miR-17~92 Promotes Progression of ABC-DLBCL Lymphoma via Regulation of Canonical NF-kB Signaling

Abstract Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoma characterized by constitutive NF-κB activation. Nevertheless, the role and mechanisms of miR-17~92 in contributing to the NF-κB activation in ABC-DLBCL are still elusive. Methods: The expression of miR-17~92 primary transcript (MIR17HG) and NF-κB target genes was determined using RNA-sequencing. The expression of miR-17~92 was performed using microarray analysis. Plasmids carrying conditional over-expression and loss-of-function of miR-17~92 were respectively constructed and dual-luciferase reporter assay was used to validate the target gene of miR-17~92. Immunoprecipitation and polyubiquitination were further used to the study of potential mechanisms.Results: Expression of MIR17HG was positively correlated with NF-κB activity, miR-17~92 activated the NF-κB signaling in ABC-DLBCL, and its over-expression promoted ABC-DLBCL cell growth, accelerated cell G1 to S phase transition and enhanced cell resistance to NF-κB inhibitor. Importantly, miR-17~92 promoted NF-κB activation through directly targeting multiple ubiquitin-editing regulators to lead to increase the K63-linked polyubiquitination and decrease the K48-linked polyubiquitination of RIP1 complex in ABC-DLBCL. We further found that miR-17~92 selectively activated IκB-α and NF-κB p65 but not NF-κB p52/p100, and high miR-17~92 expression was also associated with poorer outcome in ABC-DLBCL patients. Conclusions: Taken together, miR-17~92 selectively activate the canonical NF-κB signaling via targeting ubiquitin-editing regulators to lead to constitutively NF-κB activation and poorer outcome, which is an innovative function of miR-17~92 and previously unappreciated regulatory mechanism of NF-κB activation in ABC-DLBCL. Targeting miR-17~92 may thus provide a novel bio-therapeutic strategy for ABC-DLBCL patients.

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MiR-28-5p mediates the anti-proliferative and pro-apoptotic effects of curcumin on human diffuse large B-cell lymphoma cells

Journal of International Medical Research ◽

10.1177/0300060520943792 ◽

2020 ◽

Vol 48 (7) ◽

pp. 030006052094379

Author(s):

Tian Kang ◽

Wei-Li Sun ◽

Xiao-Fei Lu ◽

Xin-Liang Wang ◽

Lian Jiang

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Large B Cell Lymphoma ◽

Apoptotic Effects ◽

Large B Cell ◽

Dual Luciferase Reporter Assay

Objective To investigate the anti-proliferative and pro-apoptotic effects of curcumin on diffuse large B-cell lymphoma (DLBCL) cells and explore the mechanism. Methods OCI-LY7 cells were treated with curcumin (2.5, 5, 10, 20, and 40 μM) for 24, 48, or 72 hours. Cell viability and apoptosis were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide assay and TdT-mediated dUTP nick-end labeling staining, respectively. MiR-28-5p expression was detected via qRT-PCR. The binding site of miR-28-5p was predicted using online databases and verified using the dual-luciferase reporter assay. MiR-28-5p overexpression and inhibition were achieved via transfection with an miR-28-5p mimic and inhibitor, respectively. Results Curcumin decreased the viability of OCI-LY7 cells in a concentration- and time-dependent manner, and these effects were attenuated by miR-28-5p inhibition. MiR-28-5p expression was upregulated by curcumin. Curcumin increased the numbers of apoptotic cells and upregulated cleaved caspase-3 expression, and these effects were attenuated by miR-28-5p inhibition. The dual-luciferase reporter assay confirmed that miR-28-5p directly targets the 3′-untranslated region of BECN1. Curcumin downregulated BECN1 and microtubule-associated protein 1 light chain 3 beta-II/I expression and upregulated p62 expression. Conclusions Our results described the curcumin exerted anti-proliferative and pro-apoptotic effects on OCI-LY7 cells through a mechanism potentially involving miR-28-5p.

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LukS-PV-Regulated MicroRNA-125a-3p Promotes THP-1 Macrophages Differentiation and Apoptosis by Down-Regulating NF1 and Bcl-2

Cellular Physiology and Biochemistry ◽

10.1159/000485415 ◽

2017 ◽

Vol 44 (3) ◽

pp. 1093-1105 ◽

Cited By ~ 8

Author(s):

Xiao-Xi Sun ◽

Shan-Shan Zhang ◽

Chun-Yang Dai ◽

Jing Peng ◽

Qing Pan ◽

...

