BI-3406, a Potent and Selective SOS1::KRAS Interaction

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

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

2 inhibitor, is effective in KRAS-driven cancers through

3 combined MEK inhibition

4 Marco H. Hofmann1*, Michael Gmachl1*, Juergen Ramharter1*, Fabio Savarese1, Daniel 5 Gerlach1, Joseph R. Marszalek3, Michael P. Sanderson1, Dirk Kessler1, Francesca Trapani1, 6 Heribert Arnhof1, Klaus Rumpel1, Dana-Adriana Botesteanu1, Peter Ettmayer1, Thomas 7 Gerstberger1, Christiane Kofink1, Tobias Wunberg1, Andreas Zoephel1, Szu-Chin Fu4, Jessica 8 L. Teh3, Jark Böttcher1, Nikolai Pototschnig1, Franziska Schachinger1, Katharina Schipany1, 9 Simone Lieb1, Christopher P. Vellano3, Jonathan C. O’Connell2, Rachel L. Mendes2, Jurgen 10 Moll1, Mark Petronczki1, Timothy P. Heffernan3, Mark Pearson1, Darryl B. McConnell1, 11 Norbert Kraut1

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

13 2 Forma Therapeutics, Watertown, MA, USA

14 3 TRACTION Platform, Therapeutics Discovery Division, The University of Texas MD 15 Anderson Cancer Center, Houston, TX 77030, USA

16 4 Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, 17 Houston, TX 77030, USA

18 19 These authors contributed equally: Marco H. Hofmann, Michael Gmachl, and Juergen

20 Ramharter

21 Running title: Pan-KRAS SOS1 protein-protein interaction inhibitor BI-3406 22 Additional information 23 Corresponding Authors: 24 Marco H. Hofmann, Email: [email protected] and 25 Norbert Kraut, Email: [email protected] 26 Boehringer Ingelheim RCV GmbH & Co KG, 27 Dr. Boehringer-Gasse 5-11, A-1121 Vienna, 28 Austria, Tel. +431 80105-2790 29

Downloaded from cancerdiscovery.aacrjournals.org on September 25, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/2159-8290.CD-20-0142 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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

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

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

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

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

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

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

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

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

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

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

41 Cancer Center.

42 Additional Information:

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

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

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

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

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

48 Inhibitors.

49 Abstract

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

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

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

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

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

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

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

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

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

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

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

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

62 KRAS-driven tumors.

63 Significance

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

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

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

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

68 Introduction

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

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

71 signaling occurs through engagement with effector proteins that orchestrate intracellular

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

103 G12C mutant tumors (20).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

123 type cells.

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

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

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

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

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

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

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

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

132

133 Results

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

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

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

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

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

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

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

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

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

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

144 Supplementary Table S1) with the quinazoline ring pi-stacking to His905SOS1. Based on the

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

146 likely interferes with the competing Tyr884SOS1-Arg73RAS interaction and consequently

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

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

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

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

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

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

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

154 thereby significantly increasing potency. The tetrahydrofuryl substituent favorably balanced

155 solubility and metabolic stability and improved interaction with Tyr884SOS1. Synthesis of BI-

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

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

158 S1.

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

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

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

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

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

164 oncoproteins (Fig.1b).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

204

205 Association of KRAS mutation status with sensitivity to SOS1 inhibition

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

226 lines.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

243

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

268

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

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

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

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

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

274 3406 (Supplementary Fig. S2d and S2e).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

289 proliferation assays.

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

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

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

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

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

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

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

297

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

318 compensatory upregulation.

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

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

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

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

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

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

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

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

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

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

329 mutated xenograft models.

330

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

355 combination significantly improved antitumor activity as compared to both monotherapies

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

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

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

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

360 Fig. S4l-m).

361

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

389 S5k).

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

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

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

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

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

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

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

397 S5l).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

434 all KRAS-driven cancers.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

456 EGFR inhibition (43).

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

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

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

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

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

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

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

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

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

466 SOS1 inhibitor will exhibit induction of SOS2 levels.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

494 trametinib.

495 Methods

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

497

498 Cell culture

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

518 supplementary data and tables S10, S11 and S12.

