Crystal structure of menin reveals binding site for mixed lineage leukemia (MLL) protein

Menin is a tumor suppressor protein that is encoded by the MEN1 (multiple endocrine neoplasia 1) gene and controls cell growth in endocrine tissues. Importantly, menin also serves as a critical oncogenic cofactor of MLL (mixed lineage leukemia) fusion proteins in acute leukemias. Direct association of menin with MLL fusion proteins is required for MLL fusion protein-mediated leukemogenesis in vivo, and this interaction has been validated as a new potential therapeutic target for development of novel anti-leukemia agents. Here, we report the first crystal structure of menin homolog from Nematostella vectensis. Due to a very high sequence similarity, the Nematostella menin is a close homolog of human menin, and these two proteins likely have very similar structures. Menin is predominantly an α-helical protein with the protein core comprising three tetratricopeptide motifs that are flanked by two α-helical bundles and covered by a β-sheet motif. A very interesting feature of menin structure is the presence of a large central cavity that is highly conserved between Nematostella and human menin. By employing site-directed mutagenesis, we have demonstrated that this cavity constitutes the binding site for MLL. Our data provide a structural basis for understanding the role of menin as a tumor suppressor protein and as an oncogenic co-factor of MLL fusion proteins. It also provides essential structural information for development of inhibitors targeting the menin-MLL interaction as a novel therapeutic strategy in MLL-related leukemias.     

Molecular basis of the mixed lineage leukemia-menin interaction: implications for targeting mixed lineage leukemias

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) ～ 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.     

The menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis

The Mixed-Lineage Leukemia (MLL) protein is a histone methyltransferase that is mutated in clinically and biologically distinctive subsets of acute leukemia. MLL normally associates with a cohort of highly conserved cofactors to form a macromolecular complex that includes menin, a product of the MEN1 tumor suppressor gene, which is mutated in heritable and sporadic endocrine tumors. We demonstrate here that oncogenic MLL fusion proteins retain an ability to stably associate with menin through a high-affinity, amino-terminal, conserved binding motif and that this interaction is required for the initiation of MLL-mediated leukemogenesis. Furthermore, menin is essential for maintenance of MLL-associated but not other oncogene induced myeloid transformation. Acute genetic ablation of menin reverses aberrant Hox gene expression mediated by MLL-menin promoter-associated complexes, and specifically abrogates the differentiation arrest and oncogenic properties of MLL-transformed leukemic blasts. These results demonstrate that a human oncoprotein is critically dependent on direct physical interaction with a tumor suppressor protein for its oncogenic activity, validate a potential target for molecular therapy, and suggest central roles for menin in altered epigenetic functions underlying the pathogenesis of hematopoietic cancers.     

Discovery of two aminoglycoside antibiotics as inhibitors targeting the menin–mixed lineage leukaemia interface

Menin functions as an oncogenic cofactor of mixed lineage leukaemia (MLL) fusion proteins in leukaemogenesis. The menin–MLL interface is a potential therapeutic target in acute leukaemia cases. In this study, approximately 900 clinical compounds were evaluated and ranked using pharmacophore-based virtual screening, the top 29 hits were further evaluated by biochemical analysis to discover the inhibitors that target the menin–MLL interface. Two aminoglycoside antibiotics, neomycin and tobramycin, were identified as menin–MLL inhibitors with binding affinities of 18.8 and 59.9 μM, respectively. The results of thermal shift assay validated the direct interactions between the two antibiotics and menin. The results of isothermal titration calorimetry showed that the equilibrium dissociation constant between menin and neomycin was approximately 15.6 μM. We also predicted the binding modes of inhibitors at the menin–MLL interface through molecular docking analysis. The results indicated that neomycin and tobramycin competitively occupy the binding site of MLL. This study has shed light on the development of powerful probes and new therapies for MLL-mediated leukaemogenesis.     

MLL: a histone methyltransferase disrupted in leukemia

Rearrangements of the MLL gene, which is located at chromosome 11q23, are associated with aggressive acute leukemias in both children and adults. MLL regulates Hox gene expression through direct promoter binding and histone modification. MLL rearrangements occurring in leukemia include MLL fusion genes, partial tandem duplications of MLL and MLL amplification. MLL fusions and amplification upregulate Hox expression, apparently resulting in a block of hematopoietic differentiation. Future therapies for MLL-associated leukemia might involve blocking Hox gene upregulation by using fusion proteins or inhibiting the activity of Hox proteins themselves.     

The formin-binding protein 17, FBP17, binds via a TNKS binding motif to tankyrase, a protein involved in telomere maintenance

In acute myelogenous and lymphoid leukemias, rearrangements involving the MLL (mixed lineage leukemia) gene at chromosome 11q23 are frequent. The truncated MLL protein is fused in-frame to a series of partner proteins. We previously identified the formin-binding protein 17 (FBP17) as such an MLL fusion partner. In this study, we explored in vivo physiological interaction partners of FBP17 using a two-hybrid assay and found tankyrase (TNKS), an ADP-ribose polymerase protein involved in telomere maintenance and mitogen-activated protein kinase signaling. We demonstrate that FBP17 binds via a special TNKS-binding motif to tankyrase. The physiological relevance is indicated by co-immunoprecipitation of endogenous proteins in 293T cells.     

