c e l l l i n e s ( n = 3 4 1 ) · 2020-01-25 · from mettl3 inhibition p e r c e n t i n h i b i...
TRANSCRIPT
Published
Dat
a (K
O, K
D)
Acc
ent D
ata
(METTL3
KO
)
MO
LM-1
3 (In
hibito
r)
NCI-H
1650
(Inhib
itor)
0
25
50
75
100
125
Percent m6A Reductionfrom METTL3 Inhibition
Perc
en
t In
hib
itio
n m
6A
Qu
an
tifi
ed
by L
C-M
S/M
S
Mean InhibitionAccent Data (65%)
n=29
n=23
0.1 1 10 100
100
1000
10000
METTL3/14 Biochemical IC50 (nM)
Low Enzyme Assay
MO
LM
-13 m
6A
IC
50 (
nM
)
100 1000 10000
100
1000
10000
MOLM-13 m6A IC50 (nM)
MO
LM
-13 P
rolife
rati
on
48
hr
IC5
0 (
nM
)
0.01 0.1 1 10 100
0
50
100
METTL3-14 Biochemical Assay
[ATX0002405] (nM)
% In
hib
itio
n
IC50 0.1658
1 Wang et al, Molecular Cell (2016)2 Dominissini et al, Nature (2012)3 Barbieri et al, Nature (2017)4 Vu et al, Nature Medicine (2017)
METTL3/14 is a Validated, Druggable Therapeutic Target for AMLP.A. Boriack-Sjodin, A.K. Gardino, T. A. Wynn, M. Laidlaw, A. Greene-Colozzi, E.A. Sickmier, S.M. Buker,
B.L. Hodous, A.W. Case, S. Ribich, R.A. Copeland
Accent™ Therapeutics, Lexington, MA
METTL3/14: A Type I RNA Methyltransferase
• Two distinct biochemical assays utilizing an 11-mer RNA substrate containing a central DRACH sequence were developed8
• SPR assays were established for hit validation, characterization of initial hits and lead series optimization
• High resolution crystal structures of protein-inhibitor complexes are routinely generated
• Cell-based assay utilizes mass spectrometry readout of m6A from isolated mRNA after compound treatment
• Proliferation assays established in multiple AML cell lines
The authors would like to thank Chatura Jayakody at Accent for assistance with sample management and our collaborators at contract research organizations for their efforts on this program.
m6A and METTL3/14 are Implicated in AML and Additional
Aspects of Cancer Biology
METTL3, the catalytically active subunit of the METTL3/METTL14 complex, belongs to the Type I methyltransferase
superfamily1 which uses S-adenosyl-L-methionine (SAM) as a substrate. METTL3/14 adds a methyl group to adenosine
on a subset of mRNAs containing a specific sequence, most commonly 5’-GGACU-3’ also known as a DRACH motif, to
create N6-methyladenosine (m6A)2.
METTL3/14
SAM SAH
Model of mRNA Binding to METTL3/14-
SAM Complex
METTL3/14 Catalyzes the Reaction of A to m6A
METTL3/14 Preferentially Methylates DRACH
Sequences2
References
5 Wang et al, Cell Stem Cell (2018)6 Tong et al, Cell Res (2018)7 Li et al, Nature Rev Immun (2016)
8 Buker et al, SLAS Discovery (2019)9 Copeland & Boriack-Sjodin, Cell
Chem Biol (2018)
m6A modulates mRNA turnover and controls the translation of key oncogenes that confer growth advantage and migratory
behavior3-5. METTL3 is overexpressed in a subset of acute monocytic leukemia (AML) cell lines and m6A is required for in
vitro and in vivo growth in these cell types. METTL3 knockout also promotes AML differentiation and apoptosis3-5.
Additionally, m6A has been shown to be important for differentiation of multiple T-cell lineages, including Tregs6 which are
involved in immunosuppression in tumor microenvironments8.
Due to the increasing validation of m6A as important in several aspects of cancer biology and specifically in AML, there is
significant interest in METTL3/14 as a target for drug discovery efforts.
