BI-3406, a potent and selective SOS1::KRAS interaction ... · 8/19/2020 · 3406, that binds to the catalytic domain of . 57. SOS1 thereby preventi. ng. the interaction with. KRAS

BI-3406, a potent and selective SOS1::KRAS interaction 1

inhibitor, is effective in KRAS-driven cancers through 2

combined MEK inhibition 3

Marco H. Hofmann1*

, Michael Gmachl1*

, Juergen Ramharter1*

, Fabio Savarese1, Daniel 4

Gerlach1, Joseph R. Marszalek

3, Michael P. Sanderson1, Dirk Kessler

1, Francesca Trapani

1, 5

Heribert Arnhof1, Klaus Rumpel

1, Dana-Adriana Botesteanu

1, Peter Ettmayer

1, Thomas 6

Gerstberger1, Christiane Kofink

1, Tobias Wunberg

1, Andreas Zoephel

1, Szu-Chin Fu

4, Jessica 7

L. Teh3, Jark Böttcher

1, Nikolai Pototschnig

1, Franziska Schachinger

1, Katharina Schipany

1, 8

Simone Lieb1, Christopher P. Vellano

3, Jonathan C. O’Connell

2, Rachel L. Mendes

2, Jurgen 9

Moll1, Mark Petronczki

1, Timothy P. Heffernan

3, Mark Pearson

1, Darryl B. McConnell

1, 10

Norbert Kraut1 11

1 Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria 12

2 Forma Therapeutics, Watertown, MA, USA 13

3 TRACTION Platform, Therapeutics Discovery Division, The University of Texas MD 14

Anderson Cancer Center, Houston, TX 77030, USA 15

4 Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, 16

Houston, TX 77030, USA 17

18

These authors contributed equally: Marco H. Hofmann, Michael Gmachl, and Juergen 19

Ramharter 20

Running title: Pan-KRAS SOS1 protein-protein interaction inhibitor BI-3406 21

Additional information 22

Corresponding Authors: 23

Marco H. Hofmann, Email: [email protected] and 24

Norbert Kraut, Email: [email protected] 25

Boehringer Ingelheim RCV GmbH & Co KG, 26

Dr. Boehringer-Gasse 5-11, A-1121 Vienna, 27

Austria, Tel. +431 80105-2790 28

29

Research. on May 24, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/2159-8290.CD-20-0142

mailto:[email protected]

http://cancerdiscovery.aacrjournals.org/

Disclosure of conflict of interest: The authors declare competing financial interests: Marco 30

H. Hofmann, Michael Gmachl, Juergen Ramharter, Fabio Savarese, Daniel Gerlach, Michael 31

P. Sanderson, Dirk Kessler, Francesca Trapani, Heribert Arnhof, Klaus Rumpel, 32

Dana-Adriana Botesteanu, Peter Ettmayer, Thomas Gerstberger, Christiane Kofink, Tobias 33

Wunberg, Andreas Zoephel, Nikolai, Jark Böttcher, Pototschnig, Franziska Schachinger, 34

Katharina Schipany, Simone Lieb, Jurgen Moll, Mark Petronczki, Mark Pearson, Darryl B. 35

McConnell and Norbert Kraut performed the work herein reported as employees of 36

Boehringer Ingelheim RCV GmbH & Co KG. Jonathan C. O’Connell and Rachel L. Mendes 37

performed the work herein reported as employees of Forma Therapeutics. Joseph R. 38

Marszalek, Szu-Chin Fu, Jessica L. Teh, Christopher P. Vellano and Timothy P. Heffernan 39

performed the work herein reported as employees of the University of Texas MD Anderson 40

Cancer Center. 41

Additional Information: 42

This study was supported by Boehringer Ingelheim and the Austrian Research Promotion 43

Agency (FFG) with the support awards 854341, 861507, 867897 and 874517. This study was 44

also supported by the MDACC Science Park NGS Core grant: CPRIT Core Facility Support 45

Award (RP170002) and the RPPA Core facility is funded by NCI #CA16672. M.H.H., M.G., 46

J.R., F.S., D.K., C.K., and T.W., are also listed as inventors on patent applications for SOS1 47

Inhibitors. 48




Abstract 49

KRAS is the most frequently mutated driver of pancreatic, colorectal, and non-small cell lung 50

cancers. Direct KRAS blockade has proven challenging and inhibition of a key downstream 51

effector pathway, the RAF-MEK-ERK cascade, has shown limited success due to activation 52

of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, 53

a KRAS activator and important feedback node, represents an effective approach to treat 54

KRAS-driven cancers. We report the discovery of a highly potent, selective and orally 55

bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of 56

SOS1 thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-57

loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. 58

Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and 59

thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined 60

SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address 61

KRAS-driven tumors. 62

Significance 63

To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-64

3406 activity and identification of MEK inhibitors as effective combination partners provide 65

an attractive therapeutic concept for the majority of KRAS mutant cancers, including those 66

fueled by the most prevalent mutant KRAS oncoproteins G12D, G12V, G12C and G13D. 67




Introduction 68

KRAS functions as a molecular switch, cycling between inactive (GDP-bound) and active 69

(GTP-bound) states to transduce extracellular signals via cell-surface receptors. KRAS 70

signaling occurs through engagement with effector proteins that orchestrate intracellular 71

signaling cascades regulating tumor cell survival and proliferation. Aberrant activation of 72

KRAS by deregulated upstream signaling (1), loss of GTPase-activating protein function (2,3) 73

or oncogenic mutations result in increased GTP-bound KRAS and persistent downstream 74

signaling (4,5). Mutations in the KRAS gene occur in approximately 1 out of 7 of all human 75

cancers, making it the most frequently mutated oncogene (6,7). Up to 90% of pancreatic 76

tumors bear activating KRAS mutations. Mutated KRAS is also observed at high frequency in 77

other common tumors, including colorectal cancer (~44%) and non-small cell lung cancer 78

(~29%). Cancer-associated mutations in KRAS cluster in three hotspots (G12, G13, Q61), 79

with a majority (77%) of mutations causing single amino-acid substitutions at G12. The 80

KRAS missense mutation G12D is the most predominant variant in human malignancies 81

(35%), followed by G12V (29%), G12C (21%), G12A (7%), G12R (5%), and G12S (3%). 82

Besides G12, the hotspots G13 and Q61 show mutation rates of 10% and 6% respectively 83

(KRAS mutation frequencies were derived from AACR GENIE v6.1 and TCGA) (6,7). In 84

preclinical models, activated KRAS has been shown to drive both the initiation and 85

maintenance of a range of cancer types (8-11). Despite the compelling rationale to target 86

KRAS, identification of potent direct inhibitors has been challenging. Promising early results 87

from clinical trials with the two inhibitors AMG 510 and MRTX849, both targeting the 88

KRAS G12C mutant allele covalently and specifically (12,13), have been reported. These 89

inhibitors demonstrated clinical activity primarily in non-small cell lung cancer (NSCLC), 90

where the KRAS G12C mutation frequency is highest (14,15). Moreover, a nanomolar pan-91

RAS inhibitor binding to a second pocket on RAS has been described (16). 92




Despite this recent success, molecularly targeted therapies that effectively address the most 93

prevalent KRAS mutant alleles beyond G12C, including G12D and G12V, are lacking. 94

Attempts to indirectly target KRAS-driven tumors through inhibition of downstream effectors 95

of KRAS, such as members of the RAF-MEK-ERK cascade, has suffered limited clinical 96

success (17) in part due to the capacity of cancer cells to adapt by rapidly increasing KRAS-97

GTP levels. The SHP2 protein-tyrosine phosphatase is an important mediator of cellular 98

signaling through the RAS/MAP kinase pathway and is thought to act via activation of SOS1-99

regulated RAS-GTP loading. SHP2 inhibitors are being explored by several companies with 100

the most advanced inhibitors, RMC-4630 and TN0155, currently under study in phase 1 101

clinical trials (18-21). Published data show particular sensitivity to SHP2i inhibitors in KRAS 102

G12C mutant tumors (20). 103

Dynamic control of the extent and kinetics of the RAS-RAF-MEK-ERK signaling is governed 104

by positive and negative feedback loops (22). SOS1 is a key guanine exchange factor (GEF) 105

for KRAS that binds and activates GDP-bound RAS-family proteins at its catalytic binding 106

site and in this way promotes exchange of GDP for GTP. In addition to its catalytic site, 107

SOS1 can also bind GTP-bound KRAS at the allosteric site that potentiates its GEF function, 108

constituting a mechanism for positive feedback regulation (23). Depletion of SOS1 or specific 109

genetic inactivation of its GEF function has been shown to decrease the survival of tumor 110

cells harboring a KRAS mutation (24). This effect was not observed in wild-type cells that are 111

not KRAS addicted (24). Pathway activation leads to ERK-mediated phosphorylation of 112

SOS1 but not its paralog SOS2 thereby attenuating of SOS1 GEF activity (25,26). This 113

suggests that SOS1 acts as an important node in the negative feedback regulation of the 114

KRAS pathway (25,26). Based on these lines of evidence, we hypothesized that a potent and 115

selective SOS1 inhibitor would synergize with a MEK inhibitor, resulting in strong and 116

sustained pathway blockade and a robust anti-tumor efficacy in KRAS-driven cancers. 117




