Citations

Abstract Long non-coding RNAs (lncRNA) are key regulatory RNAs that do not code for protein. Recently lncRNAs have been recognized as key regulators of gene expression and chromatin organization. Although, deregulation of lncRNAs such as HOTAIR, MALAT1 and H19 has been reported in solid cancers, role of lncRNAs in lymphoma or other hematologic malignancies is not known. The aim of this study is to identify lymphoma-associated lncRNAs and elucidate their role in disease pathogenesis. Next generation RNA-sequencing and validation studies were carried out on mantle cell lymphoma (MCL) patient samples along with normal controls. As a result, hundreds of lncRNAs were found overexpressed in the MCL samples as compared with normal B cells, including RP11-24J23.2 (50 fold), RP11-12A2.3 (36 fold) and DLEU7-AS1 (19 fold). Quantitative RT-PCR (QRT-PCR) confirmed higher expression of RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 in MCL patient samples as compared to normal B cells. To investigate the biological significance of lncRNAs in MCL cells, we overexpressed RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs and analyzed effects on cell proliferation and apoptosis. To achieve overexpression lncRNAs, RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 were cloned individually in expression pcDNA3.1 generating constructs: pcDNA3RP11-24J23.2, pcDNA3RP11-12A2.3 and pcDNA3DLEU7-AS1and then transfected into MCL cell lines along with pcDNA3.1. QRT-PCR revealed effective overexpression (~100 fold increase over baseline) for each transfected lncRNAs construct in Granta and Mino cells. Analysis of proliferation and survival showed that overexpressing RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 promotes cell proliferation and survival in these cells as compared to the vector controls. Most of the lncRNAs found so far have been linked to the transcriptional regulation of the target genes through their association with chromatin remodeling factors such as Polycomb Repressive Complex 2 (PRC2). To identify the functional relevance of RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs, we examined the binding of the lymphoma specific lncRNAs with PRC2 complex, by performing RNA immunoprecipitation (RNA-IP) using specific antibodies to EZH2, EED and SUZ12. The RNA-IP results demonstrate high affinity binding of all three lncRNAs with SUZ12 and EED but a weak binding with EZH2. Furthermore, RNA-IP using antibody to histone 3-lysine 27 trimethylation (H3K27me3), a suppressive histone mark associated with PRC2 complex, also showed efficient binding with RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1. These results suggest that RP11-24J23.2, RP11-12A2.3, and DLEU7-AS1 lncRNAs can directly associated with PRC2 complex at chromatin. To understand the mechanism of increased cell growth after overexpression of lncRNAs, RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1, we analyzed expression level of important genes signature of MCL pathogenesis that include cyclin D1, cyclin D3, c-Myc, P14, and SOX11, a member of the group C SOX (SRY-related HMG-box) transcription factor family. Our results demonstrate that overexpression of lncRNAs RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 enhanced the levels of cyclin D1 (>2 fold) mRNA without any impact on cyclin D3 or c-Myc transcript levels. In contrast overexpression of RP11-24J23.2, RP11-12A2.3 or DLEU7-AS1 lncRNAs decreased SOX11 (0.08, 0.1 and 0.06 respectively) and P14 (0.06, 0.7 and 0.40 respectively) mRNA expression in Mino cells. To gain understanding of the mechanism involved in the suppression of SOX11 by the lncRNAs, we performed Chromatin immunoprecipitation assays with EZH2, EED and SUZ12 antibodies on Mino cells transfected with pcDNA3RP11-24J23.2, pcDNA3RP11-12A2.3 and pcDNA3DLEU7-AS1 or vector control. The data showed that overexpression of RP11-12A2.3 and DLEU7-AS1 significantly increased the binding of EED and to a lesser extent of EZH2 to SOX11 promoter while the binding of SUZ12 to SOX11 promoter was not changed as compared to vector alone. In conclusion, our results imply that lncRNAs are overexpressed in the MCL. Moreover, lymphoma associated lncRNAs (RP11-24J23.2, RP11-12A2.3 and DLEU7-AS1) regulate SOX11 expression via PRC2 complex contributing towards growth of MCL cells. Specific nature of lncRNAs found in lymphoma samples suggests that lncRNAs can be used as potential biomarkers for mantle cell lymphoma. Disclosures No relevant conflicts of interest to declare.

Key Points Cytokines in the BM microenvironment regulate PD-1 ligand expression and secretion in WM. Secreted PD-1 ligands modulate T-cell function in WM.

