Discovery of Potent, Selective, and Orally Available IRE1α Inhibitors Demonstrating Comparable PD Modulation to IRE1 Knockdown in a Multiple Myeloma Model.

The lack of selective and safe in vivo IRE1α tool molecules has limited the evaluation of IRE1α as a viable target to treat multiple myeloma. Focus on improving the physicochemical properties of a literature compound by decreasing lipophilicity, molecular weight, and basicity allowed the discovery of a novel series with a favorable in vitro safety profile and good oral exposure. These efforts culminated in the identification of a potent and selective in vivo tool compound, G-5758, that was well tolerated following multiday oral administration of doses up to 500 mg/kg. G-5758 demonstrated comparable pharmacodynamic effects to induced IRE1 knockdown as measured by XBP1s levels in a multiple myeloma model (KMS-11).

Activation of the PERK branch of the unfolded protein response during production reduces specific productivity in CHO cells via downregulation of PDGFRa and IRE1a signaling.

During the course of biopharmaceutical production, heterologous protein expression in Chinese hamster ovary (CHO) cells imposes a high proteostatic burden that requires cellular adaptation. To mitigate such burden, cells utilize the unfolded protein response (UPR), which increases endoplasmic reticulum (ER) capacity to accommodate elevated rates of protein synthesis and folding. In this study, we show that during production the UPR regulates growth factor signaling to modulate growth and protein synthesis. Specifically, the protein kinase R-like ER kinase (PERK) branch of the UPR is responsible for transcriptional down-regulation of platelet-derived growth factor receptor alpha (PDGFRa) and attenuation of the IRE1-alpha (IRE1a) branch of the UPR. PERK knockout (KO) cell lines displayed reduced growth and viability due to higher rates of apoptosis despite having stabilized PDGFRa levels. Knocking out PERK in an apoptosis impaired (Bax/Bak double KO) antibody-expressing cell line prevented apoptotic cell death and revealed that apoptosis was likely triggered by increased ER stress and reactive oxygen species levels in the PERK KO hosts. Our findings suggest that attenuation of IRE1a and PDGFRa signaling by the PERK branch of the UPR reduces ER protein folding capacity and hence specific productivity of CHO cells in order to mitigate UPR and prevent apoptotic cell death. Last, Bax/Bak/PERK triple KO CHO cell lines displayed 2-3 folds higher specific productivity and titer (up to 8 g/L), suggesting that modulation of PERK signaling during production processes can greatly improve specific productivity in CHO cells.

A WISP1 antibody inhibits MRTF signaling to prevent the progression of established liver fibrosis.

Fibrosis is the major risk factor associated with morbidity and mortality in patients with non-alcoholic steatohepatitis (NASH)-driven chronic liver disease. Although numerous efforts have been made to identify the mediators of the initiation of liver fibrosis, the molecular underpinnings of fibrosis progression remain poorly understood, and therapies to arrest liver fibrosis progression are elusive. Here, we identify a pathway involving WNT1-inducible signaling pathway protein 1 (WISP1) and myocardin-related transcription factor (MRTF) as a central mechanism driving liver fibrosis progression through the integrin-dependent transcriptional reprogramming of myofibroblast cytoskeleton and motility. In mice, WISP1 deficiency protects against fibrosis progression, but not fibrosis onset. Moreover, the therapeutic administration of a novel antibody blocking WISP1 halted the progression of existing liver fibrosis in NASH models. These findings implicate the WISP1-MRTF axis as a crucial determinant of liver fibrosis progression and support targeting this pathway by antibody-based therapy for the treatment of NASH fibrosis.

Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers.

Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remain unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1's lumenal domain. Phosphorylation of IRE1's kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans-autophosphorylation, which in turn drives IRE1's RNase activity.

