Gene Depletion Research Articles

Sphingosine kinase 2 is a negative regulator of inflammatory macrophage activation

Sphingosine kinases (SPHK) generate the sphingolipid sphingosine-1-phosphate, which, among other functions, is a potent regulator of inflammation. While SPHK1 produces S1P to promote inflammatory signaling, the role of SPHK2 is unclear due to divergent findings in studies utilizing gene depletion versus inhibition of catalytic activity. We sought to clarify how SPHK2 affects inflammatory signaling in human macrophages, which are main regulators of inflammation. SPHK2 expression and activity were rapidly decreased within 6 h upon stimulating primary human macrophages with lipopolysaccharide (LPS), but was upregulated after 24 h. At 24 h following LPS stimulation, targeting SPHK2 with the inhibitor ABC294640, a specific siRNA or by using Sphk2−/− mouse peritoneal macrophages increased inflammatory cytokine production. Downregulation of SPHK2 in primary human macrophages within 6 h of LPS treatment was blocked by inhibiting autophagy. SPHK2 overexpression or inhibiting autophagy 6 h after human macrophage activation with LPS suppressed inflammatory cytokine release. Mechanistically, SPHK2 suppressed LPS-triggered NF-κB activation independent of its catalytic activity and prevented increased mitochondrial ROS formation downstream of LPS. In conclusion, SPHK2 is an anti-inflammatory protein in human macrophages that is inversely coupled to inflammatory cytokine production. This needs consideration when targeting SPHK2 with specific inhibitors.

EIF3f depletion impedes mouse embryonic development, reduces adult skeletal muscle mass and amplifies muscle loss during disuse.

In muscular cells, eukaryotic initiation factor subunit f (eIF3f) activates protein synthesis by allowing physical interaction between mechanistic target of rapamycin complex 1 (MTORC1) and ribosomal protein S6 kinase 1 (S6K1), although its physiological role in animals is unknown. A knockout approach suggests that homozygous mice carrying a null mutation of the eIF3f gene fail to develop and consequently die at early embryonic stage, whereas heterozygous mice associated with a partial depletion of eIF3f gene grow normally and are phenotypically indistinguishable from wild-type mice. Heterozygous mice express reduced eIF3f mRNA and protein levels in skeletal muscles and show diminished muscle mass associated with a decrease in the protein synthesis rate and an inhibition of the MTORC1 pathway. During hindlimb immobilization, heterozygous eIF3f mice display an exacerbated immobilization-induced muscle atrophy associated with reduced protein synthesis. These results highlight the essential role of eIF3f during embryonic development and its involvement in muscular homeostasis via protein synthesis regulation. Eukaryotic translation initiation factor 3, subunit F (eIF3f), a component of eIF3 complex, plays an important role in protein synthesis regulation, although its physiological functions are unknown. We generated and analysed mice carrying a null mutation in the eIF3f gene. We showed that homozygous eIF3f knockout fail to develop and that eIF3f-/- embryos die at an early stage of development but after the pre-implantation stage. However, disrupting one eIF3f allele does not affect growth, viability and fertility of heterozygous mice but, instead, reduces eIF3f mRNA and protein levels in all tissues examined. Although heterozygous mice are phenotypically indistinguishable from wild-type mice, they present a diminished body weight and a lean mass reduction associated with normal body size. Interestingly, skeletal muscles are mainly affected and display an altered cell size without modification of fibre number. Skeletal muscles of heterozygous mice show a deficiency in polysome content, a decrease in protein synthesis rate and an inhibition of the mechanistic target of rapamycin (MTOR) pathway. We then studied the effects of hindlimb immobilization that mimic muscle disuse on heterozygous mice aiming to further explore the involvement of eIF3f in protein synthesis. We found that eIF3f partial depletion amplifies muscle atrophy compared to wild-type mice. Mass and cross-sectional area decreases were associated with reduced MTOR pathway activation and protein synthesis rate. Taken together, our data indicate that eIF3f is essential for mice embryonic development and controls adult skeletal muscle mass via protein synthesis regulation in a MTOR-dependent manner.

Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis.

