Characterization of the Activity of the PI3K/mTOR...

MS# MCT-13-0709 Pharmacodynamics and anti-tumor efficacy of XL765 (SAR245409) 1

Characterization of the Activity of the PI3K/mTOR Inhibitor XL765 (SAR245709) in Tumor Models with Diverse Genetic Alterations Impacting the PI3K Pathway Peiwen Yu, A. Douglas Laird, Xiangnan Du, Jianming Wu, Kwang-Ai Won, Kyoko Yamaguchi, Pin Pin Hsu, Fawn Qian, Christopher T. Jaeger, Wentao Zhang, Chris A. Buhr, Paula Shen, Wendy Abulafia, Jason Chen, Jenny Young, Arthur Plonowski, F. Michael Yakes, Felix Chu, Michelle Lee, Frauke Bentzien, Sanh Tan Lam, Stephanie Dale, David J. Matthews, Peter Lamb, Paul Foster Exelixis, Inc., South San Francisco, CA 94080 Running Title: Pharmacodynamics and anti-tumor efficacy of XL765 (SAR245409) Key words: XL765, SAR245409, PI3K, mTOR, inhibitor Corresponding author: A. Douglas Laird, Ph.D. Executive Director, Translational Medicine Exelixis 210 East Grand Ave South San Francisco, CA 94080 Ph: 650 837 7485 Fax: 650 837 7410 [email protected] Financial support/Conflict of interest statement: The authors are all current or former employees of Exelixis, Inc. Word count (excluding references): 8926 Total number of figures: 4 (+ 5 supplemental, including 2 added to address reviewer comments) Total number of tables: 3 (+ 3 supplemental, including one added to address reviewer comments)

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Abstract Activation of the PI3K pathway is a frequent occurrence in human tumors and is thought

to promote, growth, survival and resistance to diverse therapies. Here we report

pharmacological characterization of the pyridopyrimidinone derivative, XL765

(SAR245409), a potent and highly selective pan inhibitor of Class I PI3Ks (α, β, γ, and δ)

with activity against mTOR. Broad kinase selectivity profiling of >130 protein kinases

revealed that XL765 is highly selective for Class I PI3Ks and mTOR over other kinases.

In cellular assays, XL765 inhibits the formation of PIP3 in the membrane, and inhibits

phosphorylation of AKT, p70S6K and S6 phosphorylation in multiple tumor cell lines

with different genetic alterations impacting the PI3K pathway. In a panel of tumor cell

lines, XL765 inhibits proliferation with a wide range of potencies, with evidence of an

impact of genotype on sensitivity. In mouse xenograft models, oral administration of

XL765 results in dose-dependent inhibition of phosphorylation of AKT, p70S6K, and S6

with a duration of action of approximately 24 h. Repeat dose administration of XL765

results in significant tumor growth inhibition in multiple human xenograft models in nude

mice that is associated with anti-proliferative, anti-angiogenic, and pro-apoptotic effects.





Introduction Class I PI3 kinases convert phosphatidylinositol 4,5-bisphosphate (PIP2) to

phosphatidylinositol 3,4,5-trisphosphate (PIP3) in response to external cell stimuli (1, 2).

Activation of Class IA PI3Ks (PI3Kα, -β, and -δ) is mediated by RTKs. G-protein–

coupled hormone receptors are implicated in activation of PI3Kβ and Class IB PI3K

(PI3Kγ) (3). Ras, another important mediator of extracellular stimuli, can also promote

PI3K activation, and PI3K can mediate cellular transformation by Ras (4). Downstream

effectors of PI3K signaling, such as phosphoinositide-dependent kinase-1 (PDK1) and

AKT, bind to PIP3 at the cell membrane and are subsequently activated by

phosphorylation (1). In turn, PDK1 and AKT activate growth pathways, inhibit apoptotic

signaling and regulate transition through restriction points in the cell cycle via

phosphorylation of their respective substrates (1).

mTOR is the kinase component of two multi-subunit complexes called mTORC1

(includes mTOR/Raptor) and mTORC2 (includes mTOR/Rictor) (5,6). mTORC1 is

activated via PI3K pathway signaling and also via PI3K-independent mechanisms

involving sensing of cellular amino acid levels, AMP levels, and hypoxia, and drives

cellular growth by regulating protein translation and degradation (5,6). mTORC2 is

activated via growth factor-dependent signaling via mechanism(s)that are still being

elucidated, and regulates cell growth, proliferation, and survival via phosphorylation of

the AKT kinase (5, 6, 7).

Dysregulation of PI3K pathway components, resulting in hyperactivated PI3K and/or

mTORC1 signaling, is observed in various cancers and correlates with tumor growth and

survival (1). For example, the catalytic subunit of PI3Kα (p110α), encoded by the

PIK3CA gene, is mutated in 12% of human cancers (8). This is likely an underestimate

since many of these data are presumably generated by hot-spot sequencing. In addition,

the tumor suppressor PTEN, which serves as a critical negative regulator of PI3K

signaling by converting PIP3 back to PIP2, is frequently deleted or downregulated in

human tumors (1,9). Moreover, the tumor suppressor gene LKB-1 which negatively

regulates mTORC1 is mutated/inactivated in a variety of familial and sporadic tumors





(10). PI3K pathway signaling is implicated in tumor cell invasion, migration and

dissemination (11). Genetic and pharmacologic approaches have demonstrated that PI3K

signaling mediates vascular endothelial growth factor (VEGF) production,

neoangiogenesis, vascular permeability, and vessel integrity in preclinical tumor models

(12,13,14).

Resistance to a variety of anticancer therapies, including receptor tyrosine kinase (RTK)

inhibitors and genotoxic agents, has been attributed to ongoing activation of the PI3K/

PTEN pathway (15, 16, 17). PI3K pathway inhibitors have been shown to sensitize

cancer cells to agents targeting HER2, MET and EGFR as well as platinum drugs and

taxanes (18, 19, 20, 21, 22, 23).

mTOR has been extensively explored as an oncology target (24). The macrolide

antibiotic rapamycin is a potent inhibitor of the mTORC1 complex. Several analogs of

rapamycin have been tested clinically in various oncology indications, and evidence of

therapeutic benefit has been observed (24). For example, the FDA has approved

TORISEL™ (temsirolimus; Pfizer) for patients with advanced renal cell carcinoma

(RCC) and Afinitor™ (everolimus; Novartis) for the treatment of patients with advanced

RCC after failure of treatment with sunitinib or sorafenib, and for advanced ER-positive

breast cancer after failure of treatment with a nonsteroidal aromatase inhibitor. However,

the efficacy of these agents may be limited by the fact that rapamycin analogs do not

inhibit mTORC2 (7). In addition, mTORC1 inhibition may enhance cell survival by

upregulating PI3K/AKT signaling via inhibition of a mTORC1-dependent negative

feedback loop acting through PI3K (24). Thus, selective inhibitors of PI3K and mTOR

signaling have therapeutic potential as single agents and in combination with other

therapies for a variety of cancer indications and several such agents have entered clinical

testing in recent years (1, 24).

XL765 (SAR245409) is a potent and selective inhibitor of Class I PI3Ks. In addition,

XL765 also inhibits mTOR. In cellular assays, treatment with XL765 inhibits

phosphorylation of proteins downstream of PI3K and mTOR, including AKT and

ribosomal protein S6 (S6RP), in multiple tumor cell lines with diverse molecular





alterations impacting the PI3K pathway. In a broad panel of tumor cell lines XL765

inhibits proliferation with a wide range of potencies, which appeared to be influenced by

genetic background. Oral administration of XL765 in human xenograft tumor models in

athymic nude mice results in dose-dependent inhibition of PI3K pathway components

with a duration of action of at least 24 hours. As a single agent, XL765 shows significant

tumor growth inhibition in multiple human xenograft models at well-tolerated doses.

Taken together, these data support the ongoing clinical investigation of XL765 for the

treatment of cancer.





Materials and Methods

In vitro kinase inhibition assays

Kinase activity for PI3K isoforms was measured as the percent of ATP consumed

following the kinase reaction using luciferase-luciferin-coupled chemiluminescence as

previously described (25), with ATP concentrations approximately equal to the Km for

each respective kinase. Kinase reactions were initiated by combining test compounds,

ATP and kinase in a 20 µL volume. PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ (Upstate

Biotechnology) final enzyme concentrations were 0.5 nM, 8 nM, 20 nM, and 2 nM,

respectively. A similar assay format was utilized for DNA-PK (purchased from Promega)

and VPS34 (PIK3C3; prepared at Exelixis as an N-terminal tagged full-length human

fusion protein which was expressed in insect cells (BEVS) and affinity purified using

glutathione sepharose). VPS34 assay buffer contained 20 mM TrisHCl, pH 7.5, 3.5mM

MnCl2, 100 mM NaCl, 1 mM DTT and 0.01% cholamidopropyldimethylammonio

propanesulfonate (CHAPS). 0.5 µL DMSO containing varying concentrations of the test

compound was mixed with 10 µL enzyme solution (2X concentration). Kinase reactions

were initiated by the addition of 10 µL of liver phosphatidylinositol (PI) and ATP

solution (2X concentration). Assay concentrations for VPS34, ATP and PI were 40nM,

1µM, and 5 µM, respectively.