519

520 Synthesis of BI-3406

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

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

523

524 Protein-protein interaction assays (AlphaScreen assay technology)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

548

549 Measurement of KRAS-GTP levels

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

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

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

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

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

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

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

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

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

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

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

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

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

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

564

565 Biomarker and PK/PD analysis

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

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

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

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

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

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

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

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

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

575 (Supplementary Figure S2e).

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

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

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

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

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

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

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

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

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

585

586 RNA isolation and sequencing library preparation for expression profiling

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

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

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

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

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

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

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

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

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

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

597 Supplementary Information.

598

599 Whole-exome sequencing

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

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

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

603 retrieved from CCLE and COSMIC.

604

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

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

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

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

609 normalized to their respective GAPDH levels.

610

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

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

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

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

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

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

617

618 Cell line derived efficacy studies and biomarker studies in mice

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

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

621 Female BomTac:NMRI-Foxn1nu mice were used in all xenograft studies. For biomarker and

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

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

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

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

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

627 samples can be found in the supplementary data.

628

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

644

645 Patient derived xenograft studies

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

661 S8).

662

663 Statistical analysis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

683

684 Data availability

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

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

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

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

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

690 data is available in Supplementary Table S7.

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872 Figure Legends

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

900

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

902 mutation status with sensitivity to SOS1 inhibition

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

919 (asterisks).

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

921 xenograft models of KRAS-driven cancers

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

940 0.001, one tailed t-test).

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

942 tumors

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

959 as well Supplementary Fig. S4d and S4e).

960

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

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

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

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

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

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

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

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

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

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

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

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

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

974 in durable tumor regressions.

975

976 Acknowledgements

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

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

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

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

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

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

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

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

985 Nikolai Mischerikow.

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

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

988 Forma Therapeutics: David Richard and Adrian Saldanha

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

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

991

992 Michael P. Sanderson is a former employee of Boehringer Ingelheim RCV GmbH & Co KG, 993 Vienna, Austria now working for Merck KGaA. Jurgen Moll is a former employee of 994 Boehringer Ingelheim RCV GmbH & Co KG now working for Sanofi. Rachel L. Mendes is a 995 former employee of Forma Therapeutics now working for PerkinElmer. Jonathan C. 996 O’Connell is a former employee of Forma Therapeutics now working for Integral Health.

Figure 1 a) b) Biochemical protein-protein interaction assays 120

100

60 G12C::SOS2 40 G12D::SOS2 20 G12C::SOS1

Normalized RLU (% DMSO) G12D::SOS1 0 10 -12 10 -10 10 -8 10 -6 10 -4 BI-3406 (log, M)

c) Biochemical protein-protein interaction assays 120

F3C NH2 100

O 80

NH 60 O N 40 G12C::SOS1

N O 20 ARS-1620 Normalized RLU (% DMSO) BI-3406 BI-3406 SPR K = 9.7 nM 0 SOS1 D -10 -8 -6 -4 IC50 SOS1::KRAS = 5 nM 10 10 10 10 BI-3406 (log, M) d) Biochemical protein-protein interaction assays e) pERK modulation in MIA PaCa-2 cells 125 with SOS1 transgene expression

120 100 SOS1 wild-type 100 2.820e-008

DM SO ) 75 80 SOS1 H905V (% >1.000e-005 60 50 SOS1 H905I SOS1 wild-type Empty vector SOS1 H905V 40 G12D::SOS1 SOS1 H905I >1.000e-005 25 FLAG-SOS1 20 ARS-1620 pERK / a-Actinin ratio (% of DMSO) Normalized RLU BI-3406 a-Actinin 0 0 -10 -8 -6 -4 10 10 10 10 10-8 10-7 10-6 10-5 BI-3406 (log, M) BI-3406 (log,M)

Proliferation assay with MIA PaCa-2 cells f) 120 with SOS1 transgene expression g) GLISA assays

100 100

80 80 ed ) 60 SOS1 wild-type 60 3.192e-008 (normali z 40 SOS1 H905V 40 >1.000e-005 20 20 SOS1 H905I Normalized RLU (% DMSO)

Percentage proliferation inhibition NCI-H358 >1.000e-005 A549 0 0 10 -10 10 -8 10 -6 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 BI-3406 (log, M) BI-3406 (log, M)

