Abstract B52: Therapeutic targeting of human KRAS in pancreatic cancer using a novel method of gene-silencing: U1 adaptors

Abstract

Abstract Background: While genetic knockdown of RAS in mouse tumor models has substantiated it as a therapeutic target, there is no effective means of targeting RAS currently available in the clinic today. Numerous RNA interference-based studies targeting RAS have demonstrated therapeutic effects, however, effective delivery has been a major obstacle that has impeded this approach. U1 Adaptors are a novel technology for oligonucleotide-mediated gene silencing that act by selectively interfering with polyadenylation of messenger RNA (mRNA) inside the cell nucleus. Polyadenosine (PolyA) tail addition is an obligatory step in mRNA maturation and function, and its failure results in rapid degradation of the nascent message by endogenous nucleases. The eukaryotic U1 small nuclear ribonucleoprotein complex (U1 snRNP) is best known for its role as a pre-mRNA splicing factor, but also acts naturally to silence some genes by suppressing polyadenylation. U1 Adaptors are synthetic oligonucleotides that enable the U1 snRNP complex to stably bind to the terminal exon of any chosen pre-mRNA target, thereby interfering with polyA tail addition and causing it to be selectively degraded in the nucleus. The silencing mechanism of U1 Adaptors is distinct from those of siRNA or antisense oligonucleotides and this distinction confers an important advantage for their use as therapeutic agents. We have validated this technology in vivo demonstrating an 85% tumor growth inhibition by targeting BCL-2 and GRM-1 in human melanoma xenografts. Our in vivo proof-of-concept study relied on delivery of the U1 Adaptors non-covalently complexed with a nanoparticle comprised of a positively charged dendrimer covalently linked to a cyclic penta-peptide containing Arginine-Glycine-Aspartate referred to as the cRGD peptide, a widely-used tumor-targeting moiety. Methods/Results: We sought to translate the U1 Adaptor technology to target human KRAS. We first designed a set of U1 Adaptors for screening purposes targeting human KRAS at eight different positions along the human KRAS pre-mRNA located at the junction of the terminal exon (position 632) and untranslated region (UTR). We have evaluated these adaptors in vitro using the human pancreatic cancer cell line MIA Paca-2 (KRASG12D). Screening of these eight U1 Adaptors reveals a range of KRAS gene silencing as measured by quantitative PCR. Notably, Adaptors 2 and 3 silenced KRAS down to 27 and 24% respectively, as effective as the siRNA control. We then evaluated Adaptors 2 and 3 in human MIA Paca-2 xenografts. These adaptors were coupled to the cRGD-nanoparticle complex and administered by tail vein injection twice weekly. We observed significant tumor growth inhibition (37.3% by KRAS Adaptor 2, p=0.025, and 68.3% by KRAS Adaptor 3, p=0.0002, as compared to the vehicle control by Day 34. We also observed significant tumor growth inhibition with a U1 Adaptor targeting BCL-2 albeit to a lesser extent than KRAS Adaptor 3. Conclusion: We have demonstrated that the U1 Adaptor method of gene silencing can be successfully applied to target human KRAS both in vitro and in vivo. These results support the continued investigation of U1 Adaptor technology as a strategy for therapeutic targeting of RAS oncogenes. Citation Format: Ashley T. Tsang, Xin Yu, Rafal Goraczniak, Mark Brenneman, Samuel Gunderson, Darren R. Carpizo. Therapeutic targeting of human KRAS in pancreatic cancer using a novel method of gene-silencing: U1 adaptors. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B52. doi: 10.1158/1557-3125.RASONC14-B52