RESEARCH ARTICLE
AG-221, a First-in-Class Therapy Targeting
Acute Myeloid Leukemia Harboring Oncogenic
IDH2 Mutations
Katharine Yen
1
, Jeremy Travins
1
, Fang Wang
1
, Muriel D. David
2,3,4
, Erin Artin
1
, Kimberly Straley
1
,
Anil Padyana
1
, Stefan Gross
1
, Byron DeLaBarre
1
, Erica Tobin
1
, Yue Chen
1
, Raj Nagaraja
1
, Sung Choe
1
,
Lei Jin
1
, Zenon Konteatis
1
, Giovanni Cianchetta
1
, Jeffrey O. Saunders
1
, Francesco G. Salituro
1
,
Cyril Quivoron
2,3,4
, Paule Opolon
5
, Olivia Bawa
5
, Véronique Saada
2,3,4
, Angelo Paci
6
, Sophie Broutin
6
,
Olivier A. Bernard
2,3,4
, Stéphane de Botton
2,3,4
, Benoît S. Marteyn
7,8,9
, Monika Pilichowska
10
, YingXia Xu
11
,
Cheng Fang
11
, Fan Jiang
12
, Wentao Wei
12
, Shengfang Jin
1
, Lee Silverman
1
, Wei Liu
1
, Hua Yang
1
, Lenny Dang
1
,
Marion Dorsch
1
, Virginie Penard-Lacronique
2,3,4
, Scott A. Biller
1
, and Shin-San Michael Su
1
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MAY 2017
CANCER DISCOVERY | 479
ABSTRACT
Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2
are found in multiple hematologic and solid tumors, leading to accumulation of the
oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate–dependent
dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family,
causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided
proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and
characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme.
AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 muta-
tion–positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also
provided a statistically significant survival benefit in an aggressive IDH2
R140Q
-mutant AML xenograft
mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients
with IDH2 mutation–positive advanced hematologic malignancies.
SIGNIFICANCE: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and
contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the tar-
geted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces
differentiation of malignant blasts, supporting its clinical development.
Cancer Discov; 7(5); 478–93.
©2017 AACR.
See related commentary by Thomas and Majeti, p. 459.
See related article by Shih et al., p. 494.
1
Agios Pharmaceuticals, Inc., Cambridge, Massachusetts.
2
INSERM U1170,
Villejuif, France.
3
Gustave Roussy, Université Paris-Saclay, Villejuif, France.
4
Equipe Labellisée Ligue Contre le Cancer, Villejuif, France.
5
Plateforme
d’évaluation préclinique, Gustave Roussy, Université Paris-Saclay, Villejuif,
France.
6
Service de Pharmacologie, Département de Biologie et Pathologie
Médicales, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
7
Unité
de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France.
8
INSERM U1202, Institut Pasteur, Paris, France.
9
Laboratoire de Thérapie
Cellulaire, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
10
Department of Pathology, Tufts Medical Center, Boston, Massachusetts.
11
ShangPharma, Shanghai, China.
12
Viva Biotech Ltd., Shanghai, China.
Note: Supplementary data for this article are available at Cancer Discovery
Online (http://cancerdiscovery.aacrjournals.org/).
K. Yen, J. Travins, F. Wang, and M.D. David contributed equally to this work.
Current address for K. Straley: Vertex Pharmaceuticals, Boston, Massa-
chusetts; current address for B. DeLaBarre: The Consulting Biochemist,
LLC, Arlington, Massachusetts; current address for J.O. Saunders: Resil-
ience Therapeutics, Boston, Massachusetts; and current address for F.G.
Salituro: SAGE Therapeutics, Cambridge, Massachusetts.
Corresponding Author: Shin-San Michael Su, Agios Pharmaceuticals, Inc.,
88 Sidney Street, Cambridge, MA 02139. Phone: 617-649-8600; E-mail:
Shinsan.Su@agios.com
doi: 10.1158/2159-8290.CD-16-1034
©2017 American Association for Cancer Research.
INTRODUCTION
Metabolic reprogramming is a hallmark of cancer, contrib-
uting to the initiation and maintenance of tumors (1, 2).
The NADP
+
-dependent isocitrate dehydrogenases (IDH) are
critical metabolic enzymes that interconvert isocitrate and
α-ketoglutarate (αKG). Recurrent somatic point mutations in
active site arginine residues of IDH1 (R132) and IDH2 (R140
and R172) have been found in multiple tumors, including acute
myeloid leukemia (AML; refs. 3–9). Cancer-associated IDH1/2
mutations confer the neomorphic activity of reducing
αKG to
the oncometabolite (R)-2-hydroxyglutarate (2HG; refs. 9–11).
