Our lab aims to understand—at the molecular, cellular and organismal levels—how protein unfolding in the endoplasmic reticulum (ER) causes human disease. Using this fundamental understanding, we are pioneering new therapeutic approaches for various human diseases caused by protein unfolding in the ER.

All eukaryotic cells use an intracellular signaling pathway called the unfolded protein response (UPR) to ensure that the machinery needed to fold and assemble secreted and membrane proteins (roughly one third of all cellular proteins) is sufficient to meet secretory needs. When the ER's protein folding needs outweigh its capacity to sustain the protein folding process, cells experience a distinct form of stress—"ER stress"— that triggers the UPR pathway. UPR outputs initially reduce ER stress through augmenting the levels of ER-resident chaperones and protein folding enzymatic activities; these outputs are conserved in all eukaryotes. However, if ER stress cannot be alleviated through these adaptive outputs, in higher eukaryotes the UPR triggers a diametrically opposite strategy to instead promote destructive outcomes, including sterile inflammation, dedifferentiation, and ultimately programmed cell death (JCS, 123, 1003-6 (2010)). Over the last decade, our lab was the first to elucidate how this UPR life-death switch is controlled in mammalian cells by the master UPR sensor/effector, IRE1-alpha, a bifunctional kinase/RNAse imbedded in the ER membrane.

While several other labs have defined the adaptive benefits proceeding through IRE1-alpha, our lab was the first to show that IRE1-alpha has alternative outputs depending on the strength of upstream stresses and to mechanistically define the underlying basis of this ability—this knowledge is key to shaping therapy (see below). Specifically, in 2009, we were the first to find that IRE1-alpha has alternate RNAse outputs that are controlled by its kinase domain to promote divergent cell fates under ER stress (Cell, 138, 562–575 (2009)). In that study we showed that hyperactivated IRE1-alpha caused the ER-localized degradation of thousands of mRNAs in mammalian cells, and the consequent depletion of these mRNAs leads mammalian cells to enter apoptosis. This is the basis of how IRE1-alpha functions as a life-death switch and it is this knowledge that forms the fundamental basis for therapeutic intervention using small molecule modulators of IRE1-alpha.

The reason we are so interested in this subject is that ER stress-mediated apoptosis is being widely linked to many important human diseases. ER stress-mediated diseases occur when specialized cells in the body that evolved to secrete proteins—"professional secretory cells"— die prematurely. Over the last decade, we have focused on diabetes mellitus as a prototype of ER stress-mediated cell degenerative diseases. For example, type 2 diabetes is known to occur when about 50% of pancreatic islet beta cells (specialized cells in the pancreas that synthesize and secrete insulin) die, leading to insufficient insulin levels in the blood. We have hypothesized that pancreatic islet beta cells may become overworked as they try to counter peripheral insulin resistance in states of obesity and overnutrition (high fat and high carbohydrate diets). Overwork of these cells may cause them to experience elevated ER stress chronically. In turn, elevated ER stress puts the beta cells at heightened risk for death as the UPR starts triggering apoptosis. As more beta cells die, the remaining beta cells experience greater and greater ER stress because they have to do more work per cell. Similar logic may apply in type 1 diabetes, as beta cells are subject to autoimmune attack. This chain of events leads to a vicious cycle, as diabetes sets in (Cold Spring Harb Perspect Med. Aug 14, 2012). The process of disease progression may take many years, providing us with a long time window to reduce, and possibly even to reverse, the course of the disease.

Ultimately, our long-term goals are to advance fundamental knowledge in order to develop new drugs to reduce maladaptive and destructive responses (e.g. premature apoptosis) occurring in ER stressed cells. It is conceivable that such drugs may be efficacious in diabetes, but may also be useful for many other ER stress-mediated cell degenerative diseases. The basic questions we ask include: is there a "tipping point" of ER stress beyond which the UPR relegates beta cells to apoptosis? Can we intervene through pharmacology to adjust that tipping point, and reduce beta cell death? We aim to answer these questions mechanistically using a number of novel tools we have developed. Through such knowledge we may be able to identify UPR targets that can be drugged, and thereby develop novel approaches to treat many ER stress cell degenerative diseases, such as type 2 diabetes.