Keyword(s):

Cell Differentiation ◽

Cellular Differentiation ◽

Target Genes ◽

B Cell Lymphoma ◽

Neurofibromatosis Type ◽

Luciferase Reporter ◽

Loss Of Function ◽

Leukaemia Cell Line ◽

Reporter Assays

Background/Aims: LukS-PV is a component of Panton-Valentine leukocidin (PVL). We have previously demonstrated that LukS-PV potently promoted differentiation and induced apoptosis in THP-1 cells. However, the precise mechanisms of these actions remain unknown. MicroRNAs (miRs) play important roles in cellular differentiation and apoptosis. This study aimed to investigate the role of miR-125a-3p in LukS-PV-regulated differentiation and apoptosis and its underlying mechanism in THP-1 cells. Methods: MicroRNA profiling analyses were conducted to determine differential miRNA expression levels in THP-1 cells treated with LukS-PV. Cell differentiation and apoptosis were measured in THP-1 cells by gain-of-function and loss-of-function experiments. Bioinformatics analysis and luciferase reporter assays were used to confirm the targets of miR-125a-3p. The effects of the miR-125a-3p targets on cellular differentiation were determined by knocking them down. Results: MiR-125a-3p was up-regulated after treating the human monocytic leukaemia cell line THP-1 with LukS-PV. In addition, miR-125a-3p positively regulated apoptosis and differentiation in THP-1 cells treated with LukS-PV. Concordantly, luciferase reporter assays confirmed that neurofibromatosis type 1 (NF1) and B-cell lymphoma 2 (Bcl-2) were direct target genes of miR-125a-3p. Moreover, NF1 knockdown in THP-1 cells significantly promoted differentiation in vitro. Finally, the extracellular signal-regulated kinase (ERK) pathway, a downstream target of NF1, was activated after NF1 knockdown. Conclusions: These findings confirm that miR-125a-3p is involved in LukS-PV-mediated cell differentiation and apoptosis in THP-1 cells.

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miR-1270 enhances the proliferation, migration, and invasion of osteosarcoma via targeting cingulin

European Journal of Histochemistry ◽

10.4081/ejh.2021.3237 ◽

2021 ◽

Vol 65 (4) ◽

Author(s):

Yang Liu ◽

Weichun Guo ◽

Shuo Fang ◽

Bin He ◽

Xiaohai Li ◽

...

Keyword(s):

Therapeutic Strategy ◽

Target Genes ◽

Bioinformatics Analysis ◽

Tumor Xenograft ◽

Luciferase Reporter ◽

Migration And Invasion ◽

High Morbidity ◽

Proliferation And Apoptosis ◽

Dual Luciferase Reporter Assay

Osteosarcoma (OS), characterized by high morbidity and mortality, is the most common bone malignancy worldwide. MicroRNAs (miRNAs) play a crucial role in the initiation and development of OS. The purpose of this study was to investigate the roles of miR-1270 in OS. RT-qPCR and Western blot were applied to detect the mRNA and protein level, respectively. CCK-8, colony formation, and TUNEL assays were conducted to determine the cell viability, proliferation, and apoptosis of OS cells. Wound healing and transwell assay were performed to detect the migration and invasion ability of OS cells. Bioinformatics analysis and dual-luciferase reporter assay were used to predict the target genes of miR-1270. Tumor xenograft in vivo assay was carried out to determine miR-1270 effect on the tumor size, volume, and weight. In this study, miR-1270 was overexpressed in OS tissues and cells. However, miR-1270 knockdown inhibited the proliferation, migration and invasion, and promoted the OS cells’ apoptosis. Mechanistically, cingulin (CGN) was predicted and proved to be a target of miR-1270 and partially alleviated the effects of miR-1270 on the proliferation, migration and invasion ability of OS cells. Taken together, knockdown of miR-1270 may inhibit the development of OS via targeting CGN. This finding may provide a novel therapeutic strategy for OS.

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MiR-7e-5p downregulation promotes transformation of low-grade follicular lymphoma to aggressive lymphoma by modulating an immunosuppressive stroma through the upregulation of FasL in M1 macrophages

10.21203/rs.3.rs-38701/v3 ◽

2020 ◽

Author(s):

Xiaoli Lou ◽

Jianhong Fu ◽

Xin Zhao ◽

Xuemei Zhuansun ◽

Chao Rong ◽

...

