Pathway-specific Inhibitors Research Articles

Duration of Reduction in Enduring Stress-induced Hyperalgesia via FK506-Binding Protein 51 Inhibition Depends on Timing of Administration Relative to Traumatic Stress Exposure

Chronic pain development is a frequent outcome of severe stressor exposure, with or without tissue injury. Enduring stress-induced hyperalgesia (ESIH) is believed to play a central role, but the precise mechanisms mediating the development of chronic posttraumatic pain, and the time-dependency of these mechanisms, remain poorly understood. Clinical and pre-clinical data suggest that the inhibition of FK506-binding protein 51 (FKBP51), a key stress system regulator, might prevent ESIH. We evaluated whether peritraumatic inhibition of FKBP51 in an animal model of traumatic stress exposure, the single prolonged stress (SPS) model, reversed ESIH evaluated via daily mechanical von Frey testing. FKBP51 inhibition was achieved using SAFit2, a potent and specific small molecule inhibitor of FKBP51, administered to male and female Sprague-Dawley rats via intraperitoneal injection. To assess timing effects, FKBP51 was administered at different times relative to stress (SPS) exposure. SAFit2 administration immediately after SPS produced a complete reversal in ESIH lasting >7d. In contrast, SAFit2 administration 72h following SPS produced only temporary hyperalgesia reversal, and administration 120h following SPS had no effect. Similarly, animals undergoing SPS together with tissue injury (plantar incision) receiving SAFit2 immediately post-surgery developed acute hyperalgesia but recovered by 4d and did not develop ESIH. These data suggest that (1) FKBP51 plays an important, time-dependent role in ESIH pathogenesis, (2) time windows of opportunity may exist to prevent ESIH via FKBP51 inhibition after traumatic stress, with or without tissue injury, and (3) the use of inhibitors of specific pathways may provide new insights into chronic post-traumatic pain development. Grant support from Rita Allen Foundation Pain Scholar Award R01NS118563.

Sézary syndrome patient–derived models allow drug selection for personalized therapy

AbstractCurrent therapeutic approaches for Sézary syndrome (SS) do not achieve a significant improvement in long-term survival of patients, and they are mainly focused on reducing blood tumor burden to improve quality of life. Eradication of SS is hindered by its genetic and molecular heterogeneity. Determining effective and personalized treatments for SS is urgently needed. The present work compiles the current methods for SS patient–derived xenograft (PDX) generation and management to provide new perspectives on treatment for patients with SS. Mononuclear cells were recovered by Ficoll gradient separation from fresh peripheral blood of patients with SS (N = 11). A selected panel of 26 compounds that are inhibitors of the main signaling pathways driving SS pathogenesis, including NF-kB, MAPK, histone deacetylase, mammalian target of rapamycin, or JAK/STAT, was used for in vitro drug sensitivity testing. SS cell viability was evaluated by using the CellTiter-Glo_3D Cell Viability Assay and flow cytometry analysis. We validated one positive hit using SS patient–derived Sézary cells xenotransplanted (PDX) into NOD-SCID-γ mice. In vitro data indicated that primary malignant SS cells all display different sensitivities against specific pathway inhibitors. In vivo validation using SS PDX mostly reproduced the responses to the histone deacetylase inhibitor panobinostat that were observed in vitro. Our investigations revealed the possibility of using high-throughput in vitro testing followed by PDX in vivo validation for selective targeting of SS tumor cells in a patient-specific manner.

Duration of Reduction in Enduring Stress-Induced Hyperalgesia Via FKBP51 Inhibition Depends on Timing of Administration Relative to Traumatic Stress Exposure

