Lenalidomide and pomalidomide potently interfere with induction of myeloid-derived suppressor cells in multiple myeloma.

An increase in immunosuppressive myeloid-derived suppressor cells (MDSCs) is associated with disease progression and treatment resistance in multiple myeloma (MM). We investigated the mechanisms underlying MDSC induction, and sought to discover a strategy for prevention of MDSC induction in MM. Using a transwell co-culture system, four of nine examined human myeloma-derived cell lines (HMCLs) were potent in inducing monocytic (M)-MDSCs from normal peripheral blood mononuclear cells (PBMCs). As the results, we identified that secretion of C-C motif chemokine ligand 5 (CCL5) and macrophage migration inhibitory factor (MIF) by myeloma cells is a prerequisite for induction of MDSCs in MM. The immunomodulatory drug (IMiD) compounds, such as lenalidomide (LEN) and pomalidomide (POM), were identified as potent inhibitors of MDSC induction through bidirectional molecular effects of cereblon (CRBN)-dependent and -independent downregulation of CCL5 and MIF in myeloma cells; and downregulation of C-C motif chemokine receptor 5, a receptor for CCL5, and induction of interferon regulatory factor 8, a critical transcription factor for monocytic differentiation, in PBMCs. In the present study of the molecular mechanisms underlying MDSC induction, we identified a novel effect of LEN and POM of inhibiting MDSC induction via overlapping regulatory effects in myeloma cells and normal PBMCs.

Dual targeting of bromodomain-containing 4 by AZD5153 and BCL2 by AZD4320 against B-cell lymphomas concomitantly overexpressing c-MYC and BCL2.

Despite the recent therapeutic progress, the prognoses of diffuse large B-cell lymphomas (DLBCLs) that concomitantly overexpress c-MYC and BCL2, i.e., double hit lymphoma (DHL) and double expressing lymphoma (DEL), remain poor. This study examined triple targeting of c-MYC, BCL2 and the B-cell receptor (BCR) signaling pathway for DHL and DEL. We first used AZD5153, a novel bivalent inhibitor for bromodomain-containing 4 (BRD4), in DHL- and DEL-derived cell lines, because BRD4 regulates disease type-oriented key molecules for oncogenesis. AZD5153 was more effective than conventional monovalent BRD4 inhibitors, JQ1 and I-BET151, in inhibiting cell proliferation of a DHL-derived cell line and two DEL-derived cell lines, with at least 10-fold lower half growth inhibitory concentrations. AZD5153 caused G1/S cell cycle blockade, while the apoptosis-inducing effect was relatively modest. At the molecular level, AZD5153 was potent in downregulating various molecules for oncogenesis, such as c-MYC, AKT2 and MAP3K; those involved in the BCR signaling pathway, such as CD19, BLNK and CD79B; and those associated with B-cell development, such as IKZF1, IKZF3, PAX5, POU2AF1 and EBF1. In contrast, AZD5153 did not decrease anti-apoptotic BCL2 proteins, and did not activate pro-apoptotic BH3-only proteins, except BAD. To augment cell death induction, we added a novel BH3-mimicking BCL2 inhibitor AZD4320 to AZD5153, and found that these two agents had a mostly synergistic antitumor effect by increasing cells undergoing apoptosis in all three cell lines. These results provide a rationale for dual targeting of BRD4 and BCL2 using AZD5153 and AZD4320 as a therapeutic strategy against DHL and DEL.

Tumor-specific transcript variants of cyclin D1 in mantle cell lymphoma and multiple myeloma with chromosome 11q13 abnormalities.

