Supplementary Results to

Cancer stem cell drugs target K-ras signaling in a stemness-context

Arafath K Najumudeen et al.

Supplementary Figure 1: CSC inhibitors alter Ras distribution and PS nanoclustering.

(a) Confocal images of BHK cells transiently expressing mGFP-K-rasG12V or mGFP-H-

rasG12V treated for 24 h with either DMSO control, 1.3 µM salinomycin, 1.3 µM nigericin,

1.7 µM lasalocid or 10 nM staurosporine (STS). Shown are representative confocal images of

cells from three independent experiments. Scale bar, 20µm. (b) Nanoclustering-FRET

analysis (illustrated in scheme) in BHK cells co-expressing mGFP- and mCherry-LactC2.

Cells were treated for 24 h with either DMSO control, 1.3 µM salinomycin, 1.3 µM nigericin,

1.7 µM lasalocid or 10 nM STS. The apparent FRET-efficiency was calculated from FLIM

data (mean ± SEM, n=3). The numbers in the bars indicate the number of analyzed cells.

Statistical significance of differences between control and treated cells were examined using

one-way ANOVA tests (* p < 0.05, **** p < 0.0001).

Supplementary Figure 2: Hit cross-validation in HEK cells.

Validation of hits from the ionophore collection screen was performed with BHK and HEK

cells. FRET response of (a) K-ras NANOPS and (b) H-ras NANOPS in BHK and HEK cells

treated for 24 h with 1 µg/ml of inhibitors. Block line indicates the average Emax. Bars denote

the SEM (n≥4). Statistical significance analyzed using one-way ANOVA tests are indicated.

(c) Model of K-ras activity modulation by presence or absence of caveolae, based on work

from Ariotti et al. 1. For more explanation, please refer to the main text.

Page 1 of 11
Supplementary Figure 3: Characteristic gene expression patterns and patient survival

analysis.

(a) Similarity analysis of expression profiles of the 13 K-ras-nanoclustering-signature genes

using ESC and fibroblast expression data from the database ESTOOLS. Fibroblasts show an

expression signature that is inverted in primed ESCs. Note that there is a clear difference in

the expression pattern of naïve ESC and that there is another set of ESC with expression

patterns that seem to be in transit between the naïve and primed ESCs. Scheme on the bottom

illustrates the differentiation pathway of ESC. (b) Overall survival analysis between patient

tumor samples from TCGA with gene expression signatures similar to ESC-like cancer cells

(n=1401) vs. fibroblast-like cancer-cells (n=2631).

Supplementary Figure 4: Gene expression comparison of cancer cells, ESC and

fibroblasts.

Gene expression comparison of selected cancer cell lines including those from Figure 5 (data

extracted from the CCLE database) and ESCs and fibroblasts (data from ESTOOLS

database). Only the 13 genes from the K-ras-nanoclustering signature are compared.

Supplementary Figure 5: Additional microbial metabolite screening identifies new

candidate CSC inhibitors.

(a) Schematic illustration of the second chemical screen design and workflow. The

differential chemical screen of the MST metabolite library on H-Ras and K-Ras NANOPS

had a Z’ score of 0.60 with a combined hit rate of 10%. (b) Effector-recruitment FRET assay

(illustrated in schemes) in BHK cells expressing mGFP-K-rasG12V or mGFP-H-rasG12V

and mRFP-RBD. Cells were treated for 24 h with either DMSO control, 1.3 µM of

salinomycin, 0.2 µM of avermectin, ivermectin, conglobatin A, ophiobolin A, kazusamycin

Page 2 of 11
B, leptomycin B or streptonigrin. The apparent FRET-efficiency was calculated from FLIM

data (mean ± SEM, n=3). The numbers in the bars indicate the number of analyzed cells.

Statistical significance of differences between control and treated cells was examined using

one-way ANOVA (***, p < 0.001; ****, p < 0.0001). (c) Confocal images of MDCK cells

stably expressing GFP-K-rasG12V or GFP-H-rasG12V treated for 24 h with 0.2 µM of

inhibitors. Shown are representative confocal images of cells from three independent

experiments. Scale bar, 20 µm.

Supplementary Figure 6: Ophiobolin A is a highly potent CSC inhibitor that targets

CaM.

(a) Expression of indicated stem cell markers in MDA-MB-231, MDA-MB-436, Hs-578T

and MCF7 cells grown in adherent and non-adherent sphere forming conditions. β-actin was

used as a loading control. (b) Drug efficacy comparison of ophiobolin A on MDA-MB-231

cells grown under adherent and non-adherent, mammosphere forming conditions. Cells were

treated with different concentrations of ophiobolin A for 72 h (n=3). Percentage viability of

adherent was measured using alamarBlue and mammospheres were counted as described in

the Methods section. The values were expressed relative to the DMSO control treated cells.

(c) Analysis of siRNA mediated calmodulin 1 knockdown efficiency in MDA-MB-231 cells

used for mammosphere analysis. β-actin was used as a loading control. (d) Left,

nanoclustering-FRET analysis on HEK cells treated with 0.2 µM conglobatin A and

otherwise performed like in Figure 7b. The apparent FRET-efficiency (mean ± SEM, n=3)

was calculated from FLIM-FRET data. Statistical significance of differences between control

and treated cells was examined using one-way ANOVA (ns, not significant; ****, p <

0.0001). Right, Analysis of siRNA mediated calmodulin 1 knockdown efficiency from n=5

independent measurements in HEK293 cells. β-actin was used as a loading control. (e)

Page 3 of 11
Correlation of individual genes in the K-ras nanoclustering signature set with the drug

response to salinomycin, staurosporine and ophiobolin A across the screened cell lines (n=15

for salinomycin and staurosporine, n=14 for ophiobolin A). *, p < 0.05, Spearman rank

correlation coefficent.