Human tumor cell lines

Cell lines were obtained from American Type Culture Collection in 2001- 2005 and

maintained in culture conditions at 37°C under 5% CO2. PC 3, MCF7, and A549 cells

were maintained in DMEM (Cellgro 10-013-CV) containing 10% FBS (Heat Inactivated,

Cellgro, 35-016-CV), 1% non-essential amino acids (NEAA) (Cellgro, 30-002-CI), and

1% penicillin-streptomycin (Cellgro). U87-MG and MDA-MB-468 cells were maintained

in EMEM-Alpha (Cellgro, 10-022-CV), DMEM/F-12 (Cellgro, 15-090-CV),

respectively, supplemented with 10% FBS, 2 mM L-glutamine, 1% NEAA, and 1%

penicillin-streptomycin. LS174T cells were maintained in MEM (GIBCO, 10370-021)

containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin

streptomycin. Ramos cells were maintained in RPMI 1640 (Cellgro, 10-040-CV)





containing 10% FBS (Cellgro) and 1% penicillin-streptomycin (Cellgro). OVCAR 3 cells

were maintained in RPMI 1640 containing 20% FBS and 1% penicillin streptomycin.

Immune-Complex mTOR Kinase-Western Immunoblot Analysis

mTORC1: HEK 293 (ATCC) cells were grown in DMEM (Cellgro) containing 10% FBS

(Cellgro), 1% NEAA (Cellgro), and 1% penicillin-streptomycin (Cellgro), and lysed in

ice-cold lysis buffer containing 40 mM HEPES pH 7.5, 120 mM NaCl, 1 mM EDTA,

10 mM Na pyrophosphate, 10 mM β-glycerophosphate, 50 mM NaF, one tablet of

protease inhibitors (Complete-Mini, EDTA-free, Roche), 0.3% CHAPS, and 1.5 mM

Na3VO4. mTORC1 was incubated with anti-mTOR antibody (N-19, Santa Cruz, sc-

1549) 1.5 h to overnight. The resulting immune-complexes were immobilized on IgG

sepharose (GE Healthcare, 17-0618-01), washed sequentially three times with lysis

buffer, once with wash buffer (50 mM HEPES, pH 7.5, 40 mM NaCl, 2 mM EDTA), and

once with kinase buffer (25 mM HEPES, pH 7.5, 50 mM KCl, 20% glycerol, 10

mM MgCl2, 4 mM MnCl2, 1 mM DTT). The immune-complexes (equivalent to 1x106

cells) were pre-incubated at 30°C with XL765 or 0.1% DMSO for 10 minutes, and then

subjected to kinase reaction for 30 min in a final volume of 20 µl (including 10 µl bed

volume) containing kinase buffer, 25 µM ATP, and l µg 4EBP1 (Exelixis). Kinase

reactions were terminated by addition of 3.3 µl 4x sample buffer (Invitrogen, NP0007)

containing 7% β-mercaptoethanol and analyzed by western immunoblotting.

Nitrocellulose membranes were incubated overnight at 4°C with 1/1000 dilution of rabbit

anti-mTOR (Upstate, 07-231) in 5% non-fat milk containing TBST (TBS/0.1% Tween-

20) or with 1/500 dilution of rabbit anti-p4EBP1 (Cell Signaling Technology [CST],

#9459) in 3% BSA/TBST, followed by incubation for 1 h with a 1/5000 dilution of

secondary immunopure peroxidase conjugated goat anti-rabbit IgG (H+L) (Pierce,

31462) in 5% non-fat milk/TBST. Phospho-4EBP1 and mTOR were detected with Super

Signal West Pico Substrate (Pierce, 34080). The p4EBP1 blot was subsequently stripped

and re-probed with anti-4EBP1 antibody (1/1000, CST#9452) with the total 4EBP1

signal detected as described above. Scans were analyzed using ImageQuant software. The

DMSO control sample was used for normalization, and the IC50 value for XL765 was

determined using XLfit4 software.





mTORC2: HeLa (ATCC) cells were grown in suspension culture in EX-CELLTM HeLa

media (Sigma, 14591C) and lysed as described above with minor modifications. The

mTORC2 complex was incubated with anti-RICTOR antibody (Exelixis) for 2 h and

immune complexes (equivalent to 1×107 cells) prepared as above with minor

modifications. These were pre-incubated at 37°C with a test compound or 0.6% DMSO

for 5 min, and then subjected to a kinase reaction for 8 min in a final volume of 33 μL

(including 5 μL bed volume) containing kinase buffer, 50 μM ATP, and 0.75 μg AKT1

[full-length human AKT1 with amino-terminal Hi tag was expressed in Sf9 cells using

standard procedures, purified, then dephosphorylated with lambda protein phosphatase

(New England Biolabs, P0753L) prior to repurification and use as substrate]. Kinase

reactions were subsequently terminated and resolved as described above with minor

modifications, then transferred onto PVDF membranes at 50 V for 20 h at 4°C. The

membranes were blocked in 5% non-fat milk in TBST for 1 h and incubated overnight at

4°C with 1/1000 dilution of rabbit anti-pAKT (S473) (CST# 4060) in 3% BSA/TBST.

The membranes were washed 3 times in TBST and incubated for 1 h with a 1/10000

dilution of secondary goat anti-rabbit HRP antibody (CST# 2125) in 5% non-fat

milk/TBST. The IC50 value for XL765 was determined as described above.

PIP3 Mass Balance Assay

PC-3 (ATCC) and MCF7 (ATCC) cells were seeded at 2×106 and 2.5×106 cells,

respectively, onto 10-cm dishes in culture medium and incubated at 37°C, 5% CO2 for

24 h. Growth medium was replaced with serum-free DMEM and cells were incubated for

an additional 3 h. Serial dilutions of test compounds in fresh serum-free medium were

added to the cells in a final concentration of 0.3% DMSO (vehicle) and incubated for

23 min prior to recombinant human EGF stimulation (200 ng/ml, R&D Systems,

236-EG) for 2 min. After treatment, the medium was removed, and cellular material was

precipitated with ice cold 10% TCA and collected by centrifugation. The pellet was

washed with 3 ml of 5% TCA/1 mM EDTA. Neutral lipids were extracted from the pellet

with 3 ml of methanol:chloroform (2:1), and then the acidic lipids were extracted with

2.25 ml of methanol:chloroform:12 N HCl (80:40:1). The organic phase was separated

from the aqueous phase by the addition of 0.75 ml of chloroform and 1.35 ml of

0.1 N HCl followed by centrifugation. The organic phase was then collected into a glass





tube, dried under nitrogen gas, and resuspended by sonication in a water bath in 120 µl of

the PIP3 mass assay buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, and

1.5% sodium cholate).

Assays were conducted in 96-well plates (Perkin-Elmer, L2251692) by incubating 50 µl

of the lipid extract with 50 µl of sensor complex in detection buffer (10 mM Tris-HCl,

pH 7.2, 150 mM NaCl, 7.5 mM EDTA, 0.1% Tween-20, and 1 mM DTT) at ambient

temperature in the dark for 2 h, and plates were read using an AlphaQuest reader (Perkin-

Elmer). The sensor complex contained 15 µl of 100 nM biotinylated PIP3 (Echelon,

C-39B6), 15 µl of 100 nM GST-tagged GRP1-pleckstrin homology (PH) (Echelon,

G-1200), and 20 µl of a mixture of donor and acceptor AlphaScreen beads (GST

detection kit, Perkin-Elmer, 6760603c). The PIP3 mass present was estimated by

comparison to standard curves constructed by addition of known amounts of

diC8 PI(3,4,5)P3 standard (Echelon, P-3908) to the sensor complex.

pAKT and pS6 ELISA

pS6 ELISA was performed as previously described (26) with minor modifications. The

pAKT ELISA assay was performed as follows: PC-3 (ATCC) cells were seeded at

1.5×105 cells per well in 6-well plates (NUNC, 140685) in growth medium then

incubated at 37°C, 5% CO2 for 72 h, and the growth medium was replaced with serum-

free DMEM. Serial dilutions of the test compound in 0.3% DMSO (vehicle) were added

to the cells and incubated for 2 h and 50 min. Cells were then stimulated with 100 ng/ml

EGF (R&D Systems, 236-EG) for 10 min. Cells were washed once with ice-cold PBS,

harvested by briefly shaking in 100 µl of TENN lysis buffer (20 mM Tris-HCl, pH 7.5, 1

mM EDTA pH 8.0, 0.5% NP40, 150 mM NaCl) with protease and phosphatase inhibitors

(1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 mM EDTA, 1

mM NaF, 20 mM β-glycerophosphate, 1 mM Na-orthovanadate, and 5 mM p-nitrophenyl

phosphate), and transferred to 96-well plates. Cells were lysed on ice for 20 min by

pipetting up and down 10 times, and the supernatants were collected by centrifugation.