Abstract BACKGROUND Monocytes and macrophages (mo/mΦ) are part of composition of tumor microenvironment in B-cell non-Hodgkin lymphoma (B-NHL). CD14+HLA-DRlow monocytes are immunosuppressive and this phenotype is promoted by increased expression of IL-10 in lymphoma. Despite their association with poor outcome, monocytes retain phagocytic function particularly in the presence of monoclonal antibodies and can be associated with an improved outcome in patients treated with rituximab. The aim of this study was to determine the prevalence of CD14+HLA-DRlow mo/mΦ in B-NHL tissue compared to normal tissue, and to determine whether their phenotype suggests that they are capable of phagocytic function. METHODS Tissue mo/mΦ were isolated by negative selection from 13 diagnostic B-NHL biopsy specimens and 6 normal tissues using a monocyte enrichment kit. Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by flow cytometry, immunocytochemistry and cytometry by time-of-flight (CyTOF). The purity of isolated tissue mo/mΦ was more than 95%, being confirmed by Giemsa stain and flow cytometry. RESULTS Increased numbers of CD68+ cells in initial B-NHL biopsies when compared to normal tissue was confirmed by immunohistochemistry and flow cytometry. However, CD14 expression was substantially lower than CD68 expression in the tissues suggesting that many CD68+ mo/mΦ are CD14 negative. Using a monocyte enrichment kit to isolate all mo/mΦ, we found that CD14 negative mo/mΦ constituted half the tissue mo/mΦ. Furthermore, we found by flow cytometry, CyTOF and Giemsa stain that CD14- mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. While all cells expressed CD68, the larger cells had increased expression of CD32, CD64 and HLA-DR. The CD14+ cells typically also expressed CD163 and CD33 and were a subset of the larger monocyte population, while the population of small cells was positive only for CD68 and CD45. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αlow and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αlow cells typically constitute the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. Interestingly, while the CD14-SIRP-α- cells lack the typical phenotypic markers of mo/mΦ, they morphologically have the appearance of monocytic cells and this population appears expanded in lymphoma tissue. CONCLUSIONS We have identified a unique population of small mo/mΦ that have an immature phenotype and lack expression of CD14, SIRP-α, CD163, and other FcγR markers. This subset of mo/mΦ is prevalent in lymphoma and may have limited phagocytic function. This CD68+CD14-SIRP-α- mo/mΦ subpopulation may account for prognostic differences in the outcome of lymphoma patients treated with or without monoclonal antibodies. Citation Format: Ya-Ping Chen, Zhi-Zhang Yang, Jose C. Villasboas, Tammy Price-Troska, Anne J. Novak, Stephen M. Ansell. Expression of CD14 and SIRP-α defines distinct populations of intratumoral monocytes/macrophages in B-cell non-Hodgkin lymphomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3287.

AbstractGenetic aberrations, including trisomies 3 and 18, and well-defined IGH translocations, have been described in marginal zone lymphomas (MZLs); however, these known genetic events are present in only a subset of cases. Here, we report the cloning of an IGH translocation partner on chromosome X, t(X;14)(p11.4;q32) that deregulates expression of an poorly characterized orphan G-protein–coupled receptor, GPR34. Elevated GPR34 gene expression was detected independent of the translocation in multiple subtypes of non-Hodgkin lymphoma and distinguished a unique molecular subtype of MZL. Increased expression of GPR34 was also detected in tissue from brain tumors and surface expression of GPR34 was detected on human MZL tumor cells and normal immune cells. Overexpression of GPR34 in lymphoma and HeLa cells resulted in phosphorylation of ERK, PKC, and CREB; induced CRE, AP1, and NF-κB–mediated gene transcription; and increased cell proliferation. In summary, these results are the first to identify a role for a GPR34 in lymphoma cell growth, provide insight into GPR34-mediated signaling, identify a genetically unique subset of MZLs that express high levels of GPR34, and suggest that MEK inhibitors may be useful for treatment of GPR34-expressing tumors.

Abstract Waldenström macroglobulinemia (WM) is a rare, lymphoplasmacytic lymphoma characterized by hypersecretion of immunoglobulin M (IgM) protein and tumor infiltration into the bone marrow and lymphatic tissue. Our understanding of the mechanisms driving the development and progression of WM is currently by the shortage of representative cell models available for study. We describe here the establishment of a new WM cell line, MWCL-1. Comprehensive genetic analyses have unequivocally confirmed a clonal relationship between this novel cell line and the founding tumor. MWCL-1 cells exhibit an immunophenotype consistent with a diverse, tumor clone composed of both small B lymphocytes and larger lymphoplasmacytic cells and plasma cells: CD3−, CD19+, CD20+, CD27+, CD38+, CD49D+, CD138+, cIgM+, and κ+. Cytogenetic studies identified a monoallelic deletion of 17p13 (TP53) in both the cell line and the primary tumor. Direct DNA resequencing of the remaining copy of TP53 revealed a missense mutation at exon 5 (V143A, GTG>GCG). In accordance with primary WM tumors, MWCL-1 cells retain the ability to secrete high amounts of IgM protein in the absence of an external stimulus. The genetic, immunophenotypic, and biologic data presented here confirm the validity of the MWCL-1 cell line as a representative model of WM.