Cyclin D1 (CCND1) overexpression is an early and unifying oncogenic event in mantle cell lymphoma (MCL) and multiple myeloma (MM) with chromosome 11q13 abnormalities. Herein, we report newly discovered transcript variants of the CCND1 gene in MCL and MM cells with chromosome 11q13 abnormalities. These transcript variants, designated CCND1.tv., covered the full-length coding region of CCND1 with longer 5'-untranslated regions (5'-UTRs) of CCND1 and occasionally contained a novel exon. CCND1.tv. was specifically detectable in patient-derived primary MCL or MM cells with chromosomal translocation t(11;14)(q13;q32), but not in t(11;14)-negative cells. The lengths of the 5'-UTR sequences of CCND1.tv. differed among patients and cell lines. Introduction of CCND1.tv. led to increased expression of normal-sized CCND1 protein in HEK293 cells. Furthermore, mTOR inhibition by rapamycin or serum starvation reduced ectopic expression of CCND1.tv.-derived CCND1 protein, but not 5'-UTR less CCND1-derived CCND1 protein in HEK293 cells, suggesting that the protein expression of CCND1.tv. is regulated by the mTOR pathway. Our results suggest that the aberrant expression of CCND1.tv. may contribute to the understanding of the pathogenesis of MCL and MM with 11q13 abnormalities.

BRD4-Regulated Molecular Targets in Mantle Cell Lymphoma: Insights into Targeted Therapeutic Approach.

BACKGROUND: Since bromodomain-containing protein 4 (BRD4) facilitates the transcription of genes important for neoplastic cells in a cancer-type specific manner, BRD4-regulated molecules may also include therapeutic targets for mantle cell lymphoma (MCL), a treatment-refractory subtype of malignant lymphoma. MATERIALS AND METHODS: In order to uncover direct BRD4-regulated targets in MCL, we performed integrated analysis using the pathway database and the results of both gene-expression profiling and chromatin immunoprecipitation with parallel sequencing for BRD4. RESULTS: Treatment with BRD4 inhibitor I-BET151 exerted a dose-dependent inhibitory effect on cell proliferation in MCL cell lines. BRD4 was found to directly regulate series of genes involved in the B-cell receptor (BCR) signaling pathway, including B-cell linker (BLNK), paired box 5 (PAX5), and IKAROS family zinc finger 3 (IKZF3), and several oncogenes, such as MYB. Indeed, the combinatory inhibition of BCR pathway and IKZF showed an additive antitumor effect. CONCLUSION: Concomitant targeting multiple BRD4-regulated molecules may constitute a rational therapeutic strategy for MCL.

A novel method of amplified fluorescent in situ hybridization for detection of chromosomal microdeletions in B cell lymphoma.

Chromosomal microdeletions frequently cause loss of prognostically relevant tumor suppressor genes in hematologic malignancies; however, detection of minute deletions by conventional methods for chromosomal analysis, such as G-banding and fluorescence in situ hybridization (FISH), is difficult due to their low resolution. Here, we describe a new diagnostic modality that enables detection of chromosomal microdeletions, using CDKN2A gene deletion in B cell lymphomas (BCLs) as an example. In this method, which we refer to as amplified-FISH (AM-FISH), a 31-kb fluorescein isothiocyanate (FITC)-conjugated DNA probe encoding only CDKN2A was first hybridized with the chromosome, and then labeled with Alexa Fluor 488-conjugated anti-FITC secondary antibody to increase sensitivity. CDKN2A signals were equally identifiable by AM-FISH and conventional FISH in normal mononuclear blood cells. In contrast, when two BCL cell lines lacking CDKN2A were analyzed, CDKN2A signals were not detected by AM-FISH, whereas conventional FISH yielded false signals. Furthermore, AM-FISH detected CDKN2A deletions in two BCL patients with 9p21 microdeletions, which were not detected by conventional FISH. These results suggest that AM-FISH is a highly sensitive, specific, and simple method for diagnosis of chromosomal microdeletions.

Human herpesvirus-6 pneumonitis in a patient with follicular lymphoma following immunochemotherapy with rituximab.