2HG accumulation competitively inhibits
αKG-dependent
dioxygenases, including histone demethylases and methylcyto-
sine dioxygenases of the TET family that regulate cellular epige-
netic status (12–15). This epigenetic dysregulation is associated
with impairment of cellular differentiation in multiple cell types,
including hematopoietic cells (15–21). AGI-6780, a selective sul-
fonamide inhibitor of the mutant IDH2 enzyme, lowered 2HG
levels and induced differentiation of TF-1 erythroleukemia cells
and primary human AML cells harboring the IDH2
R140Q
muta-
tion (17), providing in vitro evidence that inhibition of the mutant
IDH2 enzyme can reverse some of the phenotypic changes it
induces. Others have reported similar findings with mutant
IDH1 inhibitor tool compounds in AML models (22).
IDH mutations are also found in premalignant disor-
ders, including myelodysplastic syndromes (MDS), and were
shown to drive leukemic transformation in cooperation with
other genetic events in IDH-mutant mouse models of AML
(23–25). It is possible that IDH-mutant cells drive clonal
hematopoiesis, sustaining a reservoir of stem cells associated
with resistance to conventional chemotherapy that needs to
be targeted via alternative mechanisms (26–28).
Here, we report the design and characterization of AG-221
(enasidenib), an orally available, selective, potent, triazine
inhibitor of the mutant IDH2 enzyme that dramatically
reduced 2HG levels in multiple models. AG-221 induced
differentiation in IDH2-mutant TF-1 cells and primary
human AML cells ex vivo, as well as in four IDH2
R140Q
-mutant
human AML xenograft mouse models in vivo, and provided
a dose-dependent, statistically significant survival benefit
in vivo in an aggressive human AML xenograft model, support-
ing its clinical development.
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Yen et al.
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RESULTS
Discovery of Allosteric Inhibitors of the
IDH2-Mutant Enzyme
In pursuit of drug candidates targeting recurrent onco-
genic IDH2 mutations, we initiated a high-throughput screen
for inhibitors of the enzyme carrying the most prevalent
IDH2 mutation in AML, IDH2
R140Q
(29–31). Several tria-
zine compounds active against the IDH2
R140Q
homodimer
emerged, and initial hit-to-lead chemistry led to compound
1, the first sub–100 nmol/L inhibitor of IDH2
R140Q
(Fig. 1A).
Although fairly potent in enzymatic and cellular assays, it
displayed high lipophilicity, leading to solubility-limited
absorption in vivo. In addition, its poor in vitro liver micro-
somal stability translated to high clearance in vivo. X-ray
crystallography revealed binding of a compound 1 molecule
to an allosteric site located within the homodimer interface
of the IDH2
R140Q
-mutant enzyme, to which the selective sul-
fonamide inhibitor AGI-6780 also binds (17). These insights
guided optimization of the substituents around the triazine
core. Through the addition of mildly polar substituents, such
as trifluoromethyl pyridine and 2-methyl-2-propanol, AG-221
(Fig. 1A and Supplementary Fig. S1A) was identified to have
excellent potency for 2HG inhibition (Table 1), improved
solubility, low clearance (0.83 l/h/kg), and good oral bio-
availability (41%) in vivo in rats (Supplementary Fig. S1B and
Supplementary Table S1).
AG-221 Is a Slow Tight Binder of the
IDH2
R140Q
-Mutant Enzyme
Because IDH2 is homodimeric, and somatic IDH mutations
found in tumors occur in a single allele, with one wild-type
(WT) allele, the mutant enzyme likely exists in cells as a mix-
ture of mutant−WT heterodimers and mutant homodimers.
As the heterodimer produces 2HG more efficiently than
mutant homodimers (32), it is an important molecular tar-
get. We therefore characterized the activity of AG-221 against
the heterodimer and mutant and WT homodimers.
AG-221 displayed a long residence time on the IDH2
R140Q
homodimer, with kinetics consistent with slow-onset tight
binding inhibition (Supplementary Fig. S2A and S2B).
