Dicer-substrate siRNA Research Articles

The PPP2R1A cancer hotspot mutant p.R183W increases clofarabine resistance in uterine serous carcinoma cells by a gain-of-function mechanism.

Uterine serous carcinoma (USC) is generally associated with poor prognosis due to a high recurrence rate and frequent treatment resistance; hence, there is a need for improved therapeutic strategies. Molecular analysis of USC identified several molecular markers, useful to improve current treatments or identify new druggable targets. PPP2R1A, encoding the Aα subunit of the tumor suppressive Ser/Thr phosphatase PP2A, is mutated in up to 40% of USCs. Here, we investigated the effect of the p.R183W PPP2R1A hotspot variant on treatment response to the nucleoside analogue clofarabine. USC cells stably expressing p.R183W Aα showed increased resistance to clofarabine treatment in vitro and, corroborated by decreased clofarabine-induced apoptosis, G1 phase arrest, DNA-damage (γH2AX) and activation of ATM and Chk1/2 kinases. Phenotypic rescue by pharmacologic PP2A inhibition or dicer-substrate siRNA (dsiRNA)-mediated B56δ subunit knockdown supported a gain-of-function mechanism of Aα p.R183W, promoting dephosphorylation and inactivation of deoxycytidine kinase (dCK), the cellular enzyme responsible for the conversion of clofarabine into its bioactive form. Therapeutic assessment of related nucleoside analogues (gemcitabine, cladribine) revealed similar effects, but in a cell line-dependent manner. Expression of two other PPP2R1A USC mutants (p.P179R or p.S256F) did not affect clofarabine response in our cell models, arguing for mutant-specific effects on treatment outcome as well. While our results call for PPP2R1A mutant and context-dependent effects upon clofarabine/nucleoside analogue monotherapy, combining clofarabine with a pharmacologic PP2A inhibitor proved synergistically in all tested conditions, highlighting a new generally applicable strategy to improve treatment outcome in USC.

The Characterization of Acute Myeloblastic Leukemia-M2 Cell Lines with Suppressed Stromal Interaction Molecule 1 (STIM1) and its Impact on Calcium/Reactive Oxygen Species Profiles

Introduction: Acute myeloid leukemia-M2 subtype (AML-M2) is a severe type of blood cancer that has a high rate of recurrence and death. Recent cancer research has linked stromal interaction molecule 1 (STIM1) and calcium/reactive oxygen species (ROS) interactions to cancer progression, drug resistance, and cancer cell self-renewal. However, the involvement of STIM1 in the modulation of calcium and ROS activities and AML-M2 cell survival is still unclear. Methods: The current study uses dicer-substrate siRNA (dsiRNA) knockdown of STIM1 to assess its functional activity in the AML-M2 cell line. Following STIM1 knockdown, the expression levels of genes involved in cell survival and ROS generation were measured by RT-qPCR. Calcium influx, ROS generation, cell proliferation, and colony formation were all evaluated. Results: Knocking down STIM1 exhibited a reduction in calcium influx and ROS generation. Kasumi-1 cell proliferation and colony formation were inhibited following STIM1 knockdown. Further transcriptomic profiling in this knockdown model revealed downregulation of KRAS, MAPK, C-MYC, Akt, NOX2, and PKC. Conclusion: The findings point to STIM1's potential role in promoting AML-M2 cell survival through calcium/ROS interplay-mediated control of KRAS and Akt-related pathways. Furthermore, it might recommend STIM1 and/or ROS for targeted therapy, which may contribute to regression of disease and improve the AML therapeutic strategy.

