Suppression Of Target Genes Research Articles

Reduced Glutamine Synthetase Activity Alters the Fecundity of Female Bactrocera dorsalis (Hendel)

Glutamine synthetase (GS) is a key enzyme in glutamine synthesis and is associated with multiple physiological processes in insects, such as embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on female fecundity in the oriental fruit fly, Bactrocera dorsalis. Based on the cloning of BdGSs, mitochondrial BdGSm and cytoplasmic BdGSc, we determined their expressions in the tissues of adult B. dorsalis. BdGSm was highly expressed in the fat body, while BdGSc was highly expressed in the head and midgut. Gene silencing by RNA interference against two BdGSs isoforms suppressed target gene expression at the transcriptional level, leading to a reduced ovarian size and lower egg production. The specific inhibitor L-methionine S-sulfoximine suppressed enzyme activity, but only the gene expression of BdGSm was suppressed. A similar phenotype of delayed ovarian development occurred in the inhibitor bioassay. Significantly lower expression of vitellogenin and vitellogenin receptor was observed when GS enzyme activity was suppressed. These data illustrate the effects of two GS genes on adult fecundity by regulating vitellogenin synthesis in different ways.

Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule-dependent Manner

The successful clinical translation of compounds that target specific oncogenic transcription factors will require an understanding of the mechanism of target suppression to optimize the dose and schedule of administration. We have previously shown trabectedin reverses the gene signature of the EWS-FLI1 transcription factor. In this report, we establish the mechanism of suppression and use it to justify the reevaluation of this drug in the clinic in patients with Ewing sarcoma.Experimental Design: We demonstrate a novel epigenetic mechanism of trabectedin using biochemical fractionation and chromatin immunoprecipitation sequencing. We link the effect to drug schedule and EWS-FLI1 downstream target expression using confocal microscopy, qPCR, Western blot analysis, and cell viability assays. Finally, we quantitate target suppression within the three-dimensional architecture of the tumor in vivo using 18F-FLT imaging. Trabectedin evicts the SWI/SNF chromatin-remodeling complex from chromatin and redistributes EWS-FLI1 in the nucleus leading to a marked increase in H3K27me3 and H3K9me3 at EWS-FLI1 target genes. These effects only occur at high concentrations of trabectedin leading to suppression of EWS-FLI1 target genes and a loss of cell viability. In vivo, low-dose irinotecan is required to improve the magnitude, penetrance, and duration of target suppression in the three-dimensional architecture of the tumor leading to differentiation of the Ewing sarcoma xenograft into benign mesenchymal tissue. These data provide the justification to evaluate trabectedin in the clinic on a short infusion schedule in combination with low-dose irinotecan with 18F-FLT PET imaging in patients with Ewing sarcoma.

Default Activation and Nuclear Translocation of the Plant Cellular Energy Sensor SnRK1 Regulate Metabolic Stress Responses and Development.

Energy homeostasis is vital to all living organisms. In eukaryotes, this process is controlled by fuel gauging protein kinases: AMP-activated kinase in mammals, Sucrose Non-Fermenting1 (SNF1) in yeast (Saccharomyces cerevisiae), and SNF1-related kinase1 (SnRK1) in plants. These kinases are highly conserved in structure and function and (according to this paradigm) operate as heterotrimeric complexes of catalytic-α and regulatory β- and γ-subunits, responding to low cellular nucleotide charge. Here, we determined that the Arabidopsis (Arabidopsis thaliana) SnRK1 catalytic α-subunit has regulatory subunit-independent activity, which is consistent with default activation (and thus controlled repression), a strategy more generally used by plants. Low energy stress (caused by darkness, inhibited photosynthesis, or hypoxia) also triggers SnRK1α nuclear translocation, thereby controlling induced but not repressed target gene expression to replenish cellular energy for plant survival. The myristoylated and membrane-associated regulatory β-subunits restrict nuclear localization and inhibit target gene induction. Transgenic plants with forced SnRK1α-subunit localization consistently were affected in metabolic stress responses, but their analysis also revealed key roles for nuclear SnRK1 in leaf and root growth and development. Our findings suggest that plants have modified the ancient, highly conserved eukaryotic energy sensor to better fit their unique lifestyle and to more effectively cope with changing environmental conditions.