To these ends, we have learned to actively manipulate the UPR pathway in living cells, via cell-permeable small molecules. Using chemical-genetics, we developed the first tools to wrest pharmacological control over the UPR in yeast through an unprecedented paradigm of kinase signaling wherein engagement of IRE1 with a kinase inhibitor bypasses phosphotransfer activity to activate the RNAse catalytic activity. (Science 302, 1533-1537 (2003)). Thus, built into IRE1 proteins there is a conformational switch that controls the RNAse through a pseudokinase module. Next, we discovered that similar engagement of mammalian IRE1-alpha confers a degree of cytoprotection in mammalian cells experiencing irremediable ER stress (Bioch Bioch Res Comm. 365 (2008—published online in 2007) 777–783).

Furthermore, we devised the first biosensors for measuring ER stress in single living cells, in real time, through employing redox-sensitive fluorescent protein sensors as a proxy for the stressed state (Cell, 135(5):933-47 (2008)). And we helped develop mathematical modeling to quantify and predict the benefit of UPR signaling (P.N.A.S. 105(51):20280-5. (2008)).

Most recently, we have identified a comprehensive signature of hyperactive UPR signaling that we call the “Terminal UPR”. Terminal UPR signaling promotes destructive outcomes in cells experiencing high or chronic ER stress. In 2012 we identified a critical node in the terminal UPR called the thioredoxin-interacting protein (TXNIP), which causes sterile inflammation in pancreatic islets to cause development of diabetes (Cell Metabolism, 16(2): 250-64 (2012). Furthermore, we have learned that TXNIP is induced post-transcriptionally by IRE1-alpha as IRE1-alpha degrades a set of inhibitory micro RNAs. Such post-transcriptional control may be widespread in the UPR.

Finally, having identified IRE1-alpha’s RNase as a life-death switch, we are optimizing small molecules that can extract desired outputs from this effector domain. As IRE1-alpha’s RNase can be controlled from a distance (allosterically) through the kinase domain, we learned in 2012 how to both turn ON or turn OFF IRE1-alpha at will: we discovered that certain small molecule kinase inhibitors of IRE1-alpha can elevate RNase activity (called type I), or attenuate it (called type II)—(Nature Chemical Biology, 12: 982-9 (2012)).

We expect that these two classes of IRE1-alpha-modulatory small molecules will allow us to tilt the outcome in ER stressed cells either towards survival or death, and therefore these kinase inhibitory compounds will be powerful tools to investigate connections between ER stress, the UPR, and apoptosis. Importantly, we are the first to develop and employ type II kinase inhibitors of IRE1-alpha (which we have named KIRAs for Kinase-Inhibiting RNAse Attenuators) as a general proof-of-concept for treating ER stress-related diseases. In 2014 we showed that in rodent ER stress models of retinal degeneration and diabetes, a first-in-class compound series of KIRAs ameliorates cell death and preserves physiological function (Cell 158: 534-48 (2014)). This was the first in vivo demonstration that hyperactivated IRE1-alpha drives myriad cell degenerative diseases (and which can be modified through small molecule inhibitors of IRE1-alpha) and represents a departure from an older, more entrenched, view that IRE1-alpha outputs are universally beneficial.

Encouraged by these positive results, we are currently advancing KIRAs through medicinal chemistry efforts to make them more drug-like and useful for animal and pre-clinical studies of ER stress-related diseases. Ultimately, we hope that our work may lead to novel disease-modifying therapies for retinal degeneration and diabetes. More generally, such compounds may be useful to treat myriad diseases proceeding from cell degeneration under unchecked ER stress. For example, several important neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Lou Gehrig's disease, are all now thought to result from protein unfolding and aggregation in different types of neurons. Hallmarks of high ER stress are apparent in the affected neuronal cells. It is therefore conceivable that small molecule drugs that reduce ER stress and terminal UPR signaling such as KIRAs may be effective at modulating progression of these deadly neurodegenerative diseases as well.

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