Keyword(s):

Follicular Lymphoma ◽

B Cell ◽

Target Gene ◽

Fas Ligand ◽

B Cell Lymphoma ◽

Differentially Expressed ◽

Aggressive Lymphoma ◽

Low Grade ◽

M1 Macrophages ◽

M1 Macrophage

Abstract Background: In follicular lymphoma (FL), histologic transformation to high-grade FL and diffuse large B-cell lymphoma (DLBCL) is a critical adverse step in disease progression. Activation of the oncogene c-MYC and tumor microenvironment remodeling account for FL progression. A panel of microRNA (miRNA) was downregulated in transformed FL. Methods: Differentially expressed miRNAs were systematically analyzed compared in eleven lymph nodes tissue samples from patients at different stages of disease. Expression of miR-7e-5p was analyzed in 46 B-cell lymphomas, including 30 FLs and 16 DLBCLs. In FL cells, transcriptional regulation of the oncogene c-MYC on its target miR-7e-5p was revealed by Chromatin Immunoprecipitation (ChIP) assay. Exosome, carrying differentially expressed miR-7e-5p was isolated and visualized by transmission electron microscope and fluorescence tracing. The effect of miR-7e-5p on recipient macrophage was determined by target gene quantification, flow cytometry, and TUNEL method in a cocultured system with miR-7e-5p-mimics or inhibitor treatment. Expression of miR-7e-5p targets, macrophage proportions, and clinical parameters were included for correlation analysis. Results: We determined that downregulation of miR-7e-5p, driven by c-MYC overexpression, was associated with poorer prognosis in FL patients. The decreased expression of miR-7e-5p in lymphoma cells led to a reduced exosomal transfer to surrounding macrophages. As a result, the target gene of miR-7e-5p, Fas ligand (FasL), was upregulated and activated the caspase signaling, which led to the apoptosis of M1 macrophages in tumor stroma. Finally, in transformed FL tissues, overexpression of FasL and activation of caspase proteins was detected in tumor stromal macrophages. Downregulation of miR-7e-5p was associated with poorer clinical outcomes. Conclusion: Downregulation of exosomal miR-7e-5p induces stromal M1 macrophage apoptosis, which leads to immunosurveillance and transformation of FL.

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miR-199a Targeting PNRC1 to Promote Keratinocyte Proliferation and Invasion in Cholesteatoma

BioMed Research International ◽

10.1155/2021/1442093 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Lihui Yao ◽

Wenjing Zhang ◽

Jian Zheng ◽

Xing Lu ◽

Fan Zhang

Keyword(s):

Target Gene ◽

Target Genes ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Keratinocyte Proliferation ◽

Qrt Pcr ◽

Candidate Target ◽

Candidate Target Gene ◽

Proliferation And Invasion ◽

Dual Luciferase Reporter Assay

Introduction. miR-199a has been reported as an oncogene of various cancers. However, the biological function and regulatory mechanism of miR-199a in keratinocytes of cholesteatoma are still unclear. Methods. Detection by qRT-PCR was conducted on miR-199a’s expression in both thirty pairs of cholesteatoma tissues and normal skins. For characterizing the function of miR-199a, this research adopted transwell assay, wound healing assay, and CCK8 assays. Under the support of qRT-PCR, efforts were made to investigate the relative expression of candidate target genes. Moreover, the evaluation of the targeting relationship between miR-199a and the candidate target gene was conducted with the dual-luciferase reporter assay. Results. The upregulation of miR-199a was found in cholesteatoma tissues, which facilitated the proliferation, migration, and invasion of HaCaT cells, while its downregulation caused opposite results. Conclusions. The findings of the present research offer more insights into the molecular mechanism of cholesteatoma progression.

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PiRNA MW557525 Promotes the Vitality and Pluripotency of Piwil2-iCSCs by Regulating NOP56.

10.21203/rs.3.rs-439016/v2 ◽

2022 ◽

Author(s):

Liming Jin ◽

Zhaoxia Zhang ◽

Zhang Wang ◽

Xiaojun Tan ◽

Zhaoying Wang ◽

...

