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.