Increases in retroelement-derived double-stranded RNAs (dsRNAs) in various types of cancer cells facilitate the activation of antitumor immune responses. The long noncoding RNA EPIC1 interacts with the histone methyltran...Increases in retroelement-derived double-stranded RNAs (dsRNAs) in various types of cancer cells facilitate the activation of antitumor immune responses. The long noncoding RNA EPIC1 interacts with the histone methyltransferase EZH2 and contributes to tumor immune evasion. Here, we found that EPIC1 in tumor cells suppressed cytoplasmic dsRNA accumulation, type I interferon (IFN) responses, and antitumor immunity. In various cancer cell lines, knockdown of EPIC1 stimulated the production of dsRNA from retroelements and an antiviral-like type I IFN response that activated immune cells. EPIC1 inhibited the expression of LINE, SINE, and LTR retroelements that were also repressed by EZH2, suggesting a potential role for the EPIC1-EZH2 interaction in regulating dsRNA production. In a humanized mouse model, in vivo delivery of EPIC1-targeting oligonucleotides enhanced dsRNA accumulation in breast cancer xenografts, reduced tumor growth, and increased the infiltration of T cells and inflammatory macrophages into tumors. Furthermore, EPIC1 knockdown improved the therapeutic efficacy of the immunotherapy drug pembrolizumab, a PD-1 inhibitor, in the humanized mouse model. Together, our findings establish EPIC1 as a key regulator of dsRNA-mediated type I IFN responses and highlight its potential as a therapeutic target to improve the efficacy of immunotherapy.
Epithelial-mesenchymal transition (EMT) occurs heterogeneously among carcinoma cells to promote chemoresistance. Identifying the signaling pathways involved will nominate drug combinations to promote chemoresponse, but c...Epithelial-mesenchymal transition (EMT) occurs heterogeneously among carcinoma cells to promote chemoresistance. Identifying the signaling pathways involved will nominate drug combinations to promote chemoresponse, but cell population-level studies can be misleading, and single-cell transcriptomics are limited to indirect ontology-based inferences. To understand EMT heterogeneity at a signaling protein level, we combined iterative indirect immunofluorescence imaging of pancreas cancer cells and tumors and mutual information (MI) analysis. Focusing first on mitogen-activated protein kinase pathways, MI indicated that cell-to-cell variation in ERK activity determined EMT heterogeneity in response to different growth factors and chemotherapeutics but that JNK compensated when MEK was inhibited. Population-level models could not capture these experimentally validated MI inferences. The dominant role of ERK was consistently indicated by MI even when the analysis was expanded to include seven potential EMT-regulating signaling nodes. More generally, this work provides an approach for studying multivariate signaling-phenotype relationships based on protein measurements in any setting.
Positive allosteric modulators of free fatty acid receptor 2 induce distinct conformations to bias G protein signaling.Positive allosteric modulators of free fatty acid receptor 2 induce distinct conformations to bias G protein signaling.
Small extracellular vesicles (sEVs) released from tumors recruit nociceptor neurons to the tumor bed. Here, we found that ablating these neurons in mouse models of head and neck carcinoma and melanoma reduced the infiltr...Small extracellular vesicles (sEVs) released from tumors recruit nociceptor neurons to the tumor bed. Here, we found that ablating these neurons in mouse models of head and neck carcinoma and melanoma reduced the infiltration of myeloid-derived suppressor cells (MDSCs). Moreover, sEV-deficient tumors failed to develop in mice lacking nociceptor neurons. We investigated the interplay between tumor-infiltrating nociceptors and immune cells in head and neck squamous cell carcinoma (HNSCC) and melanoma. Upon exposure to cancer-derived sEVs, mouse dorsal root ganglion (DRG) neurons secreted increased amounts of substance P, IL-6, and injury-associated neuronal markers. Patient-derived sEVs sensitized DRG neurons to capsaicin, implying enhanced nociceptor responsiveness. Furthermore, nociceptors cultured with sEVs induced an immunosuppressed state in CD8 T cells. Incubation with conditioned medium from cocultures of neurons and cancer cells resulted in increased expression of markers of MDSCs and suppressive function in primary bone marrow cells, and the combination of neuron-conditioned medium and cancer sEVs promoted checkpoint receptor expression on T cells. Together, these findings reveal that nociceptor neurons facilitate CD8 T cell exhaustion and bolster MDSC infiltration into HNSCC and melanoma. Consequently, targeting nociceptors may provide a strategy to disrupt detrimental neuroimmune cross-talk in cancer and potentiate antitumor immunity.
