www.sciencetranslationalmedicine.org/cgi/content/full/6/237/237ra68/DC1

Supplementary Materials for

Merlin Deficiency Predicts FAK Inhibitor Sensitivity: A Synthetic Lethal Relationship

Irina M. Shapiro, Vihren N. Kolev, Christian M. Vidal, Yuwaraj Kadariya, Jennifer E.

Ring, Quentin Wright, David T. Weaver, Craig Menges, Mahesh Padval, Andrea I. McClatchey, Qunli Xu, Joseph R. Testa, Jonathan A. Pachter*

*Corresponding author. E-mail: jpachter@verastem.com

Published 21 May 2014, Sci. Transl. Med. 6, 237ra68 (2014) DOI: 10.1126/scitranslmed.3008639

This PDF file includes:

Materials and Methods Fig. S1. Chemical structure of VS-4718. Fig. S2. Western blot analysis of lysates from cancer cell lines in Fig. 1A. Fig. S3. pFAK-Y397 levels in MDA-MB-231 and MDA-MB-468 xenograft tumor samples. Fig. S4. Average EC50 values for Fig. 2B. Fig. S5. Relative growth of MDA-MB-231/GFP and MDA-MB-231/NF2-GFP cells in response to VS-4718. Fig. S6. Quantitative polymerase chain reaction analysis of FAK cDNA in MSTO-211H and Mero-41 cells. Fig. S7. TGI and pFAK-Y397 expression analysis in MSTO-211H xenograft model. Fig. S8. Merlin and pFAK-Y397 expression in human mesothelioma PDX models. Fig. S9. pAKT and pFAK activation in Mero-48a and MSTO-211H cell lines. Fig. S10. pFAK-Y397 immunofluorescence analysis in Mero-41 and Mero-48a cells expressing NF2-GFP. Fig. S11. N-cadherin–blocking antibody treatment in H28 and MSTO-211H cells. Fig. S12. Quantification of pFAK-Y397 in H28, Mero-25, Mero-83, and Mero-41 cells. Fig. S13. Tumorsphere self-renewal and in vivo differentiation analysis of Aldefluor+ MM87 cells. Fig. S14. Effect of VS-4718 treatment on Aldefluor+ CSCs in Mero-83 and Mero-48a MPM cells.

Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/6/237/237ra68/DC1)

Table S1. Original data from in vivo studies (provided as an Excel file).

SUPPLEMENTARY MATRIALS

Materials and Methods

Cell culture

Cell lines MDA-MB-231, H2052, MSTO-211H, H28, BT549, MDA-MB-468, MDA-MB-453,

MDA-MB-436, MDA-MB-361, SUM159, SUM149, HCC1954, H2452, WM-115, WM-266-4,

G-361, C32, A375, COLO 829, HT-144, PA-1, ES-2, UWB1.289, Hs38.T, CAOV-4, OVCAR8,

OVCAR5, TOV-112D, A549, Calu-1, NCI-H1975, NCI-H358, NCI-H441, NCI-H460, HCC827,

786-O, ACHN, Caki-1, CAL-62, SW579, TT and 8505C were obtained from ATCC and were

propagated according to ATCC guidelines. COLO 679, A2058, A2780, COV318, COV434,

OV56, OV-7, OV-90, Mero-14, Mero-41, Mero-48a, Mero-83 were obtained from Sigma and

were propagated according to Sigma guidelines except Mero cell lines, as indicated below.

Mouse mesothelioma cell lines MM87 and MM129, were generated in the laboratory of one of

the authors (J.R.T.) and have been described previously (1). Cell lines Mero-41, Mero-25, Mero-

14 and Mero-83 were propagated in Ham’s F-12 media (Gibco, Life Technologies)

supplemented with 15% HI FBS (Gibco, Life Technologies), 1000 IU/ml penicillin and 1 mg/ml

streptomycin.

