| Relapse is common in acute myeloid leukemia (AML) patients due to emergence and outgrowth of minimal residual disease (MRD). New treatment strategies are needed to effectively eradicate MRD in order to improve survival. Our research focuses on how aberrant signal transduction, e.g. constitutive phospho-AKT (pAKT) expression and Nuclear Factor kappa B (NFkB) activity, and the bone marrow microenvironment (BM-ME) contribute to the emergence of MRD and thereby effect prognosis. We will study AKT and NFkB activity status in AML cell subpopulations at diagnosis, in MRD and at relapse to investigate whether changes occur during/after therapy. AML cells are protected from apoptosis when attached to the BM-ME. By co-culture of AML blasts with BM-ME we want to elucidate whether AKT/ NFkB signaling contributes to this protective effect. A FACS assay was developed for the quanti-fication of phosphorylated protein expression in AML cell subsets. U937 cells, which constitutively express pAKT, was used for validation. Without LY294002, a specific inhibitor of PI3K-dependent AKT phosphorylation, pAKT expression in U937 cells (as mean ratio MFI Ab/isotype ±sd, n=5) was 2.2±0.4. With the inhibitor, pAKT expression was absent (1.0±0.2). When using sub-optimal LY294002 concen-trations a range of pAKT expressions were detectable with FACS, which was similar to results obtained with Western Blotting. Preliminary experiments with primary patient samples showed the presence of AML cell subsets at diagnosis with differential pAKT expression after LPS stimulation. When using fibronectin, as a simple model for the BM-ME, 20% of HL60 cells were attached after 45 min of incubation. An increase in NFkB activity (factor 1.6 higher) was found in the attached cells compared to the unattached ones. Whether pAKT expression is similarly upregulated after BM-ME contact and whether pAKT expression is preceding NFkB activation is currently under investigation. We anticipate that more insight into the contribution of AKT/ NFkB signaling in the interaction between leukemic cells and the BM-ME leading to the emergence of MRD will be obtaind. This might guide the development of new treatment strategies to prevent and eradicate MRD. |