Figure 2 a) pERK modulation in tumor cells b) 3D-Proliferation assay in tumor cells 120 A375 120 A375

100 A549 100 A549

80 DLD1 80 DLD1

60 NCI-H23 60 NCI-H23

(normalized) NCI-H358 40 NCI-H358 (normalized) 40 NCI-H520 20 NCI-H520 20 Percentage pERK / total ERK

0 Percentage proliferation inhibition 0 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -10 10 -8 10 -6 10 -4 BI-3406 (log, M) BI-3406 (log, M)

c) 3D-Proliferation assay in tumor cells d) pERK modulation in NCI-H23 isogenic cells e) 3D-Proliferation assays with NCI-H23 isogenic cells 140 120 A375 140 WT / WT A549 120 120 100 G12C / WT G12C / WT DLD1 100 G13D / WT 100 G13D / WT 80 NCI-H23 NCI-H358 80 G12D / WT 80 G12D / WT 60 NCI-H520 G12V / WT G12V / WT 60 60 G12R / WT G12R / WT (normalized) (normalized) 40 (normalized) 40 G13D / G13D 40 G13D / G13D

20 20 G12D / G12D 20 G12D / G12D Percentage pERK / total ERK Q61H / Q61H Q61H / Q61H Percentage proliferation inhibition 0 0 Percentage proliferation inhibition 0 -12 -10 -8 -6 -4 -10 -8 -6 -4 10 10 10 10 10 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10 10 10 10 Panobinostat (log, M) BI-3406 (log, M) BI-3406 (log, M)

f) Sensitivity of 40 cancer cell lines treated with BI-3406 in 3D proliferation assays 10,000 Response to BI-3406 5,000 Resistant 1,000 Sensitive 500

100 Tumor type

IC50 (nM) 50 Breast carcinoma 10 Colon carcinoma 5 Melanoma NSCLC Ovarian carcinoma Pancreas carcinoma G12C G12V G12S G12A G12D G13D Q61L Q61R Q61K Q61H SCLC Uterus carcinoma KRAS * NRAS Zygosity/WT HRAS 1.00 EGFR 0.75 NF1 0.50 BRAF 0.25

A549GEO PC-9 PA-1 0.00 DLD-1 ABC-1 HT-29 A-375 SW48A-427Calu-6 SW837 SW620 LS411N NCI-H23 HCC-827 SW1417 Hs 578TNCI-H82HEC-151 NCI-H358 HCC-1438 OUMS-23COLO 205 NCI-H460NCI-H522NCI-H292 NCI-H520 NCI-H1792 NCI-H2122 MIA PaCa-2NCI-H1373NCI-H1435NCI-H1975 MEL-JUSONCI-H2347NCI-H1299NCI-H1650 NCI-H1993NCI-H2170

Figure 3 a) pERK levels in MIA PaCa-2 tumors b) pERK levels in mouse skin c) MIA PaCa-2 tumors: Gene expression profile 0.010 40 EGR1 1.5 * * ETV1 1 ** 0.008 ** FOSL1 30 0.5 SLC20A1 0.006 0 20 DUSP6 0.004 H-score SPRY4 −0.5 ETV4 10 −1 0.002 ETV5 Ratio pERK / total ERK

normalized gene expression −1.5 0 PLAUR

VehicleVehicle (0h) (4h) Vehicle (4h) BI-3406 (4h) Vehicle (4h) BI-3406 (4h) VehicleVehicle (10h)BI-3406 (24h)BI-3406 (4h)BI-3406 (10h) (24h) BI-3406 (10h)BI-3406 (24h) BI-3406 (10h)BI-3406 (24h) KRAS mutant xenograft models d) MIA PaCa-2 xenograft e) Body weight change: MIA PaCa-2 xenograft f) TGI 61 700 Vehicle Control 20 Vehicle Control ** TGI 86 10 *** BI-3406, 12 mg/kg, bid 15 BI-3406, 12 mg/kg, bid 600 TGI 89

