scholarly journals The Paradoxical Efficacy of KDM6 Inhibition in Germinal Centre B-Cell Lymphomas

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3289-3289
Author(s):  
Karina Close ◽  
Hannah Armes ◽  
Emil Kumar ◽  
Findlay Bewicke-Copley ◽  
Adam Cribbs ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Germinal Centre ◽  
Zinc Homeostasis ◽  
Global Changes ◽  
Sequencing Analysis ◽  
Gene Promoters ◽  
B Cell Lymphomas ◽  
Gain Of Function

Abstract Introduction Germinal centre (GC) lymphomas Follicular Lymphoma (FL) and Diffuse Large B Cell Lymphoma (DLBCL) are strikingly dependent on mutations affecting chromatin regulators. The majority of FL tumours harbour multiple mutated chromatin-regulatory genes, including loss-of-function mutations in KMT2D (80%), CREBBP (60%) and gain-of-function mutations in EZH2 (25%), whilst these are also frequently mutated within the GC B-cell (GCB) subtype of DLBCL. The FDA approval of EZH2-inhibitor Tazemetostat for relapsed/refractory FL exemplifies the potential and the need to broaden our search for novel avenues targeting epigenetic reprogramming in GC lymphomas. Hence, in this study, we sought to identify novel epigenetic vulnerabilities and biological interactions in GC B cell lymphomas. Methods and Results: 25 compounds targeting 18 histone binding/modifying enzymes were tested across 12 GCB-DLBCL cell lines harbouring differing degrees of epigenetic dysregulation. The two most potent cytotoxic compounds (GSK-J4 and KDOBA67) both target the H3K27me3 demethylase, KDM6. The KDM6 family consists of KDM6A (UTX) and KDM6B (JMJD3), both of which demethylate H3K27me3/H3K27me2 to lower methylation states, and KDM6C (UTY) which has negligible enzymatic activity. Indeed, KDM6 inhibition (KDM6i) with GSK-J4 increased levels of the inhibitory chromatin mark H3K27me3 and simultaneously reduced H3K27me1/me2, while we observed no global changes in H3K4me3. The induction of apoptosis in the cell lines following either inhibition of the 'eraser' KDM6 (leading to increased H3K27me3) or the opposing 'writer' EZH2 (lowering H3K27me3) appears paradoxical at first glance, given the EZH2 gain-of-function mutations seen in FL/DLBCL and known efficacy of EZH2 inhibitors. However, RNA-sequencing analysis of GSK-J4-treated cells revealed no significant overlap with published EZH2 mutation or EZH2 inhibition signatures from GC lymphoma models, implying that KDM6i-regulated H3K27me3 peaks do not overlay gene promoters targeted by EZH2 in the GC reaction. The most significant gene regulation by KDM6i was a significant up-regulation of 6 members of the metallothionein (MT) family within 4-hours of treatment, which was sustained for 72-hours (MT1E, MT1F, MT1G, MT1H, MT1X and MT2A at 4 hours log2FoldChange 5.1-12.1, average p adj 3.4E-06). These metal-binding proteins cluster on chromosome 16 and their up-regulation correlated with up-regulation of the zinc transporter SLC30A1, suggesting a potential biological relationship between KDM6 enzymes and zinc homeostasis, with both isoforms containing a zinc-binding domain. Targeted validation across 6 DLBCL cell lines also showed MT up-regulation correlated with sensitivity to GSK-J4, whilst siRNA-mediated knockdown of KDM6A or KDM6B individually recapitulated the up-regulation of MT genes, albeit to a lower extent. ChIP-qPCR profiling at the MT loci for H3K27me3 and H3K4me3 revealed no significant changes at 4 or 24 hours; considering that expression of MT genes increase within 60 minutes of drug exposure, suggesting these changes may be induced by an indirect mechanism of KDM6A and/or KDM6B activity. Using ENCODE data of 4 cell lines included in our series (DOHH-2, OCI-LY-1, SU-DHL-6, KARPAS-422), we observe a link between GSK-J4 sensitivity, up-regulation of MT genes and significantly lower basal enrichment for H3K27ac at the MT loci. This distinction may reflect altered 3D chromatin conformation that render only sensitive cell lines permissive to regulation by KDM6i, with H3K27ac having recently been shown to modulate enhancer positioning and interactions in DLBCL cell lines. Conclusion: Screening epigenetic-targeting compounds in GC lymphomas has identified KDM6 as a particularly promising candidate target. Overall, we describe up-regulation of MT genes as one potential mechanism of action, which correlates with response in DLBCL cell lines and suggests that KDM6 members may regulate H3K27me3 in a manner different from EZH2. In our current model we propose, that in GC-lymphomas KDM6A may have tumour suppressive roles whilst KDM6B is required for cell survival. Given KDM6A is frequently lost in cancer and Kabuki syndrome, a congenital developmental disorder caused by germline mutations in KMT2D or KDM6A, our results highlight the importance of establishing what roles the KDM6 family play in lymphoma pathogenesis. Disclosures Fitzgibbon: Epizyme: Research Funding.

