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Figure S1 shows single agent dose-response curves of 18 compounds employed in the screen. Figure S2 shows synergy scores of 23 compound combinations. Figure S3 shows the apoptotic effects of single agent inhibitors and their combinations on a panel of 24 cell lines. Figure S4 shows synergy scores for 6x6 concentration combinations of combined CHK1/GLUT1 or ATR/GLUT1 inhibition as well as tumor volume fold changes of A549- and HCC44-derived xenografts treated with the CHK1-inhibitor, GLUT1-inhibitor or the combination of both. Figure S5 shows synergy scores for 6x6 concentration combinations of combined CHK1/HER2 inhibition as well as tumor volume fold changes of A549- and HCC44-derived xenografts treated with the CHK1-inhibitor, HER2-inhibitor or the combination of both. Figure S6 shows that sensitive cell lines are enriched for MTORC1 signalling and sensitivity of murine KrasG12D/wt;Trp53-/--driven NSCLC and soft tissue sarcoma sarcoma. Figure S7 shows sanger sequencing results confirming driver lesions of the core cell line panel. Figure S8 shows that siRNA-mediated knockdown is efficient and validates target specifity. Figure S9 shows that drug synergy is preserved for chemically distinct inhibitors of CHK1 and ATR and on-target activity of PF477736 and VE822. Figure S10 shows that combined inhibitions of the ATR/CHK1 axis and GLUT1 synergistically induces S-phase-specific genotoxic stress and apoptosis in A549 cells and single toxicity of CHK1/ATR inhibition in A498 cells Figure S11 shows that combined inhibition of ATR/CHK1 and GLUT1 synergistically induces phosphorylation of H2AX in sensitive cells. Figure S12 shows original plots of High-content screening microscopy. Figure S13 shows that treatment regimens have no significant effects on body weights of KrasLSL.G12D/wt;Trp53fl/fl mice.

Table S4 contains all apoptotic effects of single agent inhibitors and their combinations (original data of Fig 1C and Fig. S3).

The advent of molecularly targeted therapeutic agents has opened a new era in cancer therapy. However, many tumors rely on nondruggable cancer-driving lesions. In addition, long-lasting clinical benefits from single-agent therapies rarely occur, as most of the tumors acquire resistance over time. The identification of targeted combination regimens interfering with signaling through oncogenically rewired pathways provides a promising approach to enhance efficacy of single-agent–targeted treatments. Moreover, combination drug therapies might overcome the emergence of drug resistance. Here, we performed a focused flow cytometry–based drug synergy screen and identified a novel synergistic interaction between GLUT1-mediated glucose transport and the cell-cycle checkpoint kinases ATR and CHK1. Combined inhibition of CHK1/GLUT1 or ATR/GLUT1 robustly induced apoptosis, particularly in RAS-mutant cancer cells. Mechanistically, combined inhibition of ATR/CHK1 and GLUT1 arrested sensitive cells in S-phase and led to the accumulation of genotoxic damage, particularly in S-phase. In vivo, simultaneous inhibition of ATR and GLUT1 significantly reduced tumor volume gain in an autochthonous mouse model of KrasG12D-driven soft tissue sarcoma. Taken together, these findings pave the way for combined inhibition of GLUT1 and ATR/CHK1 as a therapeutic approach for KRAS-driven cancers.Significance:Dual targeting of the DNA damage response and glucose transport synergistically induces apoptosis in KRAS-mutant cancer, suggesting this combination treatment for clinical validation in KRAS-stratified tumor patients.

Table S6 contains all p-values related to Fig. 4C.

Bacterial products can act on neurons to alter signaling and function. Here, we find that dorsal root ganglia (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes Edema Factor (EF) and Lethal Factor (LF). Intrathecal administration of Edema Toxin (ET; PA+EF) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan, and nerve injury. Analgesia depended on ANTXR2 expressed by Na(v)1.8(+) or Advillin(+) neurons. ET modulated PKA signaling in mouse sensory and human iPS-derived sensory neurons and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors which may lead to the development of novel pain therapeutics.