Neuroimmune cross-talk is emerging as an important regulator of tumor growth and progression in cancers beyond the central nervous system. In this issue of , Restaino demonstrate that tumor-derived small extracellular v...Neuroimmune cross-talk is emerging as an important regulator of tumor growth and progression in cancers beyond the central nervous system. In this issue of , Restaino demonstrate that tumor-derived small extracellular vesicles promote tumor growth by altering the secretory profile of infiltrating sensory neurons, generating a feed-forward loop that ultimately drives immunosuppression in the tumor microenvironment.
Kabella N, Bayer FP, Stamatiou K
… +15 more, Abele M, Sakhteman A, Chang YC, Wagner V, Gabriel A, Krumm J, Reinecke M, Holzner M, Aigner M, The M, Hahne H, Bassermann F, Ludwig C, Vagnarelli P, Kuster B
Mutations that activate the small GTPase KRAS are a frequent genetic alteration in cancer, and drug discovery efforts have led to inhibitors that block KRAS activity. We sought to better understand oncogenic KRAS signali...Mutations that activate the small GTPase KRAS are a frequent genetic alteration in cancer, and drug discovery efforts have led to inhibitors that block KRAS activity. We sought to better understand oncogenic KRAS signaling and the cytostatic effects of drugs that target this system. We performed proteomic analyses to investigate changes in protein abundance and posttranslational modifications in inhibitor-treated human KRAS-mutant pancreatic (KRAS G12C and G12D) and lung cancer (KRAS G12C) cells. The inhibitors used target these mutant forms of KRAS, the downstream effectors MEK and ERK, and the upstream regulators SHP2 and SOS1. Comparisons of phosphoproteomes between cell lines revealed a core KRAS signaling signature and cell line-specific signaling networks. In all cell lines, phosphoproteomes were dominated by different degrees of autonomous, oncogenic KRAS activity. Comparison of phosphoproteomes after short and long drug exposures revealed the temporal dynamics of KRAS-MEK-ERK axis inhibition that resulted in cell cycle exit. This transition to a quiescent state occurred in the absence of substantial proteome remodeling but included broad changes in protein phosphorylation and ubiquitylation. The collective data reveal insights into oncogenic KRAS signaling, place many additional proteins into this functional context, and implicate cell cycle exit as a mechanism by which cells evade death upon KRAS signaling inhibition.
Antigen-mediated aggregation of immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcεRI) initiates mast cell activation and allergic inflammation. Here, we investigated the role of ubiquitin-specific protea...Antigen-mediated aggregation of immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcεRI) initiates mast cell activation and allergic inflammation. Here, we investigated the role of ubiquitin-specific protease 5 (USP5) in IgE-mediated mast cell activation and its regulation of FcεRIγ stability. We found that USP5 knockdown inhibited the IgE-induced release of β-hexosaminidase and histamine from mast cells and attenuated allergic inflammation in mice. USP5 interacted with FcεRIγ in mast cells, leading to its deubiquitylation and stabilization. In addition, USP5 reversed the K48-linked polyubiquitylation of FcεRIγ. USP5 knockdown in mast cells or HEK293T cells increased the binding of the E3 ubiquitin ligase Cbl-b to FcεRIγ, leading to an increase in FcεRIγ polyubiquitylation and degradation. The USP5 inhibitor WP1130 attenuated IgE-mediated mast cell activation and allergic inflammation in mice. Together, these findings describe the molecular mechanism of USP5-mediated regulation of FcεRIγ stability in mast cells and identify the USP5-FcεRIγ axis as a potential drug target for the therapy of IgE/FcεRI-mediated allergic diseases.