Our cells contain many different compartments that each perform specific tasks. A cellular compartment known as the endoplasmic reticulum is responsible for making many of the proteins the cell requires and transporting them around the cell. It is important that the endoplasmic reticulum remains healthy and, therefore, cells use a protein called IRE1 that senses when this compartment is under stress. IRE1 then sends a signal to the control center of the cell (known as the nucleus) to ask for help. Previous studies suggest that IRE1 assembles into either pairs or larger groups of molecules known as oligomers when it senses that the endoplasmic reticulum is under stress. However, it remains unclear whether such assembly is the main switch that turns IRE1 on and, if so, how many molecules need to come together to flip the switch. Here, Belyy et al. genetically engineered human bone cancer cells to attach a mark known as a HaloTag to IRE1.The team developed a microscopy approach to count, in living cells, how many tagged IRE1 molecules join. The experiments indicated that IRE1 proteins were generally found as pairs in unstressed cells. When the endoplasmic reticulum experienced stress, IRE1 proteins briefly assembled into oligomers before disassembling back into pairs. Mutated versions of IRE1 revealed the exact parts of IRE1 that connect the pairs and the larger oligomers. These findings suggest that the assembly of IRE1 pairs into oligomers plays a major part in the activation of IRE1 to send a stress signal to the nucleus. IRE1 signaling is closely implicated in both cancer biology and aging, and therefore, understanding how it works may aid the development of new therapies for cancer, dementia, and other health conditions affecting older people. Furthermore, the microscopy approach developed in this work could be adapted to study other proteins that relay signals in living cells.

Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma.

Asthma and inflammatory airway diseases restrict airflow in the lung, compromising gas exchange and lung function. Inhaled corticosteroids (ICSs) can reduce inflammation, control symptoms, and improve lung function; however, a growing number of patients with severe asthma do not benefit from ICS. Using bronchial airway epithelial brushings from patients with severe asthma or primary human cells, we delineated a corticosteroid-driven fibroblast growth factor (FGF)-dependent inflammatory axis, with FGF-responsive fibroblasts promoting downstream granulocyte colony-stimulating factor (G-CSF) production, hyaluronan secretion, and neutrophilic inflammation. Allergen challenge studies in mice demonstrate that the ICS, fluticasone propionate, inhibited type 2-driven eosinophilia but induced a concomitant increase in FGFs, G-CSF, hyaluronan, and neutrophil infiltration. We developed a model of steroid-induced neutrophilic inflammation mediated, in part, by induction of an FGF-dependent epithelial-mesenchymal axis, which may explain why some individuals do not benefit from ICS. In further proof-of-concept experiments, we found that combination therapy with pan-FGF receptor inhibitors and corticosteroids prevented both eosinophilic and steroid-induced neutrophilic inflammation. Together, these results establish FGFs as therapeutic targets for severe asthma patients who do not benefit from ICS.

Antigen-derived peptides engage the ER stress sensor IRE1α to curb dendritic cell cross-presentation.

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER), bind to major histocompatibility type I (MHC-I) protein complexes, and are transported to the cell surface for cross-presentation. DCs can exhibit activation of the ER stress sensor IRE1α without ER stress, but the underlying mechanism remains obscure. Here, we show that antigen-derived hydrophobic peptides can directly engage ER-resident IRE1α, masquerading as unfolded proteins. IRE1α activation depletes MHC-I heavy-chain mRNAs through regulated IRE1α-dependent decay (RIDD), curtailing antigen cross-presentation. In tumor-bearing mice, IRE1α disruption increased MHC-I expression on tumor-infiltrating DCs and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1α inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings identify an unexpected cell-biological mechanism of antigen-driven IRE1α activation in DCs, revealing translational potential for cancer immunotherapy.

Pumilio protects Xbp1 mRNA from regulated Ire1-dependent decay.

The unfolded protein response (UPR) maintains homeostasis of the endoplasmic reticulum (ER). Residing in the ER membrane, the UPR mediator Ire1 deploys its cytoplasmic kinase-endoribonuclease domain to activate the key UPR transcription factor Xbp1 through non-conventional splicing of Xbp1 mRNA. Ire1 also degrades diverse ER-targeted mRNAs through regulated Ire1-dependent decay (RIDD), but how it spares Xbp1 mRNA from this decay is unknown. Here, we identify binding sites for the RNA-binding protein Pumilio in the 3'UTR Drosophila Xbp1. In the developing Drosophila eye, Pumilio binds both the Xbp1unspliced and Xbp1spliced mRNAs, but only Xbp1spliced is stabilized by Pumilio. Furthermore, Pumilio displays Ire1 kinase-dependent phosphorylation during ER stress, which is required for its stabilization of Xbp1spliced. hIRE1 can phosphorylate Pumilio directly, and phosphorylated Pumilio protects Xbp1spliced mRNA against RIDD. Thus, Ire1-mediated phosphorylation enables Pumilio to shield Xbp1spliced from RIDD. These results uncover an unexpected regulatory link between an RNA-binding protein and the UPR.

