Lymphangiogenic, Anti-Tumoral and Anti-Metastatic Activities

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Author Manuscript Published OnlineFirst on May 14, 2012; DOI: 10.1158/1535-7163.MCT-11-0866-T Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

SAR131675, a potent and selective VEGFR-3-TK inhibitor with anti-

lymphangiogenic, anti-tumoral and anti-metastatic activities.

Antoine Alam1, Isabelle Blanc1, Geneviève Gueguen-Dorbes1, Olivier Duclos2, Jacques

Bonnin1, Pauline Barron1, Marie-Claude Laplace1, Gaelle Morin1, Florence Gaujarengues1,

Frédérique Dol1, Jean-Pascal Hérault1, Paul Schaeffer1, Pierre Savi1 and Françoise Bono1

1Sanofi Recherche et Développement, Early to Candidate DPU, Toulouse, France,

2Sanofi Recherche et Développement, Fibrosis TSU, Chilly Mazarin, France,

Running title: SAR131675 a potent and selective VEGFR-3 inhibitor

Precis : SAR131675, a highly active and specific VEGFR-3 inhibitor, reduces

lymphangiogenesis and angiogenesis and also tumor associated macrophages infiltration and

as consequences, it reduces tumor growth and metastasis

Keywords: Angiogenesis / lymphangiogenesis / tyrosine kinase / VEGF-R3 / macrophages/

metastasis

Conflict of interest: PSch owns Sanofi stock, no potential conflicts of interest were

disclosed by other authors.

Corresponding author: Françoise Bono

Sanofi Recherche et Développement

Downloaded from mct.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 14, 2012; DOI: 10.1158/1535-7163.MCT-11-0866-T Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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Abbreviations: Erk: Extracellular signal-regulated kinases, FGF2: basic fibroblast

growth factor, GFP: green fluorescent protein, HEK: Human Embryonic Kidney,

HLMVEC: Human-Lymphatic-Micro-Vascular-Endothelial-Cells, NRP: neuropiline

receptor, PAEC: porcine aortic endothelial cells, VEGFR: Vascular Endothelial cell

Growth Factor Receptor, TAMs: Tumor Associated Macrophages

- Abstract (238 words); Text (4718 words+947 for legends); References (50),

Figures (7)

Abstract

SAR131675 is a potent and selective VEGFR-3 inhibitor. It inhibited VEGFR-3 tyrosine

kinase (TK) activity and VEGFR-3 autophosphorylation in HEK cells with IC50s of

20nM and 45nM, respectively. SAR131675 dose-dependently inhibited the proliferation

of primary human lymphatic cells, induced by the VEGFR-3 ligands VEGFC and

VEGFD, with an IC50 of about 20nM. SAR131675 was found to be highly selective for

VEGFR-3 versus 107 receptors, enzymes, ion channels and 65 kinases. However, it was

moderately active on VEGFR-2 with a VEGFR-3/VEGFR-2 ratio of about 10.

SAR131675 had no anti-proliferative activity on a panel of 30 tumors and primary cells,

further demonstrating its high specificity and indicating that SAR131675 is not a

cytotoxic or cytostatic agent. SAR131675 was very well tolerated in mice and

demonstrated a potent antitumoral effect in several orthotopic and syngenic models

including mammary 4T1 carcinoma and RIP1.Tag2 tumors. Interestingly, it significantly

reduced lymph node invasion and lung metastasis, demonstrating its anti-

lymphangiogenic activity in vivo. Moreover, treatment of mice before resection of 4T1

primary tumors was sufficient to prevent metastasis. Tumor Associated Macrophages

(TAMs) play an important role in tumor growth and metastasis. The expression of

VEGFR-3 on TAMs has been recently described. F4/80 immunostaining clearly showed

that SAR131675 significantly reduced TAM infiltration and aggregation in 4T1 tumors.

Taken together, SAR131675 is the first highly specific VEGFR-3-TK inhibitor described

to date, displaying significant anti-tumoral and anti-metastatic activities in vivo through

inhibition of lymphangiogenesis and TAM invasion.

Introduction

In most solid tumors, metastases in regional lymph nodes occur frequently in the initial

process of cancer dissemination and are a strong indicator of poor patient prognosis (1).

A number of clinical and experimental data suggest that migration of tumor cells into the

lymph nodes is greatly facilitated by tumor lymphangiogenesis (2).

One key molecule mediating tumor lymphangiogenesis is the vascular endothelial growth

factor receptor-3 (VEGFR-3), a tyrosine kinase receptor recognized and activated by

VEGFC and VEGFD, commonly expressed in malignant and tumor infiltrating stromal

cells (3). In experimental tumor models, VEGFC and VEGFD expression induces

lymphangiogenesis and correlates with lymphatic invasion and nodal metastasis (4, 5). In

addition, agents neutralizing VEGFC and VEGFD, or blocking VEGFR-3 signaling,

suppress development of new lymphatic vessels and tumor metastasis in experimental

cancer models (6, 7).

Although VEGFC and VEGFD are the sole ligands described to date as binding directly

to VEGFR-3 (8, 9), blunting of both ligands at the same time does not result in the

embryonic blood vasculature defects seen in VEGFR-3-/- mice (10-12). This suggests that

additional ligands exist or that ligand-independent signaling occurs. Indeed, whereas

VEGFA binds only to VEGFR-2, we and others have shown that VEGFA may induce

formation of VEGFR-2/-3 heterodimers (13, 14). This suggests that when cells express

both receptors, VEGFR-3 inhibitors may also block some VEGFA signaling.

In adults, VEGFR-3 is found only on endothelial cells of lymphatic vessels. However, in

malignant tumors, VEGFR-3 expression is found on vascular endothelial cells and

particularly on angiogenic sprouts. Genetic targeting of VEGFR-3 or blocking of

VEGFR-3 signaling with monoclonal antibodies results in decreased sprouting and

vascular density, suggesting that VEGFR-3 plays a crucial role in tumor angiogenesis (15,

16).

