Nana Sasaki

Galectin-3 (Gal-3) induced by leukemia microenvironment promotes drug resistance and bone marrow lodgment in chronic myelogenous leukemia

Bone marrow (BM) microenvironment (BMME) constitutes the sanctuary for leukemic cells. In this study, we investigated the molecular mechanisms for BMME-mediated drug resistance and BM lodgment in chronic myelogenous leukemia (CML). Gene-expression profile as well as signal pathway and protein analyses revealed that galectin-3 (Gal-3), a member of the β-gal–binding galectin family of proteins, was specifically induced by coculture with HS-5 cells, a BM stroma cell-derived cell line, in all five CML cell lines examined. It was also found that primary CML cells expressed high levels of Gal-3 in BM. Enforced expression of Gal-3 activated Akt and Erk, induced accumulation of Mcl-1, and promoted in vitro cell proliferation, multidrug resistance to tyrosine kinase inhibitors for Bcr-Abl and genotoxic agents as a result of impaired apoptosis induction, and chemotactic cell migration to HS-5–derived soluble factors in CML cell lines independently of Bcr-Abl tyrosine kinase. The conditioned medium from Gal-3–overexpressing CML cells promoted in vitro cell proliferation of CML cells and HS-5 cells more than did the conditioned medium from parental cells. Moreover, the in vivo study in a mice transplantation model showed that Gal-3 overexpression promoted the long-term BM lodgment of CML cells. These results demonstrate that leukemia microenvironment-specific Gal-3 expression supports molecular signaling pathways for disease maintenance in BM and resistance to therapy in CML. They also suggest that Gal-3 may be a candidate therapeutic target to help overcome BMME-mediated therapeutic resistance.

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.

FTY720 induces apoptosis of chronic myelogenous leukemia cells via dual activation of BIM and BID and overcomes various types of resistance to tyrosine kinase inhibitors

PP2A activator FTY720 has been shown to possess the anti-leukemic activity for chronic myelogenous leukemia (CML), however, the cell killing mechanism underlying its anti-leukemic activity has remained to be verified. We investigated the precise mechanisms underlying the apoptosis induction by FTY720, especially focusing on the roles of BH3-only proteins, and the therapeutic potency of FTY720 for CML. Enforced expression of either BCL2 or the dominant-negative protein of FADD (FADD.DN) partly protected CML cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways. FTY720 activates pro-apoptotic BH3-only proteins: BIM, which is essential for apoptosis by BCR-ABL1 tyrosine kinase inhibitors (TKIs), and BID, which accelerates the extrinsic apoptosis pathway. Gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the extent of cell protection was not as much as that by overexpression of either BCL2 or FADD.DN. Moreover, knockdown of both BIM and BID did not provide additional protection compared with knockdown of only BIM or BID, indicating that BIM and BID complement each other in apoptosis by FTY720, especially when either is functionally impaired. FTY720 can overcome TKI resistance caused by ABL kinase domain mutations, dysfunction of BIM resulting from gene deletion polymorphism, and galectin-3 overexpression. In addition, ABT-263, a BH3-mimetic, significantly augmented cell death induction by FTY720 both in TKI-sensitive and -resistant leukemic cells. These results provide the rationale that FTY720, with its unique effects on BIM and BID, could lead to new therapeutic strategies for CML.

Multifaceted Mechanisms for Cell Survival and Drug Targeting in Chronic Myelogenous Leukemia

Treatment outcomes for chronic myelogenous leukemia (CML) have shown major improvements as a result of the development of the tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib for the disease-specific molecular target BCR-ABL1 tyrosine kinase (TK), but a cure of CML by BCR-ABL1 TKIs has been rarely achieved. CML cells are protected from cytotoxic insults, including those by TKIs, through various collaborative BCR-ABL1- mediated and -independent mechanisms, as well as cell-intrinsic and -extrinsic molecular mechanisms. These protective mechanisms include overlapping cell signaling pathways for normal hematopoietic proliferation, modulation of molecules associated with the BCL2 family protein-regulated programmed cell death pathway, autophagic cell protection capability, bone marrow environment-mediated cell protective signaling, abnormally upregulated genetic instability and other BCR-ABL1- independent kinase activities. To develop a more effective treatment strategy for a cure by means of total leukemic cell killing, a thorough understanding of how CML cells survive and resist cytotoxic insults is essential. In this article, we review current knowledge about multifaceted BCR-ABL1-related and -unrelated mechanisms for survival and death of CML cells and present suggestions for the development of new therapeutic strategies for complete elimination of residual CML cells during TKI treatment.

