Abstract 2448: mTOR inhibitor MLN0128 Has antitumor efficacy in cell lines with intrinsic and acquired rapamycin resistance in vitro and in vivo

Introduction: The mammalian target of rapamycin (mTOR) pathway plays a central role in cancer biology. mTOR forms two multiprotein complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin and analogues are allosteric mTOR inhibitors that predominantly inhibit mTORC1. Rapamycin analogues have been FDA approved for several cancer types and are in clinical trials for treatment of many others. MLN0128 is a mTOR kinase inhibitor, that inhibit s both mTORC1 and mTORC2. We hypothesized that MLN0128 may have antitumor efficacy in cancer cell lines with intrinsic or acquired rapamycin-resistance. Methods: Twenty immortalized human cancer cell lines representing various cancer types and a range of rapamycin sensitivity were treated with Rapamycin, MLN0128 or vehicle. Sulforhodamine B assay was used to determine rapamycin and MLN0128 half maximal inhibitory concentrations (IC50), which were later confirmed with colony formation assay (CFA). Acquired resistance models were created in BT-474 and MCF-7 cell lines through culturing in progressively increasing doses of rapamycin until sustained growth at supratherapeutic (10uM) doses was achieved. Western blotting was used to characterize PI3K/mTOR signaling pathway expression. Flow cytometry and cell sorting (FACS) was used to detect and quantify apoptosis of Annexin-V-FLUOS stained cancer cell lines. Xenograft models of nu/nu mice were treated with MLN0128 and rapamycin or everolimus for 28 days. Results: Cell lines that were intrinsically rapamycin sensitive as well as those that were intrinsically rapamycin resistant were sensitive to MLN0128. MLN0128 induced cell cycle arrest; Induction of apoptosis or autophagy with MLN0128 was not detected with western blotting and annexin labeling in most cell lines tested. MLN0128 demonstrated greater inhibition of mTORC1 signaling, especially with a decrease in p4E-BP1 across all cell lines. In addition, MLN0128 demonstrated inhibition of mTORC2, with decline in pAKT473 and pAKT450. However a feedback-dependent phosphorylation of AKT S308 was observed. In cell lines with acquired rapamycin resistance, treatment with MLN0128 produced a significantly higher growth inhibition compared to rapamycin. Thus far, we have demonstrated that MLN0128 treatment resulted in increased tumor growth inhibition compared to rapamycin in xenograft models of BT474 cells with acquired rapamycin resistance. Conclusion: Cancer cell lines with intrinsic as well as acquired resistance to rapamycin