Chronic pain development is a frequent outcome of severe stressor exposure, with or without tissue injury. Enduring stress-induced hyperalgesia (ESIH) is believed to play a central role, but the precise mechanisms mediating the development of chronic post-traumatic pain, and the time-dependency of these mechanisms, remain poorly understood. Clinical and preclinical data suggest that the inhibition of FK506-binding protein 51 (FKBP51), a key stress system regulator, might prevent ESIH. We evaluated whether peritraumatic inhibition of FKBP51 in an animal model of traumatic stress exposure, the single prolonged stress (SPS) model, reversed ESIH evaluated via daily mechanical von Frey testing. FKBP51 inhibition was achieved using SAFit2, a potent and specific small molecule inhibitor of FKBP51, administered to male and female Sprague-Dawley rats via intraperitoneal injection. To assess timing effects, FKBP51 was administered at different times relative to stress (SPS) exposure. SAFit2 administration immediately after SPS produced a complete reversal in ESIH lasting >7 days. In contrast, SAFit2 administration 72 hours following SPS produced only temporary hyperalgesia reversal, and administration 120h following SPS had no effect. Similarly, animals undergoing SPS together with tissue injury (plantar incision) receiving SAFit2 immediately post-surgery developed acute hyperalgesia but recovered by 4 days and did not develop ESIH. These data suggest that: 1) FKBP51 plays an important, time-dependent role in ESIH pathogenesis, 2) time windows of opportunity may exist to prevent ESIH via FKBP51 inhibition after traumatic stress, with or without tissue injury, and 3) the use of inhibitors of specific pathways may provide new insights into chronic post-traumatic pain development. PerspectiveThe current work adds to a growing body of literature indicating that FKBP51 inhibition is a highly promising potential treatment strategy for reducing hyperalgesia. In the case of post-traumatic chronic pain, we show that such a treatment strategy would be particularly impactful if administered early after traumatic stress exposure.

LINC00891 regulated by miR-128-3p/GATA2 axis impedes lung cancer cell proliferation, invasion and EMT by inhibiting RhoA pathway.

Long non-coding RNA (lncRNA) LINC00891 knockdown is associated with poor prognosis of lung adenocarcinoma, but the underlying mechanism remains to be further explored. Here, we found that LINC00891 expression is downregulated in lung cancer tissues and cell lines compared with that in adjacent normal tissues and normal lung epithelial cells. LINC00891 overexpression impedes cell proliferation, invasion, migration and epithelial-to-mesenchymal transition (EMT) process in lung cancer cells. Mechanistic research showed that GATA2 directly binds to LINC00891 promoter and transcriptionally regulates LINC00891 expression. Meanwhile, GATA2 was identified as a target of miR-128-3p, and it is negatively regulated by miR-128-3p. Moreover, overexpression of GATA2 suppresses lung cancer cell proliferation, invasion, migration, and EMT process. Furthermore, LINC00891 restrains the RhoA pathway activity, and treatment with CCG-1423 (a specific RhoA pathway inhibitor) antagonizes the promoting effect of LINC00891 knockdown on cell malignant behaviors. Additionally, silencing of LINC00891 promotes xenograft tumor growth, which can be reversed by administration with CCG-1423. In summary, LINC00891 regulated by the miR-128-3p/GATA2 axis restrains lung cancer cell malignant progression and hinders xenograft tumor growth by suppressing the RhoA pathway.

Synovial fluid from end-stage osteoarthritis induces proliferation and fibrosis of articular chondrocytes via MAPK and RhoGTPase signaling

Alterations in the composition of synovial fluid have been associated with adverse effects on cartilage integrity and function. Here, we examined the phenotypic and proliferative behavior of human articular chondrocytes when cultured in vitro for 13 days with synovial fluid derived from end-stage osteoarthritis patients. Chondrocyte proliferation and phenotypical changes induced by osteoarthritic synovial fluid were analyzed using DNA staining, RT-qPCR, immunostainings, and immunoblotting. The molecular mechanisms by which osteoarthritic synovial fluid induced fibrosis and proliferation were studied using a phospho-protein antibody array and luciferase-based transcription factor activity assays. Specific pathway inhibitors were used to probe the involvement of pathways in fibrosis and proliferation. Prolonged stimulation with osteoarthritic synovial fluid sustained chondrocyte proliferation and induced profound phenotypic changes, favoring a fibrotic over a chondrogenic or hypertrophic phenotype. A clear loss of chondrogenic markers at both the transcriptional and protein level was observed, while expression of several fibrosis-associated markers were upregulated over time. Phospho-kinase analysis revealed activation of MAPK and RhoGTPase signaling pathways by osteoarthritic synovial fluid, which was confirmed by elevated transcriptional activity of Elk-1 and SRF. Inhibitor studies revealed that ERK played a central role in the loss of chondrocyte phenotype, while EGFR and downstream mediators p38, JNK and Rac/Cdc42 were essential for fibrosis-associated collagen expression. Finally, we identified EGF signaling as a key activator of chondrocyte proliferation. Osteoarthritic synovial fluid promoted chondrocyte fibrosis and proliferation through EGF receptor activation and downstream MAPK and RhoGTPase signaling.