In 2014, small molecules have been described which bind to a lipophilic pocket of SOS1, in 118

close proximity to the RAS binding site (27). Binding of these ligands increased SOS1-119

mediated nucleotide exchange and consequently led to activation of RAS. Recently, SOS1 120

inhibitor tool compounds were reported (28), but these non-bioavailable compounds did not 121

demonstrate the expected differential effect on KRAS-driven cancer cell lines versus wild-122

type cells. 123

In this paper, we describe the discovery of BI-3406, a potent and selective SOS1::KRAS 124

interaction inhibitor, and elucidate its mode of action both in vitro and in vivo. BI-3406 125

potently decreases the formation of GTP-loaded RAS and reduces cell proliferation of a large 126

fraction of KRAS G12C- and non-G12C-driven cancers in vitro and in vivo. BI-3406 127

attenuates feedback reactivation by MEK inhibitors and enhances sensitivity of KRAS-128

dependent cancers to MEK inhibition, resulting in tumor regressions at well-tolerated doses in 129

mouse models. Our data provide strong evidence that combined SOS1 and MEK inhibition 130

represents an attractive therapeutic concept to address KRAS-driven human tumors. 131

132

Results 133

Discovery of BI-3406, a potent and selective SOS1::KRAS interaction inhibitor 134

To discover SOS1 inhibitors, we conducted a high throughput screen of 1.7 million 135

compounds using an alpha screen and a fluorescence resonance energy transfer assay as an 136

orthogonal biochemical screen on SOS1 and KRAS G12D. Several hits containing a 137

quinazoline core were identified, best exemplified by BI-68BS (Supplementary Fig. S1a). A 138

stoichiometric and saturable dissociation constant, using surface plasmon resonance on SOS1 139

(KD = 470 nM) and the corresponding activity in a GDP-dependent KRAS-SOS1 140

displacement assay (IC50 = 1.3 µM), indicated effective disruption of the SOS1-KRAS 141

protein-protein interaction. Co-crystallization of BI-68BS and SOS1 confirmed binding to a 142




pocket (27) next to the catalytic binding site on SOS1 (Supplementary Fig. S1b, S1c and 143

Supplementary Table S1) with the quinazoline ring pi-stacking to His905SOS1

. Based on the 144

structural data, the interaction of the methoxy substituent of BI-68BS with Tyr884SOS1

most 145

likely interferes with the competing Tyr884SOS1

-Arg73RAS

interaction and consequently 146

prevents KRAS from binding to SOS1 (Supplementary Fig. S1d). In an effort to optimize BI-147

68BS, several modifications were made, which led to the discovery of BI-3406 (Fig. 1a). As 148

BI-68BS was originally synthesized as part of a project targeting EGFR, a methyl substituent 149

was incorporated in the 2-position of the quinazoline core to effectively eliminate any 150

interfering inhibition of kinase activity (tested in a panel of 324 kinases, Supplementary Table 151

S2 and S3). Introduction of a trifluoromethyl and an amino substituent at the phenethyl 152

moiety filled the pocket more effectively and formed an H-bond with M878SOS1

respectively, 153

thereby significantly increasing potency. The tetrahydrofuryl substituent favorably balanced 154

solubility and metabolic stability and improved interaction with Tyr884SOS1

. Synthesis of BI-155

3406 is described in detail in the supplementary data section (for synthesis route see also 156

Supplementary Fig. S1e and S1f.). Crystallization data can be found in Supplementary Table 157

S1. 158

A detailed biochemical characterization of BI-3406 was made possible through the analysis of 159

a variety of interaction assays using SOS1 and SOS2 recombinant proteins, in combination 160

with several KRAS mutant variants. BI-3406 was found to be a potent, single digit nanomolar 161

inhibitor binding to the catalytic site of SOS1 and thereby blocking the interaction with 162

KRAS-GDP, as exemplified in the interaction assay with KRAS G12D and G12C mutant 163

oncoproteins (Fig.1b). 164

A recently developed covalent KRAS G12C-specific inhibitor (ARS-1620) was able to 165

interfere with the SOS1-KRAS G12C protein-protein interaction but, in contrast to BI-3406, 166

had no effect on the protein-protein interaction of SOS1 with KRAS G12D (Fig. 1c, d). Upon 167




replacing SOS1 with its paralog SOS2, BI-3406 lost its ability to interfere with KRAS 168

binding, indicating that BI-3406 is a highly potent, SOS1 specific inhibitor that can address 169

multiple KRAS mutant oncoproteins (Fig. 1b). The SOS1 selectivity of BI-3406 can be 170

explained by a potential clash of the compound with Val903 and the absence of pi-interaction 171

in SOS2, which is revealed in an overlay of the published SOS2 apo structure (PDB Code 172

6EIE) with our SOS1 BI-3406 co-crystal structure (Supplementary Fig. S1g). In a 173

biochemical protein-protein interaction assay, the introduction of the mutations Y884A and 174

H905V in a recombinant SOS1 protein strongly impaired the ability of BI-3406 to disrupt the 175

interaction with KRAS G12D (Supplementary Fig. S1h). Importantly, expression of FLAG-176

SOS1 transgenes in Mia PaCa-2 and HEK293 cells revealed that the SOS1 mutations H905V 177

and H905I abrogated the ability of BI-3406 to inhibit phosphorylation of ERK (pERK) and 178

cell proliferation, demonstrating selective SOS1 on-target activity of the compound in a 179

cellular context. (Fig. 1e, f and Supplementary Fig. S1i). 180

To further investigate whether BI-3406 was capable of cellular SOS1 inhibition, cells were 181

treated with increasing concentrations of BI-3406. The compound inhibited RAS-GTP levels 182

with an IC50 of 83-231 nM in SOS1/KRAS-dependent NCI-H358 (KRAS G12C) and A549 183

(KRAS G12S) cells (Fig. 1g). Stimulation of starved NCI-H358 and MIA PaCa-2 cells with 184

EGF resulted in an increase of RAS GTP levels that could be blocked by the addition of BI-185

3406 (Supplementary Fig. S1j). Based on our mechanistic findings that BI-3406 selectively 186

targets SOS1, we next wanted to address its cellular selectivity profile. As there are no known 187

substrate differences distinguishing SOS1- and SOS2-mediated effects, we reasoned that a 188

SOS1-selective inhibitor should have an increased impact on cellular signaling in a SOS2-null 189

background. Accordingly, we generated NCI-H358 cells in which SOS2, and for comparison 190

SOS1, was genetically inactivated (Supplementary Fig. S1k) and measured RAS GTP levels 191

after treatment with BI-3406. The effect of BI-3406 on RAS-GTP levels was significantly 192

more pronounced in NCI-H358 cells harboring a SOS2 knockout when compared to the 193




parental cell line (Supplementary Fig. S1l). Moreover, the effect of BI-3406 on pERK levels 194

was enhanced in NCI-H358 SOS2 null cells compared to parental cells, while being strongly 195

reduced in SOS1 knockout cells (Supplementary Fig. S1m). The anti-proliferative effect of 196

BI-3406 was enhanced in SOS2 knockout cells, compared to parental cells (Supplementary 197

Fig. S1n). In SOS1 knockout cells, no effect on proliferation was observed following 198

treatment with BI-3406 (Supplementary Fig. S1n). Analysis of a time-course treatment of 199

NCI-H358 cells (KRAS G12C) with BI-3406 revealed a rapid reduction of RAS-GTP levels 200

that correlated with the effect on pERK levels (Supplementary Fig. S1o). RAS-GTP and 201

pERK returned to levels close to baseline at the 24 hour time point. These data further support 202

the notion that BI-3406 is a potent and SOS1-selective inhibitor. 203

204

Association of KRAS mutation status with sensitivity to SOS1 inhibition 205

The cellular activity of BI-3406 was further evaluated across a wider panel of cancer cell lines 206

driven by different KRAS pathway activating mutations. As SOS1 is uniformly expressed 207

across all tumor types, a SOS1 inhibitor could be broadly applicable in KRAS driven 208

indications (Supplementary Fig. S2a and S2b). Plotting the expression of SOS1 against SOS2 209

revealed that the cell lines used in our subsequent experiments harbored SOS1/SOS2 mRNA 210

ratios representative of ratios observed in a large dataset of human tumors (Supplementary 211

Fig. S2c). A dose-dependent partial reduction of pERK levels was observed in all RAS-212

mutated cell lines tested, with an IC50 between 17 and 57 nM (IC50 value defined as the 213

inflection point of the curve) (Fig. 2a). No pERK modulation was observed in A375 214

melanoma cells that are KRAS wild-type and harbor an activating BRAF V600E mutation 215

that likely renders them independent of KRAS signaling (Fig. 2a). 216

Cell lines expressing mutant KRAS have demonstrated variable dependencies upon KRAS for 217

viability in two-dimensional (2D) monolayer proliferation assays (29), while KRAS-218




dependency is better modelled in anchorage-independent 3D growth assays. Consistent with 219

this observation, we demonstrated that BI-3406 inhibited the 3D growth of 4 KRAS mutant 220

cancer cells with a IC50 of 16-52 nM, as half-maximum inhibitory concentration (Fig. 2b). In 221

contrast, the 3D growth of the two KRAS wild-type cancer cell lines, NCI-H520 and A375, 222

was not appreciably affected (Fig. 2b), but responded to the broad anti-proliferative agent 223

panobinostat, an HDAC inhibitor (Fig. 2c). Collectively, these data show a clear correlation 224

between signaling pathway and growth inhibition by BI-3406 in KRAS-driven cancer cell 225

lines. 226

The growth inhibitory effects of BI-3406 across different KRAS mutated cell lines could be 227

influenced by tumor lineage or co-mutations. Therefore, we evaluated the effect of SOS1 228

inhibition on a panel of isogenic cell lines, differing only in the status of their KRAS allele. 229

We used NCI-H23 cells carrying a heterozygous KRAS G12C allele and replaced the G12C 230

codon by heterozygous G12D, G12V, G12R, G13D or homozygous G12D, G13D and Q61H 231

mutations. BI-3406 showed comparable activity, independent of zygosity, with an 232

approximate 50% reduction of pERK levels in all KRAS variant isogenic cell lines (Fig. 2d). 233

