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