Antimicrobial peptide derived from insulin-like growth factor-binding protein 5 improves diabetic wound healing.

Impaired keratinocyte functions are major factors that are responsible for delayed diabetic wound healing. In addition to its antimicrobial activity, the antimicrobial peptide derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) activates mast cells and promotes keratinocyte and fibroblast proliferation and migration. However, its effects on diabetic wound healing remain unclear. Human keratinocytes were cultured in normal or high glucose milieus. The production of angiogenic growth factor and cell proliferation and migration were evaluated. Wounds in normal and streptozotocin-induced diabetic mice were monitored and histologically examined. We found that AMP-IBP5 rescued the high glucose-induced attenuation of proliferation and migration as well as the production of angiogenin and vascular endothelial growth factors in keratinocytes. The AMP-IBP5-induced activity was mediated by the epidermal growth factor receptor, signal transducer and activator of transcription 1 and 3, and mitogen-activated protein kinase pathways, as indicated by the inhibitory effects of pathway-specific inhibitors. In vivo, AMP-IBP5 markedly accelerated wound healing, increased the expression of angiogenic factors and promoted vessel formation in both normal and diabetic mice. Overall, the finding that AMP-IBP5 accelerated diabetic wound healing by protecting against glucotoxicity and promoting angiogenesis suggests that AMP-IBP5 might be a potential therapeutic target for treating chronic diabetic wounds.

The specific inhibition of glycerol synthesis and the phosphorylation of a putative Mitogen-Activated Protein Kinase give insight into the mechanism of osmotic sensing in a dinoflagellate symbiont.

Signaling pathways are fundamental for the establishment and maintenance of diverse symbioses. The symbiosis of cnidarians and dinoflagellate algae is the foundation for the ecological success of coral reefs, involving the transfer of photosynthetic products from the symbiont to host. However, signal transduction pathways for this symbiosis remain uncharacterized. Cultured and natural cnidarian symbionts can produce glycerol, one of the main translocated photosynthates. Here, we investigate whether a signal transduction pathway may be involved in inducing glycerol synthesis in cultured symbionts under an osmotic stress model. We evaluated the effect of specific inhibitors of the main transduction pathways, p38, JNK, and ERK 1/2 in Brevolium minutum, the symbiont of the Aiptasia model system. We found that glycerol production and the specific activity of the enzyme Gpdh were selectively inhibited by a p38 Mitogen-Activated Protein Kinase (MAPK) inhibitor. Additionally, the phosphorylation of a putative p38-like protein was rapidly detected. Finally, we studied the presence of each of the components of the p38 MAPK pathway in silico in genomes and transcriptomes reported up to date for different symbiont types. We propose a model for the arrangement of this pathway in the family of dinoflagellate symbionts known as Symbiodiniaceae.

BcSDR1 is involved in regulation of glucose transport and cAMP and MAPK signaling pathways in Botrytis cinerea

Botrytis cinerea is a typical necrotrophic pathogenic fungus that causes severe diseases in a wide range of plant species, leading to significant economic losses. Our previous study showed that BcSDR1 positively regulates growth, development, and pathogenicity of B. cinerea. However, the regulation mechanism of BcSDR1 and the relationship between BcSDR1 and cAMP and MAPK signaling pathways are not well understood. In this study, transcriptome data showed that BcSDR1 is involved in glucose transmembrane transport, signal transduction, secondary metabolism, and other biological processes. BcSDR1 mutant (BCt41) showed remarkably weak sensitivity to cAMP and MAPK signaling pathways specific inhibitors, SQ22536 and U0126, and significantly decreased cAMP content. The key genes of cAMP and MAPK signaling pathways, BcGB1, BcBTP1, BcBOS1, BcRAS1, and BcBMP3 were significantly upregulated, whereas BcPLC1, BcBCG1, BcCDC4, BcSAK1, BcATF1, and BcBAP1 were significantly downregulated (P<0.05). BcSDR1 was obviously upregulated in BcBCG2, BcBCG3, BcPKA1, and BcPKAR RNA interference (RNAi) mutants, but significantly downregulated in BcPKA2, BcBMP1, and BcBMP3 RNAi mutants. Thus, BcBCG2, BcBCG3, BcPKA1, and BcPKAR negatively regulate BcSDR1 expression, whereas BcPKA2, BcBMP1, and BcBMP3 positively regulate BcSDR1 expression.

