MMSET
The Histone Methyl Transferase
Affected In (t4;14) Multiple Myeloma
Jonathan D. Licht
Hematology/Oncology
Robert H. Lurie Comprehensive Cancer Center
Chromosomal Aberrations In Multiple
Myeloma Drive Oncogene Expression
Amel Dib, et al., J Natl Cancer Inst Monogr 2008;39:25
1
The MMSET Protein Contains Domains Involved In
Chromatin Function
I
PWWP
HMG
II
PWWP
HMG
PHD
PHD
PHD
PWWP
SET
PHD
SET = Suvar, Enhancer-of-Zeste and Trithorax domain; catalyzes lysine
methylation in histones and transcription factors.
HMG = High Mobility Group; Possible DNA binding domain.
PWWP = Pro-Trp-Trp-Pro; found in nuclear proteins.
PHD = Plant HomeoDomain; methyl-lysine binding module.
NLS = Nuclear Localization Sequence
MMSET Overexpressed in t(4;14)
What Are the Consequences?
t (4;14) +
t (4;14) -
KMS
H9
K
K
MM.
S
M
M
K
2
S28PE
S12B
MM2
1
9
M1
1
M
MMSET II
MB 4-2 II
MMSET
MB 4-3 II
CHR
GAPDH
MB 4-3 II
NUC
GAPDH
1
2
3
4
5
6
2
MMSET in Myeloma
· Biological Function - Is MMSET
Overexpression Important?
· Can Biochemical Analysis Lead to Insights
into Disease Pathogenesis and Therapy?
MMSET Effects on Adhesion Properties
Control
N-terminal Knock down
-DOX
+ DOX
MMSET II
PWWP NLS
HMG
PHD
PWWP
SET
PHD
MMSET I
3
MMSET Effects on Growth Properties
PWWP NLS
HMG
PHD
PWWP
SET
PHD
MMSET II
MMSET I
2
C-terminal No DOX
C-teminal DOX
)6 1.8
1.6
1.4
(x10
er 1.2
b
1
.
0.8
0.6
lNum 0.4
Cel 0.2
0
Day0
Day5
Day 8
Day 14
G0/G1
S phase
G2/M
30
+ MMSET
- MMSET
100%
25
80%
20
60%
15
40%
10
5
20%
0
0%
Control
N-terminal
C-terminal
+ MMSET
- MMSET
shRNA
MMSET in Myeloma
· Biological Function - Is MMSET
Overexpression Important?
· Can Biochemical Analysis Lead to Insights
into Disease Pathogenesis and Therapy?
4
SET Domain
Histone Post-translational Modifications
H2A-CT
H3
H4
H2B-NT
H2B-CT
H2A-NT
Khorasanizadeh S. Cell 2004;116:259-72.
5
Histone Lysine Modifications
Lysines are the most diversely modified residues in histones.
Histone Modifications are "Read"
By Specific Modules
Chromo
WDR5
H3K9
H3K4
6
Different Chromatin Modifications and Enzymes Are
Associated With Alternative Transcriptional
Outcomes
GENE ACTIVATION
GENE REPRESSION
HDAC
HxKy-Ac
HxKy
HAT
RBP2, PLU-1
H3K4-Me
H3K4
MLL, SMYD3
G9A, SUV39
H3K9
H3K9-Me
JMJD2C, LSD1
EZH2, EZH1
H3K27
H3K27-Me
JMJD2C, LSD1
Hypothesis: MMSET is a Reader and
a Writer of the Histone Code
What Mark(s) Does MMSET Read?
What Mark(s) Does MMSET Make?
How Does This Mark Affect Gene Expression?
Can MMSET Be Inhibited?
7
The N-terminal PWWP Motif Is Necessary for Tight
Chromatin Binding
DAPI
aMMSET
M1
MM.
)-4;1
t(4
M
PWWP
HMG
PHD
PHD
PHD
PWWP
SET
PHD
MMSET II
12B
MB 4-2 II
HMG
PHD
PHD
PHD
PWWP
SET
PHD
SM
MB 4-3 II
HMG
PHD
PHD
PHD
PWWP
SET
PHD
K
1
S1M
)+4
K
;1
t(4
S28PEMK
The first PWWP domain in MMSET is also a
potential DNA binding module
MMSET
MSH6
Biochemistry. 2008 Jun 10;47(23):6199207.