Page 4 of 11
 Compound
Compound name MW Activity Solvent of stock
number
1 Reveromycin A 660.8 N/A 100% DMSO
2 Antibiotic RK-682 775.1 N/A 100% DMSO
3 Nigericin sodium 746.9 K+, Rb+, Cs+, Na+ 100% DMSO
4 Deethylindanomycin 465.6 divalent cations 100% DMSO
Mg2+, Zn+ dependent
5 Antibiotic UK-1 386.4 100% DMSO
DNA binding agent
Moenomycin phosphoglycolipid
6 1583.6 100% DMSO
complex antibiotic
complexes with mono-
7 Lasalocid sodium 590.8 100% DMSO
& di-valent cations
8 Monensin A 670.8 Na+, K+, Li+, Ag+, Tl+ 100% DMSO
9 Salinomycin 751 K+ 100% DMSO
10 Enniatin complex 639.8 K+ 30% Methanol - Water
11 Indanomycin 493.7 divalent cations 100% DMSO
12 Ionomycin 709 Ca2+ 30% Methanol - Water
13 Valinomycin 1111.3 K+ 30% Methanol - Water
14 Compactin 390.51 HMG-CoA inhibitor Ethanol – PBS (1:1)

Supplementary Table 1: Collection of ionophores used in the first compound screen

with K- and H-ras-NANOPS. Compound numbers are the same as in Figure 1b. Activity:
Known ionophoric activity of the compounds is indicated. Compactin (compound 14), an
HMG-CoA inhibitor that blocks farnesylation and prevents Ras membrane anchorage, was
used as the positive control. MW denotes molecular weight. Final solvent concentration in
cells was typically less than 0.01%.

Page 5 of 11
 Name (Gene symbol) Nanoclustering (nc) associated activity References
Galectin-1 increase of GTP-H-ras nc (no binding to inactive H-ras), 2-5
(LGALS1) but decrease of GTP-K-ras nc (DA unpublished
observation)
Galectin-3 increase of K-ras nc 6,7
(LGALS3)
Nucleophosmin increase of GDP/GTP-K-ras nc 8
(NPM1)
Nucleolin binding partner of NPM1; increases membrane affinity 8
(NCL) of K-ras (not nc)
Caveolae: decrease K-ras nc by quenching plasma membrane 1
Caveolin-1 (CAV1) and phosphatidylserine (PS)
Cavin-1 (PTRF)
H-RasG12V (HRAS) decreases K-ras nc remotely, via PS perturbation 9

Supplementary Table 2: List of seven known K-ras nanocluster modulators and their
activity

References:
1 Ariotti N, Fernández-Rojo MA, Zhou Y, Hill MM, Rodkey TL, Inder KL et al. Caveolae
regulate the nanoscale organization of the plasma membrane to remotely control Ras
signaling. J Cell Biol 2014; 204: 777–792.
2 Rotblat B, Belanis L, Liang H, Haklai R, Elad-Zefadia G, Hancock JF et al. H-Ras
nanocluster stability regulates the magnitude of MAPK signal output. PLoS ONE 2010; 5:
e11991.
3 Prior IA, Muncke C, Parton RG, Hancock JF. Direct visualization of Ras proteins in
spatially distinct cell surface microdomains. J Cell Biol 2003; 160: 165–170.
4 Paz A, Haklai R, Elad-Sfadia G, Ballan E, Kloog Y. Galectin-1 binds oncogenic H-Ras to
mediate Ras membrane anchorage and cell transformation. Oncogene 2001; 20: 7486–
7493.
5 Guzmán C, Solman M, Ligabue A, Blazevitš O, Andrade DM, Reymond L et al. The
efficacy of Raf kinase recruitment to the GTPase H-ras depends on H-ras membrane
conformer-specific nanoclustering. J Biol Chem 2014; 289: 9519–9533.
6 Shalom-Feuerstein R, Plowman SJ, Rotblat B, Ariotti N, Tian T, Hancock JF et al. K-ras
nanoclustering is subverted by overexpression of the scaffold protein galectin-3. Cancer
Res 2008; 68: 6608–6616.
7 Levy R, Biran A, Poirier F, Raz A, Kloog Y. Galectin-3 mediates cross-talk between K-
Ras and Let-7c tumor suppressor microRNA. PLoS ONE 2011; 6: e27490.
8 Inder KL, Lau C, Loo D, Chaudhary N, Goodall A, Martin S et al. Nucleophosmin and
Nucleolin Regulate K-Ras Plasma Membrane Interactions and MAPK Signal
Transduction. J Biol Chem 2009; 284: 28410–28419.
9 Zhou Y, Liang H, Rodkey T, Ariotti N, Parton RG, Hancock JF. Signal Integration by
Lipid-Mediated Spatial Cross Talk between Ras Nanoclusters. Mol Cell Biol 2014; 34:
862–876.

Page 6 of 11
 Query Gene Correlated Gene
CAV1 ANXA1, ANXA2, CAV2, CD44, COL5A2, COL8A1, EMP3, FRMD6,
GLIPR1, GNG11, GREM1, LMNA, MTMR11, MYOF, NT5E, PARVA,
PEA15, RFTN1, SERPINE1, SYNJ2, TAX1BP3, TGFB1I1, TRAM2, VIM

EGFR ANPEP, LTBP2, MGLL, SQRDL

KRAS CCNB2, PAICS
LGALS1 ANXA1, ANXA2, CAV2, CD44, CD59, COL1A1, COL5A2, COL8A1,
CREB3L1, EMP3, FRMD6, GLIPR1, GNG11, ITGA5, LOX, MTMR11,
MYOF, PARVA, PEA15, RFTN1, TGFB1I1, TRAM2, VIM
LGALS3 ANPEP, FRMD6, GREM1, LOX, LTBP2, MGLL, NT5E, OPTN, PLP2,
RFTN1, SQRDL, TGFB1I1, WIPI1
NCL HNRNPAB
NPM1 CCNB2, HNRNPAB, PAICS
PTRF CD44, CD59, COL1A1, CREB3L1, EMP3, GREM1, ITGA5, LMNA, OPTN,
PLP2, SERPINE1, SYNJ2, TAX1BP3, TGFB1I1, TRAM2, WIPI1

Supplementary Table 3: List of 32 most co-expressed genes with the 10 genes in ESCs
and fibroblasts extracted from ESTOOLS database. Genes that were coregulated with
more than one of the 10 genes in the K-ras-nanoclustering signature were retained.

