cancerdiscovery.aacrjournals.org · Web viewBI-3406, a potent and selective SOS1::KRAS interaction...

BI-3406, a potent and selective SOS1::KRAS interaction

inhibitor, is effective in KRAS-driven cancers through

combined MEK inhibition

Marco H. Hofmann 1* , Michael Gmachl1*, Juergen Ramharter1*, Fabio Savarese1, Daniel

Gerlach1, Joseph R. Marszalek3, Michael P. Sanderson1, Dirk Kessler1, Francesca Trapani1,

Heribert Arnhof1, Klaus Rumpel1, Dana-Adriana Botesteanu1, Peter Ettmayer1, Thomas

Gerstberger1, Christiane Kofink1, Tobias Wunberg1, Andreas Zoephel1, Szu-Chin Fu4, Jessica

L. Teh3, Jark Beottcher, Nikolai Pototschnig1, Franziska Schachinger1, Katharina Schipany1,

Simone Lieb, Christopher P. Vellano3, Jonathan C. O’Connell2, Rachel L. Mendes2, Jurgen

Moll1, Mark Petronczki, Timothy P. Heffernan3, Mark Pearson1, Darryl B. McConnell1,

Norbert Kraut1.

1 Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria

2 Forma Therapeutics, Watertown, MA, USA

3 TRACTION Platform, Therapeutics Discovery Division, The University of Texas MD

Anderson Cancer Center, Houston, TX 77030, USA

4 Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center,

Houston, TX 77030, USA

*These authors contributed equally to the work

Supplementary Data and Tables

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Supplementary Data

Experimental procedures for synthesis of BI-3406

Unless specifically mentioned, all described reactions were performed in commercially

available laboratory glassware and standard synthetic chemistry methods were applied. All

moisture or air sensitive reactions were carried out in an inert gas atmosphere (dry nitrogen or

argon) and dried glassware was used. Commercially available starting materials were utilized

without further purification. Solvents were of commercial analytical, dry or extra-dry grade.

All other used chemicals were reagent grade. Preparative RP-HPLC was performed on

Agilent or Gilson systems. The following columns from Waters were used: Sunfire C18

OBD, 5 or 10 μm, 20 mm× 50 mm, 30 mm × 50 mm, 30 mm × 100 mm, 50 mm × 100 mm, or

50 mm × 150 mm; X-Bridge C18 OBD, 5 or 10 μm, 20 mm × 50 mm, 30 mm × 50 mm, or 50

mm × 150 mm or YMC (Triart C18, 5, 10 or 20 μm, 20 mm × 50 mm, or 30 mm × 50 mm).

Unless specifically mentioned, compounds were eluted with acetonitrile/water gradients using

either basic water (5 mL of 2 M NH4HCO3 + 2 mL of NH3 (32%) made up to 1 L with water)

or acidic water (0.2% HCOOH or TFA). All samples were analyzed on an Agilent 1260 series

LC system coupled with an Agilent 6130 mass spectrometer. Purity determination was done

via UV detection with a bandwidth of 170 nm in the range from 230-400. The following LC

parameters were used: Waters Xbridge C18 column, 2.5 μm particle size, 2.1 x 20 mm. Run

time 2.1 minutes, flow 1.4 mL/min, column temperature 45°C and 5 μl injections. Solvent A

(20 mM NH4HCO3/ NH3 pH 9), solvent B (MS grade acetonitrile). Start 5% B, gradient 5% -

95% B from 0.0 - 1.5 min, 95% B from 1.5 - 2.0 min, gradient 95% - 5% B from 2.0 – 2.1

min. Purity of all described compounds was >95%.

Bruker Avance HD 400 MHz and 500 MHz spectrometers, equipped with BBO Prodigy and

TCI cryoprobes respectively, were used for NMR experiments. All samples were dissolved in

600 μL DMSO-d6. As internal standard TMS was added. All NMR spectra were recorded at

298 Kelvin. 1D 1H spectra were recorded with 30° excitation pulses and an interpulse delay of

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4.2 sec with 64k data points and 20 ppm sweep width. 1D 13C spectra were recorded with

broadband composite pulse decoupling (WALTZ16) and an interpulse delay of 3.3 sec with

64 k data points and a sweep width of 240 ppm. Bruker Topspin 3.2 software was used to

process and analyze the 1D spectra. No zero filling was performed. After automatic baseline

correction all spectra were manually integrated. Chemical shifts are reported in ppm (δ-scale).

A Thermo Scientific Orbitrap Elite hybrid ion trap/orbitrap spectrometer system with an

Ultimate 3000 series LPG-3400XRS pump system was used to record HRMS data. Mass

calibration was done using the Pierce LTQ Velos ESI positive ion calibration solution from

Thermo Scientific.

1-(3-Nitro-5-(trifluoromethyl)phenyl)ethan-1-one ( 2 ):

1-Bromo-3-nitro-5-trifluoromethyl-benzene (1) (100.0 g, 370.4 mmol, 1.0 equiv.) is dissolved

in dry 1,4-dioxane (1.0 L). Then NEt3 (103 mL, 739.0 mmol, 2.0 equiv.) is added and the

resulting solution purged with argon for 5 min. Tributyl(1-ethoxyvinyl)tin (173.0 g,

479.0 mmol, 1.3 equiv.) and bis(triphenylphosphine)palladium(II)chloride (26.0 g,

37.0 mmol, 0.1 equiv.) are added subsequently and the resulting mixture is heated to 80 °C in

an autoclave for 12 hours. After recooling to room temperature the mixture is carefully treated

with 1.0 N aqueous HCl, EtOAc is added and the aqueous phase is extracted with EtOAc. The

combined organic layers are dried over Na2SO4, filtered and the solvent is removed in vacuo.

The crude product is purified by chromatography on silica gel using EtOAc/petrol ether as

eluent providing desired product 2 (55.0 g, 235.9 mmol) with 64% yield.

1H NMR (500 MHz, DMSO-d6): δ (ppm) 8.85-8.87 (m, 1H), 8.75 (s, 1H), 8.64 (s, 1H), 2.77

(s, 3H)

13C NMR (125 MHz, DMSO-d6): δ (ppm) 196.1, 139.6, 131.0, 131.0, 131.3, 126.7, 124.7,

123.2, 27.6

HRMS calculated for [M+H]+ (C9H7F3NO3): 234.03725, found: 234.03683

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( R,E )-2-Methyl-N-(1-(3-nitro-5-(trifluoromethyl)phenyl)ethylidene)propane-2-sulfinamide

( 3 ):

Intermediate 2 (53.0 g, 227.3 mmol; 1.0 equiv.) is dissolved in THF (600 mL).

(R)-(+)-2-methyl-2-propanesulfinamide (41.3 g; 340.8 mmol; 1.5 equiv.) and Ti(OEt)4

(129.6 g, 568.0 mmol; 2.5 equiv.) are added at room temperature and the resulting reaction

mixture is heated to 80 °C for 5 hours. The reaction mixture is cooled to room temperature

and quenched with ice water. The precipitate is dissolved in EtOAc and filtered through

celite. The combined organic layers are dried over Na2SO4, filtered and the solvent is removed

in vacuo. The crude product is purified by chromatography on silica gel using EtOAc/petrol

ether as eluent yielding desired product 3 (55.0 g, 163.5 mmol) with 72% yield.

1H NMR (500 MHz, DMSO-d6): δ (ppm) 8.85 (s, 1H), 8.66 (s, 1H), 8.55 (s, 1H), 2.82 (s, 3H),

1.26 (s, 9H)


123.2, 58.2, 22.6, 20.2

HRMS calculated for [M+H]+ (C13H16F3N2O3S): 337.08282, found: 337.08335

( R )-2-Methyl-N-(( R )-1-(3-nitro-5-(trifluoromethyl)phenyl)ethyl)propane-2-sulfinamide ( 4 ):

To a stirred solution of intermediate 3 (270.0 g, 803 mmol, 1.0 equiv.) in THF (2.5 L) and

water (50 mL) is added sodium borohydride (54.0 g, 1427 mmol, 1.8 equiv.) at -50 °C. After

5 hours and full conversion of the starting material the reaction mixture is quenched with ice

water and extracted with EtOAc. The combined organic layers are dried over Na2SO4, filtered

and the solvent is removed in vacuo. Purification by chromatography on silica gel using

EtOAc/petrol ether as eluent leads to desired diastereomer 4 (170.0 g, 0.502 mol) as main

product with 63% yield.

