MLL3 gene is mutated in FLT3-ITD AML both at DX and REL, and silencing of MLL3 in FLT3-ITD cells increased their growth in both liquid culture and clonogenic assay. (A) Schematic of the MLL3 domains and locations of the amino acid substitutions caused by somatic mutations detected by WES and TDS. Black or red triangles indicate missense mutations or nonsense mutations (either frameshift [fs] or stop-gain [X], respectively). Red arrow represents nonsense mutation identified in AML TCGA data. Structural motifs of gene: A.T hook (ATPase α/β signature), PHD (plant homeodomain), DHHC (palmitoyltransferase activity), FYR (phenylalanine tyrosine-rich domain), SET (suppressor of variegation, enhancer of zeste, trithorax). (B) Real-time PCR analysis showed reduced MLL3 mRNA in MLL3 shRNA-treated cells compared with scramble shRNA-treated cells. MLL3 shRNA3 and MLL3 shRNA4 showed approximately 50% to 60% knockdown in MV4-11 cells compared with scramble shRNA. (C) Western blot shows reduced MLL3 protein levels in MLL3 shRNA transduced cells (MV4-11) compared with scramble shRNA-treated cells. GAPDH is used as an internal control. (D) Short-term cell proliferation assays of MV4-11 cells transduced with either MLL3 shRNAs or scramble shRNA. Data represent means ± standard deviation (SD); n = 4. (E) For cell counting assay, 0.5 × 10⁵ cells were plated in 6-well plates in quadruplets. Cell proliferation was measured by counting cells over a 5-day period. Results are shown as means ± SD; n = 4. (F) Methylcellulose colony assay showed a significant increase in the number of MV4-11 colonies after cells were transfected with MLL3 shRNA compared with scramble shRNA-treated cells. Data represent means ± SD; n = 3. *P ≤ .01; **P ≤ .001.