There is considerable evidence of the expression of VEGFR-3 in several primary tumors

including colorectal, ovarian, gastric etc. (17-19) and its upregulation in breast cancer has

been shown to precede tumor cell invasion (20). Interestingly, coexpression of VEGFR-3

with VEGFC in human cancer cells was associated with increased metastasis and poorer

survival, suggesting a possible autocrine loop between VEGFC and VEGFR-3 in cancer

cells (21, 22).

Expression of VEGFR-3 has also been reported on macrophages after brain ischemia,

kidney transplantation, chronic airway inflammation or corneal injury (23-27) and also on

tumor associated macrophages (TAMs) (28, 29). TAMs are critical regulators of

angiogenesis and lymphangiogenesis; they express VEGFA and VEGFC and induce tip-

cell formation and fusion (16, 30-33). In addition they are thought to directly contribute

to lymphatic vessel formation by trans-differentiating into lymphatic endothelial cells.

(25, 28, 33-34)

Collectively, these data implicate VEGFR-3 in different processes of tumor growth and

metastasis, suggesting that therapeutic targeting of VEGFR-3 might selectively reduce

tumor growth and metastasis

In order to obtain maximal beneficial effects without causing severe side effects,

VEGFR-3 selective compounds are required. Although several marketed multikinase

inhibitors, including sunitinib and sorafinib, have been reported to present anti-VEGFR-3

activity, currently there is no specific VEGFR-3-TK inhibitor under evaluation. Here we

describe for the first time a small molecule with high selectivity for VEGFR-3 and show

that SAR131675 is able to reduce lymphangiogenesis, angiogenesis and TAM infiltration

and consequently reduce tumor growth and metastasis.

Materials and methods

Reagents: Accustain®, Drabkin and the human recombinant hemoglobin were obtained

from Sigma®. The proteome array Phospho-MAPK, the DuoSet ELISA Phospho-

ERK1/2 and the recombinant proteins bFGF, VEGFA and VEGFD were purchased from

R&D and VEGFC from ReliaTech, huVEGFR-1-TK from Upstate, and huVEGFR-3-TK

from Cell Signalling: huVEGFR-2-TK was produced internally,.

Animals: All animal treatment procedures described in this study were approved by the

Animal Care and Use Committee of sanofi. Female BALB/cByj mice and RIP1.Tag2

mice on the C57Bl/6J background were obtained from Charles River France.

Treatment: SAR131675 was dissolved on the day of use in a 0.6% methylcellulose/0.5%

Tween 80 solution.

Cell lines: Human tumor cell lines were obtained from DSMZ Braunschweig, Germany

or the American Tissue Culture Collection Frederick, MA, USA. No further

authentication was performed. Cells were cultured in the culture medium in a humidified

atmosphere of 5% CO2 and 95% air at 37°C. Cell viability was determined by trypan

blue exclusion, and always exceeded 95%.

Complete cell culture medium for human cell proliferation was RPMI 1640 (Gibco

Laboratories, Grand Island, NY) containing 10 % heat-inactivated FCS (Gibco

Laboratories), 2 mM L-glutamine, 1mM sodium pyruvate, 100 UI/ml penicillin, 100

µg/ml streptomycin (Gibco Laboratories).

Adult HLMVEC (Human Lymphatic Micro Vascular Endothelial Cells), were

purchased from Cambrex, maintained in culture as described previously (35) and were

tested and authenticated in the lab for the expression of VEGFR-3 and Prox1 as

lymphatic cell markers. Cells were used up to passage 6. 4T1 mouse mammary

adenocarcinoma cells were obtained from the ATTC (CRL-2539) and were maintained in

RPMI 1640 containing 10% heat-inactivated FCS and 2mM L-glutamine. Authentication

of the cell line was done by expression of osteopontin as described below, and used up to

passage 25.

Tyrosine kinase assay: Multiwell plates were precoated with a synthetic polymer

substrate poly-Glu-Tyr (polyGT 4:1). The reaction was performed in the presence of

kinase buffer (10X: 50 mM HEPES buffer, pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2, and

+ 0.2 mM Na3VO4) supplemented with ATP and DMSO for the positive control (C ) or

SAR131675 (ranging from 3 nM to 1000 nM). ATP was used at 30 µM for VEGFR-1

and VEGFR-3 and at 15 µM for VEGFR-2. The phosphorylated ployGT was probed with

a phosphotyrosine specific monoclonal antibody conjugated to horseradish peroxidase

(HRP) (1 / 30,000, clone PT66; Sigma) and developed in the dark with the HRP

chromogenic substrate (OPD). The reaction was then stopped by the addition of 100 µl

1.25 M H2SO4 and absorbance was determined using an Envision spectrophotometer at

492 nm.

Percent inhibition (I%) was calculated using the following formula: I%=100-

([SAR131675]-C-)/(C+-C-)*100, where [SAR131675] represents the value obtained in the

presence of the indicated concentration of SAR131675.

Autophosphorylation in HEK cells and survival and migration assays of HLMVC:

Experiments were performed as described previously (35). Twenty four hours after

transfection, the cells were treated during 1 hour with orthovanadate (100 mM),

harvested, collected and, after counting, distributed in 5 ml tubes in the presence of the

indicated concentration of SAR131675. After 30 min of incubation, the reaction was

stopped by the addition of cold PBS supplemented by orthovanadate. Cells were then

lysed with 150 µl of RIPA buffer over 15 min at 4°C, and then centrifuged for 10 min at

10,000xg. Supernatants were distributed in duplicate on 96-well plates precoated with the

anti-Flag and left for 1 hour at room temperature. After 3 washes, the anti-phospho-

tyrosine conjugated to the HRP was added and incubated for 1 hour at room temperature.

Wells were then washed 3 times with TBS buffer containing 0.5 % Tween 20 and 2mM

MgCl2. The reaction was stopped with 50µl of 2N H2SO4 and the signal was read using

an Envision spectrophotometer at 485 and 530 nm.