LIM Mineralization Protein-1 Enhances the Committed Differentiation of Dental Pulp Stem Cells through the ERK1/2 and p38 MAPK Pathways and BMP Signaling.

Tissue regeneration is the preferred treatment for dentin and bone tissue defects. Dental pulp stem cells (DPSCs) have been extensively studied for their use in tissue regeneration, including the regeneration of dentin and bone tissue. LIM mineralization protein-1 (LMP-1) is an intracellular non-secretory protein that plays a positive regulatory role in the mineralization process. In this study, an LMP-1-induced DPSCs model was used to explore the effect of LMP-1 on the proliferation and odonto/osteogenic differentiation of DPSCs, as well as the underlying mechanisms. As indicated by the cell counting kit-8 assay, the results showed that LMP-1 did not affect the proliferation of DPSCs. Overexpression of LMP-1 significantly promoted the committed differentiation of DPSCs and vice versa, as shown by alkaline phosphatase activity assay, alizarin red staining, western blot assay, quantitative real-time polymerase chain reaction assay, and in vivo mineralized tissue formation assay. Furthermore, inhibiting the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) pathways using specific pathway inhibitors showed that the ERK1/2 and p38 MAPK pathways attenuated the differentiation of DPSCs. Besides, the expression of BMP signaling pathway components were also determined, which suggested that LMP-1 could activate BMP-2/Smad1/5 signaling pathway. Our results not only indicated the underlying mechanism of LMP-1 treated DPSCs but also provided valuable insight into therapeutic strategies in regenerative medicine.

Methylation-reprogrammed CHRM3 results in vascular dysfunction in the human umbilical vein following IVF-ET†.

Assisted reproductive technology (ART) has been used globally among infertile couples. However, many epidemiological investigations have indicated that ART is associated with a range of long-term adverse health outcomes in offspring, including cardiovascular disease, obesity, and increased plasma lipid levels. Until now, direct evidence has been limited regarding the pathological changes in vascular function in fetuses with ART. In this study, human umbilical cords were collected from healthy normal pregnancies and in vitro fertilization and embryo transfer (IVF-ET) pregnancies. Vascular functional studies involving acetylcholine (ACh), antagonists of its specific receptors, and L-type calcium channel/PKC-MLC20 phosphorylation pathway specific inhibitors were conducted. Quantitative real-time PCR, Western blotting, and methylation analyses were performed on umbilical vein samples. We found that the umbilical vein constriction induced by ACh in the IVF-ET group was significantly attenuated compared with that in the healthy normal pregnancy group, which was not only associated with the hypermethylation of ACh muscarinic receptor subtype 3 (CHRM3) and decreased expression of CHRM3, PKCβ, and CaV1.2, but was also related to the reduced phosphorylation of MLC20. This study revealed that the hypermethylation of CHRM3, leading to a reduction in CHRM3 expression and downregulation of the CaV1.2/PKC-MLC20 phosphorylation pathway, was responsible for the decreased sensitivity to ACh observed in the umbilical vein under IVF-ET conditions. The hypermethylation of CHRM3 caused by IVF-ET might play an important role in altered vasoconstriction and impact cardiovascular systems in the long run.

Polypeptides IGF-1C and P24 synergistically promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro through the p38 and JNK signaling pathways.

Insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein 2 (BMP-2) both promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs). IGF-1C, the C domain peptide of IGF-1, and P24, a BMP-2-derived peptide, both have similar biological activities as their parent growth factors. This study aimed to investigate the effects and mechanisms of polypeptides IGF-1C and P24 on the osteogenic differentiation of BMSCs. The optimum concentrations of IGF-IC and P24 were explored. The effects of the two polypeptides on BMSC proliferation and osteogenic differentiation were examined using a CCK-8 assay, flow cytometry, alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red S staining, qPCR, and Western blotting. In addition, specific pathway inhibitors were utilized to explore whether the p38 and JNK pathways were involved in this process. The optimal concentration of both polypeptides was 50μg/ml. IGF-1C and P24 synergistically promoted BMSC proliferation, increased ALP activity and calcified nodule formation, upregulated the mRNA and protein levels of Osx, Runx2, Ocn, Opn, and Col1a1, and improved the phosphorylation levels of p38 and JNK proteins. Inhibition of the pathways significantly reduced p38 and JNK activation and blocked Runx2 expression while inhibiting ALP activity and calcified nodule formation. These findings suggest that IGF-1C and P24 synergistically promote the osteogenesis of BMSCs through activation of the p38 and JNK signaling pathways.