ELISA assays for pAKTT308 and total AKT were performed with the AKT[pT308]

ELISA kit (Biosource, KHO0201) and AKT ELISA kit (Biosource, KHO0101). IC50





values were determined based on the ratio of pAKT to total AKT signal in lysates from

compound-treated cells, normalized to lysates from DMSO-treated controls.

PI3K Pathway Profiling Western Immunoblot Analysis

PC 3, MCF7, A549 ,U87-MG, LS174T, MDA-MB-468, and OVCAR 3 cells were seeded

at densities of 2×106, 2×106, 1.2 ×106, 2×106, 2×106, 1.2 ×106,and 1×106 cells,

respectively, onto 10 cm dishes in in their respective culture medium and incubated at

37°C, 5% CO2 for 20-47 h. . The medium was replaced with test compounds dissolved in

the same media containing 0.3% DMSO, and the cells were incubated for 3 h. For growth

factor treatment, the medium was replaced with test compounds dissolved in serum-free

DMEM containing 0.3% DMSO. After incubation for 3 h, cells were stimulated with

100 ng/ml of EGF (R&D Systems, 236-EG) for 10 min. Cells were washed with ice-cold

PBS, and directly lysed with cell lysis buffer (Biosource International, FNN0011)

containing protease inhibitors (Complete-Mini, EDTA-free, Roche, 11836170001;

Aminoethylbenzenesulfonyl fluoride, Sigma, A8456). Protein lysates were analyzed by

western immunoblotting. PVDF membranes (Invitrogen) were incubated overnight at

4°C with primary antibodies at the indicated concentrations in 3% BSA/TBST buffer,

followed by incubation for 1 h with a 1/10000 dilution of secondary goat anti-rabbit HRP

antibody (CST#7074) or a 1/3000 dilution of secondary goat anti-mouse HRP antibody

(Amersham, NXA931) in 5% non-fat milk/TBST. Signals were detected using ECL-plus

(Amersham, RPN2132) and scanned using a Typhoon 9400 scanner (Molecular Devices).

For total protein readouts, stripped membranes were incubated with the respective

primary antibodies, with signal detection as described above. Scans were analyzed using

ImageQuant software. Phospho signals were normalized to the corresponding total

protein signals, % inhibition relative to DMSO control was determined, and IC50 values

were calculated using XLfit4 software. The following antibodies were used in Western

Immunoblot analysis: pAKT (T308) (CST#4056, 1/500 dilution), pAKT (S473)

(Biosource, 44-622G, 1/1000), AKT (Biosource, 44-607G, 1/1000 dilution), pp70S6K

(T389) (CST #9234, 1/1000 dilution), p70S6K (Bethyl, A300-510A, 1/2500 dilution),

pS6 (S240/244) (CST #2215, 1/2000 dilution), S6 (CST #2217, 1/2000 dilution),

pPRAS40 (T246) (Biosource, 44-1100G, 1/2000 dilution), PRAS40 (Biosource





AHO1031, 1/1000 dilution), pGSK3β (S9) (CST #9336, 1/1000 dilution), GSK3β (CST

#9315, 1/1000 dilution), p4EBP1 (T37/46) (CST #9459, 1/1000 dilution), 4EBP1 (CST

#9452, 1/1000 dilution), Cyclin D1 (EMD Biosciences, CC12, 1/1000 dilution), pERK

(Y204) (Santa Cruz, sc-7383, 1/1000 dilution) and ERK (CST #9102, 1/1000 dilution).

mTOR Pathway Assay in Ramos Cells

Ramos (ATCC) cells were seeded at a density of 0.3×106 cells/mL in growth medium. .

The next day, cells were centrifuged, washed with serum-free medium supplemented

with 1% bovine serum albumin (BSA, tissue culture tested, Sigma, A4919), resuspended

in RPMI supplemented with 1% BSA, and incubated for 20 h. The serum-starved cells

were centrifuged and resuspended in 5 mL of the saved media at 1×106 cells/mL. Cells

were treated with test compound administered at a final DMSO concentration of 0.3%.

For the nutrient-depletion control, the serum-starved cells were washed once with PBS,

and resuspended in 5 mL of PBS with 0.3% DMSO. After incubation for 2 h, cells were

washed once in PBS and lysed in 150 µL of Biosource cell lysis buffer containing

protease inhibitors (Complete-Mini, EDTA-free, Roche; AEBSF, Sigma). Lysates were

analyzed by western immunoblotting. PVDF membranes were incubated overnight at 4°C

with 1/500 dilution of rabbit anti-pmTOR (CST#2974), with 1/1000 dilution of rabbit

anti-pp70S6K (CST#9234), or with 1/1000 dilution of rabbit anti-p4E-BP1 (CST#9459)

in 3% BSA/TBST, followed by incubation for 1 h with a 1/10000 dilution of secondary

goat anti-rabbit HRP antibody (CST#7074) in 5% non-fat milk/TBST and signals

detected and analyzed as described above. For detection of total p70S6K and total

4E-BP1, stripped membranes were probed with rabbit anti-total p70S6K (1/2500, Bethyl,

A300-510A) and with rabbit anti-total p4E-BP1 (1/1000, CST#9452) with signals

detected and analyzed as described above.

Cell Proliferation and Cytotoxicity Assays

Cellular proliferation was assessed as previously described (27) using the Cell

Proliferation ELISA, Bromo Deoxyuridine (BrdU) chemiluminescence kit (Roche,

Applied Science). Cytotoxicity was assessed using the ATP Bioluminescence Assay as





follows: PC-3, MCF7, A549 , LS174T, MDA-MB-468, U87-MG, and OVCAR-3 cells

were plated at densities of 7×103, 1.5×104, 6×103, 7×103,7×103, 6×103, 1.5×104 cells/well,

respectively, onto 96-well microtiter plates (Corning, 3904) in culture medium, incubated

at 37°C, 5% CO2 for 18 h, and then treated with a serial dilution of compound in medium

containing a final concentration of 0.3% DMSO. Triplicate wells were used for each

compound concentration. Control wells received 0.3% DMSO in media. Cultures were

incubated at 37°C, 5% CO2 for an additional 24 h and cells were then assayed for

viability using the ViaLight™ HS kit (Cambrex, LT07-111).

Apoptosis Assay (Caspases 3/7 Assay)

PC 3, MCF7, A549, LS174T, MDA MB 468, U87 MG and OVCAR-3 cells were plated

at densities of 5×103, 1.2×104, 5×103, 6×103, 6×103, 5×103 and 1.2×104 cells/well ,

respectively, onto 96-well microtiter plates (Corning, 3904), in culture medium at 37°C,

5% CO2 for 18 h, and then treated with a serial dilution of compound in medium

containing a final concentration of 0.3% DMSO. Triplicate wells were used for each

compound concentration. Positive control wells received 5-30000 nM adriamycin (MCF7,

A549, and LS174T) or 5-30000 nM camptothecin (PC-3, U87-MG, OVCAR-3, and

MDA-MB-468) and negative control wells received 0.3% DMSO in media. Background

wells contained no cells and 0.3% DMSO in media. Following incubation at 37°C, 5%

CO2 for an additional 48 h, apoptosis was assessed using the Apo-ONETM Homogeneous

Caspase-3/7 Assay kit (Promega, G7791). EC50 values were calculated based on the

fluorescence of compound-treated wells compared to that of the corresponding positive

control.

Anchorage-Independent Growth Assay (Soft Agar Assay)

Soft agar (60 µl/well of 0.75%, BD Biosciences) was plated in a 96-well black plate

(Nalge Nunc International) and allowed to solidify at 37°C for 20 min. 4.8×103 PC-3 or

MCF7 cells in 100 µl of media containing 0.375% agar, FBS (15% for PC-3 and 20% for

MCF7), and 1x concentrations of serial dilutions of XL765 were layered over the base

agar. After 10 min, 60 µl of DMEM (GIBCO) containing FBS and 2-fold concentrated

test compounds were added over the cell layer. Following equilibration of the media and





the test compound, the final compound concentration was presumed to be 1× in all three

layers. The cultures were incubated for 14 days at 37°C, 5% CO2. At day 7, 50 µl of fresh

media containing 10% FBS without compound was added to keep the cultures from

drying out. At the completion of the incubation, the media in the top layer was removed,

and 40 µl of media containing 50% Alamar BlueTM (Biosource, DAL1025) were added to

each well followed by incubation at 37°C, 5% CO2 for 4 h and subsequent fluorescent

detection.

Migration Assay

The HGF-induced chemotaxis assay was performed as previously described (27).

PC-3, MCF7 and B16F10, Cytotoxicity Assays (Alamar Blue Assay)

PC-3 (ATCC), MCF7 (ATCC) and B16F10 (ATCC) cells were mixed with a series of

diluted compounds in serum-free DMEM (Gibco), DMEM containing 0.2% FBS, and

serum-free EBM-2 medium (Clonetics), respectively. Control wells received media with

0.25% DMSO alone. 5×103 cells were plated in each well of a 96-well plate and

incubated at 37°C, 5% CO2 for 18 h (PC-3) or 24 h (B16F10 and HMVEC-L). At the end

of the incubation, cell viability was determined using Alamar Blue solution (Biosource).

Studies in tumor-bearing mice

Tumors were collected at the indicated time-points and tumor lysates were prepared as

previously described (25). Pooled lysates were analyzed by western immunoblotting.

