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Mereau, De Rijck et al.
Supplementary information
A) Supplementary Material & Methods
Cell lines and reagents
MOLM13, (AML, MLL-AF9+); THP1, (AML, MLL-AF9+); MV4;11 (AML, MLL-AF4+);
HL-60, (AML, MLL WT); Jurkat (T-ALL); and Kasumi (AML, AML1-ETO) cell lines
were purchased from DSMZ (Braunschweig, Germany). All cells were cultured in
RPMI-1640 with glutamine, supplemented with 10% fetal calf serum (FCS) and 1%
Penicillin/streptomycin at 37°C and 5% CO2. IL3 independent Ba/F3 cells carrying
the FLT3-ITD mutation (Ba/F3 FLT3-ITD) have been described previously 1. 293T
cells (ATCC/LGC Standards, Molsheim Cedex France) were grown in Dulbecco's
modified Eagle's medium with Glutamax (Gibco, Invitrogen, Merelbeke, Belgium)
supplemented with 8% FCS and 50 µg/ml gentamicin (Gibco).
Plasmids
The pMSCV (Murine Stem Cell Virus) retroviral expression vector encoding for the
MLL-AF9 fusion was provided by J. Hess (Ann Arbor), pMSCV-MLL-ENL-neo was
provided by Robert Slany (Erlangen). The retroviral transfer plasmid expressing
eGFP (pMSCV IRES-eGFP-PGK-Puro) was derived from pLMP (Open Biosystems,
Fermentas GmbH, St Leon-Rot, Germany) where the backbone was removed by
digestion with SpeI enzyme. The missing part of the PGK sequence was replaced by
SpeI digestion of a pMSCV-PKG-Puro vector (Clontech, Saint-Germain en Laye,
France) and ligation of this fragment into the modified pLMP plasmid. To create the
MSCV plasmid expressing eGFP-LEDGF325-530 (pMSCV eGFP-LEDGF325-530 PGKPuro), eGFP-LEDGF325-530 was amplified from peGFP-LEDGF325-530 IRES Puro
2
using oligo’s DR1 and DR2 (Table 1), digested with BamHI and MfeI and subcloned
into pMSCV-PGK-Puro (Clontech, Saint-Germain en Laye, France) digested by BglII
and EcoRI. The shRNA- fragment for murine LEDGF/p75 was purchased from Open
Biosystems (V2MM_34220) and subcloned from pSMC into the pLMP retroviral
vector. To clone the lentiviral transfer plasmids pSFFV-eGFP-I-Puro-WS and pSFFVeGFP-LEDGF325-530-I-Puro-WS
expressing
eGFP
and
eGFP-LEDGF325-530
respectively, eGFP-I-Puro and eGFP-LEDGF325-530-I-Puro were amplified by PCR
from peGFP-IRES-Puro and peGFP-LEDGF325-530-IRES-Puro 2, respectively with
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1
oligo’s DR3 and DR4 (Table 1). PCR products were digested with BamHI and NheI
3
and sublconed into pSFFV-eGFP-WS-Isa
where eGFP was removed through a
BamHI/SpeI digestion. To create the LEDGF/p75 knockdown lentiviral transfer
plasmid pSFFV-eGFP–I-Puro 2xmi p75 WS, expressing eGFP and a duplicate
LEDGF/p75 microRNA cassette, the microRNA cassette was amplified by PCR with
oligo’s DR5 and DR6 (Table 1) from pSFFV-eGFP-Ires-tCD34-mirLEDGF digested
with SpeI and subcloned into peGFP-IRES-Puro
3
digested with XbaI. The resulting
eGFP-IRES-Puro-mirShLEDGF cassette was removed by NheI/BamHI digestion and
subcloned into pSFFV-eGFP-d325-2xL3
3
in which the eGFP-LEDGF325-530-2xL3
cassette was removed by XbaI/BamHI digestion. To create the plasmid for eukaryotic
expression of HA tagged Menin (pCH SFFV Menin-HA WS), the Menin coding
sequence was amplified by PCR from pGEX-Menin (kindly provided by Prof. Carlos
Casiano, Loma Linda University, USA) using oligo’s DR7 and DR8, containing the
HA-tag coding sequence and digested by BamHI/NheI. This PCR fragment was
subcloned into pSFFVeGFP-WS-Isa
3
digested with BamHI/SpeI to remove the
eGFP coding sequence. Expression vectors for various eGFP-LEDGF deletion
mutants were generated by overlap extension PCR using the LEDGF325-530 sublclone
