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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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(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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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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References 691
692
1. Moll HP, Pranz K, Musteanu M, Grabner B, Hruschka N, Mohrherr J, et al. Afatinib 693
restrains K-RAS-driven lung tumorigenesis. Science translational medicine 694
2018;10(446) doi 10.1126/scitranslmed.aao2301. 695
2. Cichowski K, Jacks T. NF1 tumor suppressor gene function: narrowing the GAP. Cell 696
2001;104(4):593-604 doi 10.1016/s0092-8674(01)00245-8. 697
3. Bollag G, Clapp DW, Shih S, Adler F, Zhang YY, Thompson P, et al. Loss of NF1 698
results in activation of the Ras signaling pathway and leads to aberrant growth in 699
haematopoietic cells. Nature genetics 1996;12(2):144-8 doi 10.1038/ng0296-144. 700
4. Cox AD, Fesik SW, Kimmelman AC, Luo J, Der CJ. Drugging the undruggable RAS: 701
Mission possible? Nature reviews Drug discovery 2014;13(11):828-51 doi 702
10.1038/nrd4389. 703
5. Stephen AG, Esposito D, Bagni RK, McCormick F. Dragging ras back in the ring. 704
Cancer cell 2014;25(3):272-81 doi 10.1016/j.ccr.2014.02.017. 705
6. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio 706
Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer 707
Genomics Data. Cancer discovery 2012;2(5):401-4 doi 10.1158/2159-8290.Cd-12-708
0095. 709
7. AACR Project GENIE: Powering Precision Medicine through an International 710
Consortium. Cancer discovery 2017;7(8):818-31 doi 10.1158/2159-8290.Cd-17-0151. 711
8. Chin L, Tam A, Pomerantz J, Wong M, Holash J, Bardeesy N, et al. Essential role for 712
oncogenic Ras in tumour maintenance. Nature 1999;400(6743):468-72 doi 713
10.1038/22788. 714
9. Fisher GH, Wellen SL, Klimstra D, Lenczowski JM, Tichelaar JW, Lizak MJ, et al. 715
Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras 716
transgene in the presence and absence of tumor suppressor genes. Genes & 717
development 2001;15(24):3249-62 doi 10.1101/gad.947701. 718
10. Ying H, Kimmelman AC, Lyssiotis CA, Hua S, Chu GC, Fletcher-Sananikone E, et al. 719
Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose 720
metabolism. Cell 2012;149(3):656-70 doi 10.1016/j.cell.2012.01.058. 721
11. Collins MA, Bednar F, Zhang Y, Brisset JC, Galban S, Galban CJ, et al. Oncogenic 722
Kras is required for both the initiation and maintenance of pancreatic cancer in mice. 723
The Journal of clinical investigation 2012;122(2):639-53 doi 10.1172/jci59227. 724
12. Hallin J, Engstrom LD, Hargis L, Calinisan A, Aranda R, Briere DM, et al. The 725
KRAS(G12C) Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility 726
of KRAS-Mutant Cancers in Mouse Models and Patients. Cancer discovery 727
2020;10(1):54-71 doi 10.1158/2159-8290.CD-19-1167. 728
13. Canon J, Rex K, Saiki AY, Mohr C, Cooke K, Bagal D, et al. The clinical 729
KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 730
2019;575(7781):217-23 doi 10.1038/s41586-019-1694-1. 731
14. Mullard A. Cracking KRAS. Nature reviews Drug discovery 2019;18(12):887-91 doi 732
10.1038/d41573-019-00195-5. 733
15. Fakih M, O'Neil B, Price TJ, Falchook GS, Desai J, Kuo J, et al. Phase 1 study 734
evaluating the safety, tolerability, pharmacokinetics (PK), and efficacy of AMG 510, a 735
novel small molecule KRASG12C inhibitor, in advanced solid tumors. Journal of 736
Clinical Oncology 2019;37(15_suppl):3003- doi 10.1200/JCO.2019.37.15_suppl.3003. 737
16. Kessler D, Gmachl M, Mantoulidis A, Martin LJ, Zoephel A, Mayer M, et al. 738
Drugging an undruggable pocket on KRAS. Proceedings of the National Academy of 739
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
Sciences of the United States of America 2019;116(32):15823-9 doi 740
10.1073/pnas.1904529116. 741
17. Lake D, Correa SA, Muller J. Negative feedback regulation of the ERK1/2 MAPK 742
pathway. Cellular and molecular life sciences : CMLS 2016;73(23):4397-413 doi 743
10.1007/s00018-016-2297-8. 744