Primary infection with human herpesvirus-6 (HHV-6) commonly occurs at an early age in children, most often at 3 years of age, and is associated with childhood diseases, such as exanthema subitum, hepatitis, febrile convulsions, or encephalitis. However, the virus occasionally reactivates from its latent state in immunosuppressed adults, especially post-transplant, resulting in serious disseminated, sometimes life-threatening end-organ complications. Herein, we report a case of a 68-year-old man with relapsed follicular lymphoma who developed HHV-6 pneumonitis. Eighteen months after achieving second complete remission by salvage immunochemotherapy with rituximab, the patient was complicated by pneumonia, with chest computed tomography finding showing disseminated nodular shadows with ground-glass opacity in both lungs. While empiric antibiotic and antifungal therapies did not improve the pneumonia, polymerase chain reaction-based viral screening tests on his bronchoalveolar lavage fluid detected the presence of HHV-6 DNA, and ganciclovir treatment quickly resolved the pneumonia, indicating that he suffered from HHV-6 pneumonitis. He had no other HHV-6-related end-organ damage, such as encephalitis. This case suggests that, although extremely rare, HHV-6 reactivation should be considered as one of the candidate pathogens for pulmonary complications of uncertain etiology in patients who have been treated with intensive immunosuppressive chemotherapy, even without hematopoietic stem cell transplantation. Furthermore, polymerase chain reaction-based viral screening testing on bronchoalveolar lavage fluid is a powerful diagnostic tool for pneumonitis due to viral reactivation, including HHV-6 reactivation.

Establishment and Characteristics of a Novel Mantle Cell Lymphoma-derived Cell Line and a Bendamustine-resistant Subline.

BACKGROUND/AIM: Bendamustine hydrochloride (BH) is a key therapeutic agent for mantle cell lymphoma (MCL), while the mechanism underlying BH-resistance has not been verified. MATERIALS AND METHODS: We compared molecular/biological characteristics of a newly-generated MCL-derived cell line KPUM-YY1 and its BH-resistant subline KPUM-YY1R. RESULTS: The growth-inhibitory IC50 for BH was 20 µM in KPUM-YY1 cells, while cell proliferation was not inhibited by up to 60 µM BH in KPUM-YY1R cells. Compared to KPUM-YY1 cells, gene expression profiling in KPUM-YY1R cells revealed up-regulation of 312 genes, including ABCB1 encoding P-glycoprotein (P-gp), and microsomal glutathione S-transferase 1 (MGST1). Addition of either a P-gp inhibitor or a GST inhibitor, at least partly, restored sensitivity to BH in KPUM-YY1R cells. In addition, KPUM-YY1R cells showed cross-resistance against various anti-MCL chemotherapeutics. CONCLUSION: BH resistance is mediated by overlapping mechanisms with overexpression of ABCB1 and MGST1, and is potentially accompanied by multidrug resistance in MCL.

Phosphoinositide-dependent protein kinase 1 is a potential novel therapeutic target in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a relatively rare subtype of B-cell non-Hodgkin lymphoma (NHL) that has a poor prognosis despite recent advances in immunochemotherapy and molecular targeted therapeutics against NHL. Therefore, the development of a new therapeutic strategy for MCL is urgently needed. In this study, we show for the first time that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), an oncogenic serine-threonine protein kinase, is commonly expressed in its phosphorylated active form in patient-derived tumor cells of various types of B-cell NHL cells, including diffuse large B-cell lymphoma, follicular lymphoma, and MCL. Blockade of PDPK1 activity by small-molecule inhibitors specific for PDPK1 (BX-912 and GSK2334470) or by RNA interference exerted antiproliferative effects in all four MCL-derived cell lines examined and these growth-inhibitory effects were mediated by both induction of apoptosis and G2/M cell cycle blockade. In addition, blockade of PDPK1 led to inactivation of its downstream effector kinase RSK2, but not AKT, suggesting the importance of the PDPK1/RSK2 signaling pathway in the proliferation and survival of MCL cells. Finally, when combined with anticancer agents, including genotoxic agents, a proteasome inhibitor, and a BH3 mimetic in vitro, the PDPK1 inhibitor BX-912 showed additive growth-inhibitory effects against MCL-derived cell lines in most settings. In particular, the combination of BX-912 and ABT-263, a BH3 mimetic, resulted in the enhancement of the induction of apoptosis. In conclusion, our results suggest that PDPK1 is a potential novel therapeutic target in MCL and indicate that clinical development of PDPK1-targeted therapy for MCL is desirable.

Chromosomal abnormality variation detected by G-banding is associated with prognosis of diffuse large B-cell lymphoma treated by R-CHOP-based therapy.

