Publications by authors named "Joshua D Webster"

58 Publications

Genetic inactivation of RIP1 kinase activity in rats protects against ischemic brain injury.

Cell Death Dis 2021 Apr 7;12(4):379. Epub 2021 Apr 7.

Department of Neuroscience, Genentech, South San Francisco, 94080, CA, USA.

RIP1 kinase-mediated inflammatory and cell death pathways have been implicated in the pathology of acute and chronic disorders of the nervous system. Here, we describe a novel animal model of RIP1 kinase deficiency, generated by knock-in of the kinase-inactivating RIP1(D138N) mutation in rats. Homozygous RIP1 kinase-dead (KD) rats had normal development, reproduction and did not show any gross phenotypes at baseline. However, cells derived from RIP1 KD rats displayed resistance to necroptotic cell death. In addition, RIP1 KD rats were resistant to TNF-induced systemic shock. We studied the utility of RIP1 KD rats for neurological disorders by testing the efficacy of the genetic inactivation in the transient middle cerebral artery occlusion/reperfusion model of brain injury. RIP1 KD rats were protected in this model in a battery of behavioral, imaging, and histopathological endpoints. In addition, RIP1 KD rats had reduced inflammation and accumulation of neuronal injury biomarkers. Unbiased proteomics in the plasma identified additional changes that were ameliorated by RIP1 genetic inactivation. Together these data highlight the utility of the RIP1 KD rats for target validation and biomarker studies for neurological disorders.
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http://dx.doi.org/10.1038/s41419-021-03651-6DOI Listing
April 2021

NINJ1 mediates plasma membrane rupture during lytic cell death.

Nature 2021 Mar 20;591(7848):131-136. Epub 2021 Jan 20.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA, USA.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1 macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1 macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1 mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.
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http://dx.doi.org/10.1038/s41586-021-03218-7DOI Listing
March 2021

Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death.

Cell Death Differ 2021 Mar 30;28(3):985-1000. Epub 2020 Sep 30.

Departments of Early Discovery Biochemistry, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA.

Receptor-interacting protein 1 (RIP1; RIPK1) is a key regulator of multiple signaling pathways that mediate inflammatory responses and cell death. TNF-TNFR1 triggered signaling complex formation, subsequent NF-κB and MAPK activation and induction of cell death involve RIPK1 ubiquitination at several lysine residues including Lys376 and Lys115. Here we show that mutating the ubiquitination site K376 of RIPK1 (K376R) in mice activates cell death resulting in embryonic lethality. In contrast to Ripk1 mice, Ripk1 mice reached adulthood and showed slightly higher responsiveness to TNF-induced death. Cell death observed in Ripk1 embryos relied on RIPK1 kinase activity as administration of RIPK1 inhibitor GNE684 to pregnant heterozygous mice effectively blocked cell death and prolonged survival. Embryonic lethality of Ripk1 mice was prevented by the loss of TNFR1, or by simultaneous deletion of caspase-8 and RIPK3. Interestingly, elimination of the wild-type allele from adult Ripk1 mice was tolerated. However, adult Ripk1 mice were exquisitely sensitive to TNF-induced hypothermia and associated lethality. Absence of the K376 ubiquitination site diminished K11-linked, K63-linked, and linear ubiquitination of RIPK1, and promoted the assembly of death-inducing cellular complexes, suggesting that multiple ubiquitin linkages contribute to the stability of the RIPK1 signaling complex that stimulates NF-κB and MAPK activation. In contrast, mutating K115 did not affect RIPK1 ubiquitination or TNF stimulated NF-κB and MAPK signaling. Overall, our data indicate that selective impairment of RIPK1 ubiquitination can lower the threshold for RIPK1 activation by TNF resulting in cell death and embryonic lethality.
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http://dx.doi.org/10.1038/s41418-020-00629-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937686PMC
March 2021

Genetic inactivation of RIP1 kinase does not ameliorate disease in a mouse model of ALS.

Cell Death Differ 2021 Mar 29;28(3):915-931. Epub 2020 Sep 29.

Department of Neuroscience, Genentech, Inc., South San Francisco, CA, 94080, USA.