Survival assay: HLMVEC were seeded in 96-well plates coated with 0.3% gelatine (5x

103 cells per well). Cells were incubated in RPMI 0.1% FCS with VEGFA (10ng/ml)

VEGFC (300ng/ml), VEGFD (300ng/ml) or FGF2 (10ng/ml) in the absence or presence

of SAR131675. Five days later, viable cells were quantified with the cell Titer-glo

luminescent cell viability assay (Promega, Madison, WI) as previously described (35).

Migration assay: The migration assay was performed with the BD BioCoat

Angiogenesis System-Endothelial Cell Migration kit (BD Biosciences, San Jose, CA).

HLMVEC (1 x 105 cells per well in RPMI) were added in triplicate in the upper

chambers. The lower chambers were loaded with RPMI alone, RPMI with 50 ng/ml

VEGF-A or 100 ng/ml VEGF-C. After 24 hours at 37°C cells were labeled with calcein

AM (Molecular probes, Inc. OR) according to the manufacturer's instructions.

Fluorescence of cells that had migrated was measured on a TECAN GENios microplate

reader (TECAN, Maennedorf, Switzerland).

Proteome Array and Erk phosphorylation: HLMVEC were plated in 6-well tissue

culture plates at a density of 2 x105 cells for 48h. Cells were then serum deprived for 2h

and stimulated or not for 10mn with VEGFC (500ng/ml) in the presence or not of the

indicated concentration of SAR131675, lysed and subjected to phospho-MAPK array or

to phospho Erk ELISA according to manufacturer’s instructions.

Quantification of VEGFR-2 phosphorylation by ELISA: Quantification of VEGFR-2

phosphorylation by ELISA was carried out as described previously using porcine aortic

endothelial cells (PAEC) expressing VEGFR-2 (35).

Blood vessel formation in zebrafish: Embryos from transgenic zebrafish (Danio rerio)

that specifically express the fluorescent protein copGFP in the vascular system were

used. The chorion of the 24 hpf (hours post fertilization) embryos were removed and

embryos were dispensed in a 96 well assay plate in embryo water mixed with

SAR131675 solubilized in DMSO. After 24 hours at 28.5°C, the embryos were

anesthetized with tricaine and the number of intersegmental vessels was analyzed by

fluorescence microscopy (2.5x magnification).

Angiogenesis and lymphangiogenesis induced in a sponge implant mouse model:

Sterile sponge discs (Cellspon®, Interchim) impregnated with 200µg of FGF2 or PBS

were sub-cutaneously introduced on the back of anaesthetized mice. FGF2 was reinjected

into the sponges the first 2 days. Daily oral treatment with SAR131675 (30, 100 and 300

mg/kg/d) started the day of sponge implantation. Seven days later, the animals were

euthanatized and the sponges were removed, harvested and lysed in RIPA buffer at 4°C.

After a centrifugation at 6,000xg, the supernatants were collected for further analysis.

RIP1.Tag2/transgenic mouse models: For the prevention study, treatment of mice

(9/group) started at 5 weeks of age for 5 weeks and then the number of Langherans islets

(red islets) was determined after retrograde perfusion with collagenase solution through

the common bile duct (36). For the intervention study, mice were treated daily from 10

weeks of age for 16 days and tumor volume was measured and calculated as for 4T1

tumors below. The tumor burden was calculated as the sum of individual tumor volumes

for each mouse. For the survival study, daily treatment (20 mice/group) started at 12

weeks of age and mice were monitored daily to detect moribund mice.

4T1 mammary carcinoma model: 4T1 cells (105) were implanted into mammary fat

pads of BALB/c mice (15/group) (37). Daily oral treatment with SAR131675 (30 and

100mg/kg/d) started at day 5. Tumors were measured 2 to 3 times weekly with calipers.

The tumor volume (V) was calculated using the formula V= 0.52 x a2 x b (a: smallest

tumor diameter and b: largest tumor diameter). At day 21, the tumors, the lungs and the

axillary lymph nodes were removed. The number of metastases at the surface of each

lung was counted. The tumors and the lymph nodes were lysed in RIPA buffer at 4°C.

The osteopontin level in lymph nodes was quantified using an ELISA kit (Assay Design).

4T1 tumor excision model: Implantation of cells and treatment was started as described

above, but primary tumors were removed at day 15 in all groups and the tumor weight

was evaluated. The mice treated with SAR131675 were then divided into two groups (12

mice/group); in the first group, treatment with SAR131675 was stopped whereas in the

second, mice were treated up to day 26. At day 26, lung metastatic foci were counted.

Immunochemistry: Tissues were fixed with 10% accustain® 24 hours, dehydrated and

embedded in paraffin. Immunostaining was done in sections incubated with anti–mouse

CD31 Ab (1/50), LYVE1 (1/1000, Santa Cruz) or F4-80 (1/50, eBiosciences, BD

PharMingen) following incubation with the Vectastain ABC kit (Vector laboratories)

appropriate to the species of primary antibody, and antigens were developed with DAB

peroxidase substrate and counterstained with hematoxylin. Images were captured with a

camera (SONY) mounted on a Nikon microscope.

Statistical Analysis:

IC50 values were obtained using internal software Biost@t-SPEED v2.0 with the 4-

parameter logistic model. For the in vivo studies, results are given as mean ± sem.

Differences between groups were examined for statistical significance using two-analysis

of variance (ANOVA) following Dunnett’s test or confidence interval calculation as

specified in the results part. Statistical analysis of data from the survival study was

performed using a log-rank test. For tumor regression analysis, two-way ANOVA with

repeated measures was performed.

Results

In vitro effect of SAR131675 on VEGFR-3 tyrosine kinase activity

SAR131675 is the result of high throughput screening and chemical optimization

(Fig.1A). Its ability to inhibit the kinase activity of VEGFR-3 was first determined by

ELISA using recombinant human (rh)-VEGFR-3, with ATP and polyGT as substrates.