1H NMR (500 MHz, DMSO-d6): δ (ppm) 8.64 (s, 1H), 8.39 (s, 1H), 8.30 (s, 1H), 6.08 (d, J =

8.5 Hz, 1H), 4.56-4.81 (m, 1H), 1.46 (d, J = 6.9 Hz, 3H), 1.14 (s, 9H)

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125.9, 125.9, 123.6, 119.3, 55.9, 54.9, 24.3, 23.0

HRMS calculated for [M+H]+ (C13H18F3N2O3S): 339.09847, found: 339.09909

( R )-1-(3-nitro-5-(trifluoromethyl)phenyl)ethan-1-amine ( 5 ):

To a stirred solution of 4 (170.0 g, 502.4 mmol, 1.0 equiv.) in 1,4-dioxane (100 mL) is added

4.0 N HCl in dioxane (250 mL, 1000 mmol, 2.0 equiv.). The reaction mixture is stirred at

room temperature and monitored by TLC. After 5 hours and complete conversion of the

starting material, the reaction mixture is concentrated in vacuo. The crude solid is washed

with Et2O to obtain desired product 5 as HCl salt (118.0 g, 436.0 mmol) with 87% yield.

1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.78 (br s, 3H), 8.50-8.54 (m, 1H), 8.47 (s, 1H),

4.75 (q, J=6.59 Hz, 1H), 1.59 (d, J=6.84 Hz, 3H)


49.5, 20.8

HRMS calculated for [M+H]+ (C9H10F3N2O2): 235.06889, found: 235.06896

( R )-3-(1-aminoethyl)-5-(trifluoromethyl)aniline ( 6 ):

The HCl salt of compound 5 (98.0 g, 362.1 mmol, 1.0 equiv.) is dissolved in MeOH

(980 mL). 10 % palladium on carbon (20.0 g, 18.79 mmol, 0.05 equiv.) is added and the

reaction purged with H2 gas (40 psi). The reaction is stirred at room temperature and

monitored by TLC. After 12 hours and complete conversion of the starting material, the

reaction mixture is filtered over celite and the filtrate concentrated in vacuo to furnish desired

product 6 as HCl salt (80.0 g, 332.4 mmol, 92%).


7.07 (s, 1H), 4.29-4.45 (m, J=5.90, 5.90, 12.10 Hz, 1H), 1.49 (d, J=6.62 Hz, 3H)


50.1, 20.9

HRMS calculated for [M+H]+ (C9H12F3N2): 205.09471, found: 205.09503

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6-Hydroxy-7-methoxy-2-methylquinazolin-4(3H)-one ( 8 ):

4.0 N HCl in dioxane (1.4 L, 5.6 mol, 7.9 equiv.) is added to a solution of compound 7

(140.0 g, 710.0 mmol, 1.0 equiv.) and acetonitrile (370 mL, 7.08 mol, 10.0 equiv.). The

resulting solution is heated to 60 °C for 5 hours until complete conversion of the starting

material is observed. After cooling to room temperature the crude product precipitates. The

solid material is collected by filtration, washed with saturated aqueous NaHCO3 and water

and finally dried to yield desired product 8 (110.0 g, 533 mmol, 75%).

1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.90 (s, 1H), 9.69 (s, 1H), 7.33 (s, 1H), 7.02 (s,

1H), 3.88 (s, 3H), 2.29 (s, 3H)


108.1, 56.2, 21.6

HRMS calculated for [M+H]+ (C10H11N2O3): 207.07642, found: 207.07656

( S )-7-Methoxy-2-methyl-6-((tetrahydrofuran-3-yl)oxy)quinazolin-4(3H)-one ( 9 ):

A suspension of compound 8 (8.50 g, 41.22 mmol, 1.0 equiv.), (R)-tetrahydrofuran-3-yl 4-

methylbenzenesulfonate (51) (11.00 g, 45.40 mmol, 1.1 equiv.) and Cs2CO3 (16.00 g,

49.11 mmol, 1.2 equiv.) in DMF (200 mL) is heated to 100 °C for 12 hours. The solvent is

removed under reduced pressure. The residue is suspended in MeOH and stirred for additional

1.5 hours. The solid material is removed by filtration. Both filtrate and precipitate contain

crude product. Purification is done for both separately. The precipitate is purified by NP

chromatography (DCM:MeOH:NH3, 19:1:0.1). The solvent of the filtrate is removed in vacuo

and the residue purified by RP chromatography. In total 7.86 g (28.45 mmol) of desired

product 9 are obtained with 69% yield.


5.00-5.17 (m, 1H), 3.87-3.90 (m, 1H), 3.83 (br s, 1H), 3.82-3.93 (m, 2H), 3.77 (br d, J=4.73

Hz, 1H), 3.36-3.38 (m, 14H), 2.31 (s, 3H), 2.20-2.29 (m, 1H), 1.96-2.04 (m, 1H)

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107.6, 78.6, 72.6, 66.9, 56.3, 32.7, 21.7

HRMS calculated for [M+H]+ (C14H17N2O4): 277.11828, found: 277.11848

( S )-7-Methoxy-2-methyl-6-((tetrahydrofuran-3-yl)oxy)quinazolin-4-yl 2,4,6-triisopropyl-

benzenesulfonate ( 10 ):

Compound 9 (6.90 g, 24.97 mmol, 1.0 equiv.), 2,4,6-triisopropylbenzenesulfonyl chloride

(9.35 g, 30.87 mmol, 1.2 equiv.), and 4-dimethylaminopyridine (427 mg, 3.495 mmol,

0.1 equiv.) are suspended in DCM (455 mL) and triethylamine (10.44 mL, 74.90 mmol,

3.0 equiv.). The reaction mixture is stirred at room temperature for 12 hours. Additional 2,4,6-

triisopropylbenzenesulfonyl chloride (2.27 g, 7.50 mmol, 0.3 equiv.) is added and the mixture

stirred for another 6 hours. The reaction is diluted with DCM, extracted with NaHCO 3 (sat.)

and the water phase washed with DCM. The combined organic layers are dried over MgSO4,

filtered and the solvent is removed in vacuo. The crude product is purified by chromatography

using cyclohexane/EtOAc to give the desired product 10 (8.75 g, 16.12 mmol, 65%).

1H NMR (500 MHz, DMSO-d6): δ (ppm) 7.44 (s, 1H), 7.10 (s, 1H), 6.94-6.97 (m, 2H), 5.16-

5.23 (m, J = 5.7, 4.7 Hz, 1H), 4.50-4.60 (m, 2H), 3.93 (s, 3H), 3.82-3.87 (m, 2H), 3.74-3.81

(m, J = 8.4, 8.4 Hz, 2H), 2.76-2.84 (m, 1H), 2.52 (br s, 3H), 2.22-2.34 (m, 1H), 1.93-2.05 (m,

1H), 1.17 (d, J = 6.9 Hz, 6H), 1.10 (d, J = 6.6 Hz, 12H)


121.9, 112.9, 108.1, 103.5, 79.0, 72.5, 66.9, 56.8, 33.7, 32.6, 28.5, 25.3, 24.3, 19.7

HRMS calculated for [M+H]+ (C29H39N2O6S): 543.25233, found: 543.25292

N -(( R )-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)-7-methoxy-2-methyl-6-((( S )-

tetrahydrofuran-3-yl)oxy)quinazolin-4-amine ( BI-3406 ):

Compound 10 (20.00 g, 36.85 mmol, 1.0 equiv.) and the HCl salt of compound 6 (13.30 g,

55.27 mmol, 1.5 equiv.) are suspended in DMSO (250 mL) and triethylamine (25.6 mL,

184.50 mmol, 5.0 equiv.) and the reaction mixture is stirred at 90 °C for 6 hours. After

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complete conversion of the starting material the mixture is diluted with MTBE (330 mL) and

extracted with saturated aqueous NaHCO3. The combined organic layers are dried over

MgSO4, filtered and the solvent is removed in vacuo. The crude product is purified by NP

chromatography (DCM:MeOH:NH3, 19:1:0.1) and RP chromatography subsequently to give

the desired product BI-3406 (13.50 g, 29.19 mmol, 79%).

1H NMR (500 MHz, DMSO-d6): δ (ppm) 7.96 (d, J=7.88 Hz, 1H), 7.70 (s, 1H), 7.05 (s, 1H),

6.88 (s, 1H), 6.84-6.87 (m, 1H), 6.70 (s, 1H), 5.56-5.62 (m, 1H), 5.53-5.56 (m, 2H), 5.11-5.22

(m, 1H), 3.98 (dd, J=4.41, 10.09 Hz, 1H), 3.89 (d, J=7.57 Hz, 1H), 3.86-3.88 (m, 3H), 3.78-

3.86 (m, 2H), 2.36 (s, 3H), 2.31 (br d, J=7.57 Hz, 1H), 1.98-2.07 (m, 1H), 1.56 (d, J=7.25 Hz,

3H)


125.1, 115.5, 110.0, 108.4, 107.5, 106.7, 105.5, 78.6, 72.6, 66.9, 56.1, 48.8, 32.8, 26.5, 22.3.

