MicroRNA-Mediated Down-Regulation of the Tumor Suppressor Gene PRDM1/Blimp-1 in Diffuse Large B-Cell Lymphomas.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3187-3187
Author(s):  
Kui Nie ◽  
Hatim Allawi ◽  
Victor Lyamichev ◽  
Mario F. Gomez ◽  
Yifang Liu ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Gene Mutations ◽  
Terminal Differentiation ◽  
B Cell Lymphomas ◽  
Down Regulation ◽  
Translation Repression ◽  
Large B Cell

Abstract PR (PRDI-BF1-RIZ-homology) domain zinc finger protein 1 (PRDM1) is a master regulator in plasma cell differentiation recently identified as a tumor suppressor target for inactivation in diffuse large B-cell lymphomas (DLBCL) of the activated B-cell (ABC) type, implying interference of B-cell terminal differentiation as a pathogenetic mechanism in DLBCL. Besides deleterious gene mutations, it has been suggested that PRDM1 may also be inactivated in DLBCL by an epigenetic mechanism. In this study, we examined the hypothesis of microRNA (miRNA)-mediated down-regulation of PRDM1 in DLBCL. 10 DLBCL cell lines and 25 clinical DLBCL samples were analyzed for PRDM1α (PRDM1 functional isoform) RNA and protein expression by quantitative real-time reverse transcriptase-PCR, Western blotting and immunohistochemistry. The clinical samples included 5 ABC-DLBCLs with PRDM1 gene deletions and inactivating mutations (Group I), 12 ABC-DLBCLs without PRDM1 mutations (Group II) and 8 germinal center B-cell (GCB) type (Group III). The myeloma cell line U266, which expresses relatively abundant PRDM1α mRNA and protein, was used as a reference standard (arbitrarily set as 1). These expression studies identify desynchrony in PRDM1α mRNA and protein expression. PRDM1α is weakly expressed or undetectable in DLBCL cell lines regardless of levels of PRDM1α transcripts. For the primary DLBCL cases, the mean levels of PRDM1α mRNA in groups I, II and III were: 2.21+0.53 (p<0.05 vs. II & III), 0.84+0.19, and 0.43+0.24, respectively. However, immunohistochemistry demonstrated that in all three DLBCL groups, including Group II which has relatively high PRDM1α mRNA and harbors no PRDM1 mutations, an average of only <5% (range: 0 to 10%) of the neoplastic B cells weakly expressed PRDM1. These results suggest epigenetic down-regulation of PRDM1 protein expression in some DLBCLs. Several lines of evidence support a role for miRNA let-7 in mediating translation repression of PRDM1 in DLBCLs: (1) let-7a levels in DLBCL cell lines and primary cases, as determined by quantitative modified Invader assays, are higher (∼2 to 30 fold) than in U266; (2) The lowest (let-7a)/(PRDM1α mRNA) ratio is found in those ABC-DLBCLs harboring PRDM1 mutations; (3) Enforced expression of let-7a caused binding site-dependent reduction in reporter gene activities of at least 50%. This reduction is due to translation repression; (4) Enforced let-7a expression reduces PRDM1α levels by ∼ 50% in U266 cell lines, suggesting functional in vivo interaction of let-7a with PRDM1 mRNA. In conclusion, PRDM1 protein levels correlate poorly with PRDM1 mRNA expression in DLBCLs. Our studies suggest miRNA-mediated down-regulation as a mechanism of lowering PRDM1 activity in DLBCL, apart from genetic mutations and transcription repression. In ABC-DLBCLs without PRDM1 gene mutations, PRDM1 inactivation is likely mediated at least in part via translation repression of PRDM1 transcripts by high levels of let-7. Those ABC-DLBCLs with PRDM1 gene mutations might have “escaped” let-7-mediated down-regulation, for example, via higher levels of induction of PRDM1 transcripts or some other mechanisms. let-7 may be considered a potential target for therapeutic inhibition to restore terminal differentiation in DLBCL cells.