Suppression of small nucleolar RNA host gene 8 (SNHG8) inhibits the progression of colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies around the world with high mortality. Accumulating evidences demonstrate that long non-coding RNAs (lncRNAs) play critical roles in CRC tumorigenesis by regulating different pathways of carcinogenesis. SNHG8 (small nucleolar RNA host gene 8), a lncRNA, is highly expressed in several cancers and acts as an oncogene that promotes cancer progression. However, the oncogenic role of SNHG8 in CRC carcinogenesis and the underlying molecular mechanisms remain unknown. In this study, we explored the role of SNHG8 in CRC cell lines by performing a series of functional experiments. Similar to the data reported in the Encyclopedia of RNA Interactome, our RT-qPCR results showed that SNHG8 expression was significantly upregulated in CRC cell lines (DLD-1, HT-29, HCT-116, and SW480) compared to the normal colon cell line (CCD-112CoN). We performed dicer-substrate siRNA transfection to knockdown the expression of SNHG8 in HCT-116 and SW480cell lines which were expressing high levels of SNHG8. SNHG8 knockdown significantly reduced CRC cell growth and proliferation by inducing autophagy and apoptosis pathways through the AKT/AMPK/mTOR axis. We performed wound healing migration assay and demonstrated that SNHG8 knockdown significantly increased migration index in both cell lines, indicating reduced migration abilities of cells. Further investigation showed that SNHG8 knockdown suppresses epithelial to mesenchymal transition and reduces cellular migratory properties of CRC cells. Taken together, our study suggests that SNHG8 acts as an oncogene in CRC through the mTOR-dependent autophagy, apoptosis, and EMT pathways. Our study provides a better understanding the role of SNHG8 in CRC at molecular level and SNHG8 might be used as novel therapeutic target for CRC management.

Vitamin D inhibits osteosarcoma by reprogramming nonsense-mediated RNA decay and SNAI2-mediated epithelial-to-mesenchymal transition.

Osteosarcomas are immune-resistant and metastatic as a result of elevated nonsense-mediated RNA decay (NMD), reactive oxygen species (ROS), and epithelial-to-mesenchymal transition (EMT). Although vitamin D has anti-cancer effects, its effectiveness and mechanism of action against osteosarcomas are poorly understood. In this study, we assessed the impact of vitamin D and its receptor (VDR) on NMD-ROS-EMT signaling in in vitro and in vivo osteosarcoma animal models. Initiation of VDR signaling facilitated the enrichment of EMT pathway genes, after which 1,25(OH)2D, the active vitamin D derivative, inhibited the EMT pathway in osteosarcoma subtypes. The ligand-bound VDR directly downregulated the EMT inducer SNAI2, differentiating highly metastatic from low metastatic subtypes and 1,25(OH)2D sensitivity. Moreover, epigenome-wide motif and putative target gene analysis revealed the VDR's integration with NMD tumorigenic and immunogenic pathways. In an autoregulatory manner, 1,25(OH)2D inhibited NMD machinery genes and upregulated NMD target genes implicated in anti-oncogenic activity, immunorecognition, and cell-to-cell adhesion. Dicer substrate siRNA knockdown of SNAI2 revealed superoxide dismutase 2 (SOD2)-mediated antioxidative responses and 1,25(OH)2D sensitization via non-canonical SOD2 nuclear-to-mitochondrial translocalization leading to overall ROS suppression. In a mouse xenograft metastasis model, the therapeutically relevant vitamin D derivative calcipotriol inhibited osteosarcoma metastasis and tumor growth shown for the first time. Our results uncover novel osteosarcoma-inhibiting mechanisms for vitamin D and calcipotriol that may be translated to human patients.

Sequence determinant of small RNA production by DICER.

RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs)1,2. However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3' overhang and a terminal loop3-11. Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER(also known as DICER1). Our analyses revealed a deeply conserved cis-acting element, termed the 'GYM motif' (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified 'ruler'-like counting mechanisms from the 5' and 3' ends of pre-miRNA3-6. Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics.

Linoleic-acid-substituted polyethylenimine to silence heat shock protein 90B1 (HSP90B1) to inhibit migration of breast cancer cells.

Breast cancer continues to be one of the leading causes of death in women, and the lack of treatment options for distant metastasis warrants the need to identify and develop more effective approaches. The aim of this study was to identify and validate targets that are associated with the survival and migration of the breast cancer cells in vitro through RNA interference (RNAi) approach. Linoleic-acid-modified polyethylenimine (PEI) polymer was used to screen a short interfering RNA (siRNA) library against numerous cell adhesion and cytoskeleton genes in MDA-MB-231 triple-negative breast cell line, and the functional outcome of silencing was determined by growth and migration inhibition with further target validation studies. Heat shock protein 90B1 (HSP90B1) was identified as a crucial gene that is known to be involved in various breast cancer machineries, including uncontrolled proliferation and brain metastasis. The success of this approach was also due to the use of hyaluronic acid (HA) additive in lipopolymer complexes that showed a profound impact in reducing the cell viability (~50%), migration (~40%), and mRNA transcript levels (~80%) with a physiologically relevant siRNA concentration of 60 nM. The use of Dicer-substrate siRNA proved to be beneficial in target silencing, and a combinational treatment of integrin-β1 (ITGB1) and HSP90B1 was effective in reducing the migration of the MDA-MB-231 and MDA-MB-436 breast cancer cells. This study demonstrates the potential to identify and silence targets using a lipid-modified PEI/siRNA system and highlights the importance of HSP90B1 in the growth and migration of breast cancer cells.