Sphingomyelins in mosquito saliva enhance flavivirus infection by preventing viral protein degradation.Sphingomyelins in mosquito saliva enhance flavivirus infection by preventing viral protein degradation.
Sun Y, Mishra AK, Chanrasekhar V
… +11 more, Door M, Kessinger CW, Xu B, Tang P, Gao Y, Kamli-Salino S, Nelson K, Delibegovic M, Abel ED, Kirk JA, Kontaridis MI
Cardiomyocytes (CMs) normally use fatty acid oxidation (FAO) as their primary energy source. In response to pathological stress, the substrate preference of CMs switches from FAO to glucose metabolism, leading to the dev...Cardiomyocytes (CMs) normally use fatty acid oxidation (FAO) as their primary energy source. In response to pathological stress, the substrate preference of CMs switches from FAO to glucose metabolism, leading to the development of heart failure. Obesity increases this pathological risk of cardiovascular disease. We focused on protein tyrosine phosphatase 1B (PTP1B), an inhibitor of insulin signaling, the abundance and activity of which are increased in brain, muscle, and adipose tissues in obese and/or diabetic animals and in obese human patients. We generated mice with CM-specific deficiency in PTP1B () to investigate the CM-specific role of PTP1B in response to cardiac dysfunction induced by high-fat diet (HFD) feeding. Although no physiological or functional cardiac differences were observed at baseline, mice were protected against development of cardiac hypertrophy, mitochondrial dysfunction, and cardiac steatosis induced by HFD feeding. Metabolomics data revealed that hearts with CM-specific deletion of PTP1B had increased FAO and lipolysis but reduced glucose metabolism. Furthermore, phosphoproteomics analyses and mechanistic studies identified an axis involving the kinases PKM2 and AMPK downstream of PTP1B in the heart, which collectively acted to promote FAO and suppress lipogenesis. Together, these results suggest that CM-specific deletion of PTP1B prevents a substrate switch from FAO to glucose metabolism, protecting the heart against the development of HFD-induced cardiac hypertrophy and dysfunction.
Aust O, Thiel MRT, Blanc E
… +13 more, Lüthen M, Hollek V, Astaburuaga-García R, Klinger B, Böhning F, Trinks A, Beule D, Papke B, Horst D, Blüthgen N, Sers C, Der CJ, Morkel M
Therapy-induced acquired resistance limits the clinical effectiveness of mutation-specific KRAS inhibitors in colorectal cancer (CRC). Here, we investigated whether broad-spectrum, active-state RAS inhibitors meet simila...Therapy-induced acquired resistance limits the clinical effectiveness of mutation-specific KRAS inhibitors in colorectal cancer (CRC). Here, we investigated whether broad-spectrum, active-state RAS inhibitors meet similar limitations. We found that KRAS-mutant CRC cell lines were sensitive to the RAS(ON) multiselective RAS inhibitor RMC-7977, given that treatment resulted in RAS-RAF-MEK-ERK pathway inhibition; halted proliferation; and, in some cases, induced apoptosis. RMC-7977 initially reduced the activity of a compartment-specific, dual-color reporter of ERK activity, with reporter reactivation emerging after long-term dose escalation. These drug-resistant cell populations exhibited distinct patterns of phospho-protein abundance, transcriptional activities, and genomic mutations, including a Y71H mutation in KRAS and an S257L mutation in RAF1. Transgenic expression of KRAS or RAF1 in drug-sensitive CRC cells induced resistance to RMC-7977. CRC cells that were resistant to RMC-7977 and harboring RAF1 exhibited synergistic sensitivity to concurrent inhibition of RAS and RAF. Our findings demonstrate the power of reporter-assisted screening together with single-cell analyses for dissecting the complex landscape of therapy resistance. The strategy offers opportunities to develop clinically relevant combinatorial treatments to counteract the emergence of resistant cancer cells.
