Single siRNAs Research Articles

Aromatic amino acid metabolites influence interferon signaling and Influenza infectivity

Abstract The gut microbiota extensively metabolizes the three aromatic amino acids. Previously we found that a tyrosine metabolite can alter type I interferon (IFN) signaling. Herein we screened 17 metabolites of these amino acids for effects on IFN signaling alone and with LPS, IFN-β, and Influenza utilizing IFN reporter human lung epithelial cells (A549) and monocytes (THP1). While the tryptophan metabolites reduced IFN signaling in both cell types, tyrosine and phenylalanine metabolites had varied effects, depending on cell type. The cells were also treated with pooled metabolites from each family and a composite treatment with all three family metabolites. The composite treatment reduced IFN signaling in both cell types, suggesting a tryptophan metabolite effect dominance. Strikingly, when pretreated with composite treatment and infected with influenza, cells had reduced viral recovery and IFN signaling in both cell types. RNA sequencing validated earlier observation of reduced viral loads and IFN signaling. Gene silencing utilizing single siRNAs for upregulated genes identified (EGR2, ATP6V0D2, SPOCK1, and IL31RA) did not completely abrogate the repressed IFN signaling. However, these targets demonstrated linkage to TGF-beta signaling, and treatment with a TGF-beta inhibitor and gene silencing of all four gene targets led to a significant reversal of IFN signaling suppression. Our in vivo work demonstrated that intranasal administration of these metabolites prior to influenza infection leads to reduced animal weight loss, viral titers, and histological inflammation. Our work implicates that microbial metabolites alter IFN signaling through TGF-beta signaling and can promote beneficial outcomes during influenza infection. The study was supported by grant from NIH (K08 AI135097-02) and Burroughs Wellcome Fund, awarded to Dr. Ashley L. Steed

Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer

BackgroundThe endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9.MethodsOVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2′-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model.ResultsApproximately 70–90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model.ConclusionsOur results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer.

Improved delivery of Mcl-1 and survivin siRNA combination in breast cancer cells with additive siRNA complexes.

This study aimed at investigating the influence of commercial transfection reagents (Prime-Fect, Leu-Fect A, and Leu-Fect C) complexed with different siRNAs (CDC20, HSP90, Mcl-1 and Survivin) in MDA-MB-436 breast cancer cells and the impact of incorporating an anionic additive, Trans-Booster, into siRNA formulations for improving in vitro gene silencing and delivery efficiency. Gene silencing was quantitatively analyzed by real-time RT-PCR while cell proliferation and siRNA uptake were evaluated by the MTT assay and flow cytometry, respectively. Amongst the investigated siRNAs and transfection reagents, Mcl-1/Prime-Fect complexes showed the highest inhibition of cell viability and the most effective siRNA delivery. The effect of various formulations on transfection efficiency showed that the additive with 1:1 ratio with siRNA was optimal achieving the lowest cell viability compared to untreated cells and negative control siRNA treatment (p < 0.05). Furthermore, the combination of Mcl-1 and survivin siRNA suppressed the growth of MDA-MB-436 cells more effectively than treatment with the single siRNAs and resulted in cell viability as low as ~ 20% (vs. non-treated cells). This aligned well with the induction of apoptosis as analyzed by flow cytometry, which revealed higher apoptotic cells with the combination treatment group. We conclude that commercial transfection reagents formulated with Mcl-1/Survivin siRNA combination could serve as a potent anti-proliferation agent in the treatment of breast cancers.

USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling.

The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R‐spondin induces auto‐ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R‐spondin by protecting ZNRF3/RNF43 from ubiquitin‐dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R‐spondin‐LGR4‐ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R‐spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface.

TIMP-2 regulates proliferation, invasion and STAT3-mediated cancer stem cell-dependent chemoresistance in ovarian cancer cells

BackgroundThe metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines.MethodsFT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence.ResultsSixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers.ConclusionsThe above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance.