Gastrodin exerts protective effects in reactive TNC1 astrocytes via regulation of the Notch signaling pathway.

BackgroundGastrodin (GAS) has been proven to play a therapeutic role in a variety of neurological diseases by affecting activated astrocytes, however, the underlying mechanisms have not been fully illustrated. This study aimed to investigate if GAS exerts the neuroprotective effect through regulating the Notch signaling pathway involved in reactive astrocytes.MethodsAstrocyte cell lines (TNC1 cells) were cultured in vitro. The hypoxic-ischemic cell model was prepared using the oxygen-glucose deprivation (OGD) method, GAS’s pretreatment concentration was 0.34 mM, intervention for 1 hour. Cell counting kit-8 (CCK-8) assay, Transwell migration assay, immunofluorescent staining (double staining), and Western blotting were used to observe the effects of OGD or GAS interference on the function of astrocytes, and the changes of key protein expressions in the Notch signaling pathway were analyzed.ResultsGAS had no obvious toxic effect on TNC1 astrocytes under physiological conditions. Following OGD, GAS can not only improve cell viability and migration, but also regulate the production of inflammatory mediators. We also found that OGD significantly increased the expression of key proteins related to the Notch signaling pathway, Notch-1, intracellular Notch receptor domain (NICD), recombining binding protein suppressor of hairless (RBP-JK), transcription factor hairy and enhancer of split-1 (Hes-1) in TNC1 astrocytes, which was significantly inhibited by GAS. In addition, GAS inhibited the OGD-induced expression of TNC1 astrocyte tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β), and enhanced the expression of nutrient factors, including brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1). The Notch signaling pathway specific inhibitor, N-[N-(3,5-Difluorophenacetyl)-1-alany1]-S-phenyglycine t-butylester (DAPT), could significantly enhance the effect of GAS on TNC1 astrocytes after OGD, such as the inhibition of inflammatory factors and the up-regulation of neurotrophic factors.ConclusionsGAS exerts dual effects on astrocytes via regulation of the Notch signaling pathway. We found that it could inhibit the pro-inflammatory factors mediated by astrocytes, and also promote the secretion of neurotrophic factors by astrocytes. These results provide a new biological mechanism for the treatment of neuroinflammatory diseases by GAS.

Shiga Toxin 2a Induces NETosis via NOX-Dependent Pathway.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is the most common cause of hemolytic uremic syndrome (HUS), one of the main causes of acute kidney injury in children. Stx plays an important role in endothelium damage and pathogenesis of STEC-HUS. However, the effects of Stx on neutrophils and neutrophil extracellular trap (NET) formation are not well understood. In this study, we investigated how Stx2a affects NET formation and NETotic pathways (NADPH or NOX-dependent and -independent) using neutrophils isolated from healthy donors and patients with STEC-HUS, during the acute and recovery phase of the disease. Stx2a dose-dependently induced NETosis in neutrophils isolated from both healthy controls and STEC-HUS patients. NETosis kinetics and mechanistic data with pathway-specific inhibitors including diphenyleneiodonium (DPI)-, ERK-, and P38-inhibitors showed that Stx2a-induced NETosis via the NOX-dependent pathway. Neutrophils from STEC-HUS patients in the acute phase showed less ROS and NETs formation compared to neutrophils of the recovery phase of the disease and in healthy controls. NETs induced by Stx2a may lead to the activation of endothelial cells, which might contribute to the manifestation of thrombotic microangiopathy in STEC-HUS.

The Antimicrobial Peptide Human β-Defensin-3 Accelerates Wound Healing by Promoting Angiogenesis, Cell Migration, and Proliferation Through the FGFR/JAK2/STAT3 Signaling Pathway.

