Cellular and Molecular Effects of NVP-BKM120 in Lymphoblastic Leukemia Cells

Background: PI3K/AKT/mTOR signaling controls most hallmarks of cancer. Constitutive activation of PIK3 pathway in T cells acute lymphoblastic leukemia (T-ALL) has been reported; in a mouse model, PI3K activation, together with MYC, cooperates in Burkitt lymphoma (BL) pathogenesis. NVP-BKM120 is an orally bioavailable 2,6-dimorpholine pyrimidine derivative, and considered a highly selective pan-class I PI3K inhibitor. In preclinical studies, it has shown efficacy in a variety of malignancies and is currently being investigated in phase I/II/III clinical testing, mainly for advanced solid tumors (clinicaltrials.gov).

Aims: Here, we described the effects of the pan-PI3K/AKT/mTOR inhibitor NVP-BKM120 on T-ALL and BL cell lines.

Methods: T-ALLcell lines, Jurkat and MOLT-4, and BL cell lines, NAMALWA and Daudi, were obtained from ATCC. NVP-BKM120 was kindly provided by Novartis, and was prepared as a 10mM stock solution in DMSO. Different concentrations of the drug were used as indicated, where cells treated only with DMSO served as control. Cell viability was measured by MTT. Colony formation was carried out in semisolid methyl cellulose medium. The induction of apoptosis was assessed by annexin-V-APC/PI and by caspase cleavage. The cell cycle was analyzed by a PI-staining method. Western blot analysis was performed by standard methods. Vital staining and flow cytometry analysis with acridine orange was performed for the detection and quantification of acidic vesicular organelles (AVOs). Comparisons between the two groups were performed by the t test. Pvalue <0.05 was considered statistically significant.

Results: Cell viability decreased in a concentration-dependent manner, with an IC₅₀ range of 7-8 μM and the clonogenic growth was significantly decreased at the concentration of 1μM and at 10µM the colony formation was completely inhibited in all cells tested. After 6 hours of NVP-BKM120 treatment, we observed an increase in apoptotic cells, as well as an increase in the cleavage of procaspase 3, 8 and 9 in Jurkat, MOLT-4 and NAMALWA cells. Compared with DMSO control, NVP-BKM120 does not have any effect during apoptosis induction in the Daudi cell line. NVP-BKM120 treatment also resulted in G₂/M arrest, associated with a decrease in the G₁population and a decrease in Cyclin B1 protein levels. Immunoblotting analysis of cells treated with the drug revealed decreased phosphorylation, in a dose-dependent manner, of AKT, P70SK6 and 4EBP1, with stable total proteins levels. Additionally, we observed a dose-dependent decrease in BAD phosphorylation, followed by an increase in BAX:BCL2 ratio. Quantification of AVOs showed a dose-dependent increase of AVOs in all cells tested, after NVP-BKM120 treatment.

Conclusions: NVP-BKM120 induced apoptosis in a dose-dependent manner in Jurkat, MOLT-4 and NAMALWA cells, while effects of the drug in the Daudi cell line were mainly cytostatic. In those cells, the induction of apoptosis suggested that the death receptor and mitochondrial pathways were activated after drug treatment. In our study, we found that NVP-BKM120 decreased the phosphorylation levels of BAD, which is linked to a pro-apoptotic activity, and up-regulated the BAX:BCL2 ratio. These results are consistent with the activation of caspase-9 and 3, related to the mitochondrial apoptosis. The accumulation of leukemia cells in the G₂/M phase of the cell cycle has been associated with enhanced apoptosis. Our results suggest that decreased Cyclin B1 protein expression might be the molecular mechanism through which NVP-BKM120 induces G₂/M arrest. The effects of NVP-BKM120 on the PI3K pathway indicate that NVP-BKM120 treatment may overcome rapamycin-induced AKT activation. P70SK6 and 4EBP1 are the two best-characterized substrates of mTOR1. Hence, the decrease in the phosphorylation levels of P70SK6 and 4EBP1 results in impaired oncogenic protein synthesis. Moreover, P70SK6 is one of the kinases whose phosphorylation by mTOR1 results in opposing autophagy. Accordingly, NVP-BKM120 resulted in increased AVOs, which is a characteristic feature of cells engaged in autophagy. In summary, our present study establishes that NVP-BKM120 effectively presents an antitumor activity against T-ALL and BL cell lines. The reduction of proliferation is possibly by down-regulation of Cyclin B1 and the increased BAX:BCL2 ratio is one of the mechanisms involved in the induction of apoptosis.