Deletion or methylation of CDKN2A/2B and PVT1 rearrangement occur frequently in highly aggressive B-cell lymphomas harboring 8q24 abnormality

Among various cytogenetic abnormalities involved in lymphomagenesis and disease progression of highly aggressive and treatment-resistant B-cell lymphomas (HABCLs), classified as the so-called aggressive B-cell lymphomas (BCLs) such as diffuse large B-cell lymphoma (DLBCL) and Burkitt-like lymphoma or Burkitt lymphoma (BL), 8q24 chromosomal abnormality is one of the major genetic/cytogenetic abnormalities, with a frequency of 5–15% [1,2]. Moreover, MYC rearrangements at 8q24 have been occasionally found in combination with a t(14;18)(q32;q21) involving BCL2 in 4% of highly aggressive “double-hit” DLBCLs (DHLs), with an extremely poor prognosis [3–5]. Indeed, MYC and BCL2 synergistically induce tumor formation, as observed in experimental models [6,7]. Therefore, better genetic/molecular diagnostic criteria are needed for the more accurate prediction of outcomes and the development of novel treatments for HABCLs. Because the complex karyotypes often found in HABCLs may cause the gene alterations associated with their biological functions, aberrations of other molecules besides MYC and BCL2 may also further affect the pathogenesis of HABCLs [4,8,9]. In this study, we subjected eight patients with aggressive BCLs with 8q24 abnormality treated at the Kyoto Prefectural University of Medicine (KPUM) between April 2006 and September 2012 (Table I) to a comprehensive molecular analysis using G-banding, spectral karyotyping (SKY), fluorescence in situ hybridization (FISH) for IGH, MYC, PVT-1 [Figure 1(A)], BCL-2, BCL-6, BACH2 and CDKN2A/2B, oligonucleotide array and methylation-specific polymerase chain reaction (PCR) (MSP) (Supplementary Materials and Methods available online at http://www.informahealthcare. com/lal/doi/10.3109/10428194.2013.790543) [8–10]. Patients 1, 2, 3, 7 and 8 were included in our previous publication on survival analysis for DLBCL [5]. We established two cell lines, KPUM-MS3 and KPUM-UH1, from patients 2 and 3, respectively [11]. This study was approved by the ethics committee of KPUM. Three patients had Burkitt-like lymphoma and five had DLBCL. As shown in Table I, all patients presented with advanced disease stage and with unfavorable International Prognostic Index scores. All patients were characterized by systemically disseminated involvements including bone marrow involvement, fluid retention, central nervous system involvement and/or multiple extranodal disease sites. As for response to immunochemotherapy incorporating rituximab and/or irradiation therapy, five patients showed disease progression, while three achieved a complete response. Patient 7 had three disease recurrences prior to a complete response to various salvage chemotherapies. The median survival of eight patients was 6.7 months. In these series, 8q24 abnormalities were detected by G-banding and SKY analysis in five patients, and by FISH in the remaining three. All patients thus showed 8q24 abnormalities, including MYC/PVT1 amplification in patient 4 [Figure 1(B)]. Based on the results obtained by FISH using both PVT1-A and PVT1-S probes, breakpoints were assigned to the PVT1 gene in two patients (2 and 5) and to the region covered by the bacterial artificial chromosome (BAC) CTD-3066D1 containing MYC in four (3, 6, 7 and 8) [Figures 1(A) and 1(C) and Supplementary Table I to be found online at http://www.informahealthcare.com/lal/doi/10.3109/1042 8194.2013.790543]. The MYC-Cg fusion gene was detected in patient 1 by long-distance PCR. Oligonucleotide array analysis demonstrated that the 8q24 rearrangement demarcated copy number changes within PVT1 in intron 1 in patient 2 (Supplementary Figure 1 to be found online at http://www. informahealthcare.com/lal/doi/10.3109/10428194.2013. L eu k L ym ph om a D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y K yo to F ur its u Id ai L ib ra ry o n 09 /2 8/ 14