Matrigel-on-top 3D culture

Cells were plated above a dense layer of Matrigel (BD Biosciences), diluted 1:1 with culture

medium. Cells were prepared as single cell suspensions in dilute Matrigel (1:50 in culture

medium) and seeded at low densities (~3000 cell/cm2) to cover the Matrigel base layer. Cells

were treated with VS-4718 at various concentrations the next day after plating. MTS assay was

used after 4 days of treatment to measure cell viability and to assess the compound effect on cell

proliferation. Nonlinear regression analysis was used to generate a graphical representation of

the results (Graphpad software).

Aldefluor assay

Cells were treated for 4 days with VS-4718, pemetrexed, cisplatin, vinorelbine or gemcitabine

prior to Aldefluor assay. An Aldefluor kit (StemCell Technologies) was used to isolate the

population with high ALDH enzymatic activity. MM87 cells were suspended in Aldefluor assay

buffer containing ALDH substrate (BAAA) and incubated for 15 min at 37°C. In each

experiment, a sample of cells was incubated with the specific ALDH inhibitor

diethylaminobenzaldehyde (DEAB) as a negative control. Hoechst dye was used to quantify the

total number of cells. Plates were imaged using Celigo (Brooks Automation), and Aldefluor-

positive and total cell numbers were quantified. The proportion of Aldefluor+ cells was

calculated relative to DEAB control and plotted as a percentage of DMSO-treated cells.

Limiting dilution assay

MM87 cells were stained using an Aldefluor kit as described above. Aldefluor-positive and

Aldefluor-negative cells were sorted using an Aria II (BD) cell sorter at the Core for Flow

Cytometry (BIMS). Cells were collected by centrifugation, washed with HBSS, re-suspended in

1 ml HBSS, and live cells were counted. Cells were mixed with 50% Matrigel (BD) prior to

implantation in 6-week old female SHrN (Harlan) mice. Tumor growth was monitored for a

maximum of 16 weeks. Tumor-initiating frequency (TIF) was calculated by L-CalcTM Software

(Stemcell Technologies Inc.).

pFAK ELISA

pFAK in tumor samples was measured as follows. Tumors were harvested within 2 hr of last

dosing and stored in RNALater at -80˚C until future use. Tumors were thawed and removed from

the RNALater for protein extraction. Thin slices were cut from representative areas of the tumor

and homogenized in lysis buffer (M-PER, 3% HALT-Protease/Phosphatase Inhibitor Cocktail,

1.5 mM EDTA and 0.05% Qiagen reagent DX (Pierce)) on the Qiashredder (Qiagen). 50 µL of

protein lysate per tumor was used to quantify total and phospho-FAK Y397 levels according to

the manufacturer’s instructions (Total FAK ELISA, FAK pY397 ELISA; Invitrogen). Data were

calculated as a ratio of pFAK to total FAK for each tumor analyzed and plotted using Prism

GraphPad 6.0 software. An unpaired t-test with Welch’s correction was used to determine the

statistical significance of the data.

pFAK in cells was measured directly in Matrigel (MoT cell culture). Briefly, 10X lysis buffer

was added directly to wells (96-well plate) containing Matrigel and cells. After 1 hr lysis on ice,

the cell plate was spun and 50 µl of lysate was transferred to an ELISA plate. pFAK ELISA was

performed as described above.

Antibodies and Western blotting

Cells were lysed in the presence of M-PER Mammalian Protein Extraction Reagent (Thermo

Scientific) on ice. Twenty micrograms of total protein from each sample were resolved on an

8%–10% SDS-PAGE Gel with Laemmli Running Buffer and transferred to PVDF membranes.

The blots were then probed with the following antibodies: anti-Merlin (clone D1D8), anti-FAK,

anti-pFAK-Y397, anti-pan AKT, anti-pAKT-S473, anti-ERK1/2, anti-pERK-T202/Y204, anti-

actin (Cell Signaling Technologies); anti-N-cadherin and anti-E-cadherin (BD Biosciences).

Blocking antibody experiments

Blocking antibody experiments were conducted with anti-β1 integrin antibody (clone Mab13;

BD Biosciences) or anti-N-cadherin antibody (Clone GC-4; Sigma). MoT assay was performed

as described above with the following modifications. Cells were pre-incubated with blocking

antibodies for 1 hr at room temperature with rotation. Blocking antibodies were added to the

dense layer of Matrigel at the bottom of the well and to the cell suspension. Purified rat IgG2a

isotype control (BD Pharmingen) was used as a negative control for the β1 integrin antibody

blocking. Mouse myeloma IgG2a (Invitrogen) was used as a negative control for the N-cadherin

antibody.