In Silico and Functional Characterization of TBL1XR1 as a Tumor Suppressor in Large B-Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 612-612 ◽  
Author(s):  
Bjoern Chapuy ◽  
Atanas Kamburov ◽  
Caroline A Coughlin ◽  
Chip Stewart ◽  
Andrew Dunford ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Cell Lines ◽  
In Silico ◽  
Functional Characterization ◽  
Genetic Alterations ◽  
Protein Crystal ◽  
B Cell Lymphomas ◽  
In Silico Modeling ◽  
Large B Cell

Abstract Large B-cell lymphomas (LBCL) exhibit multiple genetic alterations, often in specific combinations. However, little is known about the biologic consequences of these concurrent genetic features. We recently characterized the comprehensive molecular signatures of two LBCL subtypes that exclusively involve extranodal disease sites, primary central nervous system lymphoma (PCNSL) and primary testicular lymphoma (PTL). In contrast to systemic diffuse large B-cell lymphomas (DLBCLs) or most activated B-cell (ABC)-type DLBCLs, the majority of PCNSLs and PTLs exhibit oncogenic Toll-like receptor (TLR) signaling (MYD88L265P activating mutations), often with concurrent B-cell receptor (BCR) activation (CD79BY196mut) and/or additional mutations or inactivating rearrangements of TBL1XR1 (Chapuy and Roemer et al, Blood 2016; 127:869-81). TBL1XR1 is a component of the NCoR/SMRT co-repressor complex that modulates TLR/MYD88 signaling by increasing the clearance of NCoR/SMRT transcriptional co-repressors from certain TLR/MYD88 target genes. However, the functional consequences of somatic mutations in TBL1XR1 (TBL1XR1mut) or reduced TBL1XR1wt expression in LBCLs remain to be defined. For these reasons, we performed in silico modeling of the identified TBL1XR1mut and functionally characterized TBL1XR1mut and decreased TBL1XR1wt expression in model LBCL systems. First, we evaluated the frequency and types of TBL1XR1mut in our local series of PCNSLs and 151 primary DLBCLs (102 local and 49 previously published) for which we had whole exome sequenceing data. Thirty-six percent of PCNSLs and 6% of DLBCLs in this series exhibit TBL1XR1mut, over half of which occur in the context of MYD88 and/or CD79B mutations. In these PCNSLs and DLBCLs, we identifed 13 different non-synonymous TBL1XR1 mutations. All identified non-overlapping TBL1XR1 mutations were in the WD40 propellar domains, which are critical for protein-protein interactions. To gain insights into the structural consequences of TBL1XR1mut, we overlayed these alterations onto the TBL1XR1 protein crystal structure, modeled the corresponding mutations in silico and assessed protein stability changes. Eighty percent of the TBL1XR1mut resulted in significant destabilization of the mutant protein (ΔΔGibbsFreeEnergy median: 9.78 kcal/mol, range: 1.91 - 28.31). We found that mutations often perturbed the 3D protein structure by abrogating critical polar interactions (representative example TBL1XR1wt vs. TBL1XR1H390R in Figure 1). These in silico modeling data suggest that genetic perturbations of TBL1XR1mut disrupt the function of the encoded protein. For that reason, we modeled reduced TBL1XR1wt expression in informative DLBCL cell lines with or without concurrent CD79B/MYD88 mutations (TMD8 and OCI-Ly1, respectively) by genetically depleting endogenous TBL1XR1 alleles with CRISPR/Cas9. All of the resulting LBCL cell lines with loss of either one or two TBL1XR1 alleles exhibited significantly increased proliferation in comparison to the parental controls. We next assessed the role of TBL1XR1mut by generating HA-tagged versions of each identified TBL1XR1 mutation with site-directed mutagenesis. Viral particles of TBL1XR1mut constructs and the TBL1XR1wt control were used to reconstitute mutant and wild type protein in the TBL1XR1-/- Ly1 cell line. Reconstitution with each TBL1XR1mut significantly enhanced LBCL proliferation, in comparison to TBL1XR1wt. Taken together, these in silico and experimental data suggest that TBL1XR1mut in PCNSL and DLBCL are inactivating events and establish TBL1XR1 as a tumor suppressor in these diseases. Additional analysis of potential interactions between perturbed TBL1XR1 and MYD88/CD79B are underway. Disclosures Rodig: Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Epigenetic Down-Regulation of the Tumor Suppressor Gene PRDM1/Blimp-1 in Diffuse Large B Cell Lymphomas

2010 ◽  
Vol 177 (3) ◽  
pp. 1470-1479 ◽  
Author(s):  
Kui Nie ◽  
Taotao Zhang ◽  
Hatim Allawi ◽  
Mario Gomez ◽  
Yifang Liu ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Tumor Suppressor Gene ◽  
Suppressor Gene ◽  
B Cell Lymphomas ◽  
Down Regulation ◽  
Large B Cell