SAR131675 dose-dependently inhibited rh-VEGFR-3-TK activity with an IC50 of 23 ±

7nM (Fig.1B, n=4). Under the same conditions SU11248 (sunitinib) inhibited VEGFR-3-

TK activity with an IC50 of 10nM (a representative set of data with sunitinib is shown in

supplementary Fig.1). SAR131675 is an ATP-competitive inhibitor; it inhibited VEGR-

3-TK activity with a Ki of about 12nM, similar to its IC50 value (supplementary Fig.1).

The effect of SAR131675 on VEGFR-3 auto-phosphorylation was evaluated after

overexpression in HEK cells. SAR131675 was seen to be cell permeable and inhibited

VEGFR-3 autophosphorylation in a dose-dependent manner with an IC50 ranging from

30-50nM (Fig.1C). In this assay, sunitinib inhibited VEGFR-3 autophosphorylation with

an IC50 ranging from 10-30nM (not shown). SAR131675 was also evaluated on two

different variants of VEGFR-3 and also on murine flt4. The results indicated that

SAR131675 has a similar inhibitory effect on both human variants and murine VEGFR-3

(supplemental Fig.1).

The selectivity of SAR131675 towards VEGFR-1 and VEGFR-2 was evaluated using rh-

VEGFR-1 and rh-VEGFR-2 ELISA assays as for VEGFR-3. SAR131675 inhibited

VEGFR-1-TK activity with an IC50 > 3µM (I%= 31 at 3µM) and VEGFR-2-TK activity

with an IC50 of 235nM (n=2) (Table 1).

In the same assay, sunitinib inhibited VEGFR-1 and VEGFR-2 with an IC50 of 64nM and

14nM, respectively (not shown). In the autophosphorylation assay in HEK cells,

SAR131675 inhibited VEGFR-1 autophosphorylation with an IC50 of about 1µM and

VEGFR-2 with an IC50 of about 280nM (Table 1). These results confirm that SAR131675

moderately inhibits VEGFR-2 and has very little effect on VEGFR-1, demonstrating a

good selectivity for VEGFR-3, which is not the case for sunitinib.

In order to further confirm the activity of SAR131675 on VEGFR-2, we used Porcine

Aortic Endothelial Cells (PAEC) stably expressing human VEGFR-2. SAR131675

inhibited VEGFA-induced VEGFR-2 phosphorylation in a dose dependent manner, with

an IC50 of 239nM, confirming the results obtained in HEK293T cells after VEGFR-2

overexpression (Fig.1D, E).

The selectivity of SAR131675 was further confirmed as it was inactive on a panel of 65

kinases, on a panel of 107 non-kinase enzymes and receptors and on 21 ion channels

(supplementary tables 1, 2A and 2B, respectively). We ruled out the eventuality of any

non-specific cytotoxic or cytostatic effect of SAR131675 by investigating its effect on the

proliferation of tumor cell lines. SAR131675 did not significantly inhibit the proliferation

of any of the cell lines studied at concentrations up to 10µM (supplementary table 3),

thus confirming the specificity of SAR131675.

Together, these results show that SAR131675 is a potent and selective inhibitor of

VEGFR-3-TK with moderate activity towards VEGFR-2 and with no cytostatic or

cytotoxic effects on tumor or primary cells.

In vitro effect of SAR131675 on lymphatic cell survival and migration

We evaluated the effect of SAR131675 on the in vitro survival of primary human

lymphatic cells induced by specific VEGFR-3 ligands, VEGFC and VEGFD, and by

unrelated growth factors, VEGFA and FGF-2. As shown in figure 2A, SAR131675

potently inhibited lymphatic cell survival induced by VEGFC and VEGFD (IC50 of 14

and 17nM, respectively). It inhibited VEGFA-induced survival with an IC50 of 664nM,

confirming its moderate activity on VEGFR-2 and had no effect on FGF2-induced

proliferation. In the same experiment, sunitinib inhibited VEGFA, - C and -D induced

survival with an IC50 of about 5nM and, consistent with reported data (38), it had no

effect on FGF2-induced survival (supplementary Fig.1).

We then evaluated the effect of SAR131675 on HLMVEC migration induced by VEGFA

or VEGFC in Boyden’s chambers. SAR131675 potently inhibited VEGFC-induced

migration with an IC50<30nM (Fig.2B) and VEGFA-induced migration with an IC50 of

about 100nM (Fig.2C).

Using the PhosphoMAP array in HLMVEC, Erk was identified as the major

phosphorylated kinase upon VEGFC-stimulation (Fig.2D). SAR131675, significantly and

dose dependently inhibited VEGFC-induced Erk phosphorylation, with an IC50 of about

30nM (Fig.2E), confirming that SAR131675 is a potent inhibitor of VEGFR-3 signaling.

Effect of SAR131675 on angiogenesis and lymphangiogenesis in non

tumoral models In vivo

The in vivo activity of SAR131675 was first investigated in embryonic angiogenesis

using the zebrafish model (Danio rerio) expressing GFP under the control of a vascular

specific promoter. Treatment with SAR131675 started when blood flow began, 24 hours

post fertilization. Embryos treated with 3µM SAR131675 for 24h showed fewer

intersegmental vessels in the tail compared to control embryos and at the dose of 10µM

intersegmental vessels largely failed to form (Fig.3A). Thus, in this model, SAR131675

efficiently impaired embryonic vasculogenesis.

Using a corneal model, a high concentration of FGF2 has been reported to induce

VEGFR-3-dependent lymphangiogenesis (39). We therefore developed a similar

approach by subcutaneous implantation of a sterile sponge disc impregnated with FGF2

in mice (Fig.3B). In this model, FGF2 induces angiogenesis, that is measured by CD31

immunostaining and hemoglobin content (Fig.3D), as well as lymphangiogenesis that is

measured by LYVE-1 immunostaining and by VEGFR-3 quantification using ELISA

(Fig.3E). FGF2 induced a 3- to 5-fold increase of hemoglobin and VEGFR-3, thus

confirming that in these conditions, FGF2 induces angiogenesis and lymphangiogenesis

(Fig.3I).