HRMS calculated for [M+H]+ (C23H26F3N4O3): 463.19515, found: 463.19535

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Protein expression and purification

Constructs for expression of human SOS1 (residues 564-1049 (uniprot ID: Q07889) and the

respective point mutants H905V and Y884A) were obtained by gene synthesis (GeneArt,

Thermo-Fisher) in donor vector (pDONR-221) and transferred by recombinant cloning into

the glutathione S-transferase (GST) fusion (pDEST15) or His6-tag (pDEST17) vector,

respectively. The plasmids were used to transform Escherichia coli, strain BL21(DE3). After

expression in Terrific Broth (TB) media, induced with 0.25 mM IPTG at 23°C for 20 h, the

cells were harvested by centrifugation and stored at -80 °C. Cell pellets of the His6-tagged

variant, designated for crystallization, were extracted by sonication in 20 mM Tris, 300 mM

NaCl, 10 mM imidazole, containing protease inhibitors (cOmplete, EDTA-free, Merck), pH

8.0. Cell debris was removed by centrifugation for 45 min at 13,000 rpm and the supernatant

was coupled for further purification on NiNTA superflow beads (Qiagen) for 2 hours,

centrifuged and washed, put into a column and eluted with an elution buffer containing 20

mM Tris, 300 mM NaCl and 250 mM Imidazole at pH 8.0. After purification on a HiPrep

desalting 26/10 column in 20 mM Tris and 150 mM NaCl at pH 7.8, the His6-tag was cleaved

off by incubation with TEV protease overnight at 4°C. The cleaved His6-tag was removed by

passing the protein solution through a Ni-NTA column (HisTrap, GE Healthcare). Finally, the

protein was purified on a HiLoad Superdex S200 size exclusion column and concentrated to a

final concentration of 29 mg/mL. For the GST-tagged proteins used in biophysical assays,

cells pellets were solubilized in lysis buffer (20mM TRIS (pH 7.5), 300mM NaCl, 1mM

TCEP), and disrupted by sonication on ice. The sonicated lysate was clarified by

centrifugation at 15000rpm for 40min. The supernatant was loaded onto a glutathione-

Sepharose-4B affinity column (GE Healthcare) equilibrated with lysis buffer and washed until

a stable baseline was obtained. The protein was eluted with lysis buffer + 25 mM L-

Glutathione reduced. Finally, the protein was purified on a HiLoad Superdex S75 column (GE

Healthcare) and concentrated to 15-20 mg/ml for further usage.

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Protein crystallization and structure solution

Prior to crystallization, the protein at a concentration of 18.2 mg/mL was incubated for 90

minutes with 4 mM compound. Crystals were obtained after 24 hours at 277 Kelvin using the

hanging drop vapour-diffusion method with a drop ratio of 750 nL protein and 750 nL

reservoir. The condition for BI-68BS is as follows: 3 % w/v PEG 8000 and 0.1 M Imidazole

pH 7.4. Conditions for BI-3406 are slightly different with 13 % w/v PEG 8000 and 0.1 M

Imidazole pH 8.0. The cryo protectant used for flash freezing in liquid nitrogen contained 30

% v/v Ethylene glycol and 8 % v/v Glycerol.

Data were collected at the Swiss Light Source beam line X06SA (SLS, Paul Scherrer

Institute; wavelength of 1 Å using an EIGER detector and on a Rigaku home system at 1.54

Å). The images were processed with XDS (52) through the autoPROC (53) pipeline and

scaled anisotropically with STARANISO. The phase problem was solved by Molecular

Replacement using PHASER (54) with a previously determined in-house structure

(unpublished) as a search model. Model building and refinement was done using standard

protocols in CCP4 (Collaborative Computational Project, Number 4) (55), COOT (56) and

the autoBUSTER (57) software package. GRADE (GlobalPhasing, UK) was used for

calculating restraint dictionaries for ligands and pictures were created with PYMOL

(Schrödinger, USA) (56,57). The unit cell parameters for PDB code 6SCM are a = 38.0 Å, b =

78.7 Å, c = 168.9 Å and α, β ,γ = 90° with a resolution of 1.87 Å, data and the structure was

refined to Rwork and Rfree values of 19.9% and 22.0%, respectively, with 98.5% of the residues

in Ramachandran favored regions as validated with MOLPROBITY (58). For PDB code

6SFR the cell parameters are a = 84.0 Å, b = 39.29 Å, c = 176.5 Å and α, γ = 90° and β

=90.03 with a resolution of 1.91 Å. The data and the structure was refined to Rwork and Rfree

values of 20.0% and 24.5%, respectively, with 99.3% of the residues in Ramachandran

favored regions as validated with Molprobity. Statistics for data collection and refinement can

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be found in Supplementary Table S1. The coordinates and structure factors of the structure

can be accessed with PDB Code 6SCM and 6SFR in the Protein Data Bank.

Surface Plasmon Resonance

SPR experiments were performed on a Biacore T200 instrument (GE Healthcare). Human

SOS1 (564-1049) was prediluted to 0.03 mg/mL in 10 mM sodium acetate pH 5, 0.005 %

surfactant P 20, supplied with 10 uM of the SOS1 activator 4 as described by Burns et. al.

(59). 10 mM HEPES, 150 mM NaCl, pH 7.4 + 0.005 % surfactant P 20 was used as

immobilization buffer and the protein was coupled to a density of ~ 1,500 RUs onto flow cells

3 and 4 of a Biacore CM5 chip at 25°C, using EDC/NHS chemistry. Flow cell 1 was left

blank and used as a reference surface. Interaction experiments were performed in 10 mM

HEPES, 150 mM NaCl pH 7.4 + 0.005 % surfactant P 20 at a DMSO concentration of 1 %

and a temperature of 6°C. Compound binding to SOS1 was analyzed on both flow cells and

samples were measured in six independent experiments across the two flow cells. KD values

were determined using Biacore T200 evaluation software. The steady-state responses for the

different concentrations were fitted to a 1:1 interaction model. Values from the different

experiments and flow cells were averaged.

Transfection of cells with a Cas9-encoding lentivirus

NCI-H23 (ATCC #CRL-5800) and NCI-H358 (ATCC #CRL-5807) cells were cultured in

RPMI medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. For

transfection, cells were split and 0,5 x 10EXP6 cells were seeded in 6-well plates. Cells were

transfected with a Cas9 lentivirus containing plasmid MP110_Lenti_Cas9_puro_(cc60)

(GenScript) and were incubated for 20 hours. Medium was changed and cells were washed

with 1 x PBS. On the next day, puromycin selection was started by using 2 µg/mL puromycin

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(Sigma #P9620). NCI-H23_Cas9 and NCI-H358_Cas9 positive cells were expanded and used

to generate isogeneic cell lines or SOS2 knockout cells.

Generation of NCI-H23_Cas9 KRAS isogenic cell lines

gRNAs targeting exon 2 (codon 12 and 13) and exon 3 (codon 61) of the KRAS locus were

cloned into vector pX458 encoding Cas9, a gRNA expression cassette and a GFP reporter.

Donor plasmids were designed to harbor the respective KRAS codon 12/13 or 61 base pair

change, a silent mutation for the restriction enzyme HindIII-FD (Thermo Fisher #FD0504) as

well as homology arms 700-750 bp left and right of KRAS codon 12 or 61(Supplementary

Table S10). In order to generate the H23_Q61H mutation, the H23 isogenic line with

homozygous wild type G12 allel was used. All plasmids were ordered by GenScript. NCI-

H23_Cas9 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum

at 37°C and 5% CO2. 1x10EXP6 cells were transfected in a 1:1 ratio with the respective

gRNA (Supplementary Table S11) and donor plasmid via Amaxa electroporation using Kit T

(Lonza #VPA-1002) and the program setting A-023 was used according to the manufacturer’s

instructions. 48 hours after transfection, individual GFP positive cells were sorted by FACS

(SONY cell sorter S800Z) to select GFP positive cells. Subsequently, cells were cultured for

one week to allow genome editing and recovery. Recovered cells were then seeded into 96-

well plates at a single cell density. After 14-20 days of culture, single cell-derived colonies

were selected and DNA was isolated using QuickExtract™ DNA Extraction Solution

(Lucigen #101094). Verification of effective genome engineering of the KRAS alleles was

performed by PCR amplification using specific primers (Supplementary Table S12) covering

the respective regions and subsequently controlled by restriction digest, Sanger and NGS

sequencing. Clones were selected and expanded. We used NCI-H23 cells carrying a

heterozygous KRAS G12C allele and replaced the G12C codon by heterozygous G12D,

G12V, G12R, G13D or homozygous G12D, G13D or Q61H alleles.

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Generation of SOS1 or SOS2 negative cell lines

NCI-H358_Cas9 cells were cultured in RPMI or DMEM medium supplemented with 10%

fetal bovine serum at 37°C and 5% CO2, respectively. For transfection, cells were splitted and

seeded in 6-well plates to achieve a confluency of 30%. Cells were transfected with the

respective plasmid ordered from SIGMA encoding the Cas9 endonuclease, GFP and a SOS1

or SOS2 targeting gRNA using X-tremeGENE-9 DNA transfection reagent (ROCHE

#06356779001) according to the manufacturer’s instructions (Supplementary Table S11). 48

hours after transfection individual GFP positive cells were sorted by FACS (SONY cell sorter

S800Z) and seeded into 96-well plates for isolation of single cell colonies. After 20 days of

culture single cell-derived colonies were lysed directly in 96-well format using 4 x Laemmli

buffer and analyzed by Western Blot. Proteins were separated by SDS-PAGE and transferred

to nitrocellulose membranes (BioRad #1704159) according to standard protocols. Membranes

were immunoblotted with antibodies against SOS1 (Bethyl #A301-890A), SOS2 (Abcam

#ab85831) and α-Tubulin (Cell Signaling #2144) in 5% BSA in TBST blocking buffer. After

primary antibody incubation, membranes were incubated with anti-rabbit IgG secondary

antibody (Dako #P0448) and signals were detected by chemiluminescence (GE Healthcare

Life Science #RPN2106). To generate cell lines one clone showing complete knockout of the

specific target gene was selected and expanded.

