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

Abstract

Menin functions as a critical oncogenic co-factor of Mixed Lineage Leukemia (MLL) fusion proteins in 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 two N-terminal fragments of MLL. Here, we report 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 reveals 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) = 22 nM) 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, downregulation of Hoxa9 expression and differentiation. Together, our results provide the rational and essential structural basis to design next generation of inhibitors for effective targeting 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.

Full text

Page 1

doi:10.1182/blood-2012-05-429274
Prepublished online August 30, 2012;
2012 120: 4461-4469




Reddy, Maksymilian Chruszcz, Jolanta Grembecka and Tomasz Cierpicki
Aibin Shi, Marcelo J. Murai, Shihan He, George Lund, Thomas Hartley, Trupta Purohit, Gireesh
by small molecules in leukemia
Structural insights into inhibition of the bivalent menin-MLL interaction

http://bloodjournal.hematologylibrary.org/content/120/23/4461.full.html
Articles on similar topics can be found in the following Blood collections
Updated information and services can be found at:
(372 articles)Plenary Papers
?
(943 articles)Myeloid Neoplasia
?

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests
Information about reproducing this article in parts or in its entirety may be found online at:

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints
Information about ordering reprints may be found online at:

http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml
Information about subscriptions and ASH membership may be found online at:
Copyright 2011 by The American Society of Hematology; all rights reserved.
Washington DC 20036.
by the American Society of Hematology, 2021 L St, NW, Suite 900,
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly




For personal use only. at UNIV OF MICHIGAN on May 16, 2013. bloodjournal.hematologylibrary.orgFrom

Page 2

Plenary paper
Structuralinsightsintoinhibitionofthebivalentmenin-MLLinteractionbysmall
moleculesinleukemia
*Aibin Shi,1*Marcelo J. Murai,1*Shihan He,1George Lund,1Thomas Hartley,1Trupta Purohit,1Gireesh Reddy,1
Maksymilian Chruszcz,2Jolanta Grembecka,1and Tomasz Cierpicki1
1Department of Pathology, University of Michigan,AnnArbor, MI; and2Department of Molecular Physiology and Biological Physics, University of Virginia,
Charlottesville, VA
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 pro-
teins represents a very promising strat-
egy 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-
moleculeinhibitorofthemenin-MLLinter-
action, 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 (Kd? 22nM) and very
effectively disrupts the bivalent protein-
protein interaction between menin and
MLL. MI-2-2 demonstrated specific and
very pronounced activity in MLLleukemia
cells, including inhibition of cell prolifera-
tion, down-regulation of Hoxa9 expres-
sion, and differentiation. Our results pro-
vide the rational and essential structural
basis to design next generation of inhibi-
tors for effective targeting of the menin-
MLL interaction in leukemia and demon-
strate a proof of concept that inhibition of
complexmultivalentprotein-proteininter-
actions can be achieved by a small-
molecule inhibitor. (Blood. 2012;120(23):
4461-4469)
Introduction
Translocations of the MLL (mixed lineage leukemia) gene fre-
quently occur in aggressive human acute myeloid and lymphoid
leukemias in both children and adults.1Fusion of MLL with 1 of
more than 60 different genes results in chimeric MLL fusion
proteins that enhance proliferation and block hematopoietic differ-
entiation, ultimately leading to acute leukemia.2Patients 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,2leading to an overall 5-year survival rate
of only approximately 35%.4
All MLLfusion proteins preserve an N-terminal MLLfragment
approximately 1400 amino acids in length fused in-frame with the
C-terminus of the fusion partner.3,5-7Two regions in this fragment
of MLL have been shown to be indispensable for leukemogenic
transformation: the N-terminal region, which binds to menin8and
to lens epithelium–derived growth factor (LEDGF),9and the
conserved region encompassing the CXXC domain, which medi-
ates binding to nonmethylated CpG DNA10-12and interacts with the
polymerase associated factor complex (PAFc).13,14Targeting these
interactions provides new opportunities for the development of
new therapeutic agents for the MLLleukemias.15
Menin is a tumor-suppressor protein encoded by the MEN1
(multiple endocrine neoplasia 1) gene.16Mutations of MEN1 are
associated with tumors of the parathyroid glands, pancreatic islet
cells, and anterior pituitary gland.17Menin 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 MEIS1.8Loss of the ability to bind menin abolishes the
oncogenic potential of MLL fusion proteins both in vitro and
in vivo.8Disruption of the interaction between menin and MLL
fusion proteins using genetic methods blocks the development of
acute leukemia in mice,8indicating that menin functions as a
critical oncogenic cofactor of MLL fusion proteins and is required
for their leukemogenic activity. The menin-MLL interaction repre-
sents an attractive therapeutic target for the development of novel
drugs for acute leukemias with MLLrearrangements.
The development of small-molecule inhibitors of protein-
protein interactions (PPIs) is a challenging task.18Menin interacts
with 2 fragments of MLL, the high-affinity motif MBM1 (menin-
bindingmotif1,
Kd? 53nM)
(Kd? 1.4?M), located within the intrinsically unstructured
43-amino acid fragment at the N-terminus of MLL.19MBM1 and
MBM2 are separated by a 7-glycine linker and most likely bind to
adjacent sites on menin. The development of small molecules
effectively targeting the menin-MLL interaction would require the
disruption of this bivalent interaction. A point mutation within
MBM1 is sufficient to abolish the oncogenic properties of MLL
fusion proteins in vivo.8We have recently developed small
molecules that bind directly to menin and inhibit the menin-MLL
interaction in vitro and in human cells.20
To understand the molecular mechanism of the menin-MLL
interaction and to facilitate the development of novel drugs
targeting this interaction, we have determined the high-resolution
(1.46 Å) crystal structure of human menin, as well as menin in
complex with the high-affinity binding motif of MLL, MBM1. We
and low-affinityMBM2
Submitted May 9, 2012; accepted August 17, 2012. Prepublished online as
Blood First Edition paper, August 30, 2012; DOI 10.1182/blood-2012-05-429274.
*A.S., M.J.M., and S.H. contributed equally to this work.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2012 by TheAmerican Society of Hematology
4461 BLOOD, 29 NOVEMBER 2012?VOLUME 120, NUMBER 23
For personal use only. at UNIV OF MICHIGAN on May 16, 2013. bloodjournal.hematologylibrary.orgFrom