Decoding non-canonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α.

Inositol requiring enzyme 1 (IRE1) mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, in mammals RIDD seems to target only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1α-controlled mRNAs in human cells, we identify not only canonical endomotif-containing RIDD substrates, but also targets without such motifs-degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1α displays two basic endoribonuclease modalities: highly specific, endomotif-directed cleavage, minimally requiring dimers; and more promiscuous, endomotif-independent processing, requiring phospho-oligomers. An oligomer-deficient IRE1α mutant fails to support RIDDLE in vitro and in cells. Our results advance current mechanistic understanding of the UPR.

The stress-sensing domain of activated IRE1α forms helical filaments in narrow ER membrane tubes.

The signaling network of the unfolded protein response (UPR) adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1α, spans the ER membrane and activates through oligomerization. IRE1α oligomers accumulate in dynamic foci. We determined the in situ structure of IRE1α foci by cryogenic correlated light and electron microscopy combined with electron cryo-tomography and complementary immuno­electron microscopy in mammalian cell lines. IRE1α foci localized to a network of narrow anastomosing ER tubes (diameter, ~28 nm) with complex branching. The lumen of the tubes contained protein filaments, which were likely composed of arrays of IRE1α lumenal domain dimers that were arranged in two intertwined, left-handed helices. This specialized ER subdomain may play a role in modulating IRE1α signaling.

Neuronal regulated ire-1-dependent mRNA decay controls germline differentiation in Caenorhabditis elegans.

Understanding the molecular events that regulate cell pluripotency versus acquisition of differentiated somatic cell fate is fundamentally important. Studies in Caenorhabditis elegans demonstrate that knockout of the germline-specific translation repressor gld-1 causes germ cells within tumorous gonads to form germline-derived teratoma. Previously we demonstrated that endoplasmic reticulum (ER) stress enhances this phenotype to suppress germline tumor progression(Levi-Ferber et al., 2015). Here, we identify a neuronal circuit that non-autonomously suppresses germline differentiation and show that it communicates with the gonad via the neurotransmitter serotonin to limit somatic differentiation of the tumorous germline. ER stress controls this circuit through regulated inositol requiring enzyme-1 (IRE-1)-dependent mRNA decay of transcripts encoding the neuropeptide FLP-6. Depletion of FLP-6 disrupts the circuit's integrity and hence its ability to prevent somatic-fate acquisition by germline tumor cells. Our findings reveal mechanistically how ER stress enhances ectopic germline differentiation and demonstrate that regulated Ire1-dependent decay can affect animal physiology by controlling a specific neuronal circuit.

Activation of the IRE1 RNase through remodeling of the kinase front pocket by ATP-competitive ligands.

Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target.

Identification of BRaf-Sparing Amino-Thienopyrimidines with Potent IRE1α Inhibitory Activity.

Amino-quinazoline BRaf kinase inhibitor 2 was identified from a library screen as a modest inhibitor of the unfolded protein response (UPR) regulating potential anticancer target IRE1α. A combination of crystallographic and conformational considerations were used to guide structure-based attenuation of BRaf activity and optimization of IRE1α potency. Quinazoline 6-position modifications were found to provide up to 100-fold improvement in IRE1α cellular potency but were ineffective at reducing BRaf activity. A salt bridge contact with Glu651 in IRE1α was then targeted to build in selectivity over BRaf which instead possesses a histidine in this position (His539). Torsional angle analysis revealed that the quinazoline hinge binder core was ill-suited to accommodate the required conformation to effectively reach Glu651, prompting a change to the thienopyrimidine hinge binder. Resulting analogues such as 25 demonstrated good IRE1α cellular potency and imparted more than 1000-fold decrease in BRaf activity.

IRE1α Disruption in Triple-Negative Breast Cancer Cooperates with Antiangiogenic Therapy by Reversing ER Stress Adaptation and Remodeling the Tumor Microenvironment.

Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.

Misfolded proteins bind and activate death receptor 5 to trigger apoptosis during unresolved endoplasmic reticulum stress.

