Yumi Nozaki

Inhibition of hypoxia-inducible factor-1 function enhances the sensitivity of multiple myeloma cells to melphalan

Abnormal activation of hypoxia-inducible factor-1 (HIF-1), one of the most important transcription factors for the adaptation of cells to hypoxia, is frequently observed in numerous types of solid tumors. Dysregulation of HIF-1 induces tumor angiogenesis and enhances the expression of anti-apoptotic proteins and glycolysis-associated enzymes in cancer cells, which in turn leads to the promotion of tumor growth. In the present study, we examined the pathophysiologic role of HIF-1 in multiple myeloma. Furthermore, we explored the possibility that HIF-1 may be a molecular target for myeloma therapy. We identified constitutive expression of the hypoxia-inducible factor-1 α (HIF-1α)-subunit in established myeloma cell lines and in primary myeloma cells. Treatment with insulin-like growth factor-1 (IGF-1) significantly increased HIF-1α expression through activation of the AKT and mitogen-activated protein kinase signaling pathways. Inhibition of HIF-1 function either by echinomycin, a specific HIF-1 inhibitor, or a siRNA against HIF-1α resulted in enhanced sensitivity to melphalan in myeloma cells. This inhibition of HIF-1 also reversed the protective effect of IGF-1 on melphalan-induced apoptosis. Inhibition of HIF-1 drastically reduced both basal and IGF-1–induced expression of survivin, one of the most important anti-apoptotic proteins in myeloma cells. We conclude that HIF-1 inhibition may be an attractive therapeutic strategy for multiple myeloma. [Mol Cancer Ther 2009;8(8):2329–38]

NF-κB mediates aberrant activation of HIF-1 in malignant lymphoma

OBJECTIVE The goal of this study was to explore the molecular mechanisms of aberrant hypoxia inducible factor-1 (HIF-1) activation in lymphoma cells. MATERIALS AND METHODS We analyzed the expression of the α subunit of HIF-1 in three lymphoma cell lines and in normal CD19-positive B cells by Western blotting. To investigate the role of nuclear factor (NF)-κB in abnormal HIF-1α expression in lymphoma cells, we performed a reporter assay using HIF-1α promoter constructs that contained or lacked an NF-κB binding site. We also used a chromatin immunoprecipitation assay to assess whether NF-κB binds the HIF-1α promoter. In addition, we took advantage of NF-κB inhibitors. To analyze the function of HIF-1 in lymphoma cells, we established stable HIF-1α knockdown cells using short-hairpin RNA. RESULTS Malignant lymphoma cells, but not normal B cells, demonstrated constitutive expression of HIF-1α. Inhibitors of NF-κB, however, drastically suppressed this HIF-1α expression at both the messenger RNA and protein levels. Furthermore, we found that exposure of lymphoma cells to ionizing radiation clearly induced NF-κB activation and increased HIF-1α expression. Suppressing HIF-1α expression by short-hairpin RNA increased the sensitivity of lymphoma cells to ionizing radiation-induced cell death. In searching for downstream targets of the NF-κB/HIF-1 axis, we identified survivin, a member of the IAP family of anti-apoptotic proteins. CONCLUSIONS We found that aberrant activation of HIF-1 in malignant lymphoma cells was mediated, at least in part, by NF-κB activity. Our observations suggest that HIF-1 inhibition may be an effective strategy to improve the outcomes of lymphoma patients treated with radiation.

Rituximab activates Syk and AKT in CD20-positive B cell lymphoma cells dependent on cell membrane cholesterol levels

The introduction of rituximab, an anti-CD20 monoclonal antibody, has dramatically improved the treatment outcomes of patients with B cell lymphoma. Nevertheless, the clinical response to rituximab varies, and a subpopulation of patients does not respond well to this antibody. Although several molecular events have been shown to be involved in the mechanism of action of rituximab, recent studies have demonstrated that intracellular signaling pathways and the direct effects of rituximab on cell membrane components are responsible for the antilymphoma action of this drug. In the present study, we demonstrated that rituximab activated Syk and Akt, molecules with antiapoptotic functions, in several CD20-positive lymphoma cell lines. Notably, rituximab activated Syk and Akt in all the tested primary lymphoma samples from six patients. Our results show that the cholesterol levels in lymphoma cell membranes have a crucial role in the regulation of Syk and Akt. The depletion of cholesterol from the cell membrane completely blocked rituximab-induced Syk and Akt activation. Simvastatin, an inhibitor of cholesterol synthesis, also abrogated rituximab-mediated Syk and Akt activation. Finally, we report that rituximab inhibited the apoptosis induced by chemotherapeutic drugs, which was observed solely in Akt-activated cells. This work demonstrates for the first time that rituximab paradoxically works to suppress apoptosis under certain conditions in a manner that is dependent on the cell membrane cholesterol level. Our observations provide novel insights and suggest that the cell membrane cholesterol level represents a new biomarker for predicting patient response to rituximab. Furthermore, the modulation of lipid rafts could provide a new strategy for enhancing the antilymphoma action of rituximab.