Plasmid design and transgenic expression of FLAG-SOS1 variants

pMSCV-3xFLAG-SOS1wt-Puro-IRES-GFP, pMSCV-3xFLAG-SOS1-H905V-Puro-IRES-

GFP and pMSCV-3xFLAG-SOS1r-H905I-Puro-IRES-GFP were generated based on the

SOS1 cDNA sequence NCBI NM_005633, followed by cloning into the parental pMSCV

vector (GenScript, China). Ecotropic retroviral particles harboring the empty vector and

FLAG-SOS1 transgene variant were generated in Platinum-E cells (Cell Biolabs) in the

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presence of 4 μg/mL Polybrene (Merck Millipore). MIA PaCa-2 cells were transduced with a

lentiviral pRRL_RIEN (rtTA-IRES-EcoReceptor-Neomycin) vector to introduce the murine

ecotropic receptor. Geneticin (Life Technolologies)-selected cells were subsequently

transduced with the empty vector or FLAG-SOS1 variant encoding retroviruses. Stable

transgenic cell pools were selected using 2µg/mL Puromycin (SIGMA (P9620). Infection

efficiency was confirmed by assessing GFP-positive cells using a cell analyzer and by

immunodetection of the FLAG-tagged transgenes. Finally, MIA PaCa-2 cell pools were used

for BI-3406 dose-response 3D proliferation assays and phospho-ERK biomarker assays.

HEK293 cells were transiently transfected with empty vector and the FLAG-SOS1 variant

encoding plasmids (wild-type, H509V and H509I) using the X-tremeGENE 9 DNA

transfection reagent (ROCHE), according to manufacturer’s instruction.

Reduction of pERK and KRAS-GTP in the presence of EGF stimulation

Cells (NCI-H358 or MIA PaCa-2) were seeded in T175 flasks to a number of 5x10EXP5 in

duplicates or triplicates in normal supplemented media including 10% FCS. Cells were grown

for 3 days followed by starvation in media with 0,5 % FCS for one day to reduce RTK-

stimulation mediated by growth factors. Prior to harvesting, the cells were either treated with

1 µM BI-3406 for 1 hour and subsequent stimulation with 1ng/mL EGF for exactly 2 minutes

or in case of the positive control they were only stimulated with 1ng/mL EGF. For harvesting

the flask containing the cells was placed on ice, the media was aspirated and cells were

washed in 10 mL ice cold DPBS. After removal of the DPBS, cold lysis buffer (500 µL/T175

flask, 150/35 mm dish) was applied and cells were lysed for 10 minutes on ice. The cell

debris was removed in a centrifugation step and lysates were snap frozen and stored -80°C

until further analysis in three aliquots for: (i) protein concentration measurement, (ii) Western

blot analysis for pERK1/2 (Thr202/Tyr204; cell signaling) and (iii) RAS GLISA assay

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(Cytoskeleton). In case of the Western Blots, the band intensities were quantified with the

software UN-SCAN-IT and values were normalized to the intensity of the loading control.

Protein and phosphoprotein quantification in cell extracts using a capillary

immunodetection assay

Cell lysates for capillary immunodetection assays were prepared using MSD Tris Lysis Buffer

(Meso Scale Diagnostics) in combination with Halt™ Protease and Phosphatase Inhibitor

Cocktail (ThermoFisher Scientific). Antibodies against FLAG (R&D System), -Actinin,

phospho-ERK1/2 (p44/p42 MAPK, ERK1/2; Thr202/Tyr204), total ERK1/2, phospho-MEK

(phospho-MEK1/2; Ser217/221), total MEK1/2, pAkt (Ser473) and total Akt (all from Cell

Signaling) as well as GAPDH and Vinculin (Abcam) were used to detect protein levels

according to the manufacturer’s instructions (WES; Protein Simple) and analyzed using

Compass for Simple Western software.

3D proliferation

Cell proliferation assays were used to examine the potency with which compounds inhibit the

SOS1-mediated proliferation growth of cancer cell lines in vitro. Low IC50 values are

indicative of high potency in this assay setting. Cell proliferation assays were performed in

three-dimensional (3D) anchorage-independent soft-agar conditions. The assay set-up is

composed of a bottom layer consisting of medium including 1.2 % agarose, a cell-layer

consisting of medium including 0.3 % agarose and a top-layer consisting of medium including

the test compounds (without agarose). After cooling to room temperature for 1 hour, cells

were incubated overnight at 37 °C and 5 % CO2 in a humidified atmosphere. The next day the

compounds were added in triplicates. Cells were incubated at 37 °C and 5 % CO2 in a

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humidified atmosphere for 5 - 14 days, dependent of the growth kinetics of the respective cell

line. AlamarBlue suspension, as described by the provider were used for detection. Data was

fitted by iterative calculation using a sigmoidal curve analysis program (GraphPad Prism

Software) with variable hill slope to ascertain IC50 values.

3D proliferation assay with trametinib and BI-3406 to analyze synergy

A 3D proliferation assay was performed with MIA PaCa-2 (KRAS G12C) and DLD1 (KRAS

G13D) cells embedded in softagar as described above. The cells were incubated with different

concentrations of the SOS1::KRAS inhibitor BI-3406 and with different concentrations of the

MEKi trametinib (Supplementary Fig. S4a). After 7 days of incubation with the compounds

AlamarBlue suspension was used as a readout. A cell growth inhibition (CGI) of >0% and

<100% reflects a partial growth-inhibitory effect relative to vehicle-treated controls. A CGI of

100% is equivalent of complete blockade of growth and a CGI of >100% is indicative of net

cell death. The Bliss excess CGI is the difference between the experimentally observed CGI

and the predicted CGI at various compound concentration combinations (60). Bliss excess

CGI values of > 0 are indicative of more than additive effects on cell growth inhibition.

Assessment of the combinatorial effect using the Bliss independence model revealed a more

than additive anti-proliferative effect of the combined drugs, over a range of concentrations

around the individual inhibitors IC50 values.

The effect on cleaved PARP following treatment of DLD1 cells with either BI-3406,

trametinib or the combination was analyzed in cell lysates using the MSD Assay System

(Apoptosis Panel Whole Cell Lysate Kit).

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Gene expression profiling (RNA-seq) and differential gene expression analysis

Single-end sequencing reads from grafted samples were filtered into human and mouse reads

using Disambiguate (61). The filtered reads were then processed with a pipeline, building

upon the implementation of the ENCODE’ “Long RNA-seq” pipeline: filtered reads were

mapped against the Homo sapiens (human) genome hg38/GRCh38 (primary assembly,

excluding alternate contigs) or the Mus musculus (mouse) genome mm10/GRCm38 using the

STAR (v2.5.2b) (62) aligner allowing for soft clipping of adapter sequences. For

quantification, we used transcript annotation files from Ensembl version 86, which

corresponds to GENCODE 25 for human and GENCODE M11 for mouse. Samples were

quantified with the above annotations, using RSEM (v1.3.0) (63) and featureCount (v1.5.1)

(64). Quality controls were implemented using FastQC (v0.11.5) (Andrews S. 2010:

Available online at: http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc), FastQC (a quality

control tool for high throughput sequence data

(http://www.bioinformatics.babraham.ac.uk/projects/fastqc)), picardmetrics ((v0.2.4)

(Slowikowski K. (2016): Available online at: https://github.com/slowkow/picardmetrics)) and

dupRadar (v1.0.0) (65) at the respective steps. Finally, differential expression analysis was

performed on the human mapped counts derived from featureCount using limma/voom

(66,67). If not otherwise stated, we used an absolute log2 fold change cut-off of 1 and a false

discovery rate (FDR) of <0.05.

Gene expression data for TCGA data was processed starting from BAM files provided by

NCI's Genomic Data Commons on 2017-12-20 (68). BAM files were converted to paired-end

FASTQ files using samtools (PMID 19505943). From here, the gene expression pipeline was

applied as above, omitting the human/mouse filtering step. Reported gene expression values

are TPMs (Transcripts Per Million) for these TruSeq RNA samples.