Immunofluorescence of tissue sections

Paraffin blocks were cut into 5 µm sections and placed on positively charged microscope slides.

Sections were de-waxed in xylene and hydrated through a graded ethanol series. Heat-induced

antigen retrieval was performed in a sodium citrate (pH=6.0) in a microwave for 5 min. Slides

were incubated for 2 hrs with donkey serum in 3% BSA/PBS, followed by overnight incubation

with anti-ALDH1A1 antibody (Novus Biologicals). Cell nuclei were counterstained with

Hoechst 33342 (Invitrogen). Alexa Fluor 488 or 594 donkey anti-mouse or anti-rabbit IgG

(1:200, Invitrogen) was used for secondary detection. Tissue sections were imaged using a Nikon

Eclipse Ti microscope (Nikon, 20X/0.45 S Plan Fluor objective) and processed using Metamorph

software (Molecular Devices).

Cell viability and caspase activation assays

For the cell viability assay, CellTiter 96® AQueous One Solution Cell Proliferation Assay kit

(Promega), MTS) was used following the manufacturer's instructions. Briefly, 20 µl of the MTS

reagent was added into each well and cells were incubated at 37°C for 2 hr. The reaction was

stopped by addition of 25 µl of 10% SDS into each well. Absorbance was detected at 490 nm

with an EnVision Multilabel Reader (Perkin Elmer). Nonlinear regression analysis was used to

generate a graphical representation of the results (Graphpad software).

For the caspase activity assay, Caspase-Glo®3/7 Assay (Promega) was used to assess caspase3/7

activity in 96-well MoT format. Luminescence was detected with an EnVision Multilabel

Reader (Perkin Elmer). Nonlinear regression analysis was used to generate a graphical

representation of the results (Graphpad software).

Mouse models

For mouse models of mesothelioma, all animal experiments were performed in accordance

with the regulations of Fox Chase Cancer Center’s Institutional Animal Care and Use

Committee. 8-10-week-old ICR SCID mice were used for intraperitoneal (i.p.) injection of the

Nf2-/- mouse mesothelioma cells. 20 mice were each injected i.p. with 1 million cells. Two

weeks after the injections, 5 mice per group were treated with VS-4718 at 25 mg/kg, 50 mg/kg,

75 mg/kg or vehicle control (citrate buffer) twice daily (BID) by oral gavage for 10 days. 10

days after the start of the treatment regimen, mice were sacrificed, tumors excised, photographed

and measured. Tumor fragments were then stored in RNALater reagent (Qiagen) and pFAK-

Y397 and total FAK measurements were performed as described in the pFAK ELISA Methods

section.

For a lung tumor model, 8-10-week-old ICR SCID mice were used for tail vein injection

of MM87 mouse mesothelioma cells. 20 mice were each injected via tail vein with 0.5 x 106

cells. One week after injections, 10 mice per group were treated with VS-4718 at 75 mg/kg or

vehicle control (0.5% methylcellulose solution) twice daily (BID) by oral gavage for 18 days,

with one day drug holiday in the middle. Eighteen days after the start of the treatment regimen,

mice were sacrificed, lungs excised, photographed and measured. The average lung weight from

non-tumor bearing littermates was subtracted from lung weights of tumor-bearing animals to

calculate net tumor weight in each lung. An unpaired t-test with Welch’s correction was used to

determine the statistical significance of the data.

Human MPM patient derived xenograft experiments were performed at Champions

Oncology. Immune compromised mice (Harlan; nu/nu) between 5-8 weeks of age were

implanted unilaterally on the left flank with tumor fragments harvested from host animals

(passage 4). When tumors reached approximately 125-250 mm3, animals were randomized into

treatment or control groups and dosing was initiated. 10 mice per group were used in the study.