Investigational RNAi Therapeutic Targeting C5 Is Efficacious in Pre-clinical Models of Myasthenia Gravis

Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders.

Antisense yycG Regulation of Antibiotic Sensitivity of Methicillin-Resistant Staphylococcus aureus in Chronic Osteomyelitis.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an urgent medical problem in osteomyelitis. The YycFG two-component regulatory system (TCS) allows bacteria to adapt rapidly to physical, chemical, and biological stresses. The recombinant plasmid shuttle vector was used to overexpress an antisense RNA (asRNA) to inhibit target gene expression by sequence-specific double-stranded RNA complex degradation. In the current study, antisense yycG RNA (ASyycG)-overexpression MRSA clinical isolates were constructed. Methods: Bacterial growth was monitored, and biofilm biomass was determined by crystal violet microtiter assay. Quantitative reverse transcription polymerase chain reaction analysis was used to identify expression of yycF/G/H and icaA/D in MRSA and ASyycG strains. The expression of YycG protein was quantified by Western blot assays. The antibiotic resistance of ASyycG strains was compared with that of the MRSA strains. Results: The ASyycG strains showed a decrease in growth rate compared with the MRSA strains. Of note, overexpression of ASyycG led to a reduction in biofilm formation and adhesion force. ASyycG strains had decreased expressions of the yycF/G/H and icaA/D. Furthermore, Western blot data showed that expression of the YycG protein decreased by 40% in ASyycG strains compared with MRSA strains. In addition, the effect of yycG asRNA improved the susceptibility of ASyycG strains to cefoxitin. Conclusions: The ASyycG strains inhibited biofilm organization and increased antibiotic sensitivity, which may be attributed to altered intracellular polysaccharide construction.

Inhibition of prostaglandin biosynthesis leads to suppressed ovarian development in Spodoptera exigua

Prostaglandins (PGs) are a group of eicosanoids that are C20 oxygenated polyunsaturated fatty acids. PGs can mediate various physiological processes such as immunity, salivary secretion, excretion, and reproduction in insects. The objective of this study was to determine the effect of PG on oocyte development in Spodoptera exigua, a lepidopteran insect known to biosynthesize PGs. Polytrophic ovarioles of S. exigua females exhibited follicle development in germarium, in which oocytes were distinct from nurse cells. During vitellogenesis, nurse cells degenerated by losing cytoplasm called “nurse cell dumping” while oocytes showed increase in cell volume. When PG biosynthesis inhibitors such as ibuprofen or aspirin were applied, nurse cell dumping was not complete and no chorion was formed, thus preventing egg formation. However, addition of PGE2 significantly rescued such inhibition and resumed oocyte development and choriogenesis. To support the observation with genetic factor, RNA interference (RNAi) specific to peroxynectins (Pxts: Se-Pxt1 and Se-Pxt2) known to act as insect cyclooxygenase was performed to suppress PG biosynthesis. Both Se-Pxt1 and Se-Pxt2 were highly expressed in the ovary of control female. RNAi treatment against Se-Pxt1 or Se-Pxt2 specifically suppressed target genes and inhibited oocyte development. Addition of PGE2 to adults treated with RNAi rescued the suppressed development of oocytes. Results of this study suggest that PGs can stimulate oocyte development as autocrine/paracrine mediators of vitellogenesis and choriogenesis in insects.