scholarly journals Gene involved in the 3q27 translocation associated with B-cell lymphoma, BCL5, encodes a Kruppel-like zinc-finger protein

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 26-32 ◽  
Author(s):  
T Miki ◽  
N Kawamata ◽  
S Hirosawa ◽  
N Aoki
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Cdna Probe ◽  
B Cell Lymphoma ◽  
Nucleotide Sequencing ◽  
Sequencing Analysis

Abstract Chromosomal translocations involving band 3q27 are the recently described nonrandom cytogenetic abnormalities in B-cell malignancies. We have previously cloned the breakpoint region of 3q27, designated as the BCL5 locus, from the B-cell line carrying the t(3;22). The cDNA for the BCL5 gene was cloned from the human liver cDNA library. The nucleotide sequencing analysis showed that the BCL5 gene encodes a potential transcription factor containing six repeats of the Cys2-His2 zinc-finger motif resembling the Drosophila segmentation gene Kruppel. The calculated molecular weight was 78.8 kD, which was supported by an in vitro transcription and translation experiment. A part of the sequence was essentially identical to that of a genomic fragment, ZNF51, previously reported to be located at 3qter. The translocation occurred in the 5′ region of the BCL5 gene, and the protein-coding exons were fused to the Ig-lambda gene in a head-to-head configuration in the cell line carrying t(3;22). The BCL5 cDNA probe detected a major transcript of 3.8 kb in Burkitt's lymphoma cell lines and an aberrant transcript in the t(3;22) cell line, whereas no transcript was detected in myeloid, monocytoid, erythroid, T-lymphoid, and Epstein-Barr virus- immortalized B-lymphoblastoid cell lines.

Multiple Myeloma Oncogene 1 (MUM1)/Interferon Regulatory Factor 4 (IRF4) Upregulates Monokine Induced by Interferon-gamma (MIG) Gene Expression in B-Cell Malignancy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1111-1111
Author(s):  
Shinsuke Iida ◽  
Miyuki Uranishi ◽  
Takaomi Sanda ◽  
Takashi Ishida ◽  
Emi Tajima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Lines ◽  
Interferon Gamma ◽  
Zinc Finger Protein ◽  
B Cell Lymphoma ◽  
Interferon Regulatory Factor ◽  
Regulatory Factor ◽  
B Cell Lymphomas ◽  
Interferon Regulatory Factor 4

