Blocking cholesterol efflux mechanism is a potential target for antilymphoma therapy.

Cholesterol is an essential plasma membrane lipid for the maintenance of cellular homeostasis and cancer cell proliferation. Free cholesterol is harmful to cells; therefore, excessive free cholesterol must be quickly esterified by acetyl-coenzyme A:cholesterol acetyltransferase (ACAT) and exported by scavenger receptor class B member I (SR-BI) or ATP-binding cassette protein A1 from specific cells such as macrophage foam cells, which contain cholesteryl ester-derived vacuoles. Many vacuoles are present in the cytoplasm of Burkitt lymphoma cells. In this study, we observed that these vacuoles are often seen in high-grade lymphomas. Cell culture study using lymphoma cell lines found that esterified cholesterol is the main component of these vacuoles and the expression of cholesterol metabolism-related molecules was significantly upregulated in lymphoma cell lines, with SR-BI and ACAT inhibitors (BLT-1 and CI-976, respectively) impeding lymphoma cell proliferation. Cytoplasmic free cholesterol was increased by ACAT and SR-BI inhibitors, and the accumulation of free cholesterol induced lymphoma cell apoptosis by inducing endoplasmic reticulum stress. Furthermore, synergistic effects of SR-BI and ACAT inhibitors were observed in a preclinical study. Treatment with SR-BI inhibitor suppressed lymphoma progression in a tumor-bearing mouse model, whereas ACAT inhibitor did not. Therefore, SR-BI inhibitors are potential new antilymphoma therapeutics that target cholesterol metabolism.

Relationship between Serum Bortezomib Concentration and Emergence of Diarrhea in Patients with Multiple Myeloma and/or AL Amyloidosis.

(1) Background: multiple myeloma patients have benefited from bortezomib therapy, though it has often been discontinued owing to diarrhea. The objective of this study was to verify serum bortezomib concentration in the emergence of diarrhea. (2) Methods: this prospective, observational case-control, and monocentric study was performed with an approval by the Ethics Committee of Kumamoto University Hospital in 2015 (No. 1121) from February 2015 to April 2017. (3) Results: twenty-four patients with bortezomib therapy were recruited; eight patients (33.3%) developed diarrhea at day 3 as median. Median measured trough bortezomib concentration at 24 h after first or second dose for patients with or without diarrhea was 0.87 or 0.48 ng/mL, respectively (p = 0.04, Wilcoxon signed rank test). Receiver operation characteristic (ROC) analysis produced the cut-off concentration of 0.857 ng/mL (area under the ROC curve of 0.797, sensitivity of 0.625, specificity of 0.875). The survival curves between patients with and without diarrhea were similar (p = 0.667); those between patients with higher and lower concentration than median value (0.61 ng/mL) were also similar (p = 0.940). (4) Conclusions: this study indicated the possible involvement of serum bortezomib concentration in the emergence of diarrhea in bortezomib therapy in patients with multiple myeloma.

[Spontaneous remission of acquired factor XIII inhibitor concurrent to development of IgA-λ type multiple myeloma].

Coagulation factor XIII is a fibrin-stabilizing factor that leads to the crosslinking of fibrin when activated by thrombin. Acquired factor XIII inhibitor is caused when antibodies are generated against factor XIII, reducing its activity. Here we report a case of acquired factor XIII inhibitor. Although prednisolone was administered, factor XIII activity was not recovered. Interestingly, the activity normalized following the onset of multiple myeloma. The presence of inhibitors was evaluated in the patient's plasma by absorption tests and enzyme-linked immunosorbent assay. Immunoglobulin G inhibitors of factor XIII were present at admission, but later decreased with the onset of the IgA-λ-type myeloma. Thus, it is possible that the level of factor XIII inhibitors and polyclonal immunoglobulins could have been suppressed by the progression of myeloma, resulting in the normalization of factor XIII activity.

