The Dual PI3K and mTOR Kinase Inhibitor NVP-BEZ235 Induces Cell Cycle Arrest in Acute Lymphoblastic Leukemia Cells and Potentiates the Cytotoxicity of Dexamethasone, Cytarabine and Doxorubicin.
Authors: Schult Catrin , Dahlhaus Meike , Sekora Anett , Lange Sandra , Freund Mathias , Junghanss Christian 
Published: October 2019

Abstract
Abstract 4115

Introduction
Inhibition of specific signal transduction pathways in neoplastic cells has been successfully used to regulate aberrantly activated cellular processes such as proliferation. Specific target agents such as tyrosine kinase inhibitors have been successfully introduced into the treatment of several cancers. The novel dual PI3K and mTOR inhibitor, NVP-BEZ235, has shown antitumor activity in vitro and is being investigated in phase I clinical trials in solid tumors. It is currently unclear whether PI3K/Akt and mTOR inhibition might also be usefull in acute leukemia cells, and if so, what would be the best modalities for combination approaches. Here, we examined the antileukemic activity of NVP-BEZ234 in acute lymphoblastic (ALL) and myeloid leukemia (AML) cells.

Methods
ALL (SEM, RS4;11, Jurkat and MOLT-4) and AML (MV4;11, MONO-MAC-1, HL-60 and NB-4) cell lines with different cytogenetics and PI3K/Akt activity were investigated. Cells were treated for 96h with NVP-BEZ435 (1 nM, 10 nM and 100 nM) alone or in combination with cytarabine, doxorubicin or dexamethasone. Cytotoxic drugs were added simultaneously, 24h before or 24h after treatment with NVP-BEZ235. Cell count, apoptosis rates, necrosis rates, cell cycle distrubution, protein phosphorylation and metabolic activity were determined at 0.5h, 4h, 24h, 48h, 72h, and 96h by microscopy, flow cytometry, western blot and WST-1 testing, respectively.

Results
NVP-BEZ235 potently inhibited the proliferation of SEM, Jurkat, RS4;11, MV4;11, MONO-MAC-1 and NB-4 significantly. Most pronounced effects were seen after 72h. In HL-60, which has a low PI3K/Akt activation, inhibition of proliferation to a lesser degree was observed. Inhibition of PI3K and mTOR activity and G1/G0 arrest was detected at 10 nM and 100 nM in B- and T-ALL cell lines, respectively. Metabolic activity decreased significantly as early as 48h after exposure. Changes in apoptosis and necrosis rates were not observed in B-ALL and AML cells. However, in Jurkat and MOLT-4 (both T-ALL) highest doses of NVP-BEZ seemed to slightly increase. In a time and concentration dependent manner the phosphorylation status of Akt (Ser473 and Thr308), FoxO3A (Thr32) and p70S6 kinase (Thr389) decreased early (0.5-4h) after treatment. Of interest, subsequently an increase of phosphorylation of the same proteins was observed (24-48h), followed by another dephosphorylation episode. Concomittant treatment with NVP-BEZ235 and cytarabine, doxorubicin or dexamethasone enhanced the cytotoxicity in B-cells compared to single drug treatment. The most significant effects on proliferation inhibition and metabolic activity were observed for the simultaneous exposure to both drugs or pretreatment of cells with NVP-BEZ235 followed by dexamethasone, doxorubicin and cytarabine addition after 24h. No additive effect was observed when cytostatic drugs were added before inhibition of PI3K and mTOR by NVP-BEZ235 was induced.

Conclusions
NVP-BEZ235 reduces metabolic activity and induces cell cycle arrest in acute lymphoblastic and myeloid leukemia cells. Treatment of ALL cells with NVP-BEZ combined with glucocorticoids or conventional chemotherapy drugs enhances the cytotoxicity of the dual PI3K and mTOR inhibitor when NVP-BEZ235 is given upfront or simultaneously. In contrast, pretreatment with conventional chemotherapy drugs followed by NVP-BEZ235 exposure does not induce additive cytotoxicity. Our data indicate that NVP-BEZ might be a usefull drug in ALL as well AML treatment. Furthermore, simultaneous treatment with chemotherapy seems more advisable compared to NVP-BEZ interruption.

Disclosures:
No relevant conflicts of interest to declare.