RIP1 kinase is proposed to play a critical role in driving necroptosis and inflammation in neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS). Preclinical studies indicated that while pharmacological inhibition of RIP1 kinase can ameliorate axonal pathology and delay disease onset in the mutant SOD1 transgenic (SOD1-Tg) mice, genetic blockade of necroptosis does not provide benefit in this mouse model. To clarify the role of RIP1 kinase activity in driving pathology in SOD1-Tg mice, we crossed SOD1-Tgs to RIP1 kinase-dead knock-in mice, and measured disease progression using functional and histopathological endpoints. Genetic inactivation of the RIP1 kinase activity in the SOD1-Tgs did not benefit the declining muscle strength or nerve function, motor neuron degeneration or neuroinflammation. In addition, we did not find evidence of phosphorylated RIP1 accumulation in the spinal cords of ALS patients. On the other hand, genetic inactivation of RIP1 kinase activity ameliorated the depletion of the neurotransmitter dopamine in a toxin model of dopaminergic neurodegeneration. These findings indicate that RIP1 kinase activity is dispensable for disease pathogenesis in the SOD1-Tg mice while inhibition of kinase activity may provide benefit in acute injury models.
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http://dx.doi.org/10.1038/s41418-020-00625-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937687PMC
March 2021

Validating Immunohistochemistry Assay Specificity in Investigative Studies: Considerations for a Weight of Evidence Approach.

Vet Pathol 2020 Sep 25:300985820960132. Epub 2020 Sep 25.

University of Iowa, Iowa City, IA, USA.

Immunohistochemistry (IHC) is a fundamental molecular technique that provides information on protein expression in the context of spatial localization and tissue morphology. IHC is used in all facets of pathology from identifying infectious agents or characterizing tumors in diagnostics, to characterizing cellular and molecular processes in investigative and experimental studies. Confidence in an IHC assay is primarily driven by the degree to which it is validated. There are many approaches to validate an IHC assay's specificity including bioinformatics approaches using published protein sequences, careful design of positive and negative tissue controls, use of cell pellets with known target protein expression, corroboration of IHC findings with western blots and other analytical methods, and replacement of the primary antibody with an appropriate negative control reagent. Each approach has inherent strengths and weaknesses, and the thoughtful use of these approaches provides cumulative evidence, or a weight of evidence, to support the IHC assay's specificity and build confidence in a study's conclusions. Although it is difficult to be 100% confident in the specificity of any IHC assay, it is important to consider how validation approaches provide evidence to support or to question the specificity of labeling, and how that evidence affects the overall interpretation of a study's results. In this review, we discuss different approaches for IHC antibody validation, with an emphasis on the characterization of antibody specificity in investigative studies. While this review is not prescriptive, it is hoped that it will be thought provoking when considering the interpretation of IHC results.
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http://dx.doi.org/10.1177/0300985820960132DOI Listing
September 2020

Integration of innate immune signalling by caspase-8 cleavage of N4BP1.

Nature 2020 11 24;587(7833):275-280. Epub 2020 Sep 24.

Department of Physiological Chemistry, Genentech, South San Francisco, CA, USA.

Mutations in the death receptor FAS or its ligand FASL cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.
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http://dx.doi.org/10.1038/s41586-020-2796-5DOI Listing
November 2020

Ubiquitin Ligase COP1 Suppresses Neuroinflammation by Degrading c/EBPβ in Microglia.

Cell 2020 Sep 13;182(5):1156-1169.e12. Epub 2020 Aug 13.

Department of Physiological Chemistry, Genentech, South San Francisco, CA 94080, USA. Electronic address:

Dysregulated microglia are intimately involved in neurodegeneration, including Alzheimer's disease (AD) pathogenesis, but the mechanisms controlling pathogenic microglial gene expression remain poorly understood. The transcription factor CCAAT/enhancer binding protein beta (c/EBPβ) regulates pro-inflammatory genes in microglia and is upregulated in AD. We show expression of c/EBPβ in microglia is regulated post-translationally by the ubiquitin ligase COP1 (also called RFWD2). In the absence of COP1, c/EBPβ accumulates rapidly and drives a potent pro-inflammatory and neurodegeneration-related gene program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies reveal that neurotoxicity is almost entirely attributable to complement. Remarkably, loss of a single allele of Cebpb prevented the pro-inflammatory phenotype. COP1-deficient microglia markedly accelerated tau-mediated neurodegeneration in a mouse model where activated microglia play a deleterious role. Thus, COP1 is an important suppressor of pathogenic c/EBPβ-dependent gene expression programs in microglia.
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http://dx.doi.org/10.1016/j.cell.2020.07.011DOI Listing
September 2020

The Balance of TNF Mediated Pathways Regulates Inflammatory Cell Death Signaling in Healthy and Diseased Tissues.

Front Cell Dev Biol 2020 21;8:365. Epub 2020 May 21.

Departments of Pathology and Early Discovery Biochemistry, Genentech, South San Francisco, CA, United States.