8
How does MMSET Read the Histone Code?
SET
PHD
SET
PHD
PHD PHD PHD
PHD PHD PHD
How does MMSET Read the Histone Code?
MMSET II
REIIBPI
M2-PHD4
PWWP
HMG
PHD
PHD
PHD
PWWP
SET
PHD
MMSET
PHD
PWWP
SET
PHD
REIIBP
PWWP
HMG
PHD
PHD
PHD
PWWP
SET
MMSET II ­ PHD4
9
MMSET- Reads
DNA/Chromatin
· DNA Binding - Not specific
· H3K36, H3K27 Mark Binding
· PHD Domains- Possible Targets
Hypothesis: MMSET is a Reader and
a Writer of the Histone Code
What Mark(s) Does MMSET Read?
What Mark(s) Does MMSET Make?
How Does This Mark Affect Gene Expression?
Can MMSET Be Inhibited?
10
MMSET: in vitro Methyltransferase Activity
For Histone H3 and Histone H4
Catalytic
MMSET
MMSET
Methyl Transfer
SAM
SAM
HISTONE
HISTONE
MMSET+ GST
PRE SET
SET
POST SET
PHD
In Vitro- MMSET Can methylated
Many Different Histone Sites
2µg
1µg
-SET
+SET
-SET
+SET
H3K27 1me
H3K36 1me
Set Domain
Total H3
Total H3
H3
H4
-SET
+SET
H4K20 1me
11
GAL4 Tethering to Test MMSET Function
MMSET
GAL4
Multiple Repression Domains within MMSET
20000
tyvi 15000
Acti
10000
ase
fer
5000
Luci
0
47
1
2
P
P
NT
T1
CT
T2
-1
W
W
MG
4-
E
4-
E
-1
W
W
S
l4-H
al
MS
al
al4
-P
-P
G
G
MM
G
-M
al4-HDAC1
l4
Ga
G
al4
-M
G
Ga
al4
G
l4
Ga
PWWP
HMG
PHD
PHD
PHD
PWWP
SET
PHD
PWWP1
HMG
PWWP2
MMSET-NT
MMSET-CT
MMSET I
MMSET II
12
MMSET-Potential Transcriptional Repressor
HDAC1
Sin3A
HDAC2
SirT1
LSD1
X
MMSET
KRAB/ZnF
H4K20me
ACETYL
METHYL
Global Chromatin Changes in Response to MMSET
MMSET
H3K27-Me1
H3K27-Me2
H3K27-Me3
H3K36-Me1
H3K36-Me2
H3K36-Me3
Total H3
13
KNOCK OUT SYSTEM FOR MMSET
Lauring J. et al
Blood, 15 January 2008, Vol. 111, No. 2, pp. 856-864.
MMSET Knock out (KO)-Global Effects on Chromatin
MMSET
H3K27-Me1
H3K27-Me2
H3K27-Me3
H3K36-Me1
H3K36-Me2
H3K36-Me3
Total H3
14
15
MMSET Re-Addition Reverses the Switch
F1117 Y1118
F1177
F1201
SAM C
F1201
SET
F1177
Y1118
meK
Histone binding
Lysine binding
SAM binding
F1117
18A
18A
177A
1
177A
1
N
(Ming Ming Zhou)
MMSETII+
TKO
TKO
TKO+F1
TKO+Y1
TKO+F1
TKO+Y1
H3K27 3Me
H3K36 3Me
H3K36 1Me
Total H4
MMSET Methylation Activity Critical to Myeloma Growth
Control
+ MMSET II
) 7
+ Active mutant
+ Dead mutant
6
6
(x10 5
4
3
2
number
1
Cell 0
Day 0
Day 4
Day 7
Day 11
HMT Inactive Mutant
16
HMTs Gone Wild!--K36/K27 Switch
MMSET
METHYL
Hypothesis: MMSET is a Reader and
a Writer of the Histone Code
What Mark(s) Does MMSET Read?
What Mark(s) Does MMSET Make?