Reduction of pERK correlated with reduced proliferation of NCI-H23 isogenic cell lines, 234

indicating cellular sensitivities of the most prevalent KRAS G12 and G13 oncogenic variants 235

(Fig. 2d, e). Only weak inhibition of pERK levels were observed in cells carrying the Q61H 236

oncogenic variant, that was recently reported to lack intrinsic GTP hydrolysis activity and to 237

exhibit increased affinity for RAF (30). No modulation of pERK was observed in cells 238

carrying the G12R variant (Fig. 2d), a variant that was recently described not to interact with 239

the catalytic domain of SOS1 (31). In a cellular context in which the KRASG12C mutation 240

was reverted to wild-type KRAS, pERK modulation was observed following treatment with 241

BI-3406, but the wild-type cells were no longer able to grow in a 3D proliferation assay. 242

243




We further profiled BI-3406 in a larger panel of 40 solid cancer cell lines with known 244

oncogenic alterations in KRAS, NRAS, HRAS, EGFR, NF1, and BRAF (Fig. 2f and 245

Supplementary Table S4 and S5). Excitingly, BI-3406 sensitivity correlated with KRAS 246

mutation status in this larger cell panel (Fisher’s exact test, p-value 0.00337) (Fig. 2f). 247

Sensitive cell lines harbored a broad range of KRAS mutant alleles (Supplementary Table S4 248

and S5), including KRAS G12C, G12V, G12S, G12A and G13D mutations. Although no 249

difference in sensitivity could be observed based on the zygosity of the KRAS mutation, it was 250

notable that two out of the three non-responsive KRAS mutant cell lines, as well as three out 251

of five non-responsive NRAS mutant cell lines, were characterized by a Q61 mutation. NF1 is 252

a tumor suppressor and a RAS GTPase-activating protein (GAP) (2). Loss of NF1 function 253

has been shown to increase RAS-GTP levels, hyperactivate RAS/MAPK signaling and 254

contribute to a variety of human cancers (32,33). Therefore, we assessed whether NF1 255

aberrations in cell lines resulted in sensitivity to SOS1 inhibition. Interestingly 7 out of 14 cell 256

lines carrying NF1 aberrations were sensitive to BI-3406 treatment, irrespective of their KRAS 257

status. No other driver mutations in components of the RTK/KRAS/MAPK pathway could be 258

identified in several of these sensitive cell lines, suggesting NF1 aberrations are a key 259

determinant for sensitivity to BI-3406 in these lines (Supplementary Table S4). Similarly, a 260

fraction of NSCLC cell lines driven by EGFR mutations also responded to BI-3406 treatment, 261

suggesting that oncogenic RTKs can confer sensitivity to SOS1 inhibition. As none of the six 262

BRAF mutant and five NRAS mutant cell lines were sensitive to treatment with BI-3406, (Fig. 263

2f) we hypothesize that NRAS and BRAF mutations are associated with resistance to BI-3406 264

monotherapy (p-value < 0.001). Collectively, our findings highlight the critical function of 265

SOS1 in promoting KRAS/MAPK pathway activation in a large fraction of cancers driven by 266

KRAS G12C- and non-G12C alleles, NF1 aberrations, as well as EGFR mutations. 267

268




The pharmacodynamics of BI-3406 were further evaluated. In sensitive cell lines, treatment 269

with BI-3406 resulted in sustained pathway modulation of ERK1/2 phosphorylation 270

(Supplementary Fig. S2d and S2e), in contrast to insensitive cell lines, that exhibited weaker 271

and more short-lived effects (NCI-H2170 and NCI-H1299) (Supplementary Fig. S2e). 272

Compared to pERK, levels of pAkt Ser473 and Thr308 were less strongly affected by BI-273

3406 (Supplementary Fig. S2d and S2e). 274

We subsequently tested BI-3406 side-by-side with the recently reported SOS1 inhibitor 275

BAY293 (28) and the SHP2 inhibitor SHP099 (18) in 2D and 3D proliferation assays across a 276

panel of 24 cell lines, including 18 KRAS-mutated cell lines (Supplementary Table 6). The 277

three compounds demonstrated no activity in 2D proliferation assays. In 3D proliferation 278

assays, SHP099 showed the strongest anti-proliferative effects with an IC50 between 167-790 279

nM in KRAS G12C, a subset of G12D cell lines, and in one G12S cell line it yielded modest 280

effects in KRAS G13D and G12V cells (IC50:1180-4411 nM), while no effects were 281

detectable in Q61L/H and G12R KRAS mutant tumor cells. BI-3406 caused cell growth 282

inhibition in all KRAS G12 and G13 mutant cell lines (IC50: 9-220 nM) with the exception of 283

G12R and KRAS Q61L/H mutant tumor cells. The previously published SOS1 inhibitor 284

BAY293 demonstrated only a very limited potency and, in contrast to BI-3406, no sizeable 285

selectivity for KRAS mutated cells, as compared to KRAS wild-type cells (Supplementary 286

Table S6). This suggests that BI-3406 and SHP099 possess a partially overlapping yet distinct 287

profile across KRAS mutated cell lines, with BI-3406 being more broadly active in 3D 288

proliferation assays. 289

To glean first insights regarding a potential therapeutic index of BI-3406, we tested the 290

compound on primary cells and non-tumorigenic cells in vitro. BI-3406 inhibited the 291

proliferation of foreskin fibroblasts with an IC50 of 37 nM, while 2 other cell types, primary 292

smooth muscle cells and retinal pigment epithelial cells, were not impacted (IC50>5µM) 293




(Supplementary Fig. S2f-h). The extremely potent and widely used MEK inhibitor trametinib 294

affected proliferation of all 3 aforementioned cell types (retinal pigment epithelial cells IC50 295

of 12 nM, primary smooth muscle cells 843 nM, normal foreskin cells IC50 of 85 nM). 296

297

SOS1 inhibition suppresses tumor growth in xenograft models of KRAS-driven cancers 298

BI-3406 is an orally bioavailable compound (Supplementary Fig. S3a) and single 299

administration was sufficient to reduce RAS-GTP and pERK levels in A549 xenograft tumors 300

over a period of 24 hours and 7 hours, respectively (Supplementary Fig. S3b-c). At a dose of 301

50 mg/kg bid, relevant levels of unbound exposures are achieved for the first 12 hours, when 302

compared to unbound IC50 levels in A549 cells (Supplementary Fig. S3a). In MIA PaCa-2 303

tumor bearing mice, twice daily compound treatment with 50 mg/kg BI-3406 resulted in 304

pathway modulation over a period of up to 10 hours (Fig. 3a and Supplementary Fig. S3d). At 305

the 24 h time point, the compound was cleared (Supplementary Fig. S3a and S3d) and pERK 306

levels returned to baseline in both A549 and MIA PACa-2 tumors (Fig. 3a and Supplementary 307

Fig. S3b). In the same experiment, a reduction of pERK was observed by 308

immunohistochemistry in surrogate tissue (murine skin) over a similar period (Fig. 3b and 309

Supplementary Fig. S3e). As the use of phosphorylation markers can be challenging in a 310

clinical setting, effects on RAS dependent gene expression signatures were analyzed in the 311

MIA PaCa-2 xenograft model. Prolonged suppression of known pathway-related genes, such 312

as SPRY4, DUSP6, and transcriptional regulators, such as FOSL1, EGR1, ETV1, ETV4 and 313

ETV5 was observed (Fig. 3c, Supplementary Fig. S3f and Supplementary Table S7) in line 314

with published data on gene expression responses to other specific MAPK pathway inhibitors 315

(34,35). Of note, no effects on SOS2 mRNA expression were observed upon treatment with 316

BI-3406 during the period of observation (Supplementary Fig. S3g-h) suggesting no 317

compensatory upregulation. 318




Based on its potent cellular activity and favorable pharmacokinetic properties, the efficacy of 319

BI-3406 was evaluated in established, subcutaneous KRAS G12C-mutated MIA PaCa-2 320

xenografts. Twice daily treatment with either 12 or 50 mg/kg of BI-3406 was well tolerated 321

and resulted in a prolonged dose-dependent tumor growth inhibition (p<0.005 as compared to 322

vehicle control, Fig. 3d, 3e). Similar tumor growth inhibitory effects were observed in KRAS 323

G12V-mutated SW620, the KRAS G13D-mutated LoVo and the KRAS G12S-mutated A549 324

xenograft models (Fig. 3f, Supplementary Fig. S3i and S3j). No anti-tumor response was 325

observed in the BRAF mutant A375 xenograft model (Supplementary Fig. S3k), consistent 326

with the lack of effect on cell proliferation in this cell line in vitro. Thus, oral administration 327

of BI-3406 monotherapy inhibits the growth of KRAS G12C, G12V, G13D and G12S 328

mutated xenograft models. 329

330

Dual SOS1 and MEK inhibition as effective strategy to treat KRAS-mutant tumors 331

Previous work showed that many cancer models develop adaptive resistance to MEK 332

inhibitors, often due to the reactivation of SOS1 (17). Therefore, we reasoned that dual SOS1 333

and MEK inhibition could constitute an effective strategy to treat KRAS mutant tumors. 334

Consistent with this hypothesis, the combination of BI-3406 with the MEK inhibitor 335

trametinib yielded strong synergistic anti-proliferative effects in MIA PaCa-2 (KRAS G12C) 336

and DLD1 (KRAS G13D) cells in vitro (Supplementary Fig. S4a). Based on these promising 337

cellular data, we tested BI-3406 plus trametinib in both the pancreatic cancer MIA PaCa-2 338

and the colorectal LoVo (KRAS G13D) xenograft mouse models. The MEK inhibitor 339

trametinib was primarily used due to its favorable mouse pharmacokinetic properties (t1/2 = 33 340

hours) (36). The combination of 50 mg/kg BI-3406 twice daily with the clinically relevant 341

dose of trametinib (0.1-0.125 mg/kg, bid; for calculations details please see description in 342