in pBlue Script KS as template. The T3 Reverse was used as common primer to
generate the C-terminal deleted mutants and T7 Forward to generate the N-terminal
deleted fragments. The smallest fragments were generated using the same strategy
and by using the two minimal active mutants identified LEDGF 325-386 and LEDGF424530
as templates. All primers used for the generation of the LEDGF fragments are
described in Table 1. The generated LEDGF deletion mutants were then cloned back
into pLMP expression vector. To clone the plasmid for eukaryotic expression of tripleflag-tagged (Flag) MLL1-330 a codon optimized synthetic gene was ordered from Life
Technologies, Ghent, Belgium. The synthetic gene, MLL1-330 was amplified using
oligo’s DR9 and DR10 (Table 1) and digested by SalI and NheI. This PCR product
was subsequently cloned into pEGFP-C3 (Clontech, Saint-Germain-en-Laye,
France), which was digested by XhoI and XbaI. The plasmid for purification of FlagLEDGF/p75, pCPNatFlag-p75 was described before
4
To clone the plasmid for
purification of MLL1-160-GST (pET-20b MLL1-160-GST), GST, PCR amplified with
oligo’s DR 11 and DR12 and digested with EcoRI/XhoI was cloned into pET-20b
which was digested with EcoRI/XhoI. The resulting plasmid was subsequently
digested with NdeI/BamHI and used to insert MLL1-160, which was amplified by PCR
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2
with the oligos DR13 and DR14 (Table 1) from the MLL synthetic gene and digested
with NdeI/BamHI. To create the expression plasmid for His-Thioredoxin-Menin (HTrx-Menin), Menin was amplified from pGEX-Menin with oligo’s DR15 and DR16
(Table 1) and cloned into pDONR221 using the gateway system (Life Technologies)
following the protocol provided by the manufacturer. Menin was subsequently
transferred from the DONR plasmid to pHXGWA
5
using the gateway protocol. The
plasmid for MBP-LEDGF325-530 expression was described before 2. All plasmids were
sequence verified.
Lentiviral and retroviral vector production
Lentiviral vector production was performed as described earlier 6. Retroviral vector
production was performed as described previously 7.
Giemsa cytospin-staining
Murine MLL-AF9 leukemic cells were harvested 10 days post-transduction. 5x104
cells were resuspended in 100 µl PBS and spun onto glass slides using a Shandon
Cytospin-2 Centrifuge at 500 rpm for 5 min. The slides were air dried and stained
with Wright-Giemsa using the Hematek® Stain Pak Hematology Slide Strainer (Bayer
HealthCare, Zurich, Switzerland).
Apoptosis test
Murine MLL-AF9 leukemic cells were harvested 3, 5, 7 and 10 days after puromycin
selection and were stained with an Annexin-V antibody (BDBiosciences, #550475)
and DAPI (Gibco, Invitrogen; 1/10000) for 15 min at RT following the manufacturer’s
protocol. Samples were analyzed on a Cyan ADP Flow Cytometer (Dako Cytomation,
Glostrup, Denmark) using Summit software and Flowjo software.
Western Blots
Whole cell extracts of different cell lines (293T, human AML cells THP1 and the
murine hematopoietic cell lines Ba/F3 FLT3-ITD) were made in 1% SDS, separated
by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and electroblotted onto polyvinylidene difluoride membranes (Bio-Rad, Nazareth Eke,
Belgium). Membranes were blocked with milk powder in PBS-0.1% Tween 20, and
detection was carried out using specific antibodies against LEDGF/p75 (Bethyl,
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3
A302-509A), Flag (Sigma, F7425), HA (Abcam, ab9134) or GFP (Abcam, ab6673).