Abstract MUM1(multiple myeloma oncogene 1)/IRF4(interferon regulatory factor 4) is a transcription regulatory factor that is activated as a result of t(6;14)(p25;q32) in multiple myeloma. MUM1 expression is seen in various B-cell lymphomas/leukemias and has been reported to predict an unfavorable outcome in some lymphoma subtypes including diffuse large B-cell lymphoma (DLBCL) and B-cell chronic lymphocytic leukemia (B-CLL). To elucidate its role in B-cell malignancies, we prepared stably MUM1-expressing Ba/F3 cells, which proliferated at a higher rate than the parental cells, and performed cDNA microarray analysis to identify genes whose expression is regulated by MUM1. We found that the expression of four genes including FK506-binding protein 3 (FKBP3), the Monokine induced by interferon-gamma (MIG), Fas apoptotic inhibitory molecule (Faim) and Zinc finger protein 94 was altered in the MUM1-expressing cells. We then focused on MIG since its expression was immediately upregulated by MUM1 in inducible MUM1 expressing system. In reporter assays, MUM1 activated the MIG promoter in cooperation with PU.1, and the interaction between MUM1 and the MIG promoter sequence was confirmed in chromatin immunoprecipitation assay. The expression of MIG was correlated with that of MUM1 in B-CLL cell lines, and its receptor CXCR3 was also coexpressed in B-CLL cell lines that were positive for MUM1. Interestingly, treatment with neutralizing antibodies against MIG and its receptor, CXCR3, partially inhibited the proliferation of two MUM1-expressing B-CLL cell lines. These results suggest that MUM1 plays certain roles in the progression of B-cell lymphomas/leukemias by regulating the expression of various genes including MIG.

Mutations of the Tumor Suppressor Gene SOCS-1 in Classical Hodgkin Lymphoma Are Frequent and Associated with Nuclear Phospho-STAT5 Accumulation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 19-19 ◽  
Author(s):  
Marc A. Weniger ◽  
Ingo Melzner ◽  
Christiane K. Menz ◽  
Silke Wegener ◽  
Alexandra J. Bucur ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Tumor Suppressor Gene ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Suppressor Gene ◽  
Cytokine Signaling ◽  
Sequencing Analysis ◽  
Classical Hodgkin Lymphoma

Abstract The suppressors of cytokine signaling (SOCS) are critically involved in the regulation of cellular proliferation, survival, and apoptosis via cytokine-induced JAK/STAT signaling. SOCS-1 silencing by aberrant DNA methylation contributes to oncogenesis in various B-cell neoplasias and carcinomas. Recently, we showed an alternative loss of SOCS-1 function due to deleterious SOCS-1 mutations in a major subset of primary mediastinal B-cell lymphoma (PMBL) and in the PMBL line MedB-1, and a biallelic SOCS-1 deletion in PMBL line Karpas1106P (BLOOD, 105, 2535–42, 2005). For both cell lines our previous data demonstrated retarded JAK2 degradation and sustained phospho-JAK2 action leading to enhanced DNA binding of phospho-STAT5. Here we analysed SOCS-1 in laser-microdissected Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). We detected SOCS-1 mutations in HRS cells of eight of 19 cHL samples and in three of five Hodgkin lymphoma (HL)-derived cell lines by sequencing analysis. Moreover, we found a significant association between mutated SOCS-1 of isolated HRS cells and nuclear phospho-STAT5 accumulation in HRS cells of cHL tumor tissue (p<0.01). Collectively, these findings support the concept that PMBL and cHL share many overlapping features, and that defective tumor suppressor gene SOCS-1 triggers an oncogenic pathway operative in both lymphomas.

CD40L Modulates TRAIL-Induced Apoptosis in Germinal Center Derived B Cell Lymphomas.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4630-4630
Author(s):  
Marion Travert ◽  
Patricia Ame-Thomas ◽  
Thierry Fest ◽  
Céline Pangault ◽  
Gilbert Semana ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Lymphoma Cells ◽  
Over Expression ◽  
Induced Apoptosis ◽  
Transformed Cell Lines