Wolf-Hirschhorn Syndrome (WHS) is a human developmental disorder arising from a hemizygous perturbation, typically a microdeletion, on the short arm of chromosome four. In addition to pronounced intellectual disability, seizures, and delayed growth, WHS presents with a characteristic facial dysmorphism and varying prevalence of microcephaly, micrognathia, cartilage malformation in the ear and nose, and facial asymmetries. These affected craniofacial tissues all derive from a shared embryonic precursor, the cranial neural crest (CNC), inviting the hypothesis that one or more WHS-affected genes may be critical regulators of neural crest development or migration. To explore this, we characterized expression of multiple genes within or immediately proximal to defined WHS critical regions, across the span of craniofacial development in the vertebrate model system Xenopus laevis. This subset of genes, whsc1, whsc2, letm1, and tacc3, are diverse in their currently-elucidated cellular functions; yet we find that their expression demonstrates shared tissue-specific enrichment within the anterior neural tube, migratory neural crest, and later craniofacial structures. We examine the ramifications of this by characterizing craniofacial development and neural crest migration following individual gene depletion. We observe that several WHS-associated genes significantly impact facial patterning, cartilage formation, neural crest motility in vivo and in vitro, and can separately contribute to forebrain scaling. Thus, we have determined that numerous genes within and surrounding the defined WHS critical regions potently impact craniofacial patterning, suggesting their role in WHS presentation may stem from essential functions during neural crest-derived tissue formation.

Cell-intrinsic depletion of Aml1-ETO-expressing pre-leukemic hematopoietic stem cells by K-Ras activating mutation.

Somatic mutations in acute myeloid leukemia are acquired sequentially and hierarchically. First, pre-leukemic mutations, such as t(8;21) that encodes AML1-ETO, are acquired within the hematopoietic stem cell (HSC) compartment, while signaling pathway mutations, including KRAS activating mutations, are late events acquired during transformation of leukemic progenitor cells and are rarely detectable in HSC. This raises the possibility that signaling pathway mutations are detrimental to clonal expansion of pre-leukemic HSC. To address this hypothesis, we used conditional genetics to introduce Aml1-ETO and K-RasG12D into murine HSC, either individually or in combination. In the absence of activated Ras, Aml1-ETO-expressing HSC conferred a competitive advantage. However, activated K-Ras had a marked detrimental effect on Aml1-ETO-expressing HSC, leading to loss of both phenotypic and functional HSC. Cell cycle analysis revealed a loss of quiescence in HSC co-expressing Aml1-ETO and K-RasG12D, accompanied by an enrichment in E2F and Myc target gene expression and depletion of HSC self-renewal-associated gene expression. These findings provide a mechanistic basis for the observed absence of KRAS signaling mutations in the pre-malignant HSC compartment.

JUND regulates pancreatic β cell survival during metabolic stress

ObjectiveIn type 2 diabetes (T2D), oxidative stress contributes to the dysfunction and loss of pancreatic β cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation; however, the genes regulated at the level of translation are often overlooked due to the convenience of RNA sequencing technologies. Our goal is to investigate translational regulation in β cells as a means to uncover novel factors and pathways pertinent to cellular adaptation and survival during T2D-associated conditions. MethodsTranslating ribosome affinity purification (TRAP) followed by RNA-seq or RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of shRNAs to primary mouse islets or CRISPR-Cas9 to Min6 cells. Oxidative stress and apoptosis were measured in primary islets using cell-permeable dyes with fluorescence readouts of oxidation and activated cleaved caspase-3 and-7, respectively. Gene expression was assessed by RNA-seq, RT-qPCR, and western blot. ChIP-qPCR was used to determine chromatin enrichment. ResultsTRAP-seq in a PDX1-deficiency model of β cell dysfunction uncovered a cohort of genes regulated at the level of mRNA translation, including the transcription factor JUND. Using a panel of diabetes-associated stressors, JUND was found to be upregulated in mouse islets cultured with high concentrations of glucose and free fatty acid, but not after treatment with hydrogen peroxide or thapsigargin. This induction of JUND could be attributed to increased mRNA translation. JUND was also upregulated in islets from diabetic db/db mice and in human islets treated with high glucose and free fatty acid. Depletion of JUND in primary islets reduced oxidative stress and apoptosis in β cells during metabolic stress. Transcriptome assessment identified a cohort of genes, including pro-oxidant and pro-inflammatory genes, regulated by JUND that are commonly dysregulated in models of β cell dysfunction, consistent with a maladaptive role for JUND in islets. ConclusionsA translation-centric approach uncovered JUND as a stress-responsive factor in β cells that contributes to redox imbalance and apoptosis during pathophysiologically relevant stress.