Tumor necrosis factor alpha (TNF; TNFα) is a critical regulator of immune responses in healthy organisms and in disease. TNF is involved in the development and proper functioning of the immune system by mediating cell survival and cell death inducing signaling. TNF stimulated signaling pathways are tightly regulated by a series of phosphorylation and ubiquitination events, which enable timely association of TNF receptors-associated intracellular signaling complexes. Disruption of these signaling events can disturb the balance and the composition of signaling complexes, potentially resulting in severe inflammatory diseases.
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http://dx.doi.org/10.3389/fcell.2020.00365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326080PMC
May 2020

Single-Cell Transcriptome Profiling of the Kidney Glomerulus Identifies Key Cell Types and Reactions to Injury.

J Am Soc Nephrol 2020 10 10;31(10):2341-2354. Epub 2020 Jul 10.

Department of Research Biology, Genentech, South San Francisco, California

Background: The glomerulus is a specialized capillary bed that is involved in urine production and BP control. Glomerular injury is a major cause of CKD, which is epidemic and without therapeutic options. Single-cell transcriptomics has radically improved our ability to characterize complex organs, such as the kidney. Cells of the glomerulus, however, have been largely underrepresented in previous single-cell kidney studies due to their paucity and intractability.

Methods: Single-cell RNA sequencing comprehensively characterized the types of cells in the glomerulus from healthy mice and from four different disease models (nephrotoxic serum nephritis, diabetes, doxorubicin toxicity, and CD2AP deficiency).

Results: All cell types in the glomerulus were identified using unsupervised clustering analysis. Novel marker genes and gene signatures of mesangial cells, vascular smooth muscle cells of the afferent and efferent arterioles, parietal epithelial cells, and three types of endothelial cells were identified. Analysis of the disease models revealed cell type-specific and injury type-specific responses in the glomerulus, including acute activation of the Hippo pathway in podocytes after nephrotoxic immune injury. Conditional deletion of YAP or TAZ resulted in more severe and prolonged proteinuria in response to injury, as well as worse glomerulosclerosis.

Conclusions: Generation of comprehensive high-resolution, single-cell transcriptomic profiles of the glomerulus from healthy and injured mice provides resources to identify novel disease-related genes and pathways.
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http://dx.doi.org/10.1681/ASN.2020020220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609001PMC
October 2020

MAP4K4 negatively regulates CD8 T cell-mediated antitumor and antiviral immunity.

Sci Immunol 2020 03;5(45)

Department of Molecular Oncology, Genentech, South San Francisco, CA, USA.

During cytotoxic T cell activation, lymphocyte function-associated antigen-1 (LFA-1) engages its ligands on antigen-presenting cells (APCs) or target cells to enhance T cell priming or lytic activity. Inhibiting LFA-1 dampens T cell-dependent symptoms in inflammation, autoimmune diseases, and graft-versus-host disease. However, the therapeutic potential of augmenting LFA-1 function is less explored. Here, we show that genetic deletion or inhibition of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) enhances LFA-1 activation on CD8 T cells and improves their adherence to APCs or LFA-1 ligand. In addition, loss of increases CD8 T cell priming, which culminates in enhanced antigen-dependent activation, proliferation, cytokine production, and cytotoxic activity, resulting in impaired tumor growth and improved response to viral infection. LFA-1 inhibition reverses these phenotypes. The ERM (ezrin, radixin, and moesin) proteins reportedly regulate T cell-APC conjugation, but the molecular regulator and effector of ERM proteins in T cells have not been defined. In this study, we demonstrate that the ERM proteins serve as mediators between MAP4K4 and LFA-1. Last, systematic analyses of many organs revealed that inducible whole-body deletion of in adult animals is tolerated under homeostatic conditions. Our results uncover MAP4K4 as a potential target to augment antitumor and antiviral immunity.
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http://dx.doi.org/10.1126/sciimmunol.aay2245DOI Listing
March 2020

The Tumor Suppressor BAP1 Regulates the Hippo Pathway in Pancreatic Ductal Adenocarcinoma.

Cancer Res 2020 04 27;80(8):1656-1668. Epub 2020 Jan 27.