Development of transgenic okra (Abelmoschus esculentus L. Moench) lines having RNA mediated resistance to Yellow vein mosaic virus (Geminiviridae)

Begomovirus Yellow vein mosaic virus causes severe yield losses in okra and even the resistant lines developed through conventional breeding show susceptibility at various levels. This paper describes the development of YVMV resistant lines through RNAi strategy. A universal ihpRNA construct harbouring βC1 ORF from the β-satellite of the begomovirus was designed using pRNAi-LIC plasmid. Complementarity checks in sequence databases had shown no off-target effects by the target region and the success of siRNA in interference was proven using Custom Dicer-Substrate siRNA analysis. The βC1 ORF of the begomovirus was PCR amplified and sequenced using the primer combination designed. The pRNAi-LIC vector, a derivative of pCAMBIA2300 containing duplicated CaMV 35S promoter and Nos terminator from pYL44, was SmaI digested and the amplified sense and antisense strands of the βC1 region were cloned. E. coli transformed with the plasmid were screened for antibiotic resistance, and the plasmids confirmed for the sense and antisense regions through sequencing, were transferred to Agrobacterium tumefaciens strain GV3101. In planta transformation strategy was followed to transform a highly susceptible okra cv. Salkeerthi with ihpRNA-βC1 cassette. Transformation success, confirmed by the amplification of sense strand using the primers VLIC1 and VLIC5, was 11.42 %. Transcription of siRNA from the βC1 ORF in the transgenic lines was confirmed by its PCR amplification from the cDNA, using the stem loop primers designed (68 bp). When the transformed and healthy wild-type plants were co-grown with infected wild-type plants, inside an insect cage released with whiteflies and maintained within a containment facility, three of the four transgenic plants remained completely healthy throughout the crop span.

Rational design for controlled release of Dicer-substrate siRNA harbored in phi29 pRNA-based nanoparticles

Small interfering RNA (siRNA) for silencing genes and treating disease has been a dream since ranking as a top Breakthrough of the Year in 2002 by Science. With the recent FDA approval of four siRNA-based drugs, the potential of RNA therapeutics to become the third milestone in pharmaceutical drug development has become a reality. However, the field of RNA interference (RNAi) therapeutics still faces challenges such as specificity in targeting, intracellular processing, and endosome trapping after targeted delivery. Dicer-substrate siRNAs included onto RNA nanoparticles may be able to overcome these challenges. Here, we show that pRNA-based nanoparticles can be designed to efficiently harbor the Dicer-substrate siRNAs in vitro and in vivo to the cytosol of tumor cells and release the siRNA. The structure optimization and chemical modification for controlled release of Dicer-substrate siRNAs in tumor cells were also evaluated through molecular beacon analysis. Studies on the length requirement of the overhanging siRNA revealed that at least 23 nucleotides at the dweller’s arm were needed for dicer processing. The above sequence parameters and structure optimization were confirmed in recent studies demonstrating the release of functional Survivin siRNA from the pRNA-based nanoparticles for cancer inhibition in non-small-cell lung, breast, and prostate cancer animal models.

Preliminary preclinical study of Chol-DsiRNA polyplexes formed with PLL[30]-PEG[5K] for the RNAi-based therapy of breast cancer

RNA interference molecules have tremendous potential for cancer therapy but are limited by insufficient potency after i.v. administration. We previously found that Chol-DsiRNA polyplexes formed between cholesterol-modified dicer-substrate siRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase the activity of Chol-DsiRNA against a stably expressed reporter mRNA in primary murine syngeneic breast tumors after daily i.v. dosing. Here, we provide a more thorough preliminary preclinical study of Chol-DsiRNA polyplexes against the therapeutically relevant target protein, STAT3. We found that Chol-DsiSTAT3 polyplexes greatly increase plasma exposure, distribution, potency, and therapeutic activity of Chol-DsiSTAT3 in primary murine syngeneic 4T1 breast tumors after i.v. administration. Furthermore, inactive Chol-DsiCTRL polyplexes are well tolerated by healthy female BALB/c mice after chronic i.v. administration at 50 mg Chol-DsiCTRL/kg over 28 days. Thus, Chol-DsiRNA polyplexes may be a good candidate for Phase I clinical trials to improve the treatment of breast cancer and other solid tumors.