Chromosomal translocations that fuse to or cause human leukemias. In BCR-ABL and TEL-ABL fusion proteins, oligomerization and loss of an autoinhibitory myristoylation site in the SH3 domain of ABL lead to increased AB...Chromosomal translocations that fuse to or cause human leukemias. In BCR-ABL and TEL-ABL fusion proteins, oligomerization and loss of an autoinhibitory myristoylation site in the SH3 domain of ABL lead to increased ABL tyrosine kinase activity. We assessed the ability of asciminib, an allosteric inhibitor of BCR-ABL that binds to the myristoyl-binding site in the ABL kinase domain, to inhibit these fusion proteins. Although the ABL components of the two fusion proteins have identical sequences, asciminib was much less effective against TEL-ABL than it was against BCR-ABL in cell-growth assays. In contrast, ATP-competitive tyrosine kinase inhibitors, such as imatinib and ponatinib, were equally effective against both fusion proteins. A helix in the ABL kinase domain that closes over bound asciminib was required for the sensitivity of BCR-ABL to the drug but had no effect on that of TEL-ABL, suggesting that the native autoinhibitory mechanism that asciminib engages in BCR-ABL is disrupted in TEL-ABL. Single-molecule microscopy demonstrated that BCR-ABL was mainly dimeric in cells, whereas TEL-ABL formed higher-order oligomers, which promoted trans-autophosphorylation, including of a regulatory phosphorylation site (Tyr) in the SH3 domain of ABL. Nonphosphorylated TEL-ABL was intrinsically susceptible to inhibition by asciminib, but phosphorylation at Tyr disassembled the autoinhibited conformation of ABL, thereby preventing asciminib from binding. Our results demonstrate that phosphorylation determines whether an ABL fusion protein is sensitive to allosteric inhibition.
Flaviviruses pose a substantial threat to public health because of their ability to infect the central nervous system (CNS). Receptor-interacting protein kinase 3 (RIPK3) is a central coordinator that promotes neuroinfla...Flaviviruses pose a substantial threat to public health because of their ability to infect the central nervous system (CNS). Receptor-interacting protein kinase 3 (RIPK3) is a central coordinator that promotes neuroinflammation during viral infection of the CNS, a role that occurs independently of its canonical function in inducing necroptosis. Here, we used mouse genetic tools to induce astrocyte-specific deletion, overexpression, and chemogenetic activation of RIPK3 to demonstrate an anti-inflammatory function for astrocytic RIPK3. RIPK3 activation in astrocytes promoted host survival during flavivirus encephalitis by limiting immune cell recruitment to the CNS. Despite inducing a proinflammatory transcriptional program, astrocytic RIPK3 restrained neuroinflammation by increasing the abundance of the protease inhibitor SerpinA3N, which preserved blood-brain barrier integrity, reduced leukocyte infiltration, and improved survival outcomes during flavivirus encephalitis. These findings highlight a previously unappreciated role for astrocytic RIPK3 in suppressing pathologic neuroinflammation.
The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from factors that both stimulate (exogenous growth factors) and are essential for (intracellular nutrients and energy) cellular growth. Act...The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from factors that both stimulate (exogenous growth factors) and are essential for (intracellular nutrients and energy) cellular growth. Activation of the protein kinase mTOR within mTORC1 results in the phosphorylation of downstream substrates that collectively stimulate biomass accumulation to drive cell growth. Many upstream signals, especially growth factors, regulate mTORC1 by inducing the phosphorylation of the tuberous sclerosis complex 2 (TSC2) subunit of the TSC protein complex, a conserved brake on mTORC1 activation and its promotion of cell growth. Cryo-electron microscopy studies of the TSC protein complex have revealed that this phosphoregulation of TSC2 occurs almost exclusively on residues in loops that are outside of the evolutionarily conserved core structural elements and that did not resolve in these structures. These phosphorylation-rich unstructured loops evolved with metazoans, suggesting that the regulation of mTORC1 by diverse growth factors likely evolved with the emergence of complex body plans and diverse cell types to coordinate cell growth and metabolism within and across distinct tissues. Unlike the core structure of TSC2, these loops lack disease-associated missense mutations. These features suggest that the regulatory loops on TSC2 are more amenable to evolutionary changes that enable diverse signals to converge on the TSC protein complex to regulate mTORC1.
