Supplementary Tables S1-S6. Primers used in this study (S1); The statistics for the final water-refined set of NMR structures of IBD-MLL 140-160 complex (S2); The solvent-accessible surface area (BSA) of individual lBD residues buried upon IBD-HIV1-integrase (HIV IN) or IBD-MLL complex formation is expressed as a portion of the residue total solvent accessible area (BSA in %). The BSA was calculated using PDBePISA tool (S3); Effect of LEDGF/p75 mutations on the interaction with MLL as measured by AlphaScreen (S4); Effect of LEDGF/p75 mutations defective for binding to HIV-1 integrase on the interaction with MLL as measured by AlphaScreen (S5); Melting temperatures of the PWWP and IBD domain of wild type LEDGF/p75 or its mutants as determined by DSF (S6).
ARTICLE ABSTRACT
Mixed lineage leukemia (MLL) fusion–driven acute leukemias represent a genetically distinct subset of leukemias with poor prognosis. MLL forms a ternary complex with the lens epithelium–derived growth factor (LEDGF/p75) and MENIN. LEDGF/p75, a chromatin reader recognizing H3K36me3 marks, contributes to the association of the MLL multiprotein complex to chromatin. Formation of this complex is critical for the development of MLL leukemia. Available X-ray data represent only a partial structure of the LEDGF/p75–MLL–MENIN complex. Using nuclear magnetic resonance spectroscopy, we identified an additional LEDGF/p75–MLL interface, which overlaps with the binding site of known LEDGF/p75 interactors—HIV-1 integrase, PogZ, and JPO2. Binding of these proteins or MLL to LEDGF/p75 is mutually exclusive. The resolved structure, as well as mutational analysis, shows that the interaction is primarily sustained via two aromatic residues of MLL (F148 and F151). Colony-forming assays in MLL–AF9+ leukemic cells expressing MLL interaction-defective LEDGF/p75 mutants revealed that this interaction is essential for transformation. Finally, we show that the clonogenic growth of primary murine MLL-AF9–expressing leukemic blasts is selectively impaired upon overexpression of a LEDGF/p75-binding cyclic peptide CP65, originally developed to inhibit the LEDGF/p75–HIV-1 integrase interaction. The newly defined protein–protein interface therefore represents a new target for the development of therapeutics against LEDGF/p75–dependent MLL fusion–driven leukemic disorders. Cancer Res; 74(18); 5139–51. ©2014 AACR.
