Abstract A34: Therapeutic potential of the novel mTOR inhibitor Torin-2 to overcome AKT reactivation in B-precursor acute lymphoblastic leukemia (B-pre ALL).

Abstract

Abstract B-precursor acute lymphoblastic leukemia (B-pre ALL) is characterized by malignant proliferation and accumulation of early lymphoid precursor cells in the bone marrow, blood and lymphoid organs, due to acquired mutations in early B-cells. Around 20% of children and the majority of adults relapses after the treatment and the poor outcome following relapse did not significantly changed over the past twenty years. For this reason, 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. Constitutively active phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is a common feature of leukemia patients, where it negatively influences response to therapeutic treatments. Thus, targeting the PI3K/AKT /mTOR pathway is an attractive therapeutic strategy. Rapamycin, an allosteric inhibitor of mTORC1, acutely inhibits mTORC1 but not mTORC2 activity. Prolonged suppression of mTORC1 also results in disruption of a negative feedback loop and consequently results in hyperphosphorylation of Akt through activation of IRS1 and PI3K. mTOR inhibitors are currently in clinical trial for a range of hematological malignancies including ALL, mostly in combination with conventional chemotherapy. RAD001, a mTORC1 inhibitor, has been shown to affect cell cycle progression and survival with a potentially relevant therapeutic efficacy. However, prolonged inhibition of mTORC1 often leads to AKT re-phosphorylation at Ser473which may counteract RAD001 activity. A new class of ATP competitive mTOR inhibitors, such as Torin-2, have revealed that this new class of inhibitors potently targets mTORC1 and mTORC2. Together, these features have generated hope that the new generation of ATP-competitive mTOR inhibitors will exhibit broader clinical efficacy relative 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. We used a panel of B-pre-ALL to test the drugs by MTT Assay and Western Blotting after different time exposures of the cells. 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. Our data suggest an interesting cytotoxicity of Torin-2 in B-pre-ALL acting on both mTORC1 and mTORC2 as assessed by their substrate inhibition. Torin-2 alone suppresses feedback activation of PI3K/AKT, whereas RAD001 requires the addition of MK-2206 to achieve the same efficacy. These two pharmacological options targeting PI3K/Akt/mTOR at different points of the signaling pathway cascade might represent a new therapeutic potential for treatment of B-pre-ALL patients. Citation Format: Alice Cani, Carolina Simioni, Alberto M. Martelli, Giorgio Zauli, Silvano Capitani, Luca M. Neri. Therapeutic potential of the novel mTOR inhibitor Torin-2 to overcome AKT reactivation in B-precursor acute lymphoblastic leukemia (B-pre ALL). [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A34.