Promoter and Exon 1 Hypermethylation of the Tumor Suppressor Gene PRDM1/Blimp-1 indicates Its Pathogenetic Role in EBV-Positive Burkitt Lymphoma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3471-3471
Author(s):  
Kui Nie ◽  
Taotao Zhang ◽  
Carlos E Bacchi ◽  
Eduardo Queiroga ◽  
Dra. Gabriela Gualco ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
Methylation Status ◽  
Terminal Differentiation ◽  
Precursor Cell ◽  
B Cell Lymphomas ◽  
Exon 1 ◽  
Low Levels

Abstract Abstract 3471 PRDM1/Blimp1, a lymphoma tumor suppressor and master regulator of plasma cell differentiation, is inactivated by genomic mutation and deletion in a subset of activated B-cell-type diffuse large B-cell lymphomas (DLBCL), underscoring the importance of terminal differentiation impairment in lymphoma pathogenesis. The role of PRDM1 inactivation in Burkitt lymphoma (BL) is currently not known. Two findings, however, may suggest possible PRDM1 involvement in BL pathogenesis. First, although PRDM1 protein is consistently absent in BL, about 40% of BL cases are positive for IRF4/MUM1, suggesting asynchronous expression of these two proteins. Secondly, it has been hypothesized that microRNA-127 overexpression in EBV+ BL may promote BL development through down-regulation of PRDM1. Sequence analysis of the coding region of PRDM1 in BL cell lines and primary tumors did not demonstrate any mutational changes. To investigate whether epigenetic inactivation of PRDM1 may play a pathogenetic role in BL, BL cell lines and primary cases were bisulfite sequenced to assess the methylation status of 41 CG dinucleotides in a 601 base-pair region spanning the distal promoter (DP) and a CG island that extends from the proximal promoter to the first exon of PRDM1. These include (i) 11 BL cell lines, 9 EBV+ (4 latency I, 2 Wp-restricted, and 3 with latency III drift) and 2 EBV-; (ii) 7 EBV+ lymphoblastoid cell line (LCL); (iii) 62 primary BL cases, formalin-fixed, paraffin-embedded tissues (FFPE); (iv) 4 B cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCL-U), FFPE. Naïve, germinal center (GC) and memory B cells sorted by flow cytometry were also analyzed as controls. Hypermethylation in PRDM1 promoter and exon 1 was seen in all EBV+ BL cell lines except one with latency III, but not in EBV- BL lines. None of the LCL demonstrated PRDM1 hypermethylation, implying that EBV is unlikely to mediate PRDM1 hypermethylation directly. Methylation status of PRDM1 was successfully determined in 39 BLs (30 sporadic, 9 HIV-related) and 5 BCL-Us (4 sporadic, 1 HIV-related). Hypermethylation was seen in 11 BLs and 1 BCL-U (28.6% of total). All the methylated cases were EBV(+) (p=0.004). Overall, 12 of 28 (43%) EBV(+) BL and BCL-U cases exhibited hypermethylation. PRDM1 hypermethaylation was independent of BL subtype and MUM1/IRF4 expression status. To determine if PRDM1 hypermethylation can potentially repress PRDM1 transcription, BL cell lines Ramos (PRDM1 unmethylated) and Mutu1 (PRDM1 hypermethylated) are treated with IL-21 (50ng/mL), a cytokine that can mediate terminal differentiation by inducing PRDM1, for 2 to 5 days. While PRDM1 is induced by IL-21 in Ramos, levels of PRDM1 are not significantly increased in Mutu1, even though STAT3, a downstream mediator of IL-21, is activated in the latter. This finding suggests that PRDM1 hypermethylation has the potential to repress PRDM1 transcription in the presence of PRDM1-inducing signals. This function, however, may be pathogenetically more relevant in a BL precursor cell than in the final BL tumor cell. EBV+ BL is thought to derive from an EBV-infected late germinal center (GC) B cell, which harbors c-myc translocation and begins, though abortively, memory B cell differentiation and adopts an EBV latency I form. BL consistently express BCL6, a known PRDM1 transcription repressor, and harbor very low levels of PRDM1 mRNA. This suggests that high BCL6 expression, rather than PRDM1 hypermethylation, may be the primary determinant of its repressed transcription seen in BL. Indeed, PRDM1 mRNA expression is 25 to 1200 fold higher in LCLs which harbor very low levels of BCL. Furthermore, reducing expression of MTA3, which forms a complex with BCL6 and mediates PRDM1 transcription repression, in Daudi increases PRDM1 transcript levels. Therefore, we hypothesize that PRDM1 hypermethylation in BL more likely represents a memory of an epigenetic event that functions in the earlier stage of BL pathogenesis. PRDM1 hypermethylation may function to repress its transcription in a BL precursor cell as it is exposed to inducing signals, e.g. IL-21, during GC transit. Our study expands the spectrum of B cell lymphomas in which PRDM1 plays a tumor suppressor role, and supports the importance of impairment of terminal differentiation in the pathogenesis of a subset of aggressive B cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