Mogamulizumabによる肺浸潤治療後に，同種造血幹細胞移植を行った成人T細胞性白血病・リンパ腫

Adult T cell lymphoma-leukemia (ATL) is a highly aggressive disease and allogeneic hematopoietic transplantation (allo-HSCT) is the only therapeutic option for achieving a cure. However, some ATL patients cannot undergo HSCT. One of the important reasons for restricting HSCT in ATL is the high incidence of pulmonary complications associated with ATL including opportunistic infections, infiltration of ATL cells, and HTLV-1 associated bronchopneumonopathy. Herein, we report an ATL case with pulmonary infiltration of ATL cells successfully treated with allo-HSCT after improvement of pulmonary function with administration of the anti-CCR4 antibody mogamulizumab. To our knowledge, this is the first ATL case showing improvement of pulmonary invasion of ATL cells after treatment with mogamulizumab. In addition, this case suggests that mogamulizumab treatment might be useful as a bridge to allo-HSCT in ATL patients.

Gastric invasion of multiple myeloma presenting as gastrointestinal bleeding

gastric body. The patient was treated with red blood cell transfusion and a proton-pump inhibitor. Two days after the initial observation, esophagogastroduodenoscopy was performed again. As shown in Fig. 1a, irregular thickened folds were found at the gastric corpus. The biopsy specimens revealed significant invasion of round cells in the submucosal area (Fig. 1b). Immunohistochemical studies revealed that these cells were positive for CD 138 (Fig. 1c) and κ chain (Fig. 1d), and negative for CD56, CD20 and CD79a. There was no evidence of deposition of amyloids in the stomach wall. Based on these findings, we diagnosed the patient with gastrointestinal invasion of MM. Although we continued the treatment with protonpump inhibitor, we were unable to control the gastrointestinal bleeding and she developed multi-organ failure. She died 3 weeks after admission. Gastrointestinal bleeding is a common complication of hematological malignancies, including MM. A variety of factors are involved in the gastrointestinal bleeding observed in MM patients [2]. First, paraneoprotein impairs the function of platelets and coagulation factors, leading to increased bleeding tendency. Treatments for MM may also increase the risk for bleeding, including from chemotherapy-induced thrombocytopenia, steroid-induced gastric ulcers, or the administration of aspirin or anticoagulants to prevent the thromboembolic complications associated with lenalidomide or thalidomide. Additionally, the deposition of amyloid protein in the gastrointestinal system can cause severe bleeding. In the present case, we found substantial involvement of myeloma cells in the gastric mucosa. Although the incidence is rare, previous case reports have indicated that gastrointestinal involvement of multiple myeloma, including severe gastrointestinal bleeding, may occur [2–5]. The prognosis of these patients is often extremely poor [2, 3]. A 62-year-old woman was hospitalized because of melena. She had been diagnosed with Bence Jones κ-type multiple myeloma (MM) 7 years prior. At the diagnosis of multiple myeloma, chromosome analysis of bone marrow cells revealed the karyotype abnormality 46, XX, t(7;8) (q32;q13) in one of 20 cells analyzed. Following the initial diagnosis of MM, she was treated with the combination of vincristine, doxorubicin, and dexamethasone, followed by high-dose melphalan chemotherapy with autologous stem cell transplantation (ASCT) [1]. She achieved only a partial response to these treatments, and was subsequently treated with thalidomide, and then bortezomib, for 3 years. During the clinical course, she developed multiple subcutaneous plasmacytomas. She underwent palliative radiation therapy, and treatment with lenalidomide was started with prophylactic anticoagulants. Three days before the hospitalization for melena, she experienced epigastric pain. At the time of hospitalization, the patient showed marked anemia (Hb 4.8 g/dL); however, her platelet counts were only modestly decreased (141 × 10/L; normal range 155– 345 × 10/L). Emergent esophagogastroduodenoscopy was performed, and massive bleeding was observed in the

[Successful treatment with rituximab and romiplostim for thrombocytopenia associated with Waldenström's macroglobulinemia initially presenting as Evans syndrome].

A 60-year-old woman was admitted to our hospital with anemia and thrombocytopenia. Serum testing showed platelet-associated IgG elevation and she was positive on the direct and indirect Coombs tests. Together with bone marrow examination, these findings indicated a diagnosis of Evans syndrome. At diagnosis, she also had an IgM-κ type of monoclonal gammopathy of unknown significance. Initially, we administered steroids and her hemolytic anemia showed improvement. In contrast, only transient recovery of platelet counts was observed and her platelet counts rapidly decreased after steroid dose reduction. Thus, we treated her with a TPO-agonist, romiplostim. During the clinical course, she showed gradual serum IgM elevation. We thus performed another bone marrow biopsy and diagnosed her as having Waldenströms macroglobulinemia (WM). We started treatment with rituximab for WM. Together with the serum IgM reduction, she showed marked improvement of thrombocytopenia. This is a very rare case of WM initially presenting as autoimmune hemolytic anemia and immunethrombocytopenia associated with IgG class auto-antibody. Our experience suggests the usefulness of rituximab and romiplostim for the treatment of immunethrombocytopenia associated with WM.