The cytokine B cell activating factor (BAFF) and its receptor, BAFF receptor (BAFF-R), modulate signaling cascades critical for B cell development and survival. We identified a novel mutation in TNFRSF13C, the gene encoding human BAFF-R, that is present in both tumor and germline tissue from a subset of patients with non-Hodgkin lymphoma. This mutation encodes a His159Tyr substitution in the cytoplasmic tail of BAFF-R adjacent to the TRAF3 binding motif. Signaling through this mutant BAFF-R results in increased NF-κB1 and NF-κB2 activity and increased immunoglobulin production compared with the wild-type (WT) BAFF-R. This correlates with increased TRAF2, TRAF3, and TRAF6 recruitment to His159Tyr BAFF-R. In addition, we document a requirement for TRAF6 in WT BAFF-R signaling. Together, these data identify a novel lymphoma-associated mutation in human BAFF-R that results in NF-κB activation and reveals TRAF6 as a necessary component of normal BAFF-R signaling.

Abstract Waldenström's macroglobulinemia (WM) is a distinct clinicobiological entity defined as a B-cell neoplasm characterized by a lymphoplasmacytic infiltrate in bone marrow (BM) and IgM paraprotein production. Cytogenetic analyses were historically limited by difficulty in obtaining tumor metaphases, and the genetic basis of the disease remains poorly defined. Here, we performed a comprehensive analysis in 42 WM patients by using a high-resolution, array-based comparative genomic hybridization approach to unravel the genetic mechanisms associated with WM pathogenesis. Overall, 83% of cases have chromosomal abnormalities, with a median of three abnormalities per patient. Gain of 6p was the second most common abnormality (17%), and its presence was always concomitant with 6q loss. A minimal deleted region, including MIRN15A and MIRN16-1, was delineated on 13q14 in 10% of patients. Of interest, we reported biallelic deletions and/or inactivating mutations with uniparental disomy in tumor necrosis factor (TNF) receptor–associated factor 3 and TNFα-induced protein 3, two negative regulators of the nuclear factor-κB (NF-κB) signaling pathway. Furthermore, we confirmed the association between TRAF3 inactivation and increased transcriptional activity of NF-κB target genes. Mutational activation of the NF-κB pathway, which is normally activated by ligand receptor interactions within the BM microenvironment, highlights its biological importance, and suggests a therapeutic role for inhibitors of NF-κB pathway activation in the treatment of WM. [Cancer Res 2009;69(8):3579–88]

Abstract The Multiple Myeloma Research Consortium has established a tissue bank for the deposition of bone marrow samples from patients with multiple myeloma to be mailed and processed under good laboratory practices. To date, over 1,000 samples have been collected. At this time, limited information is available on shipped bone marrow aspirates in regards to cell viability, yield, purity, and subsequent RNA yield and quality. To test these determinants, we did a pilot study on behalf of the Multiple Myeloma Research Consortium where samples were drawn at Mayo Clinic Rochester (MCR) pooled and split into two equal aliquots. One-half of each sample was processed following good laboratory practices compliant standard operating procedures, immediately after sample procurement, at MCR. The CD138+ cells were stored at -80°C as a Trizol lysate. The other half of the aspirate was sent overnight to Mayo Clinic Scottsdale where they were processed using identical standard operating procedures. The RNA was extracted and analyzed in a single batch at MCR. At both locations, samples were assayed for the following quality determinants: Viability was assessed using a three-color flow cytometric method (CD45, CD38, and 7-AAD). Cell counts were done to determine plasma cell recovery and post-sort purity determined by means of a slide-based immunofluorescent assay. RNA recovery and integrity was assessed using the Agilent Bioanalyzer. Lastly, gene expression profiles were compared to determine the signature emanating from the shipment of samples. Despite minor differences, our results suggest that shipment of samples did not significantly affect these quality determinants in aggregate. (Cancer Epidemiol Biomarkers Prev 2008;17(3):666–73)