694. Targeting KRAS in Pancreatic Cancer by Gene Silencing with U1 Adaptors

Abstract

Activating mutations of the KRAS gene are key drivers of pancreatic cancer, but the KRAS protein has been refractory to small-molecule drugging. U1 Adaptors are oligonucleotides that enable the U1 small nuclear ribonucleoprotein 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 in the nucleus. Unlike siRNA or antisense oligos, U1 Adaptors can be extensively modified for nuclease resistance or targeted delivery without loss of silencing activity, offering important advantages as therapeutic agents.We sought to translate U1 Adaptor technology to suppress KRAS in pancreatic cancer. A panel of U1 Adaptors targeting human KRAS (KRAS Adaptors) was screened in vitro using the human pancreatic cancer cell line MiaPaca-2 (KRAS G12D mutant). Candidate KRAS Adaptors reduced KRAS mRNA expression by up to 76%, as effectively as an siRNA control. Knockdown of KRAS protein expression was confirmed by western blot. Inhibition of cell growth in vitro was demonstrated for MiaPaca-2 and two additional pancreatic cancer cell lines, Panc1 (KRAS G12D mutant) and BXPC3 (KRAS wildtype).We evaluated KRAS Adaptors in mice bearing subcutaneous MiaPaca-2 xenograft tumors. For in-vivo delivery, the Adaptors were initially complexed with PAMAM dendrimers linked to a tumor-targeting, cyclic RGD peptide (cRGD), and administered by tail vein injection twice weekly for three weeks. Tumor growth was inhibited by as much as 68% compared to vehicle controls (p=0.0002). Excised tumors were analyzed by qPCR and IHC, which confirmed reductions of KRAS mRNA and protein.Although U1 Adaptors complexed with cRGD-dendrimers have been efficacious in this study and others, dendrimers have technical drawbacks and reported toxicities, and dendrimer-free formulations are preferable. As an alternative, we conjugated KRAS Adaptorsdirectly to cRGD peptide or to internalizing RGD (iRGD), which is a variant RGD peptide that triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin. The cRGD- and iRGD-conjugated KRAS Adaptors were tested for efficacy against subcutaneous MiaPaca-2 xenografts, and tumor growth was inhibited to equal or greater extent as with the original cRGD-dendrimer system. iRGD may be of particular benefit for pancreatic adenocarcinomas, which have a densely fibrotic stroma that impedes drug delivery. Enhanced delivery of small- and large-molecule therapeutics into primary pancreatic adenocarcinomas in KPC mice has been achieved previously by conjugating or coinjecting iRGD peptide.We have shown that U1 Adaptors can successfully target human KRAS both in vitro and in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS in pancreatic cancer.