MLN4924 An Investigational Inhibitor Of NEDD8 Activating Enzyme (NAE) Is Active In Pre-Clinical Models Of Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4409-4409
Author(s):  
Sarah Belliotti ◽  
Juan Gu ◽  
Cory Mavis ◽  
Myron S. Czuczman ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
G1 Phase ◽  
B Cell Lymphomas ◽  
Lymphoma Cells ◽  
Down Regulation ◽  
Cycle Arrest ◽  
Large B Cell

The results of the Collaborative trial in relapsed aggressive lymphoma (CORAL) study suggest that diffuse large B-cell lymphomas (DLBCL) that relapse or fail to respond to rituximab-chemotherapy in the front-line possess a more resistant disease and represent an emerging challenge for clinicians treating aggressive B-cell lymphomas. It also stresses the need to further study and define at the molecular level the mechanisms by which DLBCL are developing resistance to chemo-immunotherapy. We previously demonstrated that the ubiquitin-proteasome system (UPS) plays an important role in the development of rituximab-chemotherapy resistance. Targeting the UPS has become an important therapeutic strategy in relapsed/refractory DLBCL. MLN4924, a NAE inhibitor selectively blocks the UPS up-stream by preventing the activation of a subset of ubiquitin ligases known as cullin-ring ligases. We evaluated the activity of MLN4924 in a panel of rituximab-chemotherapy (RSCL) sensitive and resistant (RRCL) germinal center B-cell (GCB) and ABC-DLBCL cell lines. RSCL and RRCL were exposed to MLN4924 (0.5μM and 1.0μM) for 24-72 hrs. Changes in cell viability, cell cycle and expression of key regulatory proteins of the cell cycle, Bcl-2 family members, and the UPS were evaluated using the cell titer glo assay, flow cytometry and western blotting respectively. MLN4924 induced cell death in ABC-DLBCL cell lines (both RSCL and RRCL) and to a lesser degree in GCB-DLBCL cell lines. Anti-tumor activity plateau was seen after 48 hrs of drug exposure. In MLN4924 sensitive cells we consistently observed cell cycle arrest in G1 phase, down-regulation of Bcl-XL and PARP cleavage. We also observed down regulation of NEDD8 protein across all treated cell lines. Bcl-XL down-regulation appears to be regulated at the transcriptional level. MLN4924 exposure in vitro resulted in a decrease in Bcl-XL mRNA as determined by quantitative polymerase chain reaction (qPCR), perhaps due to the inhibition of NFkB activity as demonstrated in MLN4924-exposed cells by p65 co-localization studies using the imagestream technology. Our data suggests that MLN4924 is active in ABC-DLBCL by inducing cell cycle arrest in G1 phase and rendering lymphoma cells more susceptible to apoptosis. MLN4924 lowers the apoptotic threshold of lymphoma cells by negatively regulating Bcl-XL levels at the transcription level. Selective inhibition of the NFkB transcription factor most likely play a role in the down-regulation of Bcl-XL observed in all cell lines tested. Ongoing studies aimed to further define the molecular mechanisms of action of MLN4924 can potentially assist scientists and clinicians in the optimal design of clinical trials incorporating this agent in relapsed/refractory DLBCL patients. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures: Czuczman: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

scholarly journals Complex analysis of the TP53 tumor suppressor in mantle cell and diffuse large B-cell lymphomas

2017 ◽  
Vol 38 (4) ◽  
pp. 2535-2542 ◽  
Author(s):  
Lenka Zlamalikova ◽  
Mojmir Moulis ◽  
Barbora Ravcukova ◽  
Kvetoslava Liskova ◽  
Jitka Malcikova ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Complex Analysis ◽  
B Cell Lymphomas ◽  
Mantle Cell ◽  
Large B Cell

Sensitivity of Diffuse Large B-Cell Lymphomas to DNA Methyltransferase Inhibitors Is Associated with a Specific Epigenetic Signature.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 831-831
Author(s):  
Megan Ryan ◽  
Leandro Cerchietti ◽  
Maria E. Figueroa ◽  
John Greally ◽  
Ari Melnick
Keyword(s):  
Dna Methylation ◽  
B Cell ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Serum Starvation ◽  
Gene Promoters ◽  
B Cell Lymphomas ◽  
Response Curves ◽  
Differentially Methylated Genes ◽  
Large B Cell

