The integrated stress response in pancreatic development, tissue homeostasis, and cancer.

Present in all eukaryotic cells, the integrated stress response (ISR) is a highly coordinated signaling network that controls cellular behavior, metabolism and survival in response to diverse stresses. The ISR is initiated when any one of four stress sensing kinases (PERK, GCN2, PKR, HRI) becomes activated to phosphorylate the protein translation initiation factor eIF2α, shifting gene expression toward a comprehensive rewiring of cellular machinery to promote adaptation. While the ISR has been shown to play an important role in the homeostasis of multiple tissues, evidence suggests that it is particularly crucial for the development and ongoing health of the pancreas. Among the most synthetically dynamic tissues in the body, the exocrine and endocrine pancreas relies heavily on the ISR to rapidly adjust cell function to meet the metabolic demands of the organism. The hardwiring of the ISR into normal pancreatic functions and adaptation to stress may explain why it is a commonly utilized pro-oncogenic and therapy-resistance mechanism in both pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumors (PanNETs). Here we review what is known about the key roles that the ISR plays in the development, homeostasis, and neoplasia of the pancreas.

DISSECTING TUMOR TRANSCRIPTIONAL HETEROGENEITY FROM SINGLE-CELL RNA-SEQ DATA BY GENERALIZED BINARY COVARIANCE DECOMPOSITION.

Profiling tumors with single-cell RNA sequencing (scRNA-seq) has the potential to identify recurrent patterns of transcription variation related to cancer progression, and so produce new therapeutically-relevant insights. However, the presence of strong inter-tumor heterogeneity often obscures more subtle patterns that are shared across tumors, some of which may characterize clinically-relevant disease subtypes. Here we introduce a new statistical method to address this problem. We show that this method can help decompose transcriptional heterogeneity into interpretable components - including patient-specific, dataset-specific and shared components relevant to disease subtypes - and that, in the presence of strong inter-tumor heterogeneity, our method can produce more interpretable results than existing widely-used methods. Applied to data from three studies on pancreatic cancer adenocarcinoma (PDAC), our method produces a refined characterization of existing tumor subtypes (e.g. classical vs basal), and identifies a new gene expression program (GEP) that is prognostic of poor survival independent of established prognostic factors such as tumor stage and subtype. The new GEP is enriched for genes involved in a variety of stress responses, and suggests a potentially important role for the integrated stress response in PDAC development and prognosis.

Deletion of the Unfolded Protein Response Transducer IRE1α Is Detrimental to Aging Photoreceptors and to ER Stress-Mediated Retinal Degeneration.

Purpose: The unfolded protein response (UPR) is triggered when the protein folding capacity of the endoplasmic reticulum (ER) is overwhelmed and misfolded proteins accumulate in the ER, a condition referred to as ER stress. IRE1α is an ER-resident protein that plays major roles in orchestrating the UPR. Several lines of evidence implicate the UPR and its transducers in neurodegenerative diseases, including retinitis pigmentosa (RP), a group of inherited diseases that cause progressive dysfunction and loss of rod and cone photoreceptors. This study evaluated the contribution of IRE1α to photoreceptor development, homeostasis, and degeneration. Methods: We used a conditional gene targeting strategy to selectively inactivate Ire1α in mouse rod photoreceptors. We used a combination of optical coherence tomography (OCT) imaging, histology, and electroretinography (ERG) to assess longitudinally the effect of IRE1α deficiency in retinal development and function. Furthermore, we evaluated the IRE1α-deficient retina responses to tunicamycin-induced ER stress and in the context of RP caused by the rhodopsin mutation RhoP23H. Results: OCT imaging, histology, and ERG analyses did not reveal abnormalities in IRE1α-deficient retinas up to 3 months old. However, by 6 months of age, the Ire1α mutant animals showed reduced outer nuclear layer thickness and deficits in retinal function. Furthermore, conditional inactivation of Ire1α in rod photoreceptors accelerated retinal degeneration caused by the RhoP23H mutation. Conclusions: These data suggest that IRE1α is dispensable for photoreceptor development but important for photoreceptor homeostasis in aging retinas and for protecting against ER stress-mediated photoreceptor degeneration.

Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases: Workshop Proceedings.

The Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report provides a summary of the proceedings from the workshop. The goals of the workshop were to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into six major theme areas, including 1) pancreas anatomy and physiology, 2) diabetes in the setting of exocrine disease, 3) metabolic influences on the exocrine pancreas, 4) genetic drivers of pancreatic diseases, 5) tools for integrated pancreatic analysis, and 6) implications of exocrine-endocrine cross talk. For each theme, multiple presentations were followed by panel discussions on specific topics relevant to each area of research; these are summarized here. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

Acute Recurrent Pancreatitis in a Child With INS-Related Monogenic Diabetes and a Heterozygous Pathogenic CFTR Mutation.

Given the close anatomical and physiological links between the exocrine and endocrine pancreas, diseases of 1 compartment often affect the other through mechanisms that remain poorly understood. Pancreatitis has been associated with both type 1 and type 2 diabetes, but its association with monogenic diabetes is unknown. Patients heterozygous for pathogenic CFTR variants are cystic fibrosis carriers and have been reported to have an increased risk of acute pancreatitis. We describe a 12-year-old patient with monogenic neonatal diabetes due to a pathogenic heterozygous paternally inherited mutation of the insulin gene (INS), c.94 G > A (p.Gly32Ser), who experienced 3 recurrent episodes of acute pancreatitis over 7 months in conjunction with poor glycemic control, despite extensive efforts to improve glycemic control in the past 4 years. Intriguingly, the maternal side of the family has an extensive history of adult-onset pancreatitis consistent with autosomal dominant inheritance and the proband is heterozygous for a maternally inherited, CFTR variant c.3909C > G (p.Asn1303Lys). Paternally inherited monogenic neonatal diabetes may have promoted earlier age-of-onset of pancreatitis in this pediatric patient compared to maternal relatives with adult-onset acute pancreatitis. Further study is needed to clarify how separate pathophysiologies associated with INS and CFTR mutations influence interactions between the endocrine and exocrine pancreas.

Stress and human health in diabetes: A report from the 19th Chicago Biomedical Consortium symposium.

Stress and diabetes coexist in a vicious cycle. Different types of stress lead to diabetes, while diabetes itself is a major life stressor. This was the focus of the Chicago Biomedical Consortium's 19th annual symposium, "Stress and Human Health: Diabetes," in November 2022. There, researchers primarily from the Chicago area met to explore how different sources of stress - from the cells to the community - impact diabetes outcomes. Presenters discussed the consequences of stress arising from mutant proteins, obesity, sleep disturbances, environmental pollutants, COVID-19, and racial and socioeconomic disparities. This symposium showcased the latest diabetes research and highlighted promising new treatment approaches for mitigating stress in diabetes.

SARS-CoV-2 Diverges from Other Betacoronaviruses in Only Partially Activating the IRE1α/XBP1 Endoplasmic Reticulum Stress Pathway in Human Lung-Derived Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed to be essential for viral replication. We examined the master UPR sensor IRE1α kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1α-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found that human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1α as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1α through autophosphorylation, but its RNase activity fails to splice XBP1. Moreover, while IRE1α was dispensable for replication in human cells for all coronaviruses tested, it was required for maximal expression of genes associated with several key cellular functions, including the interferon signaling pathway, during SARS-CoV-2 infection. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1α, perhaps as a strategy to eliminate detection by the host immune system. IMPORTANCE SARS-CoV-2 is the third lethal respiratory coronavirus, after MERS-CoV and SARS-CoV, to emerge this century, causing millions of deaths worldwide. Other common coronaviruses such as HCoV-OC43 cause less severe respiratory disease. Thus, it is imperative to understand the similarities and differences among these viruses in how each interacts with host cells. We focused here on the inositol-requiring enzyme 1α (IRE1α) pathway, part of the host unfolded protein response to virus-induced stress. We found that while MERS-CoV and HCoV-OC43 fully activate the IRE1α kinase and RNase activities, SARS-CoV-2 only partially activates IRE1α, promoting its kinase activity but not RNase activity. Based on IRE1α-dependent gene expression changes during infection, we propose that SARS-CoV-2 prevents IRE1α RNase activation as a strategy to limit detection by the host immune system.

