Targeting PI3K Pathway Dependent Endometrial Tumors with Allosteric AKT Inhibitors, ARQ 092 and ARQ 751Sudharshan Eathiraj*1, Brian Schwartz1, Yi Yu1, Michael J Wick2, Terence Hall1, Feng Chai1, Anthony Tolcher2, Jasgit Sachdev3, Wael Harb4, Brian Slomovitz5 and Giovanni Abbadessa1
1ArQule, Inc. Burlington, MA; 2START, LLC, San Antonio, TX; 3Scottsdale Healthcare Research Institute, Scottsdale, AZ; 4Horizon Oncology Research, Inc., Lafayette, IN; 5University of Miami Hospital and Clinics, Miami, FL
# 352
AACR-NCI-EORTC
November 2015, Boston, MA
BACKGROUND RESULTS
CONCLUSIONS
MATERIALS AND METHODSIn vitro studies
a) Biochemical profiling and kinase selectivity
Biochemical IC50 determination for ARQ 092, ARQ 751, MK-2206, and GDC-0068 against full-length
AKT1, AKT2, and AKT3 was performed at Reaction Biology (Malvern, PA). Kinase profiling of ARQ 092
and ARQ 751 was performed at 5 µM by Carna Biosciences, Inc. (Kobe, Japan).
b) OncoPanel Analysis
Anti-proliferative activity of ARQ 092 and ARQ 751 was assessed using OncoPanel 240 (Eurofins, St.
Charles, MO).
In Vivo Studies
a) In vivo acute PK/PD and xenograft studies
For acute pharmacodynamic studies, AN3CA cells were inoculated subcutaneously into female NCr nu/nu
mice three weeks prior to ARQ 092 administration. ARQ 092 was orally dosed at either 100 mg/kg or 200
mg/kg for AN3CA bearing mice (n=8) (n=4 for vehicle; n=5 for ARQ 092). Tumor tissues were collected
after 1 or 8 hours for AN3CA. For efficacy studies, AN3CA cells were inoculated into female NCr nu/nu
mice. When tumors reached a size of 75-400 mm3, mice were randomly grouped into 5 groups. Vehicle
(10% DMA in water), 50 mg/kg, 100 mg/kg, or 150mg/kg of ARQ 092 was dosed orally daily while 200
mg/kg was dosed every other day. In ARQ 751 xenograft studies, ARQ 751 was orally dosed daily at 5, 10,
20, 40, 80, or 120 mg/kg. For IHC studies, all tissue specimens were fixed for 16-24 hours in 10% neutral
buffered formalin and embedded in paraffin. IHC was performed on the tumor tissue sections with anti-
human antibodies p-AKT(S473) and p-PRAS40(T246). Rabbit on Rodent HRP-Polymer was used as a
secondary antibody. The reagent 3,3’-diaminobenzidine was used as the chromogen and the slides were
counter-stained with hematoxylin after IHC.
b) Endometrial PDX model analysis
A panel of 23 Patient-derived xenograft (START-PDX) endometrial tumor models were screened for ARQ
092 efficacy. The tumors were established from viable human tumor tissue and have been serially passaged
in immunocompromised mice. When tumors reached the appropriate Tumor Volume Initiation (TVI) range
(150-300 mm3), animals were randomized into treatment and control groups (n = 2) and ARQ 092 was
dosed once daily orally at 100 mg/kg for 14 days.
Clinical Phase I study
Endometrial cancer patients are being enrolled in the expanded cohort of ARQ 092-101 at 2 schedules:
intermittent (200mg once a day, 1 week on, 1 week off) or weekly (300mg twice daily, 1 day on, 6 days
off). ClinicalTrials.gov identifier: NCT01473095
* Presenting author
ARQ 092 and the second generation AKT inhibitor, ARQ 751, show potent AKT and AKT-dependent PI3K pathway inhibition and demonstrate
excellent anti-tumor activity in endometrial mouse xenograft and PDX models.
The preclinical observations were recapitulated in the clinic by the rapid objective response observed in the first endometrial patient with PIK3CA
mutation enrolled at full dose in the expanded cohort of the ongoing ARQ 092-101 Phase I clinical trial. Overall, these data provide a strong
mechanistic rationale for further evaluation of ARQ 092, and possibly ARQ 751, in patients with PI3K/AKT driven endometrial malignancies.
