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In most cancer models, the PI3K/Akt pathway controls the activity of the mammalian Target of Rapamycin Complex 1 (mTORC1) that regulates cell growth through the phosphorylation of P70S6K and 4E-BP1. In Acute Myeloid Leukemia (AML), both constitutive activation of PI3K (PI3K+) and of mTORC1 are detected. We previously reported in AML that PI3K activity is due to the expression of the p110d isoform of class Ia PI3K and is constitutive in 50% of samples at diagnosis. We show here in primary AML samples that the constitutive activity of mTORC1, detected by the phosphorylation of P70S6K on Thr389, is not due to PI3K/Akt activation. In PI3K+ samples, the specific inhibition of p110d with IC87114 does not inhibit mTORC1. Furthermore, a constitutive activity of mTORC1 is detected in almost all AML samples regardless of their PI3K/ Akt activation status. Similarly, the inhibition of ERK/MAPK activity with UO126 does not result in mTORC1 inhibition. Interestingly, in primary AML cells, we shows that mTORC1 is fully inhibited following leucine starvation as observed in non-transformed cells, suggesting that the kinase activity of mTOR is not deregulated and remains accessible to target inhibition. We thus focused on the links between the AMP kinase (AMPK) and mTORC1. AMPK is a physiological cellular energy sensor and becomes phosphorylated at Thr172 downstream of LKB1 in response to a decrease in cellular ATP levels. AMPK then controls multiple metabolic processes such as fatty acid oxidation, glucose production and protein synthesis. Interestingly, AMPK is usually unphosphorylated in cancer cells including AML and thereby represents a potential therapeutic target. We thus investigated the effects of metformin, a potent AMPK activator developed as an oral anti-diabetic drug, in primary AML cells. This compound induces a dose-dependent activation of AMPK that correlates with a decrease of mTORC1 activity. Primary AML blast cells cultured with metformin show a significant inhibition of proliferation, of clonogenic growth and apoptosis suggesting that AMPK may be an attractive anticancer therapeutic target in AML. However, as metformin may exerts AMPK-independent effects, we are producing a lentiviral construct expressing a constitutively activated AMPK mutant to more specifically determine the consequences of AMPK activation in mTORC1 signaling and blast cells survival.

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.

The deregulation of translation, leading to a preferential translation of oncogene-related mRNAs, markedly contributes to the malignant phenotype in human cancers. The assembly of the eukaryotic translation Initiation Factor 4F (eIF4F) complex is limiting for malignancy-related translation events and is hence an attractive anticancer therapeutic target. The Mammalian Target Of Rapamycin Complex 1 (mTORC1) is thought to positively regulate eIF4F assembly and subsequent oncogenic protein synthesis through eIF4E Binding Protein 1 (4E-BP1) phosphorylation. The mTORC1 inhibitor rapamycin has been developed as an anti-cancer agent but is of limited efficacy in the treatment of Acute Myeloid Leukemia (AML). We show in our study that in primary AML cells, 4E-BP1 phosphorylation is mTORC1-independent and is in fact controlled by the Pim- 2 serine/threonine kinase: neither the rapamycin derivate RAD001 nor Raptor siRNA inhibited 4E-BP1 phosphorylation contrary to siRNA-mediated Pim-2 knock-down. We utilized the 4E-BP1 mimetic 4EGI-1, as a direct inhibitor of translation in AML. In contrast to RAD001, 4EGI-1 totally inhibited eIF4F assembly; markedly decreased the proportion of polysome-bound c-Myc mRNA; and dramatically decreased the accumulation of oncogenic proteins that are regulated at the translational level. Moreover, 4EGI-1 demonstrated potent anti-leukemic activity ex vivo in primary AML blast cells through a selective suppression of proteins implicated in proliferation and in anti-apoptotic mechanisms. Significantly, these effects occurred with a minimal degree of toxicity against normal hematopoietic processes. Our present results therefore strongly implicate an mTORC1-independent mechanism of deregulated synthesis of oncogenic proteins in human myeloid leukemogenesis. The direct inhibition of the eIF4F translation initiating complex, by 4EGI-1, by the recently developed eIF4E antisense oligonucleotides or by specific Pim-2 inhibitors when available, thus represents an attractive option for the development of new therapies for AML.

The PI3K/Akt axis is implicated in cell proliferation and survival and appears promising for target inhibition. The constitutive activation of PI3K (PI3K+) that reflects intrinsic cytokine-independent signaling properties of blast cells is detected in 50% of AML samples at diagnosis. Several cytokines and growth factors are known to stimulate the PI3K/Akt pathway, such as FLT-3, SCF or IGF-1 but the mechanisms leading to the constitutive PI3K activation are unknown. The IGF-1/IGF-1R signaling is thought to have a crucial role in the development and progression of cancers. In AML, IGF-1 promotes cell growth and survival via PI3K signaling. As we previously reported an IGF-1 autocrine production in AML leukemic cells, we investigated whether IGF-1/IGF-1R signaling could be responsible for the constitutive activity of PI3K in AML. We show here in primary AML blast cells that IGF-1R is constantly expressed and constitutively phosphorylated in PI3K+ samples. The constitutive phosphorylation of Akt is fully inhibited in 70% of PI3K+ samples by aIR3, a neutralizing anti-IGF-1R monoclonal antibody but neither ERK nor P70S6K phosphorylation are modified by aIR3. Moreover, siRNA-mediated decrease of the IGF-1 autocrine production markedly reduces Akt phosphorylation in leukemic cells. Finally, no mutation is detected within the complete sequence of IGF-1R cDNA from PI3K+ AML samples. In conclusion, the constitutive activity of PI3K/Akt proceed from the activation of IGF-1R due to an IGF-1 autocriny in 70% of PI3K+ primary AML samples. Our current data strengthen the rational of targeted therapy against IGF-1R in AML.