Dual Inhibition of Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin with NVP-BEZ235 as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia.
Authors: Chiarini Francesca , Grimaldi Cecilia , Ricci Francesca , Tazzari Pierluigi , Evangelisti Camilla , Pession Andrea , Pagliaro Pasqualepaolo , McCubrey James , Martelli Alberto M. 
Published: October 2019

Abstract
Abstract 2025
Poster Board II-2

Introduction:
Recent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL) where it strongly influences cell proliferation and survival. Pathway activation could be due to several reasons which include Notch1 activation leading to HES1-mediated transcriptional suppression of PTEN gene, PTEN phosphorylation or oxidation, and inactivation of SHIP1 phosphatase. These findings lend compelling weight for the application of PI3K/Akt/mTOR inhibitors in T-ALL. Rapamycin and its analogues have shown some promising effects in pre-clinical models of T-ALL. However, mTOR inhibitors are mainly cytostatic and could hyperactivate Akt due to the existence of feedback loops between mTOR, p70 S6 kinase, PI3K, and Akt. Recently, dual PI3K/mTOR inhibitors have been synthesized. Here, we have analyzed the therapeutic potential of the novel, dual PI3K/mTOR inhibitor, NVP-BEZ235, an orally bioavailable imidazoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples.

Methods and Patients:
We employed a panel of cell lines with up-regulated PI3K/Akt/mTOR signaling, including CEM-R cells [which overexpress high levels of the membrane transporter, 170-kDa P-glycoprotein (P-gp)], MOLT-4 and CEM-S cells (which lack PTEN expression), Jurkat cells (which do not express both PTEN and SHIP1), and RPMI-8402 and BE-13 cells. MOLT-4, CEM, and Jurkat cells have a non-functional p53 pathway. Moreover, both Jurkat and MOLT-4 cells have aberrant Notch1 signaling. Patients samples displayed pathway activation as documented by increased levels of p-Akt, p-4E-BP1, and p-S6 ribosomal protein, as well as low/absent PTEN expression.

Results:
NVP-BEZ235 was cytotoxic to the panel of cell lines as documented by MTT assays. NVP-BEZ235 IC50 ranged from 80 to 280 nM at 24 h. A comparison between NVP-BEZ235 and the dual PI3K/mTOR inhibitor PI-103, a small synthetic molecule of the pyridofuropyrimidine class with the same targets, demonstrated that NVP-BEZ235 was more effective than PI-103 when employed at equimolar concentrations. NVP-BEZ235 did not significantly affect the proliferation of peripheral blood T-lymphocytes from healthy donors stimulated with phytohemagglutinin and interleukin-2, whereas it blocked leukemic cells in the G1 phase of the cell cycle, and this was accompanied by decreased levels of phosphorylated Retinoblastoma protein. NVP-BEZ235 treatment also resulted in apoptotic cell death (about 20-30% at 6 h of exposure, when employed at 200 nM), as documented by Annexin V/propidium iodide staining and cytofluorimetric analysis. Moreover, NVP-BEZ235 activated caspase-8 and caspase-3, as demonstrated by western blot. Western blot documented a dose- and time-dependent dephosphorylation of Akt and its downstream target, GSK-3β, in response to NVP-BEZ235. mTOR downstream targets were also efficiently dephosphorylated, including p70S6 kinase, S6 ribosomal protein, and 4E-BP1. Remarkably, NVP-BEZ235 targeted the side population (SP, identified by Hoechst 33342 staining and ABCG2 expression) of T-ALL cell lines, which might correspond to leukemia initiating cells, and synergized with several chemotherapeutic agents (dexamethasone, vincristine, cyclophosphamide, Ara-C) currently employed for treating T-ALL patients. NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells by co-culturing with MS-5 stromal cells which mimic the bone marrow microenvironment. NVP-BEZ235 was cytotoxic (IC50: 10-15 nM at 96 h) to primary lymphoblasts from patients with T-ALL, where the drug dephosphorylated 4E-BP1, at variance with rapamycin. Of note, NVP-BEZ235 targeted the SP also in T-ALL patient samples.

Conclusions:
NVP-BEZ235 was cytotoxic to T-ALL cell lines and patient lymphoblasts (including SP cells) at concentrations that have been previously reported to be achievable in vivo. Taken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR network with NVP-BEZ235, either alone or in combination with other drugs, may serve as an efficient treatment towards T-ALL cells (including those overexpressing P-gp and independently from p53 status) which require upregulation of this signaling pathway for their survival and growth.

Disclosures:
No relevant conflicts of interest to declare.