Summary Translocations involving the Mixed Lineage Leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the MEN1 gene. Here, we present the first small molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind to menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.

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...

3775 Poster Board III-711 Chromosomal translocations that affect the proto-oncogene MLL (Mixed Lineage Leukemia) occur in aggressive human acute leukemias, both AML and ALL, affecting children and adults. The normal MLL protein plays a key role in regulation of HOX genes expression, which are required for proper hematopoiesis. This function is frequently impaired by a fusion of MLL with one of 60 alternative partner genes to form a chimeric oncogene encoding MLL fusion proteins. MLL fusions upregulate HOX genes expression resulting in a blockage of blood cell differentiation that ultimately leads to acute leukemia. Patients with MLL rearrangement poorly respond to available treatments, emphasizing the urgent need to develop novel therapies to treat these leukemias. The leukemogenic activity of MLL fusions is dependent on association with menin, a protein encoded by the MEN1 (Multiple Endocrine Neoplasia I) gene. The menin binding motif is localized at the N-terminus of MLL and therefore it is retained in all MLL fusion proteins. The removal of this motif from MLL oncoproteins abrogates the ability to develop leukemia in mice. Menin functions as an essential oncogenic cofactor in MLL related leukemias and selective targeting of the menin-MLL interaction might represent a novel valuable therapeutic approach for the treatment of the MLL-related leukemias. To understand the molecular basis of how MLL-fusion proteins interact with menin, we carried out detailed in vitro characterization of menin binding to N-terminus of MLL using a collection of biochemical, biophysical and structural biology approaches. We demonstrated that 46 long N-terminal amino acid fragment of MLL very strongly associates with menin with low-nanomolar binding affinity. Employing the NMR spectroscopy, we identified the presence of two separate menin binding motifs within this MLL fragment, MBM1 (menin binding motif 1) and MBM2 (menin binding motif 2), which are separated by a poly-glycine linker. Peptides corresponding to both motifs are capable to independently interact with menin indicating the presence of two separate MLL binding sites on menin. Furthermore, the MBM1 binds to menin with 20-fold higher affinity compared to MBM2. Interestingly, we demonstrated that binding of one of the MBM peptides to menin negatively regulates binding of the second peptide most likely through the mechanism of an allosteric regulation. To aid in rational design of small molecule inhibitors of the menin-MLL interaction we characterized the conformation of the high affinity motif (MBM1) of MLL in a menin bound conformation using NMR spectroscopy. Furthermore, by applying both mutational studies and binding affinity measurements we identified that the most critical amino acids of MBM1 involved in interaction with menin comprise the RFPARP fragment of MLL. Overall, for the first time, we are providing detailed characterization and molecular basis of the MLL interaction with menin, which will be invaluable for development of therapeutically useful inhibitors selectively targeting this interaction. Disclosures: No relevant conflicts of interest to declare.