Miki Kiyota

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.

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.

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.

RSK2 Ser227 at N-Terminal Kinase Domain Is a Potential Therapeutic Target for Multiple Myeloma

Multiple myeloma is an entity of cytogenetically and genetically heterogenous plasma cell neoplasms. Despite recent improvement in the treatment outcome of multiple myeloma by novel molecular-targeted chemotherapeutics, multiple myeloma remains incurable. The identification of a therapeutic target molecule in which various signaling for cell-survival converge is a core component for the development of new therapeutic strategies against multiple myeloma. RSK2 is an essential mediator of the ERK1/2 signaling pathway for cell survival and proliferation. In this study, we discovered that RSK2Ser227, which is located at the N-terminal kinase domain and is one site responsible for substrate phosphorylation, is activated through phosphorylation regardless of the type of cytogenetic abnormalities or upstream molecular signaling in all 12 multiple myeloma–derived cell lines examined and 6 of 9 patient-derived CD138-positive primary myeloma cells. The chemical inhibition of RSK2Ser227 by BI-D1870 or gene knockdown of RSK2 inhibits myeloma cell proliferation through apoptosis induction, and this anti-myeloma effect was accompanied by downregulation of c-MYC, cyclin D, p21WAF1/CIP1, and MCL1. RSK2Ser227 inhibition resulting from BI-D1870 treatment restored lenalidomide-induced direct cytotoxicity of myeloma cells from interleukin-6–mediated cell protection, showed no cross-resistance to bortezomib, and exerted additive/synergistic antiproliferative effects in conjunction with the mTOR, histone deacetylase, and BH3-mimicking BCL2/BCLXL inhibitors. These results suggest that RSK2Ser227 is a potential therapeutic target not only for newly diagnosed but also for patients with later phase multiple myeloma who are resistant or refractory to currently available anti-myeloma therapies. Mol Cancer Ther; 11(12); 2600–9. ©2012 AACR.

Phosphoinositide Protein Kinase PDPK1 Is a Crucial Cell Signaling Mediator in Multiple Myeloma

Multiple myeloma is a cytogenetically/molecularly heterogeneous hematologic malignancy that remains mostly incurable, and the identification of a universal and relevant therapeutic target molecule is essential for the further development of therapeutic strategy. Herein, we identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a serine threonine kinase, is expressed and active in all eleven multiple myeloma-derived cell lines examined regardless of the type of cytogenetic abnormality, the mutation state of RAS and FGFR3 genes, or the activation state of ERK and AKT. Our results revealed that PDPK1 is a pivotal regulator of molecules that are essential for myelomagenesis, such as RSK2, AKT, c-MYC, IRF4, or cyclin Ds, and that PDPK1 inhibition caused the growth inhibition and the induction of apoptosis with the activation of BIM and BAD, and augmented the in vitro cytotoxic effects of antimyeloma agents in myeloma cells. In the clinical setting, PDPK1 was active in myeloma cells of approximately 90% of symptomatic patients at diagnosis, and the smaller population of patients with multiple myeloma exhibiting myeloma cells without active PDPK1 showed a significantly less frequent proportion of the disease stage III by the International Staging System and a significantly more favorable prognosis, including the longer overall survival period and the longer progression-free survival period by bortezomib treatment, than patients with active PDPK1, suggesting that PDPK1 activation accelerates the disease progression and the resistance to treatment in multiple myeloma. Our study demonstrates that PDPK1 is a potent and a universally targetable signaling mediator in multiple myeloma regardless of the types of cytogenetic/molecular profiles.

Suppression of SERPINA1-albumin complex formation by galectin-3 overexpression leads to paracrine growth promotion of chronic myelogenous leukemia cells

Galectin-3 is induced in chronic myelogenous leukemia (CML) cells by co-culture with bone marrow stromal cells, making paracrine growth promotion of CML cells in conditioned medium (CM) from galectin-3 overexpressing CML cells more potent. We used gel filtration chromatography to demonstrate that the bovine SERPINA1-fetal bovine serum albumin (BSA) complex was specifically suppressed in CM from galectin-3 overexpressing cells. The SERPINA1-BSA complex as well as human plasma SERPINA1 inhibited the growth of CML cells, while exogenous galectin-3 partly offset this effect. These findings suggest that galectin-3 overexpression promotes paracrine growth of CML cells by interfering with the action of the growth inhibitory SERPINA1-albumin complex.

Clinical manifestation of angioimmunoblastic T-cell lymphoma with exuberant plasmacytosis.

