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...
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 proteinmediated 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 ␣-helical protein with the protein core comprising three tetratricopeptide motifs that are flanked by two ␣-helical bundles and covered by a -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.
Summary Pan-NOTCH inhibitors are poorly tolerated in clinical trials because NOTCH signals are crucial for intestinal homeostasis. These inhibitors may also promote cancer as NOTCH can act as a tumor suppressor. We previously reported that the PIAS-like coactivator ZMIZ1 is frequently co-expressed with activated NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). Here, we show that similar to Notch1, Zmiz1 was important for T-cell development and controlled the expression of certain Notch target genes, such as Myc. However, unlike Notch, Zmiz1 had no major role in intestinal homeostasis or myeloid suppression. Deletion of Zmiz1 impaired the initiation and maintenance of Notch-induced T-ALL. Zmiz1 directly interacted with Notch1 via a tetratricopeptide repeat domain at a special class of Notch-regulatory sites. In contrast to the Notch cofactor Maml, which is nonselective, Zmiz1 was selective. Thus, targeting the NOTCH1-ZMIZ1 interaction may combat leukemic growth while avoiding the intolerable toxicities of NOTCH inhibitors.
During translation initiation in Saccharomyces cerevisiae, an Arg-and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2 bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2 mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2 and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA i Met:mRNA binding to the P site.
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