Control mice were treated with vehicle (0.5% CMC + 0.1% Tween 80) p.o. BID. VS-4718 was

administered at 50 mg/kg p.o. BID. Cisplatin was administered at 4 mg/kg i.p. every 7 days.

Pemetrexed was administered at 75 mg/kg i.p. once daily for 5 days followed by a 2 day holiday

on a 7 day schedule during the same period of time as when cisplatin was administered.

Cisplatin/pemetrexed treatment was performed for 14 days, at which point treatment was stopped

and tumors were allowed to regrow, or VS-4718 was administered at 50 mg/kg PO BID for

another 32 days after a 2 day break following completion of cisplatin/pemetrexed treatment. A

one-way ANOVA was used to determine statistical significance of the results.

MDA-MB-468 and MDA-MB-231 s.c. xenograft experiments were performed at

Translational Drug Development, Inc. 4-week-old female ICR-SCID mice (Harlan) were

inoculated in the right inguinal mammary fat pad with 0.1 ml of a 50% RPMI / 50% Matrigel™

(BD Biosciences) mixture containing a suspension of MDA-MB-468 or MDA-MB-231 tumor

cells (approximately 107 cells/mouse). Thirty-five days following inoculation, tumors were

measured using calipers and tumor weight was calculated using the animal study management

software (Study DirectorV.1.8.4m). Thirty mice with tumor sizes of 90-187 mg were randomized

into three groups of 10 mice each by random equilibration, using Study Director (Day 1). Dosing

was performed with vehicle control, VS-4718 at 25 mg/kg p.o. BID or 100 mg/kg p.o. BID. The

study was terminated on Day 35, upon reaching endpoint of vehicle control mean tumor weight

exceeding 750 mg. A one-way ANOVA was used to determine statistical significance of the

results.

MSTO-211H s.c. xenograft experiments were performed at Translational Drug

Development, Inc. 4-5 weeks old female athymic nude mice (Taconic) were inoculated in the

right side flank with 0.1 ml of a 50% RPMI/50% Matrigel™ (BD Biosciences) mixture

containing a suspension of MSTO-211H tumor cells (approximately 5.0 x 106 cells/mouse). 12

days following inoculation, tumors were measured using calipers and tumor weight was

calculated using the animal study management software, Study Director V.1.8.4m (Study Log).

18 mice with tumors weights of 103-195 mg were randomized into two groups of nine mice by

random equilibration, using Study Director. Dosing was performed with vehicle control or VS-

4718 at 75 mg/kg p.o. BID. The study was terminated on Day 35, upon reaching endpoint of

vehicle control mean tumor weight exceeding 750 mg. A non-parametric t-test with Welch

correction was used to determine statistical significance of the results.

Supplementary figures

Fig. S1. Chemical structure of VS-4718.

Fig. S2. Western blot analysis of lysates from cancer cell lines in Fig. 1A.

Cell lysates from MPM cell lines were analyzed by SDS-PAGE and blotted with anti-FAK and

anti-Merlin antibody, as indicated. Actin was used as a loading control.

Fig. S3. pFAK-Y397 levels in MDA-MB-231 and MDA-MB-468 xenograft tumor samples.

pFAK-Y397 levels were measured by ELISA in MDA-MB-231 (A) and MDA-MB-468 (B)

xenograft tumor samples from Fig.1, normalized to total protein and depicted as a percent of

control. (A) Data are presented as mean ±SD (N=3), representative of two technical replicates.

p=0.03 (Kruskal-Wallis test). (B) Data are presented as mean ± SD (N=6), representative of two

technical replicates. p= 0.0002 (Kruskal-Wallis test).

Fig. S4. Average EC50 values for Fig. 2B.

Fig. S5. Relative growth of MDA-MB-231/GFP and MDA-MB-231/NF2-GFP cells in

response to VS-4718.

Data are representative of two experiments and are presented as mean ±SEM (n=6).

Fig. S6. Quantitative polymerase chain reaction analysis of FAK cDNA in MSTO-211H and

Mero-41 cells.

Mero-41 cells were treated with FAK siRNA or control non-targeting siRNA, as indicated.

∆∆CT method with GAPDH as a reference was used for quantification. Data are representative

of two biological replicates.