Anti-c-myc RNAi-Based Onconanotherapeutics.

Overexpression of the c-myc proto-oncogene features prominently in most human cancers. Early studies established that inhibiting the expression of oncogenic c-myc, produced potent anti-cancer effects. This gave rise to the notion that an appropriate c-myc silencing agent might provide a broadly applicable and more effective form of cancer treatment than is currently available. The endogenous mechanism of RNA interference (RNAi), through which small RNA molecules induce gene silencing by binding to complementary mRNA transcripts, represents an attractive avenue for c-myc inhibition. However, the development of a clinically viable, anti-c-myc RNAi-based platform is largely dependent upon the design of an appropriate carrier of the effector nucleic acids. To date, organic and inorganic nanoparticles were assessed both in vitro and in vivo, as carriers of small interfering RNA (siRNA), DICER-substrate siRNA (DsiRNA), and short hairpin RNA (shRNA) expression plasmids, directed against the c-myc oncogene. We review here the various anti-c-myc RNAi-based nanosystems that have come to the fore, especially between 2005 and 2020.

Enzymatically synthesized 2′-fluoro-modified Dicer-substrate siRNA swarms against herpes simplex virus demonstrate enhanced antiviral efficacy and low cytotoxicity

Chemical modifications of small interfering (si)RNAs are used to enhance their stability and potency, and to reduce possible off-target effects, including immunogenicity. We have earlier introduced highly effective antiviral siRNA swarms against herpes simplex virus (HSV), targeting 653 bp of the essential UL29 viral gene. Here, we report a method for enzymatic production and antiviral use of 2′-fluoro-modified siRNA swarms. Utilizing the RNA-dependent RNA polymerase from bacteriophage phi6, we produced 2′-F-siRNA swarms containing either all or a fraction of modified adenosine, cytidine or uridine residues in the antisense strand of the UL29 target. The siRNA containing modified pyrimidines demonstrated high resistance to RNase A and the antiviral potency of all the UL29-specific 2′-F-siRNA swarms was 100-fold in comparison with the unmodified counterpart, without additional cytotoxicity. Modest stimulation of innate immunity signaling, including induced expression of both type I and type III interferons, as well as interferon-stimulated gene 54, by 2′-F-cytidine and 2′-F-uridine modified siRNA swarms occurred at early time points after transfection while the 2′-F-adenosine-containing siRNA was similar to the unmodified antiviral siRNA swarm in this respect. The antiviral efficacy of the 2′-F-siRNA swarms and the elicited cellular innate responses did not correlate suggesting that innate immunity pathways do not significantly contribute to the observed enhanced antiviral activity of the modified siRNAs. The results support further applications of enzymatically produced siRNA molecules with incorporated adenosine nucleotides, carrying fluoro-modification on ribose C2′ position, for further antiviral studies in vitro and in vivo.

Photoactivation of sulfonated polyplexes enables localized gene silencing by DsiRNA in breast cancer cells

Translation potential of RNA interference nanotherapeutics remains challenging due to in vivo off-target effects and poor endosomal escape. Here, we developed novel polyplexes for controlled intracellular delivery of dicer substrate siRNA, using a light activation approach. Sulfonated polyethylenimines covalently linked to pyropheophorbide-α for photoactivation and bearing modified amines (sulfo-pyro-PEI) for regulated endosomal escape were investigated. Gene knock-down by the polymer-complexed DsiRNA duplexes (siRNA-NPs) was monitored in breast cancer cells. Surprisingly, sulfo-pyro-PEI/siRNA-NPs failed to downregulate the PLK1 or eGFP proteins. However, photoactivation of these cell associated-polyplexes with a 661-nm laser clearly restored knock-down of both proteins. In contrast, protein down-regulation by non-sulfonated pyro-PEI/siRNA-NPs occurred without any laser treatments, indicating cytoplasmic disposition of DsiRNA followed a common intracellular release mechanism. Therefore, sulfonated pyro-PEI holds potential as a unique trap and release light-controlled delivery platform for on-demand gene silencing bearing minimal off target effects.