Detection was performed using chemiluminescence (ECL+; Amersham) and
horseradish peroxidase (HRP)-conjugated secondary antibodies.
Co-immunoprecipitation
7x106 293T cells were plated in a 8.5 cm petri dish and transfected with the indicated
plasmids (30 µg total) using branched PEI. After 24 hours cells were washed with
PBS and lysed with 700 µl lysis buffer (50 mM Tris/HCl, pH7.3, 250 mM NaCl, 0.5%
[v/v] Triton X-100, 10% glycerol, Complete Protease Inhibitor Cocktail [Roche,
Germany]) for 10 min on ice. The lysate was cleared by centrifugation and the
supernatant was incubated with 30 µl ANTI-FLAG® M2-agarose affinity resin (SigmaAldrich) and 10 units DNase (Roche) overnight at 4°C. The beads were collected by
centrifugation (30 s, 1800xg, 4°C) and washed 3 times in 600 µl lysis buffer.
Immunoprecipitated protein was eluted with 40 μl SDS-PAGE loading buffer and
visualized by western blotting.
Protein purification
Flag-tagged LEDGF/p75 expression and purification was essentially the same as for
the non-tagged LEDGF/p75 8. MBP-LEDGF325-530 was purified as described before 21.
All other proteins were expressed in E. Coli Rosetta2 (DE3) grown on Lysogeny
broth (LB) medium supplemented with 20 mg/ml ampicillin. Bacterial cultures were
grown at 37°C for MLL1-160-GST, His-TRX-LEDGF375-386, His-TRX-LEDGF424-435, and
His-TRX-ET production and at 28°C for His-TRX-Menin production. Protein
expression was induced with 0.5 mM isopropyl--d-1-thiogalactopyranoside (IPTG) at
an OD600 of 0.5. H-TRX-LEDGF375-386, H-TRX-LEDGF424-435 and H-TRX-ET cultures
were harvested after 1 h. MLL1-160-GST and H-TRX-Menin cultures were harvested
after 4 hours. Cells were washed in 20 mL STE buffer (10 mM Tris/HCl pH7.5; 100
mM NaCl, 0.1 mM EDTA), and pellets were stored at −20°C. For purification of HTRX tagged proteins, cell pellets were resuspended in lysis buffer (50 mM Tris–HCl
pH7.5; 150 mM NaCl, 20 mM imidazole, 1 mM PMSF and 0.1 U/ml DNase) and lysed
by sonication. The lysate was clarified by centrifugation at 19800×g for 30 minutes at
4°C and subjected to affinity chromatography using His-Select Nickel Affinity Gel
(Sigma) equilibrated with wash buffer (50 mM Tris/HCl pH7.5; 150 mM NaCl, 20 mM
imidazole). His-tagged proteins were eluted with wash buffer containing 250 mM
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4
imidazole. Purification of MLL1-160-GST was carried out by affinity chromatography on
Glutathione Sepharose-4 Fast Flow (GE Healthcare, Fairfield, CT). The resin was
equilibrated with wash buffer (50 mM Tris/HCl pH7.5, 150 mM NaCl) and GSTtagged proteins were eluted in wash buffer supplemented with 25 mM Glutathione
(GSH). Fractions were analyzed by SDS-PAGE for protein content. Peak fractions
were dialyzed against 100x excess 20 mM Tris/HCl pH7.5, 150 mM NaCl, 10%(v/v)
glycerol at 4°C over night. H-TRX-Menin was concentrated using Amicon Ultra
Concentrators (Millipore).