Page 7 of 11
 P$values0(Fishers's0exact0test)0vs0
Number0of0samples Percentage0of0samples
Total0TCGA0samples Enrichment0for0
Total0 P$value0(Fisher's0
Code Cancer0Types Total0TCGA0 ESC/0Fibro$like0
ESC$like Fibro$like TCGA0 ESC$like Fibro$like ESC$like Fibro$like exact0test)
samples vs0the0other0
samples
ACC Adrenocortical-carcinoma- 4 6 79 0.66 1.49 1.05 0.53 0.45 2.26 0.21
BLCA Bladder-Urothelial-Carcinoma- 10 6 241 1.65 1.49 3.20 0.04 0.07 1.11 1.00
BRCA Breast0invasive0carcinoma0 111 17 1041 18.35 4.23 13.81 0.01 2.42E?08 4.34 4.42E$10
Cervical-squamous-cell-carcinoma-and-
CESC endocervical-adenocarcinoma- 6 3 185 0.99 0.75 2.46 0.02 0.03 1.33 1.00
COAD Colon0adenocarcinoma0 26 3 435 4.30 0.75 5.77 0.17 9.12E?07 5.76 8.06E$04
Lymphoid-Neoplasm-Diffuse-Large-B?cell-
DLBC Lymphoma- 8 0 28 1.32 0 3.72E?01 4.40E?03 0.40 100% 0.03
GBM Glioblastoma-multiforme- 6 15 161 0.99 3.73 2.14 0.07 0.05 3.76 0.01
HNSC Head-and-Neck-squamous-cell-carcinoma- 1 10 426 0.17 2.49 5.65 9.02E?13 0.01 15.08 7.74E$04
KICH Kidney-Chromophobe- 0 3 66 0 0.75 0.88 1.45E?02 1.00 NA NA
KIRC Kidney-renal-clear-cell-carcinoma- 0 57 518 0 14.18 6.87 2.60E?17 5.38E?06 $1000% 0.00
KIRP Kidney-renal-papillary-cell-carcinoma- 1 3 172 0.17 0.75 2.28 4.47E?05 5.04E?02 4.52 0.31
LAML Acute0Myeloid0Leukemia0 98 0 173 16.20 0 2 3.69E?39 2.59E?04 100% 4.66E?21
LGG Brain0Lower0Grade0Glioma0 22 0 463 3.64 0 6 0.01 1.36E?10 100% 2.14E?05
LIHC Liver-hepatocellular-carcinoma- 2 1 191 0.33 0.25 2.54 1.09E?04 1.09E?03 1.33 1.00
LUAD Lung-adenocarcinoma- 23 40 489 3.80 9.95 6.49 0.01 0.02 2.62 3.13E$04
LUSC Lung-squamous-cell-carcinoma- 16 16 490 2.65 3.98 6.50 1.19E?04 0.06 1.50 0.27
OV Ovarian0serous0cystadenocarcinoma0 78 6 263 12.89 1.49 3.49 4.34E?18 0.03 8.64 9.07E$11
PAAD Pancreatic-adenocarcinoma- 0 3 85 0 0.75 1.13 2.66E?03 0.63 NA 0.06
PRAD Prostate-adenocarcinoma- 2 2 297 0.33 0.50 3.94 7.38E?08 5.58E?05 1.50 0.65
READ Rectum-adenocarcinoma- 10 3 164 1.65 0.75 2.18 0.47 0.07 2.22 0.26
SARC Sarcoma- 0 15 103 0 3.73 1.37 8.16E?04 1.07E?03 NA NA
SKCM Skin-Cutaneous-Melanoma- 43 144 372 7.11 35.82 4.94 0.04 2.04E?57 5.04 3.20E$21
THCA Thyroid-carcinoma- 0 31 498 0 7.71 6.61 9.52E?17 0.41 NA NA
UCEC Uterine0Corpus0Endometrial0Carcinoma0 128 18 539 21.16 4.48 7.15 1.50E?20 0.05 4.72 6.34E$12
UCS Uterine0Carcinosarcoma0 10 0 57 1.65 0 1 0.03 0.12 100% 0.01

Supplementary Table 4: Cancer-types from the TCGA database with ESC- or fibroblast-like K-ras nanoclustering expression signature.
Table shows the analysis of the different cancer tissue-types from the TCGA tumor database with ESC- or fibroblast (Fibro)-like K-ras-
nanoclustering signature. The number and percentage of samples with ESC-like (n=605), fibroblast-like signature (n=402) and total TCGA data
(n=7536) for each cancer-type is provided. P-values for difference of percentages of each cancer type against the total TCGA data from Fisher’s
exact test are provided. Differential enrichment for ESC-like denotes the ratio of ESC-like- / Fibro-like-percentages and enrichment for Fibro-like
(italics) for the inverse. P-values for this difference in percentage of cancer types in ESC-like vs. fibroblast-like signature from Fisher’s exact test
are provided in the final column. Notable values are in bold. 100% or -100% denotes that only samples of ESC- or Fibro-like signature,
respectively, were found. Cancer types are color coded with those significantly dominated by ESC-like in green and fibroblast-like in grey.

