scholarly journals Yy1 Depletion in Pancreatic Beta Cells Leads to Energy Source Switch From Glycolysis to Oxidative Phosphorylation

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A327-A327
Author(s):  
Eliana M Perez-Garcia ◽  
Ruya Liu ◽  
Vijay K Yechoor
Keyword(s):  
Gene Expression ◽  
Energy Source ◽  
Copy Number ◽  
Mitochondrial Gene ◽  
Loss Of Function ◽  
Loop Formation ◽  
Β Cells ◽  
Mitochondrial Copy Number ◽  
Gene And Protein Expression ◽  
Insulinoma Cells

Abstract Background: Gene expression is determined by structural interactions in between transcription factors, cofactors and enhancer elements, as well as enhancer-promoter interactions (1). Both YY1 and CTCF are essential, zinc finger proteins that bind hypo-methylated DNA sequences, form homodimers, and thus facilitate DNA loop formation (1). ​However, YY1 preferentially occupies interacting enhancers and promoters, whereas CTCF preferentially occupies sites distal from these regulatory elements, forming larger loops and participating in insulation (1). A sequencing study of spontaneous functional insulinomas in a Chinese cohort identified a somatic a hotspot mutation in YY1 (c.C1115G/p.T372R) in 30% of the cases, associated with increased YY1 activity (2). YY1 is a critical transcription factor involved in the regulation of proliferation and metabolism (2). Hypothesis: YY1 loss-of-function alters energy source preference in pancreatic β-cells. Methods: YY1 stable loss-of-function in mouse insulinoma cell lines was achieved by shRNA lentiviral transduction. Mitochondrial membrane potential (MMP) was measured via flow cytometry of aggregated mitochondria to monomeric mitochondria ratio. Mitostress and complex-substrate controlled respiration were measured by Seahorse analyzer. Mitochondrial copy number was assessed by mitochondrial to nuclear DNA ratio. Quantitative qPCR and Western blotting were used to assess mitochondrial gene and protein expression. Results: Our data indicated that YY1 deficient β-cells showed increased MMP and maximal respiration. No significant differences were found in basal respiration, ATP production, proton leak, non-mitochondrial oxygen consumption or coupling efficiency. We also found that YY1 deficient β-cells exhibited reduced glycolytic capacity and decreased ETC complex IV activity, with concurrent increased complex I and II activity. In addition, YY1 deficient β-cells exhibited elevated mitochondrial copy number​ and increased quantitative mRNA of mitochondrial gene expression, which could be correlated with increased PGC1-α expression. Conclusions: YY1 is critical in the metabolic regulation of β-cells, particularly in the facilitation of glycolytic metabolism. YY1 activating mutations in functional spontaneous insulinoma cells can lead to a proliferation dysregulation accompanied by a metabolic switch that favors glycolysis, while the opposite occurs in YY1 deficient β-cells. References: (1) Weintraub AS et al, Cell 2017 Dec 14; 171:1573–1588 (2) Cao Y et al, Nat Commun 2013 Dec 10; 4:2810

scholarly journals Differentiation of Sendai Virus-Reprogrammed iPSC into β Cells, Compared with Human Pancreatic Islets and Immortalized β Cell Line

2018 ◽  
Vol 27 (10) ◽  
pp. 1548-1560 ◽  
Author(s):  
Silvia Pellegrini ◽  
Fabio Manenti ◽  
Raniero Chimienti ◽  
Rita Nano ◽  
Linda Ottoboni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pancreatic Islets ◽  
Cell Line ◽  
Expression Profile ◽  
Sendai Virus ◽  
Secretory Function ◽  
Β Cells ◽  
Β Cell ◽  
Insulin Producing Cells ◽  
Gene And Protein Expression