Multiple gene silencing in STAT pathway in K562 cells

Context: Chronic myeloid leukemia (CML) is characterized by the presence of a fusion oncoprotein BCR-ABL. This mutation imparts a constitutive phosphorylation activity of tyrosine residues in the cellular proteins. One of the targets of BCR-ABL is the STAT5 protein, which when phosphorylated induces gene expression of antiapoptotic proteins such as BCL-XL. The STAT pathway has been targeted in the past by disrupting any one protein only. A multiple gene silencing has never been done in this pathway. Aim: The aim of this study was to compare the effects of downregulation of BCR-ABL, STAT5A, STAT5B, and BCL-XL, individually and simultaneously, in human CML cell line (K562 cells) through RNA interference (RNAi). Further, gene expression, inhibition of proliferation, and apoptosis induction were assessed in K562 cells. Materials and Methods: K562 cells were transfected with various combinations of small iRNA (siRNA) and the expressions of aforesaid genes were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. K562 cell proliferation and apoptosis were analyzed using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and flow cytometry, respectively. The results were compared through one-way analysis of variance. Results: qPCR and western blotting results post-siRNA transfection confirmed the targeted gene suppression and protein reduction in K562 cells. The cell proliferation assay and apoptosis assay revealed that simultaneous gene silencing of BCR-ABL, STAT5A, STAT5B, and BCL-XL had the highest killing effect on K562 cells as compared to knocking down these genes individually or in any other combinations. Conclusions: This was the first time it was shown that multiple gene silencing in STAT pathway in CML cell line K562 was better as compared to individual gene silencing.

Abstract P6-05-01: A novel cleaved cytoplasmic lncRNA LacRNA interacts with PHB2 and suppresses breast cancer metastasis via repressing MYC targets

Abstract Long noncoding RNAs (lncRNAs) have been implicated in breast cancer metastases through largely unknown mechanisms. In this study, we used microarray analysis to compare lncRNAs expression levels between matched pairs of breast lymph node metastatic tissues and primary tumors. We discovered that lncRNA LINC00478 was substantially downregulated in the metastatic tumor samples. Interestingly, we found that LINC00478 could be cleaved by RNase to simultaneously generates the mature 5' ends of cytoplasmic RNA and 3' ends of nuclear RNA by polyadenylation. We named 5' ends 791-nt RNA as LacRNA (LINC00478-assciated cytoplasmic RNA). Over expression of full-length LINC00478 and LacRNA, but not LINC00478 3' RNA, significantly inhibited breast cancer proliferation, invasion and metastasis in vitro and in vivo.We used CRISPR-dCas9 complex to mediate efficient transcriptional activation of LacRNA at endogenous genomic loci followed by RNA-seq analyses. Gene set enrichment analysis (GSEA) showed that the MYC pathway/targets were prominent gene sets negatively enriched in LacRNA-activated cells. Further study revealed that LacRNA exerted its tumor suppressive activity by directly binding with prohibitin2(PHB2) to enhance its protein stability, which promoted PHB2 competing with MYC for transcriptionally suppressing the MYC target genes (e.g., CDC20, CDC45, CCNA2 and MAD2L1). Mechanistically, LacRNA inhibits breast cancer invasion and metastasis by interacting with PHB2 through LacRNA's 1-300nt region. In addition, taking advantage of CRISPR system to knock-out and activate the expression of LacRNA, as well as rescue experiment, we uncovered the positive correlation between LacRNA and PHB2 and their role in suppressing MYC target genes and cancer metastasis. At the same time, LacRNA can attenuated the MYC induced activation of MYC targets through binding with PHB2, indicating that LacRNA plays a central role in the suppression of MYC target genes. We further explored the role of LacRNA in inhibiting lung metastasis by implanting LacRNA-activated LM2 cells into the mammary fat pads of NOD-SCID mice. Luciferase imaging and histological analysis were used to detect lung metastasis and found that LacRNA significantly suppressed lung metastasis. Immunohistochemistry were used to detect the expression of PHB2 and MYC targets in both orthotopic tumors and lung metastasis and verified their correlation in vivo. Extensive analyses of clinical data indicated that LacRNA level was substantially downregulated in metastases tumors accompanied by enrichment of MYC targets. The robustness value of LacRNA expression was further verified in two independent patient cohorts, including 530 invasive breast cancer tumors in Fudan University Shanghai Cancer Center (FUSCC) and 819 breast patients' data from TCGA. High LacRNA expression level had a significantly better clinical outcome in both cohorts and represented an independent prognostic predictor for DFS (HR=0.48, P=0.006, multivariate analysis) and OS (HR=0.32, P=0.009, multivariate analysis) in FUSCC cohort. Collectively, LacRNA functions as a tumor suppressor lncRNA that inhibits breast cancer invasion-metastasis cascade. Citation Format: Guo R, Su Y-H, Xue J-y, Si J, Chi Y-y, Wu J. A novel cleaved cytoplasmic lncRNA LacRNA interacts with PHB2 and suppresses breast cancer metastasis via repressing MYC targets [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-05-01.

