The pan-class I PI3 kinase inhibitor, NVP-BKM120, demonstrates anti-leukemic activity in acute myeloid leukemia

Background: Aberrant activation of PI3K/Akt/mTOR pathway is a common feature of acute myeloid leukemia (AML) patients contributing to chemoresistance, disease progression and unfavourable outcome. Therefore, inhibition of this pathway may represents a potential therapeutic approach in AML. NVPBKM120 (BKM120) is a pan-class I PI3K inhibitor which exerts anti-proliferative and cytotoxic effects on several solid tumors and hematological malignancies by inhibiting PI3K/Akt/mTOR activity. Aims: To evaluate the pre-clinical activity of BKM120 on AML cell lines and primary samples. Methods: BKM120 (Novartis) was tested on AML cell lines (U937, NB-4, HL- 60/Mx2, OCI-AML2, HL-60, OCI-AML3, THP-1, MOLM-13 and KG-1) and on 15 primary AML samples. Given the role of PI3K/Akt/mTOR in cell metabolism, BKM120 was then combined with dichloroacetate (DCA), a glycolitic modulator, on selected AML samples. Cell cycle and apoptosis were analyzed by Acridine-Orange and AnnexinV (AnnV)/PI staining, respectively. Signaling modulations and metabolic changes were evaluated by western blot and by the XF24 Flux analyzer, respectively. In vivo experiments were performed on non-obese diabetic severe combined immunodeficient interleukin-2 receptor g-null mice. Results: Basal expression and phosphorylation levels of PI3K/Akt/mTOR pathway components were initially assessed on AML cell lines and primary samples. Despite some heterogeneity, all cell lines shared the constitutive activation of PI3K/Akt/mTOR axis. Moreover, 7 of the 9 primary samples tested showed a higher phospho/total Akt ratio than normal mononuclear cells (MNCs) and displayed p-mTOR (S2448 and S2481) and p-4EBP1 (T37/46) overexpression, suggesting the aberrant activation of PI3K/Akt/mTOR pathway. BKM120 exposure resulted in a dose-dependent dephosphorylation of Akt (S473) and Gsk3α/β (S21/9) in all cell lines tested and affected, at higher doses, mTOR activity inducing the dephosphorylation of mTOR (S2448 and S2481), p70S6K (S371) and 4EBP1 (T37/46). Blockade of PI3K/Akt/mTOR signaling inhibited cell growth (IC50s: 0.7-1.2μM) and induced a significant (p<0.005) dose- and time-dependent apoptosis in all cell lines. Cytotoxicity was preceded by a temporary G2/M block that was rapidly followed by induction of apoptosis, as demonstrated by the increase of the subG0/G1 peak at 72h. Efficacy of BKM120 was then confirmed on primary samples: at 144h, AnnV+ cells increased from 19.5±9.6% (vehicle) to 48.0±23.6% (p<0.001) at 5μM. Dephosphorylation of Akt (S473), achieved in 3/3 primary samples, supported the target inhibition. Conversely, BKM120 failed to show considerable cytotoxicity on normal and PHA-activated MNCs (8.0% and 4.4% apoptosis net increase at 5µM, respectively). Metabolic perturbations induced by BKM120 were then assessed on AML cells demonstrating a dose-dependent reduction of basal and maximal respiration as well as ATP production in both cell lines and primary samples. Furthermore, BKM120 strongly synergized (CI<0.6) with DCA to trigger apoptosis at lower doses on AML cell lines and primary samples. Finally, in vivo administration of BKM120 in a xenotransplant mouse model of AML markedly inhibited leukemia progression and induced a significant (p<0.001) improvement of overall survival. Summary and Conclusions: Our data demonstrated that BKM120, as single agent or in combination with other drugs (i.e. glycolytic modulators), has a significant anti-leukemic activity towards AML cell lines and primary samples, thus supporting its clinical evaluation as a therapeutic agent for AML patients.