Anlotinib inhibits synovial sarcoma by targeting GINS1: a novel downstream target oncogene in progression of synovial sarcoma.

Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma with a poor prognosis owing to its resistance to radiation and chemotherapy. Thus, novel therapeutic strategies for SS are urgently required. Anlotinib, a new oral tyrosine kinase inhibitor, is designed to primarily inhibit multi-targets in vasculogenesis and angiogenesis. This study was designed to characterize its antitumor efficacy and possible mechanism in patients with advanced refractory synovial sarcoma. Anlotinib's antitumor effect was evaluated in vivo and vitro. Downstream targets of anlotinib in treating synovial sarcoma were analyzed through microarray assay. Cell proliferation and apoptosis analyses were performed to evaluate the impact of candidate downstream gene depletion in synovial sarcoma cells. Microarray assay were carried out to investigate potential signal network related with candidate downstream gene. Anlotinib significantly suppresses synovial sarcoma proliferation in PDTX model and cell lines. Additionally, GINS1 (also named as PSF1, Partner of SLD Five 1), rather than other conventional gene target, was demonstrated to be a vital target of anlotinib's antitumor effect in synovial sarcoma through microarray assay. Expression of GINS1 was remarkably higher in synovial sarcoma tumor samples and related with poor outcome. Knockdown of GINS1 expression could remarkably inhibit proliferation and promote apoptosis in vitro. Meanwhile, through microarray assay, CITED2, EGR1, SGK1 and SPP1 were identified and further validated by qPCR/WB as downstream targets of GINS1. Anlotinib might suppress proliferation of SS through a novel downstream GINS1-regulated network which plays a vital function in SS proliferation and also demonstrated that targeting the GINS1-regulated signal pathway could be a potential strategy for management of SS.

Manipulation of the microbiota to eradicate multidrug-resistant Enterobacteriaceae from the human intestinal tract

Manipulation of the microbiota to eradicate multidrug-resistant Enterobacteriaceae from the human intestinal tract

The non-canonical SMC protein SmcHD1 antagonises TAD formation and compartmentalisation on the inactive X chromosome

The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), A/B compartments and formation of two mega-domains. Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including A/B compartments and partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred following SmcHD1 knockout in a somatic cell model, but in this case, independent of Xi gene derepression. We conclude that SmcHD1 is a key factor in defining the unique chromosome architecture of Xi.

Theanine attenuates memory impairments induced by klotho gene depletion in mice.

Theanine (γ-glutamylethylamide), an amino acid in tea, is a putative neuroprotective and antioxidant compound capable of improving lifespan and cognitive function. Because we previously reported cognitive dysfunction in klotho mutant mice via down-regulation of janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3), M1 muscarinic cholinergic receptor (M1 mAChR), and ERK signaling, we, therefore, investigated whether self-administration of theanine affects memory dysfunction in response to klotho gene depletion in mice, and whether theanine modulates the JAK2/STAT3, M1 mAChR, and ERK signaling network. Theanine significantly attenuated memory impairments in klotho mutant mice. Moreover, theanine self-administration significantly attenuated inhibitions of JAK2/STAT3 phosphorylation, M1 mAChR expression, and ERK1/2 phosphorylation in the hippocampus of klotho mutant mice. Consistently, AG490, a JAK2/STAT3 inhibitor, dicyclomine, an M1 mAChR antagonist, or U0126, an ERK1/2 inhibitor, significantly counteracted theanine-induced attenuation of memory impairment induced by klotho gene depletion in mice. Our study suggests that theanine attenuates memory impairments in a genetic aging model via up-regulation of JAK2/STAT3, M1 mAChR, and ERK signaling.