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Variant calling from whole-exome sequencing data (DNA-seq)

Paired-end sequencing reads underwent adapter trimming using cutadapt (v1.15) (69) and

alignment using bwa (v0.7.17) (70) against the human genome hg38/GRCh38. We used

strelka2 (v2.8.4) (71) for mutation calling with one of the 1000 Genomes Project samples (72)

as the unmatched control samples. All mutations passing filters were annotated using the

Ensembl Variant Effect Predictor (v86 and v91) (73) to translate nucleotide to amino acid

changes and assign allele-specific population prevalences from the 1000 Genomes Project69

and the Exome Aggregation Consortium (ExAC) project (74). Allele prevalences were used

to filter putative germline variants; COSMIC was used to whitelist mutations that appear in

several independent samples. Whole-exome mutation calls from the Cell Lines Project (v83)

(75) were taken as such and fed into the variant annotation step.

Analysis of gene expression by QuantiGene single plex technology (Affymetrix)

RNA was isolated from tumors as described above. The following probes were used: DUSP6

(SA-11958) and GAPDH (SA-10001). The analysis was performed according to

manufacturer’s recommendations. The DUSP6 levels of the individual tumors were

normalized to their respective GAPDH levels.

Bioanalysis of mouse blood samples

Female BomTac:NMRI-Foxn1nu mice (Taconic) were used for the pharmacokinetic studies.

EDTA plasma was prepared by centrifugation of the collected blood at 10,000 rpm at 4°C.

Excess of acetonitrile was added for plasma precipitation and an internal standard was added.

After centrifugation for 5 min at 10,000 rpm, the supernatant was used for quantification by

liquid chromatography/tandem mass spectrometry. Quantification of the analyte was done by

high-performance liquid chromatography/tandem mass spectrometry (Agilent 1200, Applied

Biosystems API5000, ESI+) with a solvent gradient of 95% A to 10% A in 1.5 min [A: 5

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mmol/L ammonium acetate (pH 4.0), B: acetonitrile with 0.1% formic acid; XBridge BEH

C18 2.5μ 2.1 × 50].

Formulation of compounds

Vehicle – All formulations were prepared using a Vehicle comprising 0.5% Natrosol in water.

A representative procedure for preparation of 0.5% Natrosol is as follows: a 200 mL glass

bottle with stirring bar was charged with 100 mL of sterile and filtered water (Sigma, Cat:

W3500-500 mL). As the water was rapidly stirred at room temperature, 1 gram of Natrosol

was added in portions. To the cloudy solution an additional 100 mL water is added while the

solution was rapidly stirred at room temperature for 2-3 hours until a clear colorless solution

was obtained. The formulation was autoclaved for 20 minutes at 121-125 °C and 100 bar. The

autoclaved solution was transferred to a 0-5°C cold room and rapidly stirred overnight.

Solutions were stored at 2-5°C when not in use.

Formulation of BI-3406: Solid compound was weight into a clear glass. Vehicle and stirring

bars were added and formulation was stirred (700-1000 rpm) for 3 hours before use, with

occasional manual swirling, followed by sonication for 5 minutes. Formulations were

typically prepared at 5 mg/mL as uniform suspensions. The dosing suspension was then

transferred to cold room (2-5°C) and kept stirring when not in use. The compound was

applied to the mice with a volume of 10 mL/kg (cell line derived xenograft studies) or 5

mL/kg (PDX) studies. Formulation of trametinib: For a 0.1 mg/kg solution, trametinib (12 mg

for 20 fold stock solution) was weighed into a vial using an analytical balance. DMSO (1.5

mL, 5%) and a stirring bar were added and the solution stirred for 10 minutes until all of the

solid material was completely dissolved. To this, 28.5 mL of sterile-filtered water was added

and stirred. The resulting cloudy solution was a 0.4 mg/mL stock solution. To generate a 2x

solution that was then combined with SOS1i, 1.0 mL of stock solution of trametinib was

diluted 1:10 with 9 mL of 0.5% Natrosol. The stock and dosing solutions were transferred to

a cold room (2-5°C) and kept stirring when not in use. Co-formulation of BI-3406 plus

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trametinib: For co-formulation solutions of BI-3406 and trametinib (0.1 mg/kg trametinib/50

mg/kg BI-3406 in 0.5% DMSO and 0.5% Natrosol solution), 12 mg of solid trametinib were

weighed using an analytical balance into a glass vial. DMSO (3.0 mL, 10%) and stir bars

were added and the solution stirred for 10 minutes until the entire solid was completely

dissolved. Sterile-filtered water (27 mL) was then added and the mixture stirred, and the

resulting slightly cloudy 0.4 mg/mL stock solution of trametinib was obtained. Stock solution

(1.5 mL) was then transferred to another glass bottle, and then 28.5 mL of 0.5% Natrosol and

300 mg BI 3406 was add to the vial and stirred vigorously. The solution was kept stirring for

at least 4 hours prior to use and then transferred to a cold room (2-5°C) and kept stirring while

not in use.

PDX model characterization and profiling

CRC patient derived xenograft tumor models were established as previously described (76).

Tumor fragments isolated from an early passage (3-5) tumor were subjected to STR

fingerprinting as well as molecular analysis as described below to identify hotspot mutations

and deletions. For fingerprinting, PDX fragments were collected and shipped to IDEXX,

BioAnalytics (Columbia, MO 65201) for DNA fingerprinting and mycoplasma testing. The

DNA from those samples was extracted and checked with 16 markers (AMEL, CSF1PO,

D13D317, D16S539, D18S51, D21S11, D3S1358, D5S818, D7S820, D8S1179, FGA,

Penta_D, Penta_E, TH01, TPOX, and vWA). The profiles of the samples from different

studies were compared to that of the same original samples. Targeted panel sequencing was

performed on CRC PDX models at Admera Health leveraging the OncoGxOne 355 panel, a

custom panel targeting all exons for 355 genes. Admera Health implemented its standard

pipeline for removal of mouse reads, alignment, and variant calls. VCF files were annotated

with the VEP framework (73) with Ensemble V96. Variant calls were filtered by the gnomAD

variant frequency less than 1%, as matched normal material available for sequencing. For

RNA-seq, 1000 ng of DNAsed RNA samples were converted to cDNA using a TruSeq

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Stranded mRNA according to the manufacturer's protocol (Illumina, San Diego, CA). The

libraries were amplified with 8 PCR cycles and purified with AmpureXP beads (Beckman

Coulter). The purified libraries were quantified using a Kapa library quantification kit (KAPA

biosystems) and loaded on cBot (Illumina, San Diego, CA) at a final concentration of 1.5 pM

to perform cluster generation, followed by 2x76 bp sequencing on HiSeq 3000 (Illumina).

FASTQ files of RNA samples were processed using both STAR (62) following the two-step

alignment procedure and the subsequent BAM files were called for mutations using Platypus

(77). Functional annotation of the detected mutations were conducted using ANNOVAR

(http://annovar.openbioinformatics.org/en/latest/). An overview off the respective molecular

profile for KRAS, BRAF, PI3K3CA and P53 in the two PDX models C0147 and B8032 can

be found in table S13 in the supplementary data.

RPPA analysis

Tumours were isolated four hours after the second dose of trametinib + BI-3406 treatment (10

hours after the first dose, Supplementary Table S8). RPPA data were generated by the RPPA

Core facility at MD Anderson Cancer Center as previously described (78). Normalized data

were used to determine total and phospho-proteins that were differentially expressed between

vehicle-treated and BI-3406 + trametinib-treated PDX tumors. A log2 fold-change of less

than -0.5 was used as a cut-off for determining differential expression between control and

combination treated samples. Heat maps of median centered log2-transformed average group

expression RPPA data were generated using heat map function in R (v 3.6.0).

Defining the clinically equivalent trametinib dose in mice and for in vitro assays

Although trametinib is often administered in mice at daily doses of 1 mg/kg and higher (79-

81), a daily mouse dose of ~ 0.2 mg/kg matches more closely the clinical exposure achieved

with the recommended trametinib human dose, set using a 2 mg tablet of trametinib once a

day (https://www.accessdata.fda.gov/drugsatfda_docs/label/2014/204114s001lbl.pdf).

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Specifically, it was previously reported that a 2 mg repeat once-daily trametinib dose

administration leads to a total human AUC0-24 of 370 ng∙h/mL, equivalent to 601.2 nM ∙h (82)

with ~35nM (cmax) and ~20nM (cmin). Preclinically, a 0.1 mg/kg trametinib single-dose

administration in mice leads to a total mouse AUC0-24 of 586 nM ∙h. As trametinib inhibits

pERK at 2 h in NCI-H358 cells with IC50 values of 2.2 nM/0.84 nM in a 10% mouse/human

heparin plasma assay, matching the free AUC human exposure yields, a clinically equivalent

daily mouse dose of ~0.2 – 0.3 mg/kg. As BI-3406 was dosed twice daily (bid; 6 hours apart),

trametinib was dosed twice daily as well at either 0.1 or 0.125 mg/kg in all mouse xenograft

studies (Fig 4 and Supplementary Fig. S4). For in vitro assays a single-digit nM-

concentrations of trametinib matches the clinical relevant concentration considering also

binding to plasma-proteins. In the presented in vitro assays single and double-digit nM

concentrations were used not taking in account plasma-protein binding.

