scholarly journals P09.14 Blocking counterregulation of unfolded protein response by targeted protein synthesis inhibition produces highly synergistic cell death in several cancer entities

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A59.1-A59
Author(s):  
F Gsottberger ◽  
C Meier ◽  
S Petkovic ◽  
L Mellenthin ◽  
M Krumbholz ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Synthesis ◽  
Unfolded Protein Response ◽  
Therapeutic Window ◽  
Protein Synthesis Inhibition ◽  
Synthesis Inhibition ◽  
Unfolded Protein ◽  
Protein Response

BackgroundBecause tumor cells have high proliferation rates the demand for energy on the one hand and proteins on the other hand is high. In line, protein folding machinery of the ER is heavily used. 2-Deoxyglucose (2-DG) not only blocks energy synthesis by inhibiting glycolysis but also blocks synthesis of mannosyl leading to impaired N-linked glycosylation, accumulation of misfolded proteins, and increased unfolded protein response (UPR). However, due to compensatory events, UPR-induced apoptosis is hampered. Therefore, we combined 2-DG with targeted protein synthesis inhibition by immunotoxins, consisting of an antibody and pseudomonas exotoxin, to enhance UPR mediated cell death.Materials and MethodsEstablished cell lines and patient-derived B-ALL samples were treated in vitro with various protein synthesis inhibitors and UPR-inducers. Drug synergy was determined mathematically as fold-increase over additivity. Biochemical studies were performed using western blots. In vivo enhancement was tested using systemic xenograft models.ResultsThe combination of Moxetumomab and 2-DG achieved a two to nine-fold synergy in vitro. Synergy was abrogated by the addition of Mannose suggesting UPR as cause of synergistic cell death. Similarly, Moxetumomab enhanced UPR-inducers Bortezomib and tunicamycin and protein synthesis inhibition by cycloheximide and puromycin enhanced 2-DG suggesting a conserved mechanism. Using HB21, an immunotoxin targeting human transferrin-receptor, breast cancer, hepatocellular carcinoma, and glioblastoma were sensitized to 2-DG induced cell death. Biochemically, 2-DG increased XBP-1-cleavage, expression of pro-apoptotic CHOP and of anti-apoptotic BIP. Moxetumomab, however, blocked the upregulation of BIP while maintaining CHOP correlating with synergistic increase in PARP-cleavage and apoptosis. In two systemic mouse models, bone marrow (BM) lymphoma infiltration was not reduced by 2-DG or tunicamycin alone but was reduced after treatment with Moxetumomab alone by 5-fold in the JeKo-1 and by 16-fold in the Ramos model, respectively. The combination of Moxetumomab and 2-DG achieved a three-fold synergy in the JeKo-1 model and achieved MRD-negative BM status in the Ramos model. Against patient-derived B-ALL of the Burkitt’s type, 2-DG and Moxetumomab were up to 5-fold more active in vitro and up to 7-fold more active in mouse xenografts in vivo.ConclusionsCell death after persisting unfolded protein response is synergistically enhanced by tumor-cell specific inhibition of protein synthesis against four distinct tumor entities at physiologically achievable concentrations. Our approach of immunotoxin-induced targeted protein synthesis inhibition opens a novel, so far undescribed therapeutic window which may warrant clinical evaluation.Disclosure InformationF. Gsottberger: None. C. Meier: None. S. Petkovic: None. L. Mellenthin: None. M. Krumbholz: None. M. Metzler: None. A. Mackensen: None. F. Müller: None.

scholarly journals Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic β-cells under glucolipotoxic stress

2019 ◽  
Author(s):  
Sarah A. White ◽  
Lisa Zhang ◽  
Yu Hsuan Carol Yang ◽  
Dan S. Luciani
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Unfolded Protein ◽  
Protein Response

ABSTRACTER stress and apoptosis contribute to the loss of pancreatic β-cells under the pro-diabetic conditions of glucolipotoxicity. Although activation of the canonical pathway of intrinsic apoptosis is known to require Bax and Bak, their individual and combined involvement in glucolipotoxic β-cell death have not been demonstrated. It has also remained an open question if Bax and Bak in β-cells have non-apoptotic roles in mitochondrial function and ER stress signaling, as suggested in other cell types. Using mice with individual or combined β-cell deletion of Bax and Bak, we demonstrated that glucolipotoxic β-cell death in vitro happens in sequential stages; first via non-apoptotic mechanisms and later by apoptosis, which Bax and Bak were redundant in triggering. In contrast, they had non-redundant roles in mediating staurosporine-induced β-cell apoptosis. We further established that Bax and Bak do not affect normal glucose-stimulated β-cell Ca2+ responses, insulin secretion, or in vivo glucose tolerance. Finally, our experiments revealed that Bax and Bak together dampen the unfolded protein response in β-cells during the early stages of chemical- or glucolipotoxicity-induced ER stress. These findings identify novel roles of the canonical apoptosis machinery in modulating stress signals that are important for the pathobiology of β-cells in diabetes.