In addition to its antimicrobial activity, the skin-derived antimicrobial peptide human β-defensin-3 (hBD-3) promotes keratinocyte proliferation and migration to initiate the wound healing process; however, its effects on fibroblasts, which are the major cell type responsible for wound healing, remain unclear. We investigated the role of hBD-3 in cell migration, proliferation and production of angiogenic growth factors in human fibroblasts and evaluated the in vivo effect of hBD-3 on promoting wound healing and angiogenesis. Following hBD-3 treatment, the mouse wounds healed faster and showed accumulation of neutrophils and macrophages in the early phase of wound healing and reduction of these phagocytes 4 days later. hBD-3-treated wounds also displayed an increased number of fibroblasts and newly formed vessels compared to those of the control mice. Furthermore, the expression of various angiogenic growth factors was increased in the hBD-3-treated wounds. Additionally, in vitro studies demonstrated that hBD-3 enhanced the secretion of angiogenic growth factors such as fibroblast growth factor, platelet-derived growth factor and vascular endothelial growth factor and induced the migration and proliferation of human fibroblasts. The hBD-3-mediated activation of fibroblasts involves the fibroblast growth factor receptor 1 (FGFR1)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathways, as evidenced by the inhibitory effects of pathway-specific inhibitors. We indeed confirmed that hBD-3 enhanced the phosphorylation of FGFR1, JAK2 and STAT3. Collectively, the current study provides novel evidence that hBD-3 might be a potential candidate for the treatment of wounds through its ability to promote wound healing, angiogenesis and fibroblast activation.

Excessive endoplasmic reticulum stress drives aberrant mouse trophoblast differentiation and placental development leading to pregnancy loss.

Endoplasmic reticulum (ER) stress promotes placental dysmorphogenesis and is associated with poor pregnancy outcomes. We show that unfolded protein response signalling pathways located in the ER drive differentiation of mouse trophoblast stem cells into trophoblast subtypes involved in development of the placental labyrinth zone and trophoblast invasion. In a mouse model of chronic ER stress (Eif2s1tm1RjK ), higher ER stress in homozygous blastocysts is accompanied by reduced trophectoderm cell number and developmental delay and also is associated with an increased incidence of early pregnancy loss. Administration of the chemical chaperone, tauroursodeoxycholic acid, to Eif2s1+/tm1RjK heterozygous females during pregnancy alleviated ER stress in the mutant placenta, restored normal trophoblast populations and reduced the frequency of early pregnancy loss. Our results suggest that alleviation of intrauterine ER stress could provide a potential therapeutic target to improve pregnancy outcome in women with pre-gestational metabolic or gynaecological conditions. Women with pre-gestational health conditions (e.g. obesity, diabetes) or gynaecological problems (e.g. endometriosis) are at increased risk of adverse pregnancy outcomes including miscarriage, pre-eclampsia and fetal growth restriction. Increasing evidence suggests that unfavourable intrauterine conditions leading to poor implantation and/or defective placentation are a possible causative factor. The endoplasmic reticulum (ER) unfolded protein response (UPRER ) signalling pathways are a convergence point of various physiological stress stimuli that can be triggered by an unfavourable intrauterine environment. Therefore, we explored the impact of ER stress on mouse trophoblast differentiation in vitro, mouse blastocyst formation and early placenta development in the Eif2s1tm1RjK mutant mouse model of chronic ER stress. Chemically-manipulated ER stress or activation of UPRER pathways in a mouse trophoblast stem cell line promoted lineage-specific differentiation. Co-treatment with specific UPRER pathway inhibitors rescued this effect. Although the inner cell mass was unaffected, the trophectoderm of homozygous Eif2s1tm1RjK blastocysts exhibited ER stress associated with a reduced cell number. Furthermore, one-third of Eif2s1tm1RjK homozygous blastocysts exhibited severe developmental defects. We have previously reported a reduced trophoblast population and premature trophoblast differentiation in Eif2s1tm1RjK homozygous placentas at mid-gestation. Here, we demonstrate that treatment of Eif2s1+/tm1RjK heterozygous pregnant females with the chemical chaperone tauroursodeoxycholic acid alleviated ER stress, restored the trophoblast population and reduced the frequency of embryonic lethality. Our data suggest that therapeutic targeting of ER stress may improve pregnancy outcome in women with pre-gestational metabolic or gynaecological conditions.