AlphaScreen
AlphaScreen measurements were performed in a final volume of 25 µL in 384-well
Optiwell microtiter plates (PerkinElmer). All components were diluted to the indicated
concentrations in assay buffer (25 mM Tris pH7.4, 150 mM NaCl, 1 mM DTT, 0.1%
(v/v) Tween-20 and 0.1% (w/v) Bovine Serum Albumin (BSA). Optimal binding
concentrations for MLL-Menin, Menin-LEDGF/p75 and LEDGF/p75-MLL proteins
were determined in cross-titration experiments. For apparent Kd determinations,
MLL1-160-GST wild type and/or its mutants were titrated against 0.3 nM FlagLEDGF/p75 or 4 nM H-TRX-Menin. For IC50 determinations, MBP-LEDGF325-530 and
LEDGF derived peptides (untagged or tagged with H-TRX) were titrated against 10
nM MLL1-160-GST and 0.3 nM Flag-LEDGF/p75. After addition of proteins, peptides
and/or compounds, the plate was pre-incubated for 1 h at room temperature (RT). 20
µg/mL Glutathione donor and anti-Flag or Ni2+-chelate acceptor beads (PerkinElmer)
were added, bringing the final volume to 25 µL. After 1 h of incubation at RT, the
plate was analyzed on an EnVision Multi-label Reader in AlphaScreen mode
(PerkinElmer). Results were analyzed in Prism 5.0 (GraphPad software) after nonlinear regression with the appropriate equations: one-site specific binding, taking
ligand depletion into account for the apparent Kd measurements and sigmoidal doseresponse with variable slope for the IC50 determination.
Peptides
Peptides (> 95% purity, HPLC) were purchased from PeptideSynthetics, Hampshire,
UK. LEDGF424-435; KNMFLVGEGDSV, LEDGF375-386; EALDELASLQVT.
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Supplementary Table 1:
Oligo-primers used in this study
Oligo
410-530 Forward
424-530 Forward
450-530 Forward
pBlueScript II SK T3 Reverse
pBlueScript II SK T7 Forward
325-409 Reverse
325-386 Reverse
366-386 Forward
375-386 Forward
424-449 Reverse
424-435 Reverse
Sequence 5'-3'
TTTGGATCCATGGTGAGCAAGGGCGAGGA
GGGCAATTGCTAGTTATCTAGTGTAG
CATATTGGATCCATGGTGAGCAAGGGCGAGGAG
AAAGCTAGCTCAGGCACCGGGCTTG
TTTTACTAGTACTAGCGCTACCGGACTCAG
TTTTACTAGTCAACGCGTCCCGGTGGATCC
AAAGGATCC ATGGGGCTGAAGGCCGCCC
AAAAGCTAGCTCACGCGTAGTCCGGTACGTCGTACGGGTAAG
CAGCAGCGAGGCCTTTGCGCTGCCGC
TTTTGTCGACCTCAAGCTTGCGCACAGCTGTCGGTG
TTTTGCTAGCTTA GACTTTCTGGGGCTTTTCC
TTTTGAATTCGGCCTGAACGATATTTTTGAAGCGCAGAAAATTG
AATGGCATGAAGTCGAC
TCCCCTATACTAGGTTATTGGAAAATTAAG
CCCCCTCGAG CTA ATCCGATTTTGGAGGATGGTC
TTTTCATATG GCACATAGCTGTCGTTGGCG
CCCC GGATCC TCGCACTCTGACTTCTTCATCTGAG
GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGGGCTGAAGGC
CGCCCAG
GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGAGGCCTTTG
CGCTGCCGC
GCAGTCTCGAGCAGGTAATCATGGAAAAGTCTAC
GCAGTCTCGAGAAGAACATGTTCTTGGTTGGTG
GCAGTCTCGAGCATGAGGAAGCGAATAAAACC
GGTTGGCCCTCAAAAGGG
TAATACGACTCACTATAGGGC
GTACGGCGATTCAAAGTTAGTTGACAATTGATCG
TGTGACCTGAAGTGAAGCAAGTGACAATTGATCG
GCAGTCTCGAGGATAATCTTGATGTGAACAGATGCAAT
GCAGTCTCGAGGAGGCCTTGGATGAACTTGCTTCA
CTGTCTTTGTTCAGCAAGAGATTATTGACAATTGATCG
CACGGAATCTTCACCAACTGACAATTGATCG
RT-quantitative PCR primers
mouse HOXA9 forward
mouse HOXA9 reverse
mouse GAPDH forward
mouse GAPDH reverse
Sequence 5'-3
GGTTCTCCTCCAGTTGATAGAGA
GAGCGAGCATGTAGCCAGTTG
ATGACATCAAGAAGGTGGTG
CATACCAGGAAATGAGCTTG
DR1
DR2
DR3
DR4
DR5
DR6
DR7
DR8
DR9
DR10
DR11
DR12
DR13
DR14
DR15
DR16
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B) Supplementary Figures
Supplementary Figure S1
The anti-leukemic potential of LEDGF325-530 expression in MLL-AF9 transformed hematopoietic
cells is maintained upon replating. (A) Colony-forming units (CFU) per 104 MLL-AF9 murine
transformed cells expressing eGFP-LEDGF325-530 or eGFP (set to 100%). Cells were flow-sorted and
104 cells were plated in methylcellulose. Seven days later cells were harvested and 10 4 cells were
replated for a 2nd round. (B) Hoxa9 mRNA expression levels were assessed by quantitative RT-PCR
after the 1st and 2nd round in methylcellulose from the experiment described in A). The expression
levels were normalized to Gapdh and the eGFP control (set to 100%). Error bars represent the
standard deviations of two independent experiments.
