Table S1 Temsirolimus and BEZ235 IC50 and combination index (CI) for combined temsirolimus and BEZ235 treatment in the panel of eight endometrial cancer cell lines. (TIF) Table S2 Panel of molecular inhibitors explored for combina- tion therapy with temsirolimus. (TIF) References 1. Engelman JA (2009) Targeting PI3K signalling in cancer: opportunities, challenges and limitations. Nat Rev Cancer 9: 550–562. 2. Liu P, Cheng H, Roberts TM, Zhao JJ (2009) Targeting the phosphoinositide 3- kinase pathway in cancer. Nat Rev Drug Discov 8: 627–644. 3. Yap TA, Garrett MD, Walton MI, Raynaud F, de Bono JS, et al. (2008) Targeting the PI3K-AKT-mTOR pathway: progress, pitfalls, and promises. Curr Opin Pharmacol 8: 393–412. 4. Cheung LWT, Hennessy BT, Li J, Yu S, Myers AP, et al. (2011) High frequency of PIK3R1 and PIK3R2 mutations in endometrial cancer elucidates a novel mechanism for regulation of PTEN protein stability. Cancer Discovery 1: 170–185. 5. Ma XM, Blenis J (2009) Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol 10: 307–318. 6. Guertin DA, Sabatini DM (2009) The pharmacology of mTOR inhibition. Sci Signal 2: pe24. 7. Wang X, Sun SY (2009) Enhancing mTOR-targeted cancer therapy. Expert Opin Ther Targets 13: 1193–1203. 8. Breuleux M, Klopfenstein M, Stephan C, Doughty CA, Barys L, et al. (2009) Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition. Mol Cancer Ther 8: 742–753. 9. Marone R, Erhart D, Mertz AC, Bohnacker T, Schnell C, et al. (2009) Targeting melanoma with dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitors. Mol Cancer Res 7: 601–613. 10. Hsieh AC, Costa M, Zollo O, Davis C, Feldman ME, et al. (2010) Genetic dissection of the oncogenic mTOR pathway reveals druggable addiction to translational control via 4EBP-eIF4E. Cancer Cell 17: 249–261. 11. Janes MR, Limon JJ, So L, Chen J, Lim RJ, et al. (2010) Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor. Nat Med 16: 205–213. 12. Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J (2008) Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. Proc Natl Acad Sci U S A 105: 17414–17419. 13. Bansal N, Yendluri V, Wenham RM (2009) The molecular biology of endometrial cancers and the implications for pathogenesis, classification, and targeted therapies. Cancer Control 16: 8–13. 14. Salvesen HB, Carter SL, Mannelqvist M, Dutt A, Getz G, et al. (2009) Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation. Proc Natl Acad Sci U S A 106: 4834–4839. 15. Delmonte A, Sessa C (2008) Molecule-targeted agents in endometrial cancer. Curr Opin Oncol 20: 554–559. 16. O’Reilly KE, Rojo F, She QB, Solit D, Mills GB, et al. (2006) mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 66: 1500–1508. 17. Kanamori Y, Kigawa J, Itamochi H, Shimada M, Takahashi M, et al. (2001) Correlation between loss of PTEN expression and Akt phosphorylation in endometrial carcinoma. Clin Cancer Res 7: 892–895. 18. Mutter GL, Lin MC, Fitzgerald JT, Kum JB, Baak JP, et al. (2000) Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers. J Natl Cancer Inst 92: 924–930. 19. Tashiro H, Blazes MS, Wu R, Cho KR, Bose S, et al. (1997) Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignancies. Cancer Res 57: 3935–3940. 20. Neshat MS, Mellinghoff IK, Tran C, Stiles B, Thomas G, et al. (2001) Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci U S A 98: 10314–10319. 21. Steelman LS, Navolanic PM, Sokolosky ML, Taylor JR, Lehmann BD, et al. (2008) Suppression of PTEN function increases breast cancer chemotherapeutic drug resistance while conferring sensitivity to mTOR inhibitors. Oncogene 27: 4086–4095. 22. Albitar L, Pickett G, Morgan M, Davies S, Leslie KK (2007) Models representing type I and type II human endometrial cancers: Ishikawa H and Hec50co cells. Gynecol Oncol 106: 52–64. 23. Gupta M, Ansell SM, Novak AJ, Kumar S, Kaufmann SH, et al. (2009) Inhibition of histone deacetylase overcomes rapamycin-mediated resistance in diffuse large B-cell lymphoma by inhibiting Akt signaling through mTORC2. Blood 114: 2926–2935. mTOR and PI3 Kinase Inhibitor Synergy Acknowledgments We thank Kristina W. Thiel for assistance in manuscript preparation. We also thank Justin Fishbaugh in the University of Iowa Flow Cytometry Core Facility for assistance in data acquisition and analysis. Author Contributions Conceived and designed the experiments: SY XM KKL. Performed the experiments: SY XX. Analyzed the data: SY XX XM KKL. Wrote the paper: SY KKL. 24. Wang X, Yue P, Kim YA, Fu H, Khuri FR, et al. (2008) Enhancing mammalian target of rapamycin (mTOR)-targeted cancer therapy by preventing mTOR/ raptor inhibition-initiated, mTOR/rictor-independent Akt activation. Cancer Res 68: 7409–7418. 