The voltage-gated sodium NaV1.7 channel, critical for sensing pain, has been actively targeted by drug developers; however, there are currently no effective and safe therapies targeting NaV1.7. Here, we tested whether a different approach, indirect NaV1.7 regulation, could have antinociceptive effects in preclinical models. We found that preventing addition of small ubiquitin-like modifier (SUMO) on the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 functions and had antinociceptive effects in rodents. In silico targeting of the SUMOylation site in CRMP2 (Lys374) identified200 hits, of which compound 194 exhibited selective in vitro and ex vivo NaV1.7 engagement. Orally administered 194 was not only antinociceptive in preclinical models of acute and chronic pain but also demonstrated synergy alongside other analgesics—without eliciting addiction, rewarding properties, or neurotoxicity. Analgesia conferred by 194 was opioid receptor dependent. Our results demonstrate that 194 is a first-in-class protein-protein inhibitor that capitalizes on CRMP2-NaV1.7 regulation to deliver safe analgesia in rodents.

Depolarization drives neuronal plasticity. However, whether depolarization drives sensitization of peripheral nociceptive neurons remains elusive. By high-content screening (HCS) microscopy, we revealed that depolarization of cultured sensory neurons rapidly activates protein kinase A type II (PKA-II) in nociceptors by calcium influx through CaV1.2 channels. This effect was modulated by calpains but insensitive to inhibitors of cAMP formation, including opioids. In turn, PKA-II phosphorylated Ser1928 in the distal C terminus of CaV1.2, thereby increasing channel gating, whereas dephosphorylation of Ser1928 involved the phosphatase calcineurin. Patch-clamp and behavioral experiments confirmed that depolarization leads to calcium- and PKA-dependent sensitization of calcium currents ex vivo and local peripheral hyperalgesia in the skin in vivo. Our data suggest a local activity-driven feed-forward mechanism that selectively translates strong depolarization into further activity and thereby facilitates hypersensitivity of nociceptor terminals by a mechanism inaccessible to opioids.

The α2δ‐1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of α2δ‐1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/α2δ-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of α2δ-1 (α2δ-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing α2δ‐1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α2δ‐1 and suggest that gabapentin does not directly target TSP/α2δ-1 interaction to alleviate neuropathic pain.

The advent of molecularly targeted therapeutic agents has opened a new era in cancer therapy. However, many tumors rely on nondruggable cancer-driving lesions. In addition, long-lasting clinical benefits from single-agent therapies rarely occur, as most of the tumors acquire resistance over time. The identification of targeted combination regimens interfering with signaling through oncogenically rewired pathways provides a promising approach to enhance efficacy of single-agent–targeted treatments. Moreover, combination drug therapies might overcome the emergence of drug resistance. Here, we performed a focused flow cytometry–based drug synergy screen and identified a novel synergistic interaction between GLUT1-mediated glucose transport and the cell-cycle checkpoint kinases ATR and CHK1. Combined inhibition of CHK1/GLUT1 or ATR/GLUT1 robustly induced apoptosis, particularly in RAS-mutant cancer cells. Mechanistically, combined inhibition of ATR/CHK1 and GLUT1 arrested sensitive cells in S-phase and led to the accumulation of genotoxic damage, particularly in S-phase. In vivo, simultaneous inhibition of ATR and GLUT1 significantly reduced tumor volume gain in an autochthonous mouse model of KrasG12D-driven soft tissue sarcoma. Taken together, these findings pave the way for combined inhibition of GLUT1 and ATR/CHK1 as a therapeutic approach for KRAS-driven cancers. Significance: Dual targeting of the DNA damage response and glucose transport synergistically induces apoptosis in KRAS-mutant cancer, suggesting this combination treatment for clinical validation in KRAS-stratified tumor patients.

Background: The majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype (TNBC) and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype. Methods: Our group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-proficient and deficient - mouse mammary tumors. Results: We identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors. Conclusion: Taken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.

The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days. By trio whole-exome sequencing, we identified a novel homozygous missense mutation, c.662T > C, in YRDC affecting an evolutionary highly conserved amino acid (p.Ile221Thr). Functional characterization of patient-derived dermal fibroblasts revealed that this mutation impairs YRDC function and consequently results in reduced t6A modifications of tRNAs. Furthermore, we established and performed a novel and highly sensitive 3-D Q-FISH analysis based on single-telomere detection to investigate the impact of YRDC on telomere maintenance. This analysis revealed significant telomere shortening in YRDC-mutant cells. Moreover, single-cell RNA sequencing analysis of YRDC-mutant fibroblasts revealed significant transcriptome-wide changes in gene expression, specifically enriched for genes associated with processes involved in DNA repair. We next examined the DNA damage response of patient’s dermal fibroblasts and detected an increased susceptibility to genotoxic agents and a global DNA double-strand break repair defect. Thus, our data suggest that YRDC may affect the maintenance of genomic stability. Together, our findings indicate that biallelic variants in YRDC result in a developmental disorder with progeroid features and might be linked to increased genomic instability and telomere shortening.

Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Na

Summary: Cdkn1a, which encodes p21, functions as a major route for p53-mediated cell-cycle arrest. However, the consequence of Cdkn1a gene dosage on tumor suppression has not been systematically investigated. Here, we employed BAC transgenesis to generate a Cdkn1aSUPER mouse, which harbors an additional Cdkn1a allele within its natural genomic context. We show that these mice display enhanced cell-cycle arrest and reduced apoptosis in response to genotoxic stress. Furthermore, using a chemically induced skin cancer model and an autochthonous Kras-driven lung adenocarcinoma model, we show that Cdkn1aSUPER mice display a cancer protection phenotype that is indistinguishable from that observed in Tp53SUPER animals. Moreover, we demonstrate that Tp53 and Cdkn1a cooperate in mediating cancer resistance, using a chemically induced fibrosarcoma model. Overall, our Cdkn1aSUPER allele enabled us to assess the contribution of Cdkn1a to Tp53-mediated tumor suppression. : Torgovnick et al. create a mouse model, carrying a third copy of Cdkn1a (p21), which shows enhanced cell-cycle arrest capacity and protection against DNA damage-induced apoptosis. The Cdkn1aSUPER animals display delayed epithelial regeneration and a robust cancer resistance phenotype, highlighting the importance of p21 in p53-dependent tumor suppression. Keywords: Cdkn1a, p21, p53, mouse model, cancer, tumor suppressor, cell cycle arrest, apoptosis, cancer protection

Activity of endogenous protein kinase A (PKA) could never be analyzed directly in the cellular environment. Isensee et al. used antibodies to quantify conformational changes leading to an open conformation of endogenous PKA-II holoenzymes, which allowed them to analyze and model its activation cycle in primary sensory neurons., Type II isoforms of cyclic adenosine monophosphate (cAMP)–dependent protein kinase A (PKA-II) contain a phosphorylatable epitope within the inhibitory domain of RII subunits (pRII) with still unclear function. In vitro, RII phosphorylation occurs in the absence of cAMP, whereas staining of cells with pRII-specific antibodies revealed a cAMP-dependent pattern. In sensory neurons, we found that increased pRII immunoreactivity reflects increased accessibility of the already phosphorylated RII epitope during cAMP-induced opening of the tetrameric RII2:C2 holoenzyme. Accordingly, induction of pRII by cAMP was sensitive to novel inhibitors of dissociation, whereas blocking catalytic activity was ineffective. Also in vitro, cAMP increased the binding of pRII antibodies to RII2:C2 holoenzymes. Identification of an antibody specific for the glycine-rich loop of catalytic subunits facing the pRII-epitope confirmed activity-dependent binding with similar kinetics, proving that the reassociation is rapid and precisely controlled. Mechanistic modeling further supported that RII phosphorylation precedes cAMP binding and controls the inactivation by modulating the reassociation involving the coordinated action of phosphodiesterases and phosphatases.