Diffuse large B-cell lymphoma (DLBCL), which is the most prevalent disease subtype of non-Hodgkin lymphoma, is highly heterogeneous in terms of cytogenetic and molecular features. This study retrospectively investigated the clinical impact of G-banding-defined chromosomal abnormality on treatment outcomes of DLBCL in the era of rituximab-containing immunochemotherapy. Of 181 patients who were diagnosed with DLBCL and treated with R-CHOP or an R-CHOP-like regimen between January 2006 and April 2014, metaphase spreads were evaluable for G-banding in 120. In these 120 patients, 40 were found to harbor a single chromosomal aberration type; 63 showed chromosomal abnormality variations (CAVs), which are defined by the presence of different types of chromosomal abnormalities in G-banding, including 19 with two CAVs and 44 with ≥3 CAVs; and 17 had normal karyotypes. No specific chromosomal break point or numerical abnormality was associated with overall survival (OS) or progression-free survival (PFS), but the presence of ≥3 CAVs was significantly associated with inferior OS rates (hazard ratio (HR): 2.222, 95% confidence interval (CI): 1.056-4.677, P = 0.031) and tended to be associated with shorter PFS (HR: 1.796, 95% CI: 0.965-3.344, P = 0.061). In addition, ≥3 CAVs more frequently accumulated in high-risk patients, as defined by several conventional prognostic indices, such as the revised International Prognostic Index. In conclusion, our results suggest that the emergence of more CAVs, especially ≥3, based on chromosomal instability underlies the development of high-risk disease features and a poor prognosis in DLBCL.

Burkitt Lymphoma Preceded by Autoimmune Hemolytic Anemia due to Anti-D Antibody.

We herein report a rare case of Burkitt lymphoma (BL) preceded by autoimmune hemolytic anemia (AIHA) caused by autoantibodies against D antigen. After a partial response to AIHA with prednisolone (PSL) treatment for 7 months, the patient developed BL with a t(8;22)(q24;q11.2) chromosomal translocation. Intensive immunochemotherapy, including rituximab, led to a complete response (CR) of BL; however, anti-D antibody remained detectable in the plasma and antibody-dissociated solution from erythrocytes, thus continuous therapy with PSL was necessary even after achievement of the CR. BL with AIHA is extremely rare, with only one previously reported case in the literature.

Clinicopathological significance of carbonic anhydrase 9, glucose transporter-1, Ki-67 and p53 expression in oral squamous cell carcinoma.

Carbonic anhydrase 9 (CA9) is a glycoprotein present on the surface of cell membranes. It is expressed in 90% of renal cancer cells, but not in normal kidney tissue. Immunotherapy targeting CA9 is underway, and our group has also conducted a clinical trial using CA9 as a cancer vaccine and confirmed the induction of cytotoxic T lymphocytes, with efficacy in some cases. Expression of CA9 antigen in oral cancer has not been reported in Japan, but our results indicate that immunotherapy targeting CA9 might be possible. We immunohistochemically observed the expression of antigens such as CA9, Ki-67, glucose transporter-1 (GLUT-1) and p53 in 107 subjects with oral squamous cell carcinoma, and examined their correlation with clinicopathological parameters. Immunostaining analysis showed expression of CA9 in 98% of oral cancer subjects, and the survival rate was significantly lower in subjects with CA9 antigen expression in 50% or more cells (P<0.05). Subjects with poorly differentiated, T4 and lymph node metastasis, or Stage IV cancer with high CA9 expression (≥50%) had a worse outcome than those with low CA9 expression. Although GLUT-1 expression was observed in 98% of subjects, similarly to CA9 expression, no significant correlation between its expression and the survival rate was seen. However, subjects with lymph node metastasis had significantly higher GLUT-1 expression, demonstrating that GLUT-1 could be an indicator of lymph node metastasis. Ki-67 was expressed in 92% of subjects, but no correlation with outcome was observed. Expression of p53 was noted in 78% of subjects, and it was found that many oral cancers have p53 genetic abnormalities, but no correlation between p53 and outcome was observed. It was confirmed that CA9 antigen is expressed in most oral cancer subjects, suggesting the possibility of immunotherapy targeting CA9 antigen in oral cancer.