Molecular Basis of the Mixed Lineage Leukemia-Menin Interaction

Abstract

Chromosomal translocations targeting the mixed lineage leukemia (MLL) gene result in MLL fusion proteins that are found in aggressive human acute leukemias. Disruption of MLL by such translocations leads to overexpression of Hox genes, resulting in a blockage of hematopoietic differentiation that ultimately leads to leukemia. Menin, which directly binds MLL, has been identified as an essential oncogenic co-factor required for the leukemogenic activity of MLL fusion proteins. Here, we characterize the molecular basis of the MLL-menin interaction. Using 13 C-detected NMR experiments, we have mapped the residues within the intrinsically unstructured fragment of MLL that are required for binding to menin. Interestingly, we found that MLL interacts with menin with a nanomolar affinity (K d ϳ 10 nM) through two motifs, MBM1 and MBM2 (menin binding motifs 1 and 2). These motifs are located within the N-terminal 43-amino acid fragment of MLL, and the MBM1 represents a high affinity binding motif. Using alanine scanning mutagenesis of MBM1, we found that the hydrophobic residues Phe 9 , Pro 10 , and Pro 13 are most critical for binding. Furthermore, based on exchange-transferred nuclear Overhauser effect measurements, we established that MBM1 binds to menin in an extended conformation. In a series of competition experiments we showed that a peptide corresponding to MBM1 efficiently dissociates the menin-MLL complex. Altogether, our work establishes the molecular basis of the menin interaction with MLL and MLL fusion proteins and provides the necessary foundation for development of small molecule inhibitors targeting this interaction in leukemias with MLL translocations. Chromosomal translocations involving the mixed lineage leukemia (MLL)3 gene result in human acute myeloid and lymphoid leukemias, affecting both children and adults (1, 2). Fusion of MLL with one of 60 partner genes forms chimeric oncogenes encoding MLL fusion proteins, which results in enhanced proliferation and blockage of blood cell differentiation ultimately leading to the development of acute leukemia (3). Translocations of MLL are particularly prevalent in infants with acute myeloid leukemia and acute lymphoblastic leukemia and constitute up to 80% of all infant acute leukemia cases (4). Patients with leukemias harboring MLL translocations have a very poor prognosis using available therapies (20% event-free survival at 3 years), and it is clear that novel targeted therapies are urgently needed to treat these leukemias (3, 5). MLL belongs to the evolutionary conserved family of TRX (Drosophila Trithorax) proteins that positively regulate gene expression during development (6 -8). MLL has been shown to associate with promoters of Ͼ5000 human genes, suggesting that it might play a global role in transcription (9). MLL is an important regulator of Hox gene expression, which is required for normal hematopoiesis (10). Disruption of MLL by chromosomal translocations up-regulates expression of Hox genes, including Hoxa7, Hoxa9, and the Hox cofactor Meis1, r...

Structural insights into inhibition of the bivalent menin-MLL interaction by small molecules in leukemia

“…We found that both MI-2 and MI-2-2 can inhibit the interaction of menin with MLL 4-43 , which comprises the intact menin-binding fragment. 19,36 As expected, the second-generation inhibitor MI-2-2 was approximately 7-fold more potent in disrupting the menin-MLL 4-43 interaction, with an IC 50 ϭ 520nM ( Figure 4A). To assess whether these inhibitors can dissociate menin interaction with the full-length MLL-AF9 fusion protein in cells, we performed coimmunoprecipitation experiments.…”

“…Pro10 MLL binds to the site adjacent to Phe9 MLL and interacts with Phe238 and Ala242, whereas Pro13 MLL fits into a hydrophobic pocket formed by Tyr319, Tyr323, and Met322. Mutations of Phe9 MLL , Pro10 MLL , and Pro13 MLL to alanine residues result in a dramatic decrease of MBM1 binding to menin (2000-, 30-, and 50-fold decrease, respectively), 19 validating the importance of these hydrophobic interactions. Additional stabilization of the MBM1-menin complex results from a salt bridge between Arg12 MLL and Glu359 and Glu363, as well as 3 intermolecular hydrogen bonds involving the MLL backbone (residues Arg6 MLL , Trp7 MLL , and Ala11 MLL ) and menin side chains (Asn244, Asp136, and Tyr323; Figures 1B-C).…”

“…19 Deletion of a highaffinity motif MBM1 is sufficient to abolish transformation by MLL-ENL, 8 which shows that this interaction constitutes a hot spot for small-molecule development. Because MBM2 also contributes to the binding to menin, it was necessary to establish whether small molecules targeting the MBM1 site on menin are sufficient to efficiently inhibit the bivalent interaction of menin with MLL.…”

“…The longer fragment containing both motifs interacts with menin with 10nM affinity. 19 To provide an insight into the recognition of MLL by menin, we have determined the structure of menin in complex with the high-affinity motif, MBM1 (supplemental Table 1). The structure revealed that MBM1 binds to the large central cavity on menin.…”

Do We Need Small Molecule Inhibitors for the Immune Checkpoints?

“…The last decade has witnessed many successful examples of designing smallmolecule modulators for many protein-protein interactions, including orthosteric inhibitors, allosteric regulators, and interfacial binders. Recent examples include discovering small molecule inhibitors for menin and mixed lineage leukemia (MLL) interactions [30]. Some of these molecules can inhibit the menin-MLL interaction at a nanomolar concentration [31].…”