Fig. S7. TGI and pFAK-Y397 expression analysis in MSTO-211H xenograft model.

(A) Dot plot of tumor volumes in MSTO-211H s.c. xenograft model treated with VS-4718 BID

for 29 days, as indicated. Black lines represent the mean, whiskers depict 25% and 75% of the

distribution. Difference between groups is not significant (ns); p=0.092 (unpaired t-test with

Welch’s correction). (B) Dot plot of pFAK-Y397 levels measured by ELISA in MSTO-211H

xenograft tumor samples from (A). **p=0.0051 (unpaired t-test with Welch’s correction).

Fig. S8. Merlin and pFAK-Y397 expression in human mesothelioma PDX models.

A) Lysates from MPM PDX tumor samples analyzed by SDS-PAGE and probed with anti-

Merlin antibody. Actin was used as a loading control. B) and C) Bar graphs of pFAK-Y397

levels measured by ELISA in Merlin-negative (B) or Merlin-positive (C) MPM PDX tumors

normalized to total protein and depicted as a percent of control. Data are presented as mean ±

SEM (N=8). ***p=0.0003 (unpaired t-test with Welch’s correction); *p=0.038 (Mann-Whitney

test).

Fig. S9. pAKT and pFAK activation in Mero-48a and MSTO-211H cell lines.

Protein lysates from Mero-48a cells (A) or MSTO-211H cells (B) treated with 1 µM VS-4718, as

indicated, in MoT culture analyzed on SDS-PAGE and blotted with antibodies, as indicated.

Actin was used as a loading control.

Fig. S10. pFAK-Y397 immunofluorescence analysis in Mero-41 and Mero-48a cells

expressing NF2-GFP.

Cells were stained with anti-Merlin and anti-pFAK-Y397 antibodies, as indicated. Red arrows

mark focal adhesions with bright pFAK staining. Scale bar = 15 µM.

Fig. S11. N-cadherin–blocking antibody treatment in H28 and MSTO-211H cells.

A) Lysates from H28 and MSTO-211H cells analyzed on SDS-PAGE and probed with anti-N-

cadherin and anti-E-cadherin antibodies. Actin was used as a loading control. B)

Immunofluorescence analysis of MSTO-21H cells treated with control IgG or N-cadherin

blocking antibody analyzed by immunofluorescence with anti-β-catenin antibody (red),

AlexaFluor 488 conjugated phalloidin (actin, green) and DAPI to visualize nuclei (blue). Red

arrow marks cell-cell junction with bright β-catenin staining. Blue arrow marks cell-cell junction

that lost β-catenin. Scale bar = 15 µM. C) Relative growth of Merlin-positive H28 cells treated

with control IgG or N-cadherin blocking antibody in response to VS-4718, shown as percent of

control. Data are presented as mean ± SEM (N=6), representative of two biological replicates.

Fig. S12. Quantification of pFAK-Y397 in H28, Mero-25, Mero-83, and Mero-41 cells.

Bar graph of H28, Mero-25, Mero-83 and Mero-41 cells treated with IgG control or β1 integrin

blocking antibody. pFAK-Y397 levels were measured by ELISA, normalized to total FAK levels

and depicted as a percent of control. Data are presented as mean ± SD (N=4), representative of

two biological replicates.

Fig. S13. Tumorsphere self-renewal and in vivo differentiation analysis of Aldefluor+

MM87 cells.

A) Bar graphs of the numbers of primary and secondary tumorspheres formed by Aldefluor+

MM87 cells depicted as a number of spheres per 500 plated cells. For secondary tumorspheres,

primary tumorspheres were dissociated into a single cell suspension, counted and re-plated in the

fresh tumorsphere media. Data are presented as mean ± SD (N=3). B) % Aldefluor+ and

Aldefluor- negative cells in dissociated xenograft tumors formed by injecting 103 or 104 FACS

sorted Aldefluor+ MM87 MPM cells and grown for 3 weeks.

Fig. S14. Effect of VS-4718 treatment on Aldefluor+ CSCs in Mero-83 and Mero-48a MPM

cells.

Data are presented as mean ± SEM (N=6), representative of two biological replicates.