Abstract 1527: Splicing factors determine breast cancer cell mitosis through control of sister chromatid cohesion

Abstract Breast cancer is the mostly diagnosed cancer in women worldwide and is also the leading cause of death from cancer in women, mainly due to metastasis formation. Accumulating evidence suggests that RNA splicing is critical in breast cancer progression. To investigate the role of every single component of the splicing machinery (the spliceosome) in this process, we systematically evaluated the effect of siRNA-mediated knockdown of 244 splicing factors on proliferation in two highly proliferative breast cancer cell lines MDA-MB-231 and Hs578T. Hits of the primary screen were validated with single siRNAs and separated based on their effects on migration and proliferation respectively. Knockdown of nine validated proliferation hits, amongst which was SF3B1, a known breast cancer driver gene, resulted for all nine hits in a distinct poly-lobed nuclear phenotype in both cell types, accompanied by increased DNA content and higher number of cells in G1-S transition. Increased levels of phospho-Histone H3 and lack of metaphase alignment suggested a defect in mitosis, which was confirmed by decreased RNA expression of sister chromatid cohesion factors MAU2, ESPL1 and SMC1A and interestingly, increased CDCA5 RNA levels. Upon splicing factor knockdown, CDCA5 intron 1 was retained resulting in decreased CDCA5 protein levels, inaccurate metaphase alignment and finally cell death. To identify the direct partners of the splicing factors that control mitosis, we coupled pull-down of GFP fused splicing factors with mass spectrometry. Seven out of the nine splicing factors that were identified in our screen causing poly-lobed nuclear phenotypes, resided in the same protein complex together with proteins involved in snRNP assembly and some mitosis-related factors. In conclusion, we have identified several splicing factors that are critically determining breast cancer cell proliferation through modulating the expression of chromatid cohesion factors and thereby mediating the successful metaphase alignment and ultimate mitosis. Citation Format: Esmee Koedoot, John Martens, Sylvia E. Le Dévédec, Bob van de Water. Splicing factors determine breast cancer cell mitosis through control of sister chromatid cohesion [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 1527.

Liver-Specific siRNA Inhibition of Class 2a Histone Deacetylases (HDACs) Reduces Expression of Genes Regulating Gluconeogenesis in Primary Human and Mouse Hepatocytes, but Not in Mice

Gene expression control by histone-deacetylases Class 2a (HDACs 4,5,7,9) has been demonstrated to be involved in the negative regulation of hepatic gluconeogenesis and consequently inhibition of HDACs was shown to result in improved glucose and pyruvate tolerance in rodent type 2 diabetes (T2D) models. However, pan-inhibition of HDACs in all tissues leads also to severe side effects in various tissues, excluding this approach for treatment of T2D. Here, we have investigated in vitro and in vivo safety and efficacy of a liver-selective HDAC knock-down via single or combinatorial siRNAs for HDAC 4, 5 or 7. In primary mouse and human hepatocytes, specific siRNAs directed against HDAC 4, 5 or 7, as well as their combination led to a selective knock-down of the respective target gene(s) by about 80-90%, which paralleled with a significant reduction of genes involved in the regulation of gluconeogenesis. These siRNAs were administered in a liver-specific lipid-particle formulation for treatment of healthy C57BL/6J mice. Mice received five intravenous injections with either PBS, a control siRNA, one of the respective silencing siRNAs (0.75mg/kg) or all possible dual or triple combinations for 25 days. Quantitative real-time PCR demonstrated that siRNA treatment led to a significant downregulation of the respective HDACs in the liver. However no reduction of gluconeogenic genes (PCK1 and G6PC) was detected. Consequently, also no improvement in intraperitoneal pyruvate tolerance test (PTT) could be observed. In addition, no significant differences in fasting blood glucose or plasma insulin were observed between treatment groups. In summary, although active in cellular systems, liver-targeted siRNA knockdown of class 2a HDACs did not result in a clear inhibition of genes regulating gluconeogenesis in healthy mice suggesting no clear path forward for development of this approach for treatment of T2D. Disclosure S.W. Görgens: Employee; Self; Sanofi-Aventis Deutschland GmbH. S. Raichur: None. P. Wohlfart: Employee; Self; Sanofi-Aventis Deutschland GmbH. B. Brunner: Employee; Self; Sanofi. N. Tennagels: Employee; Self; Sanofi R&D. Stock/Shareholder; Self; Sanofi. M. Bielohuby: Employee; Self; Sanofi-Aventis Deutschland GmbH, Sanofi R&D.