AG-221 showed noncompetitive inhibition against the
IDH2
R140Q
homodimer for the αKG substrate and uncom-
petitive inhibition against the IDH2
R140Q
and IDH2
WT
homodimers for NADPH and NADP
+
cofactors, respectively
(Supplementary Fig. S3A–S3C). AG-221 displayed time-
dependent, nanomolar potency for inhibiting 2HG produc-
tion by the IDH2
R140Q
homodimer (IC
50
= 0.10 µmol/L at 16
hours), the IDH2
R140Q/WT
heterodimer (IC
50
= 0.03 µmol/L),
and the IDH2
R172K/WT
heterodimer (IC
50
= 0.01 µmol/L), and
time-dependent, single-digit micromolar potency for inhi-
bition of the canonical forward (oxidative) reaction in the
IDH2
WT
homodimer (IC
50
= 1.8 µmol/L at 16 hours; Table 1).
Similar potency was observed in the forward direction for
the IDH2
WT/R140Q
and IDH2
WT/R172K
heterodimers, albeit with
lower maximum percentage inhibition (range, 75%−64%).
AG-221 displayed selectivity for IDH2-mutant homo- and
heterodimers over IDH1
WT-
and IDH1
R132H
-mutant enzymes
(Table 1), a panel of kinases (Supplementary Table 2; ref.
17), and a second panel of 80 receptors, ion channels, and
enzymes (data not shown). Furthermore, AG-221 displayed
excellent potency in reducing 2HG in cell lines ectopically
expressing or overexpressing IDH2
R140Q
or IDH2
R172K
(Table
2). In these assays, AG-221 displayed higher potency against
R140Q versus R172K; this was not observed in the enzyme
assays.
AG-221 Stabilizes the Inhibitory Open Homodimer
Conformation of IDH2
R140Q
A high-resolution (1.55 Å) X-ray crystal structure of
AG-221 in complex with IDH2
R140Q
, NADPH, and Ca
2+
(IDH2
R140Q
·AG-221) confirmed that it binds to the allosteric
site enclosed within the homodimer interface, and conse-
quently the mutant enzyme adopts an open conformation.
We observed alternative conformations for AG-221 binding
in the pocket, owing to the pseudo 2-fold symmetric nature
of the pocket (Fig. 1B). To understand the molecular mecha-
nism of inhibition, we crystallized the 1.54 Å resolution X-ray
structure of IDH2
R140Q
bound to substrate αKG, NADPH,
and Ca
2+
(IDH2
R140Q
·αKG). This catalytically primed complex
adopts a compact closed homodimer conformation (Fig. 1C).
Comparing the quaternary complexes of IDH2
R140Q
·AG-221
and IDH2
R140Q
·αKG suggested that AG-221 allosterically sta-
bilizes the open homodimer conformation, preventing the
conformational change required for catalysis, consistent with
the mode of inhibition documented for IDH1
R132H
mutants
(17, 33).
AG-221 binding is anchored by multiple hydrogen bonds
and hydrophobic interactions within the pocket. The pocket
is encapsulated by four helices (
α9, α10, α9’, α10’) lining the
sides, two loops (L1 and L1’), and the Y311–D312 interaction
pairs capping the ends (Fig. 1D). Nitrogens at the 1, 3 posi-
tions on the diaminotriazine core accept hydrogen bonds
from the amino sidechain of the Q316 residues, whereas
linker amides donate hydrogen bonds to the Q316 car-
bonyl sidechain (Fig. 1E). The Q316 carbonyl also accepts
a hydrogen bond from the 2-methyl-2-propanol moiety of
AG-221. Other polar interactions include a halogen bond
between AG-221’s trifluoromethylpyridine and the D312 cap-
ping residue. In addition, van der Waals interactions from
surrounding hydrophobic residues W164, V294, V297, L298,
V315, I319, and L320 contribute to AG-221’s high inhibitory
potency. The dominant hydrophobic nature of the pocket
reveals why larger polar substituents were less favorable,
owing to a steric clash or desolvation energetic penalty. Along
with the domain motions, movement of L1 and L1’ would be
required to provide access to the binding site (17). The col-
lective structural rearrangements needed to access the deeply
buried pocket, combined with the multitude of interactions
upon binding, explain the slow-on/slow-off tight binding
kinetics of AG-221.