) + SEM BI-3406, 50 mg/kg, bid BI-3406, 50 mg/kg, bid 8 3 10 TGI 62 500 *** 5 *** 400 6 0 300 * TGI 66% -5 4 200 ** TGI 87% -10 2 100 -15 Median weight change (%) Tumour volume (mm Tumour 0 -20 Single relative tumour volume 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Day Day

LoVo VehicleLoVo BI-3406 A549 VehicleA549 BI-3406 SW620 VehicleSW620 BI-3406

MIA PaCa-2MIA PaCa-2 Vehicle BI-3406

Figure 4 a) MIA PaCa-2 (PAC, KRAS G12C) b) MIA PaCa-2 (PAC, KRAS G12C) 1,200 Vehicle Control 100 BI-3406, 50 mg/kg, bid 80 1,000 Trametinib, 0.125 mg/kg, bid Combination 60 800 40 600 20 off treatment 400 ** TGI 73% 0 *** TGI 86% -20 200 *** TGI 107% -40 Tumour volume(mm³) + SEM 0 Relativetumour volume % 0 10 20 30 40 50 60 -60 Day

c) LoVo (CRC, KRAS 13D) d) LoVo (CRC, KRAS 13D) Vehicle Control 100 800 BI-3406, 50 mg/kg, bid Trametinib, 0.125 mg/kg, bid 80 Combination 60 ) + SEM

3 600 off treatment 40 *** TGI 59% 20 400 *** TGI 62% 0 -20 200 -40 *** TGI 102% Relativetumour volume % Tumour volume(mm -60 0 0 10 20 30 40 Day

e) PDX B8032 (CRC, KRAS G12C) f) PDX C1047 (CRC, KRAS G12C) 700 Vehicle Control 1,600 Vehicle Control BI-3406, 50 mg/kg, bid, 5 on / 2 off BI-3406, 50 mg/kg, bid, 5 on / 2 off 600 1,400 Trametinib, 0.1 mg/kg, bid, 5 on / 2 off Trametinib, 0.1 mg/kg, bid, 5 on / 2off ) + SEM ) + SEM Combination 3 1,200 Combination 3 500 1,000 400 *** 800 *** 300 600 *** *** 200 400 200 100 Tumour volume(mm 0 Tumour volume(mm 0 0 5 10 15 20 25 0 10 20 30 Day Day

Figure 5 a) MIA PaCa-2 cells (2D) b) * MIA PaCa-2 cells (2D) 500 120% * 24h 48h 72h * 24h 48h 72h 400 * 100% * * * 80% 300 * * 60% * * 200

relative units * 40% 100 20% pMEK1/2 (Ser217/221) pERK (p42; Thr202/204) 0 relative to unteated controls 0%

Control Control

BI-3406 (1 µM) BI-3406 (1 µM) TrametinibBI-3406Trametinib (9 nM) (1 µM) (9 + nM)BI-3406 (1 µM) + TrametinibBI-3406Trametinib (9 nM) (1 µM) (9 + nM)BI-3406 (1 µM) + TrametinibTrametinib (25 nM) (25 nM) TrametinibTrametinib (25 nM) (25 nM)

MIA PaCa-2 tumor Growth factors c) 0.010 * d) 0.008 * RTK 0.006 GTP SOS1i

Ratio 0.004 -P SOS2 (BI-3406) GAP RAS GDP GDP 0.002 (NF1) RAS GTP SOS1 P pERK / total/ pERK ERK 0.000 MEKi MEK (Trametinib) ERK Feedback mechanism Transcription Control (4h) Dual MEK+SOS1 inhibition: Combination (4h) Proliferation / Survival Durable tumour regressions BI-3406 (4h, 50 mg/kg) Trametinib (4h, 0.1 mg/kg)

BI-3406, a potent and selective SOS1::KRAS interaction inhibitor, is effective in KRAS-driven cancers through combined MEK inhibition

Marco H Hofmann, Michael Gmachl, Juergen Ramharter, et al.

Cancer Discov Published OnlineFirst August 19, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-20-0142

Supplementary Access the most recent supplemental material at: Material http://cancerdiscovery.aacrjournals.org/content/suppl/2020/08/20/2159-8290.CD-20-0142.DC1 http://cancerdiscovery.aacrjournals.org/content/suppl/2020/08/20/2159-8290.CD-20-0142.DC2

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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