Background: New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (iβ). However, the gene expression profile and secretory function of iβ still need to be validated in comparison with native β cells. Methods: Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into iβ and compared with donor pancreatic islets and EndoC-βH1, an immortalized human β cell line. Results: Both clones of iPSCs differentiated into insulin+ cells with high efficiency (up to 20%). iβ were negative for pluripotency markers (Oct4, Sox2, Ssea4) and positive for Pdx1, Nkx6.1, Chromogranin A, PC1/3, insulin, glucagon and somatostatin. iβ basally secreted C-peptide, glucagon and ghrelin and released insulin in response either to increasing concentration of glucose or a depolarizing stimulus. The comparison revealed that iβ are remarkably similar to donor derived islets in terms of gene and protein expression profile and similar level of heterogeneity. The ability of iβ to respond to glucose instead was more related to that of EndoC-βH1. Discussion: We demonstrated that insulin-producing cells generated from iPSCs recapitulate fundamental gene expression profiles and secretory function of native human β cells.

scholarly journals Asthma and its relationship to mitochondrial copy number: Results from the Asthma Translational Genomics Collaborative (ATGC) of the Trans-Omics for Precision Medicine (TOPMed) program

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242364
Author(s):  
Maxwell P. Cocco ◽  
Evan White ◽  
Shujie Xiao ◽  
Donglei Hu ◽  
Angel Mak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Precision Medicine ◽  
Copy Number ◽  
Sequence Data ◽  
Whole Genome Sequence ◽  
Mtdna Copy Number ◽  
Respiratory Complex ◽  
Mitochondrial Copy Number ◽  
Patient Reported ◽  
Asthma Status

Background Mitochondria support critical cellular functions, such as energy production through oxidative phosphorylation, regulation of reactive oxygen species, apoptosis, and calcium homeostasis. Objective Given the heightened level of cellular activity in patients with asthma, we sought to determine whether mitochondrial DNA (mtDNA) copy number measured in peripheral blood differed between individuals with and without asthma. Methods Whole genome sequence data was generated as part of the Trans-Omics for Precision Medicine (TOPMed) Program on participants from the Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-ethnicity (SAPPHIRE) and the Study of African Americans, Asthma, Genes, & Environment II (SAGE II). We restricted our analysis to individuals who self-identified as African American (3,651 asthma cases and 1,344 controls). Mitochondrial copy number was estimated using the sequencing read depth ratio for the mitochondrial and nuclear genomes. Respiratory complex expression was assessed using RNA-sequencing. Results Average mitochondrial copy number was significantly higher among individuals with asthma when compared with controls (SAPPHIRE: 218.60 vs. 200.47, P<0.001; SAGE II: 235.99 vs. 223.07, P<0.001). Asthma status was significantly associated with mitochondrial copy number after accounting for potential explanatory variables, such as participant age, sex, leukocyte counts, and mitochondrial haplogroup. Despite the consistent relationship between asthma status and mitochondrial copy number, the latter was not associated with time-to-exacerbation or patient-reported asthma control. Mitochondrial respiratory complex gene expression was disproportionately lower in individuals with asthma when compared with individuals without asthma and other protein-encoding genes. Conclusions We observed a robust association between asthma and higher mitochondrial copy number. Asthma having an effect on mitochondria function was also supported by lower respiratory complex gene expression in this group.

scholarly journals Mitochondria-Related Nuclear Gene Expression in the Nucleus Accumbens and Blood Mitochondrial Copy Number After Developmental Fentanyl Exposure in Adolescent Male and Female C57BL/6 Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Cali A. Calarco ◽  
Megan E. Fox ◽  
Saskia Van Terheyden ◽  
Makeda D. Turner ◽  
Jason B. Alipio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nucleus Accumbens ◽  
Mitochondrial Function ◽  
Copy Number ◽  
Drug Exposure ◽  
Related Gene ◽  
Developmental Exposure ◽  
Neuronal Signaling ◽  
Mitochondrial Copy Number ◽  
The Brain