PVDF membranes were incubated overnight at 4°C with primary antibodies to the

respective phosphoepitopes at the indicated concentrations in 3% BSA/TBST buffer. The

membranes were then incubated for 1 h with a 1/10000 dilution of secondary goat

anti-rabbit HRP antibody (CST#7074) in 5% non-fat milk/TBST. Signals were detected

using ECL-plus (Amersham, RPN2132) and scanned using Typhoon (Molecular

Devices). To determine total protein levels, membranes were stripped and incubated with

the indicated primary antibodies specific for the respective total proteins. The same

procedure described above was followed to detect the total protein signal. Scans were

analyzed using Image Quant software. % inhibition was determined by normalizing the





phosphoepitope signals to the total protein signals and then calculating % inhibition

compared to vehicle control groups. The following antibodies were used in western

immunoblot analysis of tumor extracts : pAKT (T308) (CST#4056, 1/500 dilution),

pAKT (S473) (CST #9271, 1/1000), AKT (CST #9272, 1/1500 dilution), pp70S6K

(T389) (CST #9234, 1/500 dilution), p70S6K (Bethyl, A300-510A, 1/2000 dilution), pS6

(S240/244) (CST #2215, 1/2000 dilution), and S6 (CST #2217, 1/1500 dilution).

In vivo efficacy studies were performed in athymic nude mice purchased from Taconic

(Hudson, NY) and housed according to the Exelixis Institutional Animal Care and Use

Committee guidelines. Tumor cells were cultured in vitro in DMEM (Mediatech,

Manassas, VA) supplemented with 10% fetal bovine serum (20% for PC-3 and OVCAR-

3 cells), penicillin-streptomycin and nonessential amino acids at 37°C in a humidified 5%

CO2 atmosphere. On day 0, cells were harvested by brief trypsinization, and 1 to 5×106

cells in 0.1 mL ice-cold Hanks Balanced Salt Solution (HBSS) were implanted

subcutaneously (OVCAR-3) or intradermally (MCF7, U-87 MG) into the hind flank of

female athymic nude mice. In the case of the MCF7 model an estrogen pellet (IRA,

Sarasota, FL) was implanted subcutaneously at the nape of neck at the time of tumor cell

implantation. 3x106 PC-3 cells were similarly harvested and implanted subcutaneously

into the hind-flank of 5-8 week old male nude mice. Tumor growth was monitored

weekly with calipers until staging and dose initiation. During the dosing period, body and

tumor weights were assessed as previously described (27). XL765 was formulated in

sterile water/10 mM HCl or water and administered at the indicated doses and regimens

by oral gavage at a dose volume of 10 mL/kg.

Histology

After euthanasia, tumors from animals administered XL765 and/or other agents were

excised and fixed in zinc fixative (BD Pharmingen) for 24-48 h before being processed

into paraffin blocks. Five µm thin sections were cut serially to represent the largest

possible surface for each tumor and stained using standard immunohistochemical

methods to detect Ki67 nuclear antigen (Lab Vision) and CD31 positive tumor vessels

(BD Pharmingen). CD31 was detected by biotinylated secondary antibody followed by





avidin-biotin-peroxidase complex (BD Pharmingen). Ki67 was detected by Envision+

anti-rabbit peroxidase complexed polymer (DAKO). Sections were counterstained with

hematoxylin. Tumor mean vessel density and Ki67 index in tumor sections were

quantified using the ACIS™ automatic cellular imaging system (Clarient Inc., San Juan

Capistrano, CA). The mean number of tumor vessels per mm2 was determined by

analyzing eight to 15 fields across the total tumor section. Percent Ki67 positive tumor

cells was determined by sampling multiple representative fields of equal size across the

total viable tumor area of each section and dividing the number of Ki67 positive cells by

the total number of cells identified per field. Apoptosis was assessed by TUNEL as

previously described (27).The results for each IHC readout was averaged for each tumor

section, followed by averaging the results for each treatment group (n = 9-10). Statistical

analyses were performed using the standard two-tailed t-test with Bonferroni adjustment

for multiple comparisons against a single control group.





Results

XL765 is a selective inhibitor of Class I PI3Ks and of mTOR in biochemical assays

XL765 (Figure 1A) was identified following optimization of a pyridopyrimidinone

scaffold for in vivo PI3K/mTOR pathway inhibition and drug-like properties. In assays

performed using purified proteins in a luciferase-coupled chemiluminescence format,

XL765 displayed potent inhibitory activity against Class I PI3K isoforms p110α, p110β,

p110δ, and p120γ, with IC50 values of 39, 110, 43, and 9 nM, respectively (Table 1). The

IC50 value for inhibition of PI3Kα by XL765 was determined at various concentrations of

ATP, revealing XL765 to be an ATP-competitive inhibitor with an equilibrium inhibition

constant (KI) value of 13 nM.

XL765 also inhibited mTOR (IC50 values of 160 and 910 nM for mTORC1 and

mTORC2, respectively) in an immune-complex kinase assay and the PI3K-related kinase

DNA PK (IC50 value of 150 nM). In contrast, XL765 had relatively weak inhibitory

activity towards the Class III PI3K vacuolar sorting protein 34 (VPS34; IC50 value of ~

9.1 µM). XL765 was also profiled against a panel of ~130 protein kinases; no cross-

reactivity was observed at concentrations below 1.5 µM (Supplemental Table S1). All

assays were performed at ATP concentrations approximately equal to the Michaelis

constant (KM) values of the respective enzymes.

XL765 inhibits the PI3K pathway in multiple tumor cell models

MCF7 human mammary carcinoma cells and PC-3 human prostate adenocarcinoma cells

were selected for the initial assessment of the effect of XL765 on signaling downstream

of PTEN/PI3K since they each have a prevalent genetic lesion that activates the PI3K

pathway. MCF7 cells carry a heterozygous E545K activating mutation in the p110α

subunit of PI3K and PC-3 cells carry a homozygous deletion of exons 3-9 of the PTEN

tumor suppressor gene. PIP3 is the product of a class I PI3Ks acting on the physiological

substrate PIP2. Hence PIP3 levels serve as a direct assessment of PI3K activity.

Consistent with its inhibitory activity against purified PI3K proteins, XL765 inhibited

EGF induced PIP3 production in PC-3 and MCF7 cells with IC50 values of 290 and 170





nM, respectively (Table 2). The ability of XL765 to inhibit phosphorylation of key

signaling proteins downstream of PI3K was examined by assessing its effects on EGF

stimulated phosphorylation of AKT and on non-stimulated phosphorylation of S6 in PC-3

cells by cell based ELISA. XL765 inhibited these activities with IC50 values of 250 and

120 nM, respectively (Table 2).

The effects of XL765 on the PI3K signaling pathway were then examined by Western

immunoblot analysis in PC-3 and MCF7 cells (see Figure 1B and Supplemental Figure

S1). The results were consistent in both cell lines. XL765 inhibits AKT phosphorylation

at both activation sites (T308 and S473) at concentrations consistent with the IC50 values

determined by ELISA. The T308 phosphorylation site on AKT is a substrate for PDK1

(1), whereas the S473 site is a substrate for mTORC2 (6). Inhibition of AKT substrate

phosphorylation (PRAS40 and GSK3β) and inhibition of phosphorylation events

downstream of mTOR (p70S6K, S6, and 4EBP1 phosphorylation) were also evident.

XL765 induces a decrease in the levels of cyclin D1 protein, consistent with increased

GSK3β activity as a result of inhibition of AKT leading to GSK3β-mediated

phosphorylation and subsequent degradation of Cyclin D1 (Figure 1B). Overall, a similar

range of compound concentrations was required to inhibit PI3K proximal

phosphorylation events (AKT T308 phosphorylation) and phosphorylation events

downstream of mTORC1 and mTORC2 (p70S6K phosphorylation and AKT S473

phosphorylation).

The control compound ZSTK474 (an inhibitor of PI3K; Reference 28) at 10 µM robustly

decreased the levels of all the phospho readouts assessed. The TORC1 inhibitor

rapamycin at 0.1 µM did not inhibit the phosphorylation of AKT or its direct substrates

PRAS40 and GSK3β, but in fact appeared to stimulate phosphorylation of AKT. This is

consistent with relief of p70S6K-dependent negative feedback of PI3K (see

Introduction). As expected, rapamycin significantly decreased p-p70S6K and pS6 levels,

consistent with its well characterized ability to inhibit mTORC1. None of the compounds

had significant effects on ERK1/2 phosphorylation, consistent with biochemical profiling

data. XL765 was further profiled in additional cell lines bearing a variety of genetic

lesions that activate/modulate the PI3K pathway. These were OVCAR-3 (PIK3CA





amplification), U87-MG (PTEN deletion), A549 (KRAS mutation, loss-of-function

mutation in the mTOR-directed tumor suppressor gene LKB-1), MDA-MB-468 (PTEN

deletion) and LS174T (PIK3CA and KRAS mutations) cells. XL765 demonstrated

consistent activity in these cell lines with no marked differences in sensitivity being

evident (Supplemental Figures 2 and 3).