Clathrin adaptor GGA1 modulates myogenesis of C2C12 myoblasts.

During myogenesis, myogenic stem cells undergo several sequential events, including cell division, migration, and cell-cell fusion, leading to the formation of multinuclear myotubes, which are the precursors of myofibers. To understand the molecular mechanisms underlying these complex processes, an RNA interference-based gene depletion approach was used. Golgi associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1), a Golgi-resident monomeric clathrin adaptor, was found to be required for the process of myotube formation in C2C12 cells, a cultured murine myoblast cell line. Gga1 mRNA expression was upregulated during myogenesis, and Gga1 depletion prevented the formation of large multi-nucleated myotubes. Moreover, inhibition of lysosomal proteases in Gga1 knockdown myoblasts increased the amount of insulin receptor, suggesting that GGA1 is involved in the cell surface expression and sorting of the insulin receptor. These results suggested that GGA1 plays a significant role in the formation and maturation of myotubes by targeting the insulin receptor to the cell surface to establish functionally mature myofibers.

Quorum sensing LuxS/autoinducer-2 inhibits Enterococcus faecalis biofilm formation ability.

Objective:To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2.Materials and Methods: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions.Results:LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05).Conclusions:Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.

The Arabidopsis THADA homologue modulates TOR activity and cold acclimation.

Low temperature is one of the most important environmental factors that affect global survival of humans and animals and equally importantly the distribution of plants and crop productivity. Survival of metazoan cells under cold stress requires regulation of the sensor-kinase Target Of Rapamycin (TOR). TOR controls growth of eukaryotic cells by adjusting anabolic and catabolic metabolism. Previous studies identified the Thyroid Adenoma Associated (THADA) gene as the major effect locus by positive selection in the evolution of modern human adapted to cold. Here we investigate the role of THADA in TOR signaling and cold acclimation of plants. We applied BLAST searches and homology modeling to identify the AtTHADA (AT3G55160) in Arabidopsis thaliana as the highly probable orthologue protein. Reverse genetics approaches were combined with immunological detection of TOR activity and metabolite profiling to address the role of the TOR and THADA for growth regulation and cold acclimation. Depletion of the AtTHADA gene caused complete or partial loss of full-length mRNA, respectively, and significant retardation of growth under non-stressed conditions. Furthermore, depletion of AtTHADA caused hypersensitivity towards low-temperatures. Atthada displayed a lowered energy charge. This went along with decreased TOR activity, which offers a molecular explanation for the slow growth phenotype of Atthada. Finally, we used TOR RNAi lines to identify the de-regulation of TOR activity as one determinant for sensitivity towards low-temperatures. Taken together our results provide evidence for a conserved function of THADA in cold acclimation of eukaryotes and suggest that cold acclimation in plants requires regulation of TOR.

Abstract B061: Alternative RNA splicing and prostate cancer aggressiveness

Abstract Prostate cancer (PCa) is the most common cancer in men in the United States. Aggressive pathology, metastasis, and drug resistance of cancer cells are the most important prognostic factors for patients with PCa. African Americans (AAs) suffer disproportionately from PCa, with higher aggressiveness, progression, and mortality. A recently published analysis involving our laboratory reported more than 2,500 alternatively RNA spliced (ARS) genes between AA and white PCa biopsy tissue specimens. Subsequent analysis revealed that, among the race-related ARS genes in PCa tissue, 19 are also ARS in breast, lung, and liver cancer. Several of the genes that are ARS across multiple tumor types are important for cancer aggressiveness, including CD44, FN1, and LMO7. We designed PCR primers to further explore the expression of LMO7 variants in preclinical models of white and AA PCa, and found that a LMO7 variant that skips exons 10, 11, and 12 is predominantly expressed in VCaP (white) and MDA PCA 2b (AA) PCa cell lines. In addition, we detected a unique race-related LMO7 splicing event that excludes exons 10 and 11, but preserves exon 12. This event is detected in MDA PCA 2b cells, but not in VCaP cells. Importantly, both of these LMO7 splicing events are in-frame and are not frameshifts leading to truncated or extended C-terminal proteins. These LMO7 variants translate to proteins of 1297 amino acids (missing 344 amino acids coded by skipped exons 10, 11, and 12) and 1287 amino acids (missing 334 amino acids coded by skipped exons 10 and 11), which differ from the full-length LMO7 variant that translates to a protein of 1631 amino acids. Interestingly, the enrichment of these variants in cancer cells suggests an important regulatory role for the amino acids encoded by the skipped exons. Further work to study the impact of LMO7 variants as well as other candidate RNA splice variants on PCa aggressiveness is ongoing, using gene overexpression, gene depletion, and splicing manipulation by siRNA or CRISPR-cas, followed by assessment of resulting alterations in growth, migration, invasion, and drug sensitivity. In summary, we have identified genes that undergo race-related ARS in multiple tumor types and novel ARS events that may functionally contribute to increased tumor aggressiveness, including that of PCa in AA men. Citation Format: Muthana Al Abo, Steven R. Patierno, Jennifer A. Freedman. Alternative RNA splicing and prostate cancer aggressiveness [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B061.