The potency of the synthetic opioid fentanyl and its increased clinical availability has led to the rapid escalation of use in the general population, increased recreational exposure, and subsequently opioid-related overdoses. The wide-spread use of fentanyl has, consequently, increased the incidence of in utero exposure to the drug, but the long-term effects of this type of developmental exposure are not yet understood. Opioid use has also been linked to reduced mitochondrial copy number in blood in clinical populations, but the link between this peripheral biomarker and genetic or functional changes in reward-related brain circuitry is still unclear. Additionally, mitochondrial-related gene expression in reward-related brain regions has not been examined in the context of fentanyl exposure, despite the growing literature demonstrating drugs of abuse impact mitochondrial function, which subsequently impacts neuronal signaling. The current study uses exposure to fentanyl via dam access to fentanyl drinking water during gestation and lactation as a model for developmental drug exposure. This perinatal drug-exposure is sufficient to impact mitochondrial copy number in circulating blood leukocytes, as well as mitochondrial-related gene expression in the nucleus accumbens (NAc), a reward-related brain structure, in a sex-dependent manner in adolescent offspring. Specific NAc gene expression is correlated with both blood mitochondrial copy number and with anxiety related behaviors dependent on developmental exposure to fentanyl and sex. These data indicate that developmental fentanyl exposure impacts mitochondrial function in both the brain and body in ways that can impact neuronal signaling and may prime the brain for altered reward-related behavior in adolescence and later into adulthood.

scholarly journals Endoplasmic reticulum stress induces Wfs1 gene expression in pancreatic β-cells via transcriptional activation

2005 ◽  
Vol 153 (1) ◽  
pp. 167-176 ◽  
Author(s):  
Kohei Ueda ◽  
June Kawano ◽  
Komei Takeda ◽  
Toshiaki Yujiri ◽  
Katsuya Tanabe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Stress Responses ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Min6 Cells ◽  
Insulinoma Cells

Objective: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein. Homozygous WFS1 gene mutations cause Wolfram syndrome, characterized by insulin-deficient diabetes mellitus and optic atropy. Pancreatic β-cells are selectively lost from the patient’s islets. ER localization suggests that WFS1 protein has physiological functions in membrane trafficking, secretion, processing and/or regulation of ER calcium homeostasis. Disturbances or overloading of these functions induces ER stress responses, including apoptosis. We speculated that WFS1 protein might be involved in these ER stress responses. Design and methods: Islet expression of the Wfs1 protein was analyzed immunohistochemically. Induction of Wfs1 upon ER stress was examined by Northern and Western blot analyses using three different models: human skin fibroblasts, mouse pancreatic β-cell-derived MIN6 cells, and Akita mouse-derived Ins2 96Y/Y insulinoma cells. The human WFS1 gene promoter-luciferase reporter analysis was also conducted. Result: Islet β-cells were the major site of Wfs1 expression. This expression was also found in δ-cells, but not in α-cells. WFS1 expression was transcriptionally up-regulated by ER stress-inducing chemical insults. Treatment of fibroblasts and MIN6 cells with thapsigargin or tunicamycin increased WFS1 mRNA. WFS1 protein also increased in response to thapsigargin treatment in these cells. WFS1 gene expression was also increased in Ins2 96Y/Y insulinoma cells. In these cells, ER stress was intrinsically induced by mutant insulin expression. The WFS1 gene promoter-luciferase reporter system revealed that the human WFS1 promoter was activated by chemically induced ER stress in MIN6 cells, and that the promoter was more active in Ins2 96Y/Y cells than Ins2 wild/wild cells. Conclusion: Wfs1 expression, which is localized to β- and δ-cells in pancreatic islets, increases in response to ER stress, suggesting a functional link between Wfs1 and ER stress.

scholarly journals STAT3 Regulates Mitochondrial Gene Expression in Pancreatic β-Cells and its Deficiency Induces Glucose Intolerance in Obesity

2021 ◽  
Author(s):  
Anaïs Schaschkow ◽  
Lokman Pang ◽  
Valerie Vandenbempt ◽  
Bernat Elvira ◽  
Sara A. Litwak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glucose Intolerance ◽  
High Fat Diet ◽  
Cell Function ◽  
Mitochondrial Gene ◽  
High Fat ◽  
Β Cells ◽  
Β Cell ◽  
Β Cell Function ◽  
Deficient Mice