Department of Discovery Oncology, Genentech, Inc., South San Francisco, California.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk for mesothelioma and melanocytic tumors. Here, we show that pancreatic intraepithelial neoplasia driven by oncogenic mutant KrasG12D progressed to pancreatic adenocarcinoma in the absence of BAP1. The Hippo pathway was deregulated in BAP1-deficient pancreatic tumors, with the tumor suppressor LATS exhibiting enhanced ubiquitin-dependent proteasomal degradation. Therefore, BAP1 may limit tumor progression by stabilizing LATS and thereby promoting activity of the Hippo tumor suppressor pathway. SIGNIFICANCE: BAP1 is mutated in a broad spectrum of tumors. Pancreatic Bap1 deficiency causes acinar atrophy but combines with oncogenic Ras to produce pancreatic tumors. BAP1-deficient tumors exhibit deregulation of the Hippo pathway..
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1704DOI Listing
April 2020

RIP1 kinase activity is critical for skin inflammation but not for viral propagation.

J Leukoc Biol 2020 06 27;107(6):941-952. Epub 2020 Jan 27.

Early Discovery Biochemistry, Genentech, South San Francisco, California, USA.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.
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http://dx.doi.org/10.1002/JLB.3MA1219-398RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317411PMC
June 2020

Activity of caspase-8 determines plasticity between cell death pathways.

Nature 2019 11 13;575(7784):679-682. Epub 2019 Nov 13.

Department of Physiological Chemistry, Genentech, South San Francisco, CA, USA.

Caspase-8 is a protease with both pro-death and pro-survival functions: it mediates apoptosis induced by death receptors such as TNFR1, and suppresses necroptosis mediated by the kinase RIPK3 and the pseudokinase MLKL. Mice that lack caspase-8 display MLKL-dependent embryonic lethality, as do mice that express catalytically inactive CASP8(C362A). Casp8Mlkl mice die during the perinatal period, whereas Casp8Mlkl mice are viable, which indicates that inactive caspase-8 also has a pro-death scaffolding function. Here we show that mutant CASP8(C362A) induces the formation of ASC (also known as PYCARD) specks, and caspase-1-dependent cleavage of GSDMD and caspases 3 and 7 in MLKL-deficient mouse intestines around embryonic day 18. Caspase-1 and its adaptor ASC contributed to the perinatal lethal phenotype because a number of Casp8MlklCasp1 and Casp8MlklAsc mice survived beyond weaning. Transfection studies suggest that inactive caspase-8 adopts a distinct conformation to active caspase-8, enabling its prodomain to engage ASC. Upregulation of the lipopolysaccharide sensor caspase-11 in the intestines of both Casp8Mlkl and Casp8MlklCasp1 mice also contributed to lethality because Casp8MlklCasp1Casp11 (Casp11 is also known as Casp4) neonates survived more often than Casp8MlklCasp1 neonates. Finally, Casp8Ripk3Casp1Casp11 mice survived longer than Casp8MlklCasp1Casp11 mice, indicating that a necroptosis-independent function of RIPK3 also contributes to lethality. Thus, unanticipated plasticity in death pathways is revealed when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.
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http://dx.doi.org/10.1038/s41586-019-1752-8DOI Listing
November 2019

The RIPK4-IRF6 signalling axis safeguards epidermal differentiation and barrier function.

Nature 2019 10 2;574(7777):249-253. Epub 2019 Oct 2.

Department of Physiological Chemistry, Genentech, South San Francisco, CA, USA.

The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6 skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.
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http://dx.doi.org/10.1038/s41586-019-1615-3DOI Listing
October 2019

Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis.

Nature 2019 10 11;574(7778):428-431. Epub 2019 Sep 11.

Department of Physiological Chemistry, Genentech, South San Francisco, CA, USA.

The aspartate-specific cysteine protease caspase-8 suppresses necroptotic cell death mediated by RIPK3 and MLKL. Indeed, mice that lack caspase-8 die in a RIPK3- and MLKL-dependent manner during embryogenesis. In humans, caspase-8 deficiency is associated with immunodeficiency or very early onset inflammatory bowel disease. The substrates that are cleaved by caspase-8 to prevent necroptosis in vivo have not been defined. Here we show that knock-in mice that express catalytically inactive caspase-8(C362A) die as embryos owing to MLKL-dependent necroptosis, similar to caspase-8-deficient mice. Thus, caspase-8 must cleave itself, other proteins or both to inhibit necroptosis. Mice that express caspase-8(D212A/D218A/D225A/D387A), which cannot cleave itself, were viable, as were mice that express c-FLIP or CYLD proteins that had been mutated to prevent cleavage by caspase-8. By contrast, mice that express RIPK1(D325A), in which the caspase-8 cleavage site Asp325 had been mutated, died mid-gestation. Embryonic lethality was prevented by inactivation of RIPK1, loss of TNFR1, or loss of both MLKL and the caspase-8 adaptor FADD, but not by loss of MLKL alone. Thus, RIPK1(D325A) appears to trigger cell death mediated by TNF, the kinase activity of RIPK1 and FADD-caspase-8. Accordingly, dying endothelial cells that contain cleaved caspase-3 were abnormally abundant in yolk sacs of Ripk1 embryos. Heterozygous Ripk1 cells and mice were viable, but were also more susceptible to TNF-induced cell death than were wild-type cells or mice. Our data show that Asp325 of RIPK1 is essential for limiting aberrant cell death in response to TNF, consistent with the idea that cleavage of RIPK1 by caspase-8 is a mechanism for dismantling death-inducing complexes.
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http://dx.doi.org/10.1038/s41586-019-1548-xDOI Listing
October 2019