Page 3

have also obtained the first crystal structure of menin in complex
with MI-2, our recently developed small-molecule inhibitor of the
menin-MLL interaction,20and found that this compound binds to
the MLL site on menin and mimics the key interactions of MBM1.
Based on the structure of the menin–MI-2 complex, we developed
a very potent second-generation inhibitor, MI-2-2, which binds to
menin with low nanomolar affinity and is capable of potently
inhibiting the MLL-fusion protein–mediated leukemogenic trans-
formation. Our work provides an important structural insight into
menin’s function as a critical cofactor of oncogenic MLL fusion
proteins in leukemogenesis. The structures of menin complexes
with the small-molecule inhibitors MI-2 and MI-2-2 shed light on
inhibitor binding to the MLLbinding site on menin and provide the
rational and molecular basis for the development of the next
generation of menin-MLL inhibitors as novel therapies in acute
leukemias with MLLtranslocation.
Methods
Cloning, protein engineering, expression, and purification of
human menin
The gene encoding human menin was subcloned into pET32a vector
(Novagen) and the generation of deletion constructs was performed by
mutagenesis according to the QuikChange Site-Directed Mutagenesis kit
protocol to introduce different internal deletions and a stop codon. Proteins
were expressed and purified using a protocol described previously.21For
crystallization experiments, proteins were purified using the size-exclusion
column HiLoad 16/60 Superdex 75 pg resin (GE Healthcare) and 50mM
Tris-HCl, pH 8.0, 50mM NaCl, and 1mM tris(2-carboxyethyl)phosphine as
the mobile phase.
Crystallization of menin and menin complexes
For crystallization experiments, 2.5 mg/mL of menin was incubated with
MLL MBM1 peptide (MLL residues 4-15; GenScript) in a 1:1 molar ratio.
Crystals were obtained using a sitting-drop technique at 10°C in 0.2M
ammonium acetate, 0.1M HEPES, pH 7.5, and 25% wt/vol PEG 3350.
Before data collection, crystals were transferred into a cryosolution
containing 20% PEG550 MME and flash-cooled in liquid nitrogen. A
similar procedure was used for crystallization of the complexes with
MI-2 and MI-2-2.
Crystallographic data collection and structure determination
Diffraction data for menin and menin complexes were collected at the
21-ID-D and 21-ID-F beam lines at the Life Sciences CollaborativeAccess
Team at the Advanced Photon Source. Data were processed with HKL-
2000.22Structure of the free protein was determined using HKL-300023and
MOLREP24using menin from Nematostella vectensis (PDB code: 3RE2) as
a search model in molecular replacement. The model was rebuilt with
BUCCANNER25and RESOLVE26and the refinement was carried out using
HKL-3000, REFMAC,27COOT,28and the CCP4 package.29In the final
stages, refinement was performed with addition of the TLS groups defined
by the TLMSD server30or using anisotropic B-factors in the case of the
high-resolution structures. Validation of the structures was performed using
MOLPROBITY31and ADIT.32Details of data processing and refinement
are summarized in supplemental Table 1 (available on the Blood Web site;
see the Supplemental Materials link at the top of the online article).
Coordinates and structure factors have been deposited in the Protein Data
Bank under accession codes 4GPQ, 4GQ6, 4GQ3, and 4GQ4.
MLL-binding experiments
Dissociation constants for binding of MBM1 (MLL residues 4-15), MBM2
(residues 23-40), and MLL4-43(MLL residues 4-43) to full-length human
menin were determined by the fluorescence polarization method using a
protocol published previously.19
Biochemical characterization of menin-MLL inhibitors
The ability of small molecules to inhibit menin-MBM1 and menin-MLL4-43
interaction was assessed using the fluorescence polarization assay in a
protocol described previously.19,20Details of isothermal titration calorim-
etry experiments for the measurement of dissociation constants for menin
inhibitors were also described previously.20
Coimmunoprecipitation experiments
HEK293 cells were transfected with ?-actin Flag-MLL-AF9 plasmid using
Fugene 6 (Roche). Forty-eight hours after transfection, cells were treated
with compounds (0.25% final DMSO concentration) or DMSO for
12 hours. Whole-cell lysates were immunoprecipitated with anti-FLAG
M-2 Magnetic Beads (Sigma-Aldrich) and analyzed by SDS-PAGE and
Western blotting. For more details, see Grembecka et al.20
Viability assays
MLL-AF9–transduced mouse bone marrow cells (BMCs) were prepared as
described previously.13MV4;11, KOPN-8, ML-2, and MOLM-13 cells
were cultured in RPMI 1640 medium with 10% FBS, 1% penicillin/
streptomycin, and nonessential amino acids. The MTT viability assay was
carried out using a recently published protocol.20For growth curves,
3 ? 105/mL cells were plated (1 mL/well) and treated with compounds or
0.25% DMSO. Media were changed every 48 hours with viable cell
concentration restored to 3 ? 105cells/mL and compound resupplied. At a
designated time point, cell culture samples were mixed with Trypan blue
(GIBCO/Invitrogen) and counted.
Colony formation assay
The MLL-AF9–transduced murine BMCs were plated in 12-well plates at a
concentration of 5 ? 103cells/mL with 1 mL of methylcellulose medium
M3234 (StemCell Technologies) containing 20% IMDM, 1% penicillin/
streptomycin, IL-3 and 0.25% DMSO, or compounds. Six days later,
colonieswerestainedwith100 ?Lofiodonitrotetrazoliumchloride(Sigma-
Aldrich) at a final concentration of 1 mg/mL, incubated at 37°C for
30 minutes, and counted. Pictures of colonies were taken at room
temperature using an Olympus IX50 microscope with LCPIanFI 10?/0.3
Ph1 objective lenses and an Olympus DP70 camera with Olympus DP
Controller software. Pictures were resized usingAdobe Photoshop CS2.
Real-time PCR
Total RNAwas extracted from cells using the RNeasy mini kit (QIAGEN),
and then 100-1000 ng of total RNA was reverse transcribed using High
Capacity cDNAReverse Transcription Kit (Applied Biosystems) according
to the manufacturer’s protocol. Real-time PCR was performed using the
ABI Prism 7700 sequence detection system. TaqMan Gene Expression
Master Mix and TaqMan Gene Expression Assays for mouse Hoxa9
(Mm00439364_m1), Meis1 (Mm00487664_m1), ?-Actin (4352933), hu-
man HOXA9 (Hs00365956_m1), MEIS1 (Hs00180020_m1), and 18S RNA
(Hs99999901_s1) were purchased from Applied Biosystems. Relative
quantification of each gene transcript was carried out using the comparative
Ctmethod as described in theApplied Biosystems User Bulletin no. 2.
Annexin V/propidium iodide assay of inhibitor effects on
apoptosis
A total of 5 ? 105cells/mL were plated in 12-well plates (1 mL/well) and
treated with compounds (0.25% final concentration of DMSO for each
condition) or 0.25% DMSO control and incubated for 48 hours at 37°C in a
5% CO2incubator. After incubation, 1.5 ? 105cells were harvested and
resuspended in 100 ?L of 1? annexin V–binding buffer from the Annexin
V–FITC Apoptosis Kit (BD Biosciences), incubated with 4 ?L of annexin
4462SHI et alBLOOD, 29 NOVEMBER 2012?VOLUME 120, NUMBER 23
For personal use only. at UNIV OF MICHIGAN on May 16, 2013. bloodjournal.hematologylibrary.orgFrom