Abstract 893: Systematic assessment of spliceosome components as drivers of breast cancer progression

Abstract Accumulating evidence indicates that RNA splicing is involved in different steps of carcinogenesis. Although the critical role of a limited number of splicing factors has been demonstrated in several cancer studies, the function of many spliceosome components is still unknown. In breast cancer samples higher expression levels of a subset of spliceosome components consistently correlated with more aggressive tumor types in patient microarray as well as RNA sequencing data from The Cancer Genome Atlas (TCGA). We further systematically evaluated the effect of siRNA-mediated knockdown of 244 individual splicing factors on both cell migration and proliferation in two aggressive breast cancer cell lines MDA-MB-231 and Hs578T using high throughput microscopy. Primary candidates were validated with four single siRNAs and classified based on their effects on migration and proliferation. Knockdown of validated spliceosome factors such as SNRPG and SNRPA1 affected the levels of small nuclear RNAs, the main catalyzers of the spliceosome. Moreover, high expression of these key candidate hits is associated with poor breast cancer metastasis free survival. To investigate the downstream mechanisms and splicing patterns controlled by our candidate genes we performed RNA sequencing on both control and siRNA mediated knockdown cells. Interestingly, knockdown of our hits affect extracellular matrix pathways that are essential in tumor cell migration and invasion. Altogether our data indicate a key role for spliceosome factors in the control of breast cancer progression which provide new leads for future therapeutic intervention. Citation Format: Esmee Koedoot, Sylvia Le Dévédec, Bob van de Water. Systematic assessment of spliceosome components as drivers of breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 893. doi:10.1158/1538-7445.AM2017-893

RNA interference in the termite Reticulitermes speratus：a comparison of the effects of single siRNAs and a long dsRNA on the silencing of the hexamerin gene.

RNA interference in the termite Reticulitermes speratus：a comparison of the effects of single siRNAs and a long dsRNA on the silencing of the hexamerin gene.

Abstract 1735: Identification of novel synthetic lethal interactions in small cell lung cancer with the proteasome inhibitor carfilzomib

Abstract Proteasome inhibition (PI) has emerged as a valuable cancer treatment in hematological malignancies by causing a disruption of protein homeostasis. While proteasome inhibitors have been historically unsuccessful in the clinic for solid tumors, all cancer cell lines succumb to proteasome inhibition in vitro. Solid tumor cell lines have a higher threshold for disruption of protein homeostasis; as they require continuous exposure to achieve cytotoxicity similar to pulsatile exposure of PI in heme cell lines. The in-vivo half-life of proteasome inhibitors is short (∼1hr) making it challenging to mimic in vitro continuous treatment. Therefore we explored the option of sensitizing solid tumors to proteasome inhibitors through combination therapy. In order to identify novel synthetic lethal interactions with PI, we performed a screen utilizing a pooled siRNA library targeting 2240 genes of the druggable genome in combination with the proteasome inhibitor carfilzomib (CFZ) or DMSO. Cells were first transfected with the siRNA for 24 hours and then treated with CFZ or DMSO for an additional 72h to assess synergy. We performed the screen in small cell lung cancer (SCLC), an unmet medical need. Preliminary responses in SCLC patients were observed with CFZ in an all comers solid tumor trial (Papadopoulos, et al. Cancer Chemother Pharmacol, 2013). The screen was performed in NCI-H841 cells that are adherent, easy to culture, and have high transfection efficiency. The combined effect of siRNA and CFZ on viability was measured using a Sensitivity Index (SI), which ranges from -1 (antagonistic) to 1 (synergistic). Using an SI threshold >0.2, we identified 33 synthetic lethal genes when H841 cells were treated with an IC9 of CFZ. To validate screen hits, we performed experiments using single siRNA reagents (4 - 6 sequences per gene) in combination with CFZ in NCI-H841. Two hits were confirmed with two or more single siRNAs sensitizing cells to CFZ. These genes (TGFBR3 and GRK6) are of particular interest because of their involvement in proliferation signaling mechanisms. Subsequent screens have also uncovered two additional genes, involved in the protein degradation and trafficking pathway (CLPP and COG8), which meet the same validation criteria for synergy. The characterization of these hits is currently on-going in a panel of SCLC lines, and in cells engineered with CRISPR technology to achieve a near complete knockout. In parallel, we are testing commercially available reagents targeting our proteins of interest to validate our results. Preliminary data with a published GRK6 inhibitor, GF109203x, demonstrates synergy with Bliss scores of 0.35 in H841 and 0.44 in DMS114 SCLC lines (Zhou et al. J Pharmacol Exp Ther, 2001). In summary, we have identified druggable targets that would potentiate CFZ efficacy towards solid tumors and may elucidate the PI mechanism of action. Citation Format: Vincent B. Masto, Alana G. Lerner, Shirin Arastu-Kapur. Identification of novel synthetic lethal interactions in small cell lung cancer with the proteasome inhibitor carfilzomib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1735. doi:10.1158/1538-7445.AM2015-1735