Strategic Design of Dicer Substrate siRNA to Mitigate the Resistance Mediated by ABCC1 in Doxorubicin-resistant Breast Cancer

The current study involved designing of a small interfering ribonucleic acid-loaded folate functionalized chitosan-coated PLGA nanoparticles to evaluate tumor targeting potential in drug-resistant breast cancer cells. The synthesized conjugate was utilized to develop small interfering ribonucleic acid encapsulated nanoparticles. The developed siRNA nanoparticles were with a particle size of ˂250 nm and could offer protection against serum nucleases. A significant uptake of the folic acid conjugated formulation was noticed, affirming that the nanoparticles selectively targeted the drug-resistant cancer cells. Through the polymerase chain reaction and Western blot, it was evident that ABCC1 expression was significantly reduced (˂95%). The efficiency of small interfering ribonucleic acid with doxorubicin could be evaluated by cytotoxicity assay and it was found that the ABCC1 targeted small interfering ribonucleic acid minimized the resistance of doxorubicin-resistant MCF- 7 cell line to doxorubicin. The main objective in developing this folate conjugated nanoformulation was to enhance the targetability to the cancer cells and to increase the sensitivity of drug-resistant tumors to doxorubicin

Abstract 1502: KRAS RNAi therapy in preclinical models of pancreatic cancer sensitizes tumors to checkpoint blockade by affecting the tumor microenvironment

Abstract KRAS is one of the most frequently mutated oncogenes in pancreatic cancer. Recent research has revealed that activation of KRAS in cancer cells causes recruitment of immunosuppressive molecules to the tumor microenvironment (TME) and blocks T-cell mediated cytotoxicity. Despite huge progress in our understanding of KRAS and its role in tumorigenesis, direct pharmacological targeting of KRAS has thus far not been achieved. In these studies, we inhibited KRAS directly in tumor-bearing mice, using a potent and specific Dicer substrate siRNA (DsiRNA) formulated in tumor-selective lipid nanoparticles. We demonstrate that intravenously-dosed KRAS DsiRNA treatment results in reduction of multiple immunosuppressive signaling molecules, affecting recruitment of myeloid cells and regulatory T cells (Tregs) to the TME. In addition, effective KRAS inhibition reversed MEK-inhibitor-induced resistance in a model of KRAS mutant human PDAC. These data suggest that KRAS DsiRNA has potential to sensitize treatment-refractory PDAC tumors to both immune checkpoint blockade as well as conventional targeted therapeutics Note: This abstract was not presented at the meeting. Citation Format: Shanthi Ganesh, Serena Shui, Kevin Craig, Weimin Wang, Bob D. Brown, Marc Abrams. KRAS RNAi therapy in preclinical models of pancreatic cancer sensitizes tumors to checkpoint blockade by affecting the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1502.

Antitumor effect of palmitic acid-conjugated DsiRNA for colon cancer in a mouse subcutaneous tumor model.

In this study, we synthesized Dicer-substrate siRNA conjugated with palmitic acid at the 5'-end of the sense strand (C16-DsiRNA), and examined its RNAi effect on β-catenin as a target gene in a colon cancer cell line, HT29Luc, both in vitro and in vivo. We examined the in vitro RNAi effect in HT29Luc cells and found that C16-DsiRNA strongly inhibited expression of the β-catenin gene in comparison with non-modified DsiRNA. Also, high membrane permeability of C16-DsiRNA was exhibited, and it was confirmed that most of the C16-DsiRNA was localized in cytoplasm of HT29Luc cells. In regard to the in vivo RNAi effect, C16-DsiRNA complexed with Invivofectamine targeting the β-catenin gene was locally administered to a subcutaneous tumor formed by implantation of HT29Luc cells into the subcutis of nude mice; we evaluated the effect by measuring the bioluminescence increase, which reflects tumor growth, using an in vivo imaging system. As a result, C16-DsiRNA strongly inhibited the growth of tumors formed in subcutis of nude mice compared with non-modified DsiRNA, and this in vivo RNAi effect lasted up to 15days. Our results suggest that C16-DsiRNA should be vigorously pursued as a novel nucleic acid medicine for clinical treatment of cancer.