// Axel Lechner 1, 2, * , Hans Schloser 1, 3, * , Sacha I. Rothschild 1, 4, * , Martin Thelen 1 , Sabrina Reuter 1 , Peter Zentis 5 , Alexander Shimabukuro-Vornhagen 1, 6 , Sebastian Theurich 1, 6, 7 , Kerstin Wennhold 1 , Maria Garcia-Marquez 1 , Lars Tharun 8 , Alexander Quaas 8 , Astrid Schauss 5 , Jorg Isensee 9 , Tim Hucho 9 , Christian Huebbers 10 , Michael von Bergwelt-Baildon 1, 6, * and Dirk Beutner 2, * 1 Cologne Interventional Immunology, Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany 2 Department of Otorhinolaryngology, Head and Neck Surgery, University of Cologne, Cologne, Germany 3 Department of General, Visceral and Cancer Surgery, University of Cologne, Cologne, Germany 4 University Hospital Basel, Department of Internal Medicine, Medical Oncology, Basel, Switzerland 5 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany 6 Department I of Internal Medicine, Center for Integrated Oncology (CIO), University Hospital of Cologne, Cologne, Germany 7 Max-Planck-Institute for Metabolism Research, Cologne, Germany 8 Institute of Pathology, University of Cologne, Cologne, Germany 9 Department of Anesthesiology and Intensive Care Medicine, Experimental Anesthesiology and Pain Research, University Hospital of Cologne, University of Cologne, Germany 10 Jean-Uhrmacher Institute for Clinical ENT Research, University of Cologne, Cologne, Germany * These authors contributed equally to this work Correspondence to: Michael von Bergwelt-Baildon, email: michael.bergwelt@uk-koeln.de Keywords: squamous cell carcinoma, head and neck, microenvironment, T cells, immune checkpoint Received: January 10, 2017 Accepted: April 25, 2017 Published: May 16, 2017 ABSTRACT The composition of tumor-infiltrating lymphocytes (TIL) reflects biology and immunogenicity of cancer. Here, we characterize T-cell subsets and expression of immune checkpoint molecules in head and neck squamous cell carcinoma (HNSCC). We analyzed TIL subsets in primary tumors ( n = 34), blood (peripheral blood mononuclear cells (PBMC); n = 34) and non-cancerous mucosa ( n = 7) of 34 treatment-naive HNSCC patients and PBMC of 15 healthy controls. Flow cytometry analyses revealed a highly variable T-cell infiltration mainly of an effector memory phenotype (CD45RA − /CCR7 − ). Naive T cells (CD45RA + /CCR7 + ) were decreased in the microenvironment compared to PBMC of patients, while regulatory T cells (CD4 + /CD25 + /CD127 low and CD4 + /CD39 + ) were elevated. Furthermore, we performed digital image analyses of entire cross sections of HNSCC to define the ‘Immunoscore’ (CD3 + and CD8 + cell infiltration in tumor core and invasive margin) and quantified MHC class I expression on tumor cells by immunohistochemistry. Immune checkpoint molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) were increased in TILs compared to peripheral T cells in flow-cytometric analysis. Human papillomavirus (HPV) positive tumors showed higher numbers of TILs, but a similar composition of T-cell subsets and checkpoint molecule expression compared to HPV negative tumors. Taken together, the tumor microenvironment of HNSCC is characterized by a strong infiltration of regulatory T cells and high checkpoint molecule expression on T-cell subsets. In view of increasingly used immunotherapies, a detailed knowledge of TILs and checkpoint molecule expression on TILs is of high translational relevance.

Pain sensitivity is strongly modulated by intracellular signalling. The analysis of pain signalling is hampered by small numbers of neurons, high cellular heterogeneity, and near absence of transfectability. HCS microscopy allowed us to proof that nociceptive neurons express specific signalling components, to analyze signalling activity of endogenous signalling components with single cell resolution, and thereby to suggest blockers of Nav1.7 as novel therapeutic approach to boost the efficacy of opioids.

Bacterial toxins are able to act on neurons to modulate signaling and function. Here, we find that nociceptive sensory neurons that mediate pain are enriched in the receptor for anthrax toxins, ANTXR2. Anthrax Edema Toxin (ET) induced cAMP and PKA signaling in Nav1.8+nociceptive neurons and modulated painin vivo. Peripherally administered ET mediated mechanical allodynia in naïve mice and duringB. anthracisinfection. Intrathecally administered ET produced analgesic effects, potently blocking pain-like behaviors in multiple mouse models of inflammatory and chronic neuropathic pain. Nociceptor-specific ablation of ANTXR2 attenuated ET-induced signaling and analgesia. Modified anthrax toxin successfully delivered exogenous protein cargo into nociceptive neurons, illustrating utility of the anthrax toxin system as a molecular platform to target pain. ET further induced signaling in human iPSC-derived sensory neurons. Our findings highlight novel interactions between a bacterial toxin and nociceptors that may be utilized for developing new pain therapeutics.SUMMARYANTXR2 expression on nociceptive neurons allows selective targeting and modulation of pain by native and engineered anthrax toxins.

