University of Dundee PDK1-SGK1 signaling sustains AKT-independent mTORC1 activation and confers resistance to PI3K inhibition Castel, Pau; Ellis, Haley; Bago, Ruzica; Toska, Eneda; Razavi, Pedram; Carmona, F. Javier Published in: Cancer Cell DOI: 10.1016/j.ccell.2016.06.004 Publication date: 2016 Licence: CC BY-NC-ND Document Version Peer reviewed version Link to publication in Discovery Research Portal Citation for published version (APA): Castel, P., Ellis, H., Bago, R., Toska, E., Razavi, P., Carmona, F. J., kannan, S., Verma, C. S., Dickler, M., Chandarlapaty, S., Brogi, E., Alessi, D., Baselga, J., & Scaltriti, M. (2016). PDK1-SGK1 signaling sustains AKT- independent mTORC1 activation and confers resistance to PI3K inhibition. 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This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ Cancer Cell PDK1-SGK1 signaling sustains AKT-independent mTORC1 activation and confers resistance to PI3Kα inhibition --Manuscript Draft-- Manuscript Number: CANCER-CELL-D-15-00569R7 Full Title: PDK1-SGK1 signaling sustains AKT-independent mTORC1 activation and confers resistance to PI3Kα inhibition Article Type: Research Article Keywords: breast cancer; PI3K pathway; kinase; Targeted Therapy; drug resistance; PDK1; SGK1; mTOR Corresponding Author: Maurizio Scaltriti New York, UNITED STATES First Author: Pau Castel Order of Authors: Pau Castel Haley Ellis Ruzica Bago Eneda Toska Srinivasaraghavan Kannan F. Javier Carmona Pedram Razavi Chandra S Verma Maura Dickler Sarat Chandarlapaty Edi Brogi Dario R Alessi José Baselga Maurizio Scaltriti Abstract: PIK3CA, the gene encoding the alpha isoform of PI3K (PI3Kα), is frequently mutated and oncogenic in breast cancer. PI3Kα inhibitors are in clinical development and despite promising early clinical activity, primary and acquired resistance is frequent among patients. We have previously reported that residual downstream mTORC1 activity upon treatment with PI3Kα inhibitors drives resistance to these agents. However, the underlying mechanisms that mediate this phenotype are not fully understood. Here we show that in cancer cells resistant to PI3Kα inhibition, PDK1 blockade restores sensitivity to these therapies. SGK1, which is activated by PDK1, contributes to the maintenance of residual mTORC1 activity and cell survival through direct phosphorylation and inhibition of TSC2. Targeting either PDK1 or SGK1 prevents mTORC1 activation and restores the antitumoral effects of PI3Kα inhibition in resistant cancer cells. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Suggested Reviewers: Brendan Manning, PhD Harvard T.H. Chan School of Public Health [email protected] Expert in studying cellular signaling events propagated by growth factors Ricardo Biondi Frankfurt University [email protected] Expert in PDK1 signaling and regulation Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Opposed Reviewers: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Cover Letter Maurizio Scaltriti, PhD Associate Attending Molecular Biologist Department of Pathology Associate Director of Translational Science Center for Molecular-Based Therapies Memorial Sloan Kettering Cancer Center June 6, 2016 Dear Dr. Su, Please find enclosed submission of our manuscript “PDK1-SGK1 signaling sustains AKT-independent mTORC1 activation and confers resistance to PI3Kα inhibition” in accordance to our last correspondence. Regarding the 4 specific comments mentioned in the body of your last email: 1. All micrographs need to a have scale bar instead of magnification. The size of these scale bars can be stated either on the figures or in the corresponding figure legends. Several figures had scale bars in previous versions don’t have scale bars in this version. Please check all figures carefully and make all necessary revisions. We sincerely apologize for this. Now all the figures with micrographs have scale bars. 2. Supplemental References should list only references that are unique to supplemental information. Although all relevant references should be cited in the supplemental information, only those not listed in the main References should be listed in the Supplemental References. Delete the Ericson et al. (2010) one. We have deleted Ericson et al. 3. A Graphical Abstract is optional, but encouraged. The Graphical Abstract is used to summarize the main findings of the study; it cannot be used to present a model. The current Graphical Abstract is the model presented in the Figure 6, this is inappropriate. We now propose the dual PI3Kα and PDK1 inhibition model as graphical abstract, as it summarizes the main finding. If not acceptable, we would like to ask to remove the graphical abstract from our files. 