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. 2025 Mar 18;122(11):e2422943122.
doi: 10.1073/pnas.2422943122. Epub 2025 Mar 12.

SOS1 inhibitor BI-3406 shows in vivo antitumor activity akin to genetic ablation and synergizes with a KRASG12D inhibitor in KRAS LUAD

Fernando C Baltanás #  1   2 , Maximilian Kramer-Drauberg #  3 , Rósula García-Navas  1 , Enrico Patrucco  3 , Ettore Petrini  3 , Heribert Arnhof  4 , Andrea Olarte-San Juan  1 , Pablo Rodríguez-Ramos  1 , Javier Borrajo  5 , Nuria Calzada  1 , Esther Castellano  6 , Barbara Mair  4 , Kaja Kostyrko  4 , Marco H Hofmann  4 , Chiara Ambrogio  3 , Eugenio Santos  1
Affiliations

SOS1 inhibitor BI-3406 shows in vivo antitumor activity akin to genetic ablation and synergizes with a KRASG12D inhibitor in KRAS LUAD

Fernando C Baltanás et al. Proc Natl Acad Sci U S A. .

Abstract

We evaluated the in vivo therapeutic efficacy and tolerability of BI-3406-mediated pharmacological inhibition of SOS1 in comparison to genetic ablation of this universal Ras-GEF in various KRAS-dependent experimental tumor settings. Contrary to the rapid lethality caused by SOS1 genetic ablation in SOS2KO mice, SOS1 pharmacological inhibition by its specific inhibitor BI-3406 did not significantly affect animal weight/viability nor cause noteworthy systemic toxicity. Allograft assays using different KRASmut cell lines showed that treatment with BI-3406 impaired RAS activation and RAS downstream signaling and decreased tumor burden and disease progression as a result of both tumor-intrinsic and -extrinsic therapeutic effects of the drug. Consistent with prior genetic evidence and the KRASmut allografts assays in immunocompromised mice, our analyses using an in vivo model of KRASG12D-driven lung adenocarcinoma (LUAD) in immunocompetent mice showed that single, systemic BI-3406 treatment impaired tumor growth and downmodulated protumorigenic components of the tumor microenvironment comparably to SOS1 genetic ablation or to treatment with the specific KRASG12D inhibitor MRTX1133. Furthermore, markedly stronger, synergistic antitumor effects were observed upon concomitant treatment with BI-3406 and MRTX1133 in the same in vivo LUAD mouse model. Our data confirm SOS1 as an actionable therapy target in RAS-dependent cancers and suggest that BI-3406 treatment may yield clinical benefit both as monotherapy or as a potential combination partner for multiple RAS-targeting strategies.

Keywords: BI-3406; KRAS; SOS1; SOS2; TME.

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Conflict of interest statement

Competing interests statement:K.K., B.M., H.A., and M.H.H. are employees of Boehringer-Ingelheim. F.C.B., R.G.-N., and E.S. received research fee from and Boehringer-Ingelheim. C.A. received research fees from Revolution Medicines, Verastem, Roche and Boehringer-Ingelheim.