Abstract Follicular lymphoma are characterized by the rearrangement of the bcl-2 gene, present in more than 90% of patients. Over-expression of the bcl-2 protein resulting from this translocation is associated with the inability to eradicate the lymphoma, by inhibiting apoptosis. Despite the median survival ranges from 8 to 15 years, leading to the designation of indolent lymphoma, patients with advanced-stage follicular lymphoma are not cured with current therapeutic options. Numerous reports have shown that Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in a wide variety of transformed cell lines of diverse lineage, but does not appear to kill normal cells, even though TRAIL mRNA is expressed at significant levels in most normal tissues. As cell death induced by TRAIL occurs almost exclusively in tumor cells, it suggests that this drug is safe to use as an antitumor therapy. We therefore investigated the efficiency of this cytokine to induce apoptosis in germinal center derived B cell lymphoma, despite bcl-2 over-expression. Our study was also designed to evaluate the role of CD40L, one of the main differentiation signal involved in B cell maturation during the germinal center reaction, on the regulation of TRAIL-induced apoptosis. This study was performed on three germinal center derived tumor cell lines (BL2, VAL and RL), and on normal and tumor primary cells obtained from human tonsils and lymph nodes. Our data show that normal B lymphocytes obtained from tonsil biopsies are resistant to TRAIL-mediated apoptosis, when B lymphoma cells issued from lymph node of numerous patients are significantly sensitive to the cytokine. When we treat these lymphoma cells with trimeric huCD40L, we partly rescue these cells from spontaneous apoptosis which naturally occurs after few days of culture, and reverse by 50% TRAIL-mediated apoptosis when cells were co-treated with huCD40L for 16 hours. Similar results were reproduced on some germinal center derived cell lines. BL2 was indeed found highly sensitive to TRAIL-induced apoptosis following a 24 hour exposure. On the opposite, VAL and RL were almost insensitive. We have demonstrate that apoptosis is exclusively mediated by TRAIL-R1 in BL2. Analysis of signalling pathways revealed that the protection to TRAIL-induced apoptosis by CD40L is due to some specific anti-apoptotic molecules that will be described. Genes encoding these molecules are targets of the NFκB signalling pathway activated by CD40L. Our results suggest that activation of NFκB and induction of anti-apoptotic molecules by CD40L play an important role in the protection of germinal center derived B cell lymphomas against apoptosis. Then, NFκB inhibitors may be wise to use in clinical trials in conjunction with TRAIL against follicular lymphomas.

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.

High Density Lipoprotein-like Nanoparticles Synergize with Akt and Btk Inhibitors to Induce Cell Death in Diffuse Large B Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3022-3022
Author(s):  
Jonathan Scott Rink ◽  
Sol Misener ◽  
Osman Cen ◽  
Shuo Yang ◽  
Leo I. Gordon ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Density Lipoprotein ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Cholesterol Homeostasis ◽  
B Cell Lymphomas ◽  
Bcr Signaling