Studying TRP channel expressing nociceptors requires the identification of the respective subpopulations as well as the quantification of dynamic cellular events. However, the heterogeneity of sensory neurons and associated nonneuronal cells demands the analysis of large numbers of cells to reflect the distribution of entire populations. Here we report a detailed workflow how to apply high-content screening (HCS) microscopy to signaling events in TRPV1-positive neurons as well as an approach to use the selective elimination of TRPV1 positive cells from dissociated rat sensory ganglia as base for transcriptomic analysis of TRPV1-positive cells and/or as control for TRPV1 antibody specificity.

Studying TRP channel expressing nociceptors requires the identification of the respective subpopulations as well as the quantification of dynamic cellular events. However, the heterogeneity of sensory neurons and associated nonneuronal cells demands the analysis of large numbers of cells to reflect the distribution of entire populations. Here we report a detailed workflow how to apply high-content screening (HCS) microscopy to signaling events in TRPV1-positive neurons as well as an approach to use the selective elimination of TRPV1 positive cells from dissociated rat sensory ganglia as base for transcriptomic analysis of TRPV1-positive cells and/or as control for TRPV1 antibody specificity.

The advent of molecularly targeted therapeutic agents has opened a new era in cancer therapy. However, many tumors rely on nondruggable cancer-driving lesions. In addition, long-lasting clinical benefits from single-agent therapies rarely occur, as most of the tumors acquire resistance over time. The identification of targeted combination regimens interfering with signaling through oncogenically rewired pathways provides a promising approach to enhance efficacy of single-agent-targeted treatments. Moreover, combination drug therapies might overcome the emergence of drug resistance. Here, we performed a focused flow cytometry-based drug synergy screen and identified a novel synergistic interaction between GLUT1-mediated glucose transport and the cell-cycle checkpoint kinases ATR and CHK1. Combined inhibition of CHK1/GLUT1 or ATR/GLUT1 robustly induced apoptosis, particularly in

Cdkn1a, which encodes p21, functions as a major route for p53-mediated cell-cycle arrest. However, the consequence of Cdkn1a gene dosage on tumor suppression has not been systematically investigated. Here, we employed BAC transgenesis to generate a Cdkn1a

The α2δ‐1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of α2δ‐1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/α2δ-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of α2δ-1 (α2δ-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing α2δ‐1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α2δ‐1 and suggest that gabapentin does not directly target TSP/α2δ-1 interaction to alleviate neuropathic pain.

The antimetabolite 5-fluorouracil is a widely used chemotherapeutic for the treatment of several solid cancers. However, resistance to 5-fluorouracil remains a major drawback in its clinical use. In this study we report that treatment of HeLa cells with 5-fluorouracil resulted in de novo assembly of stress granules. Moreover, we revealed that stress granule assembly under stress conditions as well as disassembly is altered in cells treated with 5-fluorouracil. Notably, we discovered that RACK1, a protein mediating cell survival and apoptosis, is a component of 5-fluorouracil-induced stress granules. To explore the mode of action of 5-fluorouracil accountable for de novo stress granule assembly, we analyzed 5-fluorouracil metabolites and noticed that stress granule assembly is caused by RNA, not DNA incorporating 5-fluorouracil metabolites. Interestingly, we observed that other RNA incorporating drugs also cause assembly of stress granules. Thus, our results suggest that incorporation of chemotherapeutics into RNA may result in stress granule assembly with potential significance in chemoresistance.

Background The ion channel TRPV1 is mainly expressed in small diameter dorsal root ganglion (DRG) neurons, which are involved in the sensation of acute noxious thermal and chemical stimuli. Direct modifications of the channel by diverse signalling events have been intensively investigated, but little is known about the composition of modulating macromolecular TRPV1 signalling complexes. Here, we hypothesize that the novel adaptor protein ankyrin-rich membrane spanning protein/kinase D interacting substrate (ARMS) interacts with TRPV1 and modulates its function in rodent DRG neurons. Methods We used immunohistochemistry, electrophysiology, microfluorimetry and immunoprecipitation experiments to investigate TRPV1 and ARMS interactions in DRG neurons and transfected cells. Results We found that TRPV1 and ARMS are co-expressed in a subpopulation of DRG neurons. ARMS sensitizes TRPV1 towards capsaicin in transfected HEK 293 cells and in mouse DRG neurons in a PKA-dependent manner. Using a combination of functional imaging and immunocytochemistry, we show that the magnitude of the capsaicin response in DRG neurons depends not only on TRPV1 expression, but on the co-expression of ARMS alongside TRPV1. Conclusion These data indicate that ARMS is an important component of the signalling complex regulating the sensitivity of TRPV1. Significance The study identifies ARMS as an important component of the signalling complex regulating the sensitivity of excitatory ion channels (TRPV1) in peripheral sensory neurons (DRG neurons) and transfected cells.