Abstract DNA methyltransferase inhibitor drugs (MTIs) such as decitabine can overcome gene silencing due to aberrant hypermethylation of gene promoters. Presumably, this effect is responsible for the therapeutic activity of MTIs as clinically demonstrated in myelodysplasias (MDS) and leukemias. Other tumors such as diffuse large B-cell lymphomas (DLBCLs) can also present with aberrant promoter hypermethylation. However, it is currently difficult to prospectively identify patients likely to respond to MTIs, since specific methylation markers or signatures have not yet been identified. We predicted that decitabine would have anti-lymphoma activity in a subset of DLBCLs, and that these cases would exhibit specific methylation signatures predictive of response to these drugs. To determine whether this is the case we first exposed a panel of 7 DLBCL cell lines (Ly1, Ly7, Ly10, SU-DHL6, Farage, Pfeiffer and Toledo) to increasing concentrations of decitabine (0.5, 1, 2.5, 5, 10, 50 and 100 μM) administered after synchronization by 12 hr serum starvation. Viability was assessed after 48 hr of culture by MTS-based assay and Trypan blue exclusion. The IC25 and IC50 were calculated for all cell lines by constructing dose-response curves. The IC25 was used to discriminate sensitive (6.3 ± 1.2 μM) vs. resistant (49.4 ± 5 μM, p &lt; 0.01) cell lines. Interestingly, there was no correlation between MTI sensitivity and DLBCL subtype as defined by recent gene expression profiling classification efforts (i.e. GCB vs. ABC, or BCR vs. OxPhos). To identify the methylation signatures of these DLBCL cells we used a method that we developed for genome-wide DNA methylation quantification called HELP (HpaII tiny fragment Enrichment by LM-PCR). HELP is based on comparative Msp1 and HpaII digestion of genomic DNA, followed by size specific amplification and co-hybridization to custom high-density oligonucleotide arrays designed to provide uniform data collection over 25,000 promoters. HELP compares favorably to other high throughput methods in that it is highly reproducible (R &gt; 0.98) and has an extremely robust signal-to-noise ratio. DNA was collected from the DLBCL cells for HELP prior to drug treatment. Most significantly we found that unsupervised (i.e. unbiased) clustering of DNA methylation profiles could readily segregate decitabine resistant vs. sensitive DLBCL cell lines. Correspondence analysis clearly identified a methylation signature consisting of 133 differentially methylated genes that distinguishes between decitabine sensitive and resistant cells. Most of these appeared to be functionally relevant including such genes as Caspase-9, RARB, JUNB, and ELK1. Biological assays to determine the contribution of these genes to the phenotype are underway. Taken together, our data suggest that MTIs might be effective in a cohort of DLBCL cases that exhibit the specific methylation signature that we have identified. Prospective evaluation of the predictive value of this signature may allow optimal selection of patients for clinical trials with these agents.

Genomic Rearrangements Involving Programmed Death Ligands Are Recurrent In Primary Mediastinal Large B-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 635-635 ◽  
Author(s):  
David D. W. Twa ◽  
Fong Chun Chan ◽  
Susana Ben-Neriah ◽  
Bruce W. Woolcock ◽  
King L. Tan ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Transcript Levels ◽  
Barr Virus ◽  
Large B Cell Lymphoma ◽  
Programmed Death ◽  
Large B Cell