AG-221 Inhibited 2HG Production and Induced
Differentiation in IDH2-Mutant TF-1 Cells and
Primary Human AML Blasts
As reported previously (17), and consistent with obser-
vations in myeloblasts from patients with IDH1/2-mutant
AML (34), IDH2
R140Q
expression in the TF-1 erythroleukemia
cell line induced intracellular 2HG production to concen-
trations of 3,500 to 5,116 ng/10
6
cells; intracellular and
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AG-221 Therapy for IDH2-Mutant AML
RESEARCH ARTICLE
MAY 2017
CANCER DISCOVERY | 481
Figure 1. AG-221 structure and binding characteristics. A, Key molecules leading to candidate AG-221 and corresponding in vitro and biochemi-
cal data. B, Co-complex crystal structure of IDH2
R140Q
homodimer with AG-221 bound to an allosteric site. Protein represented as ribbon (protomers
colored cyan and green), AG-221 as magenta spheres, and NADPH as yellow sticks. C, Allosteric binding of AG-221 stabilizes inhibitory open confor-
mation of the IDH2
R140Q
active site (left) versus the catalytically primed αKG-bound IDH2
R140Q
structure (right). Solvent-accessible surface for each
protomer shown as translucent white area with cyan or yellow ribbon and solid green or yellow surface for IDH2
R140Q
:AG-221 and IDH2
R140Q
:αKG, respec-
tively. αKG and NADPH shown in ball and stick representation, and AG-221 as solid spheres. D, Detailed view of AG-221 binding site at the IDH2
R140Q
dimer interface. Secondary structures flanking the compound shown as cylinders α9 and α9’ (residues 292−299), and α10 and α10’ (residues 310−325).
Residues from each protomer shown in cyan/green. AG-221, shown as sticks (carbon in magenta, nitrogen in blue, fluorines in cyan), exhibits two pos-
sible asymmetric binding conformations. Residues Y311 and D312 (stick figures) cap one end of the pocket, and flexible loops L1 and L1’ (residues
151–168; ribbons) cap the other end. E, Molecular interactions of AG-221 in its binding site. Amino acid residues within a 3.5 Å radius of AG-221 shown
as sticks (green/cyan for carbon atoms from adjacent homodimers). Hydrogen bond interactions between AG-221 and Q316 residue shown as dotted
lines. Residues Q316, L320, I319, and V294 exhibit asymmetric alternative conformation in the binding site. E
h
, hepatic extraction ratio; HLM, human
liver microsome; inh, inhibitory.
A
IDH2
R140Q
IC
50
= 1.9 µmol/L IDH2
R140Q
+ NADPH, IC
50
at 16 h = 7 nmol/L
CI
NN
NN
N
NN
N
H
NN
H
OH
CF
3
CF
3
N
N
H
NN
H
NNN
H
Screening hit
H
O
Cell IC
50
(2HG inh) = 30 nmol/L
HLM E
h
= 0.69
Solubility (pH 2/7.4) = 2/0.8 µmol/L
IDH2
R140Q
+ NADPH, IC
50
at 16 h = 100 nmol/L
Cell IC
50
(2HG inh) = 10–20 nmol/L
HLM E
h
= 0.16
Solubility (pH 2/7.4) = 47/23 µmol/L
B
C
D
E
R140Q R140Q
NADPHNADPH
Open
AG-221
NADPH NADPH
Closed
AG-221
Y311’
Y311’
D312’
D312
Q316’
Q316
L320’
L320
I319’
L298’
L298
L160’
L160
W164
W164’
AG-221
Y311
D312’
α9’
αKG
α10’
W164’
L1’
L160’
L160
L1
W164
α9
α10
D312
Y311
Compound 1
AG-221
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Yen et al.
RESEARCH ARTICLE
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|
CANCER DISCOVERY MAY 2017 www.aacrjournals.org
extracellular 2HG levels were reduced by AG-221 treatment
(Supplementary Fig. S4A and S4B). AG-221 also inhibited
growth factor–independent proliferation and reversed his-
tone H3 hypermethylation induced by IDH2
R140Q
expression
(Supplementary Fig. S4C and S4D).
Treatment of TF-1 cells with erythropoietin (EPO) induces
expression of the genes hemoglobin alpha 1 and 2 (HBA1/2)
and erythroid Kruppel-like factor 1 (KLF1), a master regulator
of erythropoiesis, and a red color change associated with dif-
ferentiation. Such EPO-induced changes were not observed
in TF-1 cells expressing IDH2
R140Q
with elevated intracellular
2HG (3,500 ng/10
6
cells; Supplementary Fig. S5A), confirming
that this mutation blocks EPO-induced differentiation (17).
