Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia

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 multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind 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.     

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.     

Menin,the multiple endocrine neoplasia type 1 gene product,exhibits GTP-hydrolyzing activity in the presence of the tumor metastasis suppressor nm23

MEN1, the gene responsible for multiple endocrine neoplasia type 1, is a tumor suppressor gene that encodes a protein called menin, of unknown function with no homology to any known protein. Here we demonstrate that menin interacts with a putative tumor metastasis suppressor nm23H1/nucleoside diphosphate (NDP) kinase A in mammalian cells. Given the roles of nm23 as a multi-functional protein, we searched for the possible function of menin. Menin has no effect on the known activities of nm23; that is, nucleoside diphosphate kinase, protein kinase, or GTPase-activating protein for Ras-related GTPase Rad. However, we found that menin hydrolyzes GTP to GDP efficiently in the presence of nm23, whereas nm23 or menin alone shows little or no detectable GTPase activity. Furthermore, menin contains sequence motifs similar to those found in all known GTPases or GTP-binding proteins and shows low affinity but specific binding to GTP/GDP. These results suggest that menin is an atypical GTPase stimulated by nm23.     

The 32-kilodalton subunit of replication protein A interacts with menin,the product of the MEN1 tumor suppressor gene

Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.     

Menin is required in cranial neural crest for palatogenesis and perinatal viability

Menin is a nuclear protein encoded by a tumor suppressor gene that is mutated in humans with multiple endocrine neoplasia type 1 (MEN1). Menin functions as a component of a histone methyltransferase complex that regulates expression of target genes including the cell cycle inhibitor p27^kip1. Here, we show that menin plays a previously unappreciated and critical role in cranial neural crest. Tissue-specific inactivation of menin in Pax3- or Wnt1-expressing neural crest cells leads to perinatal death, cleft palate and other cranial bone defects, which are associated with a decrease in p27^kip1 expression. Deletion of menin in Pax3-expressing somite precursors also produces patterning defects of rib formation. Thus, menin functions in vivo during osteogenesis and is required for palatogenesis, skeletal rib formation and perinatal viability.     

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.     

Somatostatin stimulates menin gene expression by inhibiting protein kinase A

Somatostatin is a potent inhibitor of gastrin secretion and gene expression. Menin is a 67-kDa protein product of the multiple endocrine neoplasia type 1 (MEN1) gene that when mutated leads to duodenal gastrinomas, a tumor that overproduces the hormone gastrin. These observations suggest that menin might normally inhibit gastrin gene expression in its role as a tumor suppressor. Since somatostatin and ostensibly menin are both inhibitors of gastrin, we hypothesized that somatostatin signaling directly induces menin. Menin protein expression was significantly lower in somatostatin-null mice, which are hypergastrinemic. We found by immunohistochemistry that somatostatin receptor-positive cells (SSTR2A) express menin. Mice were treated with the somatostatin analog octreotide to determine whether activation of somatostatin signaling induced menin. We found that octreotide increased the number of menin-expressing cells, menin mRNA, and menin protein expression. Moreover, the induction by octreotide was greater in the duodenum than in the antrum. The increase in menin observed in vivo was recapitulated by treating AGS and STC cell lines with octreotide, demonstrating that the regulation was direct. The induction required suppression of protein kinase A (PKA) since forskolin treatment suppressed menin protein levels and octreotide inhibited PKA enzyme activity. Small-interfering RNA-mediated suppression of PKA levels raised basal levels of menin protein and prevented further induction by octreotide. Using AGS cells, we also showed for the first time that menin directly inhibits endogenous gastrin gene expression. In conclusion, somatostatin receptor activation induces menin expression by suppressing PKA activation.     

MEN1胰岛瘤中细胞周期蛋白cyclin B2高表达的作用和机制

多发性内分泌肿瘤1-(multiple endocrine neoplasia type 1,MEN1)是一种常染色体显性遗传的肿瘤综合征,患者常表现出多发性的内分泌器官肿瘤,包括垂体瘤、甲状旁腺瘤和胰岛瘤.抑癌基因Men1的突变导致MENl的发生,其编码的蛋白为核蛋白menin.Menin可以抑制包括胰岛β细胞在内的...     

In vitro hematopoietic differentiation of mouse embryonic stem cells requires the tumor suppressor menin and is mediated by Hoxa9

Inactivating mutations in the tumor suppressor gene MEN1 cause the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1). The ubiquitously expressed MEN1 encoded protein, menin, interacts with MLL (mixed-lineage leukemia protein), and together they are essential components of a multiprotein complex with histone methyl transferase activity. MLL is also essential for hematopoiesis, and plays a critical role in leukemogenesis via epigenetic regulation of Hoxa9 expression that also requires menin. Therefore we chose to explore the role of menin in hematopoiesis. We generated Men1^−/− embryonic stem (ES) cell lines, and induced them to differentiate in vitro. While these cells were able to form embryoid bodies (EBs) expressing the early markers Flk-1 and c-Kit, their ability to further differentiate into hematopoietic colonies was compromised. The Men1^−/− ES cells show reduced expression of Hoxa9 that can be recovered by reexpression of Menin. We demonstrate that the block in differentiation of Men1^−/− ES cell lines can be rescued not only by the expression of menin but also that of Hoxa9. These results suggest that, similar to MLL, menin is required for hematopoiesis, and this requirement may be mediated through regulation of Hoxa9 expression.