Phosphate Binder, Ferric Citrate, Attenuates Anemia, Renal Dysfunction, Oxidative Stress, Inflammation, and Fibrosis in 5/6 Nephrectomized CKD Rats.

Chronic kidney disease (CKD) causes anemia and impairs intestinal iron absorption. However, use of the phosphate binder ferric citrate (FC) increases body iron stores and hemoglobin levels in CKD patients. By intensifying oxidative stress and inflammation iron overload resulting from excessive use of intravenous iron can accelerate CKD progression. The present study explored the route of absorption and tissue distribution of iron with FC administration and its effect on renal function, histology, and inflammatory, oxidative, and fibrosis pathways in CKD rats. Male Sprague Dawley rats were randomized to sham-operated control (CTL) group and 5/6 nephrectomized (CKD) groups fed either regular or 4% FC-supplemented diets for 6 weeks. Animals were then sacrificed, and blood and target tissues were harvested and processed. The untreated CKD rats exhibited anemia, hypertension, upregulation of renal tissue inflammatory, oxidative, and fibrotic pathways, impaired nuclear translocation, and downregulation of Nrf2's target gene products and depletion of colonic epithelial tight junction proteins. FC administration raised serum iron, improved anemia, attenuated hyperphosphatemia, partially improved renal function, reduced oxidative stress, inflammation, and fibrosis, and restored colonic epithelial zonula occludens-1 protein abundance. Tissue iron staining detected presence of iron in epithelial cells and subepithelium of colon and in renal proximal tubules. In conclusion ferric citrate administration resulted in modest amelioration of renal function and histology and partial improvements of fibrosis, inflammation, and oxidative stress in the kidney tissues of CKD rats.

Abstract B58: Modeling oncogenic events in breast cancer precursor cells to study cancer evolution and genomic instability