Page 8 of 11
 Percentage deviation from control
No. FRET Emax Compound Name Class
K-ras-NANOPS H-ras-NANOPS
1 27% 18% Stauprimide Staurosporine
2 14% 11% Leptomycin analogue Leptomycin
3 13% 13% 7-Oxostaurosporine Staurosporine
4 12% 12% Leptomycin, aminolysed Leptomycin
5 10% 10% Leptomycin A Leptomycin
6 25% 7% UCN-01 Staurosporine
7 17% 5% Leptomycin analogue Leptomycin
8 13% 1% Cyclosporine D
9 14% 7% Doxorubicin Doxorubicin
10 11% 1% Leptomycin B Leptomycin
11 11% 3% Kazusamycin B Leptomycin
12 10% 5% Staurosporine Staurosporine
13 10% 9% Leptomycin A Leptomycin
14 10% 2% Avermectin Avermectin
15 10% 1% Idarubicin HCl Idarubicin
16 10% 5% Unidentified peptide
17 8% 13% Leptomycin B Leptomycin
18 -2% 11% Chromomycin A3
19 3% 10% Epothilone B
20 8% 10% Elaiophylin
21 -38% -33% Simvastatin Statin
22 -31% -31% Lovastatin Statin
23 -24% -18% Prodigiosin
24 -19% -15% Ophiobolin A Ophiobolins
25 -16% -15% Ophiobolin B Ophiobolins
26 -14% -12% 111070-14.LA5.38-63/66 Leptomycin
27 -11% -14% 8-beta-Hydroxyzearalenone
28 -12% -11% Neoantimycin
29 -12% -16% Pravastatin sodium Statin
30 -12% -3% Ivermectin Avermectin
31 -12% -7% Conglobatin A Conglobatin
32 -10% 1% Streptonigrin Streptonigrin
33 2% -13% Nonactin Nactin
34 -8% -13% Kigamicin C
35 -8% -12% Ophiobolin C Ophiobolin
36 -7% -10% Trinactin Nactin
37 -6% -10% Indanomycin
38 -8% -10% Leptomycin co-metabolite Leptomycin
39 -8% -10% Antimycin A
Supplementary Table 5: List of hits from the MST library screen with K- and H-ras-
NANOPS using cytometry-FRET. Table shows percentage change of FRET Emax value for
hit compounds as compared to the respective non-treated control values for H- and K-ras-
NANOPS. Hits that increase FRET Emax ≥10% are marked in red and those that decrease
FRET Emax ≥-10% are marked blue. Substance/chemical classes of the hits are indicated.
Hits from the same substance class are color-coded. Shown are hits satisfying the selection
threshold 10% change in FRET Emax for either H or K-ras-NANOPS compared to the
respective non-treated control.

Page 9 of 11
 Compound
No. Structure
Name
1 Leptomycin B

2 Kazusamycin B

3 Ophiobolin A

4 Conglobatin A

5 Ivermectin

6 Avermectin

7 Streptonigrin

Supplementary Table 6: Chemical structures of validated hits from the MST metabolite
screen.

Page 10 of 11
 MDA-MB- MDA-MB- Hs578T MCF7
Cell line 231 436
EGFR PIK3C2B H-rasG12D PIK3CA
K-rasG13D PIK3R1 RASGRF1 PIK3AP1
Mutations B-Raf P53 PIK3R1 CDKN2A
NF2 BRCA1 P53
P53 RB
Basal-like, Luminal Basal-like
Classification claudin-low Triple claudin-low
Luminal A
Triple negative Triple
negative negative

Percentage of
CD44+/CD24− cells 85 ± 5 72 ± 5 86 ± 5 0
(Sheridan et al., 2006)

Number of
mammospheres/2000 202 ± 15 136 ± 28 134 ± 11 133 ± 22
cells (mean ± S.D.)

Supplementary Table 7: Characteristics of breast cancer cell lines used for

mammosphere assays. Table shows the mutation status of typical oncogenes, breast cancer
type classification, reported percentage of CD44+/CD24− population and mammosphere
formation capacity in our experiments.

Page 11 of 11
 Supplementary Figure 1

a
control STS Salinomycin Nigericin Lasalocid
K-rasG12V
BHK21

H-rasG12V

b PS-clustering FRET

control 112
STS 108
*
LactC2 Salinomycin 90
PS
Nigericin 72 ****
Lasalocid 130
4 6 8 10
FRET apparent FRET efficiency, %
 c
a
un
tre
at BHK21 FRET Emax
ed
C co

0.1
0.2
0.3
0.4
0.5
R om nt
An ev pa ro

K-ras
t e c l

BHK21
D ibio rom tin
ee t
th ic R ycin
yl K A
Anind Nig -68
a
tib no eri 2
io m cin
t

p<0.02

K-ras
Supplementary Figure 2

Low Caveolae
Laic Uycin
M s K

High PS clustering
S onealo -1
In alin ns cid

signaling
da om in

PS
K-ras-NANOPS

no y A
m cin
p<0.02

yc
in

0.1
0.2
0.3
0.4
0.5
0.6

HEK293 FRET Emax

K-ras
HEK293 EBNA
b

un
tre

High Caveolae
at BHK21 FRET Emax
e

Low PS clustering
C d co
0.2
0.3
0.4
0.5
0.6

R om nt
p ro

signaling
An ev
tib ero acti l

High K-ras clustering and signaling Low K-ras clustering and signaling
D
BHK21

ee ioti my n
th c ci
R n
yl K A
Anind Nig -68
a
tib no eri 2
io m cin
t
Laic Uycin
M s K
Saonealo -1
In lin ns cid
H-ras-NANOPS

da om in
no y A
m cin
yc
in
0.2
0.3
0.4
0.5
0.6

HEK293 FRET Emax

HEK293 EBNA
 NPM1 −2
NRAS −4
HRAS 2
KRAS 0

LGALS1

LGALS3
ITGA5
EGFR

PTRF
CAV1
CAV2
NCL

VIM

SET2_ESC_H9__bFGF_1
SET2_ESC_H9__bFGF
SET2_ESC_H9_____bFGF_1
primed ESC

SET2_ESC_H9_____bFGF
SET2_ESC_H9_1_day_in_UM___4h_in_UM_GTA_rep2
(in transit)