Abstract Introduction: We previously reported that our bio-inspired, synthetic high-density lipoprotein-like nanoparticles (HDL NP) induced apoptosis in B cell lymphoma cells expressing scavenger receptor type B1 (SCARB1), the high-affinity receptor for cholesterol-rich HDLs. HDL NPs consist of a 5nm gold nanoparticle core surface functionalized with the HDL-defining apolipoprotein A1 and a phospholipid bilayer, and bind specifically to SCARB1, inducing the efflux of free cholesterol and inhibiting cholesteryl ester influx. SCARB1 is overexpressed in a subset of follicular and diffuse large B cell lymphomas (DLBCL), and resides in cholesterol-rich plasma membrane microdomains called lipid rafts, similar to the B cell receptor (BCR) and its associated signaling kinases. Upon binding to natural HDL, SCARB1 activates a number of pro-survival signaling kinases, including Akt and PI3K. Both Akt and PI3K are also involved in B cell receptor-mediated signaling in germinal center-derived (GC) DLBCL, through tonic BCR signaling, and activated B cell (ABC) DLBCL, through chronic active BCR signaling. Additionally, PI3K was recently shown to play a role in recruitment and activation of Btk, a crucial survival kinase downstream of the BCR. We hypothesized that small molecule inhibitors against pro-survival kinases, specifically Akt and Btk, will synergize with HDL NPs against B cell lymphomas. Methods: Burkitt's lymphoma (Ramos), GC DLBCL (SUDHL4) and ABC DLBCL (TMD8 and HBL-1) cell lines were treated with the Akt inhibitor GDC-0068 or the Btk inhibitor Ibrutinib, in the absence or presence of HDL NPs, and synergy was calculated using the Calcusyn software. Phos-flow was used to assay for changes in the phosphorylation status of Akt and Btk. Results: The Burkitt's lymphoma and GC DLBCL cell lines were more sensitive to HDL NP induced cell death compared to the ABC DLBCL cell lines (Ramos HDL NP IC50 = 1.5nM; SUDHL4 HDL NP IC50 = 2.1nM; TMD8 HDL NP IC50 = 31.4nM; HBL-1 HDL NP IC50 = 89nM). HDL NPs synergized with GDC-0068 in the Ramos, SUDHL4 and TMD8 cell lines (all combination indexes < 1). Correspondingly, HDL NPs dose-dependently decreased phosphorylation of Akt in Ramos and TMD8 cells. Ibrutinib synergized with the HDL NPs in all cell lines tested (all combination indexes < 1). In TMD8 cells, HDL NPs decreased p-Btk levels comparable to treatment with 10nM Ibrutinib. Addition of the PI3K inhibitor Pilaralisib (XL147) demonstrated mild synergy in the Ramos cell line, but not the SUDHL4, TMD8 or HBL-1 cell lines (all combination index values >1). Treatment of Ramos and SUDHL4 cells with an inhibitor of PTEN, a phosphatase responsible for acting in opposition to PI3K leading to inactivation of Akt, rescued the cells from HDL NP-induced cell death. TMD8 cells treated with the PTEN inhibitor demonstrated a smaller increase in survival when HDL NPs were applied, suggesting that PI3K may not play a major role in HDL NP-induced cell death in activated B cell DLBCLs. PTEN activity is influenced by the level of cholesterol and cholesteryl esters present in the cell, with increasing levels correlating with decreased PTEN activity. Cholesterol levels were higher in the ABC DLBCL cell lines compared to the other B cell lymphoma cell lines. HDL NPs significantly reduced the cholesterol content of Ramos cells, but not the TMD8 or HBL-1 cells, suggesting that the ability of the HDL NPs to alter cellular cholesterol homeostasis correlates with their ability to induce lymphoma cell death. Conclusion: HDL NPs demonstrated synergy with inhibitors to the pro-survival kinases Akt and Btk, suggesting that HDL NPs act to disrupt second messenger signaling pathways in lymphoma cells by directly altering signaling through SCARB1, modulating cellular cholesterol homeostasis, and/or through disruption of membrane raft organization. HDL NPs represent an innovative, targeted therapeutic, with great potential, to add to existing combination chemotherapy regimens. Disclosures Thaxton: Aurasense: Equity Ownership, Patents & Royalties: The patent for the HDL NPs has been licensed to Aurasense, a biotech company co-founded by C. Shad Thaxton..

scholarly journals Virally Induced Cellular MicroRNA miR-155 Plays a Key Role in B-Cell Immortalization by Epstein-Barr Virus

