcl-2 and c-Myc overexpression.

Cyclosporine A for Chemotherapy-Resistant Subcutaneous Panniculitis-Like T Cell Lymphoma with Hemophagocytic Syndrome

Subcutaneous panniculitis-like T cell lymphoma (SPTL) is a rare subtype of non-Hodgkin lymphoma for which a definitive therapeutic strategy has not been established yet. We report a case of chemotherapy-resistant SPTL with hemophagocytic syndrome (HPS) which was successfully treated with cyclosporine A (CsA) plus methylprednisolone (mPSL), and also reviewed 11 SPTL cases treated with CsA, previously reported in the literature. Our patient was a 38-year-old female with SPTL. The disease progressed despite conventional chemotherapy using cytotoxic agents including alkylators, anthracyclins or purine analogues, and, after 2 months of chemotherapy, was eventually complicated by HPS and disseminated intravascular coagulation (DIC). CsA (4 mg/kg/day) plus mPSL treatment dramatically improved HPS with DIC, reduced subcutaneous tumors within 2 weeks, and finally induced complete remission (CR) after 3 months. Currently, the patient has maintained CR while being treated with CsA for 12 months. In addition to our case, 9 of 11 SPTL cases were successfully treated with CsA, and 8 were induced to CR. Time to first response to CsA was within 2 weeks in most cases, regardless of prior treatment or the co-occurrence of HPS. Our case and this first comprehensive review on CsA for SPTL suggest that CsA may constitute a candidate treatment strategy for SPTL.

Expression of Master Regulators of Helper T-Cell Differentiation in Peripheral T-Cell Lymphoma, Not Otherwise Specified, by Immunohistochemical Analysis

The normal counterparts of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) have not been accurately identified. We immunohistochemically analyzed 10 PTCL-NOS cases to examine the expression of the master regulators of T-cell differentiation and of surface antigens, including chemokine receptors. All cases were positive for the master regulator of helper T cells (Th-POK) and the marker of effector T cells (CD45RO). Three cases each were positive for T-Bet and GATA3, which are master regulators of helper T cells (T(H) ) type 1 (T(H)1) and 2 (T(H)2), respectively. Two cases were positive for the surface antigens of central memory (Tcm) (CCR7 and CD62L), and 1 case was positive for follicular helper T-cell (TFH) phenotype (BCL6, CXCL13, and PD-1). The remaining case was negative for all markers of effector T(H) subtypes. These results suggest the postulated normal counterparts of PTCL-NOS identified in 9 of the 10 cases consist of T(H)1, T(H)2, TCM, and TFH.

Bortezomib for post-allogeneic hematopoietic stem transplantation relapse and GVHD in multiple myeloma: a single institute experience.