Abstract Introduction Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive malignancy commonly diagnosed in young adult females. In recent years, mutational and gene expression profiling has established genotypic and phenotypic similarity of PMBCL with both classical Hodgkin and diffuse large B-cell lymphoma (DLBCL). In-depth analyses of genomes and transcriptomes have highlighted several inactivating mutations (SOCS1, TP53), chromosomal amplifications (2p, 9p, Xp, Xq) and translocations (CIITA) thought to be integral in establishing and/or maintaining the PMBCL phenotype. Programmed death ligands (PDL) 1 (CD274) and 2 (PDCD1LG2), which are located on chromosome 9p24.1, are two emerging genes of interest that have been shown to be altered in PMBCL and can induce T-cell anergy by binding to the receptor, programmed death 1. Here, we describe the recurrence of chromosomal rearrangements of the PDL locus in various B-cell lymphomas and explore the association of these rearrangements with transcript levels. Methods To establish the frequency of CD274 and PDCD1LG2 aberration, we conducted fluorescence in situ hybridization (FISH) on 551 clinical samples and 20 established cell lines using in-house break-apart probes. Epstein-Barr virus encoded RNA in situ hybridization was also carried out on the clinical cohort. The clinical cases, sourced from the British Columbia Cancer Agency’s Centre for Lymphoid Cancer tissue repository, consisted of 125 PMBCLs, 216 DLBCLs, 130 primary DLBCL of the central nervous system (PCNSL), 12 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL) and 68 follicular lymphomas (FL) with diagnoses based on the WHO classification. The DLBCL cohort could be further subdivided into 134 nodal DLBCLs and 82 testicular DLBCLs (T-DLBCL). Quantitative real-time PCR (qRT-PCR) was subsequently conducted on 17 cell lines and a clinical sub-cohort of 76 samples, for which fresh-frozen material was available, to determine the effect of mutations on transcript expression. We then characterized the PDL aberrations of two clinical PMBCL cases and three cell lines (DEV, L-428, L-1236), at base pair resolution, by applying the bioinformatic tools, nFuse, deFuse and destruct to both newly produced and previously published whole genome (WGS) and whole transcriptome (RNA-seq) libraries. Results FISH revealed a PDL locus (9p24.1) break-apart frequency of 20% (25/125) in PMBCL. There were no differences in any known clinical parameters or frequency of Epstein-Barr virus positivity between positive and negative PDL break-apart cases. Break-apart frequencies in other malignancies were calculated to be 3% in DLBCL, 7% in T-DLBCL and 1% in PCNSL; no positive cases were identified in either NLPHL or FL. The proportion of break-apart positive cases was significantly higher in PMBCL as compared to the other lymphomas surveyed (P < 0.05). Further, in agreement with the published literature, we observed an amplification frequency of the PDL locus in 36% (45/125) of PMBCLs. qRT-PCR established that PDCD1LG2 transcript levels were significantly higher in cases with 9p24.1 locus rearrangements compared to copy number neutral (P = 0.0003), gain (P = 0.001) and amplified cases (P = 0.005). Likewise, CD274 transcript levels were significantly higher in rearranged cases compared to copy number neutral cases (P = 0.03). Following the analysis of WGS and RNA-seq libraries, we were able to characterize four novel fusion transcripts involving the 9p24.1 locus: PDCD1LG2-NRG1 (PMBCL clinical case), PDCD1LG2-IGHV7-81 (L-1236), CIITA-PDCD1LG2 (DEV) and KIAA1432-CLDN14 (L-428). Aberrations involving both NRG1 and CIITA have previously been implicated in breast cancer and B-cell lymphomas, respectively. We also identified a translocation in another PMBCL clinical case with breakpoints in the intergenic spaces near LRMP and CD274, though this rearrangement did not produce a fusion transcript. Conclusion Taken together, our findings show that rearrangement of the PDL locus is recurrent in PMBCL, characteristic of PMBCL and leads to overexpression of PDL transcripts. Given the well-referenced function of PDLs in repressing the anti-tumor response, these data suggest that targeting the PDL axis in a subgroup of B-cell lymphomas holds clinical promise. Disclosures: No relevant conflicts of interest to declare.

Overexpression of BCL-2 Does Not Inhibit Autophagy in Human Follicular and Diffuse Large B-Cell Lymphomas

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3007-3007
Author(s):  
Aine McCarthy ◽  
Andrew James Clear ◽  
Jacek Marzec ◽  
Rita Coutinho ◽  
Robert D. Petty ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Prognostic Significance ◽  
B Cell Lymphoma ◽  
Beclin 1 ◽  
Pcr Array ◽  
B Cell Lymphomas ◽  
Autophagy Activity ◽  
Large B Cell

