Shinya Endo

An IL-27/Stat3 axis induces expression of programmed cell death 1 ligands (PD-L1/2) on infiltrating macrophages in lymphoma.

Immune escape and tolerance in the tumor microenvironment are closely involved in tumor progression, and are caused by T‐cell exhaustion and mediated by the inhibitory signaling of immune checkpoint molecules including programmed death‐1 (PD‐1), cytotoxic T‐lymphocyte associated protein 4, and T‐cell immunoglobulin and mucin domaincontaining molecule‐3. In the present study, we investigated the expression of the PD‐1 ligand 1 (PD‐L1) in a lymphoma microenvironment using paraffin‐embedded tissue samples, and subsequently studied the detailed mechanism of upregulation of PD‐L1 on macrophages using cultured human macrophages and lymphoma cell lines. We found that macrophages in lymphoma tissues of almost all cases of adult T‐cell leukemia/lymphoma (ATLL), follicular lymphoma and diffuse large B‐cell lymphoma expressed PD‐L1. Cell culture studies showed that the conditioned medium of ATL‐T and SLVL cell lines induced increased expression of PD‐L1/2 on macrophages, and that this PD‐L1/2 overexpression was dependent on activation of signal transducer and activator of transcription 3 (Stat3). In vitro studies including cytokine array analysis showed that IL‐27 (heterodimer of p28 and EBI3) induced overexpression of PD‐L1/2 on macrophages via Stat3 activation. Because lymphoma cell lines produced IL‐27B (EBI3) but not IL‐27p28, it was proposed that the IL‐27p28 derived from macrophages and the IL‐27B (EBI3) derived from lymphoma cells formed an IL‐27 (heterodimer) that induced PD‐L1/2 overexpression. Although the significance of PD‐L1/2 expressions on macrophages in lymphoma progression has never been clarified, an IL‐27‐Stat3 axis might be a target for immunotherapy for lymphoma patients.

PU.1 is a potent tumor suppressor in classical Hodgkin lymphoma cells

PU.1 has previously been shown to be down-regulated in classical Hodgkin lymphoma (cHL) cells via promoter methylation. We performed bisulfite sequencing and proved that the promoter region and the -17 kb upstream regulatory element of the PU.1 gene were highly methylated. To evaluate whether down-regulation of PU.1 is essential for the growth of cHL cells, we conditionally expressed PU.1 in 2 cHL cell lines, L428 and KM-H2. Overexpression of PU.1 induced complete growth arrest and apoptosis in both cell lines. Furthermore, in a Hodgkin lymphoma tumor xenograft model using L428 and KM-H2 cell lines, overexpression of PU.1 led to tumor regression or stable disease. Lentiviral transduction of PU.1 into primary cHL cells also induced apoptosis. DNA microarray analysis revealed that among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428 cells after PU.1 induction. Stable knockdown of p21 rescued PU.1-induced growth arrest in L428 cells, suggesting that the growth arrest and apoptosis observed are at least partially dependent on p21 up-regulation. These data strongly suggest that PU.1 is a potent tumor suppressor in cHL and that induction of PU.1 with demethylation agents and/or histone deacetylase inhibitors is worth exploring as a possible therapeutic option for patients with cHL.

Lactate, a putative survival factor for myeloma cells, is incorporated by myeloma cells through monocarboxylate transporters 1

BackgroundLactate levels within tumors are correlated with metastases, tumor recurrence, and radioresistance, thus apparently contributing to poor outcomes in patients with various cancers. We previously reported that high-level production of lactate by multiple myeloma (MM) cell lines is associated with high-level LDH activity within such MM cells. However, the kinetics of lactate remains to be studied. In the present study, we attempted to elucidate the mechanism of lactate incorporation into MM cells.MethodsSix MM cell lines and stromal cells obtained through long-term culture of bone marrow samples from MM patients were employed. Incorporation of lactate was quantified using C14-labeled lactate. The role of MCT1, a member of the monocarboxylate transporters (MCTs), expressed on MM cells, was examined in the presence of its inhibitor (α-cyano-4-hydroxycinnamic acid: CHC) and by using gene-silencing technique.ResultsMM cell lines as well as stromal cells were found to produce lactate. Incorporation of C14-labeled lactate into MM cells occurred in all 6 MM cell lines analyzed. Inhibition of MCT1 by using CHC or MCT1-targeting siRNA reduced lactate incorporation and caused apoptosis in MM cells. This apoptosis was enhanced when the activity of pyruvate dehydrogenase kinase was blocked by dichroloacetate. Survival of normal peripheral blood mononuclear cells was not influenced by MCT1 inhibition.ConclusionsThe present data suggest that lactate is produced by MM cell lines and stromal cells, and contributes to the survival of such MM cells in autocrine or paracrine manners. Suppression of lactate incorporation by targeting MCT1 may provide a novel therapeutic strategy for MM which may be applicable for other B-cell neoplasms.