SARS-CoV-2 diverges from other betacoronaviruses in only partially activating the IRE1α/XBP1 ER stress pathway in human lung-derived cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available, or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed essential for viral replication. We examined the master UPR sensor IRE1α kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1α-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1α as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1α through autophosphorylation, but its RNase activity fails to splice XBP1. Moreover, while IRE1α was dispensable for replication in human cells for all coronaviruses tested, it was required for maximal expression of genes associated with several key cellular functions, including the interferon signaling pathway, during SARS-CoV-2 infection. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1α, perhaps as a strategy to eliminate detection by the host immune system. IMPORTANCE: SARS-CoV-2 is the third lethal respiratory coronavirus after MERS-CoV and SARS-CoV to emerge this century, causing millions of deaths world-wide. Other common coronaviruses such as HCoV-OC43 cause less severe respiratory disease. Thus, it is imperative to understand the similarities and differences among these viruses in how each interacts with host cells. We focused here on the inositol-requiring enzyme 1α (IRE1α) pathway, part of the host unfolded protein response to virus-induced stress. We found that while MERS-CoV and HCoV-OC43 fully activate the IRE1α kinase and RNase activities, SARS-CoV-2 only partially activates IRE1α, promoting its kinase activity but not RNase activity. Based on IRE1α-dependent gene expression changes during infection, we propose that SARS-CoV-2 prevents IRE1α RNase activation as a strategy to limit detection by the host immune system.

Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms.

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type I or type II. While type II CALR-mutant proteins retain many of the Ca2+ binding sites present in the wild-type protein, type I CALR-mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain unexplored. Here, we show that the loss of Ca2+ binding residues in the type I mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1α/XBP1 pathway of the unfolded protein response. Genetic or pharmacologic inhibition of IRE1α/XBP1 signaling induces cell death in type I mutant but not type II mutant or wild-type CALR-expressing cells, and abrogates type I mutant CALR-driven MPN disease progression in vivo. SIGNIFICANCE: Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265.

The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity.

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2911-919, a nine amino acid segment within a flexible interdomain region (LRRK2853-981), which we designate the "regulatory loop" (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2's association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2's interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase-dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.

Cannabidiol inhibits SARS-CoV-2 replication through induction of the host ER stress and innate immune responses.

The spread of SARS-CoV-2 and ongoing COVID-19 pandemic underscores the need for new treatments. Here we report that cannabidiol (CBD) inhibits infection of SARS-CoV-2 in cells and mice. CBD and its metabolite 7-OH-CBD, but not THC or other congeneric cannabinoids tested, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after viral entry, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD inhibits SARS-CoV-2 replication in part by up-regulating the host IRE1α RNase endoplasmic reticulum (ER) stress response and interferon signaling pathways. In matched groups of human patients from the National COVID Cohort Collaborative, CBD (100 mg/ml oral solution per medical records) had a significant negative association with positive SARS-CoV-2 tests. This study highlights CBD as a potential preventative agent for early-stage SARS-CoV-2 infection and merits future clinical trials. We caution against use of non-medical formulations including edibles, inhalants or topicals as a preventative or treatment therapy at the present time.

Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases: Workshop Proceedings.

ABSTRACT: The "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases" Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response.

The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARS-CoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.

Endoplasmic Reticulum Stress Signaling in Cancer Cells.

To survive, cancer cells must resist numerous internal and environmental insults associated with neoplasia that jeopardize proteostasis within the endoplasmic reticulum (ER). Solid and hematopoietic tumors often experience genomic instability, oncogene activation, increased protein secretion demands, and somatic mutations in proteins handled by the secretory pathway that impede their folding. Invasion or metastasis into foreign environments can expose tumor cells to hypoxia, oxidative stress, lack of growth signals, inadequate amino acid supplies, glucose deprivation, and lactic acidosis, all of which pose challenges for protein processing in the ER. Together, these conditions can promote the buildup of misfolded proteins in the ER to cause ER stress, which then activates the unfolded protein response (UPR). An intracellular signaling network largely initiated by three ER transmembrane proteins, the UPR constantly surveils protein folding conditions within the ER lumen and when necessary initiates counteractive measures to maintain ER homeostasis. Under mild or moderate levels of ER stress, the homeostatic UPR sets in motion transcriptional and translational changes that promote cell adaption and survival. However, if these processes are unsuccessful at resolving ER stress, a terminal UPR program dominates and actively signals cell suicide. This article summarizes the mounting evidence that cancer cells are predisposed to ER stress and vulnerable to targeted interventions against ongoing UPR signaling.