Most obese and insulin-resistant individuals do not develop diabetes. This is the result of the capacity of β-cells to adapt and produce enough insulin to cover the needs of the organism. The underlying mechanism of β-cell adaptation in obesity, however, remains unclear. Previous studies have suggested a role for STAT3 in mediating β-cell development and human glucose homeostasis, but little is known about STAT3 in β-cells in obesity. We observed enhanced cytoplasmic expression of STAT3 in severely obese and diabetic subjects. To address the functional role of STAT3 in adult β-cells, we generated mice with tamoxifen-inducible partial or full deletion of STAT3 in β-cells and fed them a high-fat diet before analysis. Interestingly, β-cell heterozygous and homozygous STAT3-deficient mice showed glucose intolerance when fed a high-fat diet. Gene expression analysis by RNA-Seq showed reduced expression of mitochondrial genes in STAT3 knocked down human EndoC-βH1 cells and was confirmed in FACS-purified β-cells from obese STAT3-deficient mice. Moreover, silencing of STAT3 impaired mitochondria activity in EndoC-βH1 cells and human islets, suggesting a mechanism for STAT3-modulated β-cell function. We propose STAT3 as a regulator of β-cell function, improving glucose-induced insulin secretion in obesity.

Two Cellular Mechanisms for Gamma-Globin Gene and Protein Silencing in Humans.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 374-374 ◽  
Author(s):  
Patricia A. Oneal ◽  
Joseph D. Schwartz ◽  
Nicole Gantt ◽  
Natarajan Bhanu ◽  
Y. Terry Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Beta Cells ◽  
Copy Number ◽  
Globin Gene ◽  
Beta Globin ◽  
Cellular Mechanisms ◽  
Cdna Copy ◽  
Gamma Globin ◽  
Gene And Protein Expression ◽  
Globin Gene Expression

Abstract Although the genetic processes responsible for gamma-globin gene and protein silencing are not known, the prevailing model is that gamma-globin silencing results from a gradual change within a single hematopoietic cell lineage that is governed by intrinsic properties of the cells. In order to provide a more complete characterization of the silencing phenomenon, we studied globin expression patterns directly from clinical samples using single-cell, quantitative PCR, and globin protein phenotyping. We collected blood samples from untransfused donors: umbilical cords (n=3), infants (n=11; ages 1 day to 35 months), and adults (n=3). All samples were maintained at 4°C and analyzed within 72 hours. Flow cytometry (30,000 cells per donor) and HPLC analyses were used for globin protein phenotyping. For globin gene expression, we identified reticulocytes using a strategy that required no membrane permeabilization, and sorted them as single cells directly into lysis buffer. Oligo-dT reverse transcription of mRNA was followed by real-time PCR quantitation. Globin cDNA copy numbers were calculated using standard curves from serial dilutions of a plasmid DNA. We analyzed approximately 1000 single-cell quantitative PCR amplifications for gamma- and beta-globin gene expression. In cord blood, we detected both gamma- and beta-globin gene expression in 97.4% (112/115) of the reticulocytes. The average gamma-globin cDNA copy number was 1870±1390 copies, compared with an average beta-globin cDNA copy number of 2181±2138 copies per reticulocyte. HbF and HbA were also detected in >95% of the cord blood erythrocytes. In the adult samples, HbF was detected in <5% of the circulating erythrocytes and gamma-globin gene expression in only 1.5% (3/206) of the reticulocytes. The average gamma-globin cDNA copy number in the minor population of gamma(+) adult reticulocytes was 468±198 copies, and the average beta-globin cDNA copy number in the beta(+) adult reticulocytes was 3869±3733 copies. Compared with the relatively monotonous patterns of gamma-globin gene and protein expression in cord and adult blood, we clearly detected an age-based fluctuation between those patterns in the infant blood samples. During the first three years of life, a gradual loss in the level of gamma-globin gene and protein expression was identified among the gamma(+)beta(+) reticulocytes and the HbF(+)HbA(+) erythrocytes. In addition, discrete populations of gamma(−)beta(+)reticulocytes and HbF(−)HbA(+) erythrocytes were detected. Rapid expansion of those gamma-silenced populations became apparent soon after birth. Within four months, the proportion of gamma-silenced cells eclipsed that of the gamma(+)beta(+) cells to become the predominant population. By three years after birth, the two cell populations essentially merged to become a single, gamma-silenced population similar to that found in adults. These data suggest two cellular mechanisms for gamma-globin silencing in humans: 1) a gradual loss in gamma-globin expression in the gamma(+)beta(+) cells beginning prior to delivery and continuing during infancy, and 2) replacement of the gamma(+)beta(+) cells with a population of gamma-silenced cells that rapidly accumulate after birth, possibly in response to the dramatic increase in oxygenation or other environmental changes.