Artificial MiRNA Knockdown of Platelet Glycoprotein lbα: A Tool for Platelet Gene Silencing.

In recent years, candidate genes and proteins implicated in platelet function have been identified by various genomic approaches. To elucidate their exact role, we aimed to develop a method to apply miRNA interference in platelet progenitor cells by using GPIbα as a proof-of-concept target protein. After in silico and in vitro screening of siRNAs targeting GPIbα (siGPIBAs), we developed artificial miRNAs (miGPIBAs), which were tested in CHO cells stably expressing GPIb-IX complex and megakaryoblastic DAMI cells. Introduction of siGPIBAs in CHO GPIb-IX cells resulted in 44 to 75% and up to 80% knockdown of GPIbα expression using single or combined siRNAs, respectively. Conversion of siGPIBAs to miGPIBAs resulted in reduced silencing efficiency, which could however be circumvented by tandem integration of two hairpins targeting different regions of GPIBA mRNA where 72% GPIbα knockdown was achieved. CHO GPIb-IX cells transfected with the miGPIBA construct displayed a significant decrease in their ability to aggregate characterized by lower aggregate numbers and size compared to control CHO GPIb-IX cells. More importantly, we successfully silenced GPIbα in differentiating megakaryoblastic DAMI cells that exhibited morphological changes associated with actin organization. In conclusion, we here report the successful use of miRNA technology to silence a platelet protein in megakaryoblastic cells and demonstrate its usefulness in functional assays. Hence, we believe that artificial miRNAs are suitable tools to unravel the role of a protein of interest in stem cells, megakaryocytes and platelets, thereby expanding their application to novel fields of basic and translational research.

Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir

Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs.

Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (B). Efficiency of pharmacological action

This paper examines a perspective to use newly engineered nanomaterials as effective and safe carriers for gene therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus, and carbosilane) were complexed with anticancer siRNA and the biophysical properties of the dendriplexes created were analyzed. The potential of the dendrimers as nanocarriers for anticancer Bcl-xl, Bcl-2, Mcl-1 siRNAs and additionally a scrambled sequence siRNA has been explored. Dendrimer/siRNA complexes were characterised by various methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. In this part of study, the transfection of complexes in HeLa and HL-60 cells was analyzed using both single apoptotic siRNAs and a mixture (cocktail) of them. Cocktails were more effective than single siRNAs, allowing one to decrease siRNAs concentration in treating cells. The dendrimers were compared as siRNA carriers, the most effective being the phosphorus-based ones. However, they were also the most cytotoxic on their own, so that in this regard the application of all dendrimers in anticancer therapy will be discussed.

