Endoplasmic Reticulum Stress signalling - from basic mechanisms to clinical applications

The endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one-third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling-centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes

Development of a novel companion diagnostic for IRE1α activity and its application to investigate the role of IRE1α in acute myeloid leukaemia cells

The unfolded protein response (UPR) is an evolutionarily conserved mechanism activated in response to accumulation of unfolded protein (termed ER stress). Activation of the UPR is observed during many physiological processes but also in multiple disease states, including various cancers, neurodegenerative diseases and diabetes mellitus. The UPR has both a pro-survival and pro-apoptotic role in cells, depending on the severity and duration of ER stress. The most conserved and well described transducer of the UPR is inositol-requiring enzyme 1 (IRE1) which is capable of signalling through both its kinase and RNase domains. The RNase domain of IRE1’s most studied role is in the unconventional splicing of xbox binding protein (XBP1) mRNA. Spliced XBP1 (XBP1s) mRNA allows for translation of a unique, potent transcription factor termed (XBP1s) normally associated with IRE1’s pro-survival role. Here we outline the development and application of a clinically compatible test for the detection of XBP1s and its unspliced counterpart XBP1u in cell or tissue lysates. This test, the XBP1 biochip, utilises biochip array technology (BAT) to deliver a quantitative, simultaneous result for both analytes from a single sample. We used the XBP1 biochip to investigate the effects of small molecule (MKC-8866) inhibition of the IRE1 RNase domain in cell models of acute myeloid leukaemia (AML). Investigation of extracellular effects under sub-cytotoxic ER stress revealed universal IL8 and ERdj3 release. When experiencing cytotoxic levels of ER stress due to proteasome inhibition, AML cell lines were partially protected by co-culture with immortalised bone marrow stromal cells (BMSCs). MKC-8866 was able to ablate this protection in cell lines when combined with carfilzomib (CFZ). In AML patient sample-BMSC co-cultures XBP1s expression correlated with MKC-8866 mediated enhancement of CFZ treatment. This thesis demonstrates the potential of the XBP1 biochip in research and clinical applications and CFZ with MKC-8866 co-treatment in AML.2024-08-2

Development of a novel companion diagnostic for IRE1α activity and its application to investigate the role of IRE1α in acute myeloid leukaemia cells

The unfolded protein response (UPR) is an evolutionarily conserved mechanism activated in response to accumulation of unfolded protein (termed ER stress). Activation of the UPR is observed during many physiological processes but also in multiple disease states, including various cancers, neurodegenerative diseases and diabetes mellitus. The UPR has both a pro-survival and pro-apoptotic role in cells, depending on the severity and duration of ER stress. The most conserved and well described transducer of the UPR is inositol-requiring enzyme 1 (IRE1) which is capable of signalling through both its kinase and RNase domains. The RNase domain of IRE1’s most studied role is in the unconventional splicing of xbox binding protein (XBP1) mRNA. Spliced XBP1 (XBP1s) mRNA allows for translation of a unique, potent transcription factor termed (XBP1s) normally associated with IRE1’s pro-survival role. Here we outline the development and application of a clinically compatible test for the detection of XBP1s and its unspliced counterpart XBP1u in cell or tissue lysates. This test, the XBP1 biochip, utilises biochip array technology (BAT) to deliver a quantitative, simultaneous result for both analytes from a single sample. We used the XBP1 biochip to investigate the effects of small molecule (MKC-8866) inhibition of the IRE1 RNase domain in cell models of acute myeloid leukaemia (AML). Investigation of extracellular effects under sub-cytotoxic ER stress revealed universal IL8 and ERdj3 release. When experiencing cytotoxic levels of ER stress due to proteasome inhibition, AML cell lines were partially protected by co-culture with immortalised bone marrow stromal cells (BMSCs). MKC-8866 was able to ablate this protection in cell lines when combined with carfilzomib (CFZ). In AML patient sample-BMSC co-cultures XBP1s expression correlated with MKC-8866 mediated enhancement of CFZ treatment. This thesis demonstrates the potential of the XBP1 biochip in research and clinical applications and CFZ with MKC-8866 co-treatment in AML.2024-08-2

Inhibition of IRE1α RNase activity sensitizes patient-derived acute myeloid leukaemia cells to proteasome inhibitors

Despite improvements in prognostic stratification and optimization of therapeutic intervention in acute myeloid leukaemia (AML) patients, long-term survival is low. Clinical trials suggest proteasome inhibitors may be beneficial, but further interrogation of the molecular consequences of proteasome inhibition in AML is warranted to identify novel approaches that enhance their efficacy.1 In multiple myeloma (MM), resistance to proteasome inhibitors can occur upon activation of the unfolded protein response (UPR), a stress response pathway that can control cell fate.2 Inositol-requiring enzyme 1 alpha (IRE1α) is one of three stress sensors that mediates UPR signalling. IRE1α activity occurs via its RNase domain resulting in cleavage of a 26-nucleotide intron from X-Box Binding Protein 1 (XBP1) mRNA leading to formation of a transcription factor, XBP1s. XBP1s enhances cell survival by increasing transcription of genes associated with protein folding, endoplasmic reticulum-associated degradation (ERAD) and phospholipid synthesis. We demonstrate that an IRE1 RNase inhibitor (MKC8866), in combination with proteasome inhibitors, significantly decreases XBP1s levels and increases cell death in AML cell lines and patient-derived AML cells. In addition, this combination treatment can successfully target the CD34+CD38− population and reduce clonogenic ability.</p

Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications

International audienceThe endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one-third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling-centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes

Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications

The endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one-third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling-centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes.status: publishe