Gene Expression Profiling Reveals Key Mitochondrial Gene Changes in Stored Platelets

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3559-3559 ◽  
Author(s):  
James A Bynum ◽  
Tiffani Chance ◽  
Michael Adam Meledeo ◽  
Heather F. Pidcoke
Keyword(s):  
Gene Expression ◽  
Mitochondrial Respiration ◽  
Mitochondrial Gene ◽  
Fluorescent Imaging ◽  
Mitochondrial Gene Expression ◽  
Loss Of Function ◽  
Oxygen Utilization ◽  
C Storage ◽  
Carbonyl Cyanide ◽  
Mitochondrial Respiratory

Abstract Introduction As of June 2015 the FDA has approved an alternative procedure under 21 CFR 640.120 that allows for storage of apheresis platelets at refrigerator temperature (1-6 C; 4°C) without agitation for up to 3 days for use in the resuscitation of actively bleeding patients. Understanding underlying mechanisms responsible for enhanced hemostatic function at 4°C will be critical for such improvements in platelet transfusion. We hypothesized that 4°C platelets display better mitochondrial respiratory function for up to 7 days compared to standard 5-day RT platelets and that mitochondrial gene expression differences between RT and 4°C -stored platelets will correlate with mitochondrial function. Methods Platelets were collected from healthy donors by apheresis according to an IRB-approved protocol. Apheresis platelets (AP) were rested for 1 h before allocation into platelet minibags (Blood Cell Storage, Seattle, WA) and stored for 4 storage durations (Baseline (BL), Day 3, 5, and 7). Mitochondrial respiration, maximal oxygen utilization, and individual mitochondrial complex-dependent respiration were assessed with high-resolution respirometry (O2k, Oroboros). Mitochondrial ROS generation in response to storage condition or stimulation (to assess oxidative burst capacity as a measure of function) was visualized with fluorescent imaging and assayed with flow cytometry using a superoxide stain (Life Technologies). Total RNA was extracted both immediately following apheresis (BL) and on Day 5 from RT and 4°C-stored platelets using Trizol (Molecular Research Center, Cincinnati, OH) after centrifuging the platelets at 900 x g for 10 min. Platelet RNA was quantified using the NanoDrop 2000. RNA quality was examined using gel electrophoresis with the Reliant Gel System (Cambrex, Rockland, ME). Platelet mitochondrial gene expression analysis was evaluated using the 96-well RT2 Profiler PCR Array (Qiagen, Valencia, CA) which profiled 84 mitochondria-focused targets and 12 control genes per sample. Gene expression data analysis was based on the ΔΔCt method with normalization of the raw data to housekeeping genes located on each 96-well plate. Results Mitochondrial respiration was lower in platelets stored at 4°C compared to RT on Days 3, 5, and 7 (Day 5= -57%±0.3; P < 0.05), demonstrating that refrigeration slows metabolism. Additionally, maximal mitochondrial oxygen utilization (electron transport system capacity) was better preserved in platelets stored at 4°C (Figure 1). Fluorescent imaging and flow cytometry demonstrated that mROS generation was higher in RT-stored platelets compared to 4°C, reflecting mitochondrial damage. Mitochondrial burst during de novo mROS generation due to stimulation was also preserved at 4°C. Mitochondrial gene expression studies revealed distinct differences in expression profiles for 4°C versus RT-stored platelets after 5 days of storage when normalized to BL measures. Storage at 4°C resulted in significantly greater preservation of 15 gene products at Day 5 (P<0.05). In contrast, Day 5 RT samples resulted in a marked decrease or loss of gene products when compared to BL levels of gene expression (P<0.05). Discussion Platelet mitochondrial respiratory function (mitochondrial respiration and maximal oxygen utilization) decreased in RT-stored platelets over 7 days, but the impairment was attenuated by 4°C storage. We previously noted that intracellular ROS flux was higher at room temperature, and here the gene expression analysis in combination with oximetry data showed that mitochondrial damage is likely responsible. Furthermore, gene expression profiling of mitochondrial-related genes revealed that distinct differences exist in key mitochondrial genes between the storage conditions. This work illustrates that 4°C storage of platelets preserved and enhanced critical mitochondrial genes compared to RT; this finding combined with improved mitochondrial respiratory measures and reduced ROS demonstrates a significant improvement in current efforts to mitigate platelet dysfunction. Figure 1. Maximal respiration induced by titration of the protonophore FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone) demonstrated a significant decrease in mitochondrial capacity (indicating loss of function) in RT-stored samples compared to 4 °C-stored samples by Day 7. Values are mean ± SD (n=7); *P<0.001 compared to RT. Figure 1. Maximal respiration induced by titration of the protonophore FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone) demonstrated a significant decrease in mitochondrial capacity (indicating loss of function) in RT-stored samples compared to 4 °C-stored samples by Day 7. Values are mean ± SD (n=7); *P<0.001 compared to RT. Disclosures No relevant conflicts of interest to declare.