Supplementary Data) was well tolerated (Supplementary Fig. S4b and S4c) and caused 343




substantial regressions in the entire cohort of MIA PaCa-2 tumor bearing mice (Fig. 4a and b). 344

Furthermore, following combination treatment, slow regrowth of tumors was detectable only 345

22 days after drug withdrawal (Fig. 4a). Similar results were observed in LoVo xenografts, 346

with the effect of the BI-3406 and trametinib combination therapy being significantly stronger 347

compared to both monotherapies, with sustained tumor inhibition for 7 days following drug 348

withdrawal (Fig. 4c, d). We tested two KRAS G12C-mutated colorectal cancer patient-349

derived xenograft models (PDX), one KRAS G12V and one KRAS Q61K mutant pancreatic 350

PDX model and observed improved antitumor activity using a combination of BI-3406 with 351

trametinib (Fig. 4e, f and Supplementary Fig. S4d-g). As expected based on proliferation 352

assays using KRAS Q61 mutant cells, monotherapy of BI-3406 resulted in only weak efficacy 353

in monotherapy in the KRAS Q61K mutant PDX model, yet the SOS1 and MEK inhibitor 354

combination significantly improved antitumor activity as compared to both monotherapies 355

(p=0.0026; Supplementary Fig. S4g). The combination treatment was very well tolerated 356

(Supplementary Fig. S4b-c, S4h-k). As SOS2 may promote resistance to SOS1 over time, we 357

analyzed the colorectal PDX model B8032, but found no compensatory upregulation of SOS2 358

mRNA levels upon 21 days of treatment with both SOS1 and MEK inhibitor (Supplementary 359

Fig. S4l-m). 360

361

The SOS1 inhibitor BI-3406 prevents adaptive resistance to MEK inhibition 362

The mechanism underlying the SOS1/MEK inhibitor combination efficacy was evaluated 363

with regards to the impact on modulation of the KRAS-RAF-MEK-ERK cascade. In vitro 364

MEK inhibitor treatment at clinically relevant doses, in the low nM-range (see description in 365

Supplementary Data), resulted in a progressive increase of MEK1/2 Ser217/221 366

phosphorylation, an effect termed adaptive resistance or negative feedback relief (Fig. 5a and 367

Supplementary Fig. S5a) (17). Consistent with our hypothesis that a SOS1 inhibitor might 368

counteract adaptive resistance to MEK inhibition, combination of BI-3406 and trametinib 369




antagonized the MEK inhibitor-induced increase of MEK1/2 phosphorylation in MIA PaCa-2 370

cells (Fig. 5a, and Supplementary Data Fig. 5a and b). A moderate, yet statistically significant 371

effect on pERK1/2 levels was detected after 24-72 hours of treatment with BI-3406, whereas 372

a marked reduction of ERK1/2 phosphorylation was observed with trametinib (Fig. 5b, 373

Supplementary Fig. S5c). An additional reduction in pERK levels was observed upon 374

combination of both drugs (Fig. 5b and Supplementary Fig. S5c). This effect was also 375

observed in NCI-H23 cells, a KRAS G12C mutant cell line, albeit to a lesser degree 376

(Supplementary Fig. S5d). A combination of BI-3406 and trametinib resulted in a near-377

complete reduction of pERK1/2 phosphorylation compared to the partial effects induced by 378

the two monotherapies in MIA PaCa-2 tumor bearing mice (Fig. 5c). Combination of the 379

SOS1 inhibitor and MEK inhibitor elicited a reduction of pERK and blockade of adaptive 380

resistance, measured by pMEK1/2 in MIA PaCa-2 and NCI-H23 cells, not only when grown 381

in 2D (Fig5a-b and Supplementary Fig. 5a-d) but also when cultured in 3D (Supplementary 382

Fig. S5e-h). Furthermore, we observed that the combination of MEK and SOS1 inhibition 383

resulted in an enhanced reduction of pERK and S6 phosphorylation as assessed by Reverse 384

Phase Protein Array (RPPA) analysis in two colorectal cancer patient-derived xenograft 385

models (Supplementary Fig. S5i and Table S8). In addition, the combination led to enhanced 386

reduction of DUSP6 mRNA in MIA PaCa2 tumors (Supplementary Fig. S5j) and augmented 387

induction of apoptosis as shown in the KRAS-driven cell line DLD1 (Supplementary Fig. 388

S5k). 389

Finally, we investigated whether the beneficial effect of BI-3406 described above could be 390

extended to a direct KRAS inhibitor, the clinical KRAS G12C inhibitor AMG 510. Strikingly 391

the combination of AMG 510 with BI-3406 resulted in stronger and more prolonged 392

suppression of pERK, as compared to AMG 510 monotherapy in NCI-H358 (KRAS G12C) 393

cells in vitro (Supplementary Fig. S5l). The addition of the SOS1 inhibitor to AMG 510 394

largely prevented the rebound of pERK at the 72h time point. A similar effect was observed at 395




72h upon combination of AMG 510 with the SHP2 inhibitor SHP099 (Supplementary Fig. 396

S5l). 397

In summary, the SOS1 inhibitor BI-3406 enhances the extent and duration of MAPK pathway 398

inhibition upon combination with a MEK or KRAS G12C inhibitor, suggesting it is able to 399

counteract adaptive resistance. This highlights SOS1 inhibition as a promising combination 400

option for MAPK pathway and direct KRAS inhibitors. In line with this, we show that a 401

SOS1/MEK inhibitor combination enables long-term pathway inhibition resulting in tumor 402

regressions in multiple KRAS-driven cancer models at well-tolerated doses (Fig. 5d). 403

Discussion 404

KRAS mutations are the most frequent gain-of-function alterations found in cancer patients, 405

yet KRAS-driven tumors are largely refractory to anticancer therapies. Despite more than two 406

and a half decades of research describing the central role of SOS1 in developmental and 407

oncogenic signaling pathways, most notably in the direct activation of RAS oncoproteins (37-408

40), no SOS1 inhibitor has progressed to the clinic. The previously described catalytic SOS1 409

modulator BAY293 (28) inhibited cancer cell proliferation with weak potency and 410

irrespective of KRAS status. Here we describe a highly potent and selective small molecule 411

inhibitor, BI-3406, that binds to SOS1 and thereby blocks protein-protein interaction with 412

RAS-GDP. BI-3406 is the first example of an orally bioavailable SOS1::KRAS interaction 413

inhibitor that reduces RAS-GTP levels and curtails MAPK pathway signaling in vitro and in 414

vivo. BI-3406 limits the growth of the majority of tumor cells driven by KRAS variants at 415

positions G12 and G13, as shown in 3D proliferation assays. As tumors bearing these KRAS 416

mutations are most prevalent in colorectal cancer, pancreatic cancer and non-small cell lung 417

cancer, these results provide compelling evidence that the SOS1::KRAS interface is a 418

druggable target of potential clinical importance, and highlight BI-3406 as a front-runner of a 419

new generation of GDP-KRAS-directed inhibitors with promising therapeutic potential. In 420




contrast to covalent KRAS G12C-specific inhibitors (12,14), this novel approach holds 421

promise for impact across the majority of mutant KRAS alleles, including the two most 422

prevalent variants G12D and G12V. Interestingly, our data suggest that tumors harboring 423

codon 61 mutations (such as Q61H), appear to be less sensitive to SOS1 inhibition, possibly 424

because these mutant isoforms have the lowest intrinsic GTPase activity and may require less 425

upstream signaling to remain GTP bound (41). The KRAS G12R variant, which is relatively 426

common in pancreatic cancers (~20% prevalence), showed no modulation of pERK following 427

treatment with BI-3406. This finding is in line with a recent publication describing an 428

inability of the catalytic domain of SOS1 to interact with this KRAS G12R mutant 429

oncoprotein (31). The sensitivity spectrum we have observed towards SOS1 inhibition further 430

supports the concept of oncogenic KRAS G12 and G13 variants functioning in a semi-431

autonomous manner (42) and remain susceptible to regulation by SOS1 for optimal GTP-432

loading. Collectively, our data suggest that BI-3406 will be able to impact about 80-90% of 433

all KRAS-driven cancers. 434

We have carried out a comprehensive screen for effective combination partners. Synergy was 435

observed upon combination of SOS1 with MEK inhibitors, leading to tumor regressions in 436

multiple mutant KRAS-driven cancer models at well tolerated doses. Of the two SOS 437

isoforms, SOS1 and SOS2, only SOS1 is phosphorylated by ERK, resulting in the reduction 438

of its GEF activity (26). Treatment with a MEK inhibitor reduces the activity of ERK1/2, 439

resulting in release of a negative feedback loop, thus increasing the activity of SOS1-mediated 440

formation of GTP-loaded KRAS (25,26). Combination of MEKi with BI-3406 thus blocks the 441

negative feedback release by reducing pMEK1/2 and pERK1/2 levels, supporting sustained 442

pathway inhibition and tumor regressions (Fig. 5d). Tumor stasis was observed with a 443

SOS1/MEK inhibitor combination in colorectal and pancreatic PDX models. This may 444

indicate that, in these tumor types, additional feedback and bypass mechanisms are effective, 445

and triple combinations are needed to shut down KRAS signaling and achieve tumor 446




regressions. Due to the favorable tolerability of the SOS1/MEK treatment, combinations with 447

standard of care treatments will be further evaluated with the aim to achieve tumor 448

regressions in colorectal and pancreatic cancer models. We demonstrated that, in addition to 449

the combination of BI-3406 with trametinib (MEKi), combination of BI-3406 with the 450

clinical KRAS G12C inhibitor AMG 510 (KRASG12Ci) results in enhanced and prolonged 451

