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 Boettcher1, Nikolai Pototschnig1, Franziska Schachinger1, Katharina Schipany1,

Simone Lieb1, Christopher P. Vellano3, Jonathan C. O’Connell2, Rachel L. Mendes2, Jurgen

Moll1, Mark Petronczki1, 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

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Supplementary Figures and Legends S1-S519

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Figure S1: Discovery of BI-3406, a potent and selective SOS1::KRAS interaction

inhibitor

a, AlphaScreen and TR-FRET dose response data for the confirmed actives from the

AlphaScreen HTS. Out of the 714 hits with IC50 values of ≤ 10 µM in one of the assay

formats, only 52 (7%) inhibited both read outs. Among these 52 hits were the 18 quinazoline

hits. BI-68BS is highlighted in a dark blue circle. b, Co-crystal x-ray structure of BI-68BS (in

yellow) bound to the catalytic site of SOS1 (in grey as surface representation). Key

interactions of BI-68BS with SOS1: Pi-Staking of the quinazoline core with His905,

interaction of the phenethyl substituent with a lipophilic area defined by Phe890, Leu901 and

Met878, polar interaction of the NH with Asn879 and interaction of the 6-methoxy substituent

with Tyr884. Structure and potency are shown to the lower right. c, Overlay of the co-crystal

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x-ray structures of BI-68BS and SOS1 with the previously described SOS1 activator (PDB:

4NYI) (1) and SOS1 demonstrates binding of both compounds to the same pocket. d, The

sphere representation of BI-68BS (in yellow) bound to SOS1 (key amino acid residues shown

in yellow) superimposed with the published SOS1::RAS complex (Tyr884SOS1 and Arg73RAS

shown in orange, PDB: 4NYI) clearly illustrates, that the methoxy substituent of BI-68BS

competes with Arg73RAS for interaction with Tyr884SOS1. Thus, when bound to SOS1, BI-

68BS would clash with KRAS. This prevents KRAS from binding and causes inhibition of

SOS1. e, Synthesis of key intermediate 6: i.) 1.3 eq tributyl(1-ethoxyvinyl)tin, 0.1 eq

Pd(PPh3)2Cl2, 2.0 eq NEt3, dioxane, 80°C, 12 hours, then work-up with 1N HCl, 64% yield;

ii.) 1.5 eq (R)-(+)-2-methyl-2-propane-sulfinamide, 2.5 eq Ti(OEt)4, THF, 80°C, 5 hours, 72%

yield; iii) 1.8 eq NaBH4, THF/H2O = 50/1, -50°C, 5 hours, 63% yield; iv.) 2 eq HCl in

dioxane, rt, 5 hours, 87% yield; v.) 0.05 eq Pd/C, H2, MeOH, rt, 12 hours, 92% yield. f,

Synthesis of BI-3406: vi.) 10 eq CH3CN, 8 eq HCl in dioxane, 60°C, 5 hours, 75% yield; vii.)

1.1 eq (R)-tetrahydrofuran-3-yl 4-methylbenzenesulfonate, 1.2 eq Cs2CO3, DMF, 100°C, 12

hours, 69% yield; viii.) 1.2 eq 2,4,6-triisopropylbenzenesulfonyl chloride; 0.1 eq DMAP, 3 eq

NEt3, DCM, room temperature, 65% yield; ix.) 1.5 eq intermediate 6, 5 eq NEt3, DMSO,

90°C, 6 hours, 79% yield. g, Overlay of SOS1 with BI-3406 and SOS2 (PDB ID 6EIE) in

green revealing clash of BI-3406 (with dotted surface) and Val 903 in SOS2 (spheres shown

around side chain). h, Biochemical protein-protein interaction assays (AlphaScreen). Effect of

BI 3406 on SOS1 wildtype and SOS1 binding site mutants. Recombinant wild type SOS1 and

various point mutations (Y884A, H905V and Y884A / H905V) were tested for the ability of

BI 3406 to disrupt the interaction with KRAS (KRASG12D). i, FLAG-SOS1 wild-

typeFLAG-SOS1 H905V and H905I mutant variants and empty vector control transgenes

were transiently transfected into HEK293 cells. 40 hours after transfection, cells were treated

with DMSO solvent, BI-3406 (1 µM) or trametinib (50 nM) for 4 hours. Protein lysates were

analyzed using a capillary immunodetection assay to quantify phosphoERK and loading

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control alpha-actinin signals (n=3, mean and single data points of 3 biological repeats are

displayed). Inlay: Lane views of FLAG-SOS1 transgene expression in HEK293 cells and

loading control alphaactinin. j, Cells were starved for one day in medium with 0.5% FCS.

