Abstract P6-04-09: Dual PI3K and Wnt pathway inhibition is a synergistic combination against triple negative breast cancer
Authors: Solzak JP , Atale R , Hancock BA , Radovich M 
Published: August 2014

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), 20 microdissected normal breast tissues (Komen Tissue Bank), and 10 adjacent normal tissues (TCGA), were merged and imported into Partek Genomics Suite. The merged transcript RPKMs were transformed, batch effect corrected, and analyzed for differential expression. Statistically significant genes were imported into Ingenuity Pathway Analysis (IPA) to identify therapeutic targets. For cell based studies, we tested a panel of eight PI3K/AKT/mTOR inhibitors that are either currently in clinical trials against a panel of seven TNBC cell lines. To further enhance anti-tumor efficacy, we then tested these TNBC cell lines using increasing concentrations of FH535, a WNT pathway inhibitor. Cell viability was assessed via Celltiter-Fluor.
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 and Wnt pathways. To assess anti-tumor efficacy against these pathways, we tested eight PI3K/AKT/mTOR inhibitors, and observed significant reductions in cell viability with all inhibitors. Because of the reported clinical efficacy of BKM120, a pan-isoform PI3K inhibitor, we focused our cell viability experiments with BKM120 and observed an average IC50 of 0.7uM across TNBC cell lines. We then tested FH535, a Wnt pathway inhibitor, and observed an average IC50 of 4.86μM. All TNBC cell lines were then treated with 1, 10, and 100nM BKM120 in combination with increasing doses of FH535 starting at 1nM. At 10nm BKM120, we observed significant synergy with FH535 at low doses. The average IC50 of FH535 when combined with 10nm BKM120 was 4.5nM across cell lines. While variation in sensitivity was seen across cell lines, the highest FH535 IC50 for the combination was 10nM in MDA-MB-453 cells, still demonstrating significant synergy at low doses. RNA-seq analyses of TNBC cells after treatment with BKM120 to understand the nature of this synergy are currently underway.
Conclusion: Next-generation RNA-sequencing has identified PI3K/mTOR/AKT and Wnt pathways as key targets for the treatment of TNBC and that inhibition of these pathways using small molecule inhibitors in combination is highly synergistic.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-09.