S. Liu1, J. Yu1, R. Damoiseaux2, R. Sanchez1, F. Brunicardi1 1University Of Toledo Medical Center,Department Of Surgery,Toledo, OH, USA 2David Geffen School Of Medicine, University Of California At Los Angeles,Molecular Screening Shared Resource,Los Angeles, CA, USA
Introduction: Precision cancer therapy requires matching of the tumor’s genomic profile to targeted therapy. Development of a novel targeted therapy takes years and is associated with tens of millions of dollars and low probability of success. In this study, we have tested the hypothesis that genomic profiling using RNA Seq and high throughput screening (HTS) of an FDA approved drug library using a target gene super-promoter can provide precision cancer therapy for pancreatic adenocarcinoma (PDAC) using repurposed FDA approved drugs.
Methods: RNA-Seq and Weighted Gene Coexpression Network Analysis (WGCNA) were performed on 45 human PDAC vs matched benign pancreatic specimens and networks of target genes (TG) were identified. BIRC5 was selected as the target gene for generation of a BIRC5 super promoter (BIRC5-SP). HTS of a 2000 FDA approved drug library was performed using PDAC cells stably transfected with BIRC5-SP-firefly luciferase reporter (Luc). Single vs combination of selected drugs that inhibited BIRC5-SP-Luc activity were further tested in vitro and in vivo using patient derived PDAC cell lines (PDCL 5 & 15) and commercial PDAC cell lines (PANC-1, Mia PaCa2, Capan-2, AsPC1). CMV-Gaussia luciferase stably transfected PDAC cells were used in vivo in xenograft mice to monitor tumor volume by bioluminescence imaging before and after one month of therapy ± standard chemotherapies. IHC, TUNEL assay and RNA-Seq were used for analysis of PDAC gene expression patterns before and after therapy.
Results: 14 BIRC5-SPs were generated and the optimal BIRC5-SP was selected for further studies. BIRC5-SP-Luc HTS revealed panels of FDA-approved BIRC5 inhibitory drugs and three were selected for further dose response testing in PDAC cells. Based on extensive in vitro testing, and a list of criteria to enhance translational potential, simvastatin, metformin and digoxin, (C3), were determined to be the optimal combination and then tested in three in vivo studies using PDAC xenograft mouse models. Following therapy: 1) both PDCL and commercial PDAC tumor growth was significantly suppressed vs controls (p<0.05), 2) the effect of chemotherapy on tumor suppression was significantly enhanced (p<0.05), 3) tumors displayed complete suppression of BIRC5 expression and increased apoptosis, 4) there was no toxicity in any mice. RNA Seq of PDAC tumors revealed a) decreased cell proliferation gene expression, b) decreased ATP/energy gene expression and c) markedly increase cell death gene expression after C3 therapy.
Conclusions: RNA Seq and WGCNA identified BIRC5 as a PDAC target gene. BIRC5-SP HTS identified C3, which suppressed BIRC5 expression and PDAC tumor growth in mice, and enhanced the effect of chemotherapy. Our study demonstrates a feasible cost effective strategy of repurposing FDA-approved drugs for targeted precision PDAC therapy using a super-promoter of the target gene in a clinically relevant timeframe.