MAPK pathway suppression. Our study highlights SOS1 inhibitors as promising combination 452

partners for inhibitors directly targeting KRAS, the GDP-bound form of KRAS, or 453

downstream MAPK pathway intermediates. This finding is also in line with a recent report 454

describing a marked synergy in NSCLC cell lines combining SOS1 inhibition with vertical 455

EGFR inhibition (43). 456

BI-3406 is a selective inhibitor of SOS1 and does not target the paralog GEF SOS2. 457

Simultaneous genetic inactivation of both SOS proteins leads to rapid death in mouse models, 458

in contrast to single gene perturbations (44). Thus, while the SOS1 selectivity may reduce the 459

monotherapy impact of BI-3406 on KRAS and the MAPK pathway, it can facilitate 460

combination therapies due to the expected superior tolerability of a SOS1 specific inhibitor 461

compared to a pan SOS1/SOS2 inhibitor (44,45). Furthermore, targeting SOS1 can selectively 462

exploit its key function in adaptive feedback control that is not shared with its paralogue 463

SOS2 (25,26). No upregulation of SOS2 expression was observed in our biomarker and 464

efficacy experiments. It remains to be determined whether cancer patients treated with a 465

SOS1 inhibitor will exhibit induction of SOS2 levels. 466

Recently, inhibitors targeting the protein-tyrosine phosphatase SHP2 (encoded by the gene 467

PTPN11), a common node downstream of RTKs that is required for RAS activation, have 468

been reported (18,19). Interestingly, these reports also suggest that inhibition of SHP2 can 469

attenuate adaptive MEK inhibitor resistance in KRAS-dependent cancers (46-49). Our 470

comparative analysis of the SHP2 inhibitor tool compound SHP099, suggest activity in cell 471

lines harboring G12C, a subset of G12D and possibly G12S KRAS variant driven cell lines, 472




while the SOS1 inhibitor BI-3406 demonstrates activity in all KRAS G12 and G13 mutant 473

cell lines tested, with the exception of cell lines driven by the G12R oncoprotein. While only 474

BI-3406 is active in the KRAS G13-driven context, both inhibitors lack single agent activity 475

in KRAS Q61 mutant cell lines, suggesting an overall broader impact on KRAS mutant 476

cancers by the SOS1 inhibitor. Future studies will be required to compare and contrast the 477

capabilities of SOS1 and SHP2 inhibitors to overcome adaptive resistance to 478

KRAS/RAF/MEK/ERK-targeted agents across KRAS-driven cancers. 479

While the precise mechanism by which SHP2 contributes to KRAS activation is yet to be 480

determined, SHP2 is not a direct activator of KRAS and may in part act via SOS1 (50,51). 481

Ongoing clinical evaluations will show if SOS1i/MEKi and SHP2i/MEKi combinations will 482

differ in terms of safety and response rates across tumors with different KRAS alterations. 483

Collectively, our study provides a new chemical probe for further dissection of the cellular 484

functions of SOS1 in tumorigenesis and MEK inhibitor-driven drug resistance. Importantly, 485

the pharmacological properties of BI-3406 and close analogues hold the promise of 486

developing clinical SOS1 compounds that, in combination with MEK inhibitors and 487

potentially other RTK/MAPK pathway inhibitors, could provide significant clinical benefit 488

across a broad patient population currently lacking molecularly targeted, precision medicine 489

options. A Phase 1 clinical trial has been initiated (NCT04111458) for patients with advanced 490

KRAS-mutated cancers to evaluate safety, tolerability, pharmacokinetic and 491

pharmacodynamic properties, and preliminary efficacy of BI 1701963, a SOS1::KRAS 492

inhibitor closely related to BI-3406, alone and in combination with the MEK inhibitor 493

trametinib. 494




Methods 495

Additional descriptions of methods can be found in the Supplementary Data file. 496

497

Cell culture 498

Tumor cell lines were obtained from the American Type Culture Collection (Manassas, US-499

VA) or the German Collection of Microorganisms and Cell Culture (DSMZ, Braunschweig, 500

Germany). All cell lines used in this study were cultured according to the manufacturer’s 501

instruction and authenticated by short tandem repeat (STR) analysis at Boehringer Ingelheim 502

(Supplementary Table S9). With regard to the 2D proliferation assays, cells were seeded in 503

their respective medium supplemented with 2% FCS. For the 3D proliferation assay, the cells 504

were embedded in soft agar, which required three separate layers within a well; a bottom layer 505

formed of 1% agar solution, a cell layer formed of a 0.3% agar solution and a medium layer 506

(described in detail in the supplemental data). BI-3406, trametinib or a positive control (e.g. 507

panobinostat) was added with increasing concentrations. Readout of cell proliferation was 508

adopted on cell growth properties avoiding more than 80% confluence in the control wells. 509

Dependent on the individual doubling times, readout for individual cell lines was between 5 510

and 14 days. Number of living cells were quantified through the addition of AlamarBlue® 511

reagent or CellTiter-Glo (Promega). As inhibition of the SOS1::KRAS inhibitor results in 512

most cases in only 50% reduction of proliferation, the IC50 values describe the point of 513

inflection of a curve and this does not fit in all cases with 50% inhibition. Transfection of 514

cells, generation of isogeneic NCI-H23 cells, generation of SOS1 or SOS2 negative cells lines 515

as well as the transgeneic expression of FLAG-SOS1 variants can be found in supplementary 516

data and tables S10 and S11. Details on immunodetection in cell lysate can be found in 517

supplementary data and tables S10, S11 and S12. 518

519




Synthesis of BI-3406 520

Conditions are described in the supplementary data section. A schematic representation of the 521

synthesis can be found in Supplementary Fig. S1e and S1f. 522

523

Protein-protein interaction assays (AlphaScreen assay technology) 524

Details on protein expression and purification can be found in supplementary data. 525

Measurements of various protein–protein interactions were performed using the Alpha Screen 526

technology developed by Perkin Elmer. Recombinant KRAS proteins, based on KRAS 527

isoform 4B (uniprot id P01116-2) were: KRAS G12D (1-169, N-terminal 6His-tag, C-528

terminal avi-tag) from Xtal BioStructures, Inc., KRAS G12C (1-169, C-terminal avi-tag, 529

biotinylated, mutations: C51S, C80L, C118S). Biotinylation was performed in vitro with 530

recombinant BirA biotin-protein ligase as recommended by the manufacturer (Avidity LLC, 531

Aurora, Colorado, USA). Interacting proteins such as SOS1 (564-1049, N-terminal GST-tag, 532

TEV cleavage site) and SOS2 (562-1047, N-terminal GST-tag, TEV cleavage site) were 533

expressed as glutathione S transferase (GST) fusions. Accordingly, the Alpha Screen beads 534

were glutathione coated Alpha Lisa acceptor beads (Perkin Elmer AL 109 R) and Alpha 535

Screen Streptavidin conjugated donor beads (Perkin Elmer 6760002L). Nucleotide was 536

purchased from Sigma (GDP #G7127) and Tween-20 from Biorad (#161-0781). All 537

interaction assays were carried out in PBS, containing 0.1% bovine serum albumin, 0,05% 538

Tween-20 and 10 µM GDP. Assays were carried out in white ProxiPlate-384 Plus plates 539

(Perkin Elmer #6008280) in a final volume of 20 µL. In brief, biotinylated KRAS proteins (10 540

nM final concentration) and GST-SOS1 or GST-SOS2 (10 nM final) were mixed with 541

glutathione acceptor beads (5 µg/mL final concentration) in buffer, containing GDP and were 542

incubated for 30 min at room temperature. After addition of streptavidin donor beads (5 543

µg/mL final concentration) under green light, the mixture was further incubated for 60 min in 544




the dark at room temperature. Single oxygen induced fluorescence was measured at an 545

Enspire multimode plate reader (Perkin Elmer) according to the manufacturer’s 546

recommendations. Data were analyzed using the GraphPad Prism based data software. 547

548

Measurement of KRAS-GTP levels 549

RAS-GTP levels were analyzed using a RAS G-LISA assay kit (Cytoskeleton Inc., Denver, 550

CO, USA, #BK131) according to the manufacturer’s instructions. Briefly, 600 ,000 cells 551

were seeded in 6 wells and grown to 70% confluence. Cells were washed with ice-cold 552

PBS and lysed in 80 µL ice-cold lysis buffer supplemented with the provided protease 553

inhibitor cocktail. Lysates were quickly frozen in liquid nitrogen and stored at -80°C until 554

further usage. After normalizing protein concentration, 40 µg of protein was added in 555

duplicates to wells of the RAS G-LISA plate coated with RAS GTP-binding protein and 556

incubated at 4°C for 30 minutes while shaking at 400 rpm. After washing, antigen 557

presenting buffer was added for 2 minutes. To measure bound RAS GTP levels, wells were 558

subsequently incubated with an anti-RAS primary antibody (1:50) followed by a HRP-559

labelled secondary antibody (1:500) and finally by adding a HRP detection reagent. 560

Absorbance was measured by 490 nm using an EnSpire Multimode Reader (Perkin Elmer). 561

Background was determined by a negative control well and subtracted from all samples. 562

The same assay was used to determine amount of RAS-GTP levels in tumor lysate. 563

564

Biomarker and PK/PD analysis 565

pERK and pAKT modulation in tumors was determined using the Phospho/Total ERK1/2, 566

Phospho(Ser473)/Total Akt and Phospho-Akt (Thr308) Whole Cell Lysate Kits (MesoScale, 567