(-)-Epigallocatechin Gallate (EGCG) Enhances the Sensitivity of Colorectal Cancer Cells to 5-FU by Inhibiting GRP78/NF-κB/miR-155-5p/MDR1 Pathway.

Green tea accounts for approximately 20% of the world's total tea yield. (-)-Epigallocatechin gallate (EGCG) is an active catechin in green tea, which suppresses tumor growth and enhances drug sensitivity in various cancers, but the molecular mechanism is still unclear. Chemotherapy drugs, such as 5-fluorouracil (5-FU), are a common strategy for clinical treatment of cancer patients; however, the lower response rate caused by prolonged use becomes the main reason for tumor recurrence. Therefore, discovering a safe and effective chemo-sensitizer is an urgent task required to be solved. Here, we report that EGCG reinforces the sensitivity of colon cancer cells to 5-FU, and the IC50 values of 5-FU is decreased from 40 ± 4.2 μM to 5 ± 0.36 μM in one human colon carcinoma cell line-HCT-116, and from 150 ± 6.4 μM to 11 ± 0.96 μM in the other human colon carcinoma cell line-DLD1 when these cells are cotreated with 50 μM EGCG. Consistently, compared to 5-FU or EGCG treatment alone, the combination of both significantly promotes cancer cell apoptosis and DNA damage. Further mechanism research reveals that treatment of colorectal cancer (CRC) with 50 μM EGCG inhibits GRP78 expression, activates the NF-κB (2.55 ± 0.05-fold for HCT-116 and 2.27 ± 0.08-fold for DLD1) pathway, and enhances miR-155-5p (2.12 ± 0.02-fold for HCT-116 and 2.01 ± 0.01-fold for DLD1) level. The elevated miR-155-5p strongly suppresses target gene MDR1 expression, which blocks the efflux of 5-FU. The accumulation of 5-FU resulted in caspase-3 and PARP activation, Bcl-2 reduction, and Bad increase, which ultimately lead to cancer cell apoptosis. Overall, our data show that EGCG may be act as a novel chemo-sensitizer, and the GRP78/NF-κB/miR-155-5p/MDR1 pathway plays a vital role in EGCG enhancing the sensitivity of colorectal cancer to 5-FU.

MiR-151-5p modulates APH1a expression to participate in contextual fear memory formation

ABSTRACTLong-term memory formation requires gene expression and new protein synthesis. MicroRNAs (miRNAs), a family of small non-coding RNAs that inhibit target gene mRNA expression, are involved in new memory formation. In this study, elevated miR-151-5p (miR-151) levels were found to be responsible for hippocampal contextual fear memory formation. Using a luciferase reporter assay, we demonstrated that miR-151 targets APH1a, a protein that has been identified as a key factor in γ-secretase activity, namely APH1a. Blocking miR-151 can upregulate APH1a protein levels and subsequently impair hippocampal fear memory formation. These results indicate that miR-151 is involved in hippocampal contextual fear memory by inhibiting APH1a protein expression. This work provides novel evidence for the role of miRNAs in memory formation and demonstrates the implication of APH1a protein in miRNA processing in the adult brain.

Analysis of miRNA signature differentially expressed in exosomes from adriamycin-resistant and parental human breast cancer cells.

