1

Burd et al.

Supplemental File Index

1. Figure Legends for Supplemental Materials

2. References for Supplemental Materials

3. Supplemental Figures

2

Supplemental Figure and Table Legends

Table S1. Frequency of canonical RAS mutations in human cancer. Data for each of the

indicated tumor types was downloaded from COSMIC and subsequently used to quantify the

percentage of missense mutations occurring within codons 12, 13 and 61 of K-, H- or N-RAS.

Figure S1. Generation of the LSL-N-RasQ61R allele. (A) Schematic representation of the

targeting vector used to construct the LSL-N-RasQ61R allele. The Q61R mutation is indicated by a

star. The location of the Southern probe is indicated by an orange box. SA-splice acceptor;

3xSTOP-transcriptional and translational stop sequence; Unnumbered block boxes-Flp

recombinase recognition sites (FRT); DTA-Diptheria toxin A chain (B) Southern blots of DNA

from wild type mice and heterozygous founders, pre- (Q61R+F/WT) and post-FlpE

recombination (Q61R/WT) are shown. Restriction enzyme cut sites and probe location are

indicated in ‘A’. (C) Sequencing of DNA from heterozygous LSL-N-RasQ61R embryonic stem cells

cultured in the presence of murine embryo fibroblast feeders. (D) Shown is a representative

image of PCR genotyping for the LSL-N-RasQ61R allele.

Figure S2. Conditional expression of N-Ras mutants does not alter melanocyte morphology.

(A) Isolated melanocytes, cultured as described in Materials and Methods, were stained for

tyrosinase related protein 1 (TRP-1) and analyzed using flow cytometry. Shown are

representative histograms. Visualized melanocyte staining in the absence of primary antibody

is provided as a control (2° only). (B) Sequencing of cDNA from wild type (N-RasWT/WT),

3

TpN12D/12D and TpN61R/61R melanocytes following 4-OHT treatment. (C) Shown are phase

contrast images taken at 100x magnification of TpN12D/12D and TpN61R/61R melanocytes treated

as described with 1.0 μM 4-OHT or ethanol (EtOH) vehicle and imaged 3 days later.

Figure S3. In both the heterozygous and homozygous state, NRasG12D fails to induce

melanoma formation. Kaplan-Meier curve of melanoma-free survival in an initial cohort of

syngeneic TpN12D/WT and TpN12D/12D mice.

Figure S4. Melanocyte-specific N-RASQ61R expression causes high penetrance nevus formation.

(A) Representative photographic images of nevi on the tail (bottom), paws (middle) and backs

of depilated mice. (B) In adult mice (10-24 weeks of age), the presence of hindquarter nevi was

assessed weekly by careful visual inspection. After 10 consecutive assessments by a genotype-

blinded observer, the percent of time in which a nevus was present was tabulated for each

mouse in the cohort. Each dot represents a single animal, while lines indicate the sample

mean. Each group was compared directly to the TpN61R/61R cohort using a student t-test to

determine statistical significance.

Figure S5. Characteristics of Ras-mutant murine melanocytes and melanomas. (A)

Quantitative, Taqman-based PCR was performed to detect differences in N-Ras expression

between 4-OHT treated melanocytes of the indicated genotypes. Homozygous LSL-N-

RASQ61R/Q61R melanocytes lacking CRE (No CRE) were included as a negative control. TRIA cells

harbor a melanocyte-specific transgene driving H-RASG12V and express normal levels of wild

4

type N-Ras (1). Statistical analysis was performed using a student t-test to compare TpN12D/12D

and TpN61R/61R melanocytes. (B) Growth of tumors from the indicated genotypes as scored

weekly using caliper measurements and normalized to day 1. (C) DNA was extracted from

TpN61R/61R tumors and genotyped to show CRE-mediated allelic recombination. Genotyping of

N-Ras in a K-rasG12D-driven tumor is shown as a control for the wild type allele. (D)

Quantitative, Taqman-based PCR is shown, comparing the number of N-Ras molecules in

TpN61R/61R and TpK12D/WT melanomas. Statistical analysis was performed using a student t-test.

Figure S6. Establishment and validation of TpLN61R/61R tumor cell lines. (A) Representative

images of cell lines established from TpLN61R/61R tumors and metastases. (B) Flow cytometry

showing the expression of a melanocyte marker (TRP1) on TpLN61R/61R-derived cell lines.

