We have demonstrated that 3DNA nanocarriers can specifically, effectively, and safely deliver siRNA oligos targeted against a pro-survival hub, HuR (the ELAVL1 gene) in an in vivo, pre-clinical model of pancreatic cancer. Pancreatic cancer remains one of the deadliest cancers with an overall 5 year survival rate of 9%; and is on pace to become the second leading cause of cancer related deaths in the U.S. by 2020. Pancreatic cancer is so deadly, in part, because it is highly refractory to standard chemotherapeutics. In fact, new standard of care regiments such as FOLFIRINOX have only improved survival by a few months. Our previous work has established HuR as a target in pancreatic cancer. HuR is an RNA binding protein that is primarily retained in the nucleus, but upon exposure to cancer associated stressors translocates to the cytoplasm. In the cytoplasm, HuR functions to bind, stabilize, and up-regulate expression of pro-survival target mRNAs. CRISPR-mediated deletion of HuR causes a xenograft lethal phenotype in vivo and increases sensitivity to a variety of drugs, particularly PARP inhibitor therapy, enhancing Olaparib therapy over 20-fold in vitro. In an independent doxycycline inducible knockdown model, HuR depletion combined with Olaparib treatment resulted in a 9.3 fold (p<.001) decrease in tumor volume in vivo. Based on these findings and our previous published work, we have launched into a line of investigation attempting to target HuR in pancreatic cancer cells. We have delivered siHuR oligos tethered to 3DNA nanostructures (Genisphere, LLC) in a targeted fashion using either a folic acid targeting moiety, a transferrin receptor targeted antibody, or an EGFR targeted antibody (i.e., siHuR nanotherapy). Additionally, we are working to combine siHuR nanotherapy with conventional therapeutics. 3DNA nanocarriers are constructed using stable DNA strands assembled and crosslinked in a stepwise fashion, to create a layered nanoscaffold capable of attaching multiple different targeting moieties and deliverables. The siRNA oligos hybridized to the 3DNA were modified in order to withstand systemic RNases during treatment. We first validated the modified siHuR nanotherapy in vitro by evaluating protein and RNA levels of HuR. Second, we validated the expression of our intended targeting moieties in both human cell lines and a tissue microarray consisting of patient derived pancreatic cancer tumors. For the mouse studies, athymic female nude mice were injected with 4 million MIA PaCa-2 cells per flank, and randomized to treatment arms when tumors reached 100mm3. Mice were then treated intraperitoneally twice weekly with either siHuR or siControl nanotherapies (3ug siRNA per treatment) with one of the three targeting moieties. We demonstrated efficacy of systemic targeted delivery of siHuR nanotherapy which improved average overall survival by 35% (p=.012) compared to the control arm, with an increase in survival using a Kaplan-Meier survival analysis (p=.01). We are currently setting up treatments with siHuR nanotherapy in conjunction with a targeted treatment strategy, Olaparib, which has previously been shown to work in conjunction with dox-inducible HuR silencing (i.e.,shRNA targeting) in vivo. Herein, we describe the development of siHuR nanotherapy that, as a monotherapy or most likely with clinically available drugs, may improve outcomes for patients with pancreatic cancer. Citation Format: Chris W. Schultz, Kevin O'Hayer, Kathryn M. Bormes, Teena Dhir, Samantha Brown, Avinoam Nevlar, Saswati Chand, Henry Thomsett, Wei Jiang, Jessica Bowers, Kelly Rhodes, Michael Pishvaian, Robert Getts, Jonathan Brody. Re-sensitizing Pancreatic Cancer, targeted siRNA inhibition of HuR [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B19.
HuR is an RNA binding protein involved in a coordinated cellular response to stressors. Upon insults such as chemotherapy or radiation treatment, HuR translocates from the nucleus to the cytoplasm where it binds the 3'UTR of target mRNAs. HuR's interaction with target mRNAs leads to the upregulation of target genes and ultimately treatment resistance. This is particularly relevant in the case of pancreatic ductal adenocarcinoma (PDA). PDA is highly resistant to standard chemotherapy such as FOLFIRINOX or gemcitabine/nab-paclitaxel. Using a tumor microarray (TMA), we found 79% of patient tumor samples (n=70) were positive for active cytoplasmic HuR, while little to no cytoplasmic localization was detected in normal tissue. In addition, HuR CRISPR knockout cell lines have a xenograft lethal phenotype. Previously published data also demonstrated that reduction of HuR in xenografts with a DOX-inducible shRNA system caused a 3.6 fold decrease in tumor size. HuR reduction was also shown to potentiate a PARP-inhibitor (olaparib) treatment from a 5.6-fold reduction alone to 9.3-fold reduction in tumor size when combined with shHuR, demonstrating the role of HuR in drug resistance. The aim of our current study was: A) to target HuR directly using nanoparticle delivery of siRNA against HuR; and B) to use the FDA approved small molecule pyrvinium pamoate (PP) to inhibit HuR's translocation and sensitize PDA cells to concurrent therapies. Using 3DNA, a 60nm nanoparticle composed of a sphere of crosslinked DNA, we have successfully delivered siRNA against HuR in vivo utilizing targeting moieties against receptors known to be overexpressed on the surface of PDA cells: EGFR, folic acid receptor, and transferrin receptor. Bi-weekly IP treatment of siHuR bound to 3DNA was safe and effective at extending life in a xenograft model as indicated by Kaplan Meier analysis (p=0.01). We are currently testing siHuR-3DNA dendrimer therapy's ability to sensitize PDA cells to oxaliplatin or olaparib in vivo. We are also investigating the use of PP to target HuR's localization. PP has previously been shown in bladder cancer to inhibit the translocation of HuR in vitro and in vivo. We have reproduced this finding in PDA cells, and have shown impressive drug potency with IC50s as low as 38nM in 2D cultures and 16nM in a 3D mouse PDA organoid model. Combination index (CI) values determine drugs interactions where 1 is additive, <1 is synergistic and >1 is antagonistic. We have determined that PP can enhance therapies such as gemcitabine (CI of 0.55), olaparib (CI of 0.40) and palbociclib (CI of 0.37) in vitro, and are currently validating these findings in vivo. Taken together, our data demonstrate that HuR inhibition via 3DNA delivery of siHuR and/or PP treatment can sensitize PDA cells to chemotherapy and targeted therapies. By inhibiting a resistance driver (HuR) in pancreatic cancer we aim to improve current therapies for this devastating disease. Citation Format: Christopher W. Schultz, Kevin O'Hayer, Teena Dhir, Oloruntoba Bolaji, Kathryn M. Bormes, Samantha Z. Brown, Henry Thomsett, Saswati Chand, Aditi Jain, Wei Jiang, Grace McCarthy, Charles J. Yeo, Austin Goetz, Avinoam Nevler, Jonathan R. Brody, Jordan M. Winter, Ranjan Preet, Dan Dixon, Jessica Bowers, Kelly Rhodes, Robert Getts. Gaps in the armor: Targeting HuR to sensitize pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1961.
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