scholarly journals USP19 deubiquitinating enzyme inhibits muscle cell differentiation by suppressing unfolded-protein response signaling

2015 ◽  
Vol 26 (5) ◽  
pp. 913-923 ◽  
Author(s):  
Benjamin Wiles ◽  
Miao Miao ◽  
Erin Coyne ◽  
Louise Larose ◽  
Andrey V. Cybulsky ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Unfolded Protein Response ◽  
Muscle Cell ◽  
Deubiquitinating Enzyme ◽  
Muscle Cell Differentiation ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

USP19 deubiquitinating enzyme has two isoforms, cytoplasmic and endoplasmic reticulum (ER) localized. The ER-localized isoform specifically suppresses muscle cell differentiation in vitro and appears to do so by inhibiting the unfolded-protein response that occurs during such differentiation. In vivo, loss of USP19 promotes muscle regeneration following injury.

scholarly journals A Novel Transcription Factor, ERD15 (Early Responsive to Dehydration 15), Connects Endoplasmic Reticulum Stress with an Osmotic Stress-induced Cell Death Signal

2011 ◽  
Vol 286 (22) ◽  
pp. 20020-20030 ◽  
Author(s):  
Murilo S. Alves ◽  
Pedro A. B. Reis ◽  
Silvana P. Dadalto ◽  
Jerusa A. Q. A. Faria ◽  
Elizabeth P. B. Fontes ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Transcription Factor ◽  
Osmotic Stress ◽  
Er Stress ◽  
Unfolded Protein ◽  
Eukaryotic Organisms ◽  
Protein Response

As in all other eukaryotic organisms, endoplasmic reticulum (ER) stress triggers the evolutionarily conserved unfolded protein response in soybean, but it also communicates with other adaptive signaling responses, such as osmotic stress-induced and ER stress-induced programmed cell death. These two signaling pathways converge at the level of gene transcription to activate an integrated cascade that is mediated by N-rich proteins (NRPs). Here, we describe a novel transcription factor, GmERD15 (Glycine max Early Responsive to Dehydration 15), which is induced by ER stress and osmotic stress to activate the expression of NRP genes. GmERD15 was isolated because of its capacity to stably associate with the NRP-B promoter in yeast. It specifically binds to a 187-bp fragment of the NRP-B promoter in vitro and activates the transcription of a reporter gene in yeast. Furthermore, GmERD15 was found in both the cytoplasm and the nucleus, and a ChIP assay revealed that it binds to the NRP-B promoter in vivo. Expression of GmERD15 in soybean protoplasts activated the NRP-B promoter and induced expression of the NRP-B gene. Collectively, these results support the interpretation that GmERD15 functions as an upstream component of stress-induced NRP-B-mediated signaling to connect stress in the ER to an osmotic stress-induced cell death signal.