PI3K/AKT Activation in Endometrial Cancer and incidence
of genetic alterations in endometrial carcinomas
Kinase selectivity of ARQ 092 and ARQ 751
ARQ 092 and ARQ 751 inhibit endometrial cell line growth and
anti-proliferative activity is associated with PI3K pathway alterations
ARQ 092 shows good oral bioavailability
in mouse tumor model
ARQ 092 and ARQ 751 inhibit AKT pathway in vivo ARQ 092 and ARQ 751 show excellent anti-tumor activity
in endometrial mouse xenograft model
PP
PP
p110p85
PI3K Kinase
PI2P
P P PP P
P
P
P
P
PI3P
PP P
ActiveAKT
InactiveAKT
PDK1
mTORC2
PI3P
Growth factorreceptors
Downstream Signaling
PP P
PI3P
KinaseActivation
Phosphorylation
T308
Translocationto plasma membrane
Trafficking to cytoplasm
S473
c-MetPDGRFEGFR
IGF-1RVEGFR
Plasma membrane
ARQ 092ARQ 092
PP2A
PHLPPs
Dephosphorylation
ARQ 092
Hydrophobic residues
H-bonds
ATP
‘DFG-out’PHE
H-bonding and hydrophobic interactionsATP-incompatibility
Crystal structure of AKT1/ARQ 092 complex
AKT Kinase Signaling
Trafficking to cytoplasm
Ref: Slomovitz and Coleman (2012) Clin. Cancer Res. 18:1-9
-20%
0%
20%
40%
60%
80%
100%
% T
rea
ted
/Co
ntr
ol
ARQ 092 was tested for efficacy in a panel of 23 genetically characterized
endometrial PDX models (ARQ 092, 100 mg/k, p.o. /qdx14). For TGI
analysis percent tumor growth inhibition (%TGI) values were calculated
and reported for each treatment group (T) versus control (C) using initial
(i) and final (f) tumor measurements by the formula %T⁄C = [(Tf -Ti) / (Cf-
Ci)] × 100, if the (Tf - Ti) > 0; and %T⁄C = [(Tf - Ti) / Tsd] × 100, if the (Tf
- Ti) < 0; where, Tsd is median tumor weight of the treated group on the
staging day.
ARQ 092 ARQ 751
Kinase IC50 (nM) Kinase % inh. at 5 mM
MARK4 129 Blk(h) 40
MARK3 173 Tie2 33
MARK1 180 Haspin 30
DYRK2 386 Met 28
IRAK1 806 SGK3(119-end) 28
Haspin 1160 PASK 26
Other 238 kinases <25
ARQ 092 and ARQ 751 were tested in a biochemical assay utilizing activated form of
full length AKT isoforms. In vitro inhibition of pS273-AKT1 and pPRAS40, and anti-
proliferative activity was determined in AN3CA cell line. Kinase profiling was
performed for ARQ 092 against 303 kinases and for ARQ 751 against 245 kinases. IC50
was determined for off-targets that displayed highest percent inhibition for ARQ 092.
Anti-proliferative activity of ARQ 092 and ARQ 751 was assessed in the endometrial
panel of OncoPanel 240. Mutational analysis indicates that both ARQ 092 and ARQ
751 are highly active in the PI3K pathway altered endometrial cell lines. Mutational
analysis was performed based on the data from CCLE database (Barretina, Caponigro,
Stransky et al. The Cancer Cell Line Encyclopedia enables predictive modelling of
anticancer drug sensitivity. Nature. 2012 Mar 28;483(7391):603-7)
ST
189
ST
161
9
ST
152
9
ST
259
ST
993
*
ST
040
ST
138
5
ST
328
ST
106
1
ST
413
ST
357
ST
657
ST
819
ST
100
6
ST
633
ST
609
ST
115
2
ST
013
ST
477
ST
109
4
ST
176
4
PIK3CA
AKT1
AKT2
PDX Model
Gene
10 40 4010 (mg/kg, p.o)Co
ntr
ol
Ve
hic
le
ARQ 751ARQ 092
pT308- AKT
pS473- AKT
AKT
pT246-PRAS40
PRAS40
AN3CA cells were inoculated subcutaneously into female
NCr nu/nu mice. For acute pharmacodynamics studies, ARQ
092 or ARQ 751 were orally dosed at either 10 mg/kg or 40
mg/kg. ARQ 092 was also tested at 100 mg/kg and 200
mg/kg to evaluate the tumor growth inhibition at higher
doses. Tumor samples were assessed by western blot (A) and
quantitated the phosphorylation levels (B). IHC analysis was
performed for AKT and PRAS40 activation (C).
A
B
C
AN3CA cells were inoculated into female NCr nu/nu mice. When tumors
reached a size of 250 mm3, mice were randomly grouped into 5 groups.
Vehicle (10% DMA in water), 50 mg/kg, 100 mg/kg, or 150mg/kg of ARQ
092 was dosed orally daily while 200 mg/kg was dosed every other day. In
ARQ 751 xenograft studies, ARQ 751 was orally dosed daily at 5, 10, 20,
40, 80, or 120 mg/kg.
ARQ 092 inhibits PI3K/AKT dependent kinase activation and signaling
ARQ 092 and ARQ 751 potently inhibits AKT-dependent downstream
kinase signaling and endometrial cell line growth
CompoundBiochemical IC50 (nM)
In vitro PD in
AN3CA EC50 (nM) AN3CA
GI50 (nM)AKT1 AKT2 AKT3 pAKT1 pPRAS40
ARQ 092 5 4.5 16 47 205 307
ARQ 751 0.55 0.81 1.3 3 70 11
Of the 19 enrolled patients with endometrial cancer, only one with a PIK3CA mutation (H1047R) was treated at full dose (200 QD
QOW) and obtained a durable partial response after 2 months on therapy. The subject was a 69-year-old Caucasian female with
papillary serous endometrial cancer (ER+, PR-, Her2-) diagnosed in 2012, received adjuvant and 1st line chemotherapies. She has
been receiving the treatment with ARQ 092 for 36 weeks and ongoing.
