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.
Menin functions as a critical oncogenic cofactor of mixed lineage leukemia (MLL) fusion proteins in the development of acute leukemias, and inhibition of the menin interaction with MLL fusion proteins represents a very promising strategy to reverse their oncogenic activity. MLL interacts with menin in a bivalent mode involving 2 N-terminal fragments of MLL. In the present study, we reveal the first high-resolution crystal structure of human menin in complex with a smallmolecule inhibitor of the menin-MLL interaction, MI-2. The structure shows that the compound binds to the MLL pocket in menin and mimics the key interactions of MLL with menin. Based on the menin-MI-2 structure, we developed MI-2-2, a compound that binds to menin with low nanomolar affinity (K d ؍ IntroductionTranslocations of the MLL (mixed lineage leukemia) gene frequently occur in aggressive human acute myeloid and lymphoid leukemias in both children and adults. 1 Fusion of MLL with 1 of more than 60 different genes results in chimeric MLL fusion proteins that enhance proliferation and block hematopoietic differentiation, ultimately leading to acute leukemia. 2 Patients with leukemias harboring MLL translocations have very unfavorable prognoses and respond poorly to currently available treatments. 2,3 The relapse risk is very high using conventional chemotherapy and stem cell transplantation, 2 leading to an overall 5-year survival rate of only approximately 35%. 4 All MLL fusion proteins preserve an N-terminal MLL fragment approximately 1400 amino acids in length fused in-frame with the C-terminus of the fusion partner. 3,[5][6][7] Two regions in this fragment of MLL have been shown to be indispensable for leukemogenic transformation: the N-terminal region, which binds to menin 8 and to lens epithelium-derived growth factor (LEDGF), 9 and the conserved region encompassing the CXXC domain, which mediates binding to nonmethylated CpG DNA [10][11][12] and interacts with the polymerase associated factor complex (PAFc). 13,14 Targeting these interactions provides new opportunities for the development of new therapeutic agents for the MLL leukemias. 15 Menin is a tumor-suppressor protein encoded by the MEN1 (multiple endocrine neoplasia 1) gene. 16 Mutations of MEN1 are associated with tumors of the parathyroid glands, pancreatic islet cells, and anterior pituitary gland. 17 Menin is also a highly specific binding partner for MLL and MLL fusion proteins and is required to regulate the expression of MLL target genes, including HOXA9 and MEIS1. 8 Loss of the ability to bind menin abolishes the oncogenic potential of MLL fusion proteins both in vitro and in vivo. 8 Disruption of the interaction between menin and MLL fusion proteins using genetic methods blocks the development of acute leukemia in mice, 8 indicating that menin functions as a critical oncogenic cofactor of MLL fusion proteins and is required for their leukemogenic activity. The menin-MLL interaction represents an attractive therapeutic target for the development of novel drugs for acut...
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...