scholarly journals Activation of the Unfolded Protein Response with the First-in-Class P97 Inhibitor CB-5083 Induces Stable Disease Regression and Overcomes Ara-C Resistance in AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1350-1350
Author(s):  
Steffan T. Nawrocki ◽  
Yingchun Han ◽  
Ronan LE Moigne ◽  
Valeria Visconte ◽  
Bartlomiej Przychodzen ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Board Of Directors ◽  
Cell Lines ◽  
Primary Cells ◽  
Advisory Committees ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Acute myeloid leukemia (AML) therapy has remained relatively unchanged for more than 40 years with the majority of patients not achieving long-term remission when treated with currently available agents. Novel strategies are urgently needed to improve outcomes. The constitutive dysregulation of protein synthesis/turnover contributes to disease progression and drug resistance in many forms of cancer including AML. p97 (VCP) is a master regulator of protein turnover that has been implicated in oncogenesis and malignant pathogenesis. CB-5083 is a first-in-class selective and potent orally available inhibitor of p97 that in currently being evaluated in phase I clinical trials in patients with multiple myeloma and advanced solid tumors. To assess the potential benefit of p97 inhibition as a novel approach for AML therapy, we investigated the efficacy, pharmacodynamics (PD), and pharmacokinetics (PK) of CB-5083 in a panel of human AML cell lines with diverse genetic backgrounds, primary AML specimens from both newly diagnosed and relapsed/refractory patients, and xenograft mouse models of AML. In vitro treatment with CB-5083 potently diminished the viability of AML cell lines (n = 7) and primary CD34+ blasts obtained from patients (n = 10) with IC50s significantly below 1 µM (range 200 - 700 nM) in all lines and specimens evaluated to date. Diminished viability was associated with reduced clonogenic survival and increased apoptosis in AML cell lines and primary blasts. In contrast to many conventional and experimental drugs that are less active against primary AML cells than established AML cell lines, primary cells exhibited sensitivity to CB-5083 that was similar to cell lines. Additionally, CB-5083 was highly active in 3 different cell line models of cytarabine resistance and primary cells from refractory AML patients. This suggests that CB-5083 may be effective for patients who are relapsed/refractory to conventional therapy. In vitro PD analyses demonstrated that CB-5083 rapidly triggered the accumulation of ubiquitin-conjugated proteins, activated the unfolded protein response (UPR), disrupted STAT5 signaling, reduced levels of key STAT5 targets including BCL-xL and PIM-2, and induced apoptosis. The pro-apoptotic effects of CB-5083 were associated with activation of the endoplasmic reticulum (ER) resident initiator caspase-4 and induction of the BH3-only protein NOXA, which has been previously demonstrated to be an important mediator of cell death induced by other agents that disrupt protein homeostasis. RNA sequencing (RNASeq) gene ontology (GO) analyses of MV4-11 and MOLM-13 AML cells following treatment with CB-5083 demonstrated that short-term treatment (6h) caused significant increases in multiple regulators of the unfolded protein response, protein biosynthesis, and other ubiquitin-related pathways (p<0.001). Results were confirmed by qRT-PCR. The in vivo anti-leukemic activity of CB-5083 was investigated in two different xenograft mouse models of AML: the FLT3-ITD+ MV4-11 cell line and APML HL-60 cells. Oral administration of CB-5083 (once daily, 4 days on, 3 days off) was well tolerated and induced disease regression in both xenograft models (p<0.01). In vivo PD studies demonstrated that administration of CB-5083 led to reduced AML cell proliferation (PCNA), to the induction of apoptosis (active caspase-3), and pathway inhibition as evidenced by poly-ubiquitin accumulation and elevated expression of CHOP, GRP78, and NOXA. PK-PD analyses demonstrated a correlation between the kinetics of the in vivo PD effects and drug exposure. Our collective preclinical data demonstrate that p97 inhibition is a very effective novel anti-leukemic strategy and support clinical investigation of CB-5083 in patients with relapsed/refractory AML. Disclosures LE Moigne: Cleave Biosciences: Employment. Rolfe:Cleave Biosciences: Employment. Djakovic:Cleave Biosciences: Employment. Anderson:Cleave Biosciences: Employment. Wustrow:Cleave Biosciences: Employment. Zhou:Cleave Biosciences: Employment. Wong:Cleave Biosciences: Employment. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Carew:Boehringer Ingelheim: Research Funding.

scholarly journals Unfolded Protein Response and Crohn’s Diseases: A Molecular Mechanism of Wound Healing in the Gut

2021 ◽  
Author(s):  
Chao Li
Keyword(s):  
Er Stress ◽  
Unfolded Protein Response ◽  
Immune Cell ◽  
Macrophage Polarization ◽  
Intestinal Epithelial ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Endoplasmic reticulum (ER) stress triggers a series of signaling and transcriptional events termed the unfolded protein response (UPR). Severe ER stress is associated with the development of fibrosis in different organs including lung, liver, kidney, heart, and intestine. ER stress is an essential response of epithelial and immune cells in the pathogenesis of inflammatory bowel disease (IBD) including Crohn&rsquo;s disease. Intestinal epithelial cells are susceptible to ER stress-mediated damage due to secretion of a large amount of proteins that are involved in mucosal defense. In other cells, ER stress is linked to myofibroblast activation, extracellular matrix production, macrophage polarization, and immune cell differentiation. This review focuses on the role of UPR in the pathogenesis in IBD from an immunologic perspective. The roles of macrophage and mesenchymal cells in the UPR from in vitro and in vivo animal models are discussed. The links between ER stress and other signaling pathways such as senescence and autophagy are introduced. Recent advances in the understanding of the epigenetic regulation of UPR signaling are also updated here. The future directions of development of the UPR research and therapeutic strategies to manipulate ER stress levels are also reviewed.

scholarly journals Dominant negative FADD dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy

2015 ◽  
Vol 309 (10) ◽  
pp. E861-E873 ◽  
Author(s):  
Fang Wang ◽  
Hongbo Weng ◽  
Michael J. Quon ◽  
Jingwen Yu ◽  
Jian-Ying Wang ◽  
...  
Keyword(s):  
Er Stress ◽  
Unfolded Protein Response ◽  
High Glucose ◽  
Dominant Negative ◽  
Caspase 8 ◽  
Death Domain ◽  
Unfolded Protein ◽  
Protein Response