Complexation of Chol-DsiRNA in place of Chol-siRNA greatly increases the duration of mRNA suppression by polyplexes of PLL(30)-PEG(5K) in primary murine syngeneic breast tumors after i.v. administration

RNA interference has tremendous potential for cancer therapy but is limited by the insufficient potency of RNAi molecules after i.v. administration. We previously found that complexation with PLL(30)-PEG(5K) greatly increases the potency of 3′-cholesterol-modified siRNA [Chol-siRNA] in primary murine syngeneic 4T1 breast tumors after i.v. administration but mRNA suppression decreases 24 h after the final dose. We hypothesized that complexation of cholesterol-modified Dicer-substrate siRNA (Chol-DsiRNA) in place of Chol-siRNA can increase the potency and duration of suppression by polyplexes of PLL(30)-PEG(5K) in solid tumors. We found that replacing Chol-siRNA with Chol-DsiRNA increased polyplex loading and nuclease protection, suppressed stably expressed luciferase to the same extent in primary murine 4T1-Luc breast tumors under the current dosage regimen, but maintained suppression ~72 h after the final dose. The kinetics of suppression in 4T1-Luc over 72 h, however, were similar between DsiLuc and siLuc after electroporation and between polyplexes of Chol-DsiLuc and Chol-siLuc after transfection, suggesting that Chol-DsiRNA polyplexes increase the duration of mRNA suppression through differences in polyplex activities in vivo. Thus, replacing Chol-siRNA with Chol-DsiRNA may significantly increase the duration of mRNA suppression by polyplexes of PLL(30)-PEG(5K) and possibly other PEGylated polycationic polymers in primary tumors and metastases after i.v. administration.

Electroporation of Dicer-Substrate siRNA Duplexes Targeting Endogenous TCR Enhance Tumor Killing Activity of Wilms' Tumor 1 (WT1)-Specific TCR-Redirected Cytotoxic T Cells

In adoptive cellular immunotherapy, T cells can be genetically engineered to express a novel T-cell receptor (TCR) that recognizes a tumor-associated antigen. However, mispairing between transgene and endogenous TCR chains may result in a reduction of transgene TCR expression and potentially harmful off-target reactivities. Here, we sought to develop a novel clinically safe strategy to promote transgene expression of a Wilms' tumor 1 (WT1)-specific TCR by Dicer-substrate small interfering RNA (DsiRNA)-mediated silencing of the endogenous TCR, using a double electroporation protocol.First, we isolated and cloned an HLA-A*0201-restricted WT1 peptide-specific TCR derived from a leukemia patient who demonstrated clinical benefit after receiving a WT1-targeted DC vaccine. Next, we produced a codon-optimized TCR sequence from the wild-type TCR construct and both TCR mRNAs were generated by in vitro transcription. TCR expression levels were validated by electroporation of TCR-deficient Jurkat J76.7 cells stably transduced with CD8 and an NFAT-driven GFP reporter gene. TCR functionality was confirmed by high expression levels of GFP (70% GFP+cells) upon TCR signaling after co-culture with WT1 peptide-pulsed T2 cells.In order to suppress the translation of endogenous TCR mRNA in CD8+ T cells, DsiRNA duplexes were designed to specifically target the constant regions of wild-type TCR α- and β-chains, but not the codon-optimized TCR. We further developed a double electroporation protocol combining DsiRNA and TCR mRNA transfection in which DsiRNA electroporation was performed 24 hours prior to TCR mRNA electroporation. Our results show more than 2-fold increase in WT1-specific TCR expression by HLA-A2/WT1 tetramer staining after DsiRNA treatment as compared to TCR mRNA electroporation only. This specific TCR expression was maintained at least 5 days after TCR mRNA electroporation in resting peripheral blood CD8+ lymphocytes from healthy donors. The enhanced TCR expression in DsiRNA-transfected CD8+T cells was also correlated with an increase of epitope recognition as shown by interferon (IFN)-γ ELISpot.To determine the killing capacity of DsiRNA/TCR mRNA-transfected CD8+ T cells against epitope-bearing target cells, we performed a flow cytometry-based cytotoxicity assay using WT1 peptide-pulsed T2 cells. Specific cytotoxicity, which was already present in WT1 TCR-transfected cells, was significantly enhanced in TCR mRNA-electroporated T cells following suppression of the endogenous TCR expression by DsiRNA treatment. Accordingly, DsiRNA-treated TCR mRNA transfected CD8+T cells presented higher levels of CD137 and CD69 activation markers and secretion of cytokines (IFN-γ and tumor necrosis factor-α), granzyme B, and perforin upon TCR triggering as compared to the non-DsiRNA treated T cells.In summary, we show a marked enhancement of transgene WT1-specific TCR expression upon silencing of the endogenous TCR using DsiRNA electroporation prior to TCR mRNA electroporation. Importantly, this enhancement in TCR expression was correlated with a significant increase in WT1-specific CD8+ T-cell killing activity, expression of CD69 and CD137 activation markers and cytokine secretion after recognition of WT1 peptide-bearing target cells. These results pave the way for developing a clinically safer strategy for T cell-based adoptive immunotherapy of patients with WT1-expressing malignancies. DisclosuresNo relevant conflicts of interest to declare.