2497 Chromosomal translocations that affect the MLL (Mixed Lineage Leukemia) proto-oncogene occur in aggressive acute leukemias, both in children and adults. Fusion of MLL to one of more than 50 partner genes results in generation of the MLL fusion oncoprotein, which upregulates expression of HOX genes required for normal hematopoiesis, and ultimately leads to the development of acute leukemia. Patients harboring translocations of MLL gene suffer from very aggressive leukemias and respond poorly to available therapies, emphasizing the urgent need for novel therapeutic treatments. All oncogenic MLL fusion proteins have a preserved N-terminal fragment of MLL that interacts with menin, a tumor suppressor protein encoded by MEN1 (Multiple Endocrine Neoplasia 1) gene. Importantly, the menin-MLL fusion protein interaction is critical to the leukemogenic activity of MLL fusion proteins and misregulation of HOXA9 genes, and therefore it represents a valuable molecular target for therapeutic intervention. Selective targeting of the protein-protein interaction between menin and MLL fusion proteins with small molecules could block the oncogenic activity of MLL fusion proteins and inhibit development of acute leukemia. To identify small molecule inhibitors of the menin-MLL interaction we have performed a High Throughput Screen of 350,000 compounds using a collection of biochemical assays and biophysical methods. This resulted in several classes of compounds that specifically bind to menin and inhibit the menin-MLL interaction both in vitro and in human cells. We then applied medicinal chemistry approaches to develop analogues of selected lead candidates, resulting in very potent compounds that inhibit the menin-MLL interaction with nanomolar affinities. To evaluate potency, specificity and mechanism of action of these compounds we used a broad collection of cellular assays. These compounds selectively inhibit proliferation of the MLL leukemia cells, strongly induce apoptosis and differentiation of these cells. Importantly, these compounds substantially downregulate expression of HOXA9 and MEIS1 genes that are downstream targets of MLL fusion proteins required for their leukemogenicity, and they also deplete the menin-MLL fusion protein complex from the target genes. Furthermore, the compounds that we developed specifically inhibit the MLL fusion protein mediated oncogenic transformation. All these effects closely recapitulate the effects observed upon acute loss of menin or disruption of the menin-MLL fusion protein interaction using genetic manipulations, demonstrating highly specific mode of action for these compounds. Our current efforts are focused to assess the effect of these compounds in in vivo models of MLL leukemia and evaluate their utility as future drug candidates for acute leukemias. This may provide a novel therapeutic approach for the treatment of very aggressive leukemias with MLL translocations. Disclosures: No relevant conflicts of interest to declare.
2500 Chromosomal translocations of MLL (Mixed Lineage Leukemia) gene result in aggressive acute leukemias, affecting both children and adults. Fusion of MLL to one of more than 60 partner genes results in MLL fusion oncoproteins which upregulate expression of Hox genes required for normal blood cell development, ultimately leading to development of acute leukemia. Regardless of the fusion partner, the presence of MLL translocations is associated with early relapse and poor prognosis. Survival rates are particularly low for infants and there is a pressing need for the development of targeted therapies against leukemias with MLL translocations. The oncogenic activity of MLL fusion proteins is dependent on association with LEDGF (lens epithelium-derived growth factor) and menin, both of which interact with the N-terminus of MLL retained in all MLL fusion proteins. LEDGF is a chromatin-associated protein, which interacts conjointly with MLL and menin on the chromatin of the cancer associated genes, and both interactions are required for the MLL-mediated leukemogenesis and misregulation of HOXA9 expression. Therefore, LEDGF functions as an essential oncogenic cofactor in MLL related leukemias, and may represent a valuable molecular target for therapeutic intervention with small molecules. We have performed rigorous biophysical and biochemical studies and revealed that LEDGF is involved in simultaneous interaction with menin and with the N-terminus of MLL. Interestingly, the association of LEDGF with the menin-MLL complex has relatively low affinity which limits the application of conventional screening methods for lead identification. To develop small molecule inhibitors targeting LEDGF interactions we have employed two strategies: Fragment Based Drug Discovery (FBDD) approach and High Throughput Screening (HTS). We have identified several compounds that bind directly to LEDGF. By applying NMR spectroscopy we discovered that these compounds interact with the menin binding site on LEDGF. Then we have assessed the activity of these compounds using a broad range of cell based assays. We found that compounds targeting LEDGF specifically inhibit proliferation of the MLL leukemia cells without affecting the non-MLL leukemia cells. They also induce apoptosis and differentiation of MLL leukemia cells as assessed by increased expression of CD11b differentiation marker and substantial change in morphology of these cells. Furthermore, these compounds reduce transforming properties of MLL fusion proteins and downregulate expression of Hoxa9 and Meis1 genes confirming a highly specific mode of action. Overall, our results demonstrate that targeting of LEDGF by small molecules is feasible and may results in development of potent inhibitors of LEDGF interaction with menin and MLL fusion proteins in leukemias with MLL rearrangements. Such compounds might provide a new therapeutic approach for the treatment of MLL-rearranged leukemias. Disclosures: No relevant conflicts of interest to declare.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.