Abstract 2335: Targeting MYC-enhanced glycolysis in small cell lung cancer

Abstract Small cell lung cancer (SCLC) accounts for approximately 15% of lung cancer cases with a poor five year survival rate of 6%. The National Cancer Institute has designated SCLC as one of two “recalcitrant” cancers and urges translational research to advance treatment options. In our effort to maximize treatment benefits, we have aimed our investigation at uncovering metabolic differences contributing to disease progression that may be targeted through specific pathway inhibition. Although metabolism is relatively unstudied in SCLC, metabolic reprogramming is recognized as a hallmark of cancer and can be regulated by several different mechanisms. Notably, the transcription factor MYC is overexpressed in 30% SCLC tumors and is known to modulate the balance between two major pathways of metabolism: glycolysis and mitochondrial respiration. While mitochondrial respiration is the preferred source of energy production in terminally differentiated cells, many cancer cells switch to become predominantly glycolytic in an effort to generate energy quickly and produce biomolecules and electron carriers required for proliferation—a phenomena known as the Warburg Effect. Since MYC is expressed in only a subset of highly aggressive SCLC, we applied bimodal separation to both patient and cell line SCLC datasets to establish defined MYC subsets (MYCLow; MYCHigh) and performed gene ontology pathway analysis to genes that were significantly upregulated among the MYC-expressing samples. This revealed that 37% of significantly upregulated genes were linked to metabolic processes and further investigation showed many of those genes were linked to the glycolysis pathway. We also confirmed upregulation of protein expression through reverse phase protein array (RPPA). With glycolysis proving to be increased in MYCHigh samples, we simultaneously characterized pathway utilization in cell lines at baseline and in the presence of a potent glycolysis inhibitor. MYCHigh cell lines exhibited greater glucose consumption and lactate secretion, which was significantly decreased by glycolytic inhibition. Likewise, mitochondrial analysis revealed lower oxygen consumption and ATP production that were further reduced in the presence of the glycolysis inhibitor. Although mitochondrial density was unchanged regardless of MYC expression or glycolytic inhibition, reactive oxygen species (ROS) generation was greatly enhanced in the MYCHigh subset upon suppression of glycolysis. Lastly, SCLC xenografts derived from a MYCHigh SCLC cell line showed significantly slower tumor growth whereas MYCLow derived xenografts exhibited no significant difference in tumor growth. Together, these data provide evidence of metabolic differences among SCLC subsets such that MYC expression induces reliance on glycolysis, which can be targeted for therapeutic intervention. Citation Format: Kasey R. Cargill, C. Allison Stewart, Elizabeth M. Park, Robert J. Cardnell, You Hong Fan, Qi Wang, Lixia Diao, Wai Kin Chan, Philip L. Lorenzi, Jing Wang, Lauren A. Byers. Targeting MYC-enhanced glycolysis in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2335.

Abstract 1081: Genome-wide CRISPR screens reveal Hippo pathway activation as a resistance mechanism in BRAF mutant colon cancer

Abstract BRAF V600E mutations occur in a subset of colon cancers. These are typically resistant to chemotherapy and are associated with a poor outcome. Combination treatment with BRAF and EGFR inhibitors is superior to standard chemotherapy and has recently received FDA approval, however the early emergence of drug resistance is a significant clinical problem. Clinical studies of resistant patients have identified mutations and amplifications in the MAPK pathway as important resistance drivers. To identify novel non-MAPK dependent resistance mechanisms, genome-wide CRISPR/Cas9 knockout screens were performed to identify genes causing resistance to a combination BRAF/MEK/EGFR inhibitor regimen in the BRAF mutant HT29 and LS411N colon cancer cell lines. A number of strong resistance hits were identified but importantly, only 3 genes (CSK, ARID1A and STK11) were detected as significantly enriched in both cell lines screened. Two of these, CSK and ARID1A, have been shown to play a role in activation of the Hippo signaling pathway. The generation of CSK knockout colon cell lines confirmed resistance to BRAF/MEK/EGFR inhibition both in vitro and in vivo using xenograft models. Furthermore, since CSK is a negative regulator of SRC, re-sensitisation of resistant BRAF mutant CSK knockout cells was achieved by adding Src inhibitors (Dasatinib and Saracatinib) to the combination therapy. Nuclear localisation of the transcription factors YAP1 and WWTR1 and binding to TEAD family members are required for Hippo pathway activation and we confirmed significantly increased nuclear YAP1/WWTR1 in CSK knockout cells. Furthermore, YAP1/WWTR1 nuclear localisation in these cells was reversed by treatment with Src inhibitors. Novel pharmacological TEAD inhibitors have recently been developed and will also be used to confirm that resistance can be overcome by specifically targeting the Hippo pathway. Expression profiling of CSK knockout cells revealed significant enrichment of pathways associated with Hippo signaling. Genes involved in regulating Hippo pathway activation were also identified as CRISPR screen resistance hits in both lung and head and neck cancer cell lines, suggesting that this may be an important mechanism of resistance among other tumour types and not limited to the colon. Here we show that activation of the Hippo pathway is a potential MAPK-independent resistance mechanism in BRAF mutant colon cancer, readily reversible by rational pharmacological targeting. Given the development of specific Hippo pathway inhibitors and plans for their use in clinical trials, activation of Hippo signaling should be considered in resistant BRAF mutant colon cancer where alterations in the MAPK pathway are not detected. Citation Format: Jonathan S. Brammeld, Hannah Thorpe, Mar Arias Garcia, Stacey Price, Jamie Young, Matthias Pfeifer, Barbara Lupo, Kosuke Yusa, Livio Trusolino, Mathew Garnett, Andrea Bertotti, Chris Bakal, Ultan McDermott. Genome-wide CRISPR screens reveal Hippo pathway activation as a resistance mechanism in BRAF mutant colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1081.

