A. T. Tsang1,6, L. Yi1, C. Dudgeon1, X. Yu1, R. Goraczniak5, S. Gunderson3,5, D. R. Carpizo1,2,4 6Mount Sinai St. Luke’s Roosevelt General Surgery Residency Program,New York, NY, USA 1The Cancer Institute Of New Jersey,New Brunswick, NJ, USA 2Rutgers University Robert Wood Johnson Medical School,Surgery,New Brunswick, NEW JERSEY, USA 3Rutgers University,Department Of Molecular Biology And Biochemistry,PIscataway, NEW JERSEY, USA 4Rutgers University,Department Of Pharmacology,Piscataway, NEW JERSEY, USA 5Silagene, Inc.,Hillsborough, NEW JERSEY, USA
Introduction:
One of the most significant unmet needs in pancreatic cancer therapy is targeting the most commonly mutated gene, KRAS, and its downstream mediator MYC. Small interfering RNA targeting KRAS has produced potent anti-tumor activity in preclinical studies, but technical difficulties of in-vivo delivery have impeded clinical translation. U1 Adaptors are a novel technology for oligonucleotide-mediated gene silencing that acts by blocking polyadenylation of messenger RNA. They can accommodate extensive covalent modifications for nuclease resistance, targeted delivery and in-vivo imaging without loss of silencing activity, offering important advantages over siRNA and antisense oligos as therapeutic agents. The KRAS-cMYC oncogenic axis plays a key role in the generation of self-renewing metastatic cells, making cMYC an attractive target in KRAS-driven pancreatic cancers. Genetic studies in pancreatic cancer mouse models validate the therapeutic efficacy of silencing KRAS and MYC expression.
Methods:
Candidate U1 Adaptors targeting KRAS were screened in vitro using the human pancreatic cancer cell line MIAPaCa2 (KRAS G12D mutant). The best Adaptors were applied in cell growth inhibition assays over 10 days in multiple pancreatic cancer cell lines. They were then tested for efficacy in mice bearing subcutaneous MIAPaCa2 xenograft tumors. For in-vivo delivery, Adaptors were covalently linked to a cyclic RGD-motif peptide (cRGD), a targeting ligand for integrin receptors expressed on tumor cells and endothelia, or alternately, internalizing RGD (iRGD), a variant peptide that triggers endothelial permeabilization and internalization by cells through neuropilin-1 binding. The cRGD- and iRGD-conjugated KRAS Adaptors were administered by tail vein injections twice weekly for 3 to 4 weeks once tumors reached 20mm3.. In parallel, U1 Adaptors targeting cMYC were screened in B-cell lymphoma lines and tested for efficacy in mice bearing MIA-PaCa-2 xenograft tumors using the same method.
Results:
The best KRAS Adaptors reduced KRAS mRNA expression by up to 76% – as effectively as an siRNA control. Knockdown of KRAS protein expression and its downstream effectors was confirmed by western blot. Cell growth inhibition was demonstrated for MIAPaCa2 and other established human pancreatic cancer cell lines in vitro. The potency of inhibition was dependent on mutant KRAS. Over a series of in vivo mice experiments, MIAPaCa2 xenograft tumor growth was inhibited by averages of 68% to 93% by cRGD- and iRGD-conjugated KRAS Adaptors as compared to vehicle-only controls. Tumor stasis or regression occurred in some treated mice. Remarkably, cMYC Adaptors were similarly effective in suppressing xenograft tumor growth.
Conclusion:
U1 Adaptors can successfully target human KRAS and cMYC in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS, cMYC and possibly other oncogenes in pancreatic cancer.