SET2_ESC_H9_1_day_in_UM_rep1
SET2_ESC_H9_1_day_in_UM___4h_in_MEF_CM_rep2
SET2_ESC_H9_1_day_in_UM___4h_in_MEF_CM_rep1
SET2_ESC_H9_1_day_in_UM___4h_in_UM_GTA_rep1
SET2_ESC_H9_1_day_in_UM_rep2
SET1_H9_ESC_4__O2_rep1
SET1_H9_ESC_20__O2_rep1
SET1_HUES8_ESC_rep3
SET1_HUES8_ESC_rep1
SET1_HUES8_ESC_rep2
SET2_ESC_H1_in_N2B27_ActivinA_12h_rep2
SET2_ESC_H1_in_N2B27_ActivinA_48h_rep1
SET2_ESC_H1_in_N2B27_ActivinA_36h_rep2
SET2_ESC_H1_in_N2B27_ActivinA_36h_rep1
SET1_WIBR2_ESC
SET1_WIBR1_ESC
SET1_WIBR3_ESC
SET1_WIBR3_ESC_1
SET1_ESC_H1_4__O2_rep2
SET1_ESC_H1_20__O2_rep1
SET1_ESC_H9_rep2
SET1_ESC_H9_rep1
SET1_ESC_H9_rep3
SET2_ESC_HUES7_2
SET2_ESC_HUES7_1
SET2_ESC_HUES22
SET1_ESC_rep1
SET2_ESC_HUES13_2
SET2_ESC_HUES13_1
SET1_ESC_H1
SET1_ESC_ES2_rep2
SET2_ESC_HUES21
SET2_ESC_H1_in_N2B27_ActivinA_96h_rep2
SET2_ESC_H1_in_N2B27_ActivinA_96h_rep1
naïve

SET2_ESC_H1_in_N2B27_ActivinA_72h_rep1
ESC

SET2_ESC_H1_in_N2B27_ActivinA_72h_rep2
SET2_ESC_H1_in_N2B27_ActivinA_48h_rep2
SET1_Naive_WIBR3_3_ESC
SET1_Naive_WIBR3_1_ESC
SET1_Naive_WIBR3_5_ESC
SET1_Naive_WIBR3_2_ESC
SET1_Pri_WIBR3_1_ESC
SET1_GFP__ESC_H1
SET2_ESC_HUES9
SET1_ESC_SA01_rep3_1
SET1_ESC_SA01_rep3
SET1_ESC_SA01_rep1_1
SET1_ESC_SA01_rep1
SET2_ESC_ES2
SET1_ESC_T3_rep3
SET1_ESC_T3_rep2
SET1_ESC_T3_rep1
SET1_ESC_H1_20__O2_rep2
SET1_ESC_chHES_20
SET1_ESC_H9_1
SET1_ESC_HSF1
SET1_ESC_BG01
SET2_ESC_H9_P51
SET1_ESCHD129_p6_CF
SET1_ESC_VUB01_rep1_1
SET1_ESC_VUB01_rep1
SET1_ESCs_H1_rep1
SET1_ESCs_H1_rep3
SET1_ESCs_H1_rep2
SET2_ESC_H9_P92
SET2_ESC_H9_P91
SET2_ESC_H9_P50
SET2_ESC_H9_11
SET2_ESC_H9_10
SET2_ESC_HSF6_1
SET2_ESC_H1_in_N2B27_rep2_1
SET2_ESC_H1_in_N2B27_BMP4_2h
fibroblasts

SET2_ESC_H1_in_N2B27_rep1
SET2_ESC_H1_in_N2B27_rep2
SET2_ESC_H1_in_N2B27_ActivinA_2h
SET2_ESC_H1_in_N2B27_rep1_1
SET2_ESC_ES4
SET2_ESC_CyT25
SET2_ESC_Miz4_1
SET2_ESC_ES3
SET1_ESC_rep2
SET1_ESC_VUB01_rep2_1
SET1_ESC_VUB01_rep2
SET1_ESC_VUB01_rep3_1
SET1_ESC_VUB01_rep3
SET1_ESC_HD90_p8
SET1_ESC_VUB03_DM1_rep1
(most inverted)

SET1_ESC_HS235
SET1_ESC_SA01_rep2_1
SET1_ESC_SA01_rep2
primed ESC

SET1_ESC_VUB03_DM1_rep2
SET1_ESC_VUB03_DM1_rep3
SET1_ESC_HD83_p24_abnormal_VHL
SET2_ESC_BG03_3
SET2_ESC_BG01
SET2_ESC_BG02_1
SET2_ESC_H9_3
SET2_ESC_H9_1
SET2_ESC_H9_4
SET2_ESC_H9_2
SET1_ESC_H9_rep3_1
SET1_ESC_H9_rep1_1
SET1_ESC_H9_rep2_1
SET2_ESC_CyT203
SET2_ESC_BG02_2
SET1_H9_ESC_rep3
SET1_H9_ESC_rep2
SET1_H9_ESC_rep1
SET2_ESC_Miz6_2
SET1_ESC_H1L
SET2_ESC_Miz5_2
SET2_ESC_Miz5_1
SET2_ESC_Miz6_1
SET2_ESC_Miz4_2
SET2_ESC_H1_2
primed
ESC
SET2_ESC_H1_1
SET1_ESC_ES4
SET1_ESC_ES2
SET1_ESC_H9_rep2_3
SET1_ESC_H9_rep1_3
SET1_ESC_H1_rep2
SET1_ESC_H1_rep1
SET1_ESC_HUES6_rep2
SET1_ESC_HUES6_rep1
SET1_ESC_HUES6_rep3
+TGFb/Activin
SET1_ESC_Cyt25_rep2
SET1_ESC_Cyt25_rep1
SET1_ESC_Cyt25_rep3
SET2_ESC_HSF6_2
SET1_ESC_H9_2
SET1_CA2_ESC_Ctrl_D
SET1_CA2_ESC_Ctrl_E
SET1_ESC_H13B
SET1_ESC_H7
SET1_ESC_H14A
SET1_ESC_H9
SET2_ESC_H1_in_N2B27_ActivinA_24h_rep2
SET2_ESC_H1_in_N2B27_ActivinA_24h_rep1
SET1_WIBR3_ESC_5_pO2

