mct1-mediated transport of a toxic molecule is an...

MCT1-mediated transport of a toxic molecule is an effective

strategy for targeting glycolytic tumors

Kivanc Birsoy,1,2 Tim Wang,1,2,3 Richard Possemato,1,2 Omer H. Yilmaz,1,2 Catherine E.

Koch,1,2 Walter W. Chen,1,2 Amanda W. Hutchins,1,2 Yetis Gultekin,1,2 Tim R. Peterson,1,2

Jan E. Carette,1,6 Thijn R. Brummelkamp,1,7 Clary B. Clish,3 and David M. Sabatini1,2,3,4,5

1Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA.

2Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.

3Broad Institute, Seven Cambridge Center, Cambridge, MA 02142, USA.

4David H. Koch Institute for Integrative Cancer Research at MIT, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

5Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA.

6Current address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.

7Current address: Department of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121 1066 CX, Amsterdam, The Netherlands.

To whom correspondence should be addressed. Email: [email protected]

Nature Genetics: doi:10.1038/ng.2471

cleavedCASPASE3

RPS6

Clone

BClo

ne AWT

KBM7

0.66% 3.06%

2.94%96.34% 65.16% 18.9%

97.26% 96.17%

1.93% 1.99%

1.65% 1.25%

95.42% 94.60%

3.01% 3.66%

1.26% 1.09%

7AA

D

Annexin V-PE

PBS 50uM 3-BrPa

Wild

Typ

eC

lone

AC

lone

B

a b

0 200 400 600 800 1000 1200

-log

(p_v

alue

)

Genes ranked by chromosomal position

0.1

1

10

100

1000

SLC16A1

BSG

69 insertions

26 insertions

Supplementary Figure 1 - Loss of SLC16A1 and BSG prevents 3-BrPA induced cell death.

a. SLC16A1 and BSG genes contain the highest degree of insertional enrichment in 3-BrPA selected cells compared to the unselected control cells (p=4.7E-121 and p=5E-29, respectively). Y axis represents the inverse logarithm of p values, calculated by Fisher Exact Test. The X-axis represents the insertion sites ordered by their genomic position. The diameter of the bubbles denotes the number of insertions for each gene. b. Wild type and MCT1 null KBM7 cells were treated for 3 hours with 3-BrPA (50uM) and FACS analysis have been performed using 7AAD and Annexin V staining.


0 10 20 30 40 500

50

100

150

OC

R(%

)

Clone A50uM 3-BrPa

Wild

Type

Clone

A0

200

400

600

800

AUC

Ano

va E

CAR

(mpH

)Wi

ld Ty

pe

Clone

A0

2000

4000

6000

8000

AUC

Ano

va O

CR

(pM

oles)

OXYGEN CONSUMPTION RATE

p=0.21ns

EXTRACELLULAR ACIDIFICATION RATER

elativ

e Lac

tate P

rodu

ction

Wild

Type

Clone

A0.0

0.5

1.0

1.5

a

b

LACTATE PRODUCTION

Supplementary Figure 2 - Metabolic characterization of Wild Type and MCT1 null KBM7 cells

a. Lactate production of wild type and MCT1 null KBM7 cells were measured using a colorimetric assay and normalized by cell number. ECAR and OCR reading were measured using Seahorse Extracellular Flux Analyzer. AUC (area under curve) converts OCR rate data to accumulation of total oxygen consumed upon three separate readings.b. Extracellular Flux Analysis of wild type and MCT1 null KBM7 cells upon 3-BrPA (50 uM) addition. Changes in OCR, a proxy for oxygen consumption, were monitored upon the addition of 50 uM 3-BrPA. Results are displayed as a percentage of the OCR reading immediately before 3-BrPA addition. Error bars are SEM.c. Relative cellular ATP levels during a time course following 3-BrPA treatment. 50uM 3-BrPA was added onto wild type and MCT1 null KBM7 cells. Relative ATP levels were measured using a luciferase based assay for 2 hours. Error bars are SEM.

p=0.14ns

p=0.39ns

c

*

0 25 50 75 100 1250

50

100

150

Time (min)

% A

TP le

vels

WT KBM7Clone A

WT KBM7

Time (min)


KBM7

MDA-MB-468

GAPDH

Raptor

shRNA: GAPD

H_1

GAPD

H_2

GAPD

H_1

GAPD

H_2

GFP

GFP

KBM7

PBS 3-BrPA0

50

100

150

Rela

tive C

ell N

umbe

r

shGFPshGAPDH_1shGAPDH_2

MDA-MB-468

PBS 3-BrPA0

50

100

150

Rela

tive C

ell N

umbe

r

shGFPshGAPDH_1shGAPDH_2

GAPDH

Raptor

shRNA:

Supplementary Figure 3 - Effect of GAPDH silencing on 3-BrPA sensitivity Silencing GAPDH expression in cancer cell lines using RNAi causes a decrease in cell proliferation. Cells were infected with corresponding shRNAs and grown in RPMI media in presence and absence of 3-BrPA. 50uM and 10uM 3-BrPA were used for MDA-MB-468 and KBM7, respectively. Note the synergistic effect of GAPDH inhibition and 3-BrPA treatment on cell number.


pH.7.

0pH

.5.5

3-BrPa

(10m

M)

Pyruv

ate (1

0mM)

Lacta

te (10

mM)

0

100

200

300

0.1 1 10 100 10000

50

100

150 Pyruvate

L-Lactate

D-Lactate

3-BrPa

a b

Supplementary Figure 4 - Pyruvate and Lactate can compete with 3-BrPA uptake in KBM7 cells.

a. pH dependence of MCT1 mediated 3-BrPA transport. Wild Type KBM7 cells were incubated with 100 uM of radiactively labeled 3-BrPA for 20 minutes in HBSS in presence of indicated pH conditions and various monocarboxylates (n=3). b. Dose dependent inhibition of labeled 3-BrPA transport by D-Lactate, L-Lactate and Pyruvate. Wild Type KBM7 cells were incubated with 100 uM radiactively labeled 3-BrPA for 20 minutes in presence of different concentrations of indicated monocarboxylates.