Page 4

V–FITC and 6 ?L of propidium iodide (Sigma-Aldrich) at room tempera-
ture in the dark for 10 minutes, and analyzed by flow cytometry on an LSR
II instrument. Data analysis was performed using WinList Version 3.0
software (Verity Software House Inc).
Expression of CD11b
MV4;11 cells or MLL-AF9 transduced BMCs were plated in 12-well plates
at an initial concentration of 5 ? 105cells/mL and treated with compounds
or 0.25% DMSO. Media were changed every 48 hours with viable cell
concentration restored to 5 ? 105cells/mL and compounds resupplied.
Seven days after the experiment was set up, 1.5 ? 105cells were harvested
and washed with FACS buffer (PBS, 1% FBS, and 0.1% NaN3). Cells were
resuspended in 100 ?L of FACS buffer and incubated with 2 ?L of Pacific
blue mouse anti–human CD11b Ab (BD Biosciences) or 1 ?L of Pacific
Blue rat anti–mouse CD11b Ab (BioLegend) at 4°C for 30 minutes. Cells
were then washed, resuspended in 100 ?Lof annexin V binding buffer, and
incubated with 4 ?L of annexin V–FITC (BD Biosciences) and 6 ?L of
propidium iodide (1 mg/mL; Sigma-Aldrich) at room temperature for
10 minutes before being analyzed by flow cytometry.
Cytospin/Wright-Giemsa staining
MV4;11 and mouse BMCs transduced with MLL-AF9 were plated in
12-well plates (1 mL/well) at an initial concentration of 5 ? 105/mL cells,
treated with compounds (0.25% final DMSO concentration) or 0.25%
DMSO control, and incubated at 37°C in a 5% CO2 incubator. At a
designated time point, 1 ? 105cells were harvested and placed in Shandon
EZ Single Cytofunnel (Thermo Electron). Samples were centrifuged at
550g for 5 minutes. The slides were air-dried before staining with the
Hema-3 kit (Fisher Scientific). Cytospin pictures were taken at room
temperature using an Olympus BX41 microscope with UPIanFLN 100?/
1.30 Oil objective lenses, immersion oil Immersol 518N (Carl Zeiss
Microscopy) and an Olympus DP71 camera with Olympus DP Controller
software. Pictures were resized usingAdobe Photoshop CS2.
Inhibitor effects on cell cycle of MV4;11 leukemia cells
MV4;11 cells (5 ? 105/mL) were plated in 12-well plates (1 mL/well) and
treated with MI-2-2 compound (0.25% final concentration of DMSO for
each condition) or 0.25% DMSO control and incubated for 48 hours at
37°C in a 5% CO2 incubator. After incubation, 5 ? 105cells were
harvested, washed in PBS buffer, resuspended in 1 mL of PBS buffer, and
mixed with 9 mL of 70% ethanol. Cells were kept at ?20°C for at least
24 hours, washed and resuspended in FACS buffer, and then incubated with
100 ?g/mL RNase (QIAGEN) and 10 ?g/mL of propidium iodide at
37°C for 30 minutes before flow cytometry.
Chemistry
Synthesis and characterization of the second generation of menin-MLL
inhibitors (MI-2-2 and analogs) is provided in supplemental Methods.
Results
Crystal structure of human menin and menin-MLL complex
Our initial attempts to crystallize full-length human menin failed,
most likely because of the presence of several internal fragments
predicted to be unstructured. Based on the structure of menin
homolog from N vectensis,21we deleted 3 internal fragments and
the C-terminus in human menin, which we predicted to correspond
to loops and unstructured regions (supplemental Figure 1A). This
resulted in a construct that yielded protein crystals diffracting to a
1.46 Å resolution (supplemental Table 1). The engineered protein
retains the ability to bind MLL with a similar affinity as the
wild-type menin, indicating that deletion of these fragments does
not alter the MLL-binding site (supplemental Figure 1B). Human
menin is predominantly an ?-helical protein (supplemental Figure
1C) and closely resembles the structure of N vectensis menin that
we described previously,21with only minor structural differences,
localized to the peripheral fragments and loop regions. Very
recently, the structure of a longer human menin construct has been
reported.33Interestingly, deletion of the loops resulted in a very
significant improvement in the resolution of the structure.
MLL associates with menin in a bivalent mode using 2 short
motifs, MBM1 and MBM2, separated by a polyglycine linker
(Figure 1A).19We previously reported that MBM1 and MBM2
peptides bind to menin with, respectively, 50nM and 1.4?M
affinities. The longer fragment containing both motifs interacts
with menin with 10nM affinity.19To provide an insight into the
recognition of MLLby menin, we have determined the structure of
meninincomplexwiththehigh-affinitymotif,MBM1(supplemen-
tal Table 1). The structure revealed that MBM1 binds to the large
central cavity on menin. The peptide is well ordered and its
backbone adopts a U-shaped conformation, with a single ?-turn
comprising residues 9-12 (Figure 1B and supplemental Figure 1C).
The ?-turn is stabilized by an intramolecular hydrogen bond
between the carbonyl of Phe9MLLand the backbone amide of
Arg12MLL(Figure 1C). The binding of MBM1 to menin is
maintained by several hydrophobic contacts involving the side
chains of Phe9MLL, Pro10MLL, and Pro13MLL(Figure 1B-C), which
we demonstrated to play the most important role for MBM1
binding to menin.19Phe9MLLis entirely buried in the complex and
Figure 1. Structure of the menin-MLL complex. (A) Sequence of the N-terminal
fragment of MLL with MBM1 and MBM2 motifs. (B) Details of the menin-MBM1
interaction. Structure of the MBM1 is shown in stick representation (green carbon
atoms) and MLLresidues are labeled in blue. Menin is presented as a gray ribbon and
selected side chains involved in contacts with MBM1 are shown as sticks (cyan
carbon atoms); hydrogen bonds are shown as dashed lines. (C) The most significant
contacts between MBM1 (green carbons) and selected menin side chains (cyan
carbons). (D) Probing the MBM2-binding site on menin. MBM1 (shown in sticks)
occupies a negatively charged central cavity on menin. The positions of D252 and
L289 that were mutated to lysines are labeled. The electrostatic potential was
calculated usingAPBS and mapped onto menin structure.41
INSIGHTS INTO INHIBITION OF MENIN-MLLINTERACTION 4463BLOOD, 29 NOVEMBER 2012?VOLUME 120, NUMBER 23
For personal use only. at UNIV OF MICHIGAN on May 16, 2013. bloodjournal.hematologylibrary.orgFrom