Norrin-induced activation of β-catenin-dependent signaling through the receptor frizzled4 in endothelial cells (ECs) is essential for establishing and maintaining blood-CNS barrier function. We sought to determine how th...Norrin-induced activation of β-catenin-dependent signaling through the receptor frizzled4 in endothelial cells (ECs) is essential for establishing and maintaining blood-CNS barrier function. We sought to determine how this pathway is modulated under stress or disease conditions. Specifically, we investigated the role of p53 in endothelial blood-CNS barriers because increased abundance of the transcription factor p53 in ECs correlates with leaky CNS blood vessels in type 2 diabetes. Using transcriptomic, cell-based, and mouse genetic approaches, we identified interplay between p53 and its negative regulator MDM2 and norrin/frizzled4 signaling. Mice with an EC-specific ablation of showed decreased norrin/frizzled4 signaling, reduced EC proliferation and retinal angiogenesis, and disrupted blood-retina barrier function, all of which were largely restored by concurrent deletion. Decreased norrin/frizzled4 signaling and inhibition of EC proliferation in response to p53 were associated with reduced expression of the condensin I complex component non-SMC condensin I complex subunit H (NCAPH). This study identifies a regulator of norrin/frizzled4 signaling and suggests that the clinical use of MDM2 inhibitors might impair the blood-CNS barrier. In addition, NCAPH may be a downstream effector of p53 in ECs and a candidate gene for familial exudative vitreoretinopathy (FEVR), which is caused by defects in norrin signaling.
Parkinson's disease is associated with activating mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2), which suppresses primary cilia formation in cholinergic and parvalbumin interneurons and astrocytes i...Parkinson's disease is associated with activating mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2), which suppresses primary cilia formation in cholinergic and parvalbumin interneurons and astrocytes in the striatum. As a result, there is a decrease in the production of neuroprotective glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), which normally support the viability of dopaminergic neurons. MLi-2 is a brain-penetrant, selective, and now experimental inhibitor of LRRK2. Here, we found that dietary administration of MLi-2 to young LRRK2-mutant mice for 3 months restored primary cilia formation and Hedgehog signaling in both cholinergic and parvalbumin interneurons and astrocytes. The treatment also restored the Hedgehog-responsive expression of and in the neurons. Cilia were also restored on cholinergic neurons of the pedunculopontine nucleus, where their loss correlates with severity of motor impairment in patients. Furthermore, MLi-2 increased the density of fine striatal dopaminergic processes and decreased the amount of stress-associated Sonic Hedgehog RNA expression in nigral dopaminergic neurons. Thus, pathogenic LRRK2-driven cilia loss is reversible in postmitotic neurons and astrocytes, which suggests that early administration of specific LRRK2 inhibitors may therapeutically benefit patients.
Gauron MC, Prokopenko D, Lee S
… +17 more, Wolfe SA, Hecker J, Willett J, Waqas M, Lordén G, Yang Y, Mayfield JE, Castanho I, Mullin K, Morgan S, Hahn G, Demeo DL, Hide W, Bertram L, Lange C, Newton AC, Tanzi RE
The identification of Alzheimer's disease (AD)-associated genomic variants has provided powerful insight into disease etiology. Genome-wide association studies (GWASs) of AD have successfully identified previously uniden...The identification of Alzheimer's disease (AD)-associated genomic variants has provided powerful insight into disease etiology. Genome-wide association studies (GWASs) of AD have successfully identified previously unidentified targets but have almost exclusively used additive genetic models. Here, we performed a family-based GWAS of a recessive inheritance model using whole-genome sequencing from families affected by AD. We found an association between AD risk and the variant rs7161410, which is located in an intron of the gene encoding protein kinase C eta (PKCη). In addition, a rare missense mutation, K65R, was in linkage disequilibrium with rs7161410 and was present in homozygous carriers of the rs7161410 risk allele. In vitro analysis revealed that the catalytic rate, lipid dependence, and peptide substrate binding of the purified variant were indistinguishable from those of the wild-type kinase. However, cellular studies revealed that the K65R PKCη variant had reduced cytosolic activity and, instead, enhanced localization and signaling at the Golgi. Moreover, the K65R variant had altered interaction networks in transfected cells, particularly with proteins involved in Golgi processes such as vesicle transport. In human brain tissue, the AD-associated recessive genotype of rs7161410 was associated with increased expression of , particularly in the amygdala. This association of aberrant PKCη signaling with AD and the insight into how its function is altered may lead to previously unidentified therapeutic targets for prevention and treatment.