Keyword(s):

Target Gene ◽

Opposite Effect ◽

High Throughput Sequencing ◽

Target Genes ◽

Tumor Development ◽

Luciferase Reporter ◽

Transwell Assay ◽

Kegg Analysis ◽

Detailed Function ◽

Dual Luciferase Reporter Assay

Abstract Background: CSCs play an important role in tumor development. Some studies have demonstrated that piRNAs participate in the progression of various cancers. However, the detailed function of piRNAs in CSCs requires further investigation. This study aimed to investigate the significance of some piRNAs in Piwil2-iCSCs. Methods and Results: Differentially expressed piRNAs in Piwil2-iCSCs were screened by high-throughput sequencing. Target genes were predicted by the miRanda algorithm and subjected to GO and KEGG analysis. One of the differential piRNAs, novel piRNA MW557525, was transfected and its target gene NOP56 was silenced in Piwil2-iCSCs, respectively. RT-qPCR, western blot and dual luciferase reporter assay were used to investigate the interaction of piRNA MW557525 and NOP56. We identified the effect of piRNA MW557525 and NOP56 knockdown on cell proliferation, migration, invasion, and apoptosis via CCK-8, transwell assay, and flow cytometry. The expressions of CD24, CD133, KLF4, and SOX2 were detected via WB. The results showed that piRNA MW557525 was negatively correlated with NOP56, and it promoted the proliferation, migration, invasion, and inhibited apoptosis in Piwil2-iCSCs, and it also promoted the expressions of CD24, CD133, KLF4, and SOX2, while NOP56 showed the opposite effect. Conclusions: These findings suggested that novel piRNA MW557525 might be a novel therapeutic target in Piwil2-iCSCs.

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SGK1 mutations in DLBCL generate hyperstable protein neoisoforms that promote AKT independence.

Blood ◽

10.1182/blood.2020010432 ◽

2021 ◽

Author(s):

Jie Gao ◽

Eirini Sidiropoulou ◽

Ieuan Walker ◽

Joanna Alicja Krupka ◽

Karol Mizielinski ◽

...

Keyword(s):

Therapeutic Strategy ◽

B Cell Lymphoma ◽

Suppressor Gene ◽

Kinase Domain ◽

Tumour Suppressor Gene ◽

Tumour Suppressor ◽

Protein Isoforms ◽

Loss Of Function ◽

Over Expression ◽

Frequent Finding

Serum and Glucocorticoid-regulated Kinase-1 (SGK1) is one of the most frequently mutated genes in Diffuse Large B Cell Lymphoma (DLBCL). However, little is known about its function or the consequence of its mutation. The frequent finding of truncating mutations has led to the widespread assumption that these represent loss-of-function variants and accordingly, that SGK1 must act as a tumour suppressor. Here we show that instead, the most common SGK1 mutations lead to production of aberrantly spliced mRNA neoisoforms in which translation is initiated from downstream methionines. The resulting N-terminal truncated protein isoforms show increased expression due to the exclusion of an N-terminal degradation domain. However, they retain a functional kinase domain, the over-expression of which renders cells resistant to AKT inhibition in part due to increased phosphorylation of GSK3B. These findings challenge the prevailing assumption that SGK1 is a tumour suppressor gene in DLBCL and provide the impetus to explore further the pharmacological inhibition of SGK1 as a therapeutic strategy for DLBCL.

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Alternative Splicing Generates a BCL6 Isoform Encoding a Compact Repressor.

Blood ◽

10.1182/blood.v108.11.2383.2383 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2383-2383

Author(s):

Yulei Shen ◽

Baosheng Ge ◽

Himabindu Ramachandrareddy ◽

Timothy McKeithan ◽

Wing-Chung Chan

Keyword(s):

