Effective Targeting of the PI3-K Pathway in CLL with NVP-BEZ235, a Novel Orally Available Dual PI3K/mTOR Inhibitor
Authors: Shehata Medhat , Schnabl Susanne , Demirtas Dita , Tauber Stefanie , Hilgarth Martin , Bilban Martin , Vanura Katrina , Porpaczy Edit , Fonatsch Christa , Stilgenbauer Stephan , Schwarzmeier Josef D. , Hubmann Rainer , Gaiger Alexander , Zielinski Christoph , Maira Sauveur-Michel , Garcia-Echeverria Carlos , Hackl Wolfgang , Jaeger Ulrich 
Published: April 2020

Abstract
There is growing evidence that the anti-apoptotic PI3-K/Akt pathway is involved in pathogenesis and progression of different types of cancer. We have evidence that PI3-K inhibitors such as LY294002 and wortmannin selectively induce apoptosis in CLL cells (Shehata et al Ab. Blood 2006). Recently, a new orally available PI3-K inhibitor, NVP-BEZ235 has been developed. This competitive ATP binding imidazo-quinoline derivative is already in phase I trials against solid tumors. Here we show, for the first time, the effects of NVP-BEZ235 on the viability of CLL cells in vitro. Primary CLL cells from 37 patients were investigated. Sixteen patients were in Binet stage C, 14 in B and 7 in stage A. Seventeen patients had mutated IgVH genes, 15 had unmutated IgVH and mutation status from 5 patients was not available. Fluorescence in situ hybridization (FISH) analysis showed that 23 patients had del(13q), 9 had del(17p), 8 had del(11q) and 4 patients had trisomy 12. Nineteen patients were untreated and 18 patients were previously treated. To overcome the experimental artifact due to the spontaneous apoptosis of CLL cells in vitro, which may mask the actual effect of the tested drugs, we applied a co-culture model using human bone marrow stromal fibroblasts which supports survival of CLL cells ex vivo. CLL cells were exposed to NVP-BEZ235 at different concentrations (1 nM-10 μM) and incubation times (1, 3, 7 days). Cell viability was assessed by annexin-V/propidium iodide staining, flow cytometry and MTT assays. The results showed that cell viability was significantly higher in co-cultures compared to suspension cultures (the percentage of apoptotic cells after 3 days in co-culture was 5±4 compared to 23±12 in suspension cultures, p&lt; 0,01). NVP-BEZ235 induced apoptosis in the majority of CLL samples under both experimental conditions. However, this effect tends to be more remarkable in co-culture than in suspension: 4-10 fold versus 3-fold increase in apoptosis rate respectively. The pro-apoptotic effect was dose and time dependent and could be observed within 16 hours after incubation at 10 nM. A maximum effect was obtained at a concentration of 5–10 μM. The IC50 values varied between patients and were in a range of 250–750 nM. At these concentrations, NVP-BEZ235 was significantly more effective in induction of apoptosis than LY294002. NVP-BEZ235 inhibited the adhesion of CLL cells to stromal cells suggesting that it may interfere with the survival signal provided by the lymphoid microenvironment in addition to its direct effect on the leukemic cells. FACS analysis demonstrated that NVB-BEZ235 specifically targets the leukemic CD19+ cells while a minimal effect on the viability of T cells and monocytes could be observed. The pro-apoptotic effect of NVP-BEZ235 was independent from the mutational status and cytogenetics. In addition, it induced apoptosis in vitro in CLL cells from patients resistant to fludarabine treatment. In parallel to induction of apoptosis in CLL cells, western blotting demonstrated that NVP-BEZ235 significantly inhibited Akt phosphorylation at Ser-473. This effect was also associated with dephosphorylation (activation) of the tumor suppressor PTEN at Ser-380. DNA microarray analysis using Affymetrix U133A Plus 2.0 GeneChips revealed more than 200 genes which were at least 2 fold up- or down-modulated by NVP-BEZ235 in vitro. These genes include LY9, DUSP10, CCR6, RGS2, IRS2, PI4K2A, ISG20, TFRC, EGR1, HSP90, LCK, TNFRSF17, LYZ, TGFBI and TLR10. In conclusion, the results demonstrate a significant and selective pro-apoptotic effect of NVP-BEZ235 in CLL cells. The data point also to the validity of PI3K-pathway inhibition as a novel therapeutic concept for CLL which should be evaluated in clinical trials.