Page 5

fits between the protein backbone (residues 179-181) and the side
chain of Phe238 (Figure 1C). Pro10MLLbinds to the site adjacent to
Phe9MLLand interacts with Phe238 andAla242, whereas Pro13MLL
fits into a hydrophobic pocket formed by Tyr319, Tyr323, and
Met322. Mutations of Phe9MLL, Pro10MLL, and Pro13MLLto alanine
residues result in a dramatic decrease of MBM1 binding to menin
(2000-, 30-, and 50-fold decrease, respectively),19validating the
importance of these hydrophobic interactions.Additional stabiliza-
tion of the MBM1-menin complex results from a salt bridge
between Arg12MLLand Glu359 and Glu363, as well as 3 intermo-
lecular hydrogen bonds involving the MLL backbone (residues
Arg6MLL, Trp7MLL, and Ala11MLL) and menin side chains (Asn244,
Asp136, and Tyr323; Figures 1B-C). Mutation of Arg12MLLto
alanine has a relatively modest effect on MBM1 binding (ie, a
4-fold decrease).19Remarkably, binding of the 12–amino acid
fragment of MLLdoes not cause any significant changes within the
menin structure. Very recently, the lower-resolution crystal struc-
ture of the menin-MLL complex has been shown to have a similar
interaction mode.33
We were not able to determine the crystal structure of menin
with the MBM2 fragment of MLL, presumably because of the
crystal contacts interfering with the binding. MBM1 and MBM2
are separated by a linker comprising 7 glycine residues (Figure
1A), and therefore it is possible that MBM2 occupies the space in a
close proximity to MBM1. The MBM2 is positively charged and,
using site-directed mutagenesis in menin, we probed whether it
binds to the negatively charged site in a close proximity to the
MBM1-binding site (Figure 1D). Introduction of 2 point mutations
in menin, D252K and L289K, which we assumed would cause
electrostatic repulsion with the MBM2 (Figure 1D), interfered with
binding of MBM2 without affecting protein stability or the
interaction with MBM1 (supplemental Figure 2). This indicates
that the low-affinity motif, MBM2, most likely binds in the
proximity of the MBM1-binding site and provides an additional
anchor enhancing the interaction of MLLwith menin.
Thienopyrimidine inhibitors mimic the binding mode of MBM1
We have recently developed small molecules with a thienopyrimi-
dine scaffold that bind to menin with submicromolar affinities and
disrupt the menin-MLL interaction.20One of these compounds,
MI-2 (IC50? 446 nM), showed pronounced effects in MLL
leukemia cells, representing a very promising lead compound for
the development of new antileukemic agents.20However, lack of
structural data for the MI-2 interaction with menin impeded the
development of more potent inhibitors of the menin-MLL interac-
tion. To overcome this limitation, we determined the 1.56 Å
resolution structure of MI-2 bound to menin (Figure 2A-B,
supplemental Figure 11, and supplemental Table 1), which repre-
sents the first determination of the crystal structure of menin in
complex with a small-molecule inhibitor. The electron density for
MI-2 inhibitor is very well defined (Figure 2B), enabling detailed
analysis of its interactions with menin.
We found that MI-2 binds to the same central cavity on menin
that is occupied by the MBM1 fragment of MLL. The structure of
MI-2 contains an n-propyl–substituted thienopyrimidine ring con-
nected by a piperazine linker to a dimethyl-thiazoline ring (Figure
2A-C). The interaction of MI-2 with menin is predominantly
stabilized via hydrophobic interactions and by 2 hydrogen bonds
between N1 and N3 nitrogen atoms of the thienopyrimidine ring
and the side chains of Tyr276 andAsn282, respectively (Figure 2A
and C). The n-propyl-thienopyrimidine fragment fits into a pocket
formed by the main chain residues Ser178 to His181 and the side
chains of mostly hydrophobic residues (Phe238, Leu177, Ala182,
Tyr276, Met278, Cys241, and Ser155), whereas the piperazine ring
serves mostly as a linker and approaches the side chains of Met278
and Tyr319. The dimethyl-thiazoline moiety of MI-2 fits into a site
formed by 2 orthogonally oriented tyrosine side chains (Tyr319 and
Tyr323) and the side chain of Met322 (Figure 2A).
Strikingly, MI-2 mimics the binding mode and the key interac-
tions formed by the MBM1 with menin (Figure 2D). The n-propyl
group of MI-2 fits into the pocket occupied by Phe9MLL, whereas
the dimethyl thiazoline ring binds to the same pocket as Pro13MLL.
Figure 2. Crystal structure of the menin–MI-2 com-
plex. (A) Details of MI-2 interaction with menin. Selected
menin side chains are shown in sticks (cyan carbons)
and hydrogen bonds are shown as dashed lines.
(B) Menin-MI-2 complex determined at 1.56 Å resolution
with corresponding 2Fo-Fc electron density map con-
toured at the 1? level. (C) Diagram depicting VdW
contacts and hydrogen bonds (dashed lines) between
MI-2 and menin. (D) Superposition of MI-2 (green car-
bons) with a fragment of the MBM1 motif (MLL residues
9-13, gray carbons) in a menin-bound conformation.
4464SHI et al BLOOD, 29 NOVEMBER 2012?VOLUME 120, NUMBER 23
For personal use only. at UNIV OF MICHIGAN on May 16, 2013. bloodjournal.hematologylibrary.org From

