Supplementary Figures S1-S9. Published structure of the MLL-MENIN-LEDGF/p75 complex (S1); Characterization of the murine MLL-AF9 AML model (S2); Coomassie stained SDS-PAGE gels of recombinant proteins used in this study (S3); Identification of the MLL-LEDGF/p75 interaction surface (S4); Protein melting curves of LEDGF/p75 mutants (S5); Model of the MLL-MENIN-LEDGF/p75 ternary interface (S6); Representation of the buried surface area of IBD amino acid residues upon binding either MLL or HIV-1 integrase (S7); A MLL interaction deficient LEDGF/p75 mutant cannot rescue MLL-AF9 transformation upon LEDGF/p75 knockdown (S8); CP65 interferes with LEDGF/p75-MLL but not with MENIN-MLL complex formation in vitro (S9).
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.