Endoplasmic reticulum (ER) stress and caspase 8-dependent apoptosis are two interlinked causal events in maternal diabetes-induced neural tube defects (NTDs). The inositol-requiring enzyme 1α (IRE1α) signalosome mediates the proapoptotic effect of ER stress. Diabetes increases tumor necrosis factor receptor type 1R-associated death domain (TRADD) expression. Here, we revealed two new unfolded protein response (UPR) regulators, TRADD and Fas-associated protein with death domain (FADD). TRADD interacted with both the IRE1α-TRAF2-ASK1 complex and FADD. In vivo overexpression of a FADD dominant negative (FADD-DN) mutant lacking the death effector domain disrupted diabetes-induced IRE1α signalosome and suppressed ER stress and caspase 8-dependent apoptosis, leading to NTD prevention. FADD-DN abrogated ER stress markers and blocked the JNK1/2-ASK1 pathway. Diabetes-induced mitochondrial translocation of proapoptotic Bcl-2 members mitochondrial dysfunction and caspase cleavage were also alleviated by FADD-DN. In vitro TRADD overexpression triggered UPR and ER stress before manifestation of caspase 3 and caspase 8 cleavage and apoptosis. FADD-DN overexpression repressed high glucose- or TRADD overexpression-induced IRE1α phosphorylation, its downstream proapoptotic kinase activation and endonuclease activities, and apoptosis. FADD-DN also attenuated tunicamycin-induced UPR and ER stress. These findings suggest that TRADD participates in the IRE1α signalosome and induces UPR and ER stress and that the association between TRADD and FADD is essential for diabetes- or high glucose-induced UPR and ER stress.

The role of unfolded protein response and ER-phagy in quantum dots-induced nephrotoxicity: an in vitro and in vivo study

2018 ◽  
Vol 92 (4) ◽  
pp. 1421-1434 ◽  
Author(s):  
Shengwei Jiang ◽  
Yuchun Lin ◽  
Huan Yao ◽  
Chuanli Yang ◽  
Liyin Zhang ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Unfolded Protein Response ◽  
In Vivo Study ◽  
Unfolded Protein ◽  
Protein Response

scholarly journals Unfolded Protein Response and Crohn’s Diseases: A Molecular Mechanism of Wound Healing in the Gut

2021 ◽  
Vol 3 (1) ◽  
pp. 31-43
Author(s):  
Chao Li
Keyword(s):  
Er Stress ◽  
Unfolded Protein Response ◽  
Immune Cell ◽  
Macrophage Polarization ◽  
Intestinal Epithelial ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Endoplasmic reticulum (ER) stress triggers a series of signaling and transcriptional events termed the unfolded protein response (UPR). Severe ER stress is associated with the development of fibrosis in different organs, including lung, liver, kidney, heart, and intestine. ER stress is an essential response of epithelial and immune cells in the pathogenesis of Inflammatory Bowel Disease (IBD), including Crohn’s disease (CD). Intestinal epithelial cells are susceptible to ER stress-mediated damage due to secretion of a large amount of proteins that are involved in mucosal defense. In other cells, ER stress is linked to myofibroblast activation, extracellular matrix production, macrophage polarization, and immune cell differentiation. This review focuses on the role of the UPR in the pathogenesis in IBD from an immunologic perspective. The roles of macrophage and mesenchymal cells in the UPR from in vitro and in vivo animal models are discussed. The links between ER stress and other signaling pathways, such as senescence and autophagy, are introduced. Recent advances in the understanding of the epigenetic regulation of the UPR signaling are also updated here. The future directions of development of the UPR research and therapeutic strategies to manipulate ER stress levels are also reviewed.

scholarly journals The Role of Mitochondrial Dysfunction and ER Stress in TDP-43 and C9ORF72 ALS

2021 ◽  
Vol 15 ◽  
Author(s):  
Ruxandra Dafinca ◽  
Paola Barbagallo ◽  
Kevin Talbot
Keyword(s):  
Er Stress ◽  
Mitochondrial Dysfunction ◽  
Unfolded Protein Response ◽  
Nucleocytoplasmic Transport ◽  
Cellular Processes ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants, including genetic and non-genetic factors. Despite this heterogeneity, a key pathological signature is the mislocalization and aggregation of specific proteins in the cytoplasm, suggesting that convergent pathogenic mechanisms focusing on disturbances in proteostasis are important in ALS. In addition, many cellular processes have been identified as potentially contributing to disease initiation and progression, such as defects in axonal transport, autophagy, nucleocytoplasmic transport, ER stress, calcium metabolism, the unfolded protein response and mitochondrial function. Here we review the evidence from in vitro and in vivo models of C9ORF72 and TDP-43-related ALS supporting a central role in pathogenesis for endoplasmic reticulum stress, which activates an unfolded protein response (UPR), and mitochondrial dysfunction. Disruption in the finely tuned signaling between the ER and mitochondria through calcium ions may be a crucial trigger of mitochondrial deficits and initiate an apoptotic signaling cascade, thus acting as a point of convergence for multiple upstream disturbances of cellular homeostasis and constituting a potentially important therapeutic target.

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