Direct Pharmacological Inhibition of β-Catenin by RNA Interference in Tumors of Diverse Origin.

The Wnt/β-catenin pathway is among the most frequently altered signaling networks in human cancers. Despite decades of preclinical and clinical research, efficient therapeutic targeting of Wnt/β-catenin has been elusive. RNA interference (RNAi) technology silences genes at the mRNA level and therefore can be applied to previously undruggable targets. Lipid nanoparticles (LNP) represent an elegant solution for the delivery of RNAi-triggering oligonucleotides to disease-relevant tissues, but have been mostly restricted to applications in the liver. In this study, we systematically tuned the composition of a prototype LNP to enable tumor-selective delivery of a Dicer-substrate siRNA (DsiRNA) targeting CTNNB1, the gene encoding β-catenin. This formulation, termed EnCore-R, demonstrated pharmacodynamic activity in subcutaneous human tumor xenografts, orthotopic patient-derived xenograft (PDX) tumors, disseminated hematopoietic tumors, genetically induced primary liver tumors, metastatic colorectal tumors, and murine metastatic melanoma. DsiRNA delivery was homogeneous in tumor sections, selective over normal liver and independent of apolipoprotein-E binding. Significant tumor growth inhibition was achieved in Wnt-dependent colorectal and hepatocellular carcinoma models, but not in Wnt-independent tumors. Finally, no evidence of accelerated blood clearance or sustained liver transaminase elevation was observed after repeated dosing in nonhuman primates. These data support further investigation to gain mechanistic insight, optimize dose regimens, and identify efficacious combinations with standard-of-care therapeutics. Mol Cancer Ther; 15(9); 2143-54. ©2016 AACR.

Abstract 2925: Anti-tumor activity of a MYC-targeting dicer substrate siRNA in combination with BRD4/CDK7 inhibitors

Abstract MYC is a well-characterized driver of numerous tumor types. Since the protein encoded by this gene is challenging to target via conventional modalities, progress in new therapeutic agents has been slow despite decades of research. RNA interference technology has enabled the inhibition of previously-undruggable genetic targets at the mRNA level, and has advanced to clinical development for several indications. DCR-MYC, a lipid nanoparticle (LNP)-formulated Dicer substrate siRNA (DsiRNA) targeting MYC mRNA, is currently in Phase Ib/II clinical trials and showing promising results. In this study we used an improved EnCore LNP and MYC DsiRNA, which demonstrated MYC mRNA silencing activity and efficacy in mouse models of human hepatocellular carcinoma (HCC). Small molecule inhibitors that target BRD4; JQ1 and CDK7; THZ1 has previously reported anti-proliferative effects in various cancer types and efficacy in several tumor mouse models including HCC. Treatment with both JQ1 and THZ1 induced cell cycle arrest and apoptosis in various cancer cells by repressing MYC expression. Here we observed striking anti-proliferative effects in vitro when MYC-DsiRNA was combined with THZ1 in cancer cells. In addition, when mice bearing HCC tumors were treated with MYC-DsiRNA combined with either THZ1 or JQ1, the antitumor efficacy was additive or synergistic relative to either single agent alone. We observed significantly more MYC mRNA knockdown in the tumors that had the combination treatment compared to the tumors that received either of the single agent treatment. These preclinical data suggests the possibility of a significant and practical benefit of combining MYC-DsiRNA and small molecule inhibitors that target an epigenetic regulator BRD4 (JQ1) and a global gene regulator CDK7 (THZ1). Citation Format: Edmond Chipumuro, Zakir Siddiquee, Shanthi Ganesh, Serena Shui, Anee Shah, Boyoung Kim, Dongyu Chen, Purva Pandya, Rachel Storr, Weimin Wang, Hank Dudek, Cheng Lai, Marc Abrams, Bob Brown. Anti-tumor activity of a MYC-targeting dicer substrate siRNA in combination with BRD4/CDK7 inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2925.

