IM is an inhibitor of, not a substrate for, ABCG2. (A) ABCG2 inhibition does not potentiate IM. CD34⁺ CML cells were seeded at 1 × 10⁵ cells at day 0 (indicated by dotted line), and the number of total viable cells was analyzed after 72 hours in culture with 5GF alone (filled bar; no drug), + 10 μM FTC alone (dark gray bar), + 5 μMIM (light gray bar), or both drugs (open bar). All data are mean of duplicate analyses of n = 6 samples. (B-E) To determine whether IM is a substrate or inhibitor of ABCG2, the cellular concentration of the known substrate mitoxantrone or IM and the effect of the ABCG2 inhibitor FTC or IM on drug accumulation was tested in cell lines and CML CD34⁺ cells. (B) Mitoxantrone accumulation in cell lines. The cellular concentration of ³H-mitoxantrone, a known ABCG2 substrate, was determined over a range of extracellular concentrations of the drug for both ABCG2-negative AML3 cells (solid lines, black symbols) and ABCG2-overexpressing AML6.2 cells (dashed lines, open symbols). The effect of the ABCG2 inhibitor FTC (10 μM; ▾, ▿) or 5 μM IM (□, ▪) was also determined. ***P < .01 compared with all other treatments. (C) Mitoxantrone accumulation in CML CD34⁺ cells. CD34⁺ CML cells were incubated with ³H-mitoxantrone similarly (³H-mitoxantrone alone •, + 10 μM FTC ▾, + 5 μM IM ▪). ***P < .01 compared with mitoxantrone alone. (D) IM accumulation in cell lines. The cellular concentration of ¹⁴C-IM and the effect of ABCG2 inhibition was determined over a range of extracellular concentrations of the drug for both AML3 cells (solid lines, black symbols) and AML6.2 cells (dashed lines, open symbols) in the presence (▾, ▿) and absence (•, ○) of the ABCG2 inhibitor FTC (10 μM). (E) IM accumulation in CML CD34⁺ cells. The cellular concentration of ¹⁴C IM accumulated by CD34⁺ CML cells was determined similarly (¹⁴C-IM alone •, + 10 μM FTC ▾). All data are the mean standard error from at least 3 individual patients for CML cells and 3 separate experiments for cell lines; all were analyzed in triplicate. All statistical analyses were by paired Student t test; NS indicates not significant.