Applications and considerations for the use of genetically engineered mouse models in drug development.

Cell Tissue Res 2020 May 5;380(2):325-340. Epub 2019 Sep 5.

Research Pathology, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA.

Considering high drug attrition rates in clinical studies and the overall complexity and challenging environment of drug development, it is increasingly important to understand the therapeutic molecule and target and how they intersect with disease biology as fully as possible. This requires one to use numerous tools and investigative approaches in combination. Genetically engineered mouse models are a critical component to the drug development toolbox as they can provide key insights across multiple steps of the drug development process. While knock-out and knock-in mice can inform questions of basic biology, genetically engineered mice can also be applied to model diseases for efficacy studies, to discriminate on-target and off-target effects of novel therapeutics, and to inform an array of biologic and pharmacologic questions, including pharmacodynamics, pharmacokinetics, and biomarker discovery. However, use of these models requires not only an understanding of their strengths and limitations but also a careful consideration of the context in which they are being used and the hypotheses being addressed by them. Additionally, they should not be used in isolation, but instead in combination with other biochemical, in vitro, and clinical data to create a broad understanding of the drug, target, and disease biology.
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http://dx.doi.org/10.1007/s00441-019-03101-yDOI Listing
May 2020

Unobstructed Multiscale Imaging of Tissue Sections for Ultrastructural Pathology Analysis by Backscattered Electron Scanning Microscopy.

J Histochem Cytochem 2020 01 6;68(1):9-23. Epub 2019 Aug 6.

Department of Pathology, Genentech Inc., South San Francisco, California.

Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic and investigative pathology. Evaluation of large tissue areas with suborganelle resolution is challenging because biological structures ranging from several millimeters to nanometers in size need to be identified and imaged while maintaining context over multiple scales. Imaging with field emission scanning electron microscopes (FE-SEMs) is uniquely suited for this task. We describe an efficient workflow for the preparation and unobstructed multiscale imaging of tissue sections with backscattered electron scanning electron microscopy (BSE-SEM) for applications in ultrastructural pathology. We demonstrate that a diverse range of tissues, processed by conventional electron microscopy protocols and avoiding the use of mordanting agents, can be imaged on standard glass slides over multiple scales, from the histological to the ultrastructural level, without any visual obstructions. Our workflow takes advantage of the very large scan fields possible with modern FE-SEMs that allow for the acquisition of wide-field overview images which can be explored at the ultrastructural level by digitally zooming into the images. Examples from applications in pulmonary research and neuropathology demonstrate the versatility and efficiency of this method. This BSE-SEM-based multiscale imaging procedure promises to substantially simplify and accelerate ultrastructural tissue analysis in pathology.
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http://dx.doi.org/10.1369/0022155419868992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6931167PMC
January 2020

Prognostic and predictive significance of KIT protein expression and c-kit gene mutation in canine cutaneous mast cell tumours: A consensus of the Oncology-Pathology Working Group.

Vet Comp Oncol 2019 Dec 9;17(4):451-455. Epub 2019 Jul 9.

Genentech, South San Francisco, California.

One of the primary objectives of the Oncology-Pathology Working Group (OPWG), a joint initiative of the Veterinary Cancer Society and the American College of Veterinary Pathologists, is for oncologists and pathologists to collaboratively generate consensus documents to standardize aspects of and provide guidelines for oncologic pathology. Consensus is established through critical review of peer-reviewed literature relevant to a subgroup's particular focus. Subsequent acceptance and approval of the document by the OPWG membership at large establishes consensus. The intent of this publication is to help educate practitioners and pathologists on the value of diagnostics related to the KIT receptor tyrosine kinase for canine cutaneous mast cell tumours and to provide a guide for the use of these tests in veterinary medicine. This document represents the opinions of the OPWG and the authors and does not constitute a formal endorsement by the American College of Veterinary Pathologists or the Veterinary Cancer Society.
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http://dx.doi.org/10.1111/vco.12518DOI Listing
December 2019

Ubiquitin Ligases cIAP1 and cIAP2 Limit Cell Death to Prevent Inflammation.