PDB 5K7U1 used for
modeling efforts
Acknowledgements
TM
Conclusions
Multiple Assays Developed for METTL3i Drug Discovery
-8 -6 -4
-20
0
20
40
60
80
Drug Concentration (M)% in
hib
itio
n o
f m
6A
in
mR
NA
by
LC
-MS
/MS
Inactive compound, IC50 >25,000 nM
Active compound, IC50=570 nM
CRISPR Indicates Cancer Cell Lines Are Dependent on
METTL3/14
New Biochemical Assays Developed for METTL3/148
SPR and Crystallography Enable Residence Time Optimization
and Structure-Based Design
Cell Assay Monitors m6A Decrease in mRNA
Crystal structure of METTL3/14 with
substrate analog sinefunginSAM binding to METTL3/14 by SPR
MO
LM
-13
m6A
IC
50
(nM
)
METTL3/14 Biochemical IC50 (nM)
• In vitro inhibitor potency has been optimized; compounds with biochemical IC50 values <200 pMhave been generated
• Compounds with biochemical IC50s <10 nM tested in the m6A cell assay show measurable activity in MOLM-13 cell line
• Inhibition of m6A plateaus at ~70%; residual activity may due to other m6A methyltransferases (eg, METTL16) or presence of non-coding RNAs
• Biochemical and m6A IC50s correlate with each other as expected for specific on-target inhibitor occupancy9
• Shift seen between biochemical and cellular IC50 values is expected from intracellular levels of SAM and chemical series mechanism of inhibition
• METTL3 inhibitors show antiproliferative activity in MOLM-13 cells that correlates with inhibition of m6A9
• MOLM-13 cells are dependent and sensitive to METTL3 inhibition with ~1x shift between pharmacodynamic and phenotypic IC50s
• Compound 2 dosed at 10 mM (>cellular IC90) induces apoptosis in MOLM-13 cells treated for multiple days as measured by induction of cleaved Caspase 3
IC50 = 0.166 nM
[Compound 2] (nM)
% I
nh
ibitio
n
% I
nh
ibitio
n m
6A
(m
RN
A)
n=29
Mean Inhibition
Accent Data (66%)
Correlation and 500x Shift Seen
for Biochemical and m6A IC50s
METTL3i Shows Antiproliferative Activity in AML Cell Line
MOLM-13 m6A IC50 (nM)
MO
LM
-13
Pro
lifera
tion
48
hr
IC5
0(n
M)
Correlation Seen for m6A and
Proliferation IC50s
Compounds Inhibit Methyl Mark
Deposition
Compounds Show Antiproliferative
Activity
1 10 100 1000 10000
-20
0
20
40
60
80
[ATX0002405] (nM)% in
hib
itio
n o
f m
6A
in
mR
NA
by
LC
-MS
/MS
[Compound 2] (nM)
% I
nh
ibitio
n m
6A
(m
RN
A)
% I
nh
ibitio
n m
6A
in m
RN
A
by
LC
-MS
/MS
0.1 1 10 100 1000 10000
0
50
100
[ATX0002405] (nM)
% A
nti
pro
life
rati
ve E
ffect
48 h
ou
rs
[Compound 2] (nM)
% A
ntip
rolif
era
tive
Effe
ct
48
hr
IC50 = 230 nM
IC50 = 280 nM
1x shift
Abstract # A112
• METTL3/14 is an m6A RNA methyltransferase implicated in AML through several different mechanisms
• METTL3 knockout demonstrates selective essentiality in MOLM-13 AML cell lines
• Using tools developed and optimized for drug discovery efforts, SAM-substrate competitive series has been identified and
validated; potency in this series has been optimized to <200 pM
• This pharmacophore series has demonstrated correlated inhibition of biochemical enzyme activity, intracellular m6A, and
cancer cell proliferation
• Based on genetic and small molecule inhibitor data presented, METTL3/14 is an appropriate target for further efforts in
AML
Compound Concentration (M)
• The Broad Avana CRISPR pooled screen includes cell lines from both solid and liquid tumors; this data indicates there is selective dependency on METTL3 and METTL14 (dependency graphed by CERES score (dotted line = -0.5 ))
• Individual knockout of METTL3 in the acute monocytic leukemia (AML) MOLM-13 cell line results in impaired growth compared to control cells