XL765 inhibits PI3K-independent mTOR signaling

In order to directly assess the impact of XL765 on mTOR in cells, we employed an

approach that relies on the ability of nutritional signals to activate mTOR independent of

PI3K activity. Regulation of mTOR signaling by nutrient availability is predominant in

certain transformed B cells (29), and we used the Burkitt lymphoma derived cell line

Ramos as a system to study the effect of XL765 on nutrient dependent mTOR activity

(Figure 1C). Cells were starved in serum free media for 20 h, and then treated with

compounds, DMSO, or serum free and nutrient free PBS for 2 h. Cell lysates were

prepared and analyzed by gel electrophoresis and Western immunoblotting with anti-

pmTOR, anti-p-p70S6K, and anti-p4EBP1 antibodies. Cells incubated in PBS show very

low levels of phosphorylation of p70S6K, 4EBP1 or the mTOR autophosphorylation site

S2481, consistent with low mTOR activity. Incubation of cells in serum free, nutrient

containing media results in a robust upregulation of mTOR-dependent phosphorylation

events. ZSTK474 and PI-103 (30) at 10 µM inhibit these phosphorylation events,

consistent with their ability to directly inhibit mTOR kinase activity in addition to PI3K

activity. Rapamycin treatment at 1 µM resulted in little if any decrease in mTOR

autophosphorylation, but profoundly inhibited p70S6K phosphorylation consistent with

selective inhibition of mTORC1. XL765 inhibited nutrient-dependent phosphorylation at

all sites, with IC50 values of 160, 340, and 3000 nM, for mTOR S2481, p70S6K, and

4EBP1 phosphorylation respectively. These results are consistent with the mTOR kinase

assay results presented above, and provide further evidence that XL765 is a direct

inhibitor of mTOR kinase activity.





Effects on Proliferation in a Panel of Tumor Cell Lines

In MCF7 and PC-3 cells, XL765 inhibits proliferation (monitored by BrdU incorporation)

with IC50 values of 1070 nM and 1840 nM respectively. When tested in a broad panel of

tumor cell lines with diverse origins and genetic backgrounds, XL765 was found to

inhibit proliferation with a wide range of IC50 values (200 nM to >30000 nM; Figure 2

and Supplemental Table S2). A breakdown of sensitivity by genotype suggested that

PIK3CA mutant cell lines tended to be relatively sensitive to XL765, whereas RAS or

BRAF mutant cell lines tended to be less sensitive. Interestingly, several RAS mutant cell

lines were relatively insensitive to XL765 in spite of their also harboring PIK3CA

mutations (Figure 2 and Supplemental Table S2). Cell lines with loss of PTEN showed a

range of sensitivities, with some (e.g. the prostate carcinoma lines ZR75-1, LNCap and

PC-3) being sensitive and others (e.g the glioblastoma cell lines U251, U373) being

refractory.

Anchorage-independent growth in soft agar is considered the most stringent assay for

detecting malignant transformation of cells. To further characterize the effects of XL765

on tumor cell growth, an assay monitoring the anchorage-independent growth of PC-3

and MCF7 cells in soft agar over a 14-day period was employed. XL765 inhibits colony

growth with an IC50 of 270 nM in PC-3 cells and 230 nM in MCF7 cells. These IC50

values are significantly lower than those required to inhibit growth of the cells in a

monolayer, perhaps indicating an increased reliance on PI3K pathway signaling for

growth in 3-dimensions.

To rule out direct cytotoxic effects of XL765 on tumor cells, its effects on cell viability

were determined by bioluminescent measurement of cellular ATP. XL765 did not reduce

ATP levels in cells when incubated for 24 h, indicating a lack of acute cytotoxicity

(Supplemental Table S2, footnote). Induction of cytoplasmic Caspases 3 and 7 was

examined as an indication of apoptosis induction. XL765 did not affect the activity of

these Caspases at the doses and time point tested (Supplemental Table S2, footnote). In

MCF7 cells, the anti-proliferative effects of XL765 were associated with a specific block

in the G1 phase of the cell cycle and an increase of sub G1 cell population (data not





shown). Therefore, at least in MCF7 cells, XL765 exhibits anti-proliferative effects

predominantly via blockade of the cell cycle rather than through cytotoxic or apoptotic

effects.

XL765 inhibits tumor cell migration

One of the hallmarks of aggressive tumor cells is the ability to migrate in response to

chemotactic stimuli and to invade surrounding tissue. HGF is one of the key stimulators

of these behaviors, and cell lines expressing high levels of the HGF receptor MET, are

highly invasive and metastatic in vivo. Since PI3K resides in the MET signaling pathway,

the ability of XL765 to inhibit HGF-stimulated migration was tested in vitro. Murine B16

melanoma cells express high levels of Met which becomes highly phosphorylated when

the cells are treated with HGF. In 10% serum, B16 cells plated in the top well of a

transwell chamber containing a barrier with 0.8 micron pores show very little ability to

migrate to the lower chamber side. Addition of HGF to the lower transwell chamber

greatly increases migration through the barrier over a 24 h period. XL765 blocks this

effect with an IC50 value of 601 nM (Supplemental Figure S4). The cytotoxicity IC50

value of XL765 in B16 cells is 7300 nM, 12-fold higher than the IC50 value for inhibition

of migration. Therefore, inhibition of melanoma cell migration by XL765 is unlikely to

be due to cytotoxicity.

XL765 inhibits the PI3K and mTOR pathways and displays robust anti-tumor

activity in tumor bearing mice

Lysates derived from MCF7 xenograft tumors intradermally implanted into athymic nude

mice contain high levels of constitutively phosphorylated AKT, p70S6K, and S6 proteins.

The ability of XL765 to inhibit endogenous phosphorylation of AKT, p70S6K, and S6

was examined following a single oral dose of 10, 30, 100, or 300 mg/kg. The tumors

were harvested 4 h, 24 h, or 48 h post-dose and homogenized in lysis buffer. Tumor

lysates from each animal (n=4) were then pooled for each group and analyzed for levels

of total and phosphorylated AKT, p70S6K, and S6 by western immunoblotting

(Figure 3A).





Oral administration of XL765 causes a dose-dependent decrease of phosphorylation of

AKT, p70S6K, and S6 in the tumors, reaching a maximum of 84% inhibition of S6

phosphorylation at 30 mg/kg at 4 h. The dose response relationships (not shown) derived

from the 4 h time point predict 50% inhibition of AKT, p70S6K, and S6 phosphorylation

to occur at doses of 19 mg/kg (pAKTT308 and pAKTS473), 51 mg/kg (p-p70S6K), and

18 mg/kg (pS6). Inhibition of AKT, p70S6K, and S6 phosphorylation in MCF7 tumors

following a 30 mg/kg dose of XL765 was maximal at 4 h, reaching 61-84%; however, the

level of inhibition decreased to 0-42% by 24 h, and minimal or no inhibition was evident

by 48 h (see Figure 3A). Following a 100 mg/kg dose of XL765, inhibition was also

maximal at 4 h (52-75%). However, in contrast to the 30 mg/kg dose, inhibition at 24 h

(48-71%) was almost comparable to that seen at 4 h. Partial inhibition of some

phosphoepitopes persisted through 48 h (Figure 3A).

Similarly, administration of XL765 caused a dose-dependent decrease of phosphorylation

of AKT, p70S6K, and S6 in PC-3 tumors in vivo, reaching a maximum of 93% inhibition

of AKT phosphorylation at 300 mg/kg at 4 h post-dose (Figure 3B). The dose response

relationships (not shown) derived from the 4 h time point predict 50% inhibition of AKT,

p70S6K, and S6 phosphorylation to occur at doses of 15 mg/kg (pAKTT308), 13 mg/kg

(pAKTS473), 59 mg/kg (p-p70S6K), and 48 mg/kg (pS6). Consistent with the MCF7 data,

for the 100 mg/kg dose inhibition (42-60%) persisted through 24 h post dose. In both

studies, blood was collected at the same time tumor tissue was harvested and plasma

concentrations of XL765 were assessed (Supplemental Table S3). Based on these data,

the plasma concentrations associated with inhibition of phosphorylation of AKT,

p70S6K, and S6 by 50% in these tumor models ranged from approximately 3 to 9 µM.

Hence, XL765 exhibited comparable pharmacodynamic activity in PIK3CA-mutant

MCF7 and PTEN-deficient PC-3 xenograft tumor models.

Multiple tumor models were utilized to explore the efficacy and potency of repeat-dose

oral administration of XL765 with regards to tumor growth inhibition in vivo. In addition

to the previously described MCF7 and PC-3 models, the anti-tumor efficacy of XL765

was evaluated in xenograft models including OVCAR-3 (human ovarian xenograft tumor

model exhibiting PIK3CA amplification), U-87 MG (human glioblastoma xenograft





tumor model harboring a deletion at codon 54 in the gene encoding PTEN, resulting in a

frameshift), A549 (human NSCLC xenograft tumor model harboring a homozygous

activating mutation in KRAS2 and a homozygous loss-of-function mutation in LKB1) and

Calu-6 (human NSCLC xenograft tumor model harboring an activating mutation in

KRAS2).