A major cause of failure in chemotherapy is drug resistance of cancer cells. Exosomes have been introduced to spread chemoresistance through delivering miRNAs. However, a systematic evaluation of the exosomal miRNA expression profiles responsible for chemoresistance is still lacking. In the present study, miRNA signature differentially expressed in exosomes derived from adriamycin-resistant (A/exo) and parental breast cancer cells (S/exo) were analyzed by microarray and the results were confirmed by PCR. A total of 309 miRNAs were increased and 66 miRNAs were decreased significantly in A/exo compared with S/exo. Specifically, 52 novel miRNAs with increased expression levels >16.0-fold in A/exo were identified. After prediction of target genes for 13 of 52 selected novel miRNAs, pathway analysis, gene ontology (GO) terms, and protein–protein interactions (PPIs) were constructed. The results implied that these selected exosomal miRNAs inhibited target genes involved in transcriptional misregulation in cancer, MAPK, and Wnt signaling pathways. Functional enrichment analysis demonstrated that the target genes were mainly responsible for protein phosphorylation, transcription regulation, molecular binding, and kinase activity. In summary, the current bioinformatics study of exosomal miRNAs may offer a new understanding into mechanisms of chemoresistance, which is helpful to find potential exosomal miRNAs to overcome drug insensitivity in future breast cancer treatment.

Down-regulation of AR splice variants through XPO1 suppression contributes to the inhibition of prostate cancer progression.

Emerging studies have shown that the expression of AR splice variants (ARv) lacking ligand-binding domain is associated with castrate-resistant prostate cancer (CRPC) and higher risk of tumor metastasis and recurrence. Nuclear export protein XPO1 regulates the nuclear localization of many proteins including tumor suppressor proteins. Increased XPO1 in prostate cancer is associated with a high Gleason score and bone metastasis. In this study, we found that high expression of AR splice variant 7 (AR-v7) was correlated with increased XPO1 expression. Silencing of XPO1 by RNAi or treatment with Selective Inhibitor of Nuclear Export (SINE) compounds selinexor and eltanexor (KPT-8602) down-regulated the expression of AR, AR-v7 and ARv567es at mRNA and protein levels. XPO1 silencing also inhibited the expression of AR and ARv regulators including FOXA1, Src, Vav3, MED1 and Sam68, leading to the suppression of ARv and AR target genes, UBE2C and PSA. By targeting XPO1/ARv signaling, SINE suppressed prostate cancer (PCa) growth in vitro and in vivo and potentiated the anti-cancer activity of anti-AR agents, enzalutamide and abiraterone. Therefore, XPO1 inhibition could be a novel promising agent used in combination with conventional chemotherapeutics and AR-targeted therapy for the better treatment of PCa, especially CRPC.

S-nitrosylation of the zinc finger protein SRG1 regulates plant immunity

Nitric oxide (NO) orchestrates a plethora of incongruent plant immune responses, including the reprograming of global gene expression. However, the cognate molecular mechanisms remain largely unknown. Here we show a zinc finger transcription factor (ZF-TF), SRG1, is a central target of NO bioactivity during plant immunity, where it functions as a positive regulator. NO accumulation promotes SRG1 expression and subsequently SRG1 occupies a repeated canonical sequence within target promoters. An EAR domain enables SRG1 to recruit the corepressor TOPLESS, suppressing target gene expression. Sustained NO synthesis drives SRG1 S-nitrosylation predominantly at Cys87, relieving both SRG1 DNA binding and transcriptional repression activity. Accordingly, mutation of Cys87 compromises NO-mediated control of SRG1-dependent transcriptional suppression. Thus, the SRG1-SNO formation may contribute to a negative feedback loop that attenuates the plant immune response. SRG1 Cys87 is evolutionary conserved and thus may be a target for redox regulation of ZF-TF function across phylogenetic kingdoms.