Treatment of mice with SAR131675 (30, 100 or 300 mg/kg/day) started on the day of

sponge implantation. Seven days later, SAR1316765 at 100 mg/kg/day had significantly

reduced the levels of VEGFR-3 (Fig.3H) and hemoglobin content (Fig.3I) by about 50%,

(p<0.001). Treatment with 300 mg/kg/day gave levels of VEGFR-3 and hemoglobin that

were similar to those of the control group. Thus, SAR131675 efficiently abrogates

lymphangiogenesis and angiogenesis induced in vivo by FGF2.

Furthermore, at 100mg/kg SAR131675 did not show any effect on arterial pressure

whereas at 300mg/kg, a minor and transient increase of blood pressure was observed

(supplementary Fig.2). Since the inhibition of VEGFR-2 signaling is known to increase

blood pressure, and given our in vitro results, we concluded that SAR131675 at a dose of

300mg/kg is able to inhibit both VEGFR-2 and VEGFR-3 signaling in vivo. In

consequence 100mg/kg was the highest dosage used in subsequent in vivo studies.

Effect of SAR131675 on carcinogenesis in the RIP1.Tag2 mice model

The RIP1.Tag2 transgenic mouse is a well-characterized multistep carcinogenesis model,

which arises from targeted oncogene expression in the insulin-producing beta cells (40).

We investigated the efficacy of SAR131675 on the angiogenic switch (prevention study),

on asymptomatic small tumors (intervention study) and finally on advanced near-end-

stage cancers (survival study) as illustrated in Fig.4A.

In the prevention study, 5 weeks treatment with SAR131675 was well tolerated and the

number of angiogenic islets in the pancreas of SAR131675 treated mice was significantly

decreased (42%, p<0.001) compared to the vehicle treated group (Fig.4B). In the

intervention study, daily oral administration of SAR131675 from Week 10 to Week 12.5,

caused a significant decrease in tumor burden (62%, p<0.05, Fig.4C). In these two studies

a significant decrease of CD31 positive vessels was observed in the SAR131675 treated

mice compared to control mice. In the survival study, where treatment started at Week 12

and continued until the death of the mice, mice under vehicle treatment started to die at

12 weeks with a median survival age of 13 weeks. Daily treatment with SAR131675

significantly extended the median survival age of the mice to 15 weeks (Fig.4D, p<0.01).

Taken together, these results demonstrate that SAR131675 is able to prevent the

angiogenic switch and slow tumor growth.

Effect of SAR131675 on growth and metastases of 4T1 mammary

carcinoma tumors in mice

4T1 cells were implanted orthotopically into mammary fat pads in mice who were then

orally treated with vehicle or SAR131675 (30 and 100mg/kg/day) from day 5 to day 21

after implantation.

SAR131675 was well tolerated since no loss of body weight was observed in the two

treated groups. Treatment with SAR131675 significantly reduced the tumor volume (24%

p<0.05 and 50% p<0.001 at 30 and 100mg/kg/d, respectively; Fig.5A).

Development of 4T1 tumors was associated with important vascularization and

peritumoral lymphangiogenesis as measured by CD31 and anti-LYVE1 immunostaining

(supplementary Fig.3A and C, respectively). Interestingly, as illustrated in supplementary

Fig.3B and D, SAR131675 seems to reduce both parameters. Moreover, SAR131675 at

30 and 100mg/kg/d significantly reduced VEGFR-3 levels in the tumors by 39% (p=0.05)

and 51% (p<0.01), respectively (Fig.5B), thus confirming its potent anti-

lymphangiogenic properties.

Since human breast cancer leads to high levels of osteopontin, we quantified osteopontin

levels in 4T1 cells in vitro and showed a good correlation with cell number (R2=0.998,

supplementary Fig.3E and F). Interestingly, the osteopontin content dramatically

increased in sentinel lymph nodes, indicating an infiltration by metastatic 4T1 cells

(Fig.5C). SAR131675, at 100mg/kg significantly reduced the osteopontin content in

lymph nodes (56%, p<0.01), indicating that SAR131675 has a potent effect on lymph

node invasion by 4T1 cells (Fig. 5C).

At day 21, the number of macroscopic lung metastases was reduced in the treated groups

by 17% (ns) and 28% (p<0.05) at 30 and 100 mg/kg/d, respectively (Fig.5D). Although

sunitinib strongly reduced tumor growth (82%, supplementary Fig.4A), it is interesting to

note that in this model, sunitinib at 50mg/kg/d had no effect on the number of lung

metastases (supplementary Fig.4A).

Altogether these results demonstrate that SAR131675 is a potent antitumoral agent,

acting through anti-angiogenic and anti-lymphangiogenic effects. Moreover it reduces the

migration of cancer cells into lymph nodes and lungs.

Effect of preoperative treatment with SAR131675 on distant metastasis

Recent reports suggest that anti-angiogenic compounds may promote tumor invasion

after resection of the primary tumor (41). We evaluated the effect of SAR131675 on

distant metastasis in the 4T1 model after resection of the primary tumor (Fig.6A).

Treatment was started on day 5-post cell injection, and the primary tumors were excised

by surgery at day 15. By this time the antitumoral effect of SAR131675 was already

significant (Fig.6B and C). SAR131675 induced an important decrease in the number of

lung metastases (Fig.6D), whether the treatment was pursued after primary tumor

resection or not. This indicates that SAR131675 blocks early events of tumor escape and

metastasis and suggests that treatment prior to tumor resection could be sufficient to

prevent tumor metastasis. As before, sunitinib was more efficient in reducing tumor

growth, but showed no significant effect on the number of lung metastases, whatever the

treatment schedule (supplementary Fig.4B).

Effect of SAR131675 on macrophage infiltration

TAMs play an important role in tumor promotion and metastasis (42). We analyzed

macrophage infiltration (with F4/80 immunostaining), using sections from 4T1 mammary

tumors and from the RIP1.Tag2 pancreas.