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Supplementary Tables S1-S13

Table S1. Crystallographic data collection and refinement statistics (MR)

6SCM (BI-3406) 6SFR (BI-68BS)

Data collection*

Wavelength [Å] 1.0000 1.5418

Beamline SLS X06SA RIGAKU MICROMAX-003

Data Processing

Space group P 21 21 21 P 1 21 1

Cell dimensions

a, b, c (Å) 38.0, 78.7, 168.9 84.0, 39.3, 176.5

() 90.0, 90.0 90.0 90.0, 90.03, 90.0

Resolution (Å) 84.5 – 1.87 (1.981-1.866) 84 -1.917 (2.028-1.917)

Unique Reflections 34879 (1744) 74854 (3744)

Rmerge 13.2 5.2

CC1/2 0.998 (0.342) 0.998 (0.653)

I / I 12.0 (1.1) 14.0 (1.5)

Completeness (ellipsoidal) (%) ** 90.1 (51.2) 88.2 (37.9)

Completeness (spherical) (%) 80.6 (24.9) 83.4 (27.4)

Redundancy 9.6 (5.6) 2.8 (1.5)

Refinement

Rwork / Rfree 19.9/22.0 20.0/24.5

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No. atoms (excl. hydrogens)

Protein 3925 7535

Ligands 33 66

Water 380 1400

B-factors

Protein 32.0 28.8

Ligands 24.8 24.7

Water 38.9 34.8

R.m.s. deviations

Bond lengths(Å)/ angles () 0.01/1.1 0.008/0.9

*Values in parentheses are for highest-resolution shell. ** Data were scaled anisotropically with Staraniso.

Table S2: Selectivity profile of BI-3406 in a kinase enzyme panel.

Kinase activity of BI-3406 in form of single point measurements at 10 µM on 324 kinases

(ThermoFisher, SelectScreen Kinase Profiling Services; Data is uploaded as separate file:

Table S2). Kinases inhibited stronger than 50% were evaluated in detail and IC50 values were

determined (see Supplementary Data Table S3).

Table S3: Activity of BI-3406 in selected kinases.

Kinase IC50 [µM]PLK4@IG 6.1BMPR2@IG 6.5LRRK2_FL 16.0

IC50 evaluation of kinases, which have been inhibited by BI-3406 stronger than 50% at 10 µM

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Table S4: Details of used cell lines and sensitivity following treatment with BI-3406

List of cell lines, IC50 values (3D proliferation assay), BI-3406 response class, gene symbol,

Ensembl gene identifier, Ensembl transcript identifier, DNA mutations, DNA zygosity, amino

acid mutations, and fraction of read-covered exons from Fig. 2f.

Table S5: IC50 value for BI-3406 in cell lines and re-occurring hotspot mutations for

selected genes

List of cell lines, IC50 values, BI-3406 response class, gene symbol, amino acid mutations, and

zygosity status from Fig. 2f including KRAS, NRAS, HRAS, EGFR, NF1, and BRAF.

Table S6: Proliferation assays in 2D and 3D conditions with BI-3406, BAY293 or SHP099 in a panel of cell lines. The N/KRAS status and IC50 values are shown.

List of cell lines, IC50 values following treatment with either BI-3406, BAY293 or SHP099 in

2D or 3D proliferation assays. The indicated cell lines were tested in a standard proliferation

assay after 72h (2D) or under SoftAgar / low serum conditions (2%FCS) based on cellular

growth conditions after 10 to 14 days (3D).

BI-3406 BAY293 SHP099 Gene Mutation IC50 2D 3D 2D 3D 2D 3D

NCI-H520 wt >5000 >5000 >5000 688 >5000 >5000NCI-H1437 wt >5000 3388 >5000 2510 >5000 2905NCI-H1944 wt >5000 >5000 >5000 >5000 >5000 3284

Calu-6 KRAS Q61L >5000 >5000 >5000 988 >5000 >5000NCI-H460 KRAS Q61H >5000 >5000 >5000 3077 >5000 >5000

A427 KRAS G12D >5000 220 >5000 708 >5000 3720GP2D KRAS G12D >5000 19 >5000 207 >5000 1180GP5D KRAS G12D >5000 24 >5000 1065 >5000 4093LS513 KRAS G12D >5000 66 >5000 439 >5000 4411

LCLC-97TM1 KRAS G12V >5000 17 >5000 2000 >5000 2000DLD1 KRAS G13D >5000 24 >5000 640 >5000 2510A549 KRAS G12S >5000 193 >5000 301 >5000 167

NCI-H358 KRAS G12C >5000 24 >5000 788 >5000 360NCI-H23 KRAS G12C >5000 60 >5000 861 >5000 790

NCI-H23 wt/wt KRAS wt >5000 no growth >5000 no

growth >5000 no growth

NCI-H23 G12D/wt KRAS G12D >5000 93 >5000 311 >5000 651

NCI-H23 KRAS G12D >5000 9 >5000 734 >5000 592

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G12D/G12DNCI-H23 G12V/wt KRAS G12V >5000 46 >5000 948 >5000 2596

NCI-H23 G12R/wt KRAS G12R >5000 >5000 >5000 >5000 >5000 >5000

NCI-H23 G13D/wt KRAS G13D >5000 136 >5000 1751 >5000 1432

NCI-H23 G13D/G13D KRAS G13D >5000 161 >5000 1787 >5000 2187

NCI-H23 Q61H/Q61H KRAS Q61H >5000 >5000 >5000 962 >5000 >5000

NCI-H1299 NRAS Q61L >5000 786 >5000 2631 >5000 1314NCI-H2347 NRAS Q61R >5000 >5000 >5000 2653 >5000 >5000

Table S7: Differentially expressed genes following treatment with BI-3406

List of differentially expressed genes in the MIA PaCa-2 in-vivo biomarker experiment using

BI-3406 monotherapy. Output from limma+voom for the groups BI-3406, 50 mg/kg at 4 h, 10

h, and 24 h.

Table S8 PDX sample size for RPPA analysis

C1047 B8032

Vehicle 4 4

Trametinib 4 4

BI-3406 4 4

BI-3406 + Trametinib 4 4

Table S9 Cell lines

Cell lines used in this study and authentication by short tandem repeat (STR) analysis at

Boehringer Ingelheim. The table includes cell line name, vendor/source, date of purchase,

date of STR authentication, and comments.

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Table S10. Full Donor plasmid sequences:

Plasmid name Sequence

KRAS G12D TCTCGGCTCATTGCAACCTCGGACTCCTATTTTCCCCAGAGATATTTCACACATTAAAATGTCGTCAAATATTGTTCTTCTTTGCCTCAGTGTTTAAATTTTTATTTCCCCATGACACAATCCAGCTTTATTTGACACTCATTCTCTCAACTCTCATCTGATTCTTACTGTTAATATTTATCCAAGAGAACTACTGCCATGATGCTTTAAAAGTTTTTCTGTAGCTGTTGCATATTGACTTCTAACACTTAGAGGTGGGGGTCCACTAGGAAAACTGTAACAATAAGAGTGGAGATAGCTGTCAGCAACTTTTGTGAGGGTGTGCTACAGGGTGTAGAGCACTGTGAAGTCTCTACATGAGTGAAGTCATGATATGATCCTTTGAGAGCCTTTAGCCGCCGCAGAACAGCAGTCTGGCTATTTAGATAGAACAACTTGATTTTAAGATAAAAGAACTGTCTATGTAGCATTTATGCATTTTTCTTAAGCGTCGATGGAGGAGTTTGTAAATGAAGTACAGTTCATTACGATACACGTCTGCAGTCAACTGGAATTTTCATGATTGAATTTTGTAAGGTATTTTGAAATAATTTTTCATATAAAGGTGAGTTTGTATTAAAAGGTACTGGTGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAGCTTGTGGTAGTGGGCGCCGACGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTCATGAGTACTTATTACAAGATAATTATGCTGAAAGTTAAGTTATCTGAAATGTACCTTGGGTTTCAAGTTATATGTAACCATTAATATGGGAACTTTACTTTCCTTGGGAGTATGTCAGGGTCCATGATGTTCACTCTCTGTGCATTTTGATTGGAAGTGTATTTCAGAGTTTCGTGAGAGGGTAGAAATTTGTATCCTATCTGGACCTAAAAGACAATCTTTTTATTGTAACTTTTATTTTTATGGGTTTCTTGGTATTGTGACATCATATGTAAAGGTTAGATTTAATTGTACTAGTGAAATATAATTGTTTGATGGTTGATTTTTTTAAACTTCATCAGCAGTATTTTCCTATCTTCTTCTCAACATTAGAGAACCTACAACTACCGGATAAATTTTACAAAATGAATTATTTGCCTAAGGTGTGGTTTATATAAAGGTACTATTACCAACTTTACCTTTGCTTTGTTGTCATTTTTAAATTTACTCAAGGAAATACTAGGATTTAAAAAAAAATTCCTTGAGTAAATTTAAATTGTTATCATGTTTTTGAGGATTATTTTCAGATTTTTTTAGTTTAATGAAAATTTACCAAAGTAA