Role of SUMOylation in differential ER\u03b1 transcriptional repression by tamoxifen and fulvestrant in breast cancer cells

Antiestrogens (AEs) are widely used for treatment of estrogen receptor alpha (ERα)-positive breast cancer, but display variable degrees of partial agonism in estrogen target tissues and breast cancer (BC) cells. The fact that BC cells resistant to selective ER modulators (SERMs) like tamoxifen (Tam) can still be sensitive to pure AEs, also called selective ER downregulators, suggests different mechanisms of action, some of which may contribute to the more complete suppression of estrogen target genes by pure AEs. We report herein that pure AEs such as fulvestrant induce transient binding of ERα to DNA, followed by rapid release after 30–40 min without loss of nuclear localization. Loss of DNA binding preceded receptor degradation and was not prevented by proteasome inhibition. Chromatin was less accessible in the presence of fulvestrant than with estradiol or Tam as early as 20 min following treatment, suggesting that chromatin remodeling by pure AEs at ERα target regions prevents transcription in spite of receptor binding. SUMO2/3 marks were detected on chromatin at the peak of ERα binding in cells treated with pure AEs, but not SERMs. Furthermore, decreasing SUMOylation by overexpressing the deSUMOylase SENP1 significantly delayed receptor release from DNA and de-repressed expression of estrogen target genes in the presence of fulvestrant, both in ERα-expressing MCF-7 cells and in transiently transfected ER-negative SK-BR-3 cells. Finally, mutation V534E, identified in a breast metastasis resistant to hormonal therapies, prevented ERα modification and resulted in increased transcriptional activity of estrogen target genes in the presence of fulvestrant in SK-BR-3 cells. Together, our results establish a role for SUMOylation in achieving a more complete transcriptional shut-off of estrogen target genes by pure AEs vs. SERMs in BC cells.

Inhibition of Tumor Growth via Systemic siRNA Delivery Using Reducible Bile Acid-Conjugated Polyethylenimine.

RNA interference (RNAi), mediated by small interfering RNA (siRNA), has been considered as a potential therapeutic agent for cancer owing to its ability to suppress target genes in a sequence-specific manner. In this study, a conjugate of the low molecular weight (MW) polyethylenimine (PEI) (MW 1800) and deoxycholic acid (DA) was further modified with 4-fluorothiophenol (FTP) (TP-DA-PEI) to achieve systemic siRNA delivery. The thiophenol group would be involved with disulfide bonds between the polymer chains and siRNA modified with free thiols (thiol-siRNA) to form and π–π interactions between the pendent aromatic groups and coprostane ring of the bile acid. The TP-DA-PEI conjugates could generate stable nanoparticles with thiol-siRNA. The TP-DA-PEI conjugate not only achieved enhanced intracellular uptake, serum stability, and transfection efficiency, but also showed high accumulation of TP-DA-PEI/thiol-siRNA polyplexes and significant tumor growth inhibition effect in tumor-bearing mice after systemic administration.

An Atlas of Genetic Variation Linking Pathogen-Induced Cellular Traits to Human Disease

Pathogens have been a strong driving force for natural selection. Therefore, understanding how human genetic differences impact infection-related cellular traits can mechanistically link genetic variation to disease susceptibility. Here we report the Hi-HOST Phenome Project (H2P2): a catalog of cellular genome-wide association studies (GWAS) comprising 79 infection-related phenotypes in response to 8 pathogens in 528 lymphoblastoid cell lines. Seventeen loci surpass genome-wide significance for infection-associated phenotypes ranging from pathogen replication to cytokine production. We combined H2P2 with clinical association data from patients to identify a SNP near CXCL10 as a risk factor for inflammatory bowel disease. A SNP in the transcriptional repressor ZBTB20 demonstrated pleiotropy, likely through suppression of multiple target genes, and was associated with viral hepatitis. These data are available on a web portal to facilitate interpreting human genome variation through the lens of cell biology and should serve as a rich resource for the research community.