% re

sidu

al 1

4C-3

-BrP

A si

gnal

% re

sidu

al 1

4C-3

-BrP

A si

gnal


1.0 1.5 2.0 2.5 3.00

5

10

15 Slc16a3

1.0 1.5 2.0 2.5 3.04

5

6

7

8 Slc16a7

1.0 1.5 2.0 2.5 3.03.43.63.84.04.24.44.6 Slc16a8

1.0 1.5 2.0 2.5 3.03.5

4.0

4.5

5.0 Slc5a8

1.0 1.5 2.0 2.5 3.03.03.23.43.63.84.04.2 Slc5a12

r=0.11p=0.97

r=-0.47p=0.07

r=-0.2p=0.45

r=-0.08p=0.75

r=-0.01p=0.94

MCTs

SMCTs

Supplementary Figure 5 - Correlation of other MCT transporter expression levels with 3-BrPA sensitivity

The concentration of 3-BrPA at which 50% cell growth inhibition occurred after 3 days of administration (IC50) was determined for a panel of cancer cell lines. These values were correlated with transcriptome-wide mRNA expres-sion data from the Cancer Cell Line Encyclopedia (CCLE) and the resulting Pearson correlation coefficients were plotted. Relative expression levels for known lactate transporters (MCTs and SMCTs) were correlated with the corresponding IC50 values for 3-BrPA of each cell line.

Rel

ativ

e e

xpre

ssio

n (lo

g2)

Log[IC50] R

elat

ive

exp

ress

ion

(log2

)

Rel

ativ

e e

xpre

ssio

n (lo

g2)

Rel

ativ

e e

xpre

ssio

n (lo

g2)

Rel

ativ

e e

xpre

ssio

n (lo

g2)

Log[IC50] Log[IC50]

Log[IC50] Log[IC50]


CNS

Cell L

ine

Haem

atopo

etic

Cell L

ineKid

ney

Cell L

ineLiver

Cell L

ine

Panc

reas

Cell L

ine

Pros

tate

Cell L

ine Skin

Cell L

ineTe

stis

Cell L

ine Lung

Cell L

ineBr

east

Cell L

ineCo

lon

Cell L

ine

Thyro

id

Cell L

ine

Stoma

ch

Cell L

ine

Saliva

ry

Cell L

ine

6

8

10

12

Tumor Cells and Normal Counterparts

Rel

ativ

e Ex

pres

sion

(Log

2)

a

GFP MCT1

MDA-MB-231 tumor xenograftsan

ti-M

CT1

c

d

Supplementary Figure 6 - MCT1 expression in cancers

a. IHC staining of MDA-MB-231 tumors expressing GFP and MCT1 cDNAs using an anti-MCT1 antibody.b. Time course weight measurements of tumors formed by MDA-MB-231 cells expressing the MCT1 or GFP cDNA during 3 weeks of treatment with vehicle or 3-BrPA (8 mg/kg). Error bars are ± SD (n = 5).c. Relative expression of MCT1 in cancer cell lines and their normal tissue of origins. Data was collected from CCLE and GEO browser and quantile normalized. d. Top 40 genes correlating with low (glycolytic) and high (OXPHOS) OCR/ECAR values.

TIMP2 PIM1 FN3KRP CATSPERBC12orf75 ORAI2 HS6ST3 ALDH3B2CLIP4 TNFAIP3 PPP2R2C KIAA1324RNF145 SCHIP1 ST6GALNAC2 GPR81UBE2E3 TBC1D1 TMEM141 MUM1L1BCAP29 PRKD3 SLC25A21 UCK1TSPAN3 SLC16A1 ZNF350 MYRIPNAB1 PDLIM1 LOC150622 ANKRD30ABET1 PDE8A CRAT UMODL1TMEM55A C20orf30 BRF1 MUCL1MDFIC CTSC GLS2 WDR85KLHL5 ROCK2 CNTNAP2 RAB3DGSTP1 SGCB ZNF485 PATZ1LTBP4 IFNAR2 NRK ZNF480C6orf145 ANXA11 CLCA2 ZMYND19CLN5 MTMR2 AR ZNF90PGM1 MFGE8 BDH1 RETPRKAA1 CDK6 RNF208 LOC643072IFT57 TYW3 LOC254559 AKT1BRI3 LDHB CEBPA ALDH4A1

Low

OC

R/E

CAR

Hig

h O

CR

/EC

AR

cDNA:

b

GFPMCT1GFPMCT1

cDNA Treatment3-BrPA

PBS

weeks0 1 3

0

50

100

150

200

Tum

or si

ze (

mm

3)

Start of thetreatment


Supplementary Table 1 - Primer Sequences

Cloning vector

Forward Primer Reverse Primer

pMXs-IRES-blasticidin

AGGGATCCATGCCACCAGCAGTTGGAGG AGGCGGCCGCTCAGACTGGACTTTCCTCCTCCTTG

PLJM1-puro

ATTGAATTCTATGCCACCAGCAGTTGGAGG ATTAATTCGTTCGAATCAGACTGGACTTTCCTCCTCCTTG


SUP0Supplementary Information BirsoySUP1NATGEN.aiSUP2NATGEN.aiSUP5NATGEN.aiSUP3NATGEN.aiSUP4NATGEN.aiSUP6NATGEN.ai

Cloning vector

mct1-mediated transport of a toxic molecule is an...

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