Proteomic analysis reveals Src/Stat and EGFR/MAPK pathways as potential mechanism of resistance to PI3K inhibitors in lung cancer Maria Angelica Cortez1, Lauren Averett Byers1, You Hong Fan1, Lixia Diao2, Philip Groth3, Julianne Paul3, Jing Wang2, Uma Giri1, Jayanthi Gudikote1, Hai T. Tran1, Kevin Coombes2, John D. Minna4, Ningshu Liu3, John V. Heymach1. 1Department of Thoracic and Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston,Texas, 2Department of Bioinformatics and Computational Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, 3Bayer Pharma AG, Berlin, Germany, 4Hamon Center for Therapeutic Oncology, University of Texas Southwestern Medical Center, Dallas, Texas Background The phosphoinositide 3-kinase (PI3K) signaling is the most commonly dysregulated pathway in many human cancers, including non-small cell lung cancer (NSCLC). Therefore, targeting PI3K signaling has become a promising approach in cancer therapy. Although studies have indicated development of resistance to PI3K inhibitors, the molecular mechanism of drug resistance is largely unknown in NSCLC. The goal of this study was to identify new potential mechanism of resistance to PI3K inhibitors. Figure 1. Protein profiling by reverse phase protein array (RPPA) identifies pathways and proteins dysregulated in NSCLC cell lines treated with PI3K inhibitor BAY 80-6946. C. Figure 5. Proposed mechanism of resistance to PI3K inhibitors. Suppression of mTOR pathway by PI3K inhibition upregulates Src/Stat pathway to promote cell survival. PI3K/ mTOR pathway Src/Stat EGFR/MAPK Her2.pY1248 EGFR Stat5.pY694 Src.pY416 Stat3.pY705 Met Met.pY1234.1235 Axl.pY702 mTOR.p2448 GSK3a.b.pS21.9 S6.pS240.244 S6.pS235.236 p70S6K.pT389 Akt.pT308 Akt.pS473 TSC2.pT1462 X4EBP1.pT70 X4EBP1.pS65 X4EBP1.pT37.46 Figure 3. Our analysis identified Src/Stat and EGFR/MAPK pathways as potential mechanism of resistance upon PI3K inhibition. Methods We used reverse-phase protein array (RPPA) to assess the expression levels and activation status of 137 proteins involved in signaling pathways implicated in lung cancer. Seventy-four NSCLC cell lines were treated with the PI3K inhibitor BAY 80-6946 and lysates were collected for proteomic analysis. Paired t-test was applied to each marker comparing the difference between drug and control treated cells. We evaluated the association between drug sensitivity (IC50) and RPPA expression levels at baseline in sixty NSCLC cell lines. Correlation test was applied to each marker vs drug and Wilcox Rank test was performed to compare the smallest and largest one third of all the cell lines. Conclusions Our results suggest that PI3K inhibition leads to EGFR/MAPK and Src/Stat signaling activation in vitro, suggesting a compensatory pathway that may allow NSCLC survival. Taken together, these results suggest that activation of Src/Stat pathway as an important mechanism of resistance to PI3K inhibitors in NSCLC and may be clinically relevant targets for combination therapy. Figure 2. Treatment with BAY 80-6946 significantly downregulates downstream effectors (A and B) and upregulates negative regulators (C) of mTOR pathway. A. Figure 4. Association between drug sensitivity (IC50) and RPPA expression levels at baseline. Acknowledgements This work was supported by UTSW and MDACC Lung SPORE 5 P50 CA070907; DoD PROSPECT W81XWH-07-1-0306; CCSG grant 5 P30 CA016672; Chapman Fund for Bioinformatics in Personalized Cancer Therapy, 1 U24 CA143883; Bayer Pharmaceuticals. B. A549 H441 H460 Calu1 + - + - + - + - SF FS PI3Ki (1μM) S6.pS240.244 TSC2.pT1462 p70S6K.pT389 Vinculin