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Dual inhibition of GTP-bound KRAS and mTOR in lung adenocarcinoma and squamous cell carcinoma harboring KRAS G12C

Cell Communication and Signaling volume 23, Article number: 220 (2025) Cite this article

Abstract

Background

Kirsten rat sarcoma (KRAS) mutations are somatic variants in lung adenocarcinoma. One of the most prevalent mutations, G12C, has led to the clinical approval of targeted inhibitors for advanced stages in lung cancer. Research has increasingly focused on the efficacy of combination therapies that target multiple tumorigenic pathways. Cases harboring KRAS G12C mutation are heterogenous. We explored alternative changes in genetic pathways and evaluated the effectiveness of combination therapy using several types of cell lines and KRAS inhibitors.

Methods

We comprehensively investigated genetic changes induced by KRAS G12C inhibition using RNA sequences and the candidate to inhibit in combination therapy was explored. Three lung cancer cell lines (two adenocarcinoma and one squamous cell carcinoma) and three KRAS G12C inhibitors (AMG 510, MRTX849, and ARS-1620) were used. KRAS G12C and candidate gene were simultaneously inhibited in cell lines and the efficiency of combination therapy was evaluated using clonogenic assays and MTS assay. Pathway activation was assessed via western blotting. A combination index (CI) < 0.8 was considered statistically synergistic.

Results

RNA sequences revealed treatment with two of the three KRAS G12C inhibitors led to a significant increase in mTOR expression across all three cell lines. mTOR was targeted in combination therapy; each KRAS G12C inhibitor and mTOR inhibitor (RAD001) combination exhibited synergism (CI < 0.8) in MTS and clonogenic assays. Single inhibition of mTOR induced activation of guanosine triphosphate (GTP)-RAS, thereby activating the RAS-MEK-ERK and PI3K-AKT-mTOR pathways in WB, suggesting mTOR activation is crucial for KRAS-driving lung cancer. A combination strategy targeting KRAS G12C and mTOR abrogated GTP-RAS, pmTOR (Ser2448), and pERK (Thr202/Tyr204) more efficiently.

Conclusions

KRAS G12C inhibitor plus RAD001 consistently revealed synergism. Targeting KRAS G12C and mTOR abrogates the RAS-MEK-ERK and PI3K-AKT-mTOR pathways. Our data suggests that a combined strategy targeting GTP-bound KRAS G12C and mTOR shows promise for primary lung cancers with KRAS G12C mutations. This approach may also be effective even for lung cancers harboring KRAS G12C mutation but having different profiles.

Background

Kirsten rat sarcoma (KRAS) is a major onco-driving gene in lung cancer. Somatic KRAS mutations are likely harbored in lung adenocarcinoma patients with a history of smoking [1] and can also be harbored in squamous cell carcinoma patients [2]. Targeting inhibitors for KRAS G12C have been clinically approved for advanced cases [3].

In gene target therapies, acquired resistance is inevitable and its mechanisms have been revealed in treatment targeting epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements [4,5,6,7]. Likewise, resistant mechanisms in other target therapies, secondary nucleotide changes have been reported in KRAS G12C inhibition [8, 9]. Activations of alternative or downstream pathways have been also revealed as a resistant mechanism in KRAS-driving tumors. Clinical data and in vitro studies have unveiled that secondary mutations in KRAS or amplification in MET can emerge as resistant mechanisms for KRAS G12C inhibitors [8