Glycosaminoglycans enable cancer cells to take up antiferroptotic lipoproteins.Glycosaminoglycans enable cancer cells to take up antiferroptotic lipoproteins.
Changes in the transcriptome are critical in shaping the structural plasticity of neurons, which underpins learning and long-term memory storage. Here, we explored the effect of two opposing, plasticity-associated pathwa...Changes in the transcriptome are critical in shaping the structural plasticity of neurons, which underpins learning and long-term memory storage. Here, we explored the effect of two opposing, plasticity-associated pathways-cAMP second-messenger signaling and metabotropic glutamate receptor (mGluR1 and mGluR5) signaling-on the transcriptome in hippocampal neurons and how these pathways operate in distinct and coordinated manners to induce structural changes. Integration of transcriptome data and molecular pathway analysis identified central "hub" genes that were rapidly induced by cAMP and/or mGluR1/5 in hippocampal neurons. These included the long noncoding RNA (lncRNA) Gas5, whose expression was induced specifically by cAMP and which was targeted to dendrites by the kinesin motor protein KIF1A. In the dendrites, Gas5 interacted with various proteins and coding and noncoding RNAs associated with synaptic function and plasticity, and these interactions were altered by cAMP signaling. Gas5 interacted with the microRNA miR-26a-5p and sequestered it from several of its mRNA targets associated with neuronal function and whose translation was induced by cAMP. Gas5 was critical for excitatory synaptic transmission induced by cAMP but not those induced by mGluR1/5. Furthermore, Gas5 deficiency impaired dendritic branching and synapse morphology, and Gas5 abundance was decreased in the hippocampus of a mouse model of Alzheimer's disease. Together, these findings provide insight into the transcriptional networks involved in synaptic plasticity and a lncRNA interactome that mediates dendritically localized regulation of excitatory synaptic transmission and neuronal architecture.
The large amounts of calcium (Ca) stored in the endoplasmic reticulum (ER) and the controlled release of this Ca store into the cytosol regulate many cellular functions, and altered ER Ca homeostasis induces ER stress. S...The large amounts of calcium (Ca) stored in the endoplasmic reticulum (ER) and the controlled release of this Ca store into the cytosol regulate many cellular functions, and altered ER Ca homeostasis induces ER stress. Stromal-interacting molecules 1 and 2 (STIM1/2) are homologous ER-resident Ca sensors that synergistically activate cytosolic Ca influx through Orai channels to promote Ca-dependent changes in gene expression and ER Ca refilling. Here, we demonstrated that reduced abundance of STIM2, but not that of STIM1, was associated with poor prognosis in colorectal cancer (CRC). STIM2-deficient CRC cells showed enhanced ER Ca content in a manner dependent on the ER Ca pump SERCA2, increased expression of genes associated with protein translation, and transcriptional and metabolic rewiring. STIM2 deficiency in CRC xenografts led to increased tumor size, invasion, and metastasis. STIM2 loss activated the expression of genes involved in ER stress responses in a manner dependent on the chaperone BiP and the transcription factor ATF4 and independent of Orai channels. These results suggest that loss of STIM2 may inform CRC prognosis.