In 4T1 tumors, macrophages were mainly located at the periphery of the tumor at day 12-

post implantation, whereas they infiltrated the tumor and formed clusters in more

advanced tumors (day 21, Fig.7A). Treatment with SAR131675 decreased macrophage

infiltration and clustering as illustrated in figure 7A and consistently reduced F4/80 levels

in tumors as measured by an ELISA assay (Fig.7B).

A strong decrease of F4/80 staining in the RIP1.Tag2 model was also observed in the

angiogenic islets and the tumors from SAR131675-treated mice (supplementary Fig.3G).

These results indicate that reduction of tumor growth induced by SAR131675 in the

RIP1.Tag2 model may also arise from a reduction in TAM infiltration.

Together, these results from two models corroborate the hypothesis that tumor reduction

by SAR131675 is associated with a reduction in TAM infiltration.

Discussion

It is well established that VEGFR-3 is a key player in the control of lymphangiogenesis

but also angiogenesis. In addition, an increasing number of studies have shown its

upregulation in tumor cells and also on TAMs, suggesting that selectively targeting

VEGFR-3 might be an interesting therapeutic approach to reduce tumor growth and

metastasis. Identification of such selective inhibitors could also have the advantage of

providing maximal therapeutical effect without causing severe side effects. Here we

describe for the first time a small molecule with highly restricted selectivity for VEGFR-

3. SAR131675 is a potent and selective VEGFR-3 inhibitor, with a moderate activity on

VEGFR-2. It is able to block angiogenesis, lymphangiogenesis and TAM infiltration and

in consequence, it reduces tumor growth and metastasis.

It has been reported that targeting VEGFR-3 with specific inhibitors may block new

lymphatic growth exclusively, without any effect on the survival or function of existing

lymphatic vessels in adult mice (43, 44). In agreement with these reports, SAR131675 is

able to block the formation of new lymphatic vessels in a sponge model as well during

tumor-induced lymphangiogenesis, without any sign of lymphedema. Moreover,

treatment of mice with SAR131675 for about 3 months at 100mg/kg/d did not result in

any mortality.

Vascular complications have emerged as relevant toxicities associated with angiogenesis

inhibitors. Bevacizumab, a monoclonal antibody targeting VEGFA, has been linked to

hemorrhage, arterial and venous thrombosis, as well as hypertension (45). However, at

100mg/kg, SAR131675 did not induce any significant hypertension in rats, suggesting

that inhibition of the VEGFR-3 pathway is not responsible for this adverse event. This

also shows that at 100mg/kg, any effect of SAR131675 on VEGFR-2 activity is not

sufficient to induce hypertension in vivo. This minimal activity on VEGFR-2 in vivo was

also confirmed in zebrafish embryo studies. Indeed, in agreement with results obtained

with a morpholino antisense nucleotide of VEGFC or through overexpression of a soluble

form of VEGFR-3 (46), treatment with SAR131675 affected only intersegmental vessel

development but did not give rise to side effects such as the defects in artery development

seen in the VEGFR-2 KO zebrafish (11).

Although angiogenesis inhibitors demonstrate antitumor effects in several mouse models,

they have been reported to concomitantly elicit increase of lymphatic and distant

metastasis. Indeed, an anti-VEGFR-2 neutralizing antibody reduces tumor vasculature

and volume and also increases incidence of lymph node and liver metastasis in

RIP1.Tag2 mice (47, 48). In this same model, SAR131675 reduced tumor volume in both

prevention and intervention studies without any prominent invasive fronts in the treated

animals. Increased metastasis was also observed in mice receiving short-term therapy

with sunitinib (41). In the 4T1 model, SAR131675, but not sunitinib, significantly

reduced lymph node invasion and distant metastasis. In addition, short-term treatment

prior to tumor resection with SAR131675 was sufficient to reduce lung metastasis, but

this was not the case with sunitinib.

In addition to its anti-metastatic activity, SAR131675 significantly reduced the tumor

volume in several experimental tumor models, including colon, mammary and prostate,

and in RIP1.Tag2 mice. These data are consistent with those reported previously which

showed that blocking the VEGFR-3 pathway may block primary tumor growth (49).

Three major mechanisms could explain the role of VEGFR-3 in primary tumor growth:

an autocrine effect on tumor cells, pathological angiogenesis, and TAMs.

An increasing number of studies have shown the expression of VEGFR-3 on tumor cells

in human cancer patients (5, 17-19, 21-22). However, this phenomenon has never been

reported in mouse tumor models. In agreement with this, VEGFR-3 immunostaining of

RIP1-Tag2 and 4T1 tumors confirmed that VEGFR-3 is not induced on these tumor cells

in vivo (not shown).

Although VEGFR-3 is not expressed on adult blood vessels (3), high expression of

VEGFR-3 has been reported in angiogenic sprouts and blocking VEGFR-3 signaling with

monoclonal antibodies results in decreased sprouting and vascular density in mouse

angiogenesis models (16). Thus, the reduction of tumor growth by SAR131675 could be

due to inhibition of angiogenesis. This would be consistent with the reduction of

angiogenesis in the sponge model, which expresses high levels of VEGFR-3 and also

with the decrease in vascular density in the 4T1 and the RIP1.Tag2 models. Thus

SAR131675 may block angiogenesis through different non-exclusive mechanisms: by

inhibiting VEGFR-3 signaling in endothelial cells or by blocking VEGFA- or VEGFC-

induced VEGFR-2/VEGFR-3 heterodimers. It is worth noting that the moderate VEGFR-

2 activity of SAR131675 may also contribute to its activity on tumor angiogenesis.

Finally, the anti-angiogenic effect may result from the effect of SAR131675 on TAM

infiltration. TAMs promote angiogenesis by releasing pro-angiogenic factors such as

VEGFA and VEGFC, and thereby induce tip-cell formation and fusion (16, 29, 32). It is

well established that macrophages and also TAMs express VEGFR-3 (28-30). Moreover,

VEGFR-3 inhibition decreased dendritic cell recruitment to the spleen and resulted in

prolonged rat cardiac allograft survival (50). Here we have shown that SAR131675

strongly reduced TAM infiltration in the 4T1 and the RIP1.Tag2 models. This reduction

was associated with reduced tumor growth, reduced metastasis and extended median

survival of RIP1.Tag2 mice. The effect of SAR131675 on macrophage differentiation

and recruitment and on anti-tumoral immune response is under evaluation.