Novel p97/VCP inhibitor induces endoplasmic reticulum stress and apoptosis in both bortezomib-sensitive and -resistant multiple myeloma cells.

p97/VCP is an endoplasmic reticulum (ER)-associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER-associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell-based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50 , 100-500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib-resistant cell line and primary myeloma cells purified from patients. Accumulation of poly-ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly-ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1-0.8 µmol/L) than those of DBeQ (2-5 µmol/L). In silico protein-drug-binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell-free ATPase assays, OSSL_325096 showed dose-dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti-myeloma activity, at least in part through p97/VCP inhibition.

Expression of IL-34 correlates with macrophage infiltration and prognosis of diffuse large B-cell lymphoma.

OBJECTIVES: Infiltration of macrophages through the tyrosine kinase receptor CSF1R is a poor prognosis factor in various solid tumors. Indeed, these tumors produce CSF1R ligand, macrophage colony-stimulating factor (M-CSF) or interleukin-34 (IL-34). However, the significance of these cytokines, particularly, the newly discovered IL-34 in haematological malignancies, is not fully understood. We therefore analysed the role of IL-34 in diffuse large B-cell lymphoma (DLBCL), the most common subtype of malignant lymphoma. METHODS: We analysed formalin-fixed paraffin-embedded lymphoma tissues of 135 DLBCL patients for the expression of IL-34 and the number of macrophages, and the survival of these patients. The expression of IL-34 in DLBCL cell lines and the activity of IL-34 to induce the migration of monocytic cells were also characterised. RESULTS: Several lymphoma tissues showed a clear IL-34 signal, and such signal was detectable in 36% of patients. DLBCL cell lines also expressed IL-34. Interestingly, the percentage of IL-34+ patients in the activated B-cell subtype was significantly higher than that in the germinal centre B-cell subtype. More interestingly, IL-34+ patients showed shorter survival periods and higher number of macrophages in lymphoma tissues. The recruitment of monocytes is likely the first step for the higher macrophage density in the IL-34+ lymphoma tissues. Indeed, IL-34 induced the migration of monocytic cells. CONCLUSION: Our results raise the possibility that IL-34 in lymphoma tissues of DLBCL patients recruits monocytes, leading to the higher number of macrophages in the tissues and poor prognosis of patients. IL-34 may be an additional therapeutic target of DLBCL.

Base-to-apex gradient pattern of cardiac impairment identified on myocardial T1 mapping in cardiac amyloidosis.

Late gadolinium enhancement imaging by cardiac magnetic resonance imaging (CMR) is the most reliable method for identifying cardiac involvement in patients with amyloidosis, and myocardial T1 mapping is a novel CMR technique that enables the noninvasive detection and quantification of myocardial amyloid burden. Although, base-to-apex gradient patterns of impairment in patients with cardiac amyloidosis have been reported on myocardial strain analysis using echocardiography, we could not find any other reports to demonstrate that myocardial T1 mapping on CMR can clearly identify a base-to-apex gradient pattern of cardiac impairment in a patient with cardiac amyloidosis.

Potential anti-lymphoma effect of M-CSFR inhibitor in adult T-cell leukemia/lymphoma.

The c-fms proto-oncogene is also known as macrophage colony stimulating factor receptor (M-CSFR) or colony-stimulating factor-1 receptor (CSF-1R), and is expressed on several types of malignant tumor cells and myeloid cells. In the present study, we found that overexpression of M-CSFR was present in adult T-cell leukemia/lymphoma (ATLL) cases. M-CSFR signaling was associated with lymphoma cell proliferation, and M-CSFR inhibition induced apoptosis in lymphoma cells. The ATLL cell line ATL-T expressed M-CSF/CSF-1 and interleukin (IL)-34, which are both M-CSFR ligands. M-CSF and IL-34 expression was seen in ATLL cases, and co-expression of these ligands was detected in 11 of 13 ATLL cases. M-CSFR inhibition suppressed programmed death-1 and -2 ligand in ATL-T cells and macrophages stimulated with conditioned medium from ATL-T cells. Thus, an M-CSFR inhibitor may be useful as additional therapy against ATLL due to direct and indirect mechanisms.

MUC1/KL-6 expression confers an aggressive phenotype upon myeloma cells.

