Structural insights into inhibition of the bivalent menin-MLL interaction by small molecules in leukemia.

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 small-molecule 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) = 22nM) and very effectively disrupts the bivalent protein-protein interaction between menin and MLL. MI-2-2 demonstrated specific and very pronounced activity in MLL leukemia cells, including inhibition of cell proliferation, down-regulation of Hoxa9 expression, and differentiation. Our results provide the rational and essential structural basis to design next generation of inhibitors for effective targeting of the menin-MLL interaction in leukemia and demonstrate a proof of concept that inhibition of complex multivalent protein-protein interactions can be achieved by a small-molecule inhibitor.

[1]  R. Slany,et al.  When epigenetics kills: MLL fusion proteins in leukemia , 2005, Hematological oncology.

[2]  R. Slany.  The molecular biology of mixed lineage leukemia , 2009, Haematologica.

[3]  A. Hopkins,et al.  Ligand efficiency: a useful metric for lead selection. , 2004, Drug discovery today.

[4]  Thomas A Milne,et al.  Interaction of MLL amino terminal sequences with menin is required for transformation. , 2005, Cancer research.

[5]  Akihiko Yokoyama,et al.  Menin critically links MLL proteins with LEDGF on cancer-associated target genes. , 2008, Cancer cell.

[6]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[7]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[8]  Nathan A. Baker,et al.  Electrostatics of nanosystems: Application to microtubules and the ribosome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[9]  F. Lombardo,et al.  Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. , 2001, Advanced drug delivery reviews.

[10]  Thomas Terwilliger,et al.  SOLVE and RESOLVE: automated structure solution, density modification and model building. , 2004, Journal of synchrotron radiation.

[11]  M. Lei,et al.  The same pocket in menin binds both MLL and JUND but has opposite effects on transcription , 2012, Nature.

[12]  F. Diederich,et al.  Fluorine in Pharmaceuticals: Looking Beyond Intuition , 2007, Science.

[13]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[14]  F. Lo‐Coco,et al.  Chromosomal Aberration of the 11q23 Locus in Acute Leukemia and Frequency of MLL Gene Translocation Results in 378 Adult Patients , 2004 .

[15]  P. Wright,et al.  Quantitative analysis of multisite protein-ligand interactions by NMR: binding of intrinsically disordered p53 transactivation subdomains with the TAZ2 domain of CBP. , 2012, Journal of the American Chemical Society.

[16]  L. Lally.  The CCP 4 Suite — Computer programs for protein crystallography , 1998 .

[17]  Alexei Vagin,et al.  Molecular replacement with MOLREP. , 2010, Acta crystallographica. Section D, Biological crystallography.

[18]  N. Zeleznik-Le,et al.  MLL: How complex does it get? , 2005, Journal of cellular biochemistry.

[19]  Zukang Feng,et al.  Automated and accurate deposition of structures solved by X-ray diffraction to the Protein Data Bank. , 2004, Acta crystallographica. Section D, Biological crystallography.

[20]  M. Bycroft,et al.  Solution structure of the nonmethyl‐CpG‐binding CXXC domain of the leukaemia‐associated MLL histone methyltransferase , 2006, The EMBO journal.

[21]  N. Zeleznik-Le,et al.  Structure of the MLL CXXC domain–DNA complex and its functional role in MLL-AF9 leukemia , 2010, Nature Structural &Molecular Biology.

[22]  Jay Painter,et al.  TLSMD web server for the generation of multi-group TLS models , 2006 .

[23]  J. Hess.  MLL: a histone methyltransferase disrupted in leukemia. , 2004, Trends in molecular medicine.

[24]  T. Cierpicki,et al.  Crystal Structure of Menin Reveals Binding Site for Mixed Lineage Leukemia (MLL) Protein* , 2011, The Journal of Biological Chemistry.

[25]  M. Cleary,et al.  MLL rearrangements in haematological malignancies: lessons from clinical and biological studies , 1999, British journal of haematology.

[26]  K D Cowtan,et al.  Improvement of macromolecular electron-density maps by the simultaneous application of real and reciprocal space constraints. , 1993, Acta crystallographica. Section D, Biological crystallography.

[27]  S. Marx.  Molecular genetics of multiple endocrine neoplasia types 1 and 2 , 2005, Nature Reviews Cancer.

[28]  Manfred Kansy,et al.  Fluorine Interactions at the Thrombin Active Site: Protein Backbone Fragments HCαCO Comprise a Favorable CF Environment and Interactions of CF with Electrophiles , 2004, Chembiochem : a European journal of chemical biology.

[29]  Wladek Minor,et al.  HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes. , 2006, Acta crystallographica. Section D, Biological crystallography.

[30]  Y Wang,et al.  Positional cloning of the gene for multiple endocrine neoplasia-type 1. , 1997, Science.

[31]  Andrew D Westwell,et al.  Protein–protein interactions as targets for small-molecule therapeutics in cancer , 2008, Expert Reviews in Molecular Medicine.

[32]  C. Allis,et al.  Multiple interactions recruit MLL1 and MLL1 fusion proteins to the HOXA9 locus in leukemogenesis. , 2010, Molecular cell.

[33]  Jolanta Grembecka,et al.  Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia. , 2012, Nature chemical biology.

[34]  M. Birke,et al.  The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation. , 2002, Nucleic acids research.

[35]  Alicia P. Higueruelo,et al.  Atomic Interactions and Profile of Small Molecules Disrupting Protein–Protein Interfaces: the TIMBAL Database , 2009, Chemical biology & drug design.

[36]  Jolanta Grembecka,et al.  Molecular Basis of the Mixed Lineage Leukemia-Menin Interaction , 2010, The Journal of Biological Chemistry.

[37]  J. Hess,et al.  The pathogenesis of mixed-lineage leukemia. , 2012, Annual review of pathology.

[38]  Jack Snoeyink,et al.  Nucleic Acids Research Advance Access published April 22, 2007 MolProbity: all-atom contacts and structure validation for proteins and nucleic acids , 2007 .

[39]  Jiaying Tan,et al.  The PAF complex synergizes with MLL fusion proteins at HOX loci to promote leukemogenesis. , 2010, Cancer cell.

[40]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[41]  Scott A. Armstrong,et al.  MLL translocations, histone modifications and leukaemia stem-cell development , 2007, Nature Reviews Cancer.