Abstract Background: Human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the most common forms of indolent and aggressive NHL, respectively. The t(14;18) translocation characterizes approximately 85% of FL and 20% of DLBCL and results in constitutive overexpression of the anti-apoptotic protein BCL-2. It was previously reported that BCL-2 plays dual roles in preventing apoptosis and autophagy. Autophagy is a physical and pathological process whereby cells sequester portions of cytoplasm including organelles to form autophagosomes where they are degraded and recycled. Growing evidence demonstrates that autophagy plays important roles in tumorigenesis, tumor progression, and resistance to chemotherapy. Aims: The autophagy status in human B-cell lymphomas is unknown. We hypothesized that overexpression of BCL-2 could change autophagy status and aimed to determined expression of autophagy-related genes and proteins in FL and DLBCL primary samples by PCR array and tissue microarray. We aimed to evaluate whether expression of the autophagy-related proteins p62, Beclin-1 and LC3 individually and in combination with BCL-2 protein expression could risk-stratify FL and DLBCL patients at diagnosis. Patients and methods: Using PCR array, the autophagy-related gene expression profiles were determined in purified and unpurified reactive and malignant human lymph node tissue biopsies. Diagnostic tissues from FL (n=117) and DLBCL (n=109) patients were microarrayed and autophagy protein expression was evaluated using immunohistochemistry. Univariate and multivariate analyses on both continuous and categorical variables were conducted to measure overall survival (OS), disease specific survival (DSS), and progression-free survival (PFS). Results: Seven autophagy machinery genes were up-regulated in purified FL B-cells, namely ATG9A, ATG16L1, MAP1LC3A, GABARAPL1, ULK1, LAMP1 and HDAC6 compared with reactive B-cells. Two autophagy machinery genes, MAP1LC3A and DRAM1, were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. CTSD (cathepsin D) and TGM2 (transglutaminase 2) genes and proteins were mainly up-regulated in DLBCL tumor-infiltrating macrophages. These results demonstrate that FL and DLBCL showed increased expression of autophagy-related genes, regardless of the heterogeneity of these diseases. p62, a selective autophagy substrate; LC3, an autophagosome membrane protein; and Beclin-1, an essential autophagy effector, are often used to evaluate autophagy activity in the cell. 91% FL samples were BCL-2 positive but significantly decreased expression of p62, LC3 and Beclin-1 in FL samples was observed in both intra-follicular and non-malignant inter-follicular areas. This suggests that increased basal autophagy activity in both malignant FL cells and surrounding tumor infiltrating cells, indicating that BCL-2 does not inhibit basal autophagy activity. DLBCL samples displayed heterogeneous expression patterns of BCL-2, p62, LC3 and Beclin-1. We found that decreased p62 expression confers worse OS (continuous P=0.015; and categorical P=0.003), DSS (continuous P=0.037; categorical P=0.014) and PFS (categorical P=0.002) in DLBCL patients. Decreased expression of Beclin-1 was also confers poor prognosis in both FL and DLBCL as conducted by categorical analysis, OS (DLBCL, P=0.015; FL, P=0.004), DSS (FL, P=0.006), and PFS (DLBCL, P=0.029). p62 retains prognostic significance after adjustment for the International Prognostic Index (IPI) score and levels of BCL-2, Beclin-1 and LC3 in multivariate analysis. Beclin-1 retains its prognostic significance in FL after adjusting for FLIPI scores. Low p62 plus high BCL-2 expression in DLBCL confers the worst OS (P<0.0001) and DSS (P=0.001) compared with other combinations. Conclusions: These results demonstrate that FL has increased basal autophagy activity, while it varies in DLBCL. p62 is a novel, independent prognostic biomarker for DLBCL but not for FL. Combining p62 with BCL-2 provides a more robust and reliable method to risk-stratify DLBCL patients at diagnosis. Importantly, we report for the first time that overexpression of BCL-2 in human NHL does not inhibit basal autophagy activity. We propose that increased autophagy activity could be a therapeutic target for treatment of NHL. Disclosures Gribben: Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

SYK and STAT3 Are Active in Diffuse Large B-Cell Lymphoma: Activity of Cerdulatinib, a Dual SYK/JAK Inhibitor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 926-926
Author(s):  
Y. Lynn Wang ◽  
Jiao Ma ◽  
Wei Xing ◽  
Pin Lu ◽  
Karen Dresser ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Staining Pattern ◽  
B Cell Lymphoma ◽  
Down Regulation ◽  
Dual Inhibitor ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Large B Cell

