262. Therapeutic Suppression of the KRAS-MYC Oncogenic Axis in Human Pancreatic Cancer Xenografts with U1 Adaptor Oligonucleotide / RGD Peptide Conjugates

Abstract

U1 Adaptors are synthetic oligonucleotides that enable the U1 small nuclear ribonucleoprotein (U1 snRNP) complex to stably bind within the terminal exon of a specific pre-mRNA. This interferes with the obligatory polyadenylation step in mRNA maturation, causing selective destruction of the targeted mRNA inside the nucleus. In contrast to siRNA or antisense oligos, U1 Adaptors can accept extensive covalent modifications for nuclease resistance, targeted delivery or in-vivo imaging without loss of silencing activity, offering important advantages as therapeutic agents. A panel of candidate U1 Adaptors targeting human KRAS (KRAS Adaptors) was screened in vitro using the human pancreatic cancer cell line MIA-PaCa2. The best candidates reduced KRAS mRNA expression by up to 76% - as effectively as siRNA controls. Reduced KRAS protein expression was confirmed by western blot. Inhibition of cell growth in vitro and increased apoptosis were seen for both the MIA-PaCa2 (KRASG12C) and Panc1(KRASG12D) cell lines, but not in BxPC3, a KRASwildtype pancreatic cancer cell line. In a parallel project, U1 Adaptors targeting human MYC mRNA were designed and screened in B-cell lymphoma lines, where the best candidates reduced MYC mRNA levels by over 95%. Because of the observed relationship between activating KRAS mutation and MYC overexpression in pancreatic cancers, MYC Adaptors were tested in MIA-PaCa2 cells. MYC Adaptors also inhibited cell growth and increased apoptosis in vitro. U1 Adaptors were tested for efficacy in mice bearing subcutaneous MIA-PaCa2 xenograft tumors. For in-vivo delivery, Adaptor oligos were directly conjugated to a cyclic RGD-motif peptide (cRGD), which is a targeting ligand for specific integrin-family receptors overexpressed on parenchyma and endothelial cells of many solid tumors. Alternately, U1 Adaptor oligos were linked to “internalizing” RGD (iRGD), a variant RGD peptide that also triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin-1. KRAS Adaptors linked to cRGD or iRGD were administered by tail vein injections twice weekly for three to four weeks. Over a series of experiments, tumor growth was inhibited by averages of 68% to 93%. Tumor stasis or regression occurred in some treated mice. In a pilot study, MYC Adaptors conjugated to iRGD peptide were also highly effective in suppressing tumor growth and inducing tumor regression. Excised tumors were analyzed by qPCR and western blot, which confirmed reductions of the targeted mRNAs and proteins. We have shown that U1 Adaptors conjugated to tumor-targeting / tumor-penetrating peptides can effectively target human KRAS and MYC oncogenes in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS, MYC and possibly other oncogene targets in pancreatic cancer.