Allogeneic hematopoietic stem cell transplantation (alloSCT) can result in remission and long-term survival for multiple myeloma (MM), but its effect has been often hampered by high rates of therapy-related death by graftversus-host diseases (GVHD), disease progression, or infection [1]. We retrospectively investigated the efficacy and safety of Bortezomib (Bor) alone or with dexamethasone (Dex) (BD) for MM patients with disease progression following allo-SCT. Six MM patients were treated with Bor alone or with BD as the salvage treatment for disease progression following allo-SCT, while 32 patients with relapsed/refractory MM without allo-SCT were also treated with BD at our institute between October 2003 and July 2009. The median age was 39.5 years old for the allo-SCT cohort and 67.5 for the non-allo-SCT cohort. None had prior exposure to Bor. In six post-allo-SCT patients, two underwent a conditioning regimen with high-dose cyclophosphamide (60 mg/kg) plus total body irradiation (10 Gy), while four were treated with a reduced intensity-conditioning regimen using fludarabine (90–125 mg/m) plus melphalan (140–200 mg/m) (Table 1). According to the International Myeloma Working Group response criteria, the best response for alloSCT was complete remission (CR) in one, very good partial response (VGPR) in two and stable disease (SD) in three patients. Bor (1.3, 1.0, or 0.7 mg/m/day) was administered on days 1, 4, 8 and 11, and Dex on days 1, 2, 4, 5, 8, 9, 11 and 12, every 21 days. The dosage of either agent was adjusted by the attending physician in the light of the patient’s condition. Among the six post-allo-SCT patients, one received Bor alone and five received BD, while all patients in the non-allo-SCT cohort received BD. The median number of cycles of Bor alone or BD was 3 (1–7 cycles) for the post-allo-SCT cohort and 2 for the non-allo-SCT cohort. The median interval between allo-SCT to Bor or BD was 12.5 months (6–22 months). All presented acute and chronic GVHD. Three patients received thalidomidecontaining therapy for post-allo-SCT disease progression prior to Bor (Table 2). For the six post-allo-SCT patients, the overall response rate (ORR) was 50.0%, including two VGPR and one PR, while two died of MM progression and one of bacterial pneumonia complicated with bronchiolitis obliterans, a late complication of allo-SCT. Although ORR for the allo-SCT cohort was inferior to that of the non-alloSCT cohort (68.8%), the median overall survival (OS) (912 days) and median progression-free survival (PFS) (71 days), 2-year OS (66.7%) and 2-year PFS (33.3%) from the start of Bor-containing treatment for the allo-SCT cohort did not seem significantly different from those for the non-allo-SCT cohort, although the statistical analysis was not applicable due to the small number of patients (Supplementary Fig. 1). Our findings thus seem to provide further supportive evidence that Bor or BD constitutes a promising salvage treatment even for heavily treated Electronic supplementary material The online version of this article (doi:10.1007/s12185-010-0709-3) contains supplementary material, which is available to authorized users.

Renal vein lymphoma embolism in non-Hodgkin lymphoma

This report is, to the best of our knowledge, the first description of a case of non-Hodgkin lymphoma (NHL) which was accompanied by a renal vein lymphoma embolism. A 49-year-old female was admitted to our hospital complaining of a right axillary tumor 10 cm in diameter. Biopsy disclosed that the tumor was diffusely infiltrated by abnormal immunoblastic lymphocytes, which were positive for LCA, L26, CD10 and CD79a, but negative for UCHL-1, CD3, CD5, and Ki-1. Serum levels of lactate dehydrogenase 823 IU/L (normal range 110–220) and soluble interleukin-2 receptor 6,600 U/mL (220–530) were elevated. Coagulation tests, such as prothrombin time, activated partial thromboplastin time or plasma level of fibrin degradation products, showed normal results. Systemic examination identified splenomegaly and lymph node (LN) swelling at the hepatic hilum. Bone marrow study findings were normal. According to the WHO classification and Ann Arbor criteria, the patient was diagnosed with NHL, diffuse large B cell-type (DLBCL), stage IIIAXs. Two months after the initiation of combination chemotherapy, enhanced computed tomography (CT) identified an embolus in her left renal vein as a low density lesion (Fig. 1a). We diagnosed the embolus as a thrombus, and initiated anticoagulation therapy. For 8 months, the disease was refractory to multiple courses of combination chemotherapies, including a regimen of rituximab plus CHOP therapy, after which the patient died of systemic disease dissemination. Autopsy was performed in accordance with the guidelines of Matsushita Memorial Hospital and with the informed consent of the patient’s family. Macroscopically, multiple nodular lesions were identified in the right infra-axillary lesion, para-aortic lesion, stomach, uterus, right ovary, skin, pleura and mesenterium. Microscopic examination showed that nodules were uniformly infiltrated by lymphoma cells which were positive for LCA, L26, CD10, CD79a and Bcl-2. Approximately 30% of the lymphoma cells were also positive for MIB-1. Of particular interest was that the large embolus in the left renal vein was found to be a whitish tumor partially covered by a thrombus (Fig. 1b). The tumor was microscopically confirmed as lymphoma involvement (Fig. 1c–e). Microscopic examination also showed lymphoma cell infiltration in the left renal cortex and fat tissue around the left renal vein (Fig. 1f). Lymphoma embolus was not identified in other veins. Venous tumor embolism is occasionally complicated with solid cancers, such as hepatocellular carcinoma invading the portal vein or renal cell carcinoma invading renal veins, and these cancers tend to occur in the more advanced, later clinical phase. They have an unfavorable effect on treatment outcome due to further tumor cell dissemination, organ damage by inadequate blood flow, or venous rupture. DLBCL has a well-established variant entity termed intravascular lymphoma, which is characterized by exclusive or predominant intravascular tumor growth in the lumen of small to intermediate-sized blood vessels. However, tumor embolism in large veins is extremely rare, and, according to our search results, only five NHL cases have been reported with venous tumor embolism, three in the portal vein [1–3], one in the superior vena cava [4], and one in the internal saphenous vein [5], but no reports have been published on M. Yamamoto T. Kobayashi N. Uoshima Department of Hematology, Matsushita Memorial Hospital, 5-55, Sotojima-cho, Moriguchi 570-8540, Japan