Mereau, De Rijck et al.
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Supplementary Figure S2
Expression of LEDGF/p75 or derived fragments in THP1 cells. (A) THP1 cells were transduced
with lentiviral vectors expressing the indicated eGFP fused LEDGF/p75 fragments. Fragments were
detected with an eGFP antibody. Equal loading was controlled with an anti-tubuline antibody (-Tub).
(B) Knockdown (KD) of LEDGF/p75 upon transduction with a lentiviral vector expressing a
LEDGF/p75 shRNA was quantified by Q-RT-PCR and normalized to RNaseP mRNA. Error bars
indicate standard deviations of triplicate measurements.
Mereau, De Rijck et al.
8
Supplementary Figure S3
Validation of the Alphascreen assays used in this study. (A) Coomassie stained gel of
recombinant proteins used in this study. (B) Direct interaction of MLL with Menin. Titration of MLL1-160GST or the F9A mutant against 5 nM H-TRX-Menin in AlphaScreen. Error bars represent the standard
deviation of triplicate datapoints. (C) Inhibition of the MLL-Menin interaction by small molecules. 4 nM
H-TRX-Menin and 10 nM MLL1-160-GST were preincubated in AlphaScreen. The interaction was
inhibited by adding different concentrations of the indicated small molecules. Error bars represent the
standard deviation of triplicate datapoints. (D) Direct interaction of Menin and MLL with LEDGF/p75.
Titration of H-TRX-Menin and MLL1-160-GST or the F129A, R130A, E135Q triple mutant against 0.3 nM
Flag-LEDGF/p75 in AlphaScreen. Error bars represent the standard deviation calculated from 3
independent experiments performed in triplicate.
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Supplementary Figure S4
Determination of the minimal MLL/menin interaction domain on LEDGF/p75. (A) Schematic
representation of LEDGF/p75 and the derived deletion mutants of eGFP-LEDGF325-530 used for
screening. PWWP: Pro-Trp-Trp-Pro chromatin binding domain; NLS: Nuclear localization signal; IBD:
Integrase binding domain. The numbers refer to amino acids of the reference sequence: GenBank
accession number, NP_001121689.1. (B) Colony-forming units (CFU) per 2.103 plated MLL-AF9
murine transformed cells in the presence of puromycin. Cells were transduced to express the eGFPLEDGF deletion mutants or, as controls, full-length eGFP-LEDGF325-530 or eGFP (set to 100%). Error
bars represent standard deviations of a duplicate experiment.
Mereau, De Rijck et al.
10
Supplementary Figure S5
Western Blot analysis of BAF3 cells transduced with MSCV derived vectors expressing eGFP,
eGFP-LEDGF325-530,
eGFP-LEDGF325-386,
eGFP-LEDGF424-530,
eGFP-LEDGF375-386
or
eGFP-
LEDGF424-435. Proteins were detected using an eGFP antibody. Equal loading was controlled with an
-tubuline antibody (α-Tub).
Mereau, De Rijck et al.
