The same site on IBD interacts with IBM2 and HIV-IN. (A) Superposition of MLL-IBD structure (MLL IBM2 is shown in magenta, and IBD is green) onto IBD-IN complex (IN dimer is blue; based on PDB structure 2B4J). W131 from the IN binds to the same site on IBD as F148 from IBM2 motif. (B) IN disrupts menin-MLL-IBD complex in AlphaLISA assay by sequestering IBD from menin-MLL. (C) Dual luciferase assay performed in HEK293 cells with Hoxa9-LUC reporter. Cells were cotransfected with menin, MLL-AF9, LEDGF, and increasing amounts of IN. (D-I) Expression of IN and MLL110-160 impairs transformation of BMCs with MLL-AF9. BMCs were cotransduced with MSCV-neo-MLL-AF9 and MSCV-puro containing IN, MLL110-160, and MLL110-160 F129A or MLL110-160 FLAA. Expression of IN and MLL fragments has been validated using qRT-PCR (supplemental Figure 5). (D) Growth curves normalized to MSCV-puro. (E) Expression of Hoxa9 in transformed cells (day 14). (F) Expression of c-Kit (day 14). (G) Wright-Giemsa-stained cytospins (day 14). (H) Colony counts after 3 rounds of replating. (I) Representative colonies in round 3.