Cell Rep 2019 05;27(9):2679-2689.e3

Department of Physiological Chemistry, Genentech, South San Francisco, CA 94080, USA. Electronic address:

Cellular inhibitor of apoptosis proteins cIAP1 and cIAP2 ubiquitinate nuclear factor κB (NF-κB)-inducing kinase (NIK) to suppress non-canonical NF-κB signaling and substrates such as receptor interacting protein kinase 1 (RIPK1) to promote cell survival. We investigate how these functions contribute to homeostasis by eliminating cIap2 from adult cIap1-deficient mice. cIAP1 and cIAP2 (cIAP1/2) deficiency causes rapid weight loss and inflammation, with aberrant cell death, indicated by cleaved caspases-3 and -8, prevalent in intestine and liver. Deletion of Casp8 and Ripk3 prevents this aberrant cell death, reduces the inflammation, and prolongs mouse survival, whereas Ripk3 loss alone offers little benefit. Residual inflammation in mice lacking cIap1/2, Casp8, and Ripk3 is reduced by inhibition of NIK. Loss of Casp8 and Mlkl (mixed lineage kinase domain-like), but not Mlkl loss alone, also prevents cIAP1/2-deficient mice from dying around embryonic day 11. Therefore, a major function of cIAP1/2 in vivo is to suppress caspase-8-dependent cell death.
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http://dx.doi.org/10.1016/j.celrep.2019.04.111DOI Listing
May 2019

RIP1 inhibition blocks inflammatory diseases but not tumor growth or metastases.

Cell Death Differ 2020 01 17;27(1):161-175. Epub 2019 May 17.

Department of Early Discovery Biochemistry, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA.

The kinase RIP1 acts in multiple signaling pathways to regulate inflammatory responses and it can trigger both apoptosis and necroptosis. Its kinase activity has been implicated in a range of inflammatory, neurodegenerative, and oncogenic diseases. Here, we explore the effect of inhibiting RIP1 genetically, using knock-in mice that express catalytically inactive RIP1 D138N, or pharmacologically, using the murine-potent inhibitor GNE684. Inhibition of RIP1 reduced collagen antibody-induced arthritis, and prevented skin inflammation caused by mutation of Sharpin, or colitis caused by deletion of Nemo from intestinal epithelial cells. Conversely, inhibition of RIP1 had no effect on tumor growth or survival in pancreatic tumor models driven by mutant Kras, nor did it reduce lung metastases in a B16 melanoma model. Collectively, our data emphasize a role for the kinase activity of RIP1 in certain inflammatory disease models, but question its relevance to tumor progression and metastases.
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http://dx.doi.org/10.1038/s41418-019-0347-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206119PMC
January 2020

Intrinsic apoptosis shapes the tumor spectrum linked to inactivation of the deubiquitinase BAP1.

Science 2019 Apr 18;364(6437):283-285. Epub 2019 Apr 18.

Department of Physiological Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.

Malignancies arising from mutation of tumor suppressors have unexplained tissue proclivity. For example, encodes a widely expressed deubiquitinase for histone H2A, but germline mutations are predominantly associated with uveal melanomas and mesotheliomas. We show that BAP1 inactivation causes apoptosis in mouse embryonic stem cells, fibroblasts, liver, and pancreatic tissue but not in melanocytes and mesothelial cells. Ubiquitin ligase RNF2, which silences genes by monoubiquitinating H2A, promoted apoptosis in BAP1-deficient cells by suppressing expression of the prosurvival genes and In contrast, BAP1 loss in melanocytes had little impact on expression of prosurvival genes, instead inducing Thus, BAP1 appears to modulate gene expression by countering H2A ubiquitination, but its loss only promotes tumorigenesis in cells that do not engage an RNF2-dependent apoptotic program.
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http://dx.doi.org/10.1126/science.aav4902DOI Listing
April 2019

The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification.

PLoS One 2019 5;14(4):e0214110. Epub 2019 Apr 5.

Physiological Chemistry Department, Genentech, South San Francisco, California, United States of America.