Disruption of protein folding in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR)-a signaling network that ultimately determines cell fate. Initially, UPR signaling aims at cytoprotection and restoration of ER homeostasis; that failing, it drives apoptotic cell death. ER stress initiates apoptosis through intracellular activation of death receptor 5 (DR5) independent of its canonical extracellular ligand Apo2L/TRAIL; however, the mechanism underlying DR5 activation is unknown. In cultured human cells, we find that misfolded proteins can directly engage with DR5 in the ER-Golgi intermediate compartment, where DR5 assembles pro-apoptotic caspase 8-activating complexes. Moreover, peptides used as a proxy for exposed misfolded protein chains selectively bind to the purified DR5 ectodomain and induce its oligomerization. These findings indicate that misfolded proteins can act as ligands to activate DR5 intracellularly and promote apoptosis. We propose that cells can use DR5 as a late protein-folding checkpoint before committing to a terminal apoptotic fate.

Proteins are chains of building blocks called amino acids, folded into a flexible 3D shape that is critical for its biological activity. This shape depends on many factors, but one is the chemistry of the amino acids. Because the internal and external environments of cells are mostly water-filled, correctly folded proteins often display so-called hydrophilic (or 'water-loving') amino acids on their surface, while tucking hydrophobic (or 'water-hating') amino acids on the inside. A compartment within the cell called the endoplasmic reticulum folds the proteins that are destined for the outside of the cell. It can handle a steady stream of protein chains, but a sudden increase in demand for production, or issues with the underlying machinery, can stress the endoplasmic reticulum and hinder protein folding. This is problematic because incorrectly folded proteins cannot work as they should and can be toxic to the cell that made them or even to other cells. Many cells handle this kind of stress by activating a failsafe alarm system called the unfolded protein response. It detects the presence of incorrectly shaped proteins and sends signals that try to protect the cell and restore protein folding to normal. If that fails within a certain period of time, it switches to signals that tell the cell to safely self-destruct. That switch, from protection to self-destruction, involves a protein called death receptor 5, or DR5 for short. DR5 typically triggers the cell's self-destruct program by forming molecular clusters at the cell's surface, in response to a signal it receives from the exterior. During a failed unfolded protein response, DR5 seems instead to act in response to signals from inside the cell, but it was not clear how this works. To find out, Lam et al. stressed the endoplasmic reticulum in human cells by forcing it to fold a lot of proteins. This revealed that DR5 sticks to misfolded proteins when they leave the endoplasmic reticulum. In response, DR5 molecules form clusters that trigger the cell's self-destruct program. DR5 directly recognized hydrophobic amino acids on the misfolded protein's surface that would normally be hidden inside. When Lam et al. edited these hydrophobic regions to become hydrophilic, the DR5 molecules could no longer detect them as well. This stopped the cells from dying so easily when they were under stress. It seems that DR5 decides the fate of the cell by detecting proteins that were incorrectly folded in the endoplasmic reticulum. Problems with protein folding occur in many human diseases, including metabolic conditions, cancer and degenerative brain disorders. Future work could reveal whether controlling the activation of DR5 could help to influence if and when cells die. The next step is to understand how DR5 interacts with incorrectly folded proteins at the atomic level. This could aid the design of drugs that specifically target such receptors.

Caspase-mediated cleavage of IRE1 controls apoptotic cell commitment during endoplasmic reticulum stress.

Upon detecting endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) orchestrates adaptive cellular changes to reestablish homeostasis. If stress resolution fails, the UPR commits the cell to apoptotic death. Here we show that in hematopoietic cells, including multiple myeloma (MM), lymphoma, and leukemia cell lines, ER stress leads to caspase-mediated cleavage of the key UPR sensor IRE1 within its cytoplasmic linker region, generating a stable IRE1 fragment comprising the ER-lumenal domain and transmembrane segment (LDTM). This cleavage uncouples the stress-sensing and signaling domains of IRE1, attenuating its activation upon ER perturbation. Surprisingly, LDTM exerts negative feedback over apoptotic signaling by inhibiting recruitment of the key proapoptotic protein BAX to mitochondria. Furthermore, ectopic LDTM expression enhances xenograft growth of MM tumors in mice. These results uncover an unexpected mechanism of cross-regulation between the apoptotic caspase machinery and the UPR, which has biologically significant consequences for cell survival under ER stress.