XL765 administration results in significant anti-tumor efficacy in vivo in all of these

models (Figure 4) at doses that proved well tolerated as assessed by daily monitoring of

mouse weights (Supplemental Figure S5; no or minimal impact on body weights

compared with vehicle control). The most efficacious schedules were 30 mg/kg bid and

100 mg/kg q2d, which suggests that sustained pathway inhibition is required for maximal

effect on tumor growth. These schedules generally resulted in stasis of tumor growth,

except in the PC-3 model and the Calu-6 KRAS mutant NSCLC model, where tumors

continued to grow although at a reduced rate. Immunohistochemical analyses of MCF7,

PC-3 and A549 tumors collected at the end of the dosing period revealed significant,

dose-dependent decreases in staining for Ki67, a marker of cell proliferation. Moreover,

XL765 administration was associated with increased tumor cell apoptosis in MCF7 and

A549 tumors and modestly decreased tumor vascularization in MCF7, PC-3 and A549

tumors (Table 3). Thus, inhibition of PI3K and mTOR by XL765 results in anti-

proliferative, pro-apoptotic and anti-vascular effects in xenograft tumors. Plasma

concentrations at the end of these efficacy studies were similar to those seen following

single-dose administration. For example, in the MCF7 efficacy study, average plasma

concentrations for the 30 mg/kg dose administered once daily were 8.8 μM, 5.3 μM, and

below the limit of detection at the 1 hour, 4 hour and 24 h time-points, respectively (n=3

per time-point).

The anti-tumor activity observed in the sub-cutaneous U-87 MG glioblastoma xenograft

model prompted us to examine the pharmacodynamic activity of XL765 in the mouse

brain as a measure of whether the compound could effectively cross the blood-brain

barrier. Lysates from brains of non-tumor bearing mice show significant PI3K pathway

activity as judged by levels of pAKT and pS6. Four hours following a single oral dose of





30 or 100 mg/kg XL765, pAKT and pS6 levels are substantially reduced, demonstrating

that XL765 can cross the blood-brain barrier and inhibit the PI3K pathway (Figure 4).





Discussion

Previous experience with highly selective inhibitors of signal transduction pathways has

revealed the existence of unanticipated regulatory mechanisms that can act to limit the

efficacy of pathway inhibition. An example is the upregulation of AKT phosphorylation

that occurs as the result of relief of a negative feedback loop following inhibition of

mTORC1 by the rapamycin class of mTOR inhibitors. Similarly, selective B-RAF

inhibitors can trigger a “paradoxical” activation of C-RAF in the context of activated

RAS as a result of allosteric effects on B-RAF/C-RAF dimers. Inhibition at a single node

in a pathway also allows for the development of resistance via pathway activation

downstream of the point of intervention. Combining multiple inhibitors that impact the

same pathway, or developing a single compound that inhibits multiple members of a

pathway is therefore an attractive approach to limit or circumvent these issues. For

example, emerging data suggest that combinations of B-RAF and MEK inhibitors are

superior to either agent alone for the treatment of B-RAF mutant metastatic melanoma.

The PI3K/mTOR pathway is one of the most frequently activated signaling pathways in

human cancer, in part due to the mutation, amplification or deletion of key pathway

regulatory components. Activation of the pathway promotes tumor cell proliferation,

survival and resistance to anti-cancer therapies. As a result, extensive efforts are being

devoted to identifying and developing small molecule inhibitors that impact different

nodes of the pathway. Activation of the pathway is subject to regulation at multiple

points and by a wide variety of signals, including growth factors, cellular energy levels,

nutritional status and oxygenation. We therefore elected to optimize a compound that

would inhibit two key nodes in the pathway, PI3K and mTOR, with the aim of

maximizing pathway blockade in the context of multiple genetic backgrounds and under

a variety of environmental conditions.

XL765 (SAR245709) inhibits both class I PI3Ks as well as mTOR. Although XL765

appears to be more potent against PI3Kα than against PI3Kβ when assessed by

biochemical assays using purified kinases, in cellular assays XL765 shows comparable





activity versus PI3K pathway signaling in MCF7 breast (PIK3CA mutant) and PC-3

prostate (PTEN-deleted) tumor cells. PI3Kβ is considered to be the major driver of

dysregulated PI3K pathway activity associated with PTEN deficiency (31).

Moreover, XL765 showed comparably robust and persistent pharmacodynamic activity

against these cell lines when they were grown as xenograft tumors in mice. These data

demonstrate that XL765 exhibits functionally comparable activity against PI3Kα and

PI3Kβ in cultured cells and xenograft tumors.

In biochemical assays XL765 was generally less potent against mTOR than against

Class I PI3K isoforms. In tumor cells however, XL765 inhibits mTOR-dependent

phosphorylation events and PI3K-independent, nutrient-stimulated mTOR activity with a

potency comparable to that demonstrated for PI3K-dependent signaling, suggesting

potential for concerted PI3K/mTOR inhibition in cellular and in vivo models.

Our survey of the effects on XL765 on multiple phospho-epitopes in the PI3K signaling

pathway in 6 tumor cell lines with differing genetic backgrounds revealed consistent

inhibition downstream of both PI3K and mTOR. We saw no evidence for feedback

upregulation within the pathway or with respect to ERK phosphorylation. However, it is

important to note that our data are not exhaustive with respect to either genotype or

phosphorylation sites surveyed, and are limited to a single time point. The profile of

XL765 is clearly differentiated from that of rapamycin, which in all cell lines tested is a

potent inhibitor of mTORC1-dependent p70S6K and S6 phosphorylation, but in some

cell lines augmented PI3K activity as assessed by AKT phosphorylation, consistent with

previous reports.

XL765 exhibits a wide range of anti-proliferative activity against tumor cells grown as

monolayers. In MCF7 cells, these effects were associated with a G1 arrest but not with

acute cytotoxicity or induction of apoptosis. When sensitivity to XL765 is examined in

relation to genotype, there is a trend suggesting enhanced sensitivity of cells exhibiting

PIK3CA activating mutations, consistent with similar observations previously reported

for the PI3K inhibitors GDC-0941 and CH5132799 (32, 33). Likewise, the relative

insensitivity of RAS mutant cell lines, regardless of PIK3CA status, to inhibition of





proliferation by XL765 is consistent with preclinical observations with other PI3K

pathway targeting agents (23). However, we note that a number of K-RAS mutant lines

such as the A549 NSCLC line were quite sensitive to XL765. These cells have a

concomitant deletion of the LKB1 gene which may serve to sensitize them to inhibition of

mTOR. Likewise, PTEN-deleted cell lines had a wide range of sensitivities to XL765

with some being very sensitive and some being refractory. The basis for these

differences is not understood, but presumably reflects varying degrees of dependence on

PI3K pathway signaling for proliferation as a result of alterations in other pathways that

impact growth.

In multiple xenograft tumor models, oral administration of XL765 resulted in substantial

tumor growth inhibition at well tolerated doses. The most efficacious schedules were 30

mg/kg bid or 100 mg/kg q2d, suggesting that efficacy is associated with more continuous

inhibition of the pathway. These models encompass multiple genetic lesions activating

the PI3K pathway, specifically a PIK3CA E545K mutation (MCF7), PIK3CA

amplification (OVCAR-3), PTEN deletion (PC-3 and U-87 MG), KRAS mutation (A549

and Calu-6) and LKB1 mutation (A549). The fact that efficacy was observed in all these

models suggests that XL765 may have broad utility in tumors with activation of the PI3K

pathway. Based on the IHC/IF analyses conducted on tumor xenografts following repeat

dosing of XL765, anti-tumor efficacy was associated with a combination of anti-

proliferative and pro-apoptotic effects, with a modest impact on tumor angiogenesis.

These pro-apoptotic effects in vivo, which are not evident on cultured tumor cells, likely

reflect targeting of the tumor microenviroment in addition to tumor cells themselves,

which is consistent with the antiangiogenic effects evident.

In the majority of the xenograft models, complete or near complete inhibition of tumor

growth (but not regression) was observed, with the exception of the PC-3 and Calu-6

models which were relatively resistant. Overall, our data are not extensive enough to

determine whether in vitro sensitivity is generally predictive of efficacy in xenograft

models. It is also not yet clear whether the presence of PIK3CA mutations or PTEN

deficiency will be predictive of greater clinical responsiveness to PI3K pathway

inhibitors in general, although an analysis based on combining the results of multiple





early stage trials suggested that PIK3CA H1047R mutations are associated with response

(34). Intensive molecular profiling of tumors in the ongoing XL765 clinical studies is

being performed to further explore this question.

Since we observed significant anti-tumor efficacy in the U-87 MG glioblastoma model,

we assessed the pharmacodynamic activity of XL765 in mouse brain. At the same doses

associated with efficacy in the subcutaneous xenograft model, XL765 effectively

inhibited PI3K pathway signaling in the brain, supporting its potential utility for the

treatment of CNS malignancies. Consistent with this observation, XL765 has

demonstrated significant efficacy in an orthotopic glioblastoma xenograft model, both as

a single agent and in combination with temozolomide (35). In addition, recent data from

a clinical trial in which XL765 was administered to glioblastoma patients prior to surgical

removal of recurring lesions showed significant inhibition of PI3K pathway signaling in

glioblastoma tumor tissue following XL765 dosing (36). XL765 is currently in Phase I

and II clinical studies as a single agent or in combination with other targeted or cytotoxic

agents in patients with solid tumors, lymphoma, and leukemia (NCT01390818,

NCT01410513, NCT01403636).





