Yoshitaka Kikukawa

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.

Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells

Shikonin (SHK), a natural small agent (MW 288.3), reportedly induces cell death in various tumor cells. We have found that SHK also exerts potent cytocidal effects on human multiple myeloma (MM) cells, but its anticancer mechanism in MM cells remains to be elucidated. SHK at 2.5–5 μM induced apoptosis in seven MM cell lines, including the bortezomib-resistant cell line KMS11/BTZ. The IC50 value of SHK against KMS11/BTZ was comparable to that of a parental cell line KMS11 (1.1 and 1.56 μM, respectively). SHK induces accumulation of ubiquitinated proteins and activates XBP-1 in MM cells, suggesting that SHK functions as a proteasome inhibitor, eventually inducing ER stress-associated apoptosis. SHK increases levels of HSP70/72, which protects cells from apoptosis, and exerts greater cytocidal effects in combination with the HSP70/72 inhibitor VER-155008. At higher concentrations (10–20 μM), SHK induced cell death, which was completely inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the cytocidal activity was unaffected by Z-VAD-FMK, strongly suggesting that cell death is induced by SHK at high concentrations through necroptosis. The present data show for the first time that SHK induces cell death in MM cells. SHK efficiently induces apoptosis and combination of heat shock protein inhibitor with low dose SHK enhances apoptosis, while high dose SHK induces necroptosis in MM cells. These findings together support the use of SHK as a potential therapeutic agent for MM.

Hypoxia reduces CD138 expression and induces an immature and stem cell-like transcriptional program in myeloma cells

Although CD138 expression is a hallmark of plasma cells and myeloma cells, reduced CD138 expression is occasionally found. However, the mechanisms underlying CD138 downregulation in myeloma cells remain unclear. Previous reports suggest that the bone marrow microenvironment may contribute to CD138 downregulation. Among various factors in the tumor microenvironment, hypoxia is associated with tumor progression, poor clinical outcomes, dedifferentiation and the formation of cancer stem cell niches in solid tumors. Since recent findings showed that progression of multiple myeloma (MM) delivers hypoxia within the bone marrow, we hypothesized that CD138 expression may be regulated by hypoxia. In the present study, we examined whether the expression of CD138 and transcription factors occurred in myeloma cells under hypoxic conditions. MM cell lines (KMS-12BM and RPMI 8226) were cultured under normoxic or hypoxic conditions for up to 30 days. Changes in the phenotype and the expression of surface antigens and transcription factors were analyzed using flow cytometry, RT-PCR and western blotting. All-trans retinoic acid (ATRA) was used to examine the phenotypic changes under hypoxic conditions. The expression levels of CD138, CS1 and plasma cell-specific transcription factors decreased under hypoxic conditions, while those of CD20, CXCR4 and B cell-specific transcription factors increased compared with those under normoxic conditions. Stem cell-specific transcription factors were upregulated under hypoxic conditions, while no difference was observed in ALDH activity. The reduced CD138 expression under hypoxic conditions recovered when cells were treated with ATRA, even under hypoxic conditions, along with decreases in the expression of stem cell-specific transcription factor. Interestingly, ATRA treatment sensitized MM cells to bortezomib under hypoxia. We propose that hypoxia induces immature and stem cell-like transcription phenotypes in myeloma cells. Taken together with our previous observation that decreased CD138 expression is correlated with disease progression, the present data suggest that a hypoxic microenvironment affects the phenotype of MM cells, which may correlate with disease progression.

TRAIL produced from multiple myeloma cells is associated with osteolytic markers

Skeletal complications represent major clinical problems in multiple myeloma (MM). MM cells are known to induce differentiation of osteoclasts and inhibit osteoblasts. Receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) are key molecules for osteoclastogenesis. Although OPG interacts with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the contribution of TRAIL to skeletal-related events (SRE) remains a matter of debate. In the present study, we examined the role of TRAIL in MM bone lesions. Myeloma cells were purified from 56 MM patients by CD138-immunomagnetic beads. TRAIL, DKK-1 and MIP1α RNA expression in purified MM cells was analyzed by real-time PCR. Immunohistochemistry of TRAIL was performed on paraffin-embedded plasmacytoma tissue sections. The concentration of TRAIL in the serum and bone marrow plasma from MM patients was analyzed by ELISA. TRAIL expression was significantly higher in MM cells than in plasma cells from patients with monoclonal gammopathy of undetermined significance (MGUS). TRAIL staining was detected in the cytoplasm of myeloma cells. TRAIL expression in MM cells correlated with bone marrow plasma TRAIL concentration. TRAIL expression had a positive correlation with osteolytic markers, such as serum calcium and urinary deoxypyridinoline. These results suggest that TRAIL, produced from myeloma cells, may play an important role in bone resorption of MM patients. Inhibition of this pathway may lead to development of a new therapeutic approach preventing bone resorption in MM.

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.

Isolated Pancreatic Myeloid Sarcoma Associated with t(8;21)/RUNX1-RUNX1T1 Rearrangement

No valid treatment for isolated myeloid sarcoma (IMS) has yet been established, and no thorough genetic examinations have been performed because of its low incidence and unique manner of development. We herein report a 34-year-old man with pancreatic IMS with t(8;21)/RUNX1-RUNX1T1 rearrangement. He was treated with high-dose cytarabine followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT). This is the first report of pancreatic IMS with t(8;21). Positron emission tomography/computed tomography and genetic study are useful for the diagnosis, and allo-HSCT achieved complete remission in this patient.

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.

Bortezomib plus dexamethasone vs thalidomide plus dexamethasone for relapsed or refractory multiple myeloma

A randomized phase II selection design study (JCOG0904) was carried out to evaluate the more promising regimen between bortezomib (Bor) plus dexamethasone (Dex; BD) and thalidomide (Thal) plus Dex (TD) in Bor and Thal‐naïve patients with relapsed or refractory multiple myeloma (RRMM). Patients ≥20 and <80 years old with a documented diagnosis of symptomatic multiple myeloma (MM) who received one or more prior therapies were randomized to receive BD (Bor 1.3 mg/m2) or TD (Thal 200 mg/d). In both arms, 8 cycles of induction (3‐week cycle) were followed by maintenance phase (5‐week cycle) until disease progression, unacceptable toxicity, or patient refusal. The primary end‐point was 1‐year progression‐free survival (PFS). Forty‐four patients were randomized and assigned to receive BD and TD (n = 22, each group). At a median follow‐up of 34.3 months, the 1‐year PFS in the BD and TD arms were 45.5% (95% confidence interval (CI), 24.4%‐64.3%) and 31.8% (95% CI, 14.2%‐51.1%), respectively, and the overall response rates were 77.3% and 40.9%, respectively. The 3‐year overall survival (OS) was 70.0% (95% CI, 44.9%‐85.4%) in the BD, and 48.8% (95% CI, 25.1%‐69.0%) in the TD arm. Among grade 3/4 adverse events, thrombocytopenia (54.5% vs 0.0%) and sensory peripheral neuropathy (22.7% vs 9.1%) were more frequent in BD when compared with the TD arm. Patients treated with BD had better outcomes than those treated with TD with regard to 1‐year PFS and 3‐year OS. Thus, BD was prioritized over TD for further investigations in Bor and Thal‐naïve RRMM patients. (Clinical trial registration no. UMIN000003135.)

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