Human leukocyte antigen-DR negative de novo acute myeloid leukemia with Philadelphia chromosome.

It is well known that human leukocyte antigen (HLA)-DR is present in the majority of acute myeloid leukemia (AML) [1, 2]. However, acute promyelocytic leukemia (APL) with t(15;17)(q22;q12) usually lacks the HLA-DR expression as well as CD34 expression [2, 3]. Recently, such HLA-DR/CD34 phenotype has also been described in a subset of AML with cuplike nuclear invaginations (AML-cuplike) [4]. Cytogenetically or molecular genetically, AML-cuplike is characterized with normal karyotype and high frequency of FMS-like tyrosine kinase 3 (FLT3) gene internal tandem duplication (ITD) as well as nucleophosmin (NPM1) gene mutation [4, 5]. In this paper, we reported an extremely rare case of HLA-DR/CD34 AML, which was not categorized into either APL or AMLcuplike and showed Philadelphia chromosome (Ph) at diagnosis. A 64-year-old male was admitted to the hospital because of cellulitis of left leg in April 2008. He showed neither hepatosplenomegaly nor generalized lymph node swellings. There was no bleeding tendency. Laboratory findings were as follows: white blood cells 17.7 9 10/L, with 25% blasts and 20% basophils; hemoglobin, 14.0 g/dL; platelets, 129 9 10/L. Serum lactate dehydrogenase level and D-dimer was elevated to 386 IU/L and 50.2 lg/mL, respectively. Neutrophil alkaline phosphatase (NAP) score was within the normal range. Bone marrow showed hypercellularity with 39% blasts, 7% myelocytes, 4% metamyelocytes, 13% mature neutrophils, 1% eosinophils, 25% basophils, 8% erythroblasts and 3% lymphocytes. Morphologically, blasts were medium to large, and had round or sometimes slightly indented nuclei with dispersed chromatin. Fine azurophilic granules were sometimes found in their scant basophilic cytoplasm, but typical Auer-bodies were not detected (Fig. 1). They were cytochemically positive to myeloperoxidase (MPO) staining. Therefore, he was morphologically diagnosed as having AML-M2 according to the French–American–British (FAB) classification. There were no characteristic cuplike nuclear invaginations suggesting a diagnosis of AML-cuplike on either Wright–Giemsa or MPO staining [6]. Flow cytometric immunophenotyping using the CD45 gating method showed that leukemic blasts were CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD15, CD19, CD20, CD33, CD34, CD41, CD90, CD117 and HLA-DR, suggesting HLA-DR AML (Fig. 2). More than 85% of these cells also lacked the CD56 expression. Chromosomal analysis of bone marrow using the conventional G-banding method showed t(9;22)(q34;q11) in 14 of the 20 metaphases analyzed in addition to normal variance of inv(9)(p12q13) (Fig. 3). Fluorescence in situ hybridization (FISH) also showed BCR/ABL fusion signal in 92% of T. Inaba (&) N. Fujita Department of Infection Control and Laboratory Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan e-mail: inaba178@koto.kpu-m.ac.jp