Acknowledgements:

We are grateful to Carles Callol (Biobide) for the experiment performed on zebrafish

embryos, to Sylvie Sordello (TSU Infectious Diseases, Sanofi Toulouse), for the

experiment on tumor resection, and to Natalio Vita (E2C, Sanofi, Toulouse) for his

critical review of this manuscript and to Fiona Decry (E2C, Sanofi, Chilly) for English

revision.

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Table 1: Effect of SAR131675 on human VEGFR-1 and VEGFR-2 using recombinant

enzymes or after overexpression in HEK cells. The IC50 geometric mean are represented

VEGFR-1 VEGFR-2

rhVEGFR-1 HEK- VEGFR-1 rhVEGFR- 2 HEK-VEGFR- 2

SAR131675 I%= 31 at 3µM I%= 48 at 1µM 235 nM 280 nM

Figure Legends

Fig1: Effect of SAR131675 on VEGFR Tyrosine kinases. A) Structure of SAR131675.

B) Effect of SAR131675 on kinase activity of recombinant human VEGFR-3 measured

in the presence of 30µM of ATP. C) Effect of SAR131675 on VEGFR-3 auto-

phosphorylation after overexpression in HEK cells. These data are representative of

several assays. C) Effect of VEGFA on VEGFR-2 phosphorylation in PAEC stably

expressing VEGFR-2. D) Effect of SAR131675 on VEGFA-induced VEGFR-2

phosphorylation.

Fig2. Effect of SAR131675 on human primary lymphatic cells. A) Effect on lymphatic

cell survival. Cells were plated in medium containing 0.1% FCS. Cells were then left

unstimulated or stimulated with VEGFA (10ng/ml) VEGFC (300ng/ml), VEGFD

(300ng/ml) and bFGF (10ng/ml) in the presence of increasing SAR131675 concentrations.

Data are expressed as percentage of inhibition, and plotted on SPEED software. Results

are representative of 3 independent experiments. B and C) Migration assay of HMVECs.

Cells were seeded in triplicate in the upper wells of a fluoroblok chamber. RPMI alone or

containing VEGF-A 50 ng/mL or VEGF-C 100 ng/mL was added in the bottom wells in

the presence of increasing doses of SAR131675 (30-300 nM for VEGFC and 100-

1000nM for VEGFA). The amount of migrating cells was quantified using calcein

labeling. Data are shown as mean ± standard error of the mean. D) Effect of VEGFC on

MAP-kinases. HMVEC were left unstimulated or stimulated for 10mn with VEGFC

(500ng/ml) and lysed. +: Represents positive controls, the spots corresponding to Erk and

Akt are indicated. Cell lysates were than incubated with array membranes. E) Effect of

SAR131675 on Erk phosphorylation in lymphatic cells. Cells were stimulated as

described in B in the presence of increasing concentration of SAR131675. Erk

phosphorylation was quantified by ELISA. Results are expressed as mean ± standard

error of the mean, significant differences are assessed by ANOVA followed by Dunnett

test vs VEGF-C, * p<0.05; ** p<0.01; *** p<0.001.

Fig3: In vivo effect of SAR131675 on embryonic and adult angiogenesis and

lymphangiogenesis. A) Lateral view of zebrafish embryos which specifically express the

fluorescent protein copGFP in the vascular system. Controls show the usual pattern of

intersegmental blood vessels (ISV) which failed to form after treatment with 3µM and

10µM of SAR131675 (arrowhead). B and C) Induction of angiogenesis and

lymphangiogenesis by FGF2 (200 ng) in a sponge implanted in the back of mice.

Angiogenesis and lymphangiogenesis were evaluated at day7 by immunochemistry

staining with CD31 (D) and anti LYVE-1 antibodies (E), respectively. Visual effect of

vehicle (F) and SAR131675 (100mg/kg/d, G) on FGF2-induced angiogenesis and

lymphangiogenesis. Dose effect of SAR131675 on VEGFR-3 levels (H) and on

hemoglobin content (I) in the sponge. Results are expressed as mean ± standard error of

the mean, by ANOVA followed by Dunnett test vs FGF-2 stimulated group. ** p<0.01;

*** p<0.001.

Fig.4 Effect of SAR131675 on a spontaneous multistage tumor model (RIP1.Tag2)

(A) Studies designed to target discrete stages of tumoral growth in the RIP1.Tag2

transgenic mice. In the prevention study (B), mice were treated with vehicle or with

SAR131675 (100mg/kg/d), from Week 5 to Week 10 and the number of angiogenic islets

(the transition from pre-vascular hyperplasia to highly vascularized and progressively

outgrowing tumors), were measured at 10 weeks of age. For the intervention study (C)

mice were treated with vehicle or with SAR131675 (100mg/kg/d), starting at Week 10

and the total tumor volume was calculated at Week 12 by measuring each tumor volume

with calipers. Error bars represent the SEM of data from 9 mice/group, * p<0.05 and ***

p<0.001, Student’t test vs control group. (D) Monitoring of RIP1.Tag2 mice survival

following daily treatment with vehicle or with SAR131675 (100mg/kg) starting from

Week 12 (n=20 mice/group, p=0.01, log-rank test).

Fig 5: Antitumoral effect of SAR131675 treatment on a murine mammary

carcinoma model. A) Tumor volume monitoring in Balb/C mice in control or mice

treated with SAR131675 at 30 and 100 mg/kg/d. B) Murine VEGFR3 levels were

quantified by ELISA in the tumor lysates of each treated group at the end of the

experiment (day 21).

C) Quantification of osteopontin levels by ELISA in the lymph nodes after vehicle or

SAR131675 treatment. Results are expressed as mean ± standard error of the mean,

*p<0.05; ** p<0.01; *** p<0.001, ANOVA followed by Dunnett test vs vehicle group. In

A, two-way ANOVA analysis with repeated measures was performed. D) Number of

metastases at the surface of the lungs for each treated group.