Abstract Introduction: Over recent years, our knowledge of breast cancer biology has greatly improved; however, the mechanisms of tumor initiation and genomic drivers of this disease remain poorly understood. Hormone-negative breast cancers, such as triple-negative breast cancer (TNBC), are specifically challenging to treat, since they are genetically heterogeneous, often aggressive, and lack targetable driver mutation. Most TNBCs (80%) are basal-like (BLBC) and the majority of BLBCs harbor pathogenic mutations in TP53 tumor-suppressor gene (84%), indicating that TP53 plays a critical role in the pathogenesis of this cancer. Other less frequently observed alterations include mutation or epigenetic silencing of BRCA1/2 genes (36%), amplifications of oncogenes (MYC 40%, CCNE1 9%), and mutations or deletions of tumor suppressors (PTEN 35%, RB1 20%). Unfortunately, none of these genetic aberrations is currently druggable, mostly due to the lack of specific inhibitors. However, modeling these oncogenic events in precursor cells will enable us to study cancer evolution and acquisition of genomic instability. Experimental Procedures: We used basal-like mammary epithelial cells, MCF10A and MCF12A, to generate precursor models of TNBC. We overexpressed in these cell lines a hotspot p53 mutation (R175H), which demonstrates a dominant-negative phenotype. We confirmed that ectopic expression of R175H p53MUT impairs the ability of p53WT to properly respond to DNA damage induced by hydroxyurea or etoposide. As a result, cells fail to elevate the expression of p53 downstream targets, such as CDKN1A (cell cycle inhibitor), PUMA, BAX, and MDM2 (apoptosis regulators). Using CRISPR/Cas9-mediated genome editing we further performed depletion of RB1, PTEN, BRCA1, and BRCA2 genes in derived precursor models. Depletion of olfactory receptors OR10A4 and OR2W5 was performed as a control. In addition, we overexpressed S. pyogenes Cas9 endonuclease in these cell lines, in order to utilize them in CRISPR/Cas9-based gene knockout screens. Multiplexed functional analysis of CRISPR/Cas9-induced genomic alterations can be performed with lentiviral gRNA-based libraries in Cas9-expressing cells. All modified cell lines were clonally derived and gene depletion was validated with qRT-PCR and Western blot. Cas9 efficacy was confirmed with doxycycline-inducible RB1 knockout verified by T7 Endonuclease I genotyping assay. We further performed a range of phenotypic assays, such as proliferation, migration, anchorage-independent growth, and genomic instability assays, to measure the gain of tumorigenic properties upon acquisition of different genomic alterations. Summary: We established breast cancer precursor cell line models harboring genomic alterations in tumor suppressor genes commonly altered in TNBC, such as p53, RB1, PTEN, BRCA1, and BRCA2. Conclusions: Studying early transformation events leading to development of TNBC is necessary for understanding the etiology of this disease and identifying therapeutic vulnerabilities of precancerous lesions. To our knowledge, no comprehensive repertoire of breast cancer precursor models has been derived before. Also, ectopic expression of Cas9 in these cell lines makes them suitable for CRISPR/Cas9-based genomic screens to study disease evolution. CRISPR/Cas9 screens represent powerful tools to understand pathogenicity of different mutation events in disease initiation and progression. The use of genomically stable precursor cells in such studies makes the initial identification of putative drivers easier to define, in contrast to cancer cell lines, in which various compensatory pathways have evolved to cope with high genomic instability. Thus, established precursor models can be utilized in large-scale CRISPR/Cas9 screens focused on discovery of therapeutic targets, synthetic lethality, and genomic drivers of TNBC. Citation Format: Justyna Kanska, Kruttika Dabke, Simon A. Gayther. Modeling oncogenic events in breast cancer precursor cells to study cancer evolution and genomic instability [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B58.

Genetic depletion of the Soat2 gene diminishes diet-induced hepatic steatosis and improves glucose tolerance in mice

Genetic depletion of the Soat2 gene diminishes diet-induced hepatic steatosis and improves glucose tolerance in mice

Glycogen Synthase Kinase-3β as a Putative Therapeutic Target for Bipolar Disorder.

Bipolar disorder (BD) is a debilitating mental ailment characterized by recurrent episodes of mania and depression. Primary mood-stabilizing drugs like lithium and valproate alleviate the hypomanic or mild to moderate manic episodes in patients with BD. One of the extensively studied underlying mechanisms for these pharmacological interventions is inhibition of intracellular signaling cascades associated with glycogen synthase kinase-3 beta (GSK-3β), a multi-functional serine-threonine kinase. To summarize the different mechanistic aspects associated with GSK-3β signaling involved in the pathophysiology of BD and highlights drug discovery approaches pursued for the development of GSK-3β inhibition with detailed strength, weakness, opportunity, and threat (SWOT) analysis. In this review, we endeavor to establish the correlation between neuronal GSK-3β inhibition and anti-manic response of different therapeutics used for the treatment of patients with BD. The gene depletion or pharmacological inhibition of GSK-3β reproduces some of the behavioral effects of lithium including reduction of depression- and manic-like behaviors in rodents, which attested the intracellular GSK- 3β inhibition as one of the critical steps in mediating behavioral effect of mood-stabilizers. Furthermore, converging evidence supported the participation of GSK-3β in the regulation of various neurobehavioral functions governed by neurotransmitters dopamine and serotonin. Apart from its crucial involvement in the mechanism of action of mood stabilizers, GSK-3β signaling pathways have also received attention for their role in the effects of psychoactive therapies like antidepressants, antipsychotics, and neurotrophic factors. We anticipate that the GSK-3β could be a druggable target for several incurable neuropsychiatric disorders including BD.