K15107D, K15100D and K151DYD). Tumors were homogenized using Ready Prep Mini 568

Grinders (#163-2146 BIO RAD) and lysed in MSD TRIS Lysis Buffer plus inhibitors (as 569




provided in the kit). Protein concentration was determined by Bradford analysis. 0.8 µg/µL 570

were used for pERK measurements (biological replicates) according to the recommendations 571

of the manufacturer. Signal intensities were measured using a MESO SECTOR S 600 reader. 572

The pERK to total ERK ratio was calculated and the data plotted in Graphpad PRISM. This 573

assay was used as well for measurement of PD modulation in several tumor cell lines 574

(Supplementary Figure S2e). 575

pERK levels were determined in mouse skin based on IHC staining (H-scores). 576

Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tissue, 3 577

μm sections using anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:40, CST) 578

Antibody incubation and detection were carried out at 37ºC. Antigen retrieval was performed 579

using Thermo PT module with buffer pH6 (Dako #K8005) and visualized using the EnVision 580

kit (Dako, Glostrup, Denmark). Appropriate positive and negative controls were included 581

with the study sections. Digital images of whole tissue sections were acquired using a Aperio 582

AT2 histology scanner (Leica Microsystems). Images were evaluated by a pathologist (FT) 583

and H-Score was generated using HALO software 3.0, Indica Lab©. 584

585

RNA isolation and sequencing library preparation for expression profiling 586

Cells were lysed in TRI lysis reagent (Qiagen, #79306) according to the manufacturer's 587

instructions. Instead of chloroform, 10% volume 1-bromo-2-chloropropane (SigmaAldrich, 588

#B9673) was added. Total RNA was isolated with RNAeasy Mini Kit (Qiagen, #73404). 589

Quant-seq libraries were prepared using the QuantSeq 3’ mRNA-Seq Library Prep Kit FWD 590

for Illumina from Lexogene (#015.96) according to manufacturer's instructions. Samples were 591

subsequently sequenced on an Illumina NextSeq 500 system with a single-end 76bp protocol. 592

Single-end sequencing reads from grafted samples were filtered into human and mouse reads 593

using Disambiguate (52) based on mapping to hg38 and mm10. The filtered reads were then 594




processed with a pipeline building upon and extending the implementation of the ENCODE 595

“Long RNA-seq” pipeline. Additional details on the methods are outlined in the 596

Supplementary Information. 597

598

Whole-exome sequencing 599

In-house DNA libraries were prepared using the Agilent SureSelectXT Human AllExon 50 600

Mb enrichment kit and subsequently sequenced on an Illumina HiSeq 2000 with a 100 bp 601

paired-end protocol. Sequencing data from in-house cell lines was completed with data 602

retrieved from CCLE and COSMIC. 603

604

Analysis of gene expression by QuantiGene single plex technology (Affymetrix) 605

RNA was isolated from tumors as described above. The following probes were used: DUSP6 606

(SA-11958) and GAPDH (SA-10001). The analysis was performed according to 607

manufacturer’s recommendations. The DUSP6 levels of the individual tumors were 608

normalized to their respective GAPDH levels. 609

610

Variant calling from whole-exome sequencing data (DNA-seq) 611

Paired-end sequencing reads were mapped against the human genome hg38 using bwa. We 612

used strelka2 and the Ensembl Variant Effect Predictor for variant calling and annotation. In 613

addition, data from COSMIC and CCLE was re-annotated and used to extend internal data. 614

Additional details on the methods are outlined in the Supplementary Information. Additional 615

details on the methods are outlined in the Supplementary Information. 616

617




Cell line derived efficacy studies and biomarker studies in mice 618

All animal studies were approved by the internal ethics committee and the local governmental 619

committee. Group size in efficacy studies have been selected after performing power analysis. 620

Female BomTac:NMRI-Foxn1nu

mice were used in all xenograft studies. For biomarker and 621

efficacy experiments with MIA PaCa-2 female mice were engrafted subcutaneously with 10 622

million cells suspended in Matrigel. In case of biomarker studies with MIA PaCa-2 tumors, 623

mice were randomized by tumor size in groups of 5 mice once tumors reached a size of 170-624

500 mm³. Mice were treated once at time point 0 hour and 6 hours. Tumors were explanted 625

and snap frozen to analyze biomarker modulation. Details of bioanalysis of mouse blood 626

samples can be found in the supplementary data. 627

628

In case of efficacy experiments, mice were randomized in groups (n=7 mice per treatment 629

group) by tumor size by the automated data storage system Sepia on day 7 (Fig. 3d) or 12 (Fig. 630

4.a) once tumors reached a size between 95-180 mm³. Compound treatment was initiated after 631

randomization based on body weight. Tumor size was measured by an electronic caliper and 632

body weight was monitored daily. The analysis follows largely the procedures described in 633

(53,54). Number of subcutaneous cells injected and size for tumor randomization was as 634

following with a group size of 7-10 mice: A549 (10 million cells; 62-150 mm³; n=7, Fig. 3f 635

and Supplemental data Fig. 3g and 3h), LoVo (10 million cells; 123-173 mm³; n=7, Fig. 3f 636

and 4f), SW620 (5 million cells, 80-125 mm³, Fig. 3f) and A375 (5 million cells, 64-149 mm³, 637

n=6-7, Fig. 3f). In case of the biomarker studies with A549 cells, mice were randomized once 638

tumors reached a size between 209 and 320 mm³ (Supplemental data Fig. 3b and c)). BI-3406 639

was dissolved in 0.5% Natrosol. Trametinib was dissolved in 0.5% DMSO and 0.5% Natrosol. 640

The control group was treated with 0.5% of Natrosol orally in the same frequency as in the 641




treatment groups (twice daily). All compounds were administered intragastrically by gavage 642

(10 mL/kg). Details on formulation of compounds can be found in supplementary data. 643

644

Patient derived xenograft studies 645

PDX model characterization and profiling is described in detail in the supplementary data and 646

Table S13. PDX tumor fragments (4x4x4 mm3) were implanted on the right hind flanks of 647

NSG female mice purchased from Jackson Laboratory and allowed to grow to an average 648

volume of 100-250 mm3 as monitored by calliper measurements. At enrolment, animals were 649

randomized and treated orally on a 5 days on/2 days off schedule for convenience, to avoid 650

weekend treatments, with Vehicle (0.5% Natrosol), bid (6 hours apart), BI-3406 (SOS1i) at 651

50 mg/kg, bid (6 hours apart), trametinib at 0.1 mg/kg, bid (6 hours apart), or the combination 652

thereof. Mice were 11 weeks old and treatment group sizes included at least 5-7 mice per 653

group. All animals received LabDiet 5053 chow ad libitum. trametinib was purchased from 654

ChemieTek. In the patient derived xenograft studies tumor growth was monitored two times a 655

week with calipers and the tumor volume (TV) was calculated as TV=(D X d2/2), where "D" 656

is the largest and "d" is the smallest superficial visible diameter of the tumor mass. All 657

measure are documented as mm3. Body weights were measured twice weekly and used to 658

adjust dosing volume and monitor animal health. RPPA analysis of explanted tumor material 659

is described in detail in the supplementary data section (Supplementary Figure S5i and Table 660

S8). 661

662

Statistical analysis 663

Statistical analyses and Bioinformatics analysis were performed with R version 3.5.0 & 664

Bioconductor 3.7 or GraphPad Prism. A Fisher’s exact test was used for computing the 665

associations of gene mutations with the sensitivity status of cell lines and for comparison of 666




tumor volumes from the control the group with one treatment group. For calculations of 667

tumor volume, absolute values were used for statistical analysis. Due to the observed 668

variation, nonparametric methods were applied. In case several treatment groups were 669

compared one-sided non-parametric Mann-Whitney-Wilcoxon U-tests were applied to 670

compare treatment groups with the control, as reduced tumor growth was expected following 671

treatment. The p values for the tumor volume (efficacy parameter) were adjusted for multiple 672

comparisons according to Bonferroni-Holm within each subtopic (comparisons versus control, 673

comparisons mono therapies versus combination therapy) whereas the p values of the body 674

weight (tolerability parameter) remained unadjusted in order not to overlook a possible 675

adverse effect. The level of significance was fixed at α = 5%. An (adjusted) p value of less 676

than 0.05 was considered to show a statistically significant difference between the groups and 677

differences were seen as indicative whenever 0.05 p-value < 0.10. Data are represented as 678

dot plots with bar graphs for mean ± standard deviation (s.d.) or standard error of the mean 679

(s.e.m.), as indicated. In case of the PDX experiments statistical significance was determined 680

using an unpaired t-test per row and the Holm-Sidak method to correct for multiple 681

comparisons (Fig. 4e and f and Supplementary Fig. S4d-f) 682

683

Data availability 684

Atomic coordinates and structure factors for the co-crystal x-ray structures of BI-68BS and 685

BI-3406 and SOS1 have been deposited at the Protein Data Band under accession number 686

6SFR (BI-68BS) and 6SCM (BI-3406). Data is available in Supplementary Table S1. 687

Expression data generated and analyzed in this study have been deposited in the Gene 688

Expression Omnibus (GEO) database under the accession numbers GSE128385. Processed 689

data is available in Supplementary Table S7. 690




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871




Figure Legends 872

Figure 1: Discovery of BI-3406, a potent and selective SOS1::KRAS interaction inhibitor 873

a, Co-crystal x-ray structure of BI-3406 bound to the catalytic pocket of SOS1 (ligand shown 874

in yellow, SOS1 as surface representation). The previously described catalytic RAS 875

interaction site (dark red; PDB: 1NVU) and the allosteric site (green) are highlighted. The 876

enlarged area depicts the key interactions of BI-3406 and SOS1 within the binding site. 877

Amino acids involved in the RAScat interaction are highlighted in dark red, indicating a clash 878

of BI-3406 with RAScat. Structure and potency of BI-3406 are shown at the bottom right. b, 879