The sialic glycoprotein, MUC1, is known to be involved in the pathogenesis of various types of cancers. KL-6 is one of the surface antigens of MUC1 and also a marker of interstitial pneumonitis. A fraction of patients with myeloma (3.9%) have elevated serum KL-6 levels without any evidence of interstitial pneumonitis and their myeloma cells have high MUC1 expression. We established a myeloma cell line designated EMM1 from a patient with multiple myeloma accompanied with elevated serum KL-6. EMM1 cells expressed high levels of MUC1 compared with other myeloma cell lines. Knockdown of MUC1 in EMM1 cells induced cell cycle arrest during S phase and apoptosis, suggesting that the MUC1 expression is involved in accelerated growth of EMM1 cells. RNA-seq analysis suggests that MUC1 expression activates k-ras and TNFα-induced NFκB pathways in EMM1 cells. We injected EMM1 cells subcutaneously into Rag2-/-Jak3-/- Balb/c mice to establish a mouse xenograft model. These mice had aggressive tumor growth that was accompanied by high serum KL-6 levels. In addition, MUC1 knockdown in EMM1 cells led to inhibited tumor growth. These findings demonstrate that MUC1 serves as a potential target for developing drugs for treatment of patients with KL-6+ myeloma, and EMM1 cells and EMM1-engrafted mice are useful tools for the development of such novel agents.

Differential Roles of Rad18 and Chk2 in Genome Maintenance and Skin Carcinogenesis Following UV Exposure.

Defects in DNA polymerase Eta (Polη) cause the sunlight-sensitivity and skin cancer-propensity disorder xeroderma pigmentosum variant. The extent to which Polη function depends on the upstream E3 ubiquitin ligase Rad18 is controversial and has not been investigated using mouse models. Therefore, we tested the role of Rad18 in UV-inducible skin tumorigenesis. Because Rad18 deficiency leads to compensatory DNA damage signaling by Chk2, we also investigated genetic interactions between Rad18 and Chk2 in vivo. Chk2-/-Rad18-/- mice were prone to spontaneous lymphomagenesis. Both Chk2-/- and Chk2-/-Rad18-/- mice were prone to UV-B irradiation-induced skin tumorigenesis when compared with wild-type (WT) animals, but unexpectedly Rad18-/- mice did not recapitulate the skin tumor propensity of Polη mutants. UV-irradiated Rad18-/- cells were more susceptible to G1/S arrest and apoptosis than WT cultures. Chk2 deficiency alleviated both UV-induced G1/S phase arrest and apoptosis of WT and Rad18-/- cells, but led to increased genomic instability. Taken together, our results demonstrate that the tumor-suppressive role of Polη in UV-treated skin is Rad18 independent. We also define a role for Chk2 in suppressing UV-induced skin carcinogenesis in vivo. This study identifies Chk2 dysfunction as a potential risk factor for sunlight-induced skin tumorigenesis in humans.

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.

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.

Analysis of deposited peptides in amyloid lesions of AL amyloidosis by mass spectrometry.

Although immunoglobulin light chain (LC) plays a critical role in AL amyloidosis, detailed characteristics of deposited LC are not well known. In this study, LC peptides were analyzed by a combination of laser microdissection, liquid chromatography, and mass spectrometry (LC-MS/MS) in 65 patients with AL amyloidosis. Constant regions of LC were detected in all patients. Kappa- or lambda-types were detected in 20 and 45 patients, respectively. Various types of constant regions of LC were found; however, IGLC3 and IGKC4 were the most frequently detected. Besides LC, apolipoproteins and vitronectin were repeatedly found in amyloid lesions. These results suggest marked heterogeneity in terms of subtype of constant regions of LC in amyloid lesions. Immunohistochemistry identified LC in approximately half of patients in whom LC was detected by LC-MS/MS. This finding indicates superiority of LC-MS/MS over IHC for the detection of LC.

Cell adhesion molecule-1 (CADM1) expressed on adult T-cell leukemia/lymphoma cells is not involved in the interaction with macrophages.