MLL: How complex does it get?

The mixed lineage leukemia (MLL) gene encodes a very large nuclear protein homologous to Drosophila trithorax (trx). MLL is required for the proper maintenance of HOX gene expression during development and hematopoiesis. The exact regulatory mechanism of HOX gene expression by MLL is poorly understood, but it is believed that MLL functions at the level of chromatin organization. MLL was identified as a common target of chromosomal translocations associated with human acute leukemias. About 50 different MLL fusion partners have been isolated to date, and while similarities exist between groups of partners, there exists no unifying property shared by all the partners. MLL gene rearrangements are found in leukemias with both lymphoid and myeloid phenotypes and are often associated with infant and secondary leukemias. The immature phenotype of the leukemic blasts suggests an important role for MLL in the early stages of hematopoietic development. Mll homozygous mutant mice are embryonic lethal and exhibit deficiencies in yolk sac hematopoiesis. Recently, two different MLL-containing protein complexes have been isolated. These and other gain- and loss-of-function experiments have provided insight into normal MLL function and altered functions of MLL fusion proteins. This article reviews the progress made toward understanding the function of the wild-type MLL protein. While many advances in understanding this multifaceted protein have been made since its discovery, many challenging questions remain to be answered.     

A Subset of Mixed Lineage Leukemia Proteins Has Plant Homeodomain (PHD)-mediated E3 Ligase Activity

The mixed lineage leukemia protein MLL1 contains four highly conserved plant homeodomain (PHD) fingers, which are invariably deleted in oncogenic MLL1 fusion proteins in human leukemia. Here we show that the second PHD finger (PHD2) of MLL1 is an E3 ubiquitin ligase in the presence of the E2-conjugating enzyme CDC34. This activity is conserved in the second PHD finger of MLL4, the closest homolog to MLL1 but not in MLL2 or MLL3. Mutation of PHD2 leads to MLL1 stabilization, as well as increased transactivation ability and MLL1 recruitment to the target gene loci, suggesting that PHD2 negatively regulates MLL1 activity.     

MLL targets SET domain methyltransferase activity to Hox gene promoters

MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. We show that MLL regulates target Hox gene expression through direct binding to promoter sequences. We further show that the MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins.     

The identification of novel small-molecule inhibitors targeting WDR5-MLL1 interaction through fluorescence polarization based high-throughput screening

The protein-protein interaction between WDR5 (WD40 repeat protein 5) and MLL1 (mixed-lineage leukemia 1) is important for maintaining optimal H3K4 methyltransferase activity of MLL1. Dysregulation of MLL1 catalytic function is relevant to mixed-lineage leukemia, and targeting WDR5-MLL1 interaction could be a promising therapeutic strategy for leukemia harboring MLL1 fusion proteins. To date, several peptidomimetic and non-peptidomimetic small-molecule inhibitors targeting WDR5-MLL1 interaction have been reported, yet the discovery walk of new drugs inhibiting MLL1 methytransferase activity is still in its infancy. It’s urgent to find other small-molecule WDR5-MLL1 inhibitors with novel scaffolds. In this study, through fluorescence polarization (FP)-based high throughput screening, several small-molecule inhibitors with potent inhibitory activities in vitro against WDR5-MLL1 interaction were discovered. Nuclear Magnetic Resonance (NMR) assays were carried out to confirm the direct binding between hit compounds and WDR5. Subsequent similarity-based analog searching of the 4 hits led to several inhibitors with better activity, among them, DC_M5_2 displayed highest inhibitory activity with IC₅₀ values of 9.63?±?1.46?µM. Furthermore, a molecular docking study was performed and disclosed the binding modes and interaction mechanisms between two most potent inhibitors and WDR5.     

Learning from mouse models of MLL fusion gene-driven acute leukemia

5–10% of human acute leukemias carry chromosomal translocations involving the mixed lineage leukemia (MLL) gene that result in the expression of chimeric protein fusing MLL to >80 different partners of which AF4, ENL and AF9 are the most prevalent. In contrast to many other leukemia-associated mutations, several MLL-fusions are powerful oncogenes that transform hematopoietic stem cells but also more committed progenitor cells. Here, I review different approaches that were used to express MLL fusions in the murine hematopoietic system which often, but not always, resulted in highly penetrant and transplantable leukemias that closely phenocopied the human disease. Due to its simple and reliable nature, reconstitution of irradiated mice with bone marrow cells retrovirally expressing the MLL-AF9 fusion became the most frequently in vivo model to study the biology of acute myeloid leukemia (AML). I review some of the most influential studies that used this model to dissect critical protein interactions, the impact of epigenetic regulators, microRNAs and microenvironment-dependent signals for MLL fusion-driven leukemia. In addition, I highlight studies that used this model for shRNA- or genome editing-based screens for cellular vulnerabilities that allowed to identify novel therapeutic targets of which some entered clinical trials. Finally, I discuss some inherent characteristics of the widely used mouse model based on retroviral expression of the MLL-AF9 fusion that can limit general conclusions for the biology of AML. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.