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The RNA bridge between IRE-1 and PKR leading to metaflammation: discovery and intervention in atherosclerosis

Final Report Summary - METARNAFLAMMATION (The RNA bridge between IRE-1 and PKR leading to metaflammation: discovery and intervention in atherosclerosis)

An important primer for inflammation in obesity is the chronic metabolic overloading of anabolic and catabolic organelles, such as the endoplasmic reticulum (ER) and mitochondria, leading to impairment of their function. ER serves as a critical intracellular metabolic hub for protein, lipid and calcium metabolism. The vital functions of ER are maintained by a conserved, adaptive stress response, known as the Unfolded Protein Response (UPR), that strives to re-establish homeostasis. However, irremediable ER stress causes the UPR to assemble into a signalling platform from which pro-inflammatory and pro-apoptotic signalling cascades radiate. ER stress occurs in all stages of atherosclerotic plaque formation, and is causally associated with this disease. A major challenge when beginning to think about therapeutically limiting ER stress in a chronic fashion stems from the need to dissociate UPR’s adaptive and detrimental consequences. Complete functional ablation of proximal regulators may not discriminate between these two responses. Therefore, the major goal of our studies is to uncover how ER senses lipid excess and couples to inflammation, and whether its atypical operation under metabolic stress can be suitable for therapeutic exploitation in Cardiometabolic Syndrome (CMS). This study tackles the unique modes of operation of two important players in the ER stress response that are coupled by metabolic stress, Inositol-requiring enzyme-1 (IRE-1) and double stranded RNA-sensing kinase (PKR), by taking advantage of chemical-genetics to specifically modify their activities in vitro and in vivo in atherosclerosis. IRE1, a proximal ER sensor that controls one of the UPR signalling branches, harbours dual kinase and endoribonuclease (RNase) activities. How IRE1’s RNase contributes to the pathogenesis of atherosclerosis and its RNA targets in this disease is not known. Here, we used high resolution, quantitative RNA sequencing to probe IRE1’s RNA substrates in macrophages. Our results show IRE1 regulates many pro-atherogenic genes and non-coding RNAs (ncRNA) in lipid-challenged macrophages and in atherosclerosis-prone, hypercholesterolemic mice. Furthermore, we systemically analyzed which ncRNAs functionally couple IRE-1 to PKR activation in macrophages and in atherosclerotic lesions in vivo. IRE1 selectively targets the generation of several miRNAs that alter macrophage functions and the inflammatory response. These findings show fine-tuning IRE1’s RNAse output by a small molecule inhibitor may provide a therapeutic opportunity for atherosclerosis.

In addition, we took advantage of a unique approach that couples chemical-genetics to proteomics for identifying novel substrates of PKR kinase in macrophages. These potential PKR substrates include important regulators for macrophage biology that could shed light into how PKR contributes to the atherosclerotic disease process. Intriguingly, several RNA binding proteins have been discovered as direct substrates of PKR. We systemically verified the kinase-substrate relationship in macrophages and delineated the contribution of this relationship to organelle stress and inflammatory response. We recently generated a transgenic mutant form of PKR that can be selectively and transiently inhibited by bulky ATP analogs (ATP analog sensitive kinase allele – ASKA) and have been breeding them to a mouse model of atherosclerosis. In these mice we will be able to systematically verify the PKR substrates in atherosclerotic lesions, and explore the potential of targeting PKR to mitigate the detrimental consequences of ER stress in atherosclerosis in vivo.

In summary, by combining a deep interest in the unique aspects of ER stress signalling with in vivo disease modelling, our studies aimed to provide novel pathogenic mechanisms operating in metaflammation that can be potentially targeted for the treatment of CMS. These studies shed light on a little explored but central question in the field of immunometabolism regarding how lipids engage inflammatory and stress pathways. More broadly, the new players we have discovered in the metabolic stress response expand our knowledge beyond the classical UPR. Throughout this project, we attempted to translate the fundamental findings generated by combining several approaches to the organism level to provide novel intervention strategies for atherosclerosis. Ultimately, the path-breaking nature of this study will most broadly impact a diverse biological community on the long run. Moreover, the new IRE-1 and PKR targets uncovered by this work should result in many predictions about metaflammation and pathogenesis of CMS, which will form the basis for the future work in my lab as we next explore these targets in human disease.