Peg10 (paternally expressed gene 10) is an imprinted gene that is essential for placental development. It is thought to derive from a Ty3-gyspy LTR (long terminal repeat) retrotransposon and retains Gag and Pol-like domains. Here we show that the Gag domain of PEG10 can promote vesicle budding similar to the HIV p24 Gag protein. Expressed in a subset of mouse endocrine organs in addition to the placenta, PEG10 was identified as a substrate of the deubiquitinating enzyme USP9X. Consistent with PEG10 having a critical role in placental development, PEG10-deficient trophoblast stem cells (TSCs) exhibited impaired differentiation into placental lineages. PEG10 expressed in wild-type, differentiating TSCs was bound to many cellular RNAs including Hbegf (Heparin-binding EGF-like growth factor), which is known to play an important role in placentation. Expression of Hbegf was reduced in PEG10-deficient TSCs suggesting that PEG10 might bind to and stabilize RNAs that are critical for normal placental development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214110PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450627PMC
December 2019

Ubiquitin ligase COP1 coordinates transcriptional programs that control cell type specification in the developing mouse brain.

Proc Natl Acad Sci U S A 2018 10 15;115(44):11244-11249. Epub 2018 Oct 15.

Department of Physiological Chemistry, Genentech, South San Francisco, CA 94080;

The E3 ubiquitin ligase CRL4 is active in the absence of ERK signaling, modifying the transcription factors ETV1, ETV4, ETV5, and c-JUN with polyubiquitin that targets them for proteasomal degradation. Here we show that this posttranslational regulatory mechanism is active in neurons, with ETV5 and c-JUN accumulating within minutes of ERK activation. Mice with () deleted in neural stem cells showed abnormally elevated expression of ETV1, ETV4, ETV5, and c-JUN in the developing brain and spinal cord. Expression of c-JUN target genes and was increased, whereas ETV5 and c-JUN both contributed to an expanded number of cells expressing genes associated with gliogenesis, including , , and The mice had subtle morphological abnormalities in the cerebral cortex, hippocampus, and cerebellum by embryonic day 18 and died soon after birth. Elevated c-JUN, ETV5, and ETV1 contributed to the perinatal lethality, as several -deficient mice also lacking and , or lacking and heterozygous for , were viable.
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http://dx.doi.org/10.1073/pnas.1805033115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217379PMC
October 2018

Observational Study Design in Veterinary Pathology, Part 2: Methodology.

Vet Pathol 2018 11 18;55(6):774-785. Epub 2018 Sep 18.

20 Laboratory of Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano City, Osaka, Japan.

Observational studies are a basis for much of our knowledge of veterinary pathology, yet considerations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offered advice on planning and carrying out an observational study. Part 2 of the series focuses on methodology. Our general recommendations are to consider using already-validated methods, published guidelines, data from primary sources, and quantitative analyses. We discuss 3 common methods in pathology research-histopathologic scoring, immunohistochemistry, and polymerase chain reaction-to illustrate principles of method validation. Some aspects of quality control include use of clear objective grading criteria, validation of key reagents, assessing sample quality, determining specificity and sensitivity, use of technical and biologic negative and positive controls, blinding of investigators, approaches to minimizing operator-dependent variation, measuring technical variation, and consistency in analysis of the different study groups. We close by discussing approaches to increasing the rigor of observational studies by corroborating results with complementary methods, using sufficiently large numbers of study subjects, consideration of the data in light of similar published studies, replicating the results in a second study population, and critical analysis of the study findings.
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http://dx.doi.org/10.1177/0300985818798121DOI Listing
November 2018

The Hippo pathway effector TAZ induces TEAD-dependent liver inflammation and tumors.

Sci Signal 2018 09 11;11(547). Epub 2018 Sep 11.

Department of Discovery Oncology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

The Hippo signaling pathway regulates organ size and plays critical roles in maintaining tissue growth, homeostasis, and regeneration. Dysregulated in a wide spectrum of cancers, in mammals, this pathway is regulated by two key effectors, YAP and TAZ, that may functionally overlap. We found that TAZ promoted liver inflammation and tumor development. The expression of TAZ, but not YAP, in human liver tumors positively correlated with the expression of proinflammatory cytokines. Hyperactivated TAZ induced substantial myeloid cell infiltration into the liver and the secretion of proinflammatory cytokines through a TEAD-dependent mechanism. Furthermore, tumors with hyperactivated YAP and TAZ had distinct transcriptional signatures, which included the increased expression of inflammatory cytokines in TAZ-driven tumors. Our study elucidated a previously uncharacterized link between TAZ activity and inflammatory responses that influence tumor development in the liver.
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http://dx.doi.org/10.1126/scisignal.aaj1757DOI Listing
September 2018

The tumor suppressor BAP1 cooperates with BRAFV600E to promote tumor formation in cutaneous melanoma.