Combination siRNA therapy against feline coronavirus can delay the emergence of antiviral resistance in vitro

Virulent biotypes of feline coronavirus (FCoV), commonly referred to as feline infectious peritonitis virus (FIPV), can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. We previously reported the successful in vitro inhibition of FIPV replication by synthetic siRNA mediated RNA interference (RNAi) in an immortalised cell line (McDonagh et al., 2011). A major challenge facing the development of any antiviral strategy is that of resistance, a problem which is particularly acute for RNAi based therapeutics due to the exquisite sequence specificity of the targeting mechanism. The development of resistance during treatment can be minimised using combination therapy to raise the genetic barrier or using highly potent compounds which result in a more rapid and pronounced reduction in the viral replication rate, thereby reducing the formation of mutant, and potentially resistant viruses. This study investigated the efficacy of combination siRNA therapy and its ability to delay or prevent viral escape. Virus serially passaged through cells treated with a single or dual siRNAs rapidly acquired resistance, with mutations identified in the siRNA target sites. Combination therapy with three siRNA prevented viral escape over the course of five passages. To identify more potent silencing molecules we also compared the efficacy, in terms of potency and duration of action, of canonical versus Dicer-substrate siRNAs for two previously identified effective viral motifs. Dicer-substrate siRNAs showed equivalent or better potency than canonical siRNAs for the target sites investigated, and may be a more appropriate molecule for in vivo use. Combined, these data inform the potential therapeutic application of antiviral RNAi against FIPV.

Abstract 3400: Integrated functional RNAi screening and structural genomics identifies inverse co-modulators of TP53 family and NF-kB transitional activation as potential therapeutic targets in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer, with an annual incidence of over 600,000 new cases worldwide, and 50-60% mortality. Dysregulation of tumor suppressor TP53 in HNSCC occurs in over 90% of cases. We and others have observed that TP53 related family members p63 and p73 are also deregulated, together preventing transcription of growth arrest and apoptosis genes. Conversely, we have shown that members of the NF-κB/REL family of transcription factors are aberrantly activated, and drive expression of genes that promote cell proliferation, survival, inflammation, angiogenesis,and resistance in ∼70% of HNSCC cases. We have previously published that the function of different TP53 and NF-κB family members are inversely modulated within two major subsets of HNSCC, suggesting that common molecules and pathways coordinate this modulation and may serve as critical targets for therapy. To test this hypothesis, we have developed HNSCC stable cell lines that report transcriptional activation of TP53 or NF-κB individually through a β lactamase reporter, using antibiotic selection and fluorescence activated cell sorting. In collaboration with the RNAi screening facility at NIH, kinome and druggable target genes have been screened in our NF-κB reporter cell lines. Screening was performed by 48 hour knockdown with siRNAs, followed by stimulation with TNF-α. The results have revealed known and novel targets that maintain oncogenic NF-κB signaling in HNSCC. These targets include genes participating in Toll/IKK such as IRAK1, PI3K/AKT signaling such as PIK3CA and PIK3R3, mitotic kinases such as BUB1B and AURKA, and calmodulin signaling such as CALM2 and CAMK1G. Candidate hits from RNAi screening data were first prioritized by integration with expression, mutation, and DNA copy number variation data from The Cancer Genome Atlas (TCGA) database. To further investigate the interactive signaling regulation between NF-κB and TP53 pathways, selected siRNAs that can inhibit NF-κB activation were cross screened in our TP53 reporter cell lines. Transcriptional activation of TP53 was induced with doxorubicin or cisplatin following 48 hr RNAi knockdown. We have shown that knockdown of targets in the PI3K/AKT pathway can inversely increase TP53 activation while inhibiting NF-κB activity. Selected targets were further validated by demonstration of anti-proliferative activity of single siRNAs in multiple HNSCC cell lines. Preclinical studies with PI3K-mTOR small molecule inhibitors indicate potential to co-modulate NF-κB and TP53 and inhibit HNSCC in vitro and in vivo. We predict that inhibition of targets that inversely modulate both TP53 and NF-κB signaling will be less prone to resistance and more effective in treatment of HNSCC. Supported by NIDCD intramural projects ZIADC000016, 73 and 74. Citation Format: Anthony D. Saleh, Shaleeka Cornelius, Hui Cheng, Scott Martin, Pinar Ormanoglu, Xinping Yang, Zhong Chen, Carter VanWaes. Integrated functional RNAi screening and structural genomics identifies inverse co-modulators of TP53 family and NF-kB transitional activation as potential therapeutic targets in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3400. doi:10.1158/1538-7445.AM2014-3400