scholarly journals STAT3 Regulates Mitochondrial Gene Expression in Pancreatic β-Cells and its Deficiency Induces Glucose Intolerance in Obesity

2021 ◽  
Author(s):  
Anaïs Schaschkow ◽  
Lokman Pang ◽  
Valerie Vandenbempt ◽  
Bernat Elvira ◽  
Sara A. Litwak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glucose Intolerance ◽  
High Fat Diet ◽  
Cell Function ◽  
Mitochondrial Gene ◽  
High Fat ◽  
Β Cells ◽  
Β Cell ◽  
Β Cell Function ◽  
Deficient Mice

Most obese and insulin-resistant individuals do not develop diabetes. This is the result of the capacity of β-cells to adapt and produce enough insulin to cover the needs of the organism. The underlying mechanism of β-cell adaptation in obesity, however, remains unclear. Previous studies have suggested a role for STAT3 in mediating β-cell development and human glucose homeostasis, but little is known about STAT3 in β-cells in obesity. We observed enhanced cytoplasmic expression of STAT3 in severely obese and diabetic subjects. To address the functional role of STAT3 in adult β-cells, we generated mice with tamoxifen-inducible partial or full deletion of STAT3 in β-cells and fed them a high-fat diet before analysis. Interestingly, β-cell heterozygous and homozygous STAT3-deficient mice showed glucose intolerance when fed a high-fat diet. Gene expression analysis by RNA-Seq showed reduced expression of mitochondrial genes in STAT3 knocked down human EndoC-βH1 cells and was confirmed in FACS-purified β-cells from obese STAT3-deficient mice. Moreover, silencing of STAT3 impaired mitochondria activity in EndoC-βH1 cells and human islets, suggesting a mechanism for STAT3-modulated β-cell function. We propose STAT3 as a regulator of β-cell function, improving glucose-induced insulin secretion in obesity.

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

Blood ◽  
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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