Resistance to FLT3 inhibition in an in vitro model of primary AML cells with a stem cell phenotype in a defined microenvironment.

Relapse in acute myeloid leukaemia (AML) is mediated by survival of leukaemic stem cells following remission-induction chemotherapy. It would therefore be useful to identify therapeutic agents that target leukaemic stem cells. We devised a flow cytometric chemosensitivity assay allowing 48 h culture of leukaemic blasts in a defined microenvironment followed by enumeration of viable CD34+CD38-CD123+ leukaemic stem and progenitor cells (LSPC). The assay was used to investigate the LSPC response to cytosine arabinoside (Ara-C) and to the FLT3 inhibitor AG1296. There was a 3.6-fold increase in Ara-C-treated LSPC survival under defined 'niche-like' conditions compared to culture without microenvironmental support. Nine AML samples with internal tandem duplications of FLT3 (FLT3/ITDs) were treated with AG1296. Three samples were very sensitive (>50% kill) and 4 were moderately sensitive (10-50% kill) in bulk suspension culture without microenvironmental support. However, under defined 'niche-like' conditions, the survival of LSPC was enhanced rather than inhibited by AG1296 treatment. We conclude that an interaction between LSPC and a defined in vitro microenvironment models a chemoresistant niche. Our data point to a need to investigate more novel chemotherapeutic agents under these stringent conditions to identify agents that may be suitable to target minimal residual disease in AML.