One the next day cells were either treated with 1 ng/mL EGF for exactly 2 minutes or they

were treated with BI-3406 for 1 hour before addition of 1 ng/mL of EGF. Lysate of the cells

was analyzed by Western blotting (left) for reduction of pERK levels and by GLISA (right,

n=2-3, mean±s.d.) for reduction of KRAS-GTP levels. k, Western blotting of cell lysate from

NCI-H358 cells (parental) or NCI-H358 cells carrying a genetically engineered SOS1 (NCI-

H358 SOS1 KO) or SOS2 (NCI-H358 SOS2 KO) knockout to proof absence of SOS1 or

SOS2 in the respective cell lines. The antibody for SOS2 shows two bands on the immunoblot

with one disappearing in the SOS2 KO cell. l, Effect of BI-3406 on RAS-GTP levels in NCI-

H358 cells with SOS2 knockout is more pronounced than in parental cells. NCI-H358 cells

(parental) or NCI-H358 cells carrying a genetically engineered SOS2 knockout (NCI-H358

SOS2 KO) were treated with 1 µM BI-3406 or DMSO for 2 h and RAS-GTP levels relative to

DMSO were quantified (n=2, mean±s.d.). m, Effect of BI-3406 on pERK levels is reduced in

NCI-H358 SOS1 knockout cells and enhanced in NCI-H358 SOS2 knockout cells compared

to NCI-H358 parental cells. NCI-H358 parental, SOS1 or SOS2 CRISPR engineered

knockout cells were incubated with increasing concentrations of BI-3406 for 1 h. pERK and

total ERK levels were analyzed. Dose response curves of pERK levels relative to total ERK

levels are shown (n=2, mean±s.d.). n, in vitro sensitivity of parental NCI-H358, NCI-H358

SOS1 KO or NCI-H358 SOS2 KO cells with BI-3406 in a 3D proliferation assay (n=2,

mean±s.d.). o, Time course of GTP RAS and pERK after BI 3406 treatment. NCI-H358 cells

were treated with 1µM BI 3406 and samples were harvested after 1, 3, 7 and 24h. RAS GTP

and pERK / total ERK levels were quantified.

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Figure S2: SOS1/SOS2 expression in different models/patients, phosphoprotein response

for BI-3406 inhibition, and compound effects in non-tumorous human cells

a-b, Gene expression of SOS1 and SOS2 across 33 TCGA cancer type cohorts. The dashed

line shows an expression cutoff of 3 TPM (transcripts per million). a, SOS1

(ENSG00000115904) and b, SOS2 (ENSG00000100485). c, Expression of SOS1 plotted

against expression of SOS2 for cell lines (CCLE) and tumors (TCGA). All data was processed

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with identical processing pipelines (see Material and Methods). BI-3406 sensitive and

resistant cell lines are color-coded in the figure. d, Immunoassays performed with capillary

simple western system using lysate of NCI-H358 (NSCLC) and A549 (NSCLC) cell lines

treated for 2 hours with different concentrations of BI-3406 (medium with 10% FCS) e,

Phosphoprotein levels for ERK and AKT in five cell lines and two time points (2 h and 24 h)

at four dose levels of BI-3406. The DMSO control groups are shown in grey, while BI-3406

groups are shown in shades of blue following increasing compound concentrations.

Measurements are normalized according to (p-ERK/total-ERK) / mean of (p-ERK-c1/total-

ERK-c1, p-ERK-c2/total-ERK-c2) such that the mean value in the DMSO groups is 100%.

Other measurements are then computed in relation to this standard. Each group comprises two

technical replicates (n=2). Mean values of each group are plotted as bars, while error bars

show the mean ± SD. Statistical significance was computed for the 100 nM treatment groups

using a two-sided t-test using a confidence level of 0.95 and setting the true value of the mean

to 100. Asterisks indicate p-values ≤ 0.05. f)-h) To estimate a difference in sensitivity of

tumor versus normal cells for BI-3406, we assessed compound effects on a series of normal

cells (n=3, mean±s.e.m). In vitro sensitivity was tested with BI-3406 on f) human

immortalized epithelial cells (hTERT-RPE-1) g) primary smooth muscle cells (HSMC) and h)

normal foreskin cells (BJ) in a 3D, 7 days proliferation assay in softagar.

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Figure S3: SOS1 inhibition suppresses tumor growth and KRAS/MAPK signaling in

xenograft models of KRAS-driven cancers

a, Mouse pharmacokinetic (PK) analysis in comparison to IC50 unbound in A549 cells.

