Background: The PI3K/AKT signaling pathway is frequently activated in blast cells from AML patients, and also detected in the leukemic stem cell (LSCs). The p110δ catalytic subunit of class IA PI3K is constantly expressed in leukemic cells, and IC87114, a specific p110δ inhibitor, inhibits Akt phosphorylation, reduces blast cell proliferation, but does not induce apoptosis in AML cells. The mTORC1 pathway is also activated in 50 to 70% of AML samples. Rapamycin and its derivative RAD001, inhibit mTORC1 activity, decrease the clonogenicity of leukemic progenitors, but induce low rates of apoptosis. LY294002 inhibits all p110 isoforms of PI3K as well as most kinases of the PIKK family but can not be used in clinical practice because of its toxicity. Pharmaceutical industry effort identified more selective PI3K inhibitors, and among them, PI-103, a small synthetic molecule of the pyridofuropyrimidine class, is potentially interesting as a selective PI3K inhibitor and also as an mTOR inhibitor.
Materials and methods: Bone marrow (BM) samples were obtained from 20 newly diagnosed patients with de novo AML, treated in the AML2001 trial of chemotherapy, initiated by the GOELAMS. The MOLM-14 and OCI-AML-3 leukemic cell lines were obtained from ATCC. CD34+ cells from 5 normal allogeneic donors were purified after cytapheresis.
Results: We studied the effect of PI-103 in MOLM-14 and OCI-AML-3. We observed a dose-dependent inhibition of AKT (Ser 473 and Thr 308), FOXO3a (Thr 32), and p70S6-kinase (Thr 389) phosphorylation with 0.1, 1 or 5 μM PI-103. 1μM PI-103 was as effective as 25 μM LY294002 or 10 nM RAD001 at inhibiting PI3K/AKT or mTORC1 activity, respectively. 1μM PI-103 dramatically inhibited the proliferation of both cell lines. However, PI-103 was essentially cytostatic and did not induce significant apoptosis in these cells. For subsequent experiments on primary AML blasts, the dose of 1 μM PI-103 was chosen as it was sufficient to inhibit AKT and p70S6K phosphorylations. The effect of 1μM PI-103 on PI3K/AKT and mTORC1 activation was evaluated in 5 AML samples (3 with constitutive PI3K activation (PI3K+) and 2 without constitutive PI3K activation (PI3K-)). 1 μM PI-103 inhibited completely basal and SCF or Flt3-L-stimulated AKT, FOXO3A phosphorylation and P70S6K phosphorylation in these AML samples. PI-103 dramatically inhibited both basal and growth factor-stimulated proliferation of blast cells. Interestingly, PI-103 induced significant apoptosis in 6 AML samples at 1μM and a significant decrease of CFU-L colonies in 6 other AML samples. In contrast, specific inhibition of the catalytic activity of the p110δ isoform and of the mTORC1 complex, with IC87114 and RAD001 respectively, did not induce apoptosis. Finally, excess of apoptosis was also obtained in the CD34+ CD38Low/Neg CD123+ LSCs from 4 patients in presence of PI-103 alone. Moreover, the functional effects of PI-103 on normal CD34+ cells were low, indicating that this compound may have a favourable therapeutic index.
Conclusions: Our results suggest that multi-targeted therapy toward PI3K/AKT and mTOR may be efficient in AML and particularly useful to target LSCs.
Disclosure: Research Funding: Assistance Publique des Hôpitaux de Paris/La Caisse Nationale d’Assurance Maladie (APHP/CANAM) and Fondation de France, Association Laurette Fugain, Association pour la Recherche contre le Cancer (ARC), Ligue Nationale contre le Cancer (LNCC, laboratoire associé), and Institut National du Cancer (INCA).