Genetic loss of the voltage-gated sodium channel Na(v)1.7 (Nav1.7(-/-)) results in lifelong insensitivity to pain in mice and humans. One underlying cause is an increase in the production of endogenous opioids in sensory neurons. We analyzed whether Na(v)1.7 deficiency altered nociceptive heterotrimeric guanine nucleotide–binding protein–coupled receptor (GPCR) signaling, such as initiated by GPCRs that respond to serotonin (pronociceptive) or opioids (antinociceptive), in sensory neurons. We found that the nociceptive neurons of Na(v)1.7 knockout (Na(v)1.7(-/-)) mice, but not of Na(v)1.8 knockout (Na(v)1.8(-/-)) mice, exhibited decreased pro-nociceptive serotonergic signaling through the 5-HT(4) receptors, which are Gαs-coupled GPCRs that stimulate the production of cyclic adenosine monophosphate resulting in protein kinase A (PKA) activity, as well as reduced abundance of the RIIβ regulatory subunit of PKA. Simultaneously, the efficacy of anti-nociceptive opioid signaling mediated by the Gα(i)-coupled mu opioid receptor was increased. Consequently, opioids inhibited more efficiently tetrodotoxin-resistant sodium currents, which are important for pain-initiating neuronal activity in nociceptive neurons. Thus, Na(v)1.7 controls the efficacy and balance of GPCR mediated pro- and antinociceptive intracellular signaling, such that without Na(v)1.7, the balance is shifted toward antinociception, resulting in lifelong endogenous analgesia.

The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies.

Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.Control of MRE11 association with chromatin by UBQLN4 during double-strand break repair influences repair pathway choice and can be dysregulated in tumorigenesis., This work was funded through the Dr. M. and S.G. Adelson Medical Research Foundation, The Israel Science Foundation joint ISF-NSFC Research Program and The Israel Cancer Research Fund (to Y.S.), the German-Israeli Foundation for Research and Development (I-65-412.20-2016 to Y.S. and H.C.R.), the Deutsche Forschungsgemeinschaft (KFO-286-RP2 to H.C.R., KFO-286-CP2 to M.P., JA2439/1-1 to R.D.J., DI1731/2-1 to F.D.), the Else Kröner-Fresenius Stiftung (2014-A06 to H.C.R., 2016_Kolleg.19 to R.D.J.), the Deutsche Krebshilfe (1117240 to H.C.R. and the Mildred-Scheel Professorship to M.P.), the German Ministry of Education and Research (BMBF 01GM1211B to D.W., BMBF e:Med 01ZX1303A and 01ZX1406 to M.P., BMBF e:Med 01ZX1303 and 01ZX1307 to M.F.), and R+D+I grants from the Spanish Ministry of Economy and Competitivity (SAF2013-43255-P and SAF2016-74855-P to P.H.). Y.S. is a Research Professor of the Israel Cancer Research Fund.

Background: The sexually dimorphic differentiation of the bipotential gonad into testis or ovary initiates the sexually dimorphic development of mammals and leads to divergent hormone concentrations between the sexes throughout life. However, despite the fact that anatomic and hormonal differences between the sexes are well described, only a few studies have investigated the manifestation of these differences at the transcriptional level in mammalian somatic tissue. Objective: This review focuses on basic regulatory mechanisms of sex-specific gene expression and examines recent gene expression profiling studies to outline basic differences between the sexes at the transcriptome level in somatic tissues. Methods: To identify gene expression profiling studies addressing sexually dimorphic gene expression, the PubMed database was searched using the terms sex* and dimorp* and gene expression not drosophila not elegans. Abstracts of all identified publications were screened for studies explicitly using microarrays to identify sex differences in somatic tissues of rodents or humans. The search was restricted to English-language articles published in the past 5 years. Reference lists of identified articles as well as microarray databases (Gene Expression Omnibus and ArrayExpress) were also used. Results: The application of microarray technology has enabled the systematic assessment of sex-biased gene expression on the transcriptome level, indicating that the regulatory pathways underlying sexual differentiation give rise to extensive differences in somatic gene expression across organisms. Conclusion: Sustainable annotation of sex-biased gene expression provides a key to understanding basic physiological differences between healthy males and females as well as those with diseases.