PO-295 Heterogeneity of the alpha5 integrin subunit expression in glioblastoma

IntroductionGlioblastoma (GBM) is the most aggressive primary brain tumour. Treatment failure and recurrence are explained by intratumoral heterogeneity. Our previous results showed that the integrin α5β1, the fibronectin receptor, is implicated in GBM aggressiveness and represents a pertinent therapeutic target. Recently, we observed that its expression was heterogeneous between patient tumours but also between different areas in a given tumour. We hypothesised that this intratumoral heterogeneity may be linked to different glioma initiating cells (GIC).Material and methodsGICs were grown as neurospheres in stem cell medium and their differentiation was induced by serum. We characterised α5β1 expression in 9 GICs cell lines before and after differentiation. Two cell lines were selected and were genetically modified by depletion (CrisprCas9) or transfection of the α5 integrin gene. Different clones were selected expressing or not the integrin. Aggressiveness of polyclonal lines and individual clones was analysed in vitro before and after differentiation (proliferation, migration, evasion from spheroids) and in vivo (orthotopically xenografted cells).Results and discussionsOur results show that α5 integrin is not expressed in stem cell culture conditions. However, α5 expression is induced after differentiation in about half of the cell lines supporting the notion of inter-tumoral heterogeneity of GICs. Interestingly, single cell-derived clone evaluation showed that intra-tumoral GICs heterogeneity also exists. We noticed that when GICs are programmed or forced to express α5 integrin, differentiated cells became more aggressive. Notably, differentiated cells, expressing the integrin, acquired a fibronectin-dependent motility and a proliferative phenotype. The in vivo assays demonstrated that GICs, programmed to express the integrin, were prone to form larger tumours.ConclusionOur data support the hypothesis that some GICs are programmed to express the α5 integrin subunit to form a more aggressive tumour. Further studies will be needed to explore the implication of such heterogeneity in resistance to anti-integrin therapies but also to conventional chemo/radiotherapies.

Abstract 1339: Development of a novel autophagy-inducing multikinase inhibitor for the treatment of castration-resistant prostate cancer

Abstract Inhibition of the androgen axis has revolutionized the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, mCRPC continues to be a leading cause of cancer-related deaths in men. Here, we characterized a phase I-cleared multikinase inhibitor, ESK981, in prostate cancer preclinical models. ESK981 was markedly more potent in vitro than other kinase inhibitors, including cabozantinib and crizotinib, that have been evaluated clinically in prostate cancer. Surprisingly, we observed that ESK981 was a potent inducer of cellular vacuolization that was associated with an autophagic response. ESK981-induced autophagy could be blocked by either the depletion of the autophagy vesicle formation gene, atg5, or by autophagy inhibitors such as bafilomycin. ESK981 was further shown to induce autophagy in yeast, which is an evolutionarily conserved core cellular pathway. Moreover, ESK981 triggered prostate cancer cells to release chemokines such as CXCL10 into the cell secretome, thus suggesting that ESK981 may stimulate immune surveillance while exerting its tumor inhibitory effect. Together, ESK981 is a novel and potent autophagy-inducing multikinase inhibitor that effectively inhibits CRPC growth through the activation of the autophagic cascade. Citation Format: Yuanyuan Qiao. Development of a novel autophagy-inducing multikinase inhibitor for the treatment of castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1339.

Shaping the flavivirus replication complex: It is curvaceous!

Flavivirus replication is intimately involved with remodelled membrane organelles that are compartmentalised for different functions during their life cycle. Recent advances in lipid analyses and gene depletion have identified a number of host components that enable efficient virus replication in infected cells. Here, we describe the current understanding on the role and contribution of host lipids and membrane bending proteins to flavivirus replication, with a particular focus on the components that bend and shape the membrane bilayer to induce the flavivirus‐induced organelles characteristic of infection.