Abstract mTOR is a highly conserved and widely expressed serine/threonine kinase, that is a member of the phosphatidylinositol-3 kinase–like kinase (PIKK) family. mTOR plays a pivotal role in the PI3K/Akt/mTOR signaling pathway, which senses growth factor and serves as a central regulator of fundamental cellular processes such cell growth/apoptosis, autophagy, translation, and metabolism. Hyperactivation of this pathway through loss of negative regulators, such as PTEN, or mutational activation of receptor tyrosine kinases of phosphoinositide 3-kinase (PI3K) is a frequent occurrence in leukemia patients, where it negatively influences response to therapeutic treatments. In B-precursor acute lymphoblastic leukemia (B-pre ALL) many research efforts are currently devoted to the development of targeted therapies to limit side effects of chemotherapy and to increase treatment efficacy for poor prognosis patients, i.e. poor outcome following relapse. Thus, targeting the PI3K/AKT /mTOR pathway is an attractive therapeutic strategy. Numerous inhibitors targeting these kinases are currently undergoing clinical evaluation in hematological malignancies including ALL, mostly in combination with conventional chemotherapy. Prolonged suppression of mTORC1 also results in disruption of a negative feedback loop and often results in re/hyper-phosphorylation of Akt through activation of IRS1 and PI3K, which may counteract rapalogs activity. A new class of ATP-competitive mTOR inhibitors, such as Torin-2, has revealed that these inhibitors potently targets mTORC1 and mTORC2. Together, these features have generated hope that this new generation of inhibitors will exhibit broader clinical efficacy when compared to the rapalogs. We therefore hypothesized that dual inhibition of mTORC1 and mTORC2 by Torin-2 would provide a superior outcome in ALL as compared to inhibition of mTORC1 alone with RAD001 in B-pre-ALL. We tested the capability of Torin-2 to prevent AKT reactivation after mTORC1 and mTORC2 inhibition. Furthermore we explored if dual targeting of mTORC1 and AKT may achieve results similar to those obtained with Torin-2 alone. Drugs cytotoxic activity were analyzed in a panel of B-pre-ALL cells by MTT Assay and Western Blotting at different time points. Cell cycle, apoptosis and autophagy were analyzed by flow cytometry, Western Blotting and fluorescent staining. In all the B-pre-ALL cell lines Torin-2 showed a powerful cytotoxic activity, inhibiting the growth of each cell line in a dose-dependent manner, with an IC50 in the nanomolar range as assessed by MTT assays. The major proteins along the PI3K/AKT/mTOR signaling pathway where heavily dephosphorylated after 2 hrs of drug exposure. This inhibition lasted up to at least 48 hrs at variance to RAD001, that already after 24 hrs was unable to prevent AKT reactivation. However the association of RAD001 with MK-2206, an allosteric AKT inhibitor, prevented AKT reactivation and reached a significant cytotoxicity. These data suggest an interesting cytotoxicity of Torin-2 in B-pre-ALL acting on both mTORC1 and mTORC2 as assessed by their specific substrate inhibition. Feedback activation of PI3K/AKT was suppressed by Torin-2 alone, whereas RAD001 required the addition of MK-2206 to achieve the same efficacy. These two pharmacological strategies targeting PI3K/Akt/mTOR at different points of the signaling pathway cascade might represent a new therapeutic option in B-pre-ALL patients also preventing Akt reactivation. Citation Format: Carolina Simioni, Alice Cani, Alberto M. Martelli, Giorgio Zauli, Silvano Capitani, Luca M. Neri. Activity of the novel mTOR inhibitor Torin-2 in B-precursor acute lymphoblastic leukemia and its therapeutic potential to prevent AKT reactivation. [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 B46.