Dna Binding ◽

B Cell ◽

Cell Line ◽

Target Genes ◽

B Cell Lymphoma ◽

Luciferase Reporter ◽

Single Mutation ◽

Binding Assays ◽

Protein Protein Interaction ◽

Non Hodgkin Lymphoma

Abstract BCL6 encodes a BTB/POZ zinc finger (ZF) transcriptional repressor which is essential for normal germinal center (GC) reactions. Its dysregulated expression contributes to the pathogenesis of B cell non-Hodgkin lymphoma (NHL). The BCL6 gene consists of ten exons, BCL6 translation start site is located in exon 3, and the six-ZF DNA binding domain is encoded by exons 7 to 10. BCL6S, was cloned from the cDNA of a BCL-6 positive cell line, DHL-16. DNA sequencing showed that BCL6S was a normal splicing isoform that excludes the entire exon 7 of the BCL6 gene and encodes a 650 amino acid (aa) protein. BCL6S was detected by RT-PCR in all human cell lines and tissues expressing BCL6, but was not detected in the mouse B cell lymphoma cell line A20. BCL6S accounts for 1/8 to 1/10 of total BCL6 transcripts. Luciferase reporter assays demonstrated that BCL6S could repress typical BCL6 target genes ( BCL6, CD23b, Blimp1, MIP1α ) as effectively as the full length BCL6. BCL6S retains the last four ZFs of BCL6 and localizes in the cell nucleus. Protein-protein interaction assays confirmed that BCL6 and BCL6S could form homodimers and heterodimers. DNA binding assays showed no affinity differences between BCL6 and BCL6S; however, a single mutation at the N-terminus of the POZ domain (L19 to H) not only disrupted BCL6S dimerization but also significantly decreased the DNA binding affinity, indicating that BCL6 dimerization may stabilize the ZF-DNA binding. In conclusion, we have identified a novel BCL6 splicing isoform. BCL6S, a compact repressor as potent as BCL6. POZ dimerization plays an important role in stabilization of BCL6 DNA binding whereas the first two ZFs are not absolutely required. The biological role of BCL6S in GC B cells needs to be further investigated.

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A BCL6 Target Gene Signature Predicts the Biological Behavior and Classification of Diffuse Large B-Cell Lymphoma.

Blood ◽

10.1182/blood.v108.11.616.616 ◽

2006 ◽

Vol 108 (11) ◽

pp. 616-616

Author(s):

Jose M. Polo ◽

Przemyslaw Juszczynski ◽

Leandro Cerchietti ◽

Stefano Monti ◽

Kenny Ye ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Target Gene ◽

Target Genes ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Gene Signature ◽

Transcriptional Profiles ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL), the most common lymphoid malignancy, is a heterogeneous disease. These tumors are thought to arise from normal antigen-exposed B-cells that have migrated to or through the germinal center (GC). Structural abnormalities of the BCL6 locus (chromosomal translocation and aberrant somatic hypermutation) are the most common genetic abnormalities in DLBCL, occurring in over a third of these tumors. We recently developed a potent and specific BCL6 peptide inhibitor (BPI) that disrupts the interaction between BCL6 and the SMRT co-repressor complex. BPI was cytotoxic against some, but not all, BCL6 positive primary DLBCLs and DLBCL cell lines, indicating that a subset of DLBCLs was particularly dependent on BCL6 for their survival. We predicted that such cases might be identified through a specific BCL6-dependent gene signature and utilized ChIP on chip and a 24,000 promoter genomic microarray to identify BCL6 target genes. In these studies, BCL6 bound to 431 loci. Eighty percent of these candidate target genes contained a canonical BCL6 binding site and 85% of analyzed candidates were confirmed using quantitative single-locus CHIP. GO term enrichment revealed that BCL6 targets were significantly more likely to be genes associated with transcription, ubiquitylation, response to DNA damage, cell cycle and chromatin assembly/disassembly (FDR <.05). We predicted that coordinate regulation of the BCL6 targets would serve as a signature of BCL6 activity. For this reason, we asked whether the BCL6 target gene set was differentially expressed in the recently described DLBCL comprehensive clusters, “B-cell Receptor/Proliferation (BCR),” “Oxidative Phosphorylation (OxPhos),” and Host Response (HR),” using 2 large series of primary DLBCLs with available transcriptional profiles. Since HR tumors are largely defined by infiltrating host inflammatory cells, we focused on BCR and OxPhos DLBCLs. Of interest, BCR tumors more frequently exhibit BCL6 chromosomal translocations and increased BCL6 expression. Consistent with these observations, gene set enrichment analysis (GSEA) revealed highly significant differential expression of BCL6 target genes in BCR vs. OxPhos tumors (p <.0001). In contrast, GSEA of the BCL6 targets in the same DLBCLs sorted into developmental cell-of-origin groups was not significant (“GC” vs. “ABC”/”Other”, p =.25 and “GC” vs. “ABC” only, p =.082). To assess the functional significance of the BCL6 signature in the DLBCL consensus clusters, we generated a predictive algorithm based on the transcriptional profiles of a series of DLBCL cell lines to assign the lines to BCR, OxPhos or HR comprehensive clusters. Five BCR and 3 OxPhos DLBCL cell lines were selected for additional blinded functional analyses. BPI treatment upregulated BCL6 target gene expression in BCR, but not OxPhos, DLBCLs. Furthermore, BPI was significantly more effective in BCR lines than OxPhos DLBCLs (BPI IC50s of BCR vs. Ox Phos lines, 12.7±1.7 μM vs 48.9±2.6 μM, respectively, p<. 0001). Taken together, the data indicate that BCR DLBCLs are significantly more dependent on BCL6 transcriptional repression and more sensitive to targeted BCL6 inhibition. More generally, these studies suggest that DLBCL comprehensive cluster designation will help guide the targeted therapy of biologically discrete DLBCL subsets.

