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

Analysis of factors that affect in vitro chemosensitivity of leukaemic stem and progenitor cells to gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukaemia

“…5 Using this system, we found the survival of the LSPC subset to be enhanced rather than inhibited after treatment with the FLT3 inhibitor AG1296. 5 In the current study, we have used the system to screen the effectiveness of Mylotarg against LSPC.…”

“…5 For the maintenance of LSPC phenotype in 48-h culture, we treated 21 AML samples in serum-free medium with immobilized fibronectin along with a combination of cytokines consisting of IL-3 (interleukin-3) (20 ng/ml), SDF-1 (stromal cell-derived factor 1) (100 ng/ml), SCF (stem cell factor) (50 ng/ml) and TPO (thrombopoietin) (50 ng/ml). For the chemosensitivity assay, Mylotarg was reconstituted in water (1 mg/ml) and then diluted fresh in serum-free medium (10 ng/ml) when used.…”

“…5 In trying to mimic the in vivo scenario, factors underpinning relapse in AML, such as LSPC burden, bone marrow niche microenvironment and other prognostic factors (such as FLT3 status) need to be modelled in vitro so as to understand the contributory factors that allow LSPC survival post chemotherapy. This is important to help identify appropriate therapeutic interventions that achieve lasting remissions in AML.…”

“…Flow cytometric enumeration of LSPC and bulk AML cells, as well as CD123 mean fluorescence intensities (MFI) were measured in CD34 þ CD38 À leukaemic samples as previously described. 5 Briefly, two flow cytometric analyses were used in parallel for the analysis of LSPC survival. One assay allowed reproducible measurement of the concentration of viable cells at the end of the experiment using 10 mg/ml 7-amino-actinomycin D and fixed CD45-stained normal mononuclear cells as internal standard.…”

Drug resistance in mutant FLT3-positive AML

“…A flow cytometry-based assay system demonstrated enhancement of CD34( þ )CD38(-)CD123( þ ) leukemic stem and progenitor cell survival in response to treatment with Ara-c and the FLT3 inhibitor, AG1296, respectively, when cells were drug treated under defined 'niche-like' (or microenvironmental support) conditions (Mony et al, 2008). The defined 'nichelike' microenvironment in this study was comprised of serum-free medium supplemented with IL-3, IL-6, stem cell factor and Ang-1, and also included immobilized fibronectin.…”