5Targeting Pin1 in cholangiocarcinoma tumor and stromal cells using siRNA therapeutics
Justin H Lo, Thatcher R Heumann, Nora Francini, Eva F Gbur, Michelle Zhou, Shabnam Z Eghbali, Laura W Goff, Craig L DuvallAbstract
Background and Objectives
Pin1 (peptidyl-prolyl isomerase, NIMA-interacting 1) overexpression in tumor cells and stroma has been associated with poor clinical outcomes and an immunosuppressive tumor microenvironment in pancreatic cancer. Hypothesizing that Pin1 could have a similar role in cholangiocarcinoma (CCA), we sought to investigate the therapeutic potential of silencing Pin1 using small interfering RNA (siRNA), leveraging a tumor-tropic siRNA-lipid conjugate design termed siRNA-L2.
Methods
siRNA/siRNA-L2 sequences were synthesized on a MerMade oligonucleotide synthesizer and dosed in vitro at 100 nM (with lipofectamine) or 1 μM (alone), respectively. The KRAS-p19 transgenic mouse model of intrahepatic CCA was established using hydrodynamic tail vein injection of sleeping beauty transposase, KRAS-G12D transposon, and CRISPR/Cas9+sg p19 plasmids; cancer-associated fibroblasts (CAFs) were disaggregated and isolated from this model. siRNA-L2 was dosed in vivo at 10-20 mg/kg intravenously twice/week; chemoimmunotherapy (gemcitabine, cisplatin, and anti-PD-L1 antibody) was dosed intraperitoneally twice/week. siRNA distribution was assessed using RNAscope in situ hybridization.
Results
We designed and screened siRNA sequences targeting Pin1 mRNA, with a lead sequence identified that mediates robust knockdown (>95%) as transfected siRNA and as siRNA-L2 conjugates. Pin1 siRNA slows CCA CAF proliferation in vitro. We have shown that siRNA-L2 accumulates in tumor cells and stroma in mouse models of CCA, including xenografts and transgenic tumor models. In preliminary studies, siRNA-L2 targeting Pin1 is well-tolerated at therapeutic dosing and results in tumor necrosis. Studies comparing the safety of Pin1 siRNAL2 versus alternate methods of targeting Pin1 (all-trans retinoic acid and sulfopin) are underway. Additionally, a larger study comparing the effectiveness of standard chemoimmunotherapy with or without Pin1 siRNA-L2 using the immunocompetent KRAS-p19 model is in progress. These results are anticipated by the time of the CCF Annual Conference. Finally, we are quantifying Pin1 expression by immunohistochemistry in a clinically-annotated human tumor microarray of CCA cases at our institution to determine associations with treatment responses and outcomes.
Conclusions
We have developed siRNA therapeutics that silence Pin1, distribute readily into tumor cells and stroma, and are well-tolerated in mouse models of CCA. Results from additional safety and therapeutic studies in models of CCA are forthcoming.