Figure S7. Binding of NRAS61R-mant-GMPPNP to PI3Kγ and RAF-RBD. A, left, NRASQ61R was pre-loaded

with the fluorescent GTP analog, mantGMPPNP. The ability of the BRAF-RBD to bind NRASQ61R was then

determined by measuring the nucleotide release rates in each indicated reaction. right, Shown is a plot

of the calculated NRasQ61R nucleotide dissociation rates at each BRAF-RBD concentration. To determine

the Kd for the NRasQ61R-BRAF-RBD complex, the data was fit to a standard curve. B, left, NRASQ61R was

pre-loaded with mantGMPPNP and the nucleotide release upon PI3Kγ (p110 subunit) binding measured as

described in ‘A’. right, The Kd for the NRasQ61R- PI3Kγ complex was plotted and measured as described in

‘A’.

Figure S8. NRASG12D and NRASQ61R similarly bind BRAF as measured by isothermal titration

calorimetry. Isothermal calorimetry experiments were carried out on an AutoITC200 microcalorimeter

5

(Microcal/GE Healthcare) at 26°C. 19 x 2 μL injections of 1.2 mM BRAF-RBD were automatically injected

into 200 μL of 64 µM NRASWT, NRAS61R or NRAS12D. The resulting binding isotherms were analyzed using

Microcal Origin 7.0 software package and were fit best with a single site binding model. Experiments

were performed in triplicate, and the averaged binding parameters (N, Kd, ΔH(cal/mol) and ΔS

(cal/degree/mol)) are shown (mean +/- SD).

Figure S9. Proliferation of NRAS mutant human melanoma cell lines is not codon-specific. The

percentage of proliferating cells in human melanoma cell lines grown to 60-80% confluency was

determined by flow cytometry after 6 hours of EdU labeling. Using a two-tailed student t-test, no

difference in EdU incorporation was observed between cells harboring an NRAS codon 12/13 or 61

mutation (p = 0.06). Error bars represent the mean and standard deviation within each group.

Figure S10. Activation of ERK and AKT in melanocytes is not codon-specific. A, Top, PCR

genotyping of melanocytes treated for 6 days with ethanol vehicle (E; lane 6) or 1.0 µM 4OHT

(1.0; lane 7). Genomic DNA from mice of the indicated genotypes is included as a control (lanes

3-5). Bottom, Cell lysates from melanocytes of the indicated genotypes were subjected to SDS-

PAGE and immuno-blotted for the indicated proteins. Protein expression was quantified using

LI-COR ImageStudio software. “CRE-” indicates TpN61R/61R melanocytes assayed prior to

treatment with 4-OHT. B, Top, Immortalized melanocytes were genotyped following CRE

induction with 1.0 µM 4-OHT (lanes 2,3 and 12) or adenoviral CRE infection (lanes 9-11). NL214

cells are derived from a TpLN61R tumor and serve as a positive control for recombination.

Untreated genomes are shown for comparison (lanes 4-6 and 14-16). Bottom, immortalized

melanocytes were analyzed for ERK and AKT activation as described in ‘A’.

6

Table S2. Comparative stability of GMPPCP- and GDP- bound NRAS variants.

Table S3. Summary of prior RAS-driven murine melanoma models.

Supplemental References

1. Chin L, Pomerantz J, Polsky D, Jacobson M, Cohen C, Cordon-Cardo C, et al. Cooperative effects of INK4a and ras in melanoma susceptibility in vivo. Genes & development. 1997;11:2822-34.

KRAS HRAS NRAS

12 13 61 12 13 61 12 13 61

Melanoma 961 0.02 0.00 0.00 0.00 0.00 0.01 0.07 0.05 0.84 6.59

Thyroid 333 0.13 0.06 0.02 0.07 0.02 0.11 0.01 0.01 0.58 38.40

AML 515 0.10 0.03 0.01 0.00 0.00 0.00 0.42 0.26 0.17 0.26

ALL 323 0.23 0.16 0.01 0.00 0.00 0.00 0.32 0.17 0.11 0.23

Lung Adeno 2,034 0.91 0.06 0.02 0.00 0.00 0.00 0.00 0.00 0.11 8.00

Colorectal Adeno 14,362 0.78 0.20 0.01 0.00 0.00 0.00 0.00 0.00 0.01 1.30

Pancreatic Ductal 2,975 0.99 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 N/A