18. Chen YN, LaMarche MJ, Chan HM, Fekkes P, Garcia-Fortanet J, Acker MG, et al. 745
Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine 746
kinases. Nature 2016;535(7610):148-52 doi 10.1038/nature18621. 747
19. Nichols RJ, Haderk F, Stahlhut C, Schulze CJ, Hemmati G, Wildes D, et al. RAS 748
nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, 749
NF1- and RAS-driven cancers. Nature cell biology 2018;20(9):1064-73 doi 750
10.1038/s41556-018-0169-1. 751
20. Ou SI, Koczywas M, Ulahannan S, Janne P, Pacheco J, Burris H, et al. A12 The SHP2 752
Inhibitor RMC-4630 in Patients with KRAS-Mutant Non-Small Cell Lung Cancer: 753
Preliminary Evaluation of a First-in-Man Phase 1 Clinical Trial. Journal of Thoracic 754
Oncology 2020;15(2):S15-S6 doi 10.1016/j.jtho.2019.12.041. 755
21. Mullard A. Phosphatases start shedding their stigma of undruggability. Nature reviews 756
Drug discovery 2018;17(12):847-9 doi 10.1038/nrd.2018.201. 757
22. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD 098059 is a specific 758
inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in 759
vivo. The Journal of biological chemistry 1995;270(46):27489-94 doi 760
10.1074/jbc.270.46.27489. 761
23. Freedman TS, Sondermann H, Friedland GD, Kortemme T, Bar-Sagi D, Marqusee S, 762
et al. A Ras-induced conformational switch in the Ras activator Son of sevenless. 763
Proceedings of the National Academy of Sciences of the United States of America 764
2006;103(45):16692-7 doi 10.1073/pnas.0608127103. 765
24. Jeng HH, Taylor LJ, Bar-Sagi D. Sos-mediated cross-activation of wild-type Ras by 766
oncogenic Ras is essential for tumorigenesis. Nature communications 2012;3:1168 doi 767
10.1038/ncomms2173. 768
25. Rozakis-Adcock M, van der Geer P, Mbamalu G, Pawson T. MAP kinase 769
phosphorylation of mSos1 promotes dissociation of mSos1-Shc and mSos1-EGF 770
receptor complexes. Oncogene 1995;11(7):1417-26. 771
26. Corbalan-Garcia S, Yang SS, Degenhardt KR, Bar-Sagi D. Identification of the 772
mitogen-activated protein kinase phosphorylation sites on human Sos1 that regulate 773
interaction with Grb2. Molecular and cellular biology 1996;16(10):5674-82 doi 774
10.1128/mcb.16.10.5674. 775
27. Burns MC, Sun Q, Daniels RN, Camper D, Kennedy JP, Phan J, et al. Approach for 776
targeting Ras with small molecules that activate SOS-mediated nucleotide exchange. 777
Proceedings of the National Academy of Sciences of the United States of America 778
2014;111(9):3401-6 doi 10.1073/pnas.1315798111. 779
28. Hillig RC, Sautier B, Schroeder J, Moosmayer D, Hilpmann A, Stegmann CM, et al. 780
Discovery of potent SOS1 inhibitors that block RAS activation via disruption of the 781
RAS-SOS1 interaction. Proceedings of the National Academy of Sciences of the 782
United States of America 2019;116(7):2551-60 doi 10.1073/pnas.1812963116. 783
29. Singh A, Greninger P, Rhodes D, Koopman L, Violette S, Bardeesy N, et al. A gene 784
expression signature associated with "K-Ras addiction" reveals regulators of EMT and 785
tumor cell survival. Cancer cell 2009;15(6):489-500 doi 10.1016/j.ccr.2009.03.022. 786
30. Zhou ZW, Ambrogio C, Bera AK, Li Q, Li XX, Li L, et al. KRASQ61H preferentially 787
signals through MAPK in a RAF dimer-dependent manner in non-small cell lung 788
cancer. Cancer research 2020 doi 10.1158/0008-5472.CAN-20-0448. 789
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
31. Hobbs GA, Baker NM, Miermont AM, Thurman RD, Pierobon M, Tran TH, et al. 790
Atypical KRAS(G12R) Mutant Is Impaired in PI3K Signaling and Macropinocytosis 791
in Pancreatic Cancer. Cancer discovery 2020;10(1):104-23 doi 10.1158/2159-792
8290.CD-19-1006. 793
32. Krauthammer M, Kong Y, Bacchiocchi A, Evans P, Pornputtapong N, Wu C, et al. 794
Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in 795
sun-exposed melanomas. Nature genetics 2015;47(9):996-1002 doi 10.1038/ng.3361. 796
33. Nissan MH, Pratilas CA, Jones AM, Ramirez R, Won H, Liu C, et al. Loss of NF1 in 797
cutaneous melanoma is associated with RAS activation and MEK dependence. Cancer 798
research 2014;74(8):2340-50 doi 10.1158/0008-5472.Can-13-2625. 799