Fig 6: Anti metastatic effect of SAR131675 after 4T1 tumor resection. A) Experiment

schedule. SAR131675 treatment at 100mg/kg/d starts 5 days after 4T1 cell implantation

in mammary fat pads, at day 15 primary tumors were removed in all groups and the

tumor weight was evaluated (B). In the group noted 10 days, the SAR131675 treatment

stopped at day 15 and mice were studied un-treated until day 26. In the group noted 20

days, the treatment was continued until the end of the experiment (day 26). C) Murine

VEGFR3 levels were quantified by ELISA in tumors lysates at day 15. D) The number of

metastases was counted at the lung surface at day 26. E) Illustration of the number of

lung metastases after Bouin fixation. Results are expressed as mean ± standard error of

the mean, significant differences between groups are assessed by ANOVA followed by

Dunnett test, * p<0.05; ** p<0.01; *** p<0.001.

Fig 7: Effect of SAR131675 on macrophage infiltration at different stages of tumor

development. A) Immunohistochemical staining of F4-80 in 4T1 mammary tumors at

day 12 and day 21 in control and SAR131675 treated mice (40x; 400x magnification). B)

Quantification of F4-80 levels by ELISA dosage in the 4T1 mammary tumor lysates at

day 21 in vehicle and SAR131675 treated mice (100mg/kg/d).

Fig.1

A ONH

NNNH OH 2

B C 100 100 n n n n 80 80 60 60 40 40

20 Inhibitio % 20 % Inhibitio % 0 0

1 10 100 1000 1 10 100 1000 [SAR131675] (nM) [SAR131675] (nM)

D E

A 120 100 Control

VEGF-A 80 60 Anti-VEGFR-2 200kDa 40 Inhibition

Anti-PY 200kDa % 20 0

1 10 100 1000 10000 [SAR131675](nM)

Fig.2

AB 2500

2000

120 1500 *** VEGF-D *** 1000 100 *** VEGF-C 500

80 Luminescence (AU) VEGF-A 0 NS 0 30 100 300 60 FGF-2 VEGF-C + SAR131675 (nM)

40 C

% inhibition % 2500 20 U) A A 2000 0 *** 1500 *** *** -20 1000

0 10 100 1000 500 Luminescence ( [SAR131675] (nM) 0 NS 0 100 300 1000 VEGF-A + SAR131675 (nM)

D E

+ 080.8 NS

0.6 ** ** + + 0.4

.(450nm) *** D VEGFC D

+ O. 0.2 *** *** Erk + Akt 0.0 + NS VEGF-C 1 3 10 30 100 300 500ng/ml 10mn VEGFC + SAR131675 (nM)

Fig.3

Control SAR131675 3 µM SAR131675 10 µM

B D F G

C E

H I

3.5 300 3.0 x 4 250 x 2.9 ** 2.5 ** 200 * *** 2.0 *** 150 *** 1.5 EGFR3 (pg/ml) EGFR3 *** moglobin (mg/ml) V V e 100 e

H 1.0 50 0.5 0 0 Vehicle 0 30 100 300 Vehicle 0 30 100 300

FGF-2 + SAR131675 (mg/kg/d) FGF-2 + SAR131675 (mg/kg/d)

Fig.4

†

Week 5 Week 10 Week 12

Prevention Intervention survival

B C

18 16 35 SAR131675 @ 10W § SAR131675 @12W 14 30 12 25 10 20 *** 8 r burden r burden (mm3) iogenic islets iogenic o g g 15 6 *

10 Tum 4

Nb of an 5 2

0 0 Control SAR131675 Control SAR131675

D 100

75 al v 50 SAR131675 p=0.01 Control 25 % mice survi % 0 10 12.5 15 17.5 weeks

Fig.5

B A 1800 Vehicle 1600 1600 SAR131675 30 mg/kg/d 1400 SAR131675 100 mg/kg/d 1200 1200 p=0.05 *** 1000 ** ** 800 ** 800

* (pg/ml) FR-3 or volume or volume (mm3) *** 600 G *** G *** VE Tum 400 *** 400 ** * 200 0 0 0 5 10 15 20 Vehicle SAR 131675 SAR 131675 Days 30mg/kg/d 100mg/kg/d C D 400 35 ns 350 30

300 25 * g/ml) r in lungs r in

p p 250 ** 20 200 15 150 10 100

Metastasis numbe 5 50 Osteopontin level level ( Osteopontin 0 0 Negative Vehicle SAR131675 Vehicle SAR131675 SAR131675 lymph nodes 100mg/kg/d 30mg/kg/d 100mg/kg/d

Fig.6:

Day 0: 4T1 cells A implantation Day 5: Start of Day 15: p.o. treatment Excision Day 26 Lung SAR131675 (20d treatment) Metastasis number SAR131675 (10d treatment)

B C

0.8 500 l) g) ( m m

0.6 400 *** 300 ** 0.4 200 or weightat day 15 level 15 (pg/ at day level 3 3 m m 002.2 100 Tu

0.0 VEGFR 0 Vehicle SAR131675 Vehicle SAR131675

D E 120

d26 at number 60 ** s s **

0 Lung metastasi Vehicle 10days 20days Vehicle SAR131675 SAR131675 (10 days treatment group)

Fig.7:

A B F4/80 IHC on 4T1 mammary carcinoma model Day 12 Day21 30 Control Control SAR131675

25 mor

u 20

*** 40x 40x 40x 15

10 Arbitratry Unit/ t Unit/ Arbitratry

400x 400x 400x

SAR131675 Vehicle 100mg/kg/d

SAR131675, a potent and selective VEGFR-3-TK inhibitor with antilymphangiogenic,anti-tumoral and anti-metastatic activities.

Antoine Alam, Isabelle Blanc, Geneviève Gueguen-Dorbes, et al.

Mol Cancer Ther Published OnlineFirst May 14, 2012.

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