Female NMRI-Foxn1 nude mice (n=3 per group, mean±s.e.m) were dosed with BI-3406

orally (50 mg/kg, bid) or intravenously (1 mg/kg) and the plasma concentrations (total and

unbound) were measured at the indicated time points using LC-MS. Unbound IC50 value for

an anti-proliferative effect in A549 cells are shown in comparison. b-c, Modulation of ERK

phosphorylation b, or RAS-GTP c, in subcutaneous A549 tumors following treatment with 50

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mg/kg BI-3406 prior treatment and 2h, 7h and 24h post-treatment. Treatment groups comprise

5 animals per group (mean±s.e.m). d, Following b.i.d. (t= 0 and 6h) treatment with 50mg/kg

of BI-3406 mice were sacrificed to explant the tumor to analyze biomarker modulation (see

also Fig. 3a, b, c) and blood was collected to measure plasma levels at the respective time

points (n=5 per group, mean±s.e.m).

e, Representative IHC pictures from d, of pERK in mouse skin: pERK was labeled with DAB

(Brown) Vehicle (A), BI-3406 at 4h (B), BI-3406 at 10h (C) and BI-3406 at 24h.

Magnification: 20x f, Gene expression profiling of pharmacodynamic biomarkers in a MIA

PaCa-2 CDX biomarker experiment. Subset of nine genes showing time-dependent

modulation after BI-3406 (50 mg/kg) monotherapy treatment and sampling at 4 h, 10 h, and

24 h post-first-dose. Dosing was performed b.i.d. (t= 0 and 6h). Treatment groups comprised

3-5 animals. Gene expression fold-changes are color-coded in the heat map and changes

between -1.25-fold down- and +1.25-fold up-regulation are overlaid with a white box. g-h,

Gene expression of SOS1 and SOS2 in the MIA PaCa-2 biomarker experiment described

below. Dosing was performed as described in Fig. S3f g, SOS1 (ENSG00000115904) with a

median gene expression of TPM 50 across treatment groups and h, SOS2

(ENSG00000100485) with a median gene expression of TPM 15 across treatment groups. i,

Anti-tumor effect of BI-3406 (50 mg/kg, twice daily, orally) in the A549 (KRAS

G12S/BRAF wt) non-small cell lung cancer xenograft model (n=7 mice/group, mean±s.e.m,

Student's t-test; one-tailed). j, Median body weight of mice treated as described in k). i, No

significant anti-tumor effect of BI-3406 (50 mg/kg, twice daily, orally) in the A375 (KRAS

wt/BRAF V600E) melanoma cell cancer xenograft model (n=7 mice/group).

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Figure S4: 3D Proliferation assay with BI-3406 and trametinib and suppression of

tumor growth and body weight curves in xenograft models of KRAS-driven cancers and

a, 3D Proliferation assay with BI-3406 and trametinib in MIA PaCa-2 and DLD1 cells. Cells

were treated in vitro with different concentrations of the SOS1::KRAS inhibitor BI-3406 and

with different concentrations of the MEKi trametinib. Cell growth inhibition (CGI) was

calculated. A CGI of >100% is indicative of net cell death. Bliss excess was calculated. Bliss

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

inhibition. (n=2, values calculated from the mean). b, Median body weight of mice bearing

subcutaneous MIA PaCa-2 tumors (n=7 per group). Treatment as described in Figure 4a). c,

Median body weight of mice bearing subcutaneous LoVo tumors (n=7 per group). Treatment

as described in Figure 4c. d-e, Box and whiskers graph of the relative tumor volume from d,

C1047 (KRASG12C) PDX tumors and e, B8032 (KRASG12C) PDX tumors over the course

of treatment, with measurements from individual tumors plotted. The mean tumor volume is

represented by a horizontal line within each box (*p≤0.05, **p<0.005 and ***p<0.0005,

statistical significance was determined using an unpaired t-test per row and the Holm-Sidak

method to correct for multiple comparisons). f-g) Pancreatic cancer PDX tumor growth in

mice treated with Vehicle, BI-3406 (50 mg/kg, bid), trametinib (0.1 mg/kg, bid), or the

combination for the models PATX53 (f) and PAT216 (g) tumors (n=5-7 animals per group,

means±s.e.m., statistical significance was determined using an unpaired t-test per row and the

Holm-Sidak method to correct for multiple comparisons. h-k, The average relative body

weight change for each treatment group in the PDX study with (h) C1047 (colorectal

KRASG12C), (i) with B8032 (colorectal, KRASG12C), j) with (j) PATX53 (pancreatic,

KRASG12V) and (k) with PAT216 (pancreatic, KRASQ61K) PDX tumors. l-m, Gene

expression of SOS1 and SOS2 after 21 days of treatment in the B8032 PDX efficacy study.

Dosing was performed as described in Figure 4c. (l) SOS1 (ENSG00000115904) with a

median gene expression of TPM 51.5 for the control and a median gene expression of TPM

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54.5 for the combination. (m) SOS2 (ENSG00000100485) with a median gene expression of

TPM 43 for the control and a median gene expression of TPM 49 for the combination.