Figures

Fig. 1.
Fig. 1.
Assessment of in vivo toxicities resulting from genetic ablation or pharmacological inhibition of SOS1 in mice. (A) Kaplan–Meier survival plot of TAM-induced (TAM-containing chow diet starting at 1 mo of age) SOS1/2WT, SOS1fl/fl, SOS2KO (black, pink and blue lines, respectively) and SOS1/2DKO mice (orange line). (B) Kaplan–Meier survival plot of SOS1/2WT or SOS2KO groups orally treated with vehicle (Natrosol; gavage) or BI-3406 (50 mg/kg bid; gavage) for 26 d. (C) Body weight measurements at various timepoints (days 1 to 15) during TAM treatment (starting at 1 mo of age) in SOS1/2WT, SOS1fl/fl, SOS2KO, and SOS1/2DKO groups. **P < 0.01 vs SOS1/2WT; #P < 0.05 vs SOS2KO; &&&&P < 0.0001 vs rest of groups. Data expressed as mean ± SD. The multiple t test was used. (D) Body weight measurements during BI-3406 or vehicle administration in SOS1/2WT or SOS2KO groups. ###P < 0.001 SOS1/2WT (vehicle-treated) vs SOS1/2WT (BI-3406-treated); ****P < 0.0001 SOS2KO (vehicle-treated) vs SOS2KO (BI-3406-treated); &&P < 0.01 SOS1/2WT (BI-3406-treated) vs SOS2KO (BI-3406-treated). Data expressed as mean ± SD. The multiple t test was used. For TAM treatments: SOS1/2WT (n = 7); SOS1KO (n = 9); SOS2KO (n = 8); SOS1/2DKO (n = 8). Following vehicle (Natrosol) treatment: SOS1/2WT (n = 10); SOS2KO (n = 8). Following BI-3406 treatment: SOS1/2WT (n = 21); SOS2KO (n = 30).
Fig. 2.
Fig. 2.
Allograft assays of (genetically or pharmacologically) SOS1-disrupted KRASG12C MEFs. (A) Immortalized SOS1fl/fl MEFs expressing exogenous KRASG12C were injected subcutaneously into nude mice. Once tumors reached a size of 200 mm3, animals were treated with vehicle (gavage), 4OHT (80 mg/kg; i.p), or BI-3406 (100 mg/kg, once daily; gavage) for 12 d. The mean fold-change in tumor volume relative to initial tumor volume is shown. The arrow indicates treatment starts. Waterfall plots of individual tumor responses from vehicle, 4OHT, or BI-3406-treated are depicted for day 12 and day 14. n = 5 (vehicle-treated); n = 9 (4OHT-treated); n = 10 (BI-3406-treated). The bar graph represents tumor volume fold change relative to treatment start. Error bars represent mean ± SD. **P < 0.01, ***P < 0.001 and ****P < 0.0001. One-way ANOVA was used. The panel includes representative images of SOS1fl/fl/KRASG12C allografts at day 16 after tumor implantation treated (starting at day 4; arrow) with vehicle, 4OHT, or BI-3406. (B) Quantitation of total tumor area, the percentage of Ki67-positive cells, the percentage of pERK-stained area with respect of total tumor area, the percentage of CD68-stained area, the percentage of collagen-stained area, the percentage of SMA-stained area, the number of CC3-positive cells per 20X field and the percentage of CD31-positive area. n = 5 tumors per group (for H&E studies) and n = 3 tumors per group (for the rest assays). Data shown as mean ± SD. *P < 0.05, **P < 0.01 vs vehicle-treated group; #P < 0.05 vs BI-3406-treated group. One-way ANOVA and Tukey’s test. CC3: cleaved-caspase 3; SMA: smooth muscle actin. n.s: not significant.
Fig. 3.
Fig. 3.
Allograft assays of (genetically or pharmacologically) SOS1-disrupted KRASG12D MEFs. (A) Immortalized SOS1fl/fl MEFs expressing exogenous KRASG12D were injected subcutaneously into nude mice. Once tumors reached a size of 200 mm3, animals were treated (arrow) with vehicle (gavage), 4OHT (80 mg/kg; i.p), BI-3406 (100 mg/kg, once daily; gavage), MRTX1133 (30 mg/kg/day; i.p), or combo (BI-3406+MRTX1133; gavage/i.p) for 12 d. Data shown as mean ± SD. One-way ANOVA was used. **P < 0.01 and ****P < 0.0001. n = 5 (vehicle-treated); n = 9 (4OHT-treated); n = 7 (BI-3406-treated); n = 6 (MRTX1133-treated); n = 5 (combo-treated). The mean fold-change in tumor volume relative to initial tumor volume is shown (Left side). The arrow indicates treatment start. The bar graph represents tumor volume fold change relative to treatment start. *P < 0.05 and ****P < 0.0001. One-way ANOVA was used. (B) Waterfall plots represented as Log2 of the fold change of individual tumor responses from 4OHT, BI-3406, MRTX1133, or combo-treated are depicted for day 12 and day 14. Representative images of SOS1fl/fl/KRASG12D allografts at day 16 after tumor implantation treated (starting at day 4; arrow) with vehicle, 4OHT, BI-3406, MRTX1133, or combo (Right side). *P < 0.05; ****P < 0.0001 vs vehicle-treated group. (C) The bar charts show total tumor area, the percentage of Ki67-positive cells, the percentage of pERK-stained area with respect of total tumor area, the percentage of CD68-stained area, the percentage of collagen-stained area, the percentage of SMA-stained area, the number of CC3-positive cells per 20X field and the percentage of CD31-positive area. n = 5 tumors per group (for H&E studies) and n = 3 tumors per group (for the remaining assays). Data shown as mean ± SD. One-way ANOVA and Tukey’s test. **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs MRTX1133-treated group; &P < 0.05 vs 4OHT-treated group; #P < 0.05 vs BI-3406-treated group. (D) Growth curves of KRASG12D-mutated KPB6, LKR10 or LKR13 cell cultures at 24 h and 48 h of treatment, individually, or combined, with BI-3406 (1 μM) and MRTX1133 (5 nM). DMSO was used as vehicle. n = 5 independent experiments per group. Data shown as mean ± SD. One-way ANOVA and Tukey’s test. ****P < 0.0001 vs drug-treated LUAD cells; ###P < 0.001 vs combo-treated cells; &P < 0.05 vs MRTX1133-treated cells. (E) Bar chart showing the relative levels of RAS-GTP, measured by using RAS G-LISA assay, in extracts of serum-starved (ST), vehicle-pretreated, BI-3406-pretreated, MRTX1133-pretreated, or combo-pretreated, for 2 h, KPB6, LKR10 or LKR13 LUAD cells, upon EGF stimulation (100 ng/mL) for 2 min. n = 3 independent samples per group. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA and Tukey’s test. CC3: cleaved-caspase 3; SMA: smooth muscle actin. n.s: not significant.
Fig. 4.
Fig. 4.
Impact of BI-3406 and MRTX1133 treatment on KRASG12D-driven LUAD progression. (A) Representative images of microCT from 3-mo-old SOS1/2WT/KRASG12D-mutated mice previous drug treatment (pre-treatment) and the very same animal after treatment (posttreatment) with BI-3406 administration (50 mg/kg, bid; gavage, 26 d), MRTX1133-treatment (30 mg/kg, i.p, 17 d), or combo administration (BI-3406, 50 mg/kg, bid; gavage and 30 mg/kg, i.p for 21 d). The graph’s data point corresponding to each individual mouse are identified by a distinctive color in each case. n = 4 mice per genotype (BI-3406-treated); n = 2 mice per genotype (MRTX1133-treated); n = 4 mice per genotype (combo-3406-treated). Data shown as mean ± SD. The paired t test was used. *P < 0.05. (B) Left. Representative images of paraffin-embedded sections from lung lobes of 4-mo-old, SOS1/2WT/KRASG12D-mutated mice treated with vehicle (gavage), BI-3406 (50 mg/kg; gavage; 26 d), MRTX1133 (30 mg/kg; i.p; 17 d), and combo (BI-3406, 50 mg/kg, bid; gavage and 30 mg/kg, i.p for 21 d) stained with H&E. (Scale bars, 1 mm.) Right. Quantitation of percentage of lung tumor area with respect total lung area. One-way ANOVA and Tukey’s test. (C) The bar chart shows the percentage of Ki67-positive cells into the tumor or the percentage of pERK-stained area with respect of total tumor area, the percentage of total tumor area that immunostained for SMA, the percentage of collagen in the tumor, the percentage of CD68-positive cells, the number of CD3-positive cells per 40X field in the tumor, the number of CC3-positive cells per 40X field and the percentage of CD31-stained area in vehicle-treated, BI-3406-treated, MRTX1133-treated, combo-treated samples as detailed in (B) and an additional mouse after 5 d of combo treatment. Data shown as mean ± SD. n = 4/group. *P < 0.05 vs BI-3406-treated group. n = 4/group. (B and C) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Since no tumors were found in the combo-treated group (21 d; n = 4) and because only one animal was measured in the 5 d combo-treated group, both experimental groups have not been considered for the statistical analysis. Data shown as mean ± SD. One-way ANOVA and Tukey’s test.
Fig. 5.
Fig. 5.
Determination of BI-3406 and MRTX113 sensitivity in KRASG12D mutant human LUAD cell lines. (A) Growth curves of KRASG12D-mutated A427 or SK-LU-1 cells at 12, 24, 36, and 48 h of the treatment, individually, or combined, with BI-3406 and MRTX1133 at the indicated concentrations. DMSO was used as vehicle. n = 5 independent experiments per group. Data shown as mean ± SEM. For A427 cells: **P < 0.01 vs BI-3406-treated (10 µM) cells; ***P < 0.001 vs BI-3406/MRTX1133-treated cells (1 µM/10 nM); ****P < 0.0001 vs BI-3406/MRTX1133-treated (10 µM/100 nM) cells. #P < 0.05 MRTX1133 (100 nM) vs BI-3406/MRTX1133-treated cells (1 µM/10 nM) or BI-3406-treated (10 µM) vs BI-3406/MRTX1133-treated (10 µM/100 nM); ####P < 0.0001 MRTX1133 (100 nM) vs BI-3406/MRTX1133-treated cells (10 µM/100 nM). For SK-LU-1: **P < 0.01 vs BI-3406/MRTX1133-treated (0.01 µM/0.1 nM) cells; ****P < 0.0001 vs BI-3406/MRTX1133-treated (10 µM/100 nM) cells; #P < 0.05 MRTX1133 (100 nM) vs BI-3406/MRTX1133-treated cells (0.01 µM/0.1 nM) or BI-3406-treated (10 µM) vs BI-3406/MRTX1133-treated (10 µM/100 nM); ##P < 0.01 MRTX1133 (100 nM) vs BI-3406/MRTX1133-treated cells (10 µM/100 nM). One-way ANOVA and Tukey’s test. (B) (Left) Representative western blots of active RAS pull-down assays and the corresponding signaling of pERK and pAkt, as well as the expression of SOS1 and SOS2 in each case. Tubulin was used as a loading control. (Right) Bar charts showing the relative levels of RAS-GTP, pERK, and pAkt in extracts of ST, vehicle-pretreated (EGF), BI-3406-pretreated, MRTX1133-pretreated, or combo-pretreated (for 2 h) A427 or SK-LU-1 cells, upon EGF stimulation (100 ng/mL) for 2 min. n = 3 independent samples per group. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001; &&P < 0.01 vs MRTX1133-treated cells; #P < 0.05, ###P < 0.001 vs BI-3406-treated cells. One-way ANOVA and Tukey’s test.
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