How Does This Mark Affect Gene Expression?
Can MMSET Be Inhibited?
17
Which Genes are Affected by MMSET Expression?
ab
175
MMSETII
600
303
Active
Add back
C-terminal
N-terminal
225
mutant
352
18
601
shRNA
shRNA
245
75
(>1.5 fold, p<0.05)
532
(>1.5 fold, p<0.05)
Dead
mutant
d
Ontology
p-value
Examples
Knock down
Apoptosis
5.20E-02
BCL2, HDAC4, PDCD10, STAT3
DNA repair
3.70E-04
RAB23, RRAS, ERCC1, IGF1
Chromatin packaging and remodeling
8.10E-03
MLL3, IRF7, HAT1, SETDB2
Cell cycle control
8.30E-02
BTG2, CCND2, CCNE2, GADD45A
+ MMSET II
Apoptosis
1.40E-03
BAX, ITGB1, PRKCA
Cell cycle
1.40E-03
BTG2, E2F2, CDC25A
Cell motility
2.30E-02
CXCR4, ITGB7, TUBB4, ROCK2
The HMT Activity of MMSET Yields Gene Activation
100
Regulated by WT MMSET Only
90%ile
4.5
80
Average up
5.1
60
Average down
-1.9
40
10%ile
-2.2
Change
20
#Genes
861.0
0
Fold
-20
Genes
Genes
90%ile
2.0
12
Average Up
1.9
10
Regulated by HMT Inactive Only
Average Down
-1.8
8
6
10%ile
-1.5
4
Change
#Genes
561
2
0
-2
Fold
-4
-6
Genes
18
Which Genes are Affected by MMSET Expression?
c
MMSET Knock down
MMSET Knock out
MMSET Add backs
1.2
1
JAM2
1.2
JAM2
30
JAM2
no 0.8
1
25
n
ive
o
n 20
0.6
e
0.8
at
ressi
tiv
0.6
15
Rel
0.4
la
ressi
ressio
Exp
0.4
10
0.2
Re
Relative
Exp 0.2
Exp
5
0
0
0
Control
N-terminal C-terminal
Control
MMSET
Active
Dead
+ MMSET
- MMSET
mutant
mutant
ChIP in MMSET knockout cells- GLS2
+ MMSET
MMSET
0.2
IgG
+ MMSET
- MMSET
0.15
- MMSET
0.15
ent
ent
0.1
hmic 0.1
nr
richm
E
n
%
E 0.05
0.05
%
0
0
Upstream
Exon 4
Exon 5-6
Exon 17-18
Upstream
Exon 4
Exon 5-6
Exon 17-18
+ MMSET
+ MMSET
H3K36me3
H3K36me2
- MMSET
0.2
- MMSET
0.5
0.4
0.15
0.3
hment
0.1
hment
ic
ic
0.2
Enr
Enr
%
0.05
%
0.1
0
0
Upstream
Exon 4
Exon 5-6
Exon 17-18
Upstream
Exon 4
Exon 5-6
Exon 17-18
+ MMSET
H3K27me3
- MMSET
0.8
0.7
0.6
ent
0.5
0.4
richmn 0.3
E% 0.2
0.1
0
Upstream
Exon 4
Exon 5-6
Exon 17-18
19
MMSET- Genome Wide Profiling
KMS11 cells- double crosslinking; ChIPseeqer software;
4736 Peaks Identified
MMSET+
MMSET-
MMSET+
MMSET-
20
MMSET Targets
· SET Dependent
­ Cell Cycle, Adhesion, Apoptosis Genes
Chromatin Regulators Among Targets
­ This Activity Critical for Growth
Which Targets are involved in Pathogenesis?
HMTs Gone Wild- Non
Transcriptional Effects?
MMSET
METHYL
21
MMSET effects on chromatin accessibility
Knock down
Reversed Knock down
Control
N-terminal
Control
N-terminal
Control
N-terminal
Mnase:
-
-
+
+
+
+
1000
300
200
100
50
Control
+MMSET II
+Inactive Mutant
0 Gy
5 Gy
10Gy
22
MMSET- An Effect on DNA Damage
Response
23
MMSET+ Cells Fail to Undergo Cell Cycle Arrest of DNA Damage
Hypothesis: MMSET is a Reader and
a Writer of the Histone Code
What Mark(s) Does MMSET Read?