Abstract Non-Hodgkin Lymphoma (NHL) represents about 5 percent of all cancers diagnosed in the United States. While incidence of NHL has increased slightly over the past decade, death rates have been declining steadily. These declines in mortality can be attributed to improvements in treatment that are based on an increased understanding of the biology of the disease. Diffuse large B-cell lymphoma (DLBCL) accounts for ~30% of NHLs and greater than 80% of aggressive NHLs. Recent studies including large-scale genetic analyses have demonstrated the critical roles of the B-cell receptor’s (BCR) and JAK/STAT pathways in DLBCL. Herein, we investigated the anti-lymphoma activity of cerdulatinib (aka PRT062070), a novel compound that dually targets both SYK and JAK/STAT signaling pathways. To determine whether targeting both SYK and JAK/STAT is relevant in DLBCL, we examined the expression of p-SYK (pY525/526) and p-STAT3 (pY705) on a tissue microarray of 62 DLBCL primary tumors, including 41 GCB and 21 non-GCB cases. p-SYK expression was detected in 29 (47%) cases with a characteristic peri-membrane staining pattern. Of those 29 p-SYK positive cases, 17 were GCB type (17/41, 41%) and 12 were non-GCB type (12/21, 57%). p-STAT3 exhibits a characteristic nuclear staining pattern in DLBCL cases. A total of 26 (42%) stained positive for p-STAT3; 16 were GCB type (16/41, 39%) and 10 were non-GCB type (10/21, 48%). Interestingly, there are 19 cases (31%) with reactivity for both p-SYK and p-STAT3, among which, 11 were GCB type (27%) and 8 were non-GCB type (38%). SYK and STAT3 are also phosphorylated in a panel of nine DLBCL cell lines. Immunoblotting analyses showed that ABC and GCB subtypes of DLBCL cells appear to exhibit different JAK/STAT and BCR signaling profiles. For instance, p-AKT was highly expressed in GCB cells, whereas p-STAT3 was more strongly expressed in ABC cells. Overall, the DLBCL cells are more sensitive to the dual inhibitor than to the SYK-specific inhibitor alone. In both GCB and ABC cell lines, cerdulatinib induced apoptosis via down-regulation of MCL1 protein and PARP cleavage. The compound also blocked G1/S transition and caused cell cycle arrest through inhibition of RB phosphorylation and down-regulation of cyclin E. Further analyses of the cell signaling activities showed that STAT3 phosphorylation was sensitive to inhibition by cerdulatinib in ABC cell lines while phosphorylation of SYK, PLCg2, AKT and ERK was sensitive to inhibition by cerdulatinib in GCB cell lines. Importantly, JAK/STAT and BCR signaling can be blocked by cerdulatinib in GCB and non-GCB primary human DLBCL cells, which led to cell death of these cells. Our work provided mechanistic insights into the actions of SYK/JAK dual inhibitor cerdulatinib, suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma. Disclosures Pandey: Portola Pharmaceuticals: Employment. Conley:Portola Pharmaceuticals: Employment. Coffey:Portola Pharmaceuticals: Employment.

Activation of Classical and Alternative Nuclear Factor-kappaB (NF-kB) Pathways in Diffuse Large B-Cell Lymphomas.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 29-29
Author(s):  
Botond Timar ◽  
Amy Chadburn ◽  
Daniel Knowles ◽  
Ethel Cesarman
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Target Genes ◽  
Alternative Pathway ◽  
Classical Pathway ◽  
B Cell Lymphomas ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
The Status ◽  
Large B Cell

Abstract Activation of the NF-kB pathway is involved in many human neoplasms. In this study we examined the status of the NF-kB canonical (IkB, p50/p65) and non-canonical (p52, RelB) pathways in diffuse large B-cell lymphomas (DLBCL), which are a common and heterogeneous group of lymphoid malignancies. DLBCL have been divided into activated B-cell (ABC) like, and germinal center B-cell (GCB) like subgroups, which have been reported to have high and low NF-kB activity, respectively. However, the nature of the NF-kB complexes in this lymphoma entity has not been previously evaluated. Therefore we performed Western blotting, electrophoretic mobility shift assays (EMSA) and real time quantitative RT-PCRs on nuclear and cytoplasmic protein and total RNA extracts in 20 primary tumor samples and 9 different DLBCL cell lines. In the cell lines, presence of NF-kB proteins in nuclear extracts correlated with expression of NF-kB target genes (CCR7, IkBa, CCND2, BCL-2, IRF4) as determined by RT-PCR. Therefore, these could be assigned into GCB and ABC-like categories. In primary DLBCLs, EMSA showed NF-kB binding in all but one case. The same cases (19/20) had high p52 in the nucleus indicating activation of the alternative pathway. TNF family ligand BAFF was also found to be expressed in all primary samples and most cell lines. The classical pathway, as determined by nuclear p50, was also present in these cases. Levels of p65 and RelB expression were variable, but did not correlate with the mRNA expression of NF-kB target genes. In conclusion, while DLBCL cell lines may be divided into two distinct categories, the primary samples represented a spectrum of NF-kB target gene activity, while levels of NF-kB expression were high. BAFF expression and activation of the alternative pathway may be important in the pathogenesis of DLBCL.

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