Angioimmunoblastic T-cell lymphoma (AITL) is a rare subtype of non-Hodgkin lymphoma characterized by aggressive symptoms and various abnormal laboratory test results. One of the rare immunologic abnormalities in AITL is exuberant polyclonal plasmacytosis, but its clinical significance has not been evaluated. This report concerns three AITL cases with exuberant polyclonal plasmacytosis and investigates its clinical impact by comparison with 12 patients without plasmacytosis. Our study found that the performance status (PS) of the former was significantly worse and their serum immunoglobulin levels were significantly higher. All other parameters, including B symptoms, various prognostic scores, blood cell counts other than plasmacyte, and serum levels of lactate dehydrogenase, C-reactive protein and soluble interleukin-2 receptor, showed no significant differences. More importantly, although the diagnosis of AITL with plasmacytosis was not straightforward in our series, outcomes of treatment with conventional chemotherapy or immunosuppressive therapy with cyclosporine A were favorable. To conclude, AITL should be considered a candidate underlying disease of exuberant polyclonal plasmacytosis. Provided a correct diagnosis is made early and is followed by adequate treatment, the prognosis for AITL with plasmacytosis may not be worse than that for those without plasmacytosis despite the severe exhaustion at first presentation.

Comprehensive cytogenetic study of primary cutaneous gamma-delta T-cell lymphoma by means of spectral karyotyping and genome-wide single nucleotide polymorphism array.

Primary cutaneous gamma-delta T-cell lymphoma (PCGD-TCL), which originates from activated mature gamma-delta T cells with a cytotoxic phenotype is a rare T-cell lymphoproliferative disease. The prognosis of PCGD-TCL has been rather unfavorable due to poor response to conventional chemotherapy, and its molecular features and pathophysiology underlying disease development remain unknown. We report here a case with primarily treatment-resistant PCGD-TCL featuring highly complex cytogenetic and genetic aberrations detected by spectral karyotyping and genome-wide single nucleotide polymorphism (SNP) array. Chromosomal aberrations included several chromosomal translocations involving breakpoints at 9p21, 14q11.2, 14q32.1, or 16q23.1, suggesting the involvement of WWOX, TCL gene cluster, and BCL11B, which are crucial for tumorigenesis in T-cell lymphomas. SNP analysis also identified genome copy number gains and losses in various regions, which can potently deregulate expression of various pro- and anti-oncogenic genes involved in RAS-related protein pathways, PI3K/AKT/MTOR-related pathways, MYC-related signaling, or TP53-related signaling. Thus, this case report may shed some light on the complex molecular abnormalities involved in the development of PCGD-TCL and on information that can aid the search for druggable target molecules in this disease.

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

Prognostic impact of a past or synchronous second cancer in diffuse large B cell lymphoma

Therapeutic improvements for cancers in general have resulted in a growing population of cancer survivors at risk of developing secondary primary malignancies (SPMs) due to a variety of biological factors such as cancer predisposition syndromes, environmental factors, immune impairments, and late effects of genotoxic therapies. Patients with a history of other primary malignancies have the possibility of dose-reduced treatment because of the chronic health problems induced by previous cancer treatment or previous cancer damage. In addition, when analyzing the prognostic factors, death due to other cancers before disease progression is considered a competing risk. This could also be the case with diffuse large B-cell lymphoma (DLBCL), the most prevalent subtype of nonHodgkin lymphoma. While the recent treatment progress has markedly improved overall long-term outcomes for DLBCL by the advent of the immunochemotherapy containing rituximab, the clinical and prognostic impacts of a history of past cancer or co-existence of another synchronous cancer in “secondary” or “concomitant” DLBCL has been rarely investigated in the recent era. To answer this question, we performed a retrospective analysis of 809 DLBCL patients who were diagnosed histologically and treated between January 2006 and February 2016 at institutes in the Kyoto Clinical Hematology Study Group (KOTOSG). The criteria for multiple primary malignancy (MPM) used were proposed by Warren and Gates: i) tumors have definite features of malignancy, ii) tumors are separate and distinct from the index tumor, and iii) the possibility of a tumor being a metastasis of the index tumor is ruled out. Based on the Surveillance Epidemiology and End Results Program definition for the chronicity of MPM, the term “synchronous” refers to a condition where more than two malignancies, including DLBCL, are detected within two months, while “past” indicates a metachronous condition where the tumors are detected more than two months apart. This study was approved by the Institutional Review Boards. The 809 DLBCL patients were classified into three groups: those with past cancer (i.e., patients with precedent cancer(s) that occurred more than two months before the diagnosis of DLBCL); those with synchronous cancer (i.e., patients diagnosed with DLBCL and other malignancies within two months of each other); and patients without another cancer. Among them, 123 (15.2%) were defined as having MPM, including 94 with past cancer and 29 with synchronous cancer (Supplementary Table S1). DLBCL patients with MPM were significantly older than those without MPM (75 vs. 70 years old; P< 0.001). There was no significant difference in disease stage or international prognostic index (IPI)defined disease risk between patients with and without MPM. Although small adjustments of the therapeutic regimen were allowed at the doctor’s discretion, more than 85% of patients received a rituximab plus CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) (R-CHOP)-like regimen regardless of the presence of MPM. In the 123 DLBCL patients with MPM, 103, 16, and 4 had one, two, and three other primary malignancies, respectively, before treatment for DLBCL.