What Mark(s) Does MMSET Make?
How Does This Mark Affect Gene Expression?
Can MMSET Be Inhibited?
24
BIX 01294
G9a/GLP Inhibitor
Inhibits H3K9me3
QuickTimeTM and a
decompressor
are needed to see this picture.
Reprograms Somatic to IPS cells
H3K36 Me3 Ab
n
n
4
l
A
aetoci
aetocci
T
tro
0129
h
h
M
n
C
C
Co
BIX
O
50mM
10uM
30uM
H2
10uM
HMT Inhibitor Killed MMSET+ Cell
More Readily
ATPlite BIX 01294 inhibition
90
80
MMSET+ Cells
70
60
lront 50
NTKO
fCo 40
TKO
o
% 30
20
10
0
2uM BIX
4uM BIX
6uM BIX
8uM BIX
25
5
SET Domain model for CADD
Prediction of Inhibitors
Mass Spec Based Screen for
MMSET Inhibitors
26
Self-Assembled Monolayers (SAMs) on Gold
Functional
Group
Carbon
chain
Thiol
Gold
Well-defined structure
Inert background environment
Controllable ligand density
Compatible with MALDI-TOF MS
Ac-GGVKKPHRYC-NH
Ac- GVKKPHR C-NH
2
2
+ 1 Me
12mer Peptide
+ 1 Me
7mer
Zachary A. Gurard-Levin-Mrksch Lab
27
High-Throughput Mass Spec
Methylated
1.0
Re
SAMs on gold patterned glass
peptide
Unmodified
l.
peptide
In
0.5
t.
0
m/z
Peptides arrayed
1.0
Re
l.
14 m/z
In
0.5
t.
0
m/z
Solution of HMT
1.0
Re
E
l.In
D
14 m/z
0.5
C
1. Rinse / dry
t.
B
2. SAMDI
A
0
1
2
3
4
5
6
7
8 9 10
m/z
Applied Biosystems 4800
MALDI TOF/TOF
Mrksich Lab
MMSET Inhibitor Screen Summary
·10,000 compounds screened in pools of 8
·5 uM MMSET, 2 uM peptide, 1 mM SAM, 12.5 uM each compound
·Total of 11 pools with significant inhibition (2030%)
·Deconvoluted 11 pooled hits to singletons (Data below)
·6 compounds with >40% inhibition at 10 uM
MMSET Deconvolution
1
ate 0.9
str 0.8
Single compounds in
b 0.7
assay
Su 0.6
Cutoff for Hits
ted 0.5
yla 0.4
th 0.3
0.2
Me% 0.1
0
0
204060
80
100
SAMPLE #
28
Conclusions
· MMSET Appears to be a Therapeutic Target in Myeloma
· MMSET Inhibitors exist and more will be developed
­ The HMT Activity of MMSET is Important for Biological Effects
· MMSET Can Activate and Repress Genes
­ Exact Critical Targets being determined
· MMSET May Have Effects on other Chromatin-Dependent
Processes
­ DNA Repair
­ DNA replication?
· MMSET Myeloma May Have Specific "Achilles Heels"
Northwestern University
· Dong-Joon Min
· Relja Popovic
· Eva Martinez
· Christine Will
· Teresa Ezponda
· Marianne Kim
Mayo Clinic- Scottsdale
·
Marta Chesi
·
Leif Bergsagel
National Cancer Institute
·
Lou Staudt
MSKCC
·
Dinshaw Patel
U Chicago
·
Milan Mrksch
­
Zack Guard- Levin
U Maryland
·
Alex MacKarrell
DFCI
·
Jay Bradner
29
SCOR Consortium
QuickTimeTM and a
·Jay Hess- U Mich
decompressor
are needed to see this picture.
·Dave Allis-Rockefeller
·Robert Roeder- Rockefeller
·Dinshaw Patel-MSKCC
·Ari Melnick-Weil-Cornell
QuickTimeTM and a
decompressor
are needed to see this picture.
Samuel Wax man
Cancer Resea rch Foundat ion
30