The G protein-coupled receptor 30 (GPR30) has been claimed as an estrogen receptor. However, the literature reports controversial findings and the physiological function of GPR30 is not fully understood yet. Consistent with studies assigning a role of GPR30 in the cardiovascular and metabolic systems, GPR30 expression has been reported in small arterial vessels, pancreas and chief gastric cells of the stomach. Therefore, we hypothesized a role of GPR30 in the onset and progression of cardiovascular and metabolic diseases. In order to test our hypothesis, we investigated the effects of a high-fat diet on the metabolic and cardiovascular profiles of Gpr30-deficient mice (GPR30-lacZ mice). We found that GPR30-lacZ female, rather than male, mice had significant lower levels of HDL along with an increase in fat liver accumulation as compared to control mice. However, two indicators of cardiac performance assessed by echocardiography, ejection fraction and fractional shortening were both decreased in an age-dependent manner only in Gpr30-lacZ male mice. Collectively our results point to a potential role of Gpr30 in preserving lipid metabolism and cardiac function in a sex- and age-dependent fashion. (C) 2014 Elsevier B.V. All rights reserved.

Paralogs for several proteins implicated in neurodegenerative disorders have been identified and explored to further facilitate the identification of molecular mechanisms contributing to disease pathogenesis. For the disease- causing protein in spinocerebellar ataxia type 2, ataxin-2, a paralog of unknown function, termed ataxin-2-like, has been described. We discovered that ataxin-2-like associates with known interaction partners of ataxin-2, the RNA helicase DDX6 and the poly(A)-binding protein, and with ataxin-2 itself. Furthermore, we found that ataxin-2-like is a component of stress granules. Interestingly, sole ataxin-2-like overexpression led to the induction of stress granules, while a reduction of stress granules was detected in case of a low ataxin-2-like level. Finally, we observed that overexpression of ataxin-2-like as well as its reduction has an impact on the presence of microscopically visible processing bodies. Thus, our results imply a functional overlap between ataxin-2-like and ataxin-2, and further indicate a role for ataxin-2-like in the regulation of stress granules and processing bodies.

Growth factors such as nerve growth factor and glial cell line-derived neurotrophic factor are known to induce pain sensitization. However, a plethora of other growth factors is released during inflammation and tissue regeneration, and many of them are essential for wound healing. Which wound-healing factors also alter the sensitivity of nociceptive neurons is not well known. We studied the wound-healing factor, basic fibroblast growth factor (bFGF), for its role in pain sensitization. Reverse transcription polymerase chain reaction showed that the receptor of bFGF, FGFR1, is expressed in lumbar rat dorsal root ganglia (DRG). We demonstrated presence of FGFR1 protein in DRG neurons by a recently introduced quantitative automated immunofluorescent microscopic technique. FGFR1 was expressed in all lumbar DRG neurons as quantified by mixture modeling. Corroborating the mRNA and protein expression data, bFGF induced Erk1/2 phosphorylation in nociceptive neurons, which could be blocked by inhibition of FGF receptors. Furthermore, bFGF activated Erk1/2 in a dose- and time-dependent manner. Using single-cell electrophysiological recordings, we found that bFGF treatment of DRG neurons increased the current-density of NaV1.8 channels. Erk1/2 inhibitors abrogated this increase. Importantly, intradermal injection of bFGF in rats induced Erk1/2-dependent mechanical hyperalgesia. Perspective: Analyzing intracellular signaling dynamics in nociceptive neurons has proven to be a powerful approach to identify novel modulators of pain. In addition to describing a new sensitizing factor, our findings indicate the potential to investigate wound-healing factors for their role in nociception.

G-protein-coupled receptor 30 (GPR30) has been reported to act as a membrane-bound estrogen receptor that is involved in the mediation of non-genomic estradiol signalling. In this study, we demonstrated that male, but not female, GPR30-deficient mice suffer from impaired left‑ventricular cardiac function. Left ventricles from male mutant mice were enlarged. There were no malformations in the valves or outflow tract of the heart. Both the contractility and relaxation capacity of the left ventricle were reduced, leading to increased left‑ventricular end-diastolic pressure in GPR30-deficient mice. In conclusion, our data support a role for GPR30 in the gender-specific aspects of heart failure.