Successful Treatment of Bing-Neel Syndrome Accompanying Waldenström's Macroglobulinemia with R-MPV: A Case Report.

Waldenströms macroglobulinemia (WM) is a neoplasm of lymphoplasmacytic cells that produces monoclonal IgM protein. Although hyperviscosity syndrome is a common feature of WM, central nervous system (CNS) involvement in WM is rare and is known as Bing-Neel syndrome. A 60-year-old woman was referred to our hospital with bed-bound polyneuropathy, edema, splenomegaly, IgM-λ-type monoclonal protein and CD20-positive lymphocyte infiltration in the bone marrow. She was diagnosed with WM accompanying POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal plasma cell disorder, and skin changes) and was treated with rituximab and thalidomide. She achieved partial remission of WM, and thalidomide was continued for POEMS syndrome. She visited our outpatient clinic 6 years later with sudden onset of vertigo and nausea. Magnetic resonance imaging (MRI) revealed a low-density area 4 cm in diameter in her right cerebrum and right mid-brain and she was referred to our hospital. Pathological analysis of brain biopsy samples revealed diffuse large B-cell lymphoma (DLBCL) in the CNS. Nucleic acid sequence analysis of the VDJ region using DNA obtained from the original WM tumor cells and brain tissue revealed that the DLBCL cells were derived from the original WM malignant lymphoma cells. She received five cycles of rituximab, methotrexate, procarbazine, and vincristine (R-MPV) therapy and 23.4 Gy of whole-brain irradiation followed by two cycles of high-dose cytarabine, which resolved her neurological symptoms in association with reduction of IgM levels to 367 mg/dL. MRI and computed tomography of the brain demonstrated complete remission of her CNS lymphoma.

PU.1 acts as tumor suppressor for myeloma cells through direct transcriptional repression of IRF4

We previously reported that PU.1 is downregulated in the majority of myeloma cell lines and primary myeloma cells of certain myeloma patients, and conditional expression of PU.1 in such myeloma cell lines induced cell cycle arrest and apoptosis. We found downregulation of IRF4 protein in the U266 myeloma cell line following induction of PU.1. Previous studies reported that knockdown of IRF4 in myeloma cell lines induces apoptosis, prompting us to further investigate the role of IRF4 downregulation in PU.1-induced cell cycle arrest and apoptosis in myeloma cells. PU.1 induced downregulation of IRF4 at the protein level, cell cycle arrest and apoptosis in six myeloma cell lines. Chromatin immunoprecipitation (ChIP) revealed that PU.1 directly binds to the IRF4 promoter, whereas a reporter assay showed that PU.1 may suppress IRF4 promoter activity. Stable expression of IRF4 in myeloma cells expressing PU.1 partially rescued the cells from apoptosis induced by PU.1. As it was reported that IRF4 directly binds to the IRF7 promoter and downregulates its expression in activated B cell-like subtype of diffuse large B cell lymphoma cells, we performed ChIP assays and found that IRF4 directly binds the IRF7 promoter in myeloma cells. It is known that IRF7 positively upregulates interferon-β (IFNβ) and induces apoptosis in many cell types. Binding of IRF4 to the IRF7 promoter decreased following PU.1 induction, accompanied by downregulation of IRF4 protein expression. Knockdown of IRF7 protected PU.1-expressing myeloma cells from apoptosis. Furthermore, IFNβ, which is a downstream target of IRF7, was upregulated in myeloma cells along with IRF7 after PU.1 induction. Finally, we evaluated the mRNA expression levels of PU.1, IRF4 and IRF7 in primary myeloma cells from patients and found that PU.1 and IRF7 were strongly downregulated in contrast to the high expression levels of IRF4. These data strongly suggest that PU.1-induced apoptosis in myeloma cells is associated with IRF4 downregulation and subsequent IRF7 upregulation.

Bufalin induces DNA damage response under hypoxic condition in myeloma cells

Hypoxia serves a crucial role in the development of drug resistance in various cancer cells. Therefore, many attempts targeting hypoxia are underway to overcome the drug resistance mediated by hypoxia. This strategy is useful for multiple myeloma (MM) cells, as MM cells reside within the bone marrow, where oxygen concentrations are relatively low. A natural compound library was screened to identify compounds exerting cytotoxicity in MM cells under hypoxic conditions. Bufalin exhibited marked cytotoxicity to MM cells under normoxic and hypoxic conditions. No significant toxicity was observed in lymphocytes obtained from healthy donors. Under normoxic conditions, bufalin induced a DNA double strand break (DSB) response, ROS induction and apoptosis within 24 with a rapid response compared with melphalan. Interestingly, the bufalin-induced DSB response was not impaired by low oxygen concentrations while the DSB response by melphalan was reduced. Furthermore, treatment with bufalin abolished HIF-1α expression under hypoxia, suggesting that bufalin exerts cytotoxicity under hypoxia by regulating HIF-1α. These results indicate that bufalin induces apoptosis in MM cells through DSB under hypoxic conditions by inhibiting HIF-1α, suggesting that bufalin could be useful for eradication of drug-resistant MM cells in the hypoxic microenvironment.