Page 6

The piperazine ring in MI-2 also mimics a part of the MBM1
backbone by overlapping with the main chain atoms of Arg12MLL
(Figure 2D). Overall, MI-2 is a relatively rigid molecule, and its
high binding affinity to menin is likely because of the strong shape
complementarity of this compound to the binding site on menin.
Despite its relatively small size (375 Da), MI-2 binds to menin with
only a 3-fold lower affinity than the 12–amino acid MBM1
fragment of MLL and mimics the key interactions of MBM1 with
menin. Therefore, MI-2 is a very promising candidate for further
development into a more potent inhibitor of the menin-MLL
interaction.
Structure-based design of a nanomolar inhibitor of the
menin-MLL interaction
We have exploited the structure of the menin–MI-2 complex to
design new analogs with improved binding affinities. Inspection of
the structure revealed that the n-propyl group of MI-2 does not
represent an optimal substituent for interacting with the binding
pocket on menin. The structure also rationalizes why substitution
of the n-propyl by bulky hydrophobic groups did not improve
binding affinity.20To develop more potent compounds, we used
structural information and designed several modifications of the
R1 substituent on the thienopyrimidine ring (Figure 3A). First, we
introduced oxygen, which could form a hydrogen bond with the
side chain hydroxyl of Ser155. However, addition of the methoxy-
propyl group at R1 (MI-2-1) resulted in a 3-fold decreased activity
(Figure 3A). As an alternative strategy, we synthesized several
analogs by addition of fluorine atoms to R1 to achieve optimal
shape complementarity with the binding site on menin (Figure 3A).
Indeed, the replacement of n-propyl by trifluoroethyl group (MI-
2-2) substantially improved the activity (Figure 3A-B). MI-2-2,
which has been designed based on the structure of the menin–MI-2
complex, represents a second generation of menin inhibitors and
binds to menin with Kd? 22nM as assessed by isothermal titration
calorimetry (Figure 3C). The MI-2-2 inhibits the menin-MBM1
interactionwithIC50? 46nM(Figure3B),representinganapproxi-
mately 7- to 9-fold improvement compared with MI-2
(IC50? 446nM,Kd? 158nM).Remarkably,MI-2-2bindstomenin
with a more than 2-fold higher affinity than the MBM1 fragment of
MLL(Figure 3B).
To understand the molecular basis of a significant effect caused
by introduction of the trifluoroethyl group, we have determined the
high-resolution structure (1.27 Å; supplemental Table 1) of the
MI-2-2-menin complex (Figure 3D and supplemental Figure 11).
We found that binding mode of MI-2-2 to menin is the same as that
observed for MI-2. Interestingly, one of the fluorine atoms resides
in a very close proximity (3.0-3.1 Å) to the backbone atoms of
His181 (Figure 3E). Such an orthogonal orientation of the fluorine
relative to the protein backbone results in a favorable C-F. . .C?O
dipolar interaction that has previously been identified to signifi-
cantly enhance protein-ligand interactions.34Introducing fluorine
atoms has become common in medicinal chemistry to enhance the
binding affinity of protein ligands and to improve their drug-like
properties.35Our high-resolution crystal structure of MI-2-2 bound
to menin allows for detailed analysis of the protein interactions
with fluorine, and may represent a valuable model system to better
understand such interactions and to improve the design of potent
small-molecule inhibitors of other protein-protein interactions.
Small molecules are capable of inhibiting the bivalent
menin-MLL interaction
The intrinsically unstructured N-terminus of MLL interacts with
menin via a complex mechanism, with 2 short MLL fragments
(MBM1 and MBM2) involved in binding.19Deletion of a high-
affinity motif MBM1 is sufficient to abolish transformation by
MLL-ENL,8which shows that this interaction constitutes a hot spot
for small-molecule development. Because MBM2 also contributes
to the binding to menin, it was necessary to establish whether small
molecules targeting the MBM1 site on menin are sufficient to
efficiently inhibit the bivalent interaction of menin with MLL. We
found that both MI-2 and MI-2-2 can inhibit the interaction of
menin with MLL4-43, which comprises the intact menin-binding
fragment.19,36As expected, the second-generation inhibitor MI-2-2
was approximately 7-fold more potent in disrupting the menin-
MLL4-43interaction, with an IC50? 520nM (Figure 4A). To assess
whether these inhibitors can dissociate menin interaction with the
full-length MLL-AF9 fusion protein in cells, we performed coim-
munoprecipitation experiments. We found that both MI-2 and
MI-2-2 inhibit the interaction of menin with MLL-AF9 in HEK293
cells in a dose-dependent manner at low micromolar concentrations
(Figure 4B), with MI-2-2 representing an approximately 4-fold
improvement over MI-2 (Figure 4B). These experiments provide
important evidence that small molecules that bind to menin with
Figure 3. Development of second-generation menin-
MLL inhibitors. (A) Structures and activities of new
compounds designed based on the structure of the
menin–MI-2 complex. IC50values for the inhibition of the
menin-MBM1 complex are provided in parentheses.
(B) FP experiments comparing activities of MI-2, MI-2-2
and MBM1 for disruption of the menin-MBM1 interaction
demonstrating that MI-2-2 is a more potent inhibitor than
an MLL-derived peptide. (C) Isothermal titration calorim-
etry showing the binding of MI-2-2 to menin. N represents
a stoichiometry of binding. (D) Crystal structure of the
menin–MI-2-2 complex determined at 1.27 Å resolution
with corresponding 2Fo-Fc electron density map con-
toured at 1? level. Water molecules were omitted for
clarity. (E) Orthogonal dipolar interactions between MI-
2-2 fluorine and backbone atoms of His181. The dis-
tances are shown in Å.
INSIGHTS INTO INHIBITION OF MENIN-MLLINTERACTION4465 BLOOD, 29 NOVEMBER 2012?VOLUME 120, NUMBER 23
For personal use only. at UNIV OF MICHIGAN on May 16, 2013. bloodjournal.hematologylibrary.org From

Page 7

high affinity at the MBM1 site are capable of fully dissociating the
entire menin-MLLfusion protein complex (Figure 4C).
MI-2-2 has significantly improved cellular activity compared
with MI-2
Our second-generation compound, MI-2-2, demonstrates substan-
tially improved inhibition of the menin-MLL interaction over
MI-2, and therefore we compared the effects of these 2 compounds
in mouse BMCs transformed with MLL-AF9. Both compounds
caused very significant growth inhibition at low micromolar doses
(Figure 5A). Interestingly, treatment for longer than 6 days
revealed that only the more potent compound, MI-2-2, stably
suppressed growth of MLL-AF9–transformed BMCs (Figure 5A).
We also assessed the capability of MI-2 and MI-2-2 to down-
regulate the expression of MLL fusion protein target genes.
Although treatment with 6?M MI-2 had a small effect, the same
dose of MI-2-2 caused greater than 80% down-regulation of Hoxa9
and Meis1 expression (Figure 5B-C). In the colony formation
assay, treatment with MI-2-2 resulted in a more pronounced effect
on colony number compared with MI-2, with essentially no
colonies formed at 25?M MI-2-2 (Figure 5D-E). The colonies
were also smaller and much more diffused upon treatment with
MI-2-2, reflecting a loss of transforming properties by MLL-AF9
(Figure 5E and supplemental Figure 3). Furthermore, treatment
with MI-2-2 resulted in a more pronounced hematopoietic differen-
tiation than what was observed for MI-2, as reflected by a
Figure 4. Small molecules targeting the MBM1 site
efficiently disrupt bivalent menin-MLL interaction.
(A) Fluorescence polarization experiments demonstrat-
ing displacement of MLL4-43 from menin by MI-2 and
MI-2-2. Data represent mean values from 2 experi-
ments ? SD. (B) Coimmunoprecipitation experiment in
HEK293 cells transfected with Flag-MLL-AF9 showing
that MI-2 and MI-2-2 disrupt the interaction of menin with
MLL-AF9 in human cells. Input shows the levels of menin
and MLL. The amount of menin bound to Flag-MLL-AF9
was detected by coimmunoprecipitation using anti-Flag
Ab followed by immunoblotting using menin Ab.
(C) Model of the disruption of bivalent MLL-menin interac-
tion by MI-2-2 via binding to MBM1 site in menin.
Figure 5. Second-generation inhibitor MI-2-2 exhibits
strongly enhanced cellular activities compared with
MI-2. (A) Growth curves for MLL-AF9 transduced BMC
grown in liquid culture treated with DMSO, MI-2, and
MI-2-2. The experiment was performed 2 times.
(B-C) Quantitative real-time PCR showing the expression
of Hoxa9 (B) and Meis1 (C) in MLL-AF9–transduced
BMCs over the 6 days of treatment with MI-2 and MI-2-2.
Expression of Hoxa9 and Meis1 has been normalized to
?-actin and is referenced to the DMSO-treated cells. Data
represent the mean values for duplicates ? SD. The
experiment was performed 3 times. (D) Colony counts for
methylcellulose colony assay performed with MLL-AF9–
transduced BMCs treated for 7 days with MI-2-2 and
MI-2-2. Error bars indicate SD from duplicate experi-
ments; experiments were performed 2 times. (E) Repre-
sentative colonies shown for DMSO-, MI-2-, and MI-2-2–
treated MLL-AF9–transduced
methylcellulose. Black lines represent the scale bars
(500 ?m). (F) Quantification of CD11b expression in
MLL-AF9 transduced BMC treated for 6 days with the
menin-MLLinhibitors as detected by flow cytometry. Data
represent the mean values for duplicates ? SD. The
experiment was performed 2 times. (G) Wright-Giemsa–
stained cytospins for MLL-AF9–transformed BMCs after
7 days of treatment with DMSO, MI-2 (12?M), and MI-2-2
(12?M). Black lines represent the scale bars (50 ?m).
Statistical analysis and calculation of P values was
performed using 2-wayANOVA.
BMCs platedon
4466 SHI et alBLOOD, 29 NOVEMBER 2012?VOLUME 120, NUMBER 23
For personal use only. at UNIV OF MICHIGAN on May 16, 2013. bloodjournal.hematologylibrary.orgFrom