AN3CA xenograft mice were dosed iv at 5 mg/kg or PO
with 100 mg/kg with ARQ 092. Plasma concentration vs
time profiles and PK parameters were determined following
5 mg/kg single IV and 100 mg/mg single PO ARQ 092
administration to mice.
CT scan results of subject 072-0085 with partial response with a genetic mutation of PIK3CA-H1047R
Ma. X., Ma. CX., Wang. J., (2014) Endometrial carcinogenesis and molecular signaling pathways. American J. Mol. Biol. 4, 134-149
Yu Y, Savage RE, Eathiraj S, Meade J, Wick MJ, Hall T, Abbadessa G, Schwartz B. (2015) Targeting AKT1-E17K and the PI3K/AKT pathway with an allosteric AKT
inhibitor, ARQ 092. PLoS One. 10:e0140479
Slomovitz BM and Coleman RL. (2012) The PI3K/AKT/mTOR pathway as a therapeutic target in endometrial cancer. Clin. Cancer Res. 18:5856-64
Clinical Phase I StudyIn vivoIn vitro Tumor assignment of endometrial patients treated with ARQ 092
REFERENCES
Gene PTEN PIK3CA PIK3R1 AKT1 HER2 EGFR FGFR2 KRAS
Alterations Mut Loss Mut Amp Mut Mut Amp Amp Mut Mut
Percentage 70 75 46 2-14 21-43 2 3 38-46 12-16 13-26
Mut: Mutation; Amp: Amplification;
ARQ 092 is efficacious in endometrial PDX models
0.01
0.1
1
10
0 2 4 6 8 10 12 14 16
Pla
sm
a c
on
c. (
µM
)
Time (hr)
Plasma Level of ARQ 092 vs Time Profile(Female Ncr-M NUDE mice)
IV 5 mg/kg
PO 100 mg/kg
Cmax = 1.75 µM
The Cmax of patients enrolled at the intermittent and weekly schedules were 1.3 and 1.5 µM, respectively, these values are comparable
to plasma Cmax measured in mice bearing the AN3CA xenograft model.
0.001
0.01
0.1
1
10
AN
3C
A
SKU
T1
RL9
52
KLE
HEC
1A
MFE
28
0
MFE
29
6
EN
HEC
1B
GI 5
0(m
M)
ARQ 751
ARQ 092
PIK3CA WT R88Q WT WT G1049R H1047YP539R,I20
M
T1025A,T1
031AG1049R
PIK3CB WT WT WT WT WT WT WT L915P WT
PIK3CD WT R229Q WT WT A215V WT WT WT WT
PIK3R1557_561RE
IDK>QWT
R639*,R38
6GWT WT WT WT S279_splice WT
PTEN R130fsT321fs,V31
7fs
M134I,R17
3HWT WT WT
T321fs,R13
0QWT WT
MTORG803*,R12
01QA41T WT WT R2254M WT R1482C Y861H WT
HRAS F82L R73C Q61H WT WT WT WT WT R123C
KRAS WT WT WT WT G12D WT WT WT G12D
Cell line
Gene
0
500
1000
1500
2000
2500
3000
0 2 4 6 8 10
Tu
mo
r vo
lum
e (
mm
3)
Days post treatment
ARQ 092 in AN3CA tumor modelUntreated control
50 mg/kg Q1Dx10
100 mg/kg Q1Dx10
150 mg/kg Q1Dx8
200 mg/kg Q2Dx5
0
500
1000
1500
2000
2500
0 1 4 6 8 10
Tu
mo
r vo
lum
e (
mm
3)
Days post treatment
ARQ 751 in AN3CA tumor model
Untreated control
5 mg/kg
10 mg/kg
20 mg/kg
40 mg/kg
80 mg/kg
120 mg/kg
AKT1-E17K
PIK3CA-H1047R
Not PI3K pathway
activating mutation
Pending
-100.0
-80.0
-60.0
-40.0
-20.0
0.0
20.0
40.0
Tum
or
size
ch
ange
fro
m b
ase
ling
(%)
Patient # 00
89
00
93
00
41
00
22
00
99
01
03
00
29
00
35
00
07
00
27
00
18
00
39
01
05
00
85
Dose Level (mg) 200 200 160 60 200 200 80 120 10 60 80 160 300 200
BIDQD
QOW
QD
QOW
QD
QOWQD
QD
QOW
QD
QOW
QD
QOW
QD
QOWQD QD QD
QD
QOWQW
QD
QOW
Weeks on treatment 5 3 7 8 12 7 15 7 24 9 24 21 3 36
*Sequence information is not available.
