The phosphoinositide 3-kinase (PI3-kinase) signalling pathway plays a key role in the regulation of cell survival and proliferation. We show that the PI3-kinase/Akt pathway is constitutively active in primary acute myeloid leukaemia (AML) cells and that blockade by the selective inhibitor LY294002 reduces survival of the total blast population (mean 52%). The ERK/MAPK module is also constitutively active and treatment with the MAPKK inhibitor U0126 reduces cell survival by 22%. In 10 of 18 samples, PI3-kinase contributes to MAPK activation as incubation with LY294002 leads to a marked reduction in its phosphorylation. PI3-kinase inhibition reduces survival of the CD34 þ 38À AML progenitor subset by 44%, whereas MAPKK inhibition has little effect. Reporter assays in primary AML cells show that blocking PI3-kinase leads to a marked reduction of constitutive NF-kB activity and promotes p53-mediated transcription. This is associated with a synergistic interaction between LY294002 and Ara-C. An inducible activated form of Akt protects normal myeloid cells from Ara-C and etoposide-mediated apoptosis. These results show that blocking PI3-kinase has direct antileukaemic effects and potentiates the response to conventional cytotoxics via a number of targets including NF-kB, p53 and MAPK. Inhibitors of PI3-kinase and Akt may be useful in the treatment of AML.

The b-catenin protein is at the core of the canonical Wnt signalling pathway. Wnt stimulation leads to b-catenin accumulation, nuclear translocation and interaction with T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors to regulate genes important for embryonic development and proliferation. Wnt/b-catenin can promote stem cell self-renewal and is dysregulated in colon carcinoma. We have examined the role of the Wnt pathway in the development of acute myeloid leukaemia (AML) and find that the b-catenin protein is readily detected in primary AML samples. Using transfection of a TCF/LEF reporter construct into primary AML cells and normal human progenitors, we find increased reporter activity in 16/25 leukaemia samples. Retrovirally mediated expression of a mutant active b-catenin in normal progenitors preserves CD34 expression and impairs myelomonocytic differentiation. Activation of TCF/LEF signalling decreases factor withdrawal-induced apoptosis of normal progenitors. A significant proportion of AML cases show aberrant expression of components of the Wnt pathway including Wnt-1, Wnt-2b and LEF-1. These results provide evidence for the involvement of the Wnt/b-catenin pathway in the pathogenesis of AML.

Current therapy for acute myeloid leukaemia (AML) is suboptimal with a high incidence of relapse. There is strong evidence that constitutive phosphoinositide 3-kinase (PI3K) activity plays a significant role in the pathophysiology of AML. PI3K products are derived from the activity of a number of PI3K catalytic isoforms (class I, II and III) but the relative contribution of these enzymes in AML remains unknown. As non-isoformselective inhibitors of PI3K such as LY294002 may produce unwanted toxicity to normal tissues, we have investigated the role of the leukocyte-restricted p110d PI3K isoform in 14 cases of AML. p110d was detected in all cases whereas the expression levels of the other class I PI3Ks varied more widely, and were often undetectable. The p110d-selective compound IC87114 inhibited constitutive phosphorylation of the PI3K target Akt/PKB and reduced cell number to a mean of 6675% (range 14-88%). In eight cases, the combination of IC87114 and VP16 (a topoisomerase II inhibitor) was synergistic in reducing viable cell number, and was associated with a reduction in constitutive NF-jB activity. IC87114 did not have direct adverse effects or enhance the activity of VP16 on the proliferation and survival of normal haemopoietic progenitors. Overall, our results identify the p110d isoform as a potential therapeutic target in AML and support a clinical approach to use isoform-selective over broad-spectrum PI3K inhibitors.