4. Both Figure S1 and Figure S2 are related to Figure 1, the panels in these two supplemental figures can be more balanced distributed so that panels don’t need to be that small. Maurizio Scaltriti Associate Lab Member; Human Oncology and Pathogenesis Program Associate Attending Biologist, Department of Medicine [email protected] 1275 York Avenue, Box 20, New York, NY 10065 T 646-888-3519 F 646-422-0247 www.mskcc.org NCI-designated Comprehensive Cancer Center Page 2 of 2 We would prefer to keep Figure S1 and Figure S2 as they are now because Figure S1 is only about the siRNA screening while Figure S2 is only about the validation of the screening. We understand the concern about the unbalanced distribution between these two figures; however, it would not be trivial to decide which panels we should move keeping Figure S2 seamless. Regarding the comments included in “Castel et al-suggestions”: Italicizing the gene names in the gene expression arrays showed in Figure 2 and Figure S3 would require an intense computational work. These names are automatically listed by the program utilized by the bioinformatician and we would have to repeat the analyses changing this parameter to correct this. We did italicize the gene names in Figure 2 panel C manually. In addition, the paragraph titled “SGK1 is upregulated in BYL719-resistant cell lines” became “SGK1 is upregulated in BYL719-resistant cell lines and in tumors from patients refractory to PI3Kα inhibition”. This change was made to better reflect the content of this paragraph. We believe we have corrected all the other issues. Best regards Maurizio Scaltriti JoséBaselga Response to Reviewers No response to Reviewers is needed at this time. GraphicalAbstract Click here todO\tJnload GraphicalAbstract Graphicalabstract.pdf PDK1I - PDK1 TSC2 inhibitors l 9 Fox03-dependent genes Tumor Regression Manuscript PDK1-SGK1 signaling sustains AKT-independent mTORC1 activation and confers resistance to PI3Kα inhibition Pau Castel1, Haley Ellis1, Ruzica Bago2, Eneda Toska1, Pedram Razavi1, F. Javier Carmona1, Srinivasaraghavan Kannan3, Chandra S. Verma3,4,5, Maura Dickler6, Sarat Chandarlapaty1,6, Edi Brogi7, Dario R. Alessi2, JoséBaselga 1,6, Maurizio Scaltriti1,7. 1Human Oncology & Pathogenesis Program (HOPP), Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 20, New York, NY 10065 2MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland 3Bioinformatics Institute (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671 4School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 5Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 6Department of Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 20, New York, NY 10065 7Department of Pathology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 20, New York, NY 10065, USA Corresponding Authors: Maurizio Scaltriti, PhD Department of Pathology Memorial Sloan Kettering Cancer Center 1275 York Avenue, Box 20 New York, NY 10065 Tel: 646-888-3519 Fax: 646-422-0247 E-mail: [email protected] JoséBaselga, MD, PhD Department of Medicine Memorial Sloan Kettering Cancer Center 1275 York Avenue - Suite M2015 New York, NY 10065 Phone: 212 639-8000 Fax: 212 794-3182 E-mail: [email protected] 1 Summary PIK3CA, which encodes the p110subunit of PI3K, is frequently mutated and oncogenic in breast cancer. PI3Kα inhibitors are in clinical development and despite promising early clinical activity, intrinsic resistance is frequent among patients. We have previously reported that residual downstream mTORC1 activity upon treatment with PI3Kα inhibitors drives resistance