Disruption of IRE1α through its kinase domain attenuates multiple myeloma.

Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.

Tetravalent biepitopic targeting enables intrinsic antibody agonism of tumor necrosis factor receptor superfamily members.

Agonism of members of the tumor necrosis factor receptor superfamily (TNFRSF) with monoclonal antibodies is of high therapeutic interest due to their role in immune regulation and cell proliferation. A major hurdle for pharmacologic activation of this receptor class is the requirement for high-order clustering, a mechanism that imposes a reliance in vivo on Fc receptor-mediated crosslinking. This extrinsic dependence represents a potential limitation of virtually the entire pipeline of agonist TNFRSF antibody drugs, of which none have thus far been approved or reached late-stage clinical trials. We show that tetravalent biepitopic targeting enables robust intrinsic antibody agonism for two members of this family, OX40 and DR5, that is superior to extrinsically crosslinked native parental antibodies. Tetravalent biepitopic anti-OX40 engagement co-stimulated OX40low cells, obviated the requirement for CD28 co-signal for T cell activation, and enabled superior pharmacodynamic activity relative to native IgG in a murine vaccination model. This work establishes a proof of concept for an engineering approach that addresses a major gap for the therapeutic activation of this important receptor class.

Coordination between Two Branches of the Unfolded Protein Response Determines Apoptotic Cell Fate.

The kinases PERK and IRE1 alleviate endoplasmic reticulum (ER) stress by orchestrating the unfolded protein response (UPR). If stress mitigation fails, PERK promotes cell death by activating pro-apoptotic genes, including death receptor 5 (DR5). Conversely, IRE1-which harbors both kinase and endoribonuclease (RNase) modules-blocks apoptosis through regulated IRE1-dependent decay (RIDD) of DR5 mRNA. Under irresolvable ER stress, PERK activity persists, whereas IRE1 paradoxically attenuates, by mechanisms that remain obscure. Here, we report that PERK governs IRE1's attenuation through a phosphatase known as RPAP2 (RNA polymerase II-associated protein 2). RPAP2 reverses IRE1 phosphorylation, oligomerization, and RNase activation. This inhibits IRE1-mediated adaptive events, including activation of the cytoprotective transcription factor XBP1s, and ER-associated degradation of unfolded proteins. Furthermore, RIDD termination by RPAP2 unleashes DR5-mediated caspase activation and drives cell death. Thus, PERK attenuates IRE1 via RPAP2 to abort failed ER-stress adaptation and trigger apoptosis.

Confirming a critical role for death receptor 5 and caspase-8 in apoptosis induction by endoplasmic reticulum stress.

Several studies implicate specific death receptors (DRs) and caspase-8 in mediating apoptosis in response to endoplasmic reticulum (ER) stress; however, a recent paper challenges this conclusion. Here we validate the importance of DR5 and caspase-8 as critical signal conduits for apoptosis activation upon ER stress.

From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors.

Members of the B cell lymphoma 2 (BCL-2) gene family have a central role in regulating programmed cell death by controlling pro-apoptotic and anti-apoptotic intracellular signals. In cancer, apoptosis evasion through dysregulation of specific BCL-2 family genes is a recurring event; accordingly, selective inhibition of specific anti-apoptotic BCL-2 family proteins represents an exciting therapeutic opportunity. A combination of nuclear magnetic resonance (NMR)-based screening and structure-based drug design has yielded the first bona fide BCL-2 homology 3 (BH3) mimetics, including the BCL-2 and BCL-XL dual antagonist navitoclax, which is the first BCL-2 family inhibitor to show efficacy in patients with cancer. Clinical experience with navitoclax prompted the generation of the highly selective BCL-2 inhibitor venetoclax, which is now approved in the United States for the treatment of patients with chronic lymphocytic leukaemia with 17p deletion who have received at least one prior therapy. Recent advances have also been made in the development of potent and selective inhibitors of BCL-XL and myeloid cell leukaemia 1 (MCL1), which are additional BCL-2 family members with established anti-apoptotic roles in cancer. Here we review the latest progress in direct and selective targeting of BCL-2 family proteins for cancer therapy.