Cell adhesion molecule 1 (CADM1) is a cell adhesion molecule that is expressed in brain, liver, lung, testis, and some kinds of cancer cells including adult T-cell leukemia/lymphoma (ATLL). Recent studies have indicated the involvement of CADM1 in cell-cell contact between cytotoxic T-lymphocytes and virus infected cells. We previously reported that cell-cell interaction between lymphoma cells and macrophages induces lymphoma cell proliferation. In the present study, we investigated whether CADM1 is associated with cell-cell interaction between several human lymphoma cell lines and macrophages.CADM1 expression was observed in the ATLL cell lines, ATN-1, ATL-T, and ATL-35T, and in the B cell lymphoma cell lines, TL-1, DAUDI, and SLVL, using western blotting. Significant cell-cell interaction between macrophages and ATN-1, ATL-T, ATL-35T and MT-2, DAUDI, and SLVL cells, as assessed by induction of cell proliferation, was observed. Immunohistochemical analysis of human biopsy samples indicated CADM1 expression in 10 of 14 ATLL cases; however, no case of follicular lymphoma or diffuse large B-cell lymphoma was positive for CADM1. Finally, the interaction of macrophages with cells of the CADM1-negative ED ATLL cell line and CADM1-transfected ED cells was tested. However, significant cell-cell interaction between macrophage and CADM1-transfected ED cells was not observed. We conclude that CADM1 was not associated with cell-cell interaction between lymphoma cells and macrophages, although CADM1 may be a useful marker of ATLL for diagnostic procedures.

A xenograft model reveals that PU.1 functions as a tumor suppressor for multiple myeloma in vivo.

We previously demonstrated that PU.1 expression is down-regulated in the majority of myeloma cell lines and primary myeloma cells from patients. We introduced the tet-off system into the human myeloma cell lines U266 and KMS12PE that conditionally express PU.1 and demonstrated that PU.1 induces cell cycle arrest and apoptosis in myeloma cells in vitro. Here, we established a mouse xenograft model of myeloma using these cell lines to analyze the effects of PU.1 on the phenotype of myeloma cells in vivo. When doxycycline was added to the drinking water of mice engrafted with these myeloma cells, all mice had continuous growth of subcutaneous tumors and could not survived more than 65 days. In contrast, mice that were not exposed to doxycycline did not develop subcutaneous tumors and survived for at least 100 days. We next generated mice engrafted with subcutaneous tumors 5-10 mm in diameter that were induced by exposure to doxycycline. Half of the mice stopped taking doxycycline-containing water, whereas the other half kept taking the water. Although the tumors in the mice taking doxycycline continued to grow, tumor growth in the mice not taking doxycycline was significantly suppressed. The myeloma cells in the tumors of the mice not taking doxycycline expressed PU.1 and TRAIL and many of such cells were apoptotic. Moreover, the expression of a cell proliferation marker Ki67 was significantly decreased in tumors from the mice not taking doxycycline, compared with that of tumors from the mice continuously taking doxycycline. The present data strongly suggest that PU.1 functions as a tumor suppressor of myeloma cells in vivo.

Successful Treatment of Amyloid Light-chain Amyloidosis in a Charcot-Marie-Tooth Disease Patient with Lenalidomide, Cyclophosphamide, and Dexamethasone.

A 70-year-old woman with Charcot-Marie-Tooth disease (CMT) suffered from nephrotic syndrome and a renal biopsy revealed non-AA amyloid depositions that contained immunoglobulin light chain λ. Her serum λ free LC was elevated to 80.8 mg/L and she was diagnosed with primary amyloid light-chain (AL) amyloidosis. She was subsequently treated with lenalidomide, cyclophosphamide, and dexamethasone (RCD). After 14 cycles of RCD, she achieved complete remission. Her serum albumin levels gradually normalized to 3.1 g/dL. No exacerbation of neurologic symptoms related to CMT was observed. Thus, RCD may be a well-tolerated and effective regimen for treating AL amyloidosis in patients with CMT disease.

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.

Successful treatment of TAFRO syndrome, a variant type of multicentric Castleman disease with thrombotic microangiopathy, with anti-IL-6 receptor antibody and steroids.