• Knockout in additional AML cell lines is in progress
Multiple Cancer Cell Lines are Dependent on METTL3 and METTL14
- 1 . 5
- 1 . 0
- 0 . 5
0 . 0
0 . 5 M E T T L 3
C e l l L i n e s ( n = 3 4 1 )
R a n k e d h i g h e s t t o l o w e s t s e n s i t i v i t y
CE
RE
S V
alu
e
- 1 . 5
- 1 . 0
- 0 . 5
0 . 0
0 . 5 M E T T L 1 4
C e l l L i n e s ( n = 3 4 1 )
R a n k e d h i g h e s t t o l o w e s t s e n s i t i v i t y
CE
RE
S V
alu
e
- 1 . 5
- 1 . 0
- 0 . 5
0 . 0
0 . 5 M E T T L 3
C e l l L i n e s ( n = 3 4 1 )
R a n k e d h i g h e s t t o l o w e s t s e n s i t i v i t y
CE
RE
S V
alu
e
- 1 . 5
- 1 . 0
- 0 . 5
0 . 0
0 . 5 M E T T L 1 4
C e l l L i n e s ( n = 3 4 1 )
R a n k e d h i g h e s t t o l o w e s t s e n s i t i v i t y
CE
RE
S V
alu
e
METTL14METTL3
Cell Lines (n = 341)
Ranked highest to lowest sensitivity
Cell Lines (n = 341)
Ranked highest to lowest sensitivity
CE
RE
S V
alu
e
CE
RE
S V
alu
e
• Multiple transcripts, including MYC,
MYB, SP1, SP2, PTEN, BCL2, and
TNFRSF2 are methylated by the
METTL3/14 complex to generate m6A
• In some instances, such as for the
mRNA encoding the pro-apoptotic TNF-
a receptor TNFRSF2, m6A is read by
YTH-domain containing protein YTHDF2
to promote transcript degradation and
thereby prevent apoptosis of leukemic
stem cells (LSCs)
• In other transcripts, m6A is bound by the
m6A readers YTHDF1/3 or EIF3h to
promote translation of key oncogenes
that promote the proliferation and
prevent differentiation of leukemic blasts
The Role of METTL3/14 Generated m6A in AML
NT (PCDHA1 sgRNA) PCNA sgRNA METTL3 sgRNA
0
2
4
6
8
10
7-daycell proliferation
n c
ells
da
y 7
/n c
ells
day 0
In Vitro Characterization of METTL3/14 Inhibitor Series
0 2 4 6 8 100.00
0.05
0.10
0.15
0.20
0.25
Compound IC50 Comparisons With Varying [SAM]
[SAM] (X of 0.37 uM)
IC50 (m
M)
Com
pou
nd
1 I
C5
0(m
M)
[SAM]/KM
Time (min)
Cou
nts
Per
Min
ute
[Compound 1] (mM)
% I
nh
ibitio
n
Inhibition is Not Time Dependent
Compound Binding Confirmed by
SPRCompounds are Competitive With SAM and Mutually Exclusive with
Product SAH
Inhibition is Reversable
[Compound 1] (mM)
v/v 0
Time (sec)
Resp
on
se
IC50 = 30 nM
Compound 1
KD = 23 nM
• Hit-finding activities resulted in identification and validation of a SAM-competitive series for METTL3/14
• Rapid optimization from mM to nM affinity proved the hit series was chemically tractable
Compounds Synthesized with pM
Potency
Compounds Synthesized with pM
Potency
Biochemical and m 6A Cell Potencies Correlate for METTL3i
60-70% m6A Reduction Obtained
From Knockdown and METTL3i
Compounds Show Extended Residence Time by SPR
and in Cells
GAPDH
Inhibitor Treatment Induces Apoptosis
Cleaved
Caspase 3
Treatment
Time (hr) 24 48 72 96
Compounds Have Slow Dissociation
from METTL3/14 by SPR
Compound 3: IC50 = 0.60 nM
KD = 0.41 nM
Residence Time = 59 min
m6A Inhibition in Cells is
Maintained After Washout
% I
nh
ibitio
n m
6A
(m
RN
A)
3X PBS wash
• KD values measured by SPR are similar to IC50 values from biochemical assays and significant on-target residence time is seen
• Cells treated with Compound 3 (residence time ~1 hour) show maximum inhibition of m6A is achieved within 8 hours and target inhibition persists at least 8 hours after washout
Compound 3 used for
cell washout experiment
METTL3 Knockout in MOLM-13 Growth Impairment Seen in
Cells with METTL3 Knockout
- control sgRNA + control sgRNA METTL3 sgRNA# C
ells (
Day 7
) /
# C
ells (D
ay 0
)
BIT
S
Time (sec)
Resp
on
se
(PCDHA1) (PCNA)
Time (hr) Time After Washout (hr)
t½ = 1.1 hr t½ = 58.5 hr
Linear Regression:
Slope = 694
95% CI = 592 – 797
R2 = 0.91