Pancytopenia and Myelodysplastic Changes in Aceruloplasminemia: A Case with a Novel Pathogenic Variant in the Ceruloplasmin Gene

A 72-year-old Japanese woman suffered from mild pancytopenia 3 years before her initial hospitalization. On admission, the levels of trace elements, particularly copper, and ceruloplasmin were significantly decreased in her blood serum. Abdominal lymphadenopathy and bone marrow dysplasia were detected. Hemosiderin deposition was observed in her lymph nodes and bone marrow, and magnetic resonance imaging suggested its deposition in various organs. A novel missense pathogenic variant (c.T1670G) was detected in the ceruloplasmin gene, resulting in an amino acid change (p.M557R). When copper deficiency is accompanied by cytopenia and dysplasia in a patient, it is worthwhile to consider a differential diagnosis of aceruloplasminemia.

Safety of mogamulizumab for relapsed ATL after allogeneic hematopoietic cell transplantation

Adult T-cell leukemia-lymphoma (ATL) is a peripheral T cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1) infection. Although antiviral therapy (such as Zidovudine plus interferon alpha) or combination chemotherapies are used for the treatment of patients with aggressive ATL (acute and lymphoma types), the prognosis of these patients is still very poor [1, 2]. On the other hand, some allogeneic hematopoietic cell transplantation (alloHCT) recipients with aggressive ATL have achieved longterm survival, suggesting the presence of graft-versus-ATL effects after allo-HCT [3]. However, relapse after allo-HCT is still a major obstacle to cure in recipients of allo-HCT [4]. Mogamulizumab (Mog), an anti CC chemokine receptor 4 (CCR4) antibody, was developed for use in patients with aggressive ATL, and previous studies showed that Mog was safe and effective in this population [5]. However, CCR4 is also highly expressed by regulatory T cells (Tregs), which play pivotal roles in the reconstitution of immune tolerance after allo-HCT [6]. Therefore, a major concern is that administration of Mog before or after allo-HCT could potentially increase the risk of graft-versus-host disease (GVHD) by depletion of Tregs. We previously reported that the use of Mog before allo-HCT increased severe acute GVHD and non-relapse mortality (NRM) [7, 8]. However, it remains unknown whether administration of Mog after allo-HCT increases the risk of subsequent GVHD. Hence, we conducted a retrospective analysis of the safety and efficacy of Mog in patients with relapsed aggressive ATL after allo-HCT. We analyzed the clinical data of six patients with aggressive ATL who received Mog for relapsed ATL after allo-HCT at Kumamoto University Hospital from 2014 to 2017. In five of the six patients, we analyzed ATL cells, Tregs and human leukocyte antigen (HLA) in peripheral blood (PB) by multi-color flow cytometry (FCM). Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Paque Plus (GE Healthcare), then stained with the following fluorescent-labeled antibodies: APC-CD3 (clone HIT3a), Brilliant Violet 510-CD4 (clone OKT4), APC-Cy7CD25 (clone BC96), Brilliant Violet 421-CD127 (clone A019D5), PE-Cy7-CCR4 (clone 2G12), PE-HLA-A2 (clone BB7.2) (BioLegend) and FITC-HLA-A9 (clone REA127) (Miltenyi Biotec). Flow cytometric analysis was performed using a BD FACSVerse flow cytometer (BD Biosciences). The patient and transplantation characteristics are shown in Table 1. The median time from allo-HCT to relapse was 79 days (range, 56–168 days). The types of relapse were systemic lymphadenopathy without ATL cells in PB in patients 1, 2 and 6, systemic lymphadenopathy with ATL cells in PB in patient 3 and 5 and focal lymphadenopathy with ATL cells in PB in patient 4. The median time from allo-HCT to the administration of Mog was 97 days (range, 83–295 days). In 3 patients (patients 3, 4 and 5), ATL cells in PB promptly disappeared after Mog administration. Meanwhile, in five patients with systemic lymphadenopathy, lymph node lesions grew larger or new lesions appeared even after Mog administration. Patients 1 and 3 died soon after the final administration of Mog due to PD, however, patient 2, 5 and 6 derived some benefit from the combination chemotherapies or radiotherapy after Mog administration. Patient 4 received radiotherapy for focal lymph node lesions before the administration of Mog. Thereafter, she achieved a * Yoshitaka Inoue yinoue1110@gmail.com