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TRAF3 Loss Drives Alternative NF-κB Pathway Activation in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2020-141126 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Michael Y. Li ◽

Lauren C. Chong ◽

Elizabeth Chavez ◽

Bruce W Woolcock ◽

Adele Telenius ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

B Cell ◽

Cell Lines ◽

Copy Number ◽

Research Funding ◽

B Cell Lymphoma ◽

Luciferase Reporter ◽

Published Data ◽

Loss Of Function

Introduction: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor family that regulates gene expression programs contributing to inflammation and cell survival. NF-κB signaling occurs via two branches: classical and alternative, and is often enriched in somatic mutations of key pathway members in several lymphoid malignancies. Here, we reveal deregulation and constitutive activation of the alternative NF-κB pathway in a subset of DLBCL patients with recurrent genomic loss of the gene encoding tumor necrosis factor receptor-associated factor 3 (TRAF3), a regulator of the NF-κB signaling pathway. Methods and Results: To uncover novel driver mutations of DLBCL pathogenesis and tumor maintenance, we performed Affymetrix SNP6.0 copy number analysis on 347 de novo DLBCL samples from patients uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). We observed frequent, focal genomic loss of chr:14q32.31-32 which included TRAF3 and RCOR1 (7%, 22/313) in the minimally deleted region and an enrichment of activated B-cell-like (ABC) subtype cases over germinal center B-cell-like (GCB) subtype cases, confirming previously published data (Chan et al, Blood 2014). RNAseq of these DLBCL samples revealed a significant reduction of TRAF3 mRNA in chr:14q32.31-32 deleted cases compared to copy number neutral cases (p=0.002). Next, we focused on characterizing the phenotypic consequences of TRAF3 loss in DLBCL. We used CRISPR/Cas9 gene editing to knock out TRAF3 in 2 GCB-DLBCL (DOHH2, OCI-LY1) and 2 ABC-DLBCL (HBL1, OCI-LY3) cell lines. We performed immunoblotting analysis of NF-κB pathway members on cell fractionated samples of TRAF3 knockout cells and found increased levels of the NF-κB inducing kinase NIK (a direct target of TRAF3-mediated ubiquitin-proteasome degradation) and a concomitant increased nuclear translocation of NF-κB transcription factor complex subunits RelB and p52. Proteasome blockade restored RelB cytoplasmic localization and reduced processed p52 protein in TRAF3 knockout GCB-DLBCL lines only, indicating other factors may contribute to alternative NF-κB activation in ABC-DLBCL. Moreover, classical NF-κB activation remained unaffected, highlighting the specific role of TRAF3 regulation on the alternative NF-κB pathway in DLBCL. Consistent with these findings, TRAF3 knockout cells exhibited NF-κB-dependent transcriptional upregulation by luciferase reporter activity and elevated pro-inflammatory cytokine production (IL-6, TNF-β) by Luminex and ELISA. To study transcriptome changes as a result of TRAF3 loss-of-function, we performed RNAseq and differential gene expression analysis on wildtype and TRAF3 knockout DLBCL cell lines as well as primary DLBCL samples (N=347). We found enrichment of NIK and NF-κB associated pathways in TRAF3 deficient DLBCL and uncovered additional enriched gene sets including those involved in cell cycle regulation, cell division and metabolism, suggesting a potential proliferative and survival advantage. Conclusion: Our findings link TRAF3 loss-of-function to clinical and gene expression phenotypes in DLBCL and highlight alternative NF-κB activation as a pathogenically important pathway in both GCB and ABC subtypes. Future studies will be directed towards comprehensive evaluation of NF-κB inhibitors for effective blockade of constitutive alternative NF-κB activation in DLBCL. Disclosures Scott: NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; AbbVie: Consultancy.

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