+bFGF
SET2_ESC_H1_in_N2B27_ActivinA_12h_rep1
SET2_ESC_H1_in_N2B27_0h_rep2
SET2_ESC_H1_in_N2B27_0h_rep1
SET1_BGO1_Nanog_transgenic_ESC_line
SET1_BGO1_ESC_mTESR
SET1_BGO1_ESC

5 10 15 20 25 30
SET2_ESC_BG03_1
SET1_BJ1_fibroblast_47
SET1_ESC_H9_3

naïve

Fibroblast-like cancer cells

ESC
SET1_CA1_ESC_Ctrl_H
SET2_ESC_BG03_4
SET2_ESC_BG03_2

Cox PH P-value =8.8×10−4

SET1_ESC_CSES4p37_mRNA
SET1_CA1_ESC_Ctrl_I
SET2_ESC_H9_rep2
SET2_ESC_H9_rep1
SET2_ESC_H9_rep3

ESC-like cancer cells

SET1_ESC_H9_rep3_2
SET1_ESC_H9_rep1_2
SET1_ESC_H9_rep2_2
SET1_ESC_HS181_p50
SET2_Fibroblast_HS27Fib_5
SET2_Fibroblast_HS27Fib_3
SET2_Fibroblast_SC30BFib
SET2_Fibroblast_SC30AFib

Time in years
SET2_Fibroblast_HS27Fib_4
SET1_Foreskin_fibroblast_rep2
SET1_Foreskin_fibroblast_rep1
SET2_Fibroblast_HS27Fib_2
SET2_Fibroblast_HS27Fib_1
SET1_Fibroblast_unaffected

Stat3/LiF
SET1_Fibroblast_SMA
SET1_Parental_foreskin_fibroblast_rep2
SET1_BJ_fibroblast_rep3

c-Myc
SET1_BJ_fibroblast_rep2
SET1_BJ_fibroblast_rep1

Klf4
SET1_Fibroblast_NHDF3535p61_mRNA
SET1_BJ1_fibroblast_48
Supplementary Figure 3

SET1_Fibroblast_MBD_rep1
SET1_MRC5_fibroblast_59

fibroblasts
SET1_dH1f_fibroblast_44
SET1_MRC5_fibroblast_40
SET1_dH1f_fibroblast_58
SET1_WI38_fibroblast_rep2
SET1_WI38_fibroblast_rep1
SET1_WI38_fibroblast_rep3
SET1_D551_fibroblast_rep3
SET1_D551_fibroblast_rep2
SET1_D551_fibroblast_rep1
SET1_Fibroblast
SET1_Fibroblast_PDB
SET1_Parent_fibroblast_for_1_8_and_3_2
SET1_Parent_fibroblast_for_2_4
SET1_HDF
SET1_Fibroblast_rep3
SET1_Fibroblast_rep1
SET1_Fibroblast_rep2
SET1_Fibroblast_with_ctrl_vector_18_days
SET2_Fibroblast
SET2_ESC_fibroblast
SET1_Fibroblast_1
SET1_dH1cf16_fibroblast
SET1_Fibroblast_4_PD

0
SET1_Fibroblast_2_GD
SET1_Fibroblast_6_fetal_lung
SET1_Fibroblast_3_fetal_skin

1.0
0.8
0.6
0.4
0.2
0.0
SET1_Fibroblast_1_MBD
SET1_Fibroblast_5_SBDS
SET1_HFF1
SET2_ESC_HUES20
SET2_Fibroblast_SC33BFib
SET2_Fibroblast_SC33AFib
SET1_hTFC_rep2
SET1_hTFC_rep1
SET1_hTFC_rep3
SET1_H1_OGN_hES_cells
SET2_ESC_H9_6
Survival probability
SET2_ESC_H9_5
a

b
 EGFR −1
HRAS −2
−3
3
KRAS 2
NRAS 1
0

LGALS1

LGALS3
ITGA5
NPM1

PTRF
CAV2

CAV1
NCL

VIM

SET1_WIBR2.ESC
SET1_WIBR1.ESC
SET1_WIBR3.ESC
SET1_WIBR3.ESC.1
SET1_ESC.HS181.p50
SET1_H9.ESC.4..O2.rep1
SET1_H9.ESC.20..O2.rep1
SET1_BJ1.fibroblast.47
SET1_ESC.H1.4..O2.rep2
SET1_ESC.H1.20..O2.rep1
SET1_ESC.H9.rep2.3
SET1_ESC.H9.rep1.3
SET1_ESC.H1.rep2
SET1_ESC.H1.rep1
SET1_ESC.rep1
SET1_ESC.HUES6.rep2
SET1_ESC.HUES6.rep1
SET1_ESC.HUES6.rep3
SET1_ESC.Cyt25.rep2
SET1_ESC.Cyt25.rep1
SET1_ESC.Cyt25.rep3
SET1_ESC.rep2
SET1_ESC.H1.20..O2.rep2
SET1_ESC.chHES.20
SET1_ESC.H9.1
SET1_ESC.HSF1
SET1_ESC.BG01
SET1_ESC.SA01.rep3.1
SET1_ESC.SA01.rep3
primed ESC

SET1_ESC.SA01.rep1.1
SET1_ESC.SA01.rep1
(in transit)