Pigment Cell Melanoma Res 2019 03 24;32(2):269-279. Epub 2018 Sep 24.

Department of Discovery Oncology, Genentech, Inc., South San Francisco, California.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAF ) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1-null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild-type counterparts. Molecularly, Bap1-null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1-null tumors are completely responsive to BRAF- and MEK-targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.
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http://dx.doi.org/10.1111/pcmr.12735DOI Listing
March 2019

Detection of Necroptosis by Phospho-RIPK3 Immunohistochemical Labeling.

Methods Mol Biol 2018 ;1857:153-160

Department of Physiological Chemistry, Genentech, South San Francisco, CA, USA.

Activation of the kinase RIPK3 (receptor interacting protein kinase 3) is a hallmark of cells dying by necroptosis. RIPK3 phosphorylates both itself and the pseudokinase MLKL (mixed lineage kinase-like) resulting in MLKL translocation to membranes and cell lysis. Antibodies recognizing RIPK3 autophosphorylation or the RIPK3-dependent phosphorylation sites on MLKL have therefore been used to monitor necroptosis induction. Here we describe immunohistochemical labeling for autophosphorylated mouse RIPK3 as a means of detecting cells undergoing necroptosis in mouse tissues.
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http://dx.doi.org/10.1007/978-1-4939-8754-2_15DOI Listing
May 2019

Kinase domain dimerization drives RIPK3-dependent necroptosis.

Sci Signal 2018 08 21;11(544). Epub 2018 Aug 21.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Necroptosis, an inflammatory form of cell death, is initiated by the activation of receptor-interacting protein kinase 3 (RIPK3), which depends on its interaction with RIPK1. Although catalytically inactive, the RIPK3 mutant D161N still stimulates RIPK1-dependent apoptosis and embryonic lethality in RIPK3 D161N homozygous mice. Whereas the absence of RIPK1 rescues RIPK3 D161N homozygous mice, we report that the absence of RIPK1 leads to embryonic lethality in RIPK3 D161N heterozygous mice. This suggested that the kinase domain of RIPK3 had a noncatalytic function that was enhanced by a conformation induced by the D161N mutation. We found that the RIPK3 kinase domain homodimerized through a surface that is structurally similar to that of the RAF family members. Mutation of residues at the dimer interface impaired dimerization and necroptosis. Kinase domain dimerization stimulated the activation of RIPK3 through cis-autophosphorylation. This noncatalytic, allosteric activity was enhanced by certain kinase-deficient mutants of RIPK3, including D161N. Furthermore, apoptosis induced by certain RIPK3 inhibitors was also dependent on the kinase dimerization interface. Our studies reveal that the RIPK3 kinase domain exhibits catalytically independent function that is important for both RIPK3-dependent necroptosis and apoptosis.
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http://dx.doi.org/10.1126/scisignal.aar2188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310155PMC
August 2018

Observational Study Design in Veterinary Pathology, Part 1: Study Design.

Vet Pathol 2018 09 2;55(5):607-621. Epub 2018 Aug 2.

20 Laboratory of Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano City, Osaka, Japan.

Observational studies are the basis for much of our knowledge of veterinary pathology and are highly relevant to the daily practice of pathology. However, recommendations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offer advice on planning and conducting an observational study with examples from the veterinary pathology literature. Investigators should recognize the importance of creativity, insight, and innovation in devising studies that solve problems and fill important gaps in knowledge. Studies should focus on specific and testable hypotheses, questions, or objectives. The methodology is developed to support these goals. We consider the merits and limitations of different types of analytic and descriptive studies, as well as of prospective vs retrospective enrollment. Investigators should define clear inclusion and exclusion criteria and select adequate numbers of study subjects, including careful selection of the most appropriate controls. Studies of causality must consider the temporal relationships between variables and the advantages of measuring incident cases rather than prevalent cases. Investigators must consider unique aspects of studies based on archived laboratory case material and take particular care to consider and mitigate the potential for selection bias and information bias. We close by discussing approaches to adding value and impact to observational studies. Part 2 of the series focuses on methodology and validation of methods.
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http://dx.doi.org/10.1177/0300985818785705DOI Listing
September 2018

OTULIN limits cell death and inflammation by deubiquitinating LUBAC.

Nature 2018 07 27;559(7712):120-124. Epub 2018 Jun 27.

Department of Physiological Chemistry, Genentech, South San Francisco, CA, USA.

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease and embryonic lethality during mouse development. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.
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http://dx.doi.org/10.1038/s41586-018-0256-2DOI Listing
July 2018