KRAB-containing zinc finger protein ZNF496 inhibits breast cancer cell proliferation by selectively repressing ERα activity

KRAB-containing zinc finger proteins (KZNF) constitute the largest family of transcriptional regulators in humans and play critical roles in normal development and tumorigenesis. However, the function and mechanism of most KZNFs remain unclear. Here, we report that ZNF496, a KZNF family member, interacts with the DNA binding domain (DBD) of estrogen receptor alpha (ERα) via its C2H2 domain. This interaction decreases ERα binding to chromatin DNA and results in the repression of ERα transactivation, the selective suppression of ERα target genes, and ultimately in a reduction of ERα-positive cell growth in the presence of E2. An analysis of clinical data revealed that the downregulation of ZNF496 expression is observed only in ERα-positive and not in ERα-negative breast cancer tissues when compared with that in matched adjacent tissues. Lastly, we also observed that the downregulation of ZNF496 is associated with poor recurrence-free survival among patients with breast cancer. Collectively, our findings demonstrate that ZNF496 is a novel ERα-binding protein that acts as a target gene-specific ERα corepressor and inhibits the growth of ERα-positive breast cancer cells.

Engineered Flock House Virus for Targeted Gene Suppression Through RNAi in Fruit Flies (Drosophila melanogaster) in Vitro and in Vivo.

RNA interference (RNAi) is a powerful tool to study functional genomics in insects and the potential of using RNAi to suppress crop pests has made outstanding progress. However, the delivery of dsRNA is a challenging step in the development of RNAi bioassays. In this study, we investigated the ability of engineered Flock House virus (FHV) to induce targeted gene suppression through RNAi under in vitro and in vivo condition. As proxy for fruit flies of agricultural importance, we worked with S2 cells as derived from Drosophila melanogaster embryos, and with adult stages of D. melanogaster. We found that the expression level for all of the targeted genes were reduced by more than 70% in both the in vitro and in vivo bioassays. Furthermore, the cell viability and median survival time bioassays demonstrated that the recombinant FHV expressing target gene sequences caused a significantly higher mortality (60–73% and 100%) than the wild type virus (24 and 71%), in both S2 cells and adult insects, respectively. This is the first report showing that a single stranded RNA insect virus such as FHV, can be engineered as an effective in vitro and in vivo RNAi delivery system. Since FHV infects many insect species, the described method could be exploited to improve the efficiency of dsRNA delivery for RNAi-related studies in both FHV susceptible insect cell lines and live insects that are recalcitrant to the uptake of naked dsRNA.

Abstract 3674: Pharmacodynamic assessment in whole blood for the BET bromodomain inhibitor CPI-0610 of target engagement in patients with progressive lymphoma

Abstract Defining the relationship between small molecule exposure, clinical efficacy and target engagement via pharmacodynamic (PD) biomarkers is a key element of phase 1 clinical development. CPI-0610 is an orally bioavailable inhibitor of the bromodomain and extra-terminal (BET) domain family of proteins currently being evaluated in a number of phase I trials. To support the clinical development of CPI-0610, we sought to identify BET target genes in whole blood that function as PD markers for target engagement in patients. The utilization of whole blood rather than tumor samples for PD assessments allows for a high level of compliance for sample collection and assessment of serial samples to understand the kinetics of optimal gene regulation. This is important for the mechanism of action of BET bromodomain inhibitors, as it has been shown that target gene suppression is rapidly reversible upon removal of drug. A preliminary list of BET target genes was first identified after ex vivo treatment of healthy donor blood samples with a BET inhibitor followed by transcriptional profiling. Further characterization was carried out using blood samples recovered from in vivo studies in mice, as well as rat and dog toxicology studies for CPI-0610. Blood samples collected from patients with progressive lymphoma participating in a Phase I clinical study (NCT01949883) were analyzed for changes in the BET target genes and correlated with CPI-0610 dose, pharmacokinetic (PK) exposure and clinical activity. This enabled further refinement of the BET target gene signature to identify the most robust gene expression changes in patient samples. IL8 and CCR1 were the most strongly regulated genes, showing an exposure-dependent downregulation in patient samples. Using these target genes, we were able to identify a minimum threshold of exposure required for BET target engagement as well as show that BET target engagement can be achieved at doses below the maximum tolerated dose. Furthermore, ≥50% downregulation of IL8 was consistent with anti-lymphoma activity in patients from our trial. CCR2, FN1, CSF1R and THBS1 were identified and validated preclinically but in patient samples were difficult to interpret due to lack of robust regulation or low basal levels of expression. In summary, a BET target gene signature assay using whole blood samples was established and implemented in a Phase I lymphoma study which demonstrated that CPI-0610 regulates direct BET target genes in blood cells in a robust and dose-dependent manner. Citation Format: Jennifer A. Mertz, Kristie A. Blum, Anas Younes, Jeremy S. Abramson, Michael B. Maris, Ian W. Flinn, Andre Goy, Darrell R. Borger, Michael R. Cooper, Robert J. Sims. Pharmacodynamic assessment in whole blood for the BET bromodomain inhibitor CPI-0610 of target engagement in patients with progressive lymphoma [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 3674.

