A novel therapeutic approach for acute leukemia by targeting LEDGF/p75.

Mixed-lineage leukemias (MLL) represent a genetically distinct subset of acute leukemias characterized by chromosomal translocations in the MLL gene. Balanced rearrangements result in the formation of new fusion proteins inducing myeloid as well as lymphoblastic leukemias with poor prognosis. All N-terminal MLL fusions (MLLNT-fusions) form a 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 MLLNT-fusion multiprotein complexes to chromatin and interacts with MLL-MENIN via its integrase binding domain (IBD). Knockdown of LEDGF/p75 or the expression of an LEDGF/p75 C-terminal fragment containing the IBD, but lacking the chromatin binding domain (LEDGF325-530) impairs clonogenic growth of MLL-fusion gene transformed cells in vitro and in a mouse model. Therefore, the interaction of LEDGF/p75 with MLL/MENIN is considered a promising target for MLL leukemia treatment.

Through NMR spectroscopy and mutational analysis, we identified, mapped and validated a non-resolved MLL-LEDGF/p75 interface. The MLL-LEDGF/p75 interaction interface is maintained mainly by two phenylalanine side chains occupying two hydrophobic pockets on the IBD surface. From a structural point of view these two pockets represent the only obvious druggable site on the interface between LEDGF/p75 and MLL/MENIN. Overexpression of a LEDGF/p75 IBD binding peptide (CP65) impaired clonogenic growth of primary murine MLL-AF9 expressing leukemic blasts. The selective anti-leukemic activity of CP65 provides a direct rationale for the design of small molecules targeting the newly defined LEDGF/p75-MLL interface. Based on these results, we performed the first steps towards the development of small molecule LEDGF/p75-MLL interaction inhibitors. We employed a high throughput AlphaScreen based assay to screen a diverse library of over 72 000 drug-like compounds. Currently we are in the early hit-to-lead optimization phase of the first MLL-LEDGF/p75 interaction inhibitors.

Next to ongoing drug development efforts, we also characterized the interaction of the LEDGF/p75 IBD with its other known cellular interaction partners. Beside MLL, the LEDGF/p75 IBD is known to interact with several other cellular proteins like JPO2, PogZ and ASK. Using NMR spectroscopy, we characterized the interaction of LEDGF/p75 with the transcriptional repressor JPO2 and the domesticated transposase PogZ. This resulted in the identification of an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75 including MLL. The importance of the IBM to maintain the interaction was verified through mutational analysis. In addition, based on IBM conservation, we identified and validated IWS1 as a novel LEDGF/p75 interaction partner. The similar binding mode of physiological LEDGF/p75 interaction partners represents a challenge for the development of selective interaction inhibitors.