SET1_ESC.T3.rep3
SET1_ESC.T3.rep2
SET1_ESC.T3.rep1
SET1_ESCHD129.p6.CF
SET1_ESC.HD90.p8
SET1_ESC.HD83.p24.abnormal.VHL
SET1_ESC.VUB01.rep1.1
SET1_ESC.VUB01.rep1
SET1_ESCs.H1.rep1
SET1_ESCs.H1.rep3
SET1_ESCs.H1.rep2
SET1_ESC.VUB01.rep3.1
SET1_ESC.VUB01.rep3
SET1_ESC.VUB01.rep2.1
SET1_ESC.VUB01.rep2
SET1_ESC.VUB03.DM1.rep1
SET1_ESC.HS235
SET1_ESC.SA01.rep2.1
SET1_ESC.SA01.rep2
SET1_ESC.VUB03.DM1.rep2
SET1_ESC.VUB03.DM1.rep3
SET1_BGO1.Nanog.transgenic.ESC.line
SET1_BGO1.ESC.mTESR
SET1_BGO1.ESC
SET1_H9.ESC.rep3
SET1_H9.ESC.rep2
SET1_H9.ESC.rep1
SET1_ESC.H9.rep2.1
SET1_ESC.H9.rep1.1
SiTayeb_ESC.H9.rep3
SET1_ESC.H9.rep3.1
SiTayeb_ESC.H9.rep2
SiTayeb_ESC.H9.rep1
SET1_ESC.H9.rep2
SET1_ESC.H9.rep1
SET1_ESC.H9.rep3
SET1_ESC.CSES4p37.mRNA
SET1_CA1.ESC.Ctrl.I
SET1_ESC.H9.rep3.2
SET1_ESC.H9.rep1.2
SET1_ESC.H9.rep2.2
SET1_HUES8.ESC.rep3
SET1_HUES8.ESC.rep1
SET1_HUES8.ESC.rep2
SET1_CA2.ESC.Ctrl.E
SET1_CA2.ESC.Ctrl.D
SET1_WIBR3.ESC.5.pO2
SET1_ESC.H9.2
SET1_ESC.H9.3
SET1_CA1.ESC.Ctrl.H
SET1_ESC.H13B
SET1_ESC.H7
naïve

SET1_ESC.H14A
ESC

SET1_ESC.H9
SET1_ESC.H1L
SET1_Naive.WIBR3.3.ESC
SET1_Naive.WIBR3.1.ESC
SET1_Naive.WIBR3.5.ESC
SET1_Naive.WIBR3.2.ESC
SET1_Pri.WIBR3.1.ESC
SET1_GFP..ESC.H1
SET1_ESC.H1
SET1_ESC.ES2.rep2
SET2_ESC.HUES9
SET2_ESC.HSF6.2
SET2_ESC.HUES7.2
SET2_ESC.HUES22
SET2_ESC.HUES7.1
SET2_ESC.H9.6
SET2_ESC.H9.5
SET2_ESC.HUES13.2
SET2_ESC.HUES13.1
SET2_ESC.HUES20
SET2_ESC.HUES21
SET2_ESC.H9..bFGF.1
SET2_ESC.H9..bFGF
SET2_ESC.H9.....bFGF.1
SET2_ESC.H9.....bFGF
SET2_ESC.H9.1.day.in.UM...4h.in.MEF.CM.rep1
SET2_ESC.H9.1.day.in.UM.rep1
SET2_ESC.H9.1.day.in.UM...4h.in.MEF.CM.rep2
SET2_ESC.H9.1.day.in.UM.rep2
(most inverted)

SET2_ESC.H9.1.day.in.UM...4h.in.UM.GTA.rep2
SET2_ESC.H9.1.day.in.UM...4h.in.UM.GTA.rep1
SET2_ESC.BG03.4
primed ESC

SET2_ESC.BG03.2
SET2_ESC.BG03.1
SET2_ESC.H9.rep3
SET2_ESC.H9.rep1
SET2_ESC.H9.rep2
SET2_ESC.CyT203
SET2_ESC.BG01
SET2_ESC.H9.3
SET2_ESC.H9.1
SET2_ESC.BG03.3
SET2_ESC.H9.4
SET2_ESC.H9.2
SET2_ESC.BG02.1
SET2_ESC.BG02.2
SET2_ESC.H9.P92
SET2_ESC.H9.P91
SET2_ESC.H9.P50
SET2_ESC.H9.P51
SET2_ESC.H9.11
SET2_ESC.H9.10
SET2_ESC.Miz4.1
SET2_ESC.HSF6.1
SET2_ESC.H1.in.N2B27.ActivinA.2h
SET2_ESC.H1.in.N2B27.rep1.1
SET2_ESC.H1.in.N2B27.rep2.1
SET2_ESC.H1.in.N2B27.BMP4.2h
SET2_ESC.H1.in.N2B27.rep2
SET2_ESC.H1.in.N2B27.rep1
SET2_ESC.ES4
SET2_ESC.ES3
SET2_ESC.ES2
SET2_ESC.CyT25
SET2_ESC.H1.in.N2B27.ActivinA.24h.rep1
SET2_ESC.H1.in.N2B27.ActivinA.12h.rep1
SET2_ESC.H1.in.N2B27.ActivinA.24h.rep2
SET2_ESC.H1.in.N2B27.0h.rep2
SET2_ESC.H1.in.N2B27.0h.rep1
SET2_ESC.H1.in.N2B27.ActivinA.36h.rep2
SET2_ESC.H1.in.N2B27.ActivinA.36h.rep1
SET2_ESC.H1.in.N2B27.ActivinA.12h.rep2
SET2_ESC.Miz5.2
SET2_ESC.Miz4.2
SET2_ESC.Miz6.1
SET2_ESC.Miz5.1
SET2_ESC.Miz6.2
SET2_ESC.H1.2
SET2_ESC.H1.1
SET2_ESC.H1.in.N2B27.ActivinA.48h.rep2
SET2_ESC.H1.in.N2B27.ActivinA.48h.rep1
SET2_ESC.H1.in.N2B27.ActivinA.72h.rep2
SET2_ESC.H1.in.N2B27.ActivinA.72h.rep1
SET2_ESC.H1.in.N2B27.ActivinA.96h.rep2
SET2_ESC.H1.in.N2B27.ActivinA.96h.rep1
SNU398
COLO320
DOHH2
ESC-like cancer cells
CMLT1
A2780
OVSAHO
HUTU80
HGC27
MEWO
CALU6
SKMEL2
SET1_ESC.ES4
SET1_ESC.ES2
NCIH1395
HS675T
A498
fibroblast-like cancer cells
NCIH1975
SKMEL24
MDAMB231
NCIH196
HS281T
U2OS
A375
U87MG
SET2_Fibroblast.SC30BFib
SET2_Fibroblast.SC30AFib
SET2_Fibroblast.HS27Fib.4
SET2_Fibroblast.HS27Fib.5
SET2_Fibroblast.HS27Fib.3
SET2_Fibroblast.SC33BFib
SET2_Fibroblast.SC33AFib
SET2_Fibroblast
SET2_ESC.fibroblast
SET2_Fibroblast.HS27Fib.2
SET2_Fibroblast.HS27Fib.1
SET1_WI38.fibroblast.rep2
Supplementary Figure 4

