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Burd et al.
Supplemental File Index
1. Figure Legends for Supplemental Materials
2. References for Supplemental Materials
3. Supplemental Figures
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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),
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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
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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
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(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’.
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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
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