MLLDN expression specifically inhibits the initiation of MLL leukemic cells. (A-C) Proliferation assays were performed with lin⁻c-kit⁺ mouse bone marrow cells grown in double selective media following transduction with combinations of the MLL-AF9 or E2A-HLF fusion proteins and empty MSCV puro or MLLDN after 1 day in selective media (mean ± SD; n = 2 independent experiments performed in duplicate (P > .05); 2-way ANOVA). (D-E) qRT-PCR was performed on RNA collected from either Neo-Puro– or Neo-MLLDN–transduced bone marrow cells at days 0, 3, and 6 following plating described in (A-C). Arrows indicate time points for RNA collection. Expression levels for Hoxa9 and Meis1 are shown relative to Neo-Puro–transduced cells at day 0. MLLDN expression is shown relative to Neo-MLLDN–transduced cells at day 0 (mean ± SD; n = 2 independent experiments performed in triplicate; ***P < .001, *P < .05; unpaired 2-tailed Student t test). (G-I) Colony-forming assays were performed using the methods described in (A-C). Double-transduced cells were grown in double-selective semisolid media for 7 days for each round. Colony numbers from each successive round of replating are shown. Results suggest a selective sensitivity of MLL-AF9–transformed cells to disruption of the MLL-PAFc interaction. (G) Cells transduced with empty MSCV-neo and either MSCV puro or MSCV-MLLDN. (H) Cells transduced with MSCV-MLL-AF9 and either MSCV puro or MSCV-MLLDN. (I) Cells transduced with MSCV-E2A-HLF and either MSCV puro or MSCV-MLLDN (mean ± SD; n = 2 independent experiments performed in duplicate; *P < .05, ****P < .0001 (P > .05); unpaired 2-tailed Student t test).