Acknowledgements

We are grateful to Coumaran Egile for critical reading of the manuscript. Requests

relating to provision of XL765 (SAR245409) should be direct to Coumaran Egile at

sanofi ([email protected]).





References

1. Liu P, Cheng H, Roberts TM, Zhao JJ. Targeting the phosphoinositide 3-kinase

pathway in cancer. Nat Rev Drug Discov. 2009;8:627-44.

2. Zhao L, Vogt PK. Class I PI3K in oncogenic cellular transformation. Oncogene.

2008;27:5486-96.

3. Guillermet-Guibert J, Bjorklof K, Salpekar A, Gonella C, Ramadani F, Bilancio A,

et al. The p110beta isoform of phosphoinositide 3-kinase signals downstream of

G protein-coupled receptors and is functionally redundant with p110gamma. Proc

Natl Acad Sci U S A. 2008;105:8292-7.

4. Rodriguez-Viciana P, Warne PH, Khwaja A, Marte BM, Pappin D, Das P, et al.

Role of phosphoinositide 3-OH kinase in cell transformation and control of the

actin cytoskeleton by Ras. Cell. 1997;89:457-67.

5. Dancey J. mTOR signaling and drug development in cancer. Nat Rev Clin Oncol.

2010;7:209-19.

6. Guertin DA and Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell.

2007;12:9-22.

7. Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-Bromage H,

et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive

and raptor-independent pathway that regulates the cytoskeleton. Curr Biol.

2004;14:1296-302.

8. Febuary 2014 search of COSMIC database,

http://www.sanger.ac.uk/genetics/CGP/cosmic/

9. Hollander MC, Blumenthal GM, Dennis PA. PTEN loss in the continuum of

common cancers, rare syndromes and mouse models. Nat Rev Cancer.

2011;11:289-301.

10. van Veelen W, Korsse SE, van de Laar L, Peppelenbosch MP. The long and

winding road to rational treatment of cancer associated with

LKB1/AMPK/TSC/mTORC1 signaling. Oncogene. 2011;30:2289-303.

11. Jiang BH, Liu LZ. PI3K/PTEN signaling in angiogenesis and tumorigenesis. Adv

Cancer Res. 2009;102:19-65.





12. Yuan TL, Choi HS, Matsui A, Benes C, Lifshits E, Luo J, et al. Class 1A PI3K

regulates vessel integrity during development and tumorigenesis. Proc Natl Acad

Sci U S A. 2008;105:9739-44.

13. Schnell CR, Stauffer F, Allegrini PR, O'Reilly T, McSheehy PM, Dartois C, et al.

Effects of the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin

inhibitor NVP-BEZ235 on the tumor vasculature: implications for clinical

imaging. Cancer Res. 2008;68:6598-607.

14. Zhang S, Yu D. PI(3)king apart PTEN's role in cancer. Clin Cancer Res.

2010;16:4325-30.

15. Kolasa IK, Rembiszewska A, Felisiak A, Ziolkowska-Seta I, Murawska M, Moes

J, Timorek A, et al. PIK3CA amplification associates with resistance to

chemotherapy in ovarian cancer patients. Cancer Biol Ther. 2009;8:21-6.

16. O'Brien NA, Browne BC, Chow L, Wang Y, Ginther C, Arboleda J, et al.

Activated phosphoinositide 3-kinase/AKT signaling confers resistance to

trastuzumab but not lapatinib. Mol Cancer Ther. 2010;9:1489-502.

17. Faber AC, Li D, Song Y, Liang MC, Yeap BY, Bronson RT, et al. Differential

induction of apoptosis in HER2 and EGFR addicted cancers following PI3K

inhibition. Proc Natl Acad Sci U S A. 2009;106:19503-8.

18. Qi J, McTigue MA, Rogers A, Lifshits E, Christensen JG, Jänne PA, Engelman

JA. Multiple mutations and bypass mechanisms can contribute to development of

acquired resistance to MET inhibitors. Cancer Res. 2011;71:1081-91.

19. Chakrabarty A, Sánchez V, Kuba MG, Rinehart C, Arteaga CL. Breast Cancer

Special Feature: Feedback upregulation of HER3 (ErbB3) expression and activity

attenuates antitumor effect of PI3K inhibitors. Proc Natl Acad Sci U S A. 2012;

109:2718-23.

20. Klein S, Levitzki A. Targeting the EGFR and the PKB pathway in cancer. Curr

Opin Cell Biol. 2009;21:185-93.

21. Priulla M, Calastretti A, Bruno P, Azzariti A, Paradiso A, Canti G, et al.

Preferential chemosensitization of PTEN-mutated prostate cells by silencing the

Akt kinase. Prostate. 2007;67:782-9.





22. Santiskulvong C, Konecny GE, Fekete M, Chen KY, Karam A, Mulholland D, et

al. Dual targeting of phosphoinositide 3-kinase and mammalian target of

rapamycin using NVP-BEZ235 as a novel therapeutic approach in human ovarian

carcinoma. Clin Cancer Res. 2011;17:2373-84.

23. Engelman JA. Targeting PI3K signalling in cancer: opportunities, challenges and

limitations. Nat Rev Cancer. 2009;9:550-62.

24. García-Echeverría C. Allosteric and ATP-competitive kinase inhibitors of mTOR

for cancer treatment. Bioorg Med Chem Lett. 2010;20:4308-12.

25. Gendreau SB, Ventura R, Keast P, Laird AD, Yakes FM, Zhang W, et al.

Inhibition of the T790M gatekeeper mutant of the epidermal growth factor

receptor by EXEL-7647. Clin Cancer Res. 2007;13:3713-23.

26. Bussenius J, Anand NK, Blazey CM, Bowles OJ, Bannen LC, Chan DS, et al.

Design and evaluation of a series of pyrazolopyrimidines as p70S6K inhibitors.

Bioorg Med Chem Lett. 2012;22:2283-6.

27. Yakes FM, Chen J, Tan J, Yamaguchi K, Shi Y, Yu P, et al. Cabozantinib

(XL184), a novel MET and VEGFR2 inhibitor, simultaneously suppresses

metastasis, angiogenesis, and tumor growth. Mol Cancer Ther. 2011;10:2298-308.

28. Yaguchi S, Fukui Y, Koshimizu I, Yoshimi H, Matsuno T, Gouda H, et al.

Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor. J

Natl Cancer Inst. 2006;98:545-56.

29. Wlodarski, P., Kasprzycka, M., Liu, X., Marzec, M., Robertson, E. S., Slupianek,

A., et al. Activation of mammalian target of rapamycin in transformed B

lymphocytes is nutrient dependent but independent of Akt, mitogen-activated

protein kinase/extracellular signal-regulated kinase kinase, insulin growth factor-I,

and serum. Cancer Res 2005; 65: 7800-7808.

30. Fan QW, Knight ZA, Goldenberg DD, Yu W, Mostov KE, Stokoe D, et al. A dual

PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma. Cancer Cell

2006; 9: 341-349.

31. Jia S, Roberts TM, Zhao JJ. Should individual PI3 kinase isoforms be targeted in

cancer? Curr Opin Cell Biol. 2009;21:199-208.





32. O'Brien C, Wallin JJ, Sampath D, GuhaThakurta D, Savage H, Punnoose EA, et

al. Predictive biomarkers of sensitivity to the phosphatidylinositol 3' kinase

inhibitor GDC-0941 in breast cancer preclinical models. Clin Cancer Res.

2010;16:3670-83.

33. Tanaka H, Yoshida M, Tanimura H, Fujii T, Sakata K, Tachibana Y, et al. The

selective class I PI3K inhibitor CH5132799 targets human cancers harboring

oncogenic PIK3CA mutations. Clin Cancer Res. 2011;17:3272-81.

34. Janku F, Wheler JJ, Naing A, Falchook GS, Hong DS, Stepanek VM, et al.

PIK3CA mutation H1047R is associated with response to PI3K/AKT/mTOR

signaling pathway inhibitors in early-phase clinical trials. Cancer Res.

2013;73:276-84.

35. Prasad G, Sottero T, Yang X, Mueller S, James CD, Weiss WA, et al. Inhibition

of PI3K/mTOR pathways in glioblastoma and implications for combination

therapy with temozolomide. Neuro Oncol. 2011;13:384-92.

36. Cloughesy TF, Mischel PS, Omuro AMP, Prados M, Wen PY, Wu B, et al.

Tumor pharmacokinetics (PK) and pharmacodynamics (PD) of SAR245409

(XL765) and SAR245408 (XL147) administered as single agents to patients with

recurrent glioblastoma (GBM): An Ivy Foundation early-phase clinical trials

consortium study. J Clin Oncol 31, 2013 (suppl; abstr 2012).