SiRNA Combinations Mediate Greater Suppression of Hepatitis B virus Replication in Mice

Hepatitis B virus (HBV) infection is a major world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome shortcomings of established drugs. In this study, the inhibitory effect mediated by combination of siRNAs targeting different sites of HBV in transgenic mice was analyzed. HBsAg and HBeAg in the sera of the mice were analyzed by enzyme-linked immunoadsorbent assay, HBV DNA by real-time PCR and HBV mRNA by RT-PCR. Our data demonstrated that all the three siRNAs employed showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, combination of siRNAs compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA used for therapy was the same. Secreted HBsAg and HBeAg in the serum of mice treated with siRNA combination were reduced by 96.7 and 96.6 %, respectively. Immunohistochemical detection of liver tissue revealed 91 % reduction of HBsAg-positive cells in the combination therapy group. The combination of siRNAs caused a greater inhibition in the levels of viral mRNA and DNA (90 and 87.7 %) relative to the control group. It was noted that the siRNA3 showed stronger inhibition of cccDNA (78.6 %). Our results revealed that combination of siRNAs mediated a stronger inhibition of viral replication and antigen expression in transgenic mice than single siRNAs.

Epithelial Sodium Channel Silencing as a Strategy to Correct the Airway Surface Fluid Deficit in Cystic Fibrosis

In the respiratory system, Na(+) absorption and Cl(-) secretion are balanced to maintain an appropriate airway surface fluid (ASF) volume and ensure efficient mucociliary clearance. In cystic fibrosis (CF), this equilibrium is disrupted by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in the absence of functional CFTR-dependent Cl(-) secretion. The consequences of defective Cl(-) transport are worsened by the persistence of Na(+) absorption, which contributes to airway surface dehydration. We asked whether normal ASF can be restored to an equal extent by recovering Cl(-) secretion from mutated CFTR or by reducing Na(+) absorption. This is highly relevant in the selection of the best strategy for the treatment of patients with CF. We analyzed the ASF thickness of primary cultured bronchial CF and non-CF epithelia after silencing the epithelial Na(+) channel (ENaC) with specific short, interfering RNAs (siRNAs) and after the pharmacological stimulation of CFTR. Our results indicate that (1) single siRNAs complementary to ENaC subunits are sufficient to reduce ENaC transcripts, Na(+) channel activity, and fluid transport, but only silencing both the α and β ENaC subunits at the same time leads to an increase of ASF (from nearly 7 µm to more than 9 µm); (2) the ASF thickness obtained in this way is about half that measured after maximal CFTR stimulation in non-CF epithelia (10-14 µm); and (3) the pharmacological rescue of mutant CFTR increases the ASF to the same extent as ENaC silencing. Our results indicate that CFTR rescue and ENaC silencing both produce a significant and long-lasting increase of airway hydration in vitro.

Simultaneous siRNA-mediated silencing of pairs of genes coding for enzymes involved in glycosaminoglycan synthesis.

It has been demonstrated recently that it is possible to decrease expression of genes coding for enzymes involved in synthesis of glycosaminoglycans (GAGs) by using specific siRNAs which interfere with stability of particular mRNAs. This procedure has been proposed as a potential treatment for patients suffering from mucopolysaccharidoses, a group of inherited metabolic diseases caused by dysfunction of enzymes required for GAG degradation, and resultant storage of these compounds in cells of affected persons. Here, we asked if the simultaneous use two species of specific siRNAs aimed at silencing two genes involved in particular steps of GAG synthesis may be more effective than the use of single siRNA. We found that inhibition of GAG synthesis in cells treated with two siRNAs is generally more effective than using single siRNAs. However, the differences were not statistically significant, therefore the potential benefit from the use of two siRNAs over the use of a single siRNA is doubtful in the light of the cost-benefit ratio and possibly stronger side-effects of the putative therapy.

