Abstract P6-13-09: Dual PI3K and Wnt pathway inhibition is a synergistic combination against triple negative breast cancer
Authors: Solzak JP , Atale R , Hancock B , Radovich M 
Published: May 2016

Abstract
Introduction: Triple negative breast cancer (TNBC) accounts for 15% of all breast cancer cases in the United States, and despite its lower incidence, contributes to a disproportionately higher rate of morbidity and mortality compared to other breast cancer subtypes. Because these tumors lack expression of the estrogen, progesterone, or HER-2 receptors ("triple negative"), TNBC patients do not respond to targeted therapies that have been successfully used against tumors that over-express these proteins. Thus, there exists a critical need to improve the outcomes of TNBC patients through the implementation of novel targeted agents.
Methods: RNA-seq data from 94 TNBCs (from Indiana University and TCGA) and 20 microdissected normal breast tissues (Komen Tissue Bank) were merged and analyzed using Partek Genomics Suite. Statistically significant genes were imported into Ingenuity Pathway Analysis (IPA) to identify therapeutic targets. For in vitro studies, we tested a panel of TNBC cell lines using Buparlisib (a PI3K pathway inhibitor) and WNT974 (a WNT pathway inhibitor), individually and in combination. Cell viability was assessed via Celltiter-Fluor. Synergy between the two drugs was calculated using the Chou-Talalay method. In vivo studies were performed using the TMD-231 cell line and a patient derived xenograft (PDX) from the Jackson Laboratory. Dosing of the mice was performed using 30 mg/kg and 3 mg/kg of Buparlisib and WNT974 respectively, both in combination and individually.
Results: Using next-generation RNA sequencing data of TNBCs and microdissected normal breast tissue, Ingenuity Pathway Analysis identified over-expression and hyper-activation of the PI3K/AKT/mTOR pathway. When treating cell lines with the PI3K inhibitor Buparlisib, RNA sequencing revealed compensatory induction of the WNT pathway across TNBC cell lines. In particular, we observed induction of Porcupine, a Wnt maturation protein, which is the protein target of the Wnt pathway inhibitor, WNT974. When anti-tumor efficacy against these pathways was assessed, a significant reduction in cell viability using Buparlisib and WNT974 in combination was observed. Using the Chou-Talalay method, we found for MDA-MB-231 and Hs578T cell lines, a ∼50% reduction in cell viability at 100nM concentration of each drug that was highly synergistic (Combination Index = 0.33, and 0.36 respectively). In our in vivo PK study of the combination, there was no drug-drug interaction observed. Furthermore, in our PD analysis using a TMD-231 xenograft, we observed a ∼40% reduction in tumor volume after seven days. In a subsequent in vivo PDX experiment, we observed an 80% survival rate using combination therapy compared to 40% when administering the drugs individually or with vehicle control after 30 days.
Conclusion: PI3K/mTOR/AKT and Wnt pathways are strong candidates for the development of novel targeted agents for TNBC. Using two small molecule inhibitors that are currently in clinical trials as single agents (Buparlisib and WNT974) we observe significant in vitro synergy when inhibiting both pathways at low nanomolar doses. Furthermore, confirmatory in vivo xenograft studies display a similar synergy for the combination compared to single agent and vehicle controls.
Citation Format: Solzak JP, Atale R, Hancock B, Radovich M. Dual PI3K and Wnt pathway inhibition is a synergistic combination against triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-09.