# Cannonical

RAS

Mutations

Recorded

NRAS 61 :

NRAS 12+13

Burd_Table. S1

SacI + StuI

-5,558bp

-6,662bp -7,312bp

A

C

D

0 2 1 3 6 5 4

(*Q61R)

SA 3x STOP NEO Targeted

Allele

0 2 1 3 6 5 4

0 2 1 3 DTA

(*Q61R)

3,797 bp

SA 3x STOP NEO

1,046 bp

N-rasWT

Targeting

Construct

- 371 bp (LSL) - 487 bp (WT)

Burd_Fig. S1

B

D T A G Q E E Y AA:

DNA: GACACAGCTGGACAAGAGGAGTAC

SacI StuI

StuI StuI

SacI

LSL-N-ras61R 0 2 1 3 6 5 4

(*Q61R)

SA 3x STOP

+ FlpE

A

Co

un

t

Trp1

2° Only

αTrp1

0.62%

99.9 ± 0.08 %

n=6

C

V A G AA:

DNA:

D G V G

AA:

DNA:

T G A R E E Y

N-rasWT/WT

N-ras12D/12D

V A G AA:

DNA:

G G V G

Cod

on

12

Cod

on

61

N-ras61R/61R

AA:

DNA:

T G A Q E E Y

N-rasWT/WT

B

Burd_Fig. S2

Tp

N61R

/61R

+EtOH +4OHT

Tp

N12D

/12D

Burd_Fig. S3

Age in Weeks

M e l

a n o m

a F

r e e

S u r v

i v a l

0 20 40 60 80 0

20

40

60

80

100

TpN G12D/WT (n=14)

TpN G12D/G12D (n=14)

Tp TpK12D/WT TpN12D/12D TpN61R/61R A

Burd_Fig. S4

B

Tp TpN 12D/12D

TpN 61R/61R

TpK 12D/WT

0

20

40

60

80

100

%

Tim

e N

evu

s P

osi

tiv

e

H20

N-R

asW

T

TpN61R/61R

- LSL-Q61R - WT N-Ras - Q61R

1 2 3 4 5 6 7

B

D

Burd_Fig. S5

A

C

0

2

4

6 p<0.001

Lo

g10 N

-Ra

s M

ole

cule

s

0 2 4 6

0

200

400

600

800

1000

TpK12D/WT

TpN61R/61R

TpN12D/12D

Weeks

% I

ncr

ease

(T

um

or

Siz

e)

# M

ole

cule

s N

-Ra

s/3

ng

RN

A

TpN 61R/61R

TpK 12D/WT

0

1000

2000

3000

4000 NS

Co

un

t

αTrp1

NL216

NL214

NL212

NL146

NL145

IgG Control

NL146

NL214

NL212

NL216

Burd_Fig. S6 A

B

Burd_Fig. S7

A

B

Time (min)

Molar Ratio

µca

l/se

c K

Cal

/Mo

le In

ject

ant

NRASWT NRAS61R

µca

l/se

c K

Cal

/Mo

le In

ject

ant

Time (min)

Molar Ratio

NRAS12D

Molar Ratio

µca

l/se

c K

Cal

/Mo

le In

ject

ant

Kd = 0.31 +/- 0.30µM N= 0.86 +/- 0.03 ΔH = -3,005 +/- 83 ΔS = 20.4 +/- 2.12

Kd = 0.28 +/- 0.10 µM N= 0.84 +/- 0.01 ΔH = -2,637 +/- 117 ΔS = 21.3 +/- 1.21

Kd = 0.35 +/- 0.07 µM N= 0.82 +/- 0.0 ΔH = -3,097 +/- 176 ΔS = 19.2 +/- 0.14

Time (min)

Burd_Fig. S8

Burd_Fig. S9

% E

d U

P o s i

t i v e

Codon 12/13 Codon 61 0

20

40

60

Mel224

WM1366 VMM39

SKMel147

WM3629

WM3670

MaMel27II

NZM63

NZM24

Primary Melanocytes

Immortalized Melanocytes

1 2 3 5 4OHT: - - - Lane:

