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Acquired resistance to molecular targeted therapy represents a major challenge for the effective treatment of cancer. Hyperactivation of the PI3K/AKT pathway is frequently observed in virtually all human malignancies, and numerous PI3K and AKT inhibitors are currently under clinical evaluation. However, mechanisms of acquired resistance to AKT inhibitors have yet to be described. Here, we use a breast cancer preclinical model to identify resistance mechanisms to a small molecule allosteric AKT inhibitor, MK2206. Using a step-wise and chronic high-dose exposure, breast cancer cell lines harboring oncogenic PI3K resistant to MK2206 were established. Using this model, we reveal that AKT3 expression is markedly upregulated in AKT inhibitor–resistant cells. Induction of AKT3 is regulated epigenetically by the bromodomain and extra terminal domain proteins. Importantly, knockdown of AKT3, but not AKT1 or AKT2, in resistant cells restores sensitivity to MK2206. AKT inhibitor–resistant cells also display an epithelial to mesenchymal transition phenotype as assessed by alterations in the levels of E-Cadherin, N-Cadherin, and vimentin, as well as enhanced invasiveness of tumor spheroids. Notably, the invasive morphology of resistant spheroids is diminished upon AKT3 depletion. We also show that resistance to MK2206 is reversible because upon drug removal resistant cells regain sensitivity to AKT inhibition, accompanied by reexpression of epithelial markers and reduction of AKT3 expression, implying that epigenetic reprogramming contributes to acquisition of resistance. These findings provide a rationale for developing therapeutics targeting AKT3 to circumvent acquired resistance in breast cancer. Mol Cancer Ther; 15(8); 1964–74. ©2016 AACR.

Resistance to targeted therapy poses a major challenge for successful treatment of cancers. Despite a number of drugs targeting the PI 3-K/Akt pathway are in clinical development, mechanisms of resistance to drugs targeting this pathway have not been studied extensively. To determine how breast cancer cells develop resistance to Akt inhibitors (Akti), we generated Akti-resistant lines using dose escalation regimens with pan-Akt inhibitors (ATP-competitive: GDC0068, GSK690693; Allosteric: MK2206). Interestingly, in multiple Akti-resistant breast cancer lines, we observed a robust increase in the protein expression of Akt3, but not Akt1 or Akt2. We were intrigued by this observation, as we have recently demonstrated that Akt3 has a predominant function in regulating growth of triple negative breast cancer cells in three-dimensional spheroids and in xenograft models. The increased expression of Akt3 in Akti-resistant cells occurs via a genetic event, since the levels of Akt3 remain elevated after 3 weeks of Akt inhibitor removal. Moreover, upon Akt inhibitor removal in resistant lines, cells remain resistant to Akt inhibition. The enhanced Akt3 expression is observed in both luminal and basal-like triple-negative breast cancer resistant lines. Importantly, knockdown of Akt3, but not Akt1 or Akt2, results in re-sensitization of resistant cells to Akti. Mechanistically, using anti-phosphotyrosine receptor antibody array (RTK array), we observed increased phosphorylation of several RTKs in resistant cells, including EGFR, Her2, and ROR1. Akt3 knockdown in resistant cells results in reduction of pEGFR, pHer3 and pHer2, suggesting that Akt3 plays a role in the regulation of multiple RTKs in resistant cells. Epithelial-mesenchymal transition (EMT) is implicated in therapeutic resistance of cancers. Interestingly, several of our resistant lines display EMT properties including downregulation of E-Cadherin, and upregulation of N-Cadherin as well as Vimentin. We propose that enhanced Akt3 expression is a general mechanism by which breast cancer cells acquire resistance to Akti targeted therapy. Current efforts are aimed at investigating the mechanistic basis for Akt3-mediated resistance, and demonstrating the physiological relevance of these findings in clinical samples as well as in mouse models. The role of Akt3 in regulating EMT during establishment of resistance will also be explored. These studies will provide insights into molecular mechanisms of therapeutic resistance to drugs targeting the PI 3-K/Akt pathway, as well as information for designing combination strategies and next generation therapy. Citation Format: Y Rebecca Chin, Alex Toker. Increased Akt3 expression as a resistance mechanism to targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A42.