Biochemical protein-protein interaction assays (AlphaScreen) between recombinant SOS1 or 880

SOS2 and recombinant KRAS G12C or KRAS G12D conducted under incubation with 881

increasing concentrations of BI-3406 (dose-response curves as relative fluorescence units 882

(RFUs) means±s.e.m., n=2). c-d, Biochemical protein-protein interaction assays 883

(AlphaScreen) between recombinant SOS1 and recombinant KRAS G12C (c) or KRAS 884

G12D (d) carried out under increasing concentrations of BI-3406 or the covalent KRAS 885

G12C inhibitor ARS-1620 (c, and d, n=2, means±s.e.m.). Dose response curves as in (b). e, 886

MIA PaCa-2 stably transduced with FLAG-tagged wild-type SOS1 or the indicated mutant 887

SOS1 transgenes were exposed to different concentrations of BI-3406 for 2 hours. Phospho-888

ERK levels were subsequently quantified in cell lysates by capillary immunodetection using 889

alpha-actinin as a loading control and normalized to the levels measured in DMSO solvent 890

treated samples (n=3 independent biological replicates). IC50 values (nM) are shown in the 891

legend. Inlay: Lane view of FLAG-SOS1 transgene expression in comparison to alpha-892

actinin in stably transduced MIA PaCa-2 cells from a representative capillary 893

immunodetection experiment. f, Cell proliferation assay using MIA PaCa-2 transgenic cell 894

pools expressing the indicated FLAG-SOS1 transgenes (data points are derived from two 895

independent biological replicates each containing three technical replicates). IC50 values (nM) 896

are shown in the legend. g, Dose-dependent, cellular effect of BI-3406 on RAS-GTP levels 897




(n=2, means±s.e.m.) in standard 2D / 10% serum conditions with increasing concentrations of 898

BI-3406 for 2 h. RAS-GTP levels were quantified relative to DMSO controls (RAS G-LISA). 899

900

Figure 2: Drug sensitivity profiling of cancer cell lines uncovers an association of KRAS 901

mutation status with sensitivity to SOS1 inhibition 902

a, Inhibition of pERK activity by BI-3406 after 1 hour in 2D assay conditions in a cancer cell 903

line panel quantified by Western blotting (n=2, means±s.d.). A375 (KRAS wt, BRAF V600E), 904

A549 (KRAS G12S), DLD-1 (KRAS G13D), NCI-H23 (KRAS G12C), NCI-H358 (KRAS 905

G12C), and NCI-H520 (KRAS wt and BRAF wt). b, Inhibition of cell proliferation by BI-906

3406 in a cancer cell line panel in 3D proliferation assays (n=3, means±s.d.). c, In vitro 907

sensitivity of a panel of cell lines to the positive control panobinostat (Sigma Aldrich) in a 3D 908

proliferation assay (n=3, means±s.d.). d) Effect of BI-3406 on pERK levels in a panel of 909

isogenic NCI-H23 cell lines. Values were normalized to total ERK protein (n=2, means±s.d.). 910

e) In vitro sensitivity of a panel of isogenic cell lines treated with BI-3406 in a 3D 911

proliferation assay (n=3, means±s.d.). f, In vitro sensitivity of 40 cancer cell lines treated with 912

BI-3406 in 3D proliferation assays. Panels depict the proliferation data (n=2), the respective 913

cancer type, and the mutation status of selected genes. Cell lines are grouped based on an IC50 914

cut-off of 100 nmol/L. The mutation status and zygosity is shown by a continuous color-915

coding scheme, blue boxes reflect wild-type status, while light blue boxes indicate an 916

unknown status. Only re-occurring hotspot mutations are reported for KRAS, NRAS, HRAS, 917

EGFR, and BRAF (Supplementary Table S5). NCI-H2347 carries a KRAS L19F mutation 918

(asterisks). 919




Figure 3: SOS1 inhibition suppresses tumor growth and KRAS/MAPK signaling in 920

xenograft models of KRAS-driven cancers 921

a, pERK levels analyzed by a multiplexed immunoassay in explanted MIA PaCa-2 tumors 922

treated with 50 mg/kg BI-3406 twice daily at the time point 0 h and 6 h. (n=5 animals per 923

group, means±s.e.m, two-tailed t-test). b, pERK levels in mouse skin (treatment as in a) 924

assessed by IHC staining (H-scores) (n=5 animals per group, means±s.d., two-tailed t-test). c, 925

Gene expression profiling of pharmacodynamic biomarkers in a MIA PaCa-2 in vivo 926

biomarker experiment (n=4-5 animals per group, medians of normalized gene expression). A 927

subset of nine genes shows time-dependent modulation after BI-3406 (50 mg/kg) treatment, 928

visualized as a color-coded expression heatmap. d, Anti-tumor effect of BI-3406 in the 929

MIA PaCa-2 xenograft model (n=7 animals per group, means±s.e.m., one-tailed t-test) e, 930

Median body weight change of mice bearing subcutaneous MIA PaCa-2 xenografts 931

administered as described in (d) (n=7 animals per group, medians). f, Responses of different 932

xenograft models after treatment with BI-3406 (50 mg/kg bid) or vehicle (control). Tumor 933

growth inhibition (TGI) was determined based on tumor size after 20-23 days of continuous 934

treatment (n=7-9 animals per group, means±s.d.). Genotypes of tested xenograft models: 935

SW620 colorectal (KRAS G12V, BRAF wt), LoVo colorectal (KRAS G13D, BRAF wt), 936

MIA PaCa-2 pancreas (KRAS G12C, BRAF wt), and A549 non-small cell lung cancer 937

(KRAS G12S, BRAF wt). Significant TGI was achieved in all tested KRAS mutant xenograft 938

models, with the exception of the KRAS wild-type model A375 (*≤0.05 ** < 0.01, *** < 939

0.001, one tailed t-test). 940




Figure 4: Combined SOS1 and MEK inhibition leads to regressions in KRAS-mutant 941

tumors 942

a, Tumor volumes of mice injected subcutaneously with MIA PaCa-2 cells. All mice were 943

treated bid (with a delta of 6 hours) with vehicle (control), trametinib (0.125 mg/kg), BI-3406 944

(50 mg/kg) for 22 days, or the combination of both agents for 29 days (n=7 animals per group, 945

means±s.e.m.) followed by an off-treatment period until day 57. b, Relative tumor volume of 946

MIA PaCa-2 are indicated as percent change from baseline at day 22. Values smaller than 947

zero percent indicate tumor regressions. c, Efficacy of the combination of BI-3406 and 948

trametinib in the LoVo xenograft model. Continuous treatment with trametinib or BI-3406 949

alone or in combination for 23 days, followed by an off-treatment period until day 34 (n=7 950

animals per group, means±s.e.m.). (*≤0.05 ** < 0.01, *** < 0.001; a and c, one tailed 951

Student's t-test comparing control with treatment groups). d, Relative tumor volume for the 952

LoVo model are indicated as percent change from baseline at day 22. e-f Tumor growth of 953

colorectal cancer (CRC) PDX xenografts in mice treated with vehicle, BI-3406 (50 mg/kg, 954

bid), trametinib (0.1 mg/kg, bid), or the combination for the models B8032 (e) and C1047 (f) 955

tumors (n=5-7 animals per group, means±s.e.m. For convenience in PDX models mice were 956

treated in a 5 days on / 2 days off schedule. Statistical significance was determined using an 957

unpaired t-test per row and the Holm-Sidak method to correct for multiple comparisons, see 958

as well Supplementary Fig. S4d and S4e). 959

960




Figure 5: Biomarker modulation upon combined SOS1 and MEK inhibition 961

a, Western blot analysis of pMEK1/2 in MIA PaCa-2 cells grown in vitro in 2D and treated 962

with BI-3406, trametinib, or the combination for the indicated time periods (n=3, 963

means±s.e.m.). b, Western blot analysis of pERK in MIA PaCa-2 cells grown in vitro in 2D 964

as in a; (a and b, one tailed t-test p=0.05; two-tailed t-test: p=0.1). c, Multiplexed 965

immunoassay measurements of pERK and total ERK in MIA PaCa-2 tumor xenografts at 4 h 966

post-treatment (n=4 animals per group, means±s.d.; two tailed t-test). d, Proposed model of 967

the effects of combined MEK and SOS1 inhibition. Inhibition of MEK results in the 968

attenuation of negative feedback control leading to increased SOS1 activity KRAS-GTP 969

loading driving reactivation of downstream signals. Based on these adaptive responses, 970

effects of MEK inhibitors on cell proliferation and survival are limited. Adaptive responses 971

can be abrogated through combined blockade of MEK and SOS1, which prevents MEK 972

inhibitor-induced KRAS-GTP loading and reduces signaling downstream of KRAS, resulting 973

in durable tumor regressions. 974

975




Acknowledgements 976

The authors thank Neal Rosen for critical review of the manuscript. The authors also thank 977

the following colleagues who supported this work in the following institutions: 978

Boehringer Ingelheim: Alexandra Beran, Doris Brantl, Silke Brandl , Fischerauer Bernhard, 979

Ida Dinold, Wolfgang Egermann, Wolfgang Hela, Astrid Jeschko, Thomas Karner, Matthias 980

Klemencic, Matthew Kennedy, Lyne Lamarre, Silvia Munico Martinez, Reiner Meyer, 981

Thomas Pecina, Vanessa Roessler, Christian Salamon Renate Schnitzer, Andreas Schrenk, 982

Heinz Stadtmueller, Harald Studensky, Sandra Strauss, Gabriela Siszler, Elisabeth Traxler, 983

Bernhard Wolkerstorfer, Jens Quant, Vittoria Zinzalla, Mark Petronczki, Tao You and 984