Thrombotic thrombocytopenic purpura associated with myelodysplastic syndrome

Thrombotic thrombocytopenic purpura (TTP), a lifethreatening disseminated thrombotic microangiopathy (TMA) syndrome, is characterized by bicytopenias with thrombocytopenia and hemolytic anemia (HA) with schistocytes, neurological disturbances, acute renal failure, and pyrexia. It has an incidence of 2–7 per million person per year with a peak incidence between the ages of 30 and 40 [1, 2]. Myelodysplastic syndrome (MDS) is also characterized by multilineage cytopenias due to ineffective hematopoiesis, and predominantly affects the elderly with a peak incidence between the ages of 60 and 75. Compared with other disorders, such as aplastic anemia, phenomena secondary to leukemias, autoimmune disorders, such as systemic lupus erythematosus or Evans syndrome, viral infection, or adverse drug effects, both MDS and TTP are less common as causatives for multilineage cytopenias in young patients. This report describes a rare case of MDS associated with TTP in a young female presented with multilineage cytopenias. A 21-year-old female was referred to our hospital with complaints of purpura, petechiae, headache, numbness and pancytopenia. She was not pregnant, showed no sign of recent infection and had no recent history of drug exposure. Peripheral blood count findings were pancytopenia with white blood cells at 2.4 9 10/L (3.4–7.3 9 10) (neutrophils 47%, lymphocytes 44%, abnormal cells 0%), red blood cells at 2.21 9 10/L (3.62–4.99 9 10), hemoglobin at 7.3 g/dL (11.7–15.1) and platelet count of 20.0 9 10/L (160–327 9 10). Blood examination also demonstrated the presence of HA, shown by elevated serum levels of total bilirubin 2.2 mg/dL (0.2–1.0), and lactate dehydrogenase 453 IU/L (114–243), serum haptoglobin reduced to below detectable levels and the presence of schistocytes. Serum vitamin B12 and folic acid were normal, 384 ng/L (180–914) and 3.9 lg/L ([3.1), respectively. Coombs test, Ham test, and the cell surface expression of glycosyl phosphatidilinositol-anchored proteins were all negative. Other examinations showed no evidence of autoimmune disorders. Bone marrow (BM) examination revealed hypercellular marrow with 300 9 10/L BM nucleated cell counts, and multilineage dysplasia, such as pseudo-Pelger neutrophils or micromegakaryocytes, but with no abnormal increase of myeloblasts at 1.6% of all BM nucleated cells (Fig. 1). Chromosome aberration was not identified by either Gbanding or spectral karyotyping analysis. According to the WHO classification criteria, the patient was diagnosed as MDS, comprising refractory cytopenia with multilineage dysplasia (RCMD) [3]. One month later, she suddenly showed various neuropsychological symptoms, such as disorientation, aphypnia, and anxiety neurosis. Serum urea nitrogen and creatinine levels were normal, 21.1 mg/dL (8.0–23.0) and 0.7 mg/dL (0.4–1.2), respectively. Coagulation tests, such as prothrombin time, activated partial thromboplastin time, or plasma levels of fibrinogen or fibrin degradation product, were normal (data not shown), while a decrease in ADAMTS13 activity (\0.5%) and an increase in the autoantibody for ADAMTS13 (14 Bethesda unit/ml), which functions as an inhibitor of ADAMTS13, were detected in her serum. We diagnosed her as having N. Sasaki J. Kuroda (&) M. Taniwaki Division of Hematology and Oncology, Department of Medicine, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan e-mail: junkuro@koto.kpu-m.ac.jp

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.