Validation and Structural Characterization of the LEDGF/p75–MLL Interface as a New Target for the Treatment of MLL-Dependent Leukemia

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 þ 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 fusiondriven leukemic disorders. Cancer Res; 74(18); 5139-51. Ó2014 AACR.

Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif

“…3d). Highly similar results were obtained previously for mapping of the IBD-MLL1 interaction 14 , indicating that JPO2, PogZ and MLL1 share a common interaction interface on IBD (Supplementary Fig. 3d).…”

“…Alignment of the sequences encompassing these residues with the recently published IBD-interacting residues of MLL1 (residues 140-155) 14 revealed a conserved consensus sequence ((E/D)-X-E-X-F-X-G-F), which we refer to as the IBM (Fig. 4e).…”

“…4) or MLL1 (ref. 14). To assess the importance of these residues in LEDGF/p75 interactions, we mutated them to alanines.…”

“…To provide a more detailed insight into the actual organization of the interaction interface, initially mapped by backbone NMR signal perturbations, we solved the solution structure of the PogZ-derived peptide in complex with the IBD (PDB ID: 2n3a), as a representative of the IBM. In particular, we determined the solution structure of the PogZ-derived peptide (PogZ 1389-1404 ) in complex with the IBD using the previously published approach 14 . Comprehensive backbone and side-chain resonance assignments for the PogZ 1389-1404 bound 13 C/ 15 N labelled LEDGF/p75 IBD were obtained using established triple-resonance experiments and for the bound unlabelled PogZ 1389-1404 peptide using 13 C/ 15 N filtered homonuclear total correlation spectroscopy and nuclear Overhauser enhancement spectroscopy (NOESY) experiments.…”

“…Recently, we and others identified a second menin-independent MLL1 binding site on LEDGF/p75 (refs 14,15). Both interfaces were shown to be important for MLL fusion-mediated transformation and represent valuable novel therapeutic targets 14,15 .…”