KRAS_G12V TCTCGGCTCATTGCAACCTCGGACTCCTATTTTCCCCAGAGATATTTCACACATTAAAATGTCGTCAAATATTGTTCTTCTTTGCCTCAGTGTTTAAATTTTTATTTCCCCATGACACAATCCAGCTTTATTTGACACTCATTCTCTCAACTCTCATCTGATTCTTACTGTTAATATTTATCCAAGAGAACTACTGCCATGATGCTTTAAAAGTTTTTCTGTAGCTGTTGCATATTGACTTCTAACACTTAGAGGTGGGGGTCCACTAGGAAAACTGTAACAATAAGAGTGGAGATAGCTGTCAGCAACTTTTGTGAGGGTGTGCTACAGGGTGTAGAGCACTGTGAAGTCTCTACATGAGTGAAGTCATGATATGATCCTTTGAGAGCCTTTAGCCGCCGCAGAACAGCAGTCTGGCTATTTAGATAGAACAACTTGATTTTAAGATAAAAGAACTGTCTATGTAGCATTTATGCATTTTTCTTAAGCGTCGATGGAGGAGTTTGTAAATGAAGTACAGTTCATTACGATACACGTCTGCAGTCAACTGGAATTTTCATGATTGAATTTTGTAAGGTATTTTGAAATAATTTTTCATATAAAGGTGAGTTTGTATTAAAAGGTACTGGTGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTG

566

CTGAAAATGACTGAATATAAGCTTGTGGTAGTGGGCGCCGTGGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTCATGAGTACTTATTACAAGATAATTATGCTGAAAGTTAAGTTATCTGAAATGTACCTTGGGTTTCAAGTTATATGTAACCATTAATATGGGAACTTTACTTTCCTTGGGAGTATGTCAGGGTCCATGATGTTCACTCTCTGTGCATTTTGATTGGAAGTGTATTTCAGAGTTTCGTGAGAGGGTAGAAATTTGTATCCTATCTGGACCTAAAAGACAATCTTTTTATTGTAACTTTTATTTTTATGGGTTTCTTGGTATTGTGACATCATATGTAAAGGTTAGATTTAATTGTACTAGTGAAATATAATTGTTTGATGGTTGATTTTTTTAAACTTCATCAGCAGTATTTTCCTATCTTCTTCTCAACATTAGAGAACCTACAACTACCGGATAAATTTTACAAAATGAATTATTTGCCTAAGGTGTGGTTTATATAAAGGTACTATTACCAACTTTACCTTTGCTTTGTTGTCATTTTTAAATTTACTCAAGGAAATACTAGGATTTAAAAAAAAATTCCTTGAGTAAATTTAAATTGTTATCATGTTTTTGAGGATTATTTTCAGATTTTTTTAGTTTAATGAAAATTTACCAAAGTAA

KRAS_G12R TCTCGGCTCATTGCAACCTCGGACTCCTATTTTCCCCAGAGATATTTCACACATTAAAATGTCGTCAAATATTGTTCTTCTTTGCCTCAGTGTTTAAATTTTTATTTCCCCATGACACAATCCAGCTTTATTTGACACTCATTCTCTCAACTCTCATCTGATTCTTACTGTTAATATTTATCCAAGAGAACTACTGCCATGATGCTTTAAAAGTTTTTCTGTAGCTGTTGCATATTGACTTCTAACACTTAGAGGTGGGGGTCCACTAGGAAAACTGTAACAATAAGAGTGGAGATAGCTGTCAGCAACTTTTGTGAGGGTGTGCTACAGGGTGTAGAGCACTGTGAAGTCTCTACATGAGTGAAGTCATGATATGATCCTTTGAGAGCCTTTAGCCGCCGCAGAACAGCAGTCTGGCTATTTAGATAGAACAACTTGATTTTAAGATAAAAGAACTGTCTATGTAGCATTTATGCATTTTTCTTAAGCGTCGATGGAGGAGTTTGTAAATGAAGTACAGTTCATTACGATACACGTCTGCAGTCAACTGGAATTTTCATGATTGAATTTTGTAAGGTATTTTGAAATAATTTTTCATATAAAGGTGAGTTTGTATTAAAAGGTACTGGTGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAGCTTGTGGTAGTGGGCGCCCGGGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTCATGAGTACTTATTACAAGATAATTATGCTGAAAGTTAAGTTATCTGAAATGTACCTTGGGTTTCAAGTTATATGTAACCATTAATATGGGAACTTTACTTTCCTTGGGAGTATGTCAGGGTCCATGATGTTCACTCTCTGTGCATTTTGATTGGAAGTGTATTTCAGAGTTTCGTGAGAGGGTAGAAATTTGTATCCTATCTGGACCTAAAAGACAATCTTTTTATTGTAACTTTTATTTTTATGGGTTTCTTGGTATTGTGACATCATATGTAAAGGTTAGATTTAATTGTACTAGTGAAATATAATTGTTTGATGGTTGATTTTTTTAAACTTCATCAGCAGTATTTTCCTATCTTCTTCTCAACATTAGAGAACCTACAACTACCGGATAAATTTTACAAAATGAATTATTTGCCTAAGGTGTGGTTTATATAAAGGTACTATTACCAACTTTACCTTTGCTTTGTTGTCATTTTTAAATTTACTCAAGGAAATACTAGGATTTAAAAAAAAATTCCTTGAGTAAATTTAAATTGTTATCATGTTTTTGAGGATTATTTTCAGATTTTTTTAGTTTAATGAAAATTTACCAAAGTAA

KRAS_G13D TCTCGGCTCATTGCAACCTCGGACTCCTATTTTCCCCAGAGATATTTC

ACACATTAAAATGTCGTCAAATATTGTTCTTCTTTGCCTCAGTGTTTAAATTTTTATTTCCCCATGACACAATCCAGCTTTATTTGACACTCATTCTCTCAACTCTCATCTGATTCTTACTGTTAATATTTATCCAAGAGAACTACTGCCATGATGCTTTAAAAGTTTTTCTGTAGCTGTTGCATATTGACTTCTAACACTTAGAGGTGGGGGTCCACTAGGAAAACTGTAACAATAAGAGTGGAGATAGCTGTCAGCAACTTTTGTGAGGGTGTGCTACAGGGTGTAGAGCACTGTGAAGTCTCTACATGAGTGAAGTCATGATATGATCCTTTGAGAGCCTTTAGCCGCCGCAGAACAGCAGTCTGGCTATTTAGATAGAACAACTTGATTTTAAGATAAAAGAACTGTCTATGTAGCATTTATGCATTTTTCTTAAGCGTCGATGGAGGAGTTTGTAAATGAAGTACAGTTCATTACGATACACGTCTGCAGTCAACTGGAATTTTCATGATTGAATTTTGTAAGGTATTTTGAAATAATTTTTCATATAAAGGTGAGTTTGTATTAAAAGGTACTGGTGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAGCTTGTGGTAGTGGGCGCCGGTGACGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTCATGAGTACTTATTACAAGATAATTATGCTGAAAGTTAAGTTATCTGAAATGTACCTTGGGTTTCAAGTTATATGTAACCATTAATATGGGAACTTTACTTTCCTTGGGAGTATGTCAGGGTCCATGATGTTCACTCTCTGTGCATTTTGATTGGAAGTGTATTTCAGAGTTTCGTGAGAGGGTAGAAATTTGTATCCTATCTGGACCTAAAAGACAATCTTTTTATTGTAACTTTTATTTTTATGGGTTTCTTGGTATTGTGACATCATATGTAAAGGTTAGATTTAATTGTACTAGTGAAATATAATTGTTTGATGGTTGATTTTTTTAAACTTCATCAGCAGTATTTTCCTATCTTCTTCTCAACATTAGAGAACCTACAACTACCGGATAAATTTTACAAAATGAATTATTTGCCTAAGGTGTGGTTTATATAAAGGTACTATTACCAACTTTACCTTTGCTTTGTTGTCATTTTTAAATTTACTCAAGGAAATACTAGGATTTAAAAAAAAATTCCTTGAGTAAATTTAAATTGTTATCATGTTTTTGAGGATTATTTTCAGATTTTTTTAGTTTAATGAAAATTTACCAAAGTAA