Abstract 2492: Down-regulation of AR splice variants through XPO1 suppression contributes to the inhibition of prostate cancer progression

Abstract Increased XPO1 (a key nuclear exporter of many tumor suppressor proteins [TSPs]) expression in prostate cancer (PCa) has been found to be associated with a high Gleason score and bone metastasis. However, it is unknown whether aberrant XPO1 activity regulates AR and AR splice variants (Arv) signaling in PCa progression. In this study, using 22Rv1 and VCaP prostate cancer cells which harbor full length AR and ARv, we conduct in vitro cellular and molecular biological experiments and in vivo animal studies to elucidate the role XPO1 inhibition plays in PCa progression through regulation of XPO1/AR/ARv signaling. We found that high expression of AR splice variant 7 (AR-v7) was correlated with increased XPO1 expression. Silencing of XPO1 by RNAi or treatment with Selective Inhibitor of Nuclear Export (SINE) compounds (selinexor and KPT-8602/eltanexor) down-regulated the expression of AR, AR-v7 and ARv567es at the mRNA and protein levels. Mechanistic studies showed that XPO1 silencing also inhibited the expression of AR/ARv regulators including FOXA1, Src, Vav3, MED1 and Sam68, leading to the suppression of ARv and AR target genes, UBE2C and PSA. SINE compound treatment of cells retained the eIF4E protein (translation initiation factor and nuclear transporter of capped depended mRNAs) in nuclear compartment, leading to nuclear retention of AR-v7 and PSA mRNAs. The nuclear localization of AR-v7 mRNA resulted in the reduction in the ARv protein. Furthermore, SINE compound treatment retained TSPs including Rb, p21, p53, APC and SMAD4 in the nucleus leading to inhibition of cell proliferation and induction of apoptosis. Moreover, by targeting XPO1/ARv signaling, SINE compounds suppressed prostate cancer growth in vitro and in vivo and potentiated the anti-cancer activity of conventional chemotherapeutic agent docetaxel and anti-AR agents (enzalutamide and abiraterone) through the inhibition of AR, AR-v7, FOXA1, PSA and UBE2C. From these results, we conclude that there is a causal relationship between XPO1 and AR splice variants. High expression of both XPO1 and AR-v7 could lead to constitutively activated AR signaling, CRPC development and progression, and anti-AR drug resistance. By targeting XPO1/AR/ARv signaling and increasing anti-AR sensitivity, SINE compounds could be novel agents used in combination with conventional chemotherapeutics and AR-targeted therapy for the treatment of PCa, especially CRPC. Citation Format: Irfana Muqbil, Yiwei Li, Erkan Baloglu, William Senapedis, Yosef Landesman, Christian Argueta, Michael Kauffman, Sharon Friedlander, Hua Chang, Sharon Shacham, Elisabeth Heath, Asfar S. Azmi. Down-regulation of AR splice variants through XPO1 suppression contributes to the inhibition of prostate cancer progression [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 2492.