Parallel Signaling through IRE1α and PERK Regulates Pancreatic Neuroendocrine Tumor Growth and Survival.

Master regulators of the unfolded protein response (UPR), IRE1α and PERK, promote adaptation or apoptosis depending on the level of endoplasmic reticulum (ER) stress. Although the UPR is activated in many cancers, its effects on tumor growth remain unclear. Derived from endocrine cells, pancreatic neuroendocrine tumors (PanNET) universally hypersecrete one or more peptide hormones, likely sensitizing these cells to high ER protein-folding stress. To assess whether targeting the UPR is a viable therapeutic strategy, we analyzed human PanNET samples and found evidence of elevated ER stress and UPR activation. Genetic and pharmacologic modulation of IRE1α and PERK in cultured cells, xenograft, and spontaneous genetic (RIP-Tag2) mouse models of PanNETs revealed that UPR signaling was optimized for adaptation and that inhibiting either IRE1α or PERK led to hyperactivation and apoptotic signaling through the reciprocal arm, thereby halting tumor growth and survival. These results provide a strong rationale for therapeutically targeting the UPR in PanNETs and other cancers with elevated ER stress. SIGNIFICANCE: The UPR is upregulated in pancreatic neuroendocrine tumors and its inhibition significantly reduces tumor growth in preclinical models, providing strong rationale for targeting the UPR in these cancers.

Elastase 3B mutation links to familial pancreatitis with diabetes and pancreatic adenocarcinoma.

While improvements in genetic analysis have greatly enhanced our understanding of the mechanisms behind pancreatitis, it continues to afflict many families for whom the hereditary factors remain unknown. Recent evaluation of a patient with a strong family history of pancreatitis sparked us to reexamine a large kindred originally reported over 50 years ago with an autosomal dominant inheritance pattern of chronic pancreatitis, diabetes and pancreatic adenocarcinoma. Whole exome sequencing analysis identified a rare missense mutation in the gene encoding pancreas-specific protease Elastase 3B (CELA3B) that cosegregates with disease. Studies of the mutant protein in vitro, in cell lines and in CRISPR-Cas9 engineered mice indicate that this mutation causes translational upregulation of CELA3B, which upon secretion and activation by trypsin leads to uncontrolled proteolysis and recurrent pancreatitis. Although lesions in several other pancreatitic proteases have been previously linked to hereditary pancreatitis, this is the first known instance of a mutation in CELA3B and a defect in translational control contributing to this disease.

A Medical Student Inquiry Behavior Assessment Tool: Development and Validity Evidence.

PURPOSE: The University of California, San Francisco (UCSF) School of Medicine's Bridges curriculum is designed to teach inquiry: the process of approaching problems with curiosity, challenging current concepts, and creating new knowledge. The authors aimed to develop and gather validity evidence for a tool to guide development of medical student inquiry behaviors in small groups. METHOD: The authors reviewed the literature to identify inquiry behaviors, verified findings with an expert focus group, and synthesized the results into 40 behaviors. In a modified two-round Delphi survey in 2016, faculty and students rated the behaviors for inclusion in the tool. Feedback from cognitive interviews and a pilot helped refine the tool. In 2016-2017, the authors implemented the final tool for 152 first-year UCSF medical students in inquiry small groups as a faculty assessment and a student self-assessment each quarter. RESULTS: The two-round response rate was 77% (36/47). Five behaviors were selected for inclusion in the tool: select relevant questions to pursue; justify explanations with evidence; critically evaluate his/her explanation in light of alternative possibilities; allow for the possibility that his/her own knowledge may not be completely correct; and collaborate well with peers. During implementation, faculty and student scores increased on most items, indicating skills development over time. Content, response process, internal structure, and consequential validity evidence is presented. CONCLUSIONS: The tool's five items are observable, measurable core inquiry behaviors. The tool is ready for use by small-group facilitators within inquiry-based curricula to promote student self-assessment and guide feedback to students.

CPEB4 links the clock and the UPR to protect the liver.

Under misfolded protein stress, the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to restore homeostasis, or commits the cell to apoptosis. A study now uncovers how the UPR is governed by the circadian clock to adjust ER protein-folding capacity to metabolic demand and protect against liver damage.