KRAS_WT TCTCGGCTCATTGCAACCTCGGACTCCTATTTTCCCCAGAGATATTTCACACATTAAAATGTCGTCAAATATTGTTCTTCTTTGCCTCAGTGTTTAAATTTTTATTTCCCCATGACACAATCCAGCTTTATTTGACACTCATTCTCTCAACTCTCATCTGATTCTTACTGTTAATATTTATCCAAGAGAACTACTGCCATGATGCTTTAAAAGTTTTTCTGTAGCTGTTGCATATTGACTTCTAACACTTAGAGGTGGGGGTCCACTAGGAAAACTGTAACAATAAGAGTGGAGATAGCTGTCAGCAACTTTTGTGAGGGTGTGCTACAGGGTGTAGAGCACTGTGAAGTCTCTACATGAGTGAAGTCATGATATGATCCTTTGAGAGCCTTTAGCCGCCGCAGAACAGCAGTCTGGCTATTTAGATAGAACAACTTGATTTTAAGATAAAAGAACTGTCTATGTAGCATTTATGCATTTTTCTTAAGCGTCGATGGAGGAGTTTGTAAATGAAGTACAGTTCATTACGATACACGTCTGCAGTCAACTGGAATTTTCATGATTGAATTTTGTAAGGTATTTTGAAATAATTTTTCATATAAAGGTGAGTTTGTATTAAAAGGTACTGGTGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAGCTTGTGGTAGTGGGCGCCGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGA

CCATTTTCATGAGTACTTATTACAAGATAATTATGCTGAAAGTTAAGTTATCTGAAATGTACCTTGGGTTTCAAGTTATATGTAACCATTAATATGGGAACTTTACTTTCCTTGGGAGTATGTCAGGGTCCATGATGTTCACTCTCTGTGCATTTTGATTGGAAGTGTATTTCAGAGTTTCGTGAGAGGGTAGAAATTTGTATCCTATCTGGACCTAAAAGACAATCTTTTTATTGTAACTTTTATTTTTATGGGTTTCTTGGTATTGTGACATCATATGTAAAGGTTAGATTTAATTGTACTAGTGAAATATAATTGTTTGATGGTTGATTTTTTTAAACTTCATCAGCAGTATTTTCCTATCTTCTTCTCAACATTAGAGAACCTACAACTACCGGATAAATTTTACAAAATGAATTATTTGCCTAAGGTGTGGTTTATATAAAGGTACTATTACCAACTTTACCTTTGCTTTGTTGTCATTTTTAAATTTACTCAAGGAAATACTAGGATTTAAAAAAAAATTCCTTGAGTAAATTTAAATTGTTATCATGTTTTTGAGGATTATTTTCAGATTTTTTTAGTTTAATGAAAATTTACCAAAGTAA

KRAS_Q61H TGGAGCATTTTGGATTTCAGATTTTTGGATTAACCTGCATTAATGCTCAACCTATATGAAATTTTATTCCTTTTTATGGCTGAATAATGTTCCACTGTATGTATATACTACATTTTGTTTATCCATTCATCTGTTAACAGACACTTAAGTTATTTCCACATTTTGGGTATTATAAATAGTGCTGCTGCGAACATTGGTGTACATGTATCTGTTTGAGTCCCTGTTTTTAGTTATTTTGGTTATATACCTAGGAATGGAATTGCTGATCATATGGTAATTCTGTGTTTAACTTTTTGAGGAACTACCACTGTTTTCCACAATGGCATCACCATTTTACATTCCCACCAGCAATGCACAAAGATTTCAGTGTCTGTATCCTTGCTAACACTTATTTTCCATTTTTTGAGTTTTTTTGTTTTGTTTTTTTAATAATAGCCAATCCTAATGGGTATGTGGTAGCATCTCATGGTTTTGATTTTATTTTCCTGACTATTGATGATGTTGAGCATCTTTTCAGGTGCTTAGTGGCCATTTGTCCGTCATCTTTGGAGCAGGAACAATGTCTTTTCAAGTCCTTTGCCCATTTTTAAATTGAATTTTTTGTTGTTGAGTTGTATATAACACCTTTTTTGAAGTAAAAGGTGCACTGTAATAATCCAGACTGTGTTTCTCCCTTCTCAGGATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACCGCGGGTCACGAGGAGTACAGCGCTATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAATCATTTGAAGATATTCACCATTATAGGTGGGTTTAAATTGAATATAATAAGCTGACATTAAGGAGTAATTATAGTTTTTATTTTTTGAGTCTTTGCTAATGCCATGCATATAATATTTAATAAAAATTTTTAAATAATGTTTATGAGGTAGGTAATATCCCTGTTTTATAAATGAAGTTCTTGGGGGATTAGAGCAGTGGAGTAACTTGCTCCAGACTGCATCGGTAGTGGTGGTGCTGGGATTGAAACCTAGGCCTGTTTGACTCCACAGCCTTCTGTACTCTTGACTATTCTACAAAAGCAAGACTTTAAACTTTTTAGATACATCATTAAAAAAGAAAACCATAAAAAAGAATATGAAAAGATGATTTGAGATGGTGTCACTTTAACAGTCTTAAAAGCAATCGTGTGTATAGCATAGAATTGCTTGGATTGGATAAACAGTGGCATTATATATTTTAAAAAATAAAAGTTTTGAAAGATTGAAGAATTTGGGCATTACAGTTCTCTTAAATCTGACAAAGCTGCATAAAACTATTAAAATAATCATTATTATACTATTTTATATTCTATTTCTTTGAGGGTTTAGTTTTCCAAAAACTACATATTAAGCAAATGAATCACTCAGTGGCTATGTCATATAATAACGAGTTAGCCTAGTTATAAGAA

567

Table S11. Primers and plasmids used to generate the donor plasmids

Target ID Purpose Target

Gene

Target Site /Sequence Vector Lot Nr.

generated cell

line

HS0000319342 Primer SOS1 GAGAACGCGCCCAAGTGG

CGGG

U6gRNA

-Cas9-

2A-GFP

10171609M

N

HS0000340122 Primer SOS2 ATCTTGAACAGTCCTTGGC

TGG

U6gRNA

-Cas9-

2A-GFP

10171609M

N

HS0000340129 Primer SOS2 AATCCTCTTTTACTGCCTGT

GG

U6gRNA

-Cas9-

2A-GFP

10171609M

N

#1 gRNA KRAS

exon3

TTGGATATTCTCGACACAG

C

pSpCas

9_BB-

2A-GFP

(pX458)

#2 gRNA KRAS

exon3

TCTCGACACAGCAGGTCAA

G

pSpCas

9_BB-

2A-GFP

(pX458)

#3 gRNA KRAS

exon3

AAGAGGAGTACAGTGCAA

TG

pSpCas

9_BB-

2A-GFP

(pX458)

#6 gRNA KRAS

exon 2

CTTGTGGTAGTTGGAGCTT

G

pSpCas

9_BB-

568

2A-GFP

(pX458)

#7 gRNA KRAS

exon 2

(G12C

specific)

GTAGTTGGAGC

TTGTGGCGT

pSpCas

9_BB-

2A-GFP

(pX458)

KRAS

Donor

plasmid

KRAS_

G12D

GAC / GGC

(Codon 12 / 13 sequence)

pUC57-

mini

NCI-

H23_Cas9_

G12D

KRAS

Donor

plasmid

KRAS_

G12V

GTG / GGC


pUC57-

mini

NCI-

H23_Cas9_

G12V

KRAS

Donor

plasmid

KRAS_

G12G_G

13D

GGT / GAC


pUC57-

mini

NCI-

H23_Cas9_

G12G_G13

D

KRAS

Donor

plasmid

KRAS_

Q61H

CAA / CAC

(Codon 61)

pUC57-

mini

NCI-

H23_Cas9_

Q61H_Q61

H

Table S12. KRAS codon 12 PCR primer information and PCR conditions

Name Sequence 5’ – 3’ gRNA site

Tm° % GC

bp

KRAS_Fwd_3_codon 12GGGGGTCCACTAGGAAA

ACTG#6, 7 65,6 57,1 21

569

570

KRAS_Rev_3_codon 12TCAGCGCAAAGTGTCTC

TTCA#6, 7 66,2 47,6 21

KRAS_sequencing_codon 12

TTTGTGAGGGTGTGCTACAGG

#6, 7 65,0 52,4 21

KRAS_Fwd_2_codon 61 ACCAGCATGTACACTTGCATT

# 1,2,3 62,0 42,8 21

KRAS_Rev_2_codon 61 CACCACCACTACCGATGCAG # 1,2,3 66,6 60 20KRAScodon 61_Fwd_1_sequ

GGTGCACTGTAATAATCCAGACTGT

# 1,2,3 64,0 44 25

KRAS codon 61_Rev_1_sequ

TGCATGGCATTAGCAAAGACTC

# 1,2,3 66,1 45,4 22

KRAS codon 61_Fwd_2_sequ

TCCAGACTGTGTTTCTCCCTTCTCA

# 1,2,3 69,1 48 25

Table: KRAS codon 12 / 61 PCR primer information

Table S13 PDX molecular profiling.

C1047 B8032 PATX53 PATX216

KRAS G12C G12C G12V Q61K

BRAF R444W G469A wild-type wild-type

PIK3CA wild-type wild-type wild-type wild-type

APC R213*;Q1406* E1464*;E1573* wild-type V830M

P53 A248W;A63G;A159V;C135* R175H R306*;R213* V216M

SMAD4 wild-type wild-type wild-type A199S

CDKN2A wild-type wild-type wild-type unknown

571

572

573

574

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