SET1_WI38.fibroblast.rep1
SET1_WI38.fibroblast.rep3
SET1_D551.fibroblast.rep3
SET1_D551.fibroblast.rep2
SET1_D551.fibroblast.rep1
SET1_Fibroblast.1

fibroblasts
SET1_BJ1.fibroblast.48
SET1_Fibroblast.MBD.rep1
SET1_Fibroblast.unaffected
SET1_Fibroblast.SMA
SET1_MRC5.fibroblast.59
SET1_BJ.fibroblast.rep3
SET1_BJ.fibroblast.rep2
SET1_BJ.fibroblast.rep1
SET1_dH1cf16.fibroblast
SET1_MRC5.fibroblast.40
SET1_dH1f.fibroblast.58
SET1_dH1f.fibroblast.44
SiTayeb_Fibroblast.rep1
SET1_Fibroblast.rep1
SiTayeb_Fibroblast.rep3
SET1_Fibroblast.rep3
SiTayeb_Fibroblast.rep2
SET1_Fibroblast.rep2
SET1_Fibroblast.with.ctrl.vector.18.days
SET1_Parent.fibroblast.for.1.8.and.3.2
SET1_Parent.fibroblast.for.2.4
SET1_HDF
SET1_Fibroblast
SET1_Fibroblast.PDB
SET1_Fibroblast.NHDF3535p61.mRNA
SET1_Fibroblast.4.PD
SET1_Fibroblast.2.GD
SET1_Parental.foreskin.fibroblast.rep2
SET1_Fibroblast.6.fetal.lung
SET1_Fibroblast.3.fetal.skin
SET1_Fibroblast.1.MBD
SET1_Fibroblast.5.SBDS
SET1_Foreskin.fibroblast.rep2
SET1_Foreskin.fibroblast.rep1
SET1_HFF1
SET1_H1.OGN.hES.cells
SET1_hTFC.rep2
SET1_hTFC.rep1
SET1_hTFC.rep3
 Supplementary Figure 5

a
cells expressing MST Library
NANOPS 384 compounds

NANOPS-FRET assay

primary hits ≥10% change in FRET efficiency

H-Ras K-Ras
NANOPS 11 14 14 NANOPS

validated hits 7

b
K-rasG12V - RBD recruitment FRET H-rasG12V - RBD recruitment FRET

control 121 control 207

Salinomycin 67 **** Salinomycin 125
Avermectin 116 *** Avermectin 180 **
K-ras RBD Ivermectin 131 ** H-ras RBD Ivermectin 182 *
Conglobatin 83 ns Conglobatin 182 **
Ophiobolin A 26 ** Ophiobolin A 174
Kazusamycin B 81 ** Kazusamycin B 85 ns
FRET Leptomycin B 67 *** FRET Leptomycin B 90
Streptonigrin 58 ** Streptonigrin 62 ns
5 10 15 5 10 15
apparent FRET efficiency (%) apparent FRET efficiency (%)

c
control Avermectin Ivermectin Conglobatin A Ophiobolin A Kazusamycin B Leptomycin B Streptonigrin
K-rasG12V
stable MDCK
H-rasG12V
 Supplementary Figure 6

a b c
MDA-MB-231

MDA-MB-436

MDA-MB-231
MDA-MB-436
MDA-MB-231 MDA-MB-231
Hs-578T

Hs-578T
MCF7

MCF7
150

scrambled
to control (%)
1.0

CaM/β-Actin
Oct 3/4

relative
100

siCaM
Sox 2
0.5
NANOG 50 Adherent CaM
β−Actin Spheres
0 0.0 β−Actin
Adherent Spheres 0.01 0.1 1

(6 d

)
nM
aM ble
concentration [μM]

0
ra
sc
C
si
d K-rasG12V nanoclustering-FRET
control
Conglobatin A ****

siRNA CaM1 ****

1.0
scrambled
CaM/β-Actin

CongA siCaM

siCaM + wt-CaM
− ns
0.5
+ ****
− ns CaM
siCaM + mut-CaM + ****
0.0 β−Actin
5 10 15
)
ed
nM

apparent FRET efficiency (%)

bl
0
m
(6
ra
aM
sc
C
si

e Salinomycin
1.0
Staurosporine
Ophiobolin A
Correlation coefficient

* * *
0.5

0.0

-0.5
*
*
KRAS
NCL
NPM1
NRAS
LGALS1
VIM
HRAS
PTRF
ITGA5
EGFR
LGALS3
CAV1
CAV2

-1.0