34. Loboda A, Nebozhyn M, Klinghoffer R, Frazier J, Chastain M, Arthur W, et al. A 800
gene expression signature of RAS pathway dependence predicts response to PI3K and 801
RAS pathway inhibitors and expands the population of RAS pathway activated tumors. 802
BMC medical genomics 2010;3:26 doi 10.1186/1755-8794-3-26. 803
35. Wagle MC, Kirouac D, Klijn C, Liu B, Mahajan S, Junttila M, et al. A transcriptional 804
MAPK Pathway Activity Score (MPAS) is a clinically relevant biomarker in multiple 805
cancer types. NPJ precision oncology 2018;2(1):7 doi 10.1038/s41698-018-0051-4. 806
36. Gilmartin AG, Bleam MR, Groy A, Moss KG, Minthorn EA, Kulkarni SG, et al. 807
GSK1120212 (JTP-74057) is an inhibitor of MEK activity and activation with 808
favorable pharmacokinetic properties for sustained in vivo pathway inhibition. 809
Clinical cancer research : an official journal of the American Association for Cancer 810
Research 2011;17(5):989-1000 doi 10.1158/1078-0432.Ccr-10-2200. 811
37. Rogge RD, Karlovich CA, Banerjee U. Genetic dissection of a neurodevelopmental 812
pathway: Son of sevenless functions downstream of the sevenless and EGF receptor 813
tyrosine kinases. Cell 1991;64(1):39-48 doi 10.1016/0092-8674(91)90207-f. 814
38. Bonfini L, Karlovich CA, Dasgupta C, Banerjee U. The Son of sevenless gene product: 815
a putative activator of Ras. Science (New York, NY) 1992;255(5044):603-6 doi 816
10.1126/science.1736363. 817
39. Chardin P, Camonis JH, Gale NW, van Aelst L, Schlessinger J, Wigler MH, et al. 818
Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. 819
Science (New York, NY) 1993;260(5112):1338-43 doi 10.1126/science.8493579. 820
40. Egan SE, Giddings BW, Brooks MW, Buday L, Sizeland AM, Weinberg RA. 821
Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase 822
signal transduction and transformation. Nature 1993;363(6424):45-51 doi 823
10.1038/363045a0. 824
41. Hunter JC, Manandhar A, Carrasco MA, Gurbani D, Gondi S, Westover KD. 825
Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations. 826
Molecular cancer research : MCR 2015;13(9):1325-35 doi 10.1158/1541-7786.Mcr-827
15-0203. 828
42. Bivona TG. Dampening oncogenic RAS signaling. Science (New York, NY) 829
2019;363(6433):1280-1 doi 10.1126/science.aav6703. 830
43. Theard PLS, E.; Sealover, N.E.; Linke, A.J.; Pratico, D.J.; and Kortum, R.L. Marked 831
Synergy by Vertical Inhibition of EGFR signaling in NSCLC: SOS1 as a therapeutic 832
target in EGFR-mutated cancer. . eLife 2020;accepted. 833
44. Baltanas FC, Perez-Andres M, Ginel-Picardo A, Diaz D, Jimeno D, Liceras-Boillos P, 834
et al. Functional redundancy of Sos1 and Sos2 for lymphopoiesis and organismal 835
homeostasis and survival. Molecular and cellular biology 2013;33(22):4562-78 doi 836
10.1128/MCB.01026-13. 837
45. Esteban LM, Fernandez-Medarde A, Lopez E, Yienger K, Guerrero C, Ward JM, et al. 838
Ras-guanine nucleotide exchange factor sos2 is dispensable for mouse growth and 839
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
development. Molecular and cellular biology 2000;20(17):6410-3 doi 840
10.1128/mcb.20.17.6410-6413.2000. 841
46. Ruess DA, Heynen GJ, Ciecielski KJ, Ai J, Berninger A, Kabacaoglu D, et al. Mutant 842
KRAS-driven cancers depend on PTPN11/SHP2 phosphatase. Nature medicine 843
2018;24(7):954-60 doi 10.1038/s41591-018-0024-8. 844
47. Mainardi S, Mulero-Sanchez A, Prahallad A, Germano G, Bosma A, Krimpenfort P, et 845
al. SHP2 is required for growth of KRAS-mutant non-small-cell lung cancer in vivo. 846
Nature medicine 2018;24(7):961-7 doi 10.1038/s41591-018-0023-9. 847
48. Wong GS, Zhou J, Liu JB, Wu Z, Xu X, Li T, et al. Targeting wild-type KRAS-848
amplified gastroesophageal cancer through combined MEK and SHP2 inhibition. 849
Nature medicine 2018;24(7):968-77 doi 10.1038/s41591-018-0022-x. 850
49. Fedele C, Ran H, Diskin B, Wei W, Jen J, Geer MJ, et al. SHP2 Inhibition Prevents 851
Adaptive Resistance to MEK Inhibitors in Multiple Cancer Models. Cancer discovery 852
2018;8(10):1237-49 doi 10.1158/2159-8290.Cd-18-0444. 853
50. Torres-Ayuso P, Brognard J. Shipping Out MEK Inhibitor Resistance with SHP2 854