TAFRO syndrome is a rare variant type of multicentric Castleman disease, which is characterized by thrombocytopenia, anasarca, reticulin fibrosis of bone marrow, renal dysfunction and organomegaly. Here, we report a case of TAFRO syndrome that was successfully treated with tocilizumab. A 50-year-old man, who presented with fever, epigastric pain, abdominal fullness, and massive edema of the extremities, was admitted to our hospital. Computed tomography revealed bilateral pleural effusions, ascites, and lymphadenopathy. Laboratory data showed renal dysfunction, anemia, and thrombocytopenia. Examination of bone marrow and cervical lymph nodes led to a diagnosis of hyaline vascular-type Castleman disease. The level of serum interleukin (IL)-6 was extremely high. TAFRO syndrome was finally diagnosed. The patient was treated weekly with tocilizumab, an anti-IL-6 receptor antibody and steroids. In 4 weeks, all symptoms disappeared and serum IL-6 level returned to normal. Activity of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which was significantly decreased (9.9 %) prior to treatment, increased after treatment with tocilizumab. The present case suggests that tocilizumab is an effective therapeutic agent for TAFRO syndrome. We suggest that hypercytokinemia in TAFRO syndrome inhibits ADAMTS13 activity, thereby inducing thrombotic microangiopathy.

Immunomodulatory drugs act as inhibitors of DNA methyltransferases and induce PU.1 up-regulation in myeloma cells.

Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide are efficacious in the treatment of multiple myeloma and significantly prolong their survival. However, the mechanisms of such effects of IMiDs have not been fully elucidated. Recently, cereblon has been identified as a target binding protein of thalidomide. Lenalidomide-resistant myeloma cell lines often lose the expression of cereblon, suggesting that IMiDs act as an anti-myeloma agent through interacting with cereblon. Cereblon binds to damaged DNA-binding protein and functions as a ubiquitin ligase, inducing degradation of IKZF1 and IKZF3 that are essential transcription factors for B and T cell development. Degradation of both IKZF1 and IKZF3 reportedly suppresses myeloma cell growth. Here, we found that IMiDs act as inhibitors of DNA methyltransferases (DMNTs). We previously reported that PU.1, which is an ETS family transcription factor and essential for myeloid and lymphoid development, functions as a tumor suppressor in myeloma cells. PU.1 induces growth arrest and apoptosis of myeloma cell lines. In this study, we found that low-dose lenalidomide and pomalidomide up-regulate PU.1 expression through inducing demethylation of the PU.1 promoter. In addition, IMiDs inhibited DNMT1, DNMT3a, and DNMT3b activities in vitro. Furthermore, lenalidomide and pomalidomide decreased the methylation status of the whole genome in myeloma cells. Collectively, IMiDs exert demethylation activity through inhibiting DNMT1, 3a, and 3b, and up-regulating PU.1 expression, which may be one of the mechanisms of the anti-myeloma activity of IMiDs.

Rituximab Monotherapy and Rituximab-Containing Chemotherapy Were Effective for Paraneoplastic Pemphigus Accompanying Follicular Lymphoma, but not for Subsequent Bronchiolitis Obliterans.

A 60-year-old male patient suffered from mild exertional dyspnea, wheezing, and systemic blisters. He was diagnosed with paraneoplastic pemphigus (PNP) with follicular lymphoma in the pancreas head and pelvic cavity. He was first treated with eight cycles of rituximab; his blisters and erosions gradually improved and highly elevated levels of auto-antibodies related to PNP gradually decreased to normal levels. However, obstructive and restrictive respiratory failure still progressed. Computed tomography of the inspiratory and expiratory phases revealed obstructive pulmonary disorder, leading to a diagnosis of bronchiolitis obliterans (BO). The patient underwent plasma exchange and was repeatedly treated with rituximab monotherapy and rituximab-containing chemotherapies, but died 7 months after the diagnosis of BO. Early introduction of rituximab-containing regimens may be necessary to prevent the development of BO accompanying PNP. However, when a diagnosis of PNP-related BO is made, lung transplantation may also be considered for patients in whom rituximab-containing regimens are effective for PNP.