Akt/foxo signaling pathway enforces the differentiation blockade in myeloid leukemias

Acute myeloid leukemia (AML) is a heterogeneous blood cancer comprised of different sub types that are categorized by genetic and molecular alterations. The complex heterogeneity of AMLs has made it difficult to identify molecular candidates that could provide the basis for designing universal or broad spectrum target based AML therapies. Interestingly, the activity of the oncogenic kinase AKT has a bimodal distribution in AML, where 50% of patient samples display elevated AKT activity. We aimed to clarify the molecular mechanisms that contribute to the dichotomous pattern of AKT signaling in AML. AKT phosphorylates numerous substrates includingn members of the FOXO family of transcription factors. FOXOs are exported from the nucleus and thereby inactivated upon AKT mediated phosphorylation. Therefore, we examined the cellular distribution of FOXOs in primary human AML samples. Of the 9 samples evaluated, 8 displayed a wide range of nuclear FOXO3 (10 70%). To determine the global status of FOXO activity in AML we used a hematopoietic specific gene signature of FOXO activity to perform hierarchical clustering analysis of gene expression data from a large cohort (n=436) of primary human AML samples. This analysis showed that AML stratifies into two distinct clusters based on FOXO activity (Cluster 1 representing lower FOXO (n=253) activity than Cluster 2 (n=183)) stronglyn indicating that FOXOs play an important role in AML. To determine the functional relevance of FOXOs in AML, we introduced FOXO3 specific shRNA into both primary and cultured AML cells. Inhibition of FOXO3 expression resulted in diminished growth, enhanced differentiation and subsequent death of AML cells. Furthermore, ablation of FoxO1/3/4 in a murine model of AML (driven by MLL AF9) resulted in decreased leukemic burden (pl0.0001), increased survival (p=0.0009) and reduced leukemia initiating cell activity (p=0.0007) in vivo. Although deletion of FoxO1/3/4 extended latency, the majority of animals transplanted with leukemia null for FoxO1/3/4 eventually develop overt AML. Therefore, we predicted that a molecular switch exists that allows leukemia cells to survive FOXO inactivation. Examination of oncogenic signaling pathways associated with AML revealed that the stressn activated kinase JNK and its downstream substrate cn JUN are consistently activated in murine AMLs nulln for FOXOs. Further analysis of the aforementionedn 436 primary AML sample gene expression datan revealed that AMLs that display lower FOXO activity exhibit elevated c JUN expression (cluster 1) and vicen versa (pl0.0001). Remarkably,n pharmacological inhibition of JNK cooperates with FOXO inhibition ton induce apoptosis in murine and human AML cells. These data identify previouslyn unrecognized roles ofn AKT, FOXOs, JNK and cJUN in AML and unveil a signalingn network that could potentially haven diagnostic and therapeutic implications.n