Page 8

substantial increase in the level of CD11b, a differentiation marker
of myeloid cells (Figure 5F), and very pronounced change in cell
morphology. Treatment for 7 days with 12?M MI-2-2 was
sufficienttocauseterminalmonocyticdifferentiationofMLL-AF9–
transformed BMCs, whereas MI-2 did not cause such a pronounced
effect (Figure 5G and supplemental Figure 4). In summary, the
structure-based design inhibitor MI-2-2 represents a significant
improvement over MI-2, as reflected by its substantially more
potent cellular activity in MLL-AF9–transduced BMCs.
MI-2-2 exhibits potent activities in human MLL leukemia cells
We also tested the activity of MI-2-2 in the MV4;11 human MLL
leukemia cell line, which harbors a MLL-AF4 translocation.
Similar to the effects observed in MLL-AF9–transduced BMCs,
treatment of MV4;11 cells with MI-2-2 caused growth inhibition of
these cells (GI50? 3?M; Figure 6A), whereas weak or no activity
was shown in non-MLL leukemia cells (supplemental Figure 5).
The MI-2-2 compound also resulted in a substantial and dose-
dependent increase in the number of cells undergoing apoptosis
(Figure 6B) and in G0/G1cell-cycle arrest (Figure 6C-D). The
MI-2-2 also exhibited strong down-regulation of HOXA9 and
MEIS1 expression in MV4;11 cells (Figure 6E) and induced
differentiation in these cells, as manifested by an increase in the
expression of CD11b and the formation of multilobed nuclei and
highly vacuolated cytoplasm (Figure 6F-G and supplemental
Figure 6). Remarkably, the effect of MI-2-2 on growth inhibition
(Figure 6H) and differentiation of MV4;11 cells was significantly
more pronounced compared with the first-generation compound
MI-2.20We also observed very similar activities of MI-2-2 on
growth inhibition and differentiation in 3 other cell lines harboring
various MLL translocations: ML-2, MOLM-13, and KOPN8
(supplemental Figures 8-10).Overall, these results demonstrate that
the second-generation inhibitor MI-2-2 exhibits very pronounced
effects in human leukemia cells carrying the MLLtranslocation, which
isconsistentwiththeimprovedinhibitionofthemenin-MLLinteraction.
Discussion
The development of low-molecular-weight compounds targeting
PPIs is generally considered a challenging task.18Recently, we
have provided a proof of principle that targeting the PPI between
menin and MLL by small molecules is feasible.20In the present
study, we carried out structural studies to characterize human
menin and its interaction with MLL, which provides an essential
molecular basis to understand the function of menin as an
oncogenic cofactor of MLL fusion proteins in acute leukemias. We
have also determined the crystal structure of menin in complex
with the small-molecule inhibitor MI-2, and found that this
compound binds to menin in a mode that mimics the key
interactions of MBM1 with menin. The structure of the menin–
MI-2 complex was crucial to the development of a second-
generation inhibitor, MI-2-2, with 8-fold improved binding affinity
toward menin. The MI-2-2 compound binds to menin with
Kd? 22nM and is capable of inhibiting both the interaction of
menin with MBM1 (IC50? 46nM) and with the bivalent fragment
of MLL that comprises both MBM1 and MBM2 (IC50? 520 nM).
The MI-2-2 exhibits very pronounced activities at low micromolar
concentrations in BMCs transformed with MLL-AF9 and in
MV4;11, a human leukemia cell line harboring the MLL-AF4
translocation. This also indicates that this compound has high
cellular permeability and might be used as a valuable chemical
probe with which to study the function of the menin-MLL and
menin-MLLfusion protein interactions.
The difficulty in developing potent small-molecule inhibitors
of menin-MLL arises from the relatively high binding affinity
(Kd? 10 nM) due to the bivalent interaction mode, where
2 MLL fragments (MBM1 and MBM2) are involved in binding to
menin.19Our previous studies demonstrated that the entire
N-terminal fragment of MLLbinds to menin with an approximately
5-fold stronger affinity than MBM1 alone due to the presence of a
Figure 6. MI-2-2 exhibits pronounced activity in
MV4;11 human leukemia cells with MLL-AF4 translo-
cation. (A) Inhibition of cell proliferation in MV4;11 cells
induced by MI-2-2 after 72 hours of treatment, as de-
tected by the MTT cell viability assay. Data represent
mean values for 4 samples ? SD. The experiment was
performed 3 times. (B) Apoptosis and cell death induced
by MI-2-2 in MV4;11 cells as detected by flow cytometry
using Annexin V/propidium iodide (PI) staining. Data
represent mean values for duplicates ? SD. (C) Histo-
grams from cell-cycle analysis performed by FACS after
PI staining in MV4;11 cells treated with DMSO and
MI-2-2. (D) Dose-dependent effect of MI-2-2 on cell-cycle
progression measured by FACS in MV4;11 cells after PI
staining. Data represent the mean values for 4 experi-
ments ? SD. (E) Expression of the HOXA9 and MEIS1
genes normalized to 18S rRNA determined by quantita-
tive RT-PCR in MV4;11 cells treated for 4 days with
MI-2-2 referenced to DMSO-treated cells. Data represent
the mean values for duplicates ? SD. (F) Quantification
of CD11b expression in MV4;11cells treated for 7 days
with the MI-2-2 as detected by flow cytometry. Data
represent the mean values for duplicates ? SD. The
experiment was performed 2 times. (G) Wright-Giemsa–
stained cytospins demonstrating differentiation of MV4;
11 cells after 10 days of treatment with MI-2-2 and
compared with DMSO. (H). Comparison of growth curves
for MV4;11 cells treated with DMSO, MI-2, and MI-2-2.
INSIGHTS INTO INHIBITION OF MENIN-MLLINTERACTION4467 BLOOD, 29 NOVEMBER 2012?VOLUME 120, NUMBER 23
For personal use only. at UNIV OF MICHIGAN on May 16, 2013. bloodjournal.hematologylibrary.org From