On AG-221 treatment, dose-dependent increases in KLF1 and
HBA1/2 expression were seen in IDH2
R140Q
cells, along with
the color change indicative of cellular differentiation (Sup-
plementary Fig. S5A–S5C). Treatment of IDH2
R140Q
-mutant
TF-1 cells with AG-221 did not induce apoptosis, as shown
Table 1. In vitro potency of AG-221 against IDH2-mutant, IDH2
WT
, and IDH1
WT
enzymes
Enzyme
IC
50
(µmol/L)
a
mean ± SD
(max % inh) forward (oxidative)
IC
50
(µmol/L)
a
mean ± SD
(max % inh) reverse (reductive)
IDH2
IDH2
R140Q
homodimer + NADPH @ 1 h 0.32 ± 0.05 (99 ± 2)
IDH2
R140Q
homodimer + NADPH @ 16 h 0.10 ± 0.03 (110 ± 4)
IDH2
R172K
homodimer + NADPH @ 1 h 0.20 ± 0.07 (86 ± 5)
IDH2
R172K
homodimer + NADPH @ 16 h 0.40 ± 0.14 (89 ± 5)
IDH2
WT
homodimer + NADP
+
@ 1 h 39.83 ± 9.08
b
IDH2
WT
homodimer + NADP
+
@ 16 h 1.80 ± 0.32 (88 ± 2)
IDH2
WT/R140Q
heterodimer + NADP
+
/NADPH @ 1 h 0.38 ± 0.19 (73 ± 2) 0.31 ± 0.17 (96 ± 4)
IDH2
WT/R140Q
heterodimer + NADP
+
/NADPH @ 16 h 0.04 ± 0.02 (75 ± 2) 0.03 ± 0.02 (89 ± 8)
IDH2
WT/R172K
heterodimer + NADP
+
/NADPH @ 1 h 0.18 ± 0.09 (71 ± 0) 0.11 ± 0.01 (107 ± 4)
IDH2
WT/R172K
heterodimer + NADP
+
/NADPH @ 16 h 0.03 ± 0.02 (64 ± 3) 0.01 ± 0.01 (100 ± 2)
IDH1
IDH1
WT
homodimer + NADP
+
@ 1 h 1.12 ± 0.68 (77 ± 2)
IDH1
WT
homodimer + NADP
+
@ 16 h 0.45 ± 0.31 (75 ± 1)
IDH1
R132H
homodimer + NADPH @ 1 h 77.64 ± 11.99
b
IDH1
R132H
homodimer + NADPH @ 16 h 48.40 ± 10.20
b
NOTE: For activity against enzyme, the enzyme, cofactor, and compound were preincubated for 1 or 16 hours as described in Methods. For all enzyme
assessments, n ≥ 3. The forward (oxidative) reaction refers to conversion of isocitrate and NADP
+
to αKG and NADPH, and the reverse (reductive)
reaction to conversion of αKG and NADPH to 2HG and NADP
+
.
Abbreviations: h, hour; inh, inhibitory; max, maximum.
a
Consistent with the mechanism of action, IC
50
measurements were carried out in the presence of NADP
+
cofactor for the IDH1
WT
and IDH2
WT
homodimers; NADPH for the IDH2
R140Q
, IDH2
R172K
, and IDH1
R132H
homodimers; and a mixture of NADP
+
/NADPH cofactors for the
IDH2
WT/R140Q
and IDH2
WT/R172K
heterodimers.
b
Fit to 100% (assay does not reach 100% inhibition at 100 µmol/L maximum compound concentration).
Table 2. In vitro potency of AG-221 for 2HG suppression
Cell line Cell origin Mutation n
IC
50
(µmol/L)
Mean ± SD (max % inh)
HCT-116 KI
a
Human colorectal carcinoma IDH2
R172K
9
0.53 ± 0.26 (84)
TF-1 pLVX
b
Human erythroleukemia IDH2
R140Q
3
0.02 ± 0.01 (85)
TF-1 pLVX
b
Human erythroleukemia IDH2
R172K
3
0.98 ± 0.18 (80)
U87MG pLVX
b
Human glioblastoma IDH2
R172K
5
1.59 ± 0.42 (58)
U87MG pLVX
b
Human glioblastoma IDH2
R140Q
9
0.01 ± 0.00 (96)
NOTE: Potency of AG-221 for 2HG suppression in cell lines with endogenous or ectopically expressed
IDH2
R140Q
or IDH2
R172K
mutations was assessed based on 2HG levels in culture medium. Values are
normalized to IDH2-mutant samples treated with DMSO (control).
Abbreviations: inh, inhibitory; max, maximum.
a
Ectopic expression (knock-in mutation).
b
Overexpression.
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