In Vitro Characterization of the Innate Immune Pathways Engaged by Live and Inactivated Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) infection can lead to inflammation of the central nervous system. The disease can be effectively prevented by whole inactivated virus vaccines. Here, we investigated the innate immune profile induced in vitro by the antigen component of the vaccines, inactivated TBEV (I-TBEV), to gain insights into the mechanism of action of the TBE vaccine as compared to the live virus. To this end, we exposed human peripheral blood mononuclear cells (PBMCs) to inactivated and live TBEV and assessed cellular responses by RNA sequencing. Both inactivated and live TBEV significantly induced an interferon-dominated gene signature and an increased RIG-I-like receptor (RLR) expression. Using pathway-specific inhibitors, we assessed the involvement of pattern recognition receptors in the sensing of inactivated or live TBEV. Only RLR pathway inhibition significantly suppressed the downstream cascade induced by I-TBEV, while responses to the replicating virus were impacted by the inhibition of RIG-I-like, as well as Toll-like, receptors. Our results show that inactivated and live TBEV predominantly engaged an interferon response in our in vitro PBMC platform, and indicate RLRs as the main pattern recognition receptors involved in I-TBEV sensing.

Discovery of an alternative pathway of peptidoglycan biosynthesis: A new target for pathway specific inhibitors.

Peptidoglycan in bacterial cell walls is a biopolymer consisting of sugars and amino acids and plays important role in maintaining cell integrity from the environment. Its biosynthesis is a major target for antibiotics and the genes and enzymes involved in the biosynthetic pathway have been well studied. However, we recently identified an alternative pathway in the early stage of peptidoglycan biosynthesis in Xanthomonas oryzae, a plant pathogen causing bacterial blight disease of rice. The distribution of the alternative pathway is limited to relatively few bacterial genera that contain many pathogenic species, including Xylella and Stenotrophomonas, besides Xanthomonas. Thus, the alternative pathway is an attractive target for the development of narrow-spectrum antibiotics specific to pathogens. In this minireview, we summarize the discovery of the alternative pathway and identification of its specific inhibitors.

Extracellular succinate hyperpolarizes M2 macrophages through SUCNR1/GPR91-mediated Gq signaling.

Succinate functions both as a classical TCA cycle metabolite and an extracellular metabolic stress signal sensed by the mainly Gi-coupled succinate receptor SUCNR1. In the present study, we characterize and compare effects and signaling pathways activated by succinate and both classes of non-metabolite SUCNR1 agonists. By use of specific receptor and pathway inhibitors, rescue in G-protein-depleted cells and monitoring of receptor G protein activation by BRET, we identify Gq rather than Gi signaling to be responsible for SUCNR1-mediated effects on basic transcriptional regulation. Importantly, in primary human M2 macrophages, in which SUCNR1 is highly expressed, we demonstrate that physiological concentrations of extracellular succinate act through SUCNR1-activated Gq signaling to efficiently regulate transcription of immune function genes in a manner that hyperpolarizes their M2 versus M1 phenotype. Thus, sensing of stress-induced extracellular succinate by SUCNR1 is an important transcriptional regulator in human M2 macrophages through Gq signaling.