11
Supplementary Figure S6
JURKAT
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
0
Number of cells (X103) / ml
Number of cells (X103) / ml
Control (eGFP)
eGFP
eGFP-LEDGF325-530
eGFP-d325
eGFP-LEDGF375-386
eGFP-375
eGFP-LEDGF424-435
eGFP-424
180
Control (eGFP)
eGFP
160
eGFP-LEDGF325-530
eGFP-d325
140
eGFP-LEDGF375-386
eGFP-375
120
eGFP-LEDGF424-435
eGFP-424
100
80
60
40
20
0
1
C
KASUMI
B
2
3
4
5
Time (days)
6
7
D
HL60
4500
4000
Control
(eGFP)
eGFP
(control)
3500
eGFP-LEDGF
LEDGF
325-530
325-530
3000
eGFP-LEDGF
LEDGF
375-386
375-386
2500
eGFP-LEDGF
LEDGF
424-435
424-435
2000
1500
1000
500
0
1
2
3
4
Time (days)
1
Number of cells (X103) / ml
Number of cells (X103) / ml
A
5
6
7
2
3
4
5
Time (days)
6
7
MV4;11
1800
Control
(eGFP)
eGFP
(control)
1600
eGFP-LEDGF
LEDGF
325-530325-530
1400
eGFP-LEDGF
LEDGF
375-386375-386
1200
eGFP-LEDGF
LEDGF
424-435424-435
1000
800
600
400
200
0
1
2
3
4
5
Time (days)
6
7
Expression of the LEDGF/p75 fragments significantly impairs growth of MLL-fusion positive
MV4;11 cells but not of other acute human acute leukemia cell lines. (A-D) Cell proliferation of
Jurkat (T-ALL), Kasumi (AML1-ETO+; AML), HL-60 (c-myc amplification; AML) and MV4;11 (MLLAF4+; AML) cells, transduced with lentiviral vectors expressing the indicated eGFP fused LEDGF/p75
fragments. Error bars represent the standard deviation of two independent experiments.
Mereau, De Rijck et al.
12
Supplementary Figure S7
A
B
Control (eGFP)
LEDGF325-530
LEDGF375-386
LEDGF424-435
80
70
% of cells
60
50
40
30
20
10
3 days
Living
5 days
7 days
Early Apoptosis
5
43
4-
42
5
43
4-
e
DG GFP
LE F3
DG 25-5
3
LE F3 0
DG 75-3
F 86
LE
LE
42
e
DG GFP
LE F3
DG 25-5
3
LE F3 0
DG 75-3
F 86
5
43
4-
42
e
DG GFP
LE F3
DG 25-5
3
LE F3 0
DG 75-3
F 86
LE
5
43
4-
LE
42
e
DG GFP
LE F3
DG 25-5
3
LE F3 0
DG 75-3
F 86
0
10 days
Late apoptosis/Necrosis
Expression of eGFP fused LEDGF-fragments induced morphological changes in MLL-AF9 murine
cells without increasing apoptosis. (A) Representative Wright-Giemsa-stained cytospin preparation of
MLL-AF9 murine leukemic cells 10 days after transduction with eGFP-LEDGF325-530, eGFP-LEDGF375-386 or
eGFP-LEDGF424-435 vs. vector control, selected in puromycin. Cells were visualized with an Olympus (Tokyo,
Japan) BX61 microscope magnification 60X (Olympus UPlanApo 60X/1,35 NA oil) and pictures were
acquired with Cell^P software and ColorviewIII imaging system (Olympus). For each picture, the black scale
bare in the bottom left is representative of 20 µm.
(B) Evaluation of the proportion of MLL-AF9 cells
expressing eGFP fused LEDGF fragments or eGFP control with the living (Annexin-V-/DAPI-), early
apoptotic (Annexin-V+/DAPI-) and late apoptotic /necrotic (Annexin-V+/DAPI+) cell fractions. Cells were
grown in the presence of puromycin (2g/ml) and analyzed at the indicated time points.
Mereau, De Rijck et al.
13
Supplementary Figure S8
Cartoon representation of the published MLL/Menin-IBD co-crystal structure
10.
LEDGF375-386
and the resolved part of LEDGF424-435 are indicated in dark blue.
Mereau, De Rijck et al.
14
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