Inhibitors. Cancer discovery 2018;8(10):1210-2 doi 10.1158/2159-8290.Cd-18-0915. 855
51. Mai TT, Lito P. A treatment strategy for KRAS-driven tumors. Nature medicine 856
2018;24(7):902-4 doi 10.1038/s41591-018-0111-x. 857
52. Ahdesmaki MJ, Gray SR, Johnson JH, Lai Z. Disambiguate: An open-source 858
application for disambiguating two species in next generation sequencing data from 859
grafted samples. F1000Research 2016;5:2741 doi 10.12688/f1000research.10082.2. 860
53. Waizenegger IC, Baum A, Steurer S, Stadtmuller H, Bader G, Schaaf O, et al. A 861
Novel RAF Kinase Inhibitor with DFG-Out-Binding Mode: High Efficacy in BRAF-862
Mutant Tumor Xenograft Models in the Absence of Normal Tissue Hyperproliferation. 863
Molecular cancer therapeutics 2016;15(3):354-65 doi 10.1158/1535-7163.Mct-15-864
0617. 865
54. Sanderson MP, Hofmann MH, Garin-Chesa P, Schweifer N, Wernitznig A, Fischer S, 866
et al. The IGF1R/INSR Inhibitor BI 885578 Selectively Inhibits Growth of IGF2-867
Overexpressing Colorectal Cancer Tumors and Potentiates the Efficacy of Anti-VEGF 868
Therapy. Molecular cancer therapeutics 2017;16(10):2223-33 doi 10.1158/1535-869
7163.Mct-17-0336. 870
871
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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
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(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
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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
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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
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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
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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
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g)
d)
b)
c)
BI-3406SPRSOS1 KD = 9.7 nMIC50 SOS1::KRAS = 5 nM
a)
10 -12 10 -10 10 -8 10 -6 10 -40
20
40
60
80
100
120
Nor
mal
ized
RLU
(%D
MSO
)
G12C::SOS2G12D::SOS2G12C::SOS1G12D::SOS1
BI-3406 (log, M)
10 -9 10 -8 10 -7 10 -6 10 -5 10 -40
20
40
60
80
100
A549NCI-H358
BI-3406 (log, M)
10-10 10-8 10-6 10-40
20
40
60
80
100
120
ARS-1620BI-3406N
orm
aliz
ed R
LU (%
DM
SO)
10-10 10-8 10-6 10-40
20
40
60
80
100
120
ARS-1620BI-3406N
orm
aliz
edR
LU(%
DM
SO)
Nor
mal
ized
RLU
(%D
MSO
)
BI-3406 (log, M)
BI-3406 (log, M)
G12D::SOS1
G12C::SOS1N
N O
O
NH
O
F3C NH2
0
25
50
75
100
125
BI-3406 (log,M)
pER
K / a
-Act
inin
ratio
(% o
f DM
SO)
SOS1 wild-type
SOS1 H905V
SOS1 H905I
2.820e-008
>1.000e-005
>1.000e-005FLAG-SOS1
a-Actinin
f)
e)
0
20
40
60
80
100
120
BI-3406 (log, M)
Perc
enta
ge p
rolif
erat
ion
inhi
bito
n(n
orm
alize
d)
SOS1 wild-type
SOS1 H905V
SOS1 H905I
3.192e-008
>1.000e-005
>1.000e-005
10 -10 10 -8 10 -6
10-8 10-7 10-6 10-5
Empt
y ve
ctor
SOS1
H90
5I
SOS1
H90
5V
SOS1
wild
-type
Biochemical protein-protein interaction assays
Biochemical protein-protein interaction assays
Biochemical protein-protein interaction assays
GLISA assays
pERK modulation in MIA PaCa-2 cellswith SOS1 transgene expression
Proliferation assay with MIA PaCa-2 cellswith SOS1 transgene expression
Figure 1
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10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -40
20
40
60
80
100
120 A375
A549
BI-3406 (log, M)
Perc
enta
ge p
ERK
/ tot
al E
RK
(nor
mal
ized
) DLD1
NCI-H520
NCI-H358
NCI-H23
10 -10 10 -8 10 -6 10 -40
20
40
60
80
100
120 A375
A549
DLD1
NCI-H358
NCI-H23
NCI-H520
BI-3406 (log, M)
Per
cent
age
prol
ifera
ton
inhi
bitio
n(n
orm
aliz
ed)
a) b)
f)
510
50100
5001,000
5,00010,000
BRAFNF1
EGFRHRASNRASKRAS
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
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-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
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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
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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
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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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