25. Jin N, Jiang T, Rosen DM, Nelkin BD, Ball DW (2009) Dual inhibition of mitogen-activated protein kinase kinase and mammalian target of rapamycin in differentiated and anaplastic thyroid cancer. J Clin Endocrinol Metab 94: 4107–4112. 26. Yazbeck VY, Buglio D, Georgakis GV, Li Y, Iwado E, et al. (2008) Temsirolimus downregulates p21 without altering cyclin D1 expression and induces autophagy and synergizes with vorinostat in mantle cell lymphoma. Exp Hematol 36: 443–450. 27. Maira SM, Stauffer F, Brueggen J, Furet P, Schnell C, et al. (2008) Identification and characterization of NVP-BEZ235, a new orally available dual phosphati- dylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Mol Cancer Ther 7: 1851–1863. 28. Fu L, Kim YA, Wang X, Wu X, Yue P, et al. (2009) Perifosine inhibits mammalian target of rapamycin signaling through facilitating degradation of major components in the mTOR axis and induces autophagy. Cancer Res 69: 8967–8976. 29. Degtyarev M, De Maziere A, Klumperman J, Lin K (2009) Autophagy, an Achilles’ heel AKTing against cancer? Autophagy 5: 415–418. 30. Brachmann SM, Hofmann I, Schnell C, Fritsch C, Wee S, et al. (2009) Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells. Proc Natl Acad Sci U S A 106: 22299–22304. 31. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G (2007) Self-eating and self- killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8: 741–752. 32. Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, et al. (2004) LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci 117: 2805–2812. 33. Werzowa J, Koehrer S, Strommer S, Cejka D, Fuereder T, et al. (2011) Vertical inhibition of the mTORC1/mTORC2/PI3K pathway shows synergistic effects against melanoma in vitro and in vivo. J Invest Dermatol 131: 495–503. 34. Dan S, Yoshimi H, Okamura M, Mukai Y, Yamori T (2009) Inhibition of PI3K by ZSTK474 suppressed tumor growth not via apoptosis but G0/G1 arrest. Biochem Biophys Res Commun 379: 104–109. 35. Baumann P, Mandl-Weber S, Oduncu F, Schmidmaier R (2009) The novel orally bioavailable inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin, NVP-BEZ235, inhibits growth and proliferation in multiple myeloma. Exp Cell Res 315: 485–497. 36. Burris H, Rodon J, Sharma S, Herbst RS, Tabernero J, et al. (2010) First-in- human phase I study of the oral PI3K inhibitor BEZ235 in patients (pts) with advanced solid tumors. J Clin Oncol 28: abstr 3005. 37. Yaguchi S, Fukui Y, Koshimizu I, Yoshimi H, Matsuno T, et al. (2006) Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor. J Natl Cancer Inst 98: 545–556. 38. Terakawa N, Kanamori Y, Yoshida S (2003) Loss of PTEN expression followed by Akt phosphorylation is a poor prognostic factor for patients with endometrial cancer. Endocr Relat Cancer 10: 203–208. 39. Konecny GE, Venkatesan N, Yang G, Dering J, Ginther C, et al. (2008) Activity of lapatinib a novel HER2 and EGFR dual kinase inhibitor in human endometrial cancer cells. Br J Cancer 98: 1076–1084. 40. Shoji K, Oda K, Nakagawa S, Hosokawa S, Nagae G, et al. (2009) The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas. Br J Cancer 101: 145–148. 41. Oda K, Okada J, Timmerman L, Rodriguez-Viciana P, Stokoe D, et al. (2008) PIK3CA cooperates with other phosphatidylinositol 39-kinase pathway muta- tions to effect oncogenic transformation. Cancer Res 68: 8127–8136. 42. Garrido-Laguna I, Janku F, Tsimberidou A, Wheler J, Falchook GS, et al. (2010) Phosphatase and tensin homologue (PTEN) loss and response to phase I trials targeting PI3K/AKT/mTOR pathway in patients with advanced cancer. J Clin Oncol 28: abstr e13018. 43. Shoji K, Oda K, Nakagawa S, Ikeda Y, Kuramoto H, et al. (2010) Activity of dual PI3K/mTOR inhibitor, NVP-BEZ235, and mTOR inhibitor, RAD001 (everolimus), in endometrial cancer cell lines. J Clin Oncol 28: abstr 5074. PLoS ONE | www.plosone.org 12 October 2011 | Volume 6 | Issue 10 | e26343