2010 ◽  
Vol 84 (22) ◽  
pp. 11670-11678 ◽  
Author(s):  
Sarah D. Linnstaedt ◽  
Eva Gottwein ◽  
Rebecca L. Skalsky ◽  
Micah A. Luftig ◽  
Bryan R. Cullen
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
B Cell Lymphoma ◽  
Selective Inhibition ◽  
B Cell Lymphomas ◽  
Cell Infection ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Infection of resting primary human B cells by Epstein-Barr virus (EBV) results in their transformation into indefinitely proliferating lymphoblastoid cell lines (LCLs). LCL formation serves as a model for lymphomagenesis, and LCLs are phenotypically similar to EBV-positive diffuse large B-cell lymphomas (DLBCLs), which represent a common AIDS-associated malignancy. B-cell infection by EBV induces the expression of several cellular microRNAs (miRNAs), most notably miR-155, which is overexpressed in many tumors and can induce B-cell lymphomas when overexpressed in animals. Here, we demonstrate that miR-155 is the most highly expressed miRNA in LCLs and that the selective inhibition of miR-155 function specifically inhibits the growth of both LCLs and the DLBCL cell line IBL-1. Cells lacking miR-155 are inefficient in progressing through S phase and spontaneously undergo apoptosis. In contrast, three other B-cell lymphoma lines, including two EBV-positive Burkitt's lymphoma cell lines, grew normally in the absence of miR-155 function. These data identify the induction of cellular miR-155 expression by EBV as critical for the growth of both laboratory-generated LCLs and naturally occurring DLBCLs and suggest that targeted inhibition of miR-155 function could represent a novel approach to the treatment of DLBCL in vivo.

scholarly journals TCL1: A Shared Tumor-Associated Antigen for Immunotherapy Against B-Cell Lymphomas

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 105-105
Author(s):  
Jinsheng Weng ◽  
Seema Rawal ◽  
Hyun Jun Park ◽  
Rakesh Sharma ◽  
Sattva S. Neelapu
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Primary Lymphoma ◽  
B Cell Lymphomas ◽  
Therapeutic Vaccines ◽  
Normal Tissues ◽  
Tumor Associated Antigen

Abstract Abstract 105 Rituximab-based chemotherapy regimens have improved complete response rates, progression-free survival, and overall survival of B-cell non-Hodgkin lymphoma patients. However, most patients relapse and die of their lymphoma. To further improve clinical outcome, novel strategies that eradicate minimal residual disease (MRD) after induction therapy are needed. Therapeutic vaccines may induce antitumor antibody and T-cell responses and may eradicate MRD by complementary mechanisms. A recent randomized phase III clinical trial showed that patient-specific idiotype vaccination improves disease-free survival when administered in the setting of MRD in follicular lymphoma (FL), providing proof of principle that therapeutic vaccines can improve clinical outcome (Schuster et al, J Clin Oncol, 2011). However, generation of a custom-made vaccine formulation for each patient can be expensive and time consuming. To overcome these difficulties, identification of novel shared lymphoma-associated antigens is necessary. The T-cell leukemia/lymphoma 1 (TCL1) oncoprotein encoded by the TCL1 gene is a co-activator of Akt and promotes cell proliferation and survival. Ectopic expression of TCL1 in B cells in transgenic mice results in the development of B-cell malignancies and TCL1 was reported to be aberrantly expressed in multiple human B cell malignancies. Here, we determined whether TCL1 can serve as a novel shared tumor-associated antigen in B cell lymphomas. We analyzed the expression pattern of TCL1 in human B cell lymphomas and normal tissues by real-time PCR, flow cytometry, immunohistochemistry, and Western blotting. TCL1 mRNA transcripts were hyperexpressed in multiple types of primary B cell lymphomas [chronic lymphocytic leukemia (CLL), n=7; mantle cell lymphoma (MCL), n=6; FL, n=12, diffuse large B-cell lymphoma (DLBCL), n=5] as compared with B cells derived from normal donors. TCL1 mRNA was not detected in other normal tissues that included T cells, adipose tissue, esophagus, ovary, spleen, bladder, heart, placenta, brain, kidney, prostate, liver, muscle, thyroid, colon, lung, intestine, trachea, thymus, and cervix. Very low TCL1 mRNA was detected in testis. By flow cytometry, immunohistochemistry, and/or Western blotting we observed that the TCL1 protein is hyperexpressed in CLL, MCL, DLBCL, Burkitts lymphoma, and FL, but not splenic marginal zone lymphoma. To determine whether TCL1 is immunogenic, we synthesized overlapping 15-mer peptides spanning the entire length of the TCL1 protein and stimulated PBMC from HLA-A*0201+ (HLA-A2+) normal donors to generate peptide-specific T cells. We found that TCL165–79 peptide (TQIGPSLLPIMWQLY) consistently induced T cells that secreted significant amount of IFN-γ from 3 normal donors. By intracellular cytokine assay, we determined that the TCL165–79 peptide-specific IFN-g was produced by CD8+ T cells but not CD4+ T cells. Using a panel of peptide-pulsed EBV-transformed B-cell lines that were mismatched at the MHC Class I locus and MHC Class I blocking antibodies, we determined that HLA-A2 is the restriction element of the TCL165–79 peptide-specific T cells. Consistent with this, TCL165–79 peptide-specific CD8+ T cells lysed TCL1 expressing HLA-A2+ MCL cell lines but did not lyse, TCL1 expressing HLA-A2− myeloma cell line. Using a panel of peptides truncated progressively by one amino acid at the N- and C-termini of the TCL165–79peptide, we determined that TCL171–78(LLPIMWQL) is the minimal epitope that bound to HLA-A2. Furthermore, cytotoxic T lymphocytes (CTL) specific to this peptide efficiently killed lymphoma cell lines and primary lymphoma cells from CLL, MCL, DLBCL, and FL in an HLA-A2-restricted manner. Using TCL171–78 peptide-specific HLA-A2 tetramers, we found that TCL1 epitope-specific CTLs were present in the peripheral blood and/or tumor-infiltrating lymphocytes of B-cell lymphoma patients (n=8). More importantly, these TCL1 epitope-specific CTLs could be expanded in vitro and lysed autologous and allogeneic tumor cells in an HLA-A2-restricted manner. In conclusion, our results suggest that TCL1 is naturally processed and presented on the surface of primary lymphoma cells for recognition by CTL and can serve as a novel, shared tumor-associated antigen for therapeutic vaccine development against common B-cell lymphomas including CLL, MCL, DLBCL, and FL. Disclosures: Neelapu: Biovest International, Inc.: Research Funding.