The female sex hormone estradiol plays an important role in reproduction, mammary gland development, bone turnover, metabolism, and cardiovascular function. The effects of estradiol are mediated by two classical nuclear receptors, estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). In 2005, G-protein-coupled receptor 30 (GPR30) was claimed to act as a non-classical estrogen receptor that was also activated by the ERalpha and ERbeta antagonists tamoxifen and fulvestrant (ICI 182780). Despite many conflicting results regarding the potential role of GPR30 as an estrogen receptor, the official nomenclature was changed to GPER (G-protein-coupled estrogen receptor). This review revisits the inconsistencies that still exist in the literature and focuses on selected publications that basically address the following two questions: what is the evidence for and against the hypothesis that GPR30 acts as an estrogen receptor? What is the potential in vivo role of GPR30? Thus, in the first part we focus on conflicting results from in vitro studies analysing the subcellular localization of GPR30, its ability to bind (or not to bind) estradiol and to signal (or not to signal) in response to estradiol. In the second part, we discuss the strengths and limitations of four available GPR30 mouse models. We elucidate the potential impact of different targeting strategies on phenotypic diversity.

Multiple reports implicated the function of G protein-coupled receptor (GPR)-30 with nongenomic effects of estrogen, suggesting that GPR30 might be a G-protein coupled estrogen receptor. However, the findings are controversial and the expression pattern of GPR30 on a cell type level as well as its function in vivo remains unclear. Therefore, the objective of this study was to identify cell types that express Gpr30 in vivo by analyzing a mutant mouse model that harbors a lacZ reporter (Gpr30-lacZ) in the Gpr30 locus leading to a partial deletion of the Gpr30 coding sequence. Using this strategy, we identified the following cell types expressing Gpr30: 1) an endothelial cell subpopulation in small arterial vessels of multiple tissues, 2) smooth muscle cells and pericytes in the brain, 3) gastric chief cells in the stomach, 4) neuronal subpopulations in the cortex as well as the polymorph layer of the dentate gyrus, 5) cell populations in the intermediate and anterior lobe of the pituitary gland, and 6) in the medulla of the adrenal gland. In further experiments, we aimed to decipher the function of Gpr30 by analyzing the phenotype of Gpr30-lacZ mice. The body weight as well as fat mass was unchanged in Gpr30-lacZ mice, even if fed with a high-fat diet. Flow cytometric analysis revealed lower frequencies of T cells in both sexes of Gpr30-lacZ mice. Within the T-cell cluster, the amount of CD62L-expressing cells was clearly reduced, suggesting an impaired production of T cells in the thymus of Gpr30-lacZ mice.

The prevalence and clinical manifestation of several cardiovascular diseases vary considerably with sex and age. Thus, a better understanding of the molecular basis of these differences may represent a starting point for an improved gender-specific medicine. Despite the fact that sex-specific differences have been observed in the cardiovascular system of humans and animal models, systematic analyses of sexual dimorphisms at the transcriptional level in the healthy heart are missing. Therefore we performed gene expression profiling on mouse and human cardiac samples of both sexes and young as well as aged individuals and verified our results for a subset of genes using real-time polymerase chain reaction in independent left ventricular samples. To tackle the question whether sex differences are evolutionarily conserved, we also compared sexually dimorphic genes between both species. We found that genes located on sex chromosomes were the most abundant ones among the sexually dimorphic genes. Male-specific expression of Y-linked genes was observed in mouse hearts as well as in the human myocardium (e.g. Ddx3y, Eif2s3y and Jarid1d). Higher expression levels of X-linked genes were detected in female mice for Xist, Timp1 and Car5b and XIST, EIF2S3X and GPM6B in women. Furthermore, genes on autosomal chromosomes encoding cytochromes of the monoxygenase family (e.g. Cyp2b10), carbonic anhydrases (e.g. Car2 and Car3) and natriuretic peptides (e.g. Nppb) were identified with sex- and/or age-specific expression levels. This study underlines the relevance of sex and age as modifiers of cardiac gene expression. Electronic supplementary material The online version of this article (doi:10.1007/s00109-007-0240-z) contains supplementary material, which is available to authorized users.