Abstract 3795: BubR1 function is required for the antiproliferative activity of the novel microtubule-targeting drug BAL27862 (active moiety of the prodrug BAL101553)

Abstract Background: BAL27862 is a novel small molecule that induces apoptosis in cancer cells through microtubule destabilization associated with a distinct microtubule phenotype. BAL27862 has broad antitumor activity in preclinical models of human cancer, and has recently entered Phase I clinical trials in advanced cancer patients administered as a highly soluble lysine prodrug (BAL101553). Methods: Combinatorial RNAi screening using HeLa cervical carcinoma cells and a siRNA library targeting the human kinome/phosphatome was followed by image analysis of cell number and fraction with normal morphology after optimal BAL27862 treatment (50nM, 24h). BubR1 siRNA effects were also assessed by immunoblotting (IB) and 48h crystal violet proliferation assays. Drug-resistant tumor lines were selected in vitro by incubation with increasing BAL27862 concentrations. Patient-derived tumors were maintained in mice and analyzed by immunohistochemistry (IHC). Results: Combinatorial RNAi screening identified BubR1 (BUB1B gene) as a protein required for the antiproliferative and phenotypic effect of BAL27862. This finding was confirmed using single independent siRNAs. BubR1, an evolutionary conserved protein kinase controlling cell division, is a core component of the spindle assembly checkpoint. Consistent with this, a more detailed siRNA analysis across a panel of lines derived from different histological backgrounds (breast, colon, lung, brain) confirmed the essential role of BubR1 in the in vitro response of diverse tumor lines to BAL27862 treatment. For example, non-targeting control siRNA (NTC)-treated MDA-MB-453 breast carcinoma and H460 NSCLC cells entered a cell death program following BAL27862 treatment (∼60 % & ∼40 % cell death resp. vs. starting cell number); a response clearly attenuated by BubR1 down regulation (∼80% cell proliferation. vs. DMSO controls). Further IB analysis of tumor cell variants selected for stable drug resistance to BAL27862 (∼3-24-fold resistant), indicated that BubR1 protein expression was down regulated in 3 of 5 variants as compared to parental lines (SKOV3 ovarian, A549 & H460 non-small cell lung cancer [NSCLC]). Hence, reduced BubR1 expression may modulate development of resistance to BAL27862 under treatment. Strikingly, IHC analysis of paired patient-derived tumors derived from gastric and NSCLC carcinomas, demonstrated that intrinsic resistance to BAL27862 treatment ex vivo can also be associated with reduced BubR1 tumor expression. Conclusions: Reduced BubR1 expression correlates with resistance to BAL27862 treatment in a number of experimental models. Further work to understand the functional significance of BubR1 to the mechanism of action of BAL27862 is ongoing to explore the potential of using this biomarker to support patient stratification efforts during the clinical development of BAL101553. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3795. doi:1538-7445.AM2012-3795

Common Seed Analysis to Identify Off-Target Effects in siRNA Screens

Genome-scale small interfering RNA (siRNA) screens have become an increasingly popular approach to new target identification and pathway elucidation. However, the large data sets generated from siRNA screens have demonstrated high false-positive rates and the requirement for extensive experimental triage to distinguish true hits. A number of groups have independently reported the presence of siRNAs with identical seed sequences among their top screening hits. Based on these observations, we have developed a comprehensive technique for detecting and visualizing seed-based off-target effects in siRNA screening data. This is accomplished by analyzing the behavior of siRNAs that share identical seed sequences, which we refer to as common seed analysis (CSA). By applying these techniques to primary screening data of the Wnt pathway, we identify 158 distinct seed sequences that have a statistically significant effect on the assay. The promiscuous seed sequences identified in this manner can then be discounted in the analysis of follow-up experiments using single siRNAs. The ability to detect off-target effects when sufficient numbers of siRNAs share a common seed has significant implications for the design of siRNA screening experiments, data analysis, hit selection, and library design.