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...

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