Tables

Table 1

Kinase Inhibition Profile of XL765

Family Kinase

XL765

IC50 (nM)

PI3K

Class IA

PI3Kα 39 ± 10

PI3Kβ 110 ± 30

PI3Kδ 43 ± 3

Class IB PI3Kγ 9 ± 3

Class III VPS34 9060

PI3K-related

mTORC1 160 a

mTORC2 910 a

DNA-PK 150

DNAPK, DNA protein kinase; IC50, concentration required for 50% target inhibition; mTORC1/2, mammalian target of rapamycin complex 1/2; PI3K, phosphatidylinositol 3-kinase; VPS34, vacuolar sorting protein 34.

a Immunoprecipitation kinase assay using cell lysates.

Table 2

Effects of XL765 on PIP3 Production and AKT and S6 Phosphorylation

Cell Line PIP3 IC50 (nM) pAKT IC50 (nM)a pS6 IC50 (nM)a

PC-3 290 250 120

MCF7 170 ndb ndb

See Materials and Methods for details. aIC50 values determined using ELISA assay. bnd, not determined





Table 3

Immunohistochemical Analysis of Proliferation, Vascularization and Apoptosis in

MCF7, PC-3, and A549 Xenograft Tumors

Group

Ki67 Analysis CD31 Analysis Apoptotic Index

(TUNEL)

% Positive

Cellsa

%

Reductionb MVDc

%

Reductionb

% Positive

Cells

Fold

Increaseb

MCF7

Vehicle, 10 ml/kg PO QD 43 ± 4 na 50 ± 7 nd 1.0 ± 0.5 na

XL765, 10 mg/kg PO QD 37 ± 5 15 (ns) 28 ± 4 44 3.8 ± 2.5 4 (ns)

XL765, 30 mg/kg PO QD 27 ± 6 39 33 ± 12 34 (ns) 5.3 ± 1.6 6

XL765, 30 mg/kg PO BID 23 ± 3 48 23 ± 7 54 3.5 ± 1.8 4

XL765, 100 mg/kg PO Q2D 7 ± 8 84 41d 18 36 ± 8 37

PC-3

Vehicle, 10 ml/kg PO QD 21 ± 2 na 34 ± 4 na nd nd

XL765, 30 mg/kg PO QD 13 ± 4 37 28 ± 6 17 (ns) nd nd

XL765, 30 mg/kg PO BID 10 ± 2 51 27 ± 4 19 nd nd

XL765, 100 mg/kg PO Q2D 18 ± 2 16 26 ± 3 22 nd nd

A549

Vehicle, 10 ml/kg PO QD 32 ± 5 na 37 ± 8 na 1.0 ± 0.3 na

XL765, 30 mg/kg PO QD 21 ± 3 33 35 ± 10 6 (ns) 3.8 ± 0.7 3.8

XL765, 30 mg/kg PO BID 16 ± 3 50 29 ± 6 22 3.1 ± 0.3 3.1

XL765, 100 mg/kg PO Q2D 20 ± 2 39 31 ± 8 18 (ns) 7.1 ± 0.7 7.1

XL765, 100 mg/kg PO BIW 22 ± 4 33 34 ± 6 8 (ns) 6.1 ± 1.7 6.1 aValues are mean ± standard deviation. bValues are relative to vehicle control (in all cases p < 0.05 except where indicated as ns, not significant). cMean Vessel Density. dBased on one evaluable tumor (insufficient viable tissue to score in 8 of 9 tumors).

na, not applicable; nd, not determined.





Figure Legends Figure 1

(A) Chemical structure of XL765 (SAR245409). (B) XL765 inhibits PI3K pathway

signaling in EGF-stimulated MCF7 cells. After incubation with XL765 at the indicated

concentrations, PI-103 (10 μM), ZSTK474 (10 μM), or rapamycin (0.1 μM), MCF7 cells

were stimulated with 100 ng/ml of EGF for 10 min. The cells were then lysed and effects

of compound on PI3K pathway signaling assessed by western immunoblotting. (C)

XL765 inhibits nutrient-dependent mTOR signaling pathway in Ramos cells. Cells were

starved in serum free media for 20 h, then treated with compounds at the indicated

concentrations, DMSO, or serum- and nutrient-free PBS for 2 h. Cell lysates were

prepared and analyzed by western immunoblotting

Figure 2

Relative sensitivity of tumor cells to XL765 as a function of genetic status. a Cell

proliferation IC50 values are presented normalized to that for BT474 (most sensitive cell

line). See Materials and Methods and Supplemental Table S2 for details.

Figure 3

Administration of XL765 inhibits PI3K pathway signaling in MCF7 and PC-3 tumors. A

single dose of XL765 or vehicle was administered by oral gavage to MCF7 (A) or PC-3

(B). Tumors were resected at the indicated times post dose and the effects of XL765 on

phosphorylation of AKT, p70S6K, and S6 were assessed by western immunoblotting.

Figure 4

XL765 administration results in tumor growth inhibition of established xenograft tumors.

(A) MCF7, (B) PC-3, (C) OVCAR-3, (D) U87-MG, (E) A549 or (F) Calu-6 tumor cells

were implanted and when tumors reached ~100 mg in size administration of vehicle or

XL765 was initiated at the indicated doses and regimens (Day 0/1 = day of grouping;

dosing was initiated on Day 1). Data points represent the mean ± standard error for each

treatment group (N = 9–10). (D) The inset panel in the U-87 MG tumor growth inhibition





graph shows the effect of XL765 at the indicated oral dose on pAKT and pS6 levels in

normal mouse brain at 4 h post dose.




N

N

ON

N

A

N

NN

B

a0O K

+ EGF

O 03

XL765 (nM)

Rap

a

3000

0

6000

9.6

240

481200

DM

SO

ZS

TK

DM

SO

PI-

10

pAKTT308

pAKTS473

AKT

pp70S6KT389

pS6S240/S244

S6

PRAS40

p70S6K

pPRAS40T246

4EBP1

GSK3β

Cyclin D1

pGSK3βS9

p4EBP1T37/T46

C

10µ

M

100n

M

OO

XL765 (µM)

pERKY204

ERK

Cyclin D1

pmTORS2481

pp70S6KT389

4EBP1T37/T46

p70S6K

ZS

TK

Rap

a

DM

SO

PB

S

0.3

30 10 3 1 0.1

DM

SO

Yu et al, Pharmacodynamics and anti-tumor efficacy of XL765 (SAR245409)

Figure 1. (A) Chemical structure of XL765 (SAR245409). (B) Effects of XL765 on PI3K Signaling Pathway in EGF-Stimulated MCF7 Cells. (C) XL765 inhibits Nutrient-Dependent mTOR Signaling Pathway in Ramos Cells

p4EBP1T37/T46

4EBP1




120

140RAS MutBRAF MutPTEN Mut

PIK3CA Mut/Amp

PIK3CA/RAS PTEN/BRAFPTEN/RAS

60

80

100

mal

ized

IC50

a

20

40

No

rm

0

BT474T47D

OVCAR-3

MCF7H508

IGROV-1

MDA-MB-453

SK-OV-3AGS

LS174T

HCT116H460

ZR75-1

LNCaPPC3

JRT3-T3.5BC-3

U87-MG

OPM-2 H4

H446U937

786-O

MDA-MB-468U251U373

MOLT4

WM-266

A2058

SK-MEL-28

SK-MEL-3

Colo-205

SK-HEP-1A375

HT29

OCI-AML3LoVo

RPMI8226

HL-60A549

AsPC-1H441

Capan-1

MDA-MB-231T

HT-1080

PANC-1

DLD-1

OVCAR-5

MiaPaCa

CALU-6

TALL-1

SW480

SHP-77


Figure 2Sensitivity to proliferation inhibition by genotype




MCF-7

AA XL765

Dose (mg/kg) 10 30 100 10 30 100 10 30 100Time (h) 4 4 4 4 24 24 24 24 48 48 48 48

pAKTT308

AKT

XL765 XL765

p-p70S6KT389

p70S6K

pS6S240/S244

pAKTS473

AKT

PC-3

S6

p

B

pAKTT308

AKT

Dose (mg/kg) 10 30 100 300 10 30 100 300

Time (h) 4 4 4 4 4 24 24 24 24 24

XL765 XL765

p-p70S6KT389

p70S6K

pS6S240/S244

AKT

pAKTS473

AKT

S6

pS6S240/S244


Figure 3Inhibition of AKT, p70S6K, and S6 Phosphorylation in MCF7 and PC-3 XenograftTumors after a Single Oral Dose




A B

DC

Vehicle 10 30 100 mg/kg

normal brain

pS6S240/S244

pAKTT308

pAKTS473

AKT

S6

g g

FE

Figure 4Anti-tumor efficacy of XL765Yu et al, Pharmacodynamics and anti-tumor efficacy of XL765 (SAR245409)




Published OnlineFirst March 14, 2014.Mol Cancer Ther Peiwen Yu, A. Douglas Laird, Xiangnan Du, et al. Alterations Impacting the PI3K PathwayXL765 (SAR245709) in Tumor Models with Diverse Genetic Characterization of the Activity of the PI3K/mTOR Inhibitor

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