Abstract LB-272: Histidine decarboxylase (Hdc)-expressing myeloid cells support Foxp3+ Treg cells and promote colorectal cancer progression

Abstract The tumor microenvironment contains a diverse population of myeloid cells that are recruited from bone marrow and converted to immunosuppressive cells, thus mediating tumor cell escape from immune checkpoint. We have identified a subset of dynamic bone marrow myeloid cells, which can be identified by histidine decarboxylase (Hdc) mRNA and GFP expression in Hdc-GFP transgenic mice. Gene expression profiling showed that Hdc-GFP+ CD11b+Gr1+ myeloid cells express higher levels of cell cycle promoting genes such as Ccnd2, Ccnd3 and Cdc14a, while cell proliferation repressors including Cdc14b, Cdc16 and Cdk4 were downregulated in Hdc-GFP+ myeloid cells. To further elucidate the role of Hdc+ myeloid cells in tumor progression, we performed lineage-tracing studies using Hdc-creERT2;R26-LSL-TdTomato reporter mice. Pulsed with tamoxifen, the majority of TdTomato+ cells were localized initially to a group of CD11b+Gr1+ myeloid cells representing the highest Hdc mRNA expression in bone marrow, spleen and blood. Later on, TdTomato+CD11b+Gr1- F4/80+ macrophages can be detected, indicating a hierarchy of Hdc+ myeloid cells in which Hdc+CD11b+Gr1+ myeloid cells reside at the apex. In general, CD11b+ myeloid cells have a relative short lifespan (<2 weeks); however, treatment with dextran sodium sulfate (DSS) in the drinking water increased the longevity and frequency of Hdc+ myeloid cells, also promoted the differentiation of Gr1+ immature myeloid cells into neutrophils and macrophages. In a mouse model of azoxymethane (AOM)/DSS colorectal carcinogenesis, Hdc-creERT2;R26-LSL-TdTomato;R26-LSL-DTA mice were injected with 10 mg/kg AOM and followed by 3 circles of 10 days 1.5% DSS ad libitum in drinking water. We found that Hdc-TdTomato labeled a proportion of tumor infiltrating CD11b+Gr1+ myeloid cells that expressed higher levels of Arg-1, Cox2, and Pdl1 transcripts. Continuous depletion of Hdc+ myeloid cells by administration of tamoxifen chow to induce DTA (diphtheria toxin subunit A) killing in Hdc-expressing myeloid cells abrogated half of the tumor-infiltrating MDSCs and released tumoricidal CD8+ T cells (> 15-fold), leading to decreased tumor number. This tumor suppression could be rescued by Hdc+ CD11b+Gr1+ cell adoptive transfer. Serum chemokine profiling revealed that Hdc+ DTA mediated myeloid depletion also decreased serum chemokine levels, among which Cxcl13 decreased the most (>30-fold). Cxcl13 protein and Cxcl13 mRNA also decreased in the colonic tumor tissue in the Hdc+ myeloid cell depleted AOM/DSS treatment group. Along with reduction in Cxcl13 levels, we also detected a significant reduction of Foxp3+ Treg cells in the tumor frozen sections stained with antibody against Foxp3 compared to R26-LSL-TdTomato;R26-LSL-DTA controls received the same treatments. Pre-knockdown of Cxcl13 by Dicer-substrate SiRNA (DsiRNAs) in a co-culture of splenic Hdc+ CD11b+Gr1+ myeloid cells from colon tumor-bearing mice with splenic Foxp3-GFP+ Treg cells induced apoptosis and decreased numbers of GFP+ cells compared to the scramble knockdown control group. Taken together, our data suggest that Hdc marks a distinct subset of myeloid cells with greater potency for promoting tumorigenicity, in part through supporting Tregs and suppressing CD8+ Tcells in the tumor microenvironment. Citation Format: Xiaowei Chen, Yoshihiro Takemoto, Karan K. Nagar, Timothy H. Chu, Zhengyu Jiang, Wenju Chang, Richard A. Friedman, Yagnesh H. Tailor, Daniel L. Worthley, Timothy C. Wang. Histidine decarboxylase (Hdc)-expressing myeloid cells support Foxp3+ Treg cells and promote colorectal cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-272.