IκB Kinase Overcomes PI3K to Control FoxO3a Subcellular Localization in Acute Myeloid Leukemia.

Abstract 478

The prognosis of Acute Myeloid Leukaemia (AML) remains globally poor. Aberrant activation of signalling pathways is frequently found in AML and leads to uncontrolled cell growth and survival. Effective targeting of these pathways with new therapeutics may result in suppression of leukemic cell proliferation and survival. In AML, constitutive PI3K activity, mainly due to the expression of the class IA PI3K p110δ isoform, is detected in 50% of patients at diagnosis (PI3K+ AML samples) (Sujobert, Bardet et al., Blood 2005; Tamburini, Elie et al., Blood 2007). The PI3K/Akt axis represents therefore an attractive therapeutic target in this disease. Accordingly, inhibition of PI3K activity with IC87114, a specific p110δ inhibitor decreases AML blast cells proliferation (Sujobert, Bardet et al., Blood 2005). However, IC87114 failed to induce significant apoptosis (Park, Chapuis et al., Leukemia 2008). The PI3K/Akt network controls different targets implicated in the regulation of cell survival. Among them, the FoxO transcription factors which include FoxO1, FoxO3a, FoxO4 and FoxO6 up regulate the expression of target genes involved in apoptosis, such Fas-L and Bim. Akt, by phosphorylating FoxO proteins at three conserved sites (T32, S253 and S315 on FoxO3a), negatively regulates their transcriptional activity by inducing their nuclear export. Given that IC87114 did not induce apoptosis, we hypothesized that FoxO proteins may escape to PI3K/Akt control in AML cells. For that purpose, we investigated their mechanisms of regulation in both primary AML cells and a MV4-11 human leukemic cell line engineered to stably overexpress a FoxO3awt-GPF fusion protein introduced by lentiviral infection. We focused especially on FoxO3a as we found, using real-time RT-PCR and WB analysis, that it was the only FoxO protein constantly expressed in primary AML cells. First, we examined the relationship between FoxO3a subcellular localization using immunofluorescence and PI3K activity in primary AML cells. As expected in PI3K+ AML cells (n=6), FoxO3a was phosphorylated on T32 and S253 and constantly localized in the cytoplasm. However, although Akt S473 and FoxO3a T32 and S253 phosphorylations were fully inhibited by IC87114, FoxO3a did not translocate into the nucleus of blast cells. Accordingly, Fas-L and Bim expression tested by qRT-PCR were not induced by IC87114. Identical results were obtained from the same analysis of the MV4-11FoxO3awt-GFP cell line. Furthermore, in PI3K- AML cells (n=5), neither Akt S473 nor FoxO3a T32 and S253 phosphorylations were detected but FoxO3a was still confined in the cytoplasm of these cells. Altogether, these results clearly demonstrate that an Akt-independent mechanism contributes to the nuclear exclusion of FoxO3a in AML cells. In addition to Akt, the IκB Kinase (IKK) may regulate FoxO3a subcellular localization by phosphorylation on S644 in AML, as it has previously been shown in breast cancer (Hu, Lee et al., Cell 2004). As the oncogenic IκB Kinase is known to be activated in almost all AML samples (Guzman, Neering et al., Blood 2001), we investigated whether IKK could maintain FoxO3a in the cytoplasm of AML cells. Interestingly, we found that inhibition of IKK with a specific IKKγ/NEMO-antagonistic peptide (Agou, Courtois et al., J Biol Chem 2004) induced a strong nuclear translocation of FoxO3a and an efficient apoptosis in both primary AML blast cells (n=5) and MV4-11FoxO3awt-GFP cell line. To confirm this result, MV4-11 cells were infected with a lentivirus expressing a FoxO3aS644A-GFP mutant protein in which the IKK phosphorylation is abrogated. We found that the FoxO3aS644A-GFP mutant protein primarily localized within the nucleus of MV4-11 cells and induced a decrease of cell proliferation and a moderate pro-apoptotic effect. We thus conclude that IKK, by phosphorylating FoxO3a on S644, leads to the constant nuclear export of FoxO3a in AML cells. Consequently, the therapeutical potential value of IKK targeted inhibition in AML could be due not only to the inhibition of NF-κB activity but also to the FoxO3a nuclear import. Moreover, the PI3K-independent FoxO3a regulation probably contributes to the low potential of specific PI3K inhibitors to induce apoptosis in AML cells.