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Figure S5: Biomarker modulation upon combined blockade of MEK and SOS1

a, PD marker modulation upon MEKi/SOS1 combination treatment in vitro. MIA PaCa-2

cells (2D growth) were treated with BI-3406 (1 µM), trametinib (25 nM) or the combination

for 2, 4, 8 hours. Whole cell extracts were subjected to Western blot analysis and compared

with untreated cells using pMEK1/2 (Ser217/221) antibodies. b, Lysate of MIA PaCa-2 cells

(2D growth) treated with BI-3406 (1 µM), trametinib (9 nM and 25 nM) or the combination

for 24, 48, 72 hours was analyzed for modulation of total MEK protein. c, PD marker

modulation upon MEKi/SOS1i combination treatment in vitro. MIA PaCa-2 cells (2D

growth) were treated with BI-3406 (1 µM), trametinib (1, 3 or 9 nM) or the combination for 2,

4, 8 hours. Whole cell extracts were subjected to Western blot analysis and compared with

untreated cells using antibodies against pERK1/2 (Thr202/Tyr204) and the total protein of

ERK1/2. d, PD marker modulation upon MEKi/SOS1i combination treatment in vitro. NCI-

23 cells (2D growth) were treated with BI-3406 (1 µM), trametinib (1, 3 or 9 nM) or the

combination for 2, 4, 8 hours. Whole cell extracts were subjected to Western blot analysis and

compared with untreated cells using antibodies against pERK1/2 (Thr202/Tyr204) and the

total protein of ERK1/2 (a-d) n=3, means±s.e.m., Student's t-test; one-tailed). e-h, PD marker

modulation of ERK and pMEK analyzed with capillary immunodetection assay. e,

Immunoblot of cell lysate of MIA PaCa-2 cells grown in 3D on coated plates (medium with

1%FCS) treated with BI-3406. f, Immunoblot of untreated MIA PaCa-2 cells grown in 3D on

coated plates, and cells treated with 10nM of trametinib alone or in combination with 100 or

300 nM of BI-3406. g, Immunoblot of cell lysate of NCI-H23 cells grown in 3D on coated

plates (medium with 1%FCS) treated with BI-3406. h, Immunoblot of cell lysates of untreated

NCI-H23 cells grown in 3D on coated plates, and cells treated with 10nM of trametinib alone

or in combination with 100 or 300 nM of BI-3406. i, PDX tumor bearing (C1047 or B8032)

mice were treated with Vehicle, trametinib (0.1 mg/kg), BI-3406 (50 mg/kg), or the

combination on a bid schedule, with each dose given 6 hours apart (Supplementary Table

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S12, n=4 per group). Samples were collected 4 hours after the second dose and tumors were

harvested for RPPA analysis (Supp. A heatmap of log2 normalized protein expression for all

four markers averaged from both samples within treatment groups is shown, with the numbers

denoting fold-change expression over the corresponding Vehicle control. Samples were

analyzed for phospho-ERK (Thr202/Tyr204), phospho-S6 (Ser235/236) and Phospho-AKT

(Ser473). j, MIA PaCa-2 tumor bearing mice (n = 4 mice per group) were treated with either a

vehicle (0.5% Natrosol), 50 mg/kg of BI-3406, 0.1 mg/kg of trametinib or the combination.

Tumors were explanted four hours post treatment and DUSP6 and GAPDH mRNA levels

were measured by QuantiGene single plex assays (one-tailed, t-test). The DUSP6 levels of the

individual tumors were normalized to their respective GAPDH levels (means±s.d.). k, DLD1

cells were treated in vitro for 24, 48 or 72 hours with trametinib, BI-3406 or the combination.

Cells lysate was analyzed for cleaved PARP using the MSD Assay System (mean±s.d.)

Apoptosis Panel Whole Cell Lysate Kit). l, NCI-H358 cells (KRAS G12C) were treated with

solvent control DMSO, the KRAS G12 inhibitor AMG 510 (20 nM), BI-3406 (1 µM), or the

SHP2 inhibitor SHP099 (5 µM) alone or in combinations of AMG 510 with BI-3406 or

SHP099. pERK and alpha-actinin loading control levels were quantified by capillary

immunodection assays in protein lysates were prepared at the indicated time points and

analyzed. The pERK to alpha-actinin signal ratio is displayed in the graph over time (n=2

mean ± SD).

References for supplementary figures and legends

1. Burns MC, Sun Q, Daniels RN, Camper D, Kennedy JP, Phan J, et al. Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange. Proceedings of the National Academy of Sciences of the United States of America 2014;111(9):3401-6 doi 10.1073/pnas.1315798111.

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