Page 9

second, low-affinity binding motif.19Mutational analysis indicates
that deletion of MBM1 is sufficient to abolish development of
leukemia in vivo,8and therefore the menin-MBM1 interaction
constitutes a primary target for inhibitor development. The small-
molecule compound we developed, MI-2-2, binds to menin with an
affinity comparable to the 40–amino acid–long MLL fragment
encompassing both MBM1 and MBM2.19More importantly,
MI-2-2 can efficiently disrupt not only the binding of MBM1, but
also the entire MLLfusion protein to menin (Figure 4).
Multisite binding modes have also been described for the
interactions of other intrinsically unstructured proteins.37There-
fore, the development of small molecules that would efficiently
target such interactions represents an additional difficulty for PPI
inhibitors. Our work provides an important proof of concept that
the inhibition of a complex with multivalent interactions can be
achieved by a low-molecular-weight compound if that the ligand
binds with high affinity to the PPI hot spot.
Structural analysis of protein-ligand complexes revealed that
inhibitors of PPIs tend to be large molecules forming extensive
hydrophobic contacts with relatively few hydrogen bonds.38The
MI-2-2 has relatively low molecular weight (415 Da) and lipophi-
licity (cLogP ? 3.9), and is fully compliant with the Lipinski rule
of 5 for orally bioavailable drugs.39Furthermore, MI-2-2 has a very
favorable ligand efficiency index (LE ? 0.39),40which is much
better than the average value of 0.24 reported for PPI inhibitors.38
In summary, MI-2-2 has several advantageous qualities as a
protein-protein inhibitor, including nanomolar affinity and favor-
able drug-like properties. The availability of the high-resolution
(1.27 Å) crystal structure of the MI-2-2 in complex with menin
provides an excellent foundation to develop novel drugs for
effective targeting of menin-MLL interaction in acute leukemias
with MLLrearrangements.
Acknowledgments
TheauthorsthankJayL.HessandAndrewG.Munteanforcelllines.
This work was funded by anAmerican Cancer Society Research
Scholar Grant (RSG-11-082-01-DMC to T.C.), a Leukemia &
Lymphoma Society TRP grant (6116-12 to J.G.), and a National
Institutes of Health grant (1R01CA160467 to J.G.). Use of the
Advanced Photon Source was supported by the US Department of
Energy, Office of Science, Office of Basic Energy Sciences under
contract number DE-AC02-06CH11357. Use of the LS-CAT
Sector 21 was supported by the Michigan Economic Development
Corporation and the Michigan Technology Tri-Corridor for the
support of this research program (grant 085P1000817).
Authorship
Contribution: A.S. synthesized the compounds; M.J.M., T.H., and
G.R. crystallized the menin and performed the biochemical experi-
ments; S.H. and T.P. performed the cellular assays; G.L. and M.C.
determined the crystal structures; and J.G. and T.C. planned the
experiments and wrote the manuscript with input from all authors.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
The current affiliation for M.C. is Department of Chemistry and
Biochemistry, University of South Carolina, Columbia, SC.
Correspondence: Tomasz Cierpicki or Jolanta Grembecka,
University of Michigan, Department of Pathology, 1150 W Medi-
cal Center Dr, MSRB1, Rm 4510, Ann Arbor, MI 48109; e-mail:
tomaszc@umich.edu or jolantag@umich.edu.
References
1. Cox MC, Panetta P, Lo-Coco F, et al. Chromo-
somal aberration of the 11q23 locus in acute leu-
kemia and frequency of MLL gene translocation:
results in 378 adult patients. Am J Clin Pathol.
2004;122(2):298-306.
2. Slany RK. When epigenetics kills: MLL fusion
proteins in leukemia. Hematol Oncol. 2005;23(1):
1-9.
3. Popovic R, Zeleznik-Le NJ. MLL: how complex
does it get? J Cell Biochem. 2005;95(2):234-242.
4. Dimartino JF, Cleary ML. Mll rearrangements in
haematological malignancies: lessons from clini-
cal and biological studies. Br J Haematol. 1999;
106(3):614-626.
5. Hess JL. MLL: a histone methyltransferase dis-
rupted in leukemia. Trends Mol Med. 2004;
10(10):500-507.
6. KrivtsovAV, Armstrong SA. MLL translocations,
histone modifications and leukaemia stem-cell
development. Nat Rev Cancer. 2007;7(11):823-
833.
7. Slany RK. The molecular biology of mixed lineage
leukemia. Haematologica. 2009;94(7):984-993.
8. YokoyamaA, Somervaille TC, Smith KS,
Rozenblatt-Rosen O, Meyerson M, Cleary ML.
The menin tumor suppressor protein is an essen-
tial oncogenic cofactor for MLL-associated leuke-
mogenesis. Cell. 2005;123(2):207-218.
9. YokoyamaA, Cleary ML. Menin critically links
MLL proteins with LEDGF on cancer-associated
target genes. Cancer Cell. 2008;14(1):36-46.
10. Cierpicki T, Risner LE, Grembecka J, et al. Struc-
ture of the MLL CXXC domain-DNAcomplex and
its functional role in MLL-AF9 leukemia. Nat
Struct Mol Biol. 2010;17(1):62-68.
11. Birke M, Schreiner S, Garcia-Cuellar MP, Mahr K,
Titgemeyer F, Slany RK. The MT domain of the
proto-oncoprotein MLL binds to CpG-containing
DNAand discriminates against methylation.
Nucleic Acids Res. 2002;30(4):958-965.
12. Allen MD, Grummitt CG, Hilcenko C, et al. Solu-
tion structure of the nonmethyl-CpG-binding
CXXC domain of the leukaemia-associated MLL
histone methyltransferase. EMBO J. 2006;25(19):
4503-4512.
13. MunteanAG, Tan J, Sitwala K, et al. The PAF
complex synergizes with MLL fusion proteins at
HOX loci to promote leukemogenesis. Cancer
Cell. 2010;17(6):609-621.
14. Milne TA, Kim J, Wang GG, et al. Multiple interac-
tions recruit MLL1 and MLL1 fusion proteins to
the HOXA9 locus in leukemogenesis. Mol Cell.
2010;38(6):853-863.
15. MunteanAG, Hess JL. The pathogenesis of
mixed-lineage leukemia. Annu Rev Pathol. 2012;
7:283-301.
16. Chandrasekharappa SC, Guru SC, Manickam P,
et al. Positional cloning of the gene for multiple
endocrine neoplasia-type 1. Science. 1997;
276(5311):404-407.
17. Marx SJ. Molecular genetics of multiple endo-
crine neoplasia types 1 and 2. Nat Rev Cancer.
2005;5(5):367-375.
18. WhiteAW, WestwellAD, Brahemi G. Protein-
protein interactions as targets for small-molecule
therapeutics in cancer. Expert Rev Mol Med.
2008;10:e8.
19. Grembecka J, BelcherAM, Hartley T, Cierpicki T.
Molecular basis of the mixed lineage leukemia-
menin interaction: implications for targeting mixed
lineage leukemias. J Biol Chem. 2010;285(52):
40690-40698.
20. Grembecka J, He S, ShiA, et al. Menin-MLL in-
hibitors reverse oncogenic activity of MLL fusion
proteins in leukemia. Nat Chem Biol. 2012;8:277-
284.
21. Murai MJ, Chruszcz M, Reddy G, Grembecka J,
Cierpicki T. Crystal structure of menin reveals
binding site for mixed lineage leukemia (MLL)
protein. J Biol Chem. 2011;286(36):31742-31748.
22. Otwinowski Z, Minor W. Processing of X-ray dif-
fraction data collected in oscillation mode. Meth-
ods Enzymol. 1997;276:307-326.
23. Minor W, Cymborowski M, Otwinowski Z,
Chruszcz M. HKL-3000: the integration of data
reduction and structure solution–from diffraction
images to an initial model in minutes. Acta Crys-
tallogr D Biol Crystallogr. 2006;62(8):859-866.
24. VaginA, TeplyakovA. Molecular replacement with
MOLREP. Acta Crystallogr D Biol Crystallogr.
2010;66(1):22-25.
25. Cowtan KD, Main P. Improvement of macromo-
lecular electron-density maps by the simultane-
ous application of real and reciprocal space con-
straints. Acta Crystallogr D Biol Crystallogr. 1993;
49(1):148-157.
26. Terwilliger T. SOLVE and RESOLVE: automated
structure solution, density modification and model
building. J Synchrotron Radiat. 2004;11(1):49-52.
27. Murshudov GN, VaginAA, Dodson EJ. Refine-
ment of macromolecular structures by the
maximum-likelihood method. Acta Crystallogr D
Biol Crystallogr. 1997;53(3):240-255.
28. Emsley P, Cowtan K. Coot: model-building tools
for molecular graphics. Acta Crystallogr D Biol
Crystallogr. 2004;60(12 pt 1):2126-2132.
4468 SHI et alBLOOD, 29 NOVEMBER 2012?VOLUME 120, NUMBER 23
For personal use only. at UNIV OF MICHIGAN on May 16, 2013. bloodjournal.hematologylibrary.org From