Nikolai Mischerikow. 985

MD Anderson Cancer Center: Christopher Bristow, Ningping Feng, Scott Kopetz, Mikhila 986

Mahendra, Robert Mullinax, Jianhua Zhang, Giulio Draetta and Andy Zuniga 987

Forma Therapeutics: David Richard and Adrian Saldanha 988

Some results shown here are in whole or part based upon data generated by the TCGA 989

Research Network: https://www.cancer.gov/tcga.” 990

991

Michael P. Sanderson is a former employee of Boehringer Ingelheim RCV GmbH & Co KG, 992

Vienna, Austria now working for Merck KGaA. Jurgen Moll is a former employee of 993

Boehringer Ingelheim RCV GmbH & Co KG now working for Sanofi. Rachel L. Mendes is a 994

former employee of Forma Therapeutics now working for PerkinElmer. Jonathan C. 995

O’Connell is a former employee of Forma Therapeutics now working for Integral Health. 996



https://www.cancer.gov/tcga


g)

d)

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BI-3406SPRSOS1 KD = 9.7 nMIC50 SOS1::KRAS = 5 nM

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20

40

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Proliferation assay with MIA PaCa-2 cellswith SOS1 transgene expression

Figure 1




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NCI-H17

92

NCI-H35

8

SW837

NCI-H23

SW620

NCI-H21

22A54

9GEO

MIA PaCa-2DLD

-1

NCI-H13

73

HCC-1438

NCI-H14

35ABC-1

NCI-H19

75

HCC-827PC-9

OUMS-23HT-29

COLO 20

5

SW1417A-37

5

LS41

1N

Hs 578

T

NCI-H82

MEL-JUSO

HEC-151

NCI-H23

47PA-1

NCI-H12

99

NCI-H16

50SW48

A-427

Calu-6

NCI-H46

0

NCI-H52

2

NCI-H29

2

NCI-H19

93

NCI-H21

70

NCI-H52

0

IC50

(nM

)

Reponse to BI-3406ResistantSensitive

Tumor typeBreast carcinomaColon carcinomaMelanomaNSCLCOvarian carcinomaPancreas carcinomaSCLCUterus carcinoma

Zygosity/WT

0.000.250.500.751.00

G12C G12V G12S G12A G12D G13D Q61L Q61R Q61K Q61H

*

10-12 10-10 10-8 10-6 10-40

20

40

60

80

100

120 A375A549

Panobinostat (log, M)

DLD1

NCI-H358NCI-H23

NCI-H520

d) e)

Per

cent

age

prol

ifera

ton

inhi

bitio

n(n

orm

aliz

ed)

10-10 10-9 10-8 10-7 10-6 10-5 10-40

20

40

60

80

100

120

140WT / WT

G13D / G13D

G12C / WT

G12D / WT

G12V / WT

G13D / WT

G12R / WT

G12D / G12D

Q61H / Q61H

BI-3406 (log, M)

Perc

enta

ge p

ERK

/ tot

al E

RK

(nor

mal

ized

)

Per

cent

age

prol

ifera

ton

inhi

bitio

n(n

orm

aliz

ed)

BI-3406 (log, M)10 -10 10 -8 10 -6 10 -40

20

40

60

80

100

120

140c)

G13D / G13D

G12C / WT

G12D / WT

G12V / WT

G13D / WT

G12R / WT

G12D / G12D

Q61H / Q61H

pERK modulation in NCI-H23 isogenic cells 3D-Proliferation assays with NCI-H23 isogenic cells

pERK modulation in tumor cells 3D-Proliferation assay in tumor cells

3D-Proliferation assay in tumor cells

Sensitivity of 40 cancer cell lines treated with BI-3406 in 3D proliferation assays

Figure 2




-20-15-10

-505

101520

0 5 10 15 20 25 30 35 40Day

Med

ian

wei

ght c

hang

e (%

)e)

BI-3406, 12 mg/kg, bidBI-3406, 50 mg/kg, bid

Vehicle Control

0100

200

300

400

500

600

700

0 5 10 15 20 25 30 35 40

BI-3406, 12 mg/kg, bidBI-3406, 50 mg/kg, bid

Vehicle Control

Day

d)

Rat

io p

ERK

/ tot

al E

RK

0

0.002

0.004

0.006

0.008

0.010

f)

a) b) c)

H-s

core

Vehicle

(4h)

BI-340

6 (4h

)

BI-340

6 (24

h)

BI-340

6 (10

h)

30

20

40

10

Vehicle

(4h)

BI-340

6 (4h

)

BI-340

6 (24

h)

BI-340

6 (10

h)

Tum

our v

olum

e (m

m3 )

+ SE

M

SW620 V

ehicle

SW620 B

I-340

6

LoVo V

ehicle

LoVo B

I-340

6

MIA PaCa-2

Vehicle

MIA PaCa-2

BI-340

6

A549 V

ehicle

A549 B

I-340

6

**

***

TGI 61

TGI 62

TGI 86

TGI 89

EGR1ETV1FOSL1SLC20A1DUSP6SPRY4ETV4ETV5PLAUR −1.5

−1

−0.5

0

0.5

1

1.5

Vehicle

(4h)

BI-340

6 (4h

)

Vehicle

(10h

)

Vehicle

(24h

)

Vehicle

(0h)

norm

aliz

ed g

ene

expr

essi

on

* TGI 66%

** TGI 87%

****

* *

pERK levels in MIA PaCa-2 tumors pERK levels in mouse skin MIA PaCa-2 tumors: Gene expression profile

MIA PaCa-2 xenograft Body weight change: MIA PaCa-2 xenograftKRAS mutant xenograft models

0

2

4

6

8

10

Sing

le re

lativ

e tu

mou

r vol

ume

***

***

BI-340

6 (24

h)

BI-340

6 (10

h)

Figure 3




Day

Vehicle Control

Combination

BI-3406, 50 mg/kg, bidTrametinib, 0.125 mg/kg, bid

0

200

400

600

800

1,000

1,200

0 10 20 30 40 50 60

Tum

our v

olum

e (m

m³)

+ SE

M

a)

0

200

400

600

800

0 10 20 30 40

Tum

our v

olum

e (m

m3 )

+ SE

M

-60

-40

-20

0

20

40

60

80

100

Rel

ativ

e tu

mou

r vol

ume

%

-60

-40

-20

0

20

40

60

80

100R

elat

ive

tum

our v

olum

e %

d)

b)

e)

c)

0 5 10 15 20 250

200400600800

1,0001,2001,4001,600

Day

0 10 20 300

100

200

300

400

500

600

700

Day

f)

off treatment

off treatment

Vehicle Control

Combination

BI-3406, 50 mg/kg, bidTrametinib, 0.125 mg/kg, bid

MIA PaCa-2 (PAC, KRAS G12C)

Tum

our v

olum

e (m

m3 )

+ SE

M

Tum

our v

olum

e (m

m3 )

+ SE

M

MIA PaCa-2 (PAC, KRAS G12C)

LoVo (CRC, KRAS 13D)LoVo (CRC, KRAS 13D)

PDX B8032 (CRC, KRAS G12C) PDX C1047 (CRC, KRAS G12C)

Day

Vehicle Control

Combination

BI-3406, 50 mg/kg, bid, 5 on / 2 offTrametinib, 0.1 mg/kg, bid, 5 on / 2off

Vehicle Control

Combination

BI-3406, 50 mg/kg, bid, 5 on / 2 offTrametinib, 0.1 mg/kg, bid, 5 on / 2 off

** TGI 73%*** TGI 86%*** TGI 107%

*** TGI 59%*** TGI 62%

*** TGI 102%

***

***

***

***

Figure 4




0.0000.0020.0040.0060.0080.010

Rat

iopE

RK

/ tot

alER

K

a) b)

c)

Contro

l (4h)

BI-340

6 (4h

, 50 m

g/kg)

Trameti

nib (4

h, 0.1

mg/k

g)

Combin

ation

(4h)

Dual MEK+SOS1 inhibition: Durable tumour regressions

Growth factors

Proliferation / Survival

RTK

MEKi (Trametinib)

MEK

ERKTranscription

Feedback mechanism

RAS GDPRAS GTP SOS1

GPDGTP SOS1i

(BI-3406)-P SOS2GAP(NF1)

d)

P

0

100

200

300

400

500

Contro

l

BI-3406

(1 µM

)

Trameti

nib (9

nM)

BI-3406

(1 µM

) +

Trameti

nib (9

nM)

Trameti

nib (2

5 nM)

BI-3406

(1 µM

) +

Trameti

nib (2

5 nM)

pMEK

1/2

(Ser

217/

221)

rela

tive

units

0%

20%

40%

60%

80%

100%

120%MIA PaCa-2 cells (2D)

pER

K (p

42; T

hr20

2/20

4)

rela

tive

to u

ntea

ted

cont

rols

MIA PaCa-2 cells (2D)

24h 48h 72h 24h 48h 72h

**

**

*

*

**

*

*

*

**

*

MIA PaCa-2 tumors

Contro

l

BI-3406

(1 µM

)

Trameti

nib (9

nM)

BI-3406

(1 µM

) +

Trameti

nib (9

nM)

Trameti

nib (2

5 nM)

BI-3406

(1 µM

) +

Trameti

nib (2

5 nM)

Figure 5




Published OnlineFirst August 19, 2020.Cancer Discov Marco H Hofmann, Michael Gmachl, Juergen Ramharter, et al. combined MEK inhibitioninhibitor, is effective in KRAS-driven cancers through BI-3406, a potent and selective SOS1::KRAS interaction

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BI-3406, a potent and selective SOS1::KRAS interaction ... · 8/19/2020 · 3406, that binds to the catalytic domain of . 57. SOS1 thereby preventi. ng. the interaction with. KRAS