scholarly journals Gene involved in the 3q27 translocation associated with B-cell lymphoma, BCL5, encodes a Kruppel-like zinc-finger protein

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 26-32 ◽  
Author(s):  
T Miki ◽  
N Kawamata ◽  
S Hirosawa ◽  
N Aoki
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Cdna Probe ◽  
B Cell Lymphoma ◽  
Nucleotide Sequencing ◽  
Sequencing Analysis

Chromosomal translocations involving band 3q27 are the recently described nonrandom cytogenetic abnormalities in B-cell malignancies. We have previously cloned the breakpoint region of 3q27, designated as the BCL5 locus, from the B-cell line carrying the t(3;22). The cDNA for the BCL5 gene was cloned from the human liver cDNA library. The nucleotide sequencing analysis showed that the BCL5 gene encodes a potential transcription factor containing six repeats of the Cys2-His2 zinc-finger motif resembling the Drosophila segmentation gene Kruppel. The calculated molecular weight was 78.8 kD, which was supported by an in vitro transcription and translation experiment. A part of the sequence was essentially identical to that of a genomic fragment, ZNF51, previously reported to be located at 3qter. The translocation occurred in the 5′ region of the BCL5 gene, and the protein-coding exons were fused to the Ig-lambda gene in a head-to-head configuration in the cell line carrying t(3;22). The BCL5 cDNA probe detected a major transcript of 3.8 kb in Burkitt's lymphoma cell lines and an aberrant transcript in the t(3;22) cell line, whereas no transcript was detected in myeloid, monocytoid, erythroid, T-lymphoid, and Epstein-Barr virus- immortalized B-lymphoblastoid cell lines.

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