Page 10

29. The CCP4 suite: programs for protein crystallog-
raphy. Acta Crystallogr D Biol Crystallogr. 1994;
50(Pt 5):760-763.
30. Painter J, Merritt EA. TLSMD web server for the
generation of multi-group TLS models. J Appl
Crystallogr. 2006;39:109-111.
31. Davis IW, Leaver-FayA, Chen VB, et al. MolPro-
bity: all-atom contacts and structure validation for
proteins and nucleic acids. Nucleic Acids Res.
2007;35(Web server issue):W375-383.
32. YangH,GuranovicV,DuttaS,FengZ,BermanHM,
Westbrook JD.Automated and accurate deposi-
tion of structures solved by X-ray diffraction to the
Protein Data Bank. Acta Crystallogr D Biol Crys-
tallogr. 2004;60(10):1833-1839.
33. Huang J, Gurung B, Wan B, et al. The same
pocket in menin binds both MLL and JUND but
has opposite effects on transcription. Nature.
2012;482(7386):542-546.
34. Olsen JA, Banner DW, Seiler P, et al. Fluorine
interactions at the thrombin active site: protein
backbone fragments H-C(alpha)-C?O comprise
a favorable C-F environment and interactions of
C-F with electrophiles. Chembiochem. 2004;5(5):
666-675.
35. Mu ¨ller K, Faeh C, Diederich F. Fluorine in phar-
maceuticals: looking beyond intuition. Science.
2007;317(5846):1881-1886.
36. CasliniC,YangZ,El-OstaM,MilneTA,SlanyRK,
Hess JL. Interaction of MLL amino terminal se-
quences with menin is required for transforma-
tion. Cancer Res. 2007;67(15):7275-7283.
37. Arai M, Ferreon JC, Wright PE. Quantitative anal-
ysis of multisite protein-ligand interactions by
NMR: binding of intrinsically disordered p53
transactivation subdomains with the TAZ2 do-
main of CBP. J Am Chem Soc. 2012;134(8):3792-
3803.
38. HiguerueloAP,SchreyerA,BickertonGR,PittWR,
Groom CR, Blundell TL.Atomic interactions and
profile of small molecules disrupting protein-
protein interfaces: the TIMBAL database. Chem
Biol Drug Des. 2009;74(5):457-467.
39. LipinskiCA,LombardoF,DominyBW,FeeneyPJ.
Experimental and computational approaches to
estimate solubility and permeability in drug dis-
covery and development settings. Adv Drug Deliv
Rev. 2001;46(1-3):3-26.
40. HopkinsAL, Groom CR, AlexA. Ligand efficiency:
a useful metric for lead selection. Drug Discov
Today. 2004;9(10):430-431.
41. Baker NA, Sept D, Joseph S, Holst MJ,
McCammon JA. Electrostatics of nanosystems:
application to microtubules and the ribosome.
Proc Natl Acad Sci U S A. 2001;98(18):10037-
10041.
INSIGHTS INTO INHIBITION OF MENIN-MLLINTERACTION4469BLOOD, 29 NOVEMBER 2012?VOLUME 120, NUMBER 23
For personal use only. at UNIV OF MICHIGAN on May 16, 2013. bloodjournal.hematologylibrary.orgFrom