NG12D

NQ61R

4 - E

6 7 1.0

- LSL - WT

+CRE

- LSL - WT

+CRE

AKT

β-ACTIN

β-ACTIN

pERK

(T202/Y204)

ERK

pAKT

(S473)

- LSL - WT

+CRE

CRE: + + - - - -

1 Lane: 2 3 4 5 6

NG12D

7

LSL/LSL#1

NQ61R

8 CRE:

Lane: + 9

+ 10

+ 11

+ 12

+ 13

- 14

- 16

- LSL - WT

+CRE

- 15

- 17

G12D #1 #1

AKT

β-ACTIN

β-ACTIN

pERK

(T202/Y204)

ERK

pAKT (S473)

#2 #2 Q61R

pERK/ERK:

ERK/ β -ACTIN:

pAKT/AKT: AKT/ β -ACTIN:

pERK/ERK: ERK/ β -ACTIN:

pAKT/AKT:

AKT/ β -ACTIN:

0.004 0.007 0.015 0.011

1.53 1.14 1.54 1.45

5.98 2.62 5.52 9.73

0.042 0.066 0.027 0.007

0.026 0.031 0.032 0.043

4.13 4.08 4.80 3.99

1.84 4.48 2.87 22.1

0.027 0.031 0.012 0.003

A B Burd_Fig. S10

Burd_Table. S2

RAS

Mutant Tm (°C)

NRASWT 67 ± 3

NRASG12D 70 ± 3

NRASQ 61R

(GDP bound)74 ± 4

NRASQ 61R

(GMPPCP bound)80 ± 5

Burd_Table. S3

RAS Additional Alleles Tumor

Mutant or Treatments Penetrance Metastases Citation(s)

None None N/A Powell et al ., 1995; Hacker et al ., 2005

UVR, DMBA, or TPA Variable Varies (Lung, LN) Powell et al ., 1999; Hacker et al ., 2005

p16-/-

35-~50% NR Sharpless et al ., 2003; Kannan et al ., 2003

Arf-/-

~50-52% NR Sharpless et al ., 2003; Kannan et al ., 2003

HRasG12V

p53-/-

26% None Bardeesy et al ., 2001

Ink4/Arf-/-

60% None Chin et al. , 1997

Ink4/Arf-/-

& Pten-/-

75% Rare Nogueira et al. , 2010

Ink4/Arf-/-

& Ikkb-/- 4.6% NR Yang et al ., 2010

Cdk4R24C

33-58% 0/~50% (LN) Chawla et al ., 2010; Hacker et al ., 2006

P1A, Ink4/ArfL/L

33% 25% (LN) Hujibers et al ., 2006

iHRasG12V

p16-/-

25% NR Chin et al. , 1999

KRasG12V

None 85% None Milagre et al .,2010

KRasG12D

Viral RAS, Ink4/ArfL/L

None N/A VanBrocklin et al ., 2010

None 29% 75% (Lung, LN) Ackermann et al ., 2005

Cdk4R24C

100% NR Ferguson et al. , 2010

NRasQ61K

p16-/-

94% 76% (Lung, Liver, LN) Ackermann et al ., 2005

Arf-/-

~25% NR Ferguson et al. , 2010

p53L/L

100% NR Ferguson et al. , 2010

B-cateninsta

85% NR Delmas et al ., 2007

iNRasQ61K

Cdkn2a-/-

50% NR Kwong et al ., 2012

NRasQ61R

Viral RAS, Ink4/ArfL/L

36% With passage VanBrocklin et al ., 2010

Oncogenic RAS Transgenics

Endogenous RAS Knock-Ins

None None NR Monahan et al ., 2010; Liu et al. , 2012

p53L/L

45% NR Monahan et al ., 2010

p16L/L

73% NR Monahan et al ., 2010

p53L/L

& p16L/L

100% NR Monahan et al ., 2010

KRasG12D

BRafD594A

100% NR Heidorn et al ., 2010

Lkb1L/L 100%

100% (LN, Lung,

Liver, Spleen)Liu et al. , 2012

p53L/L

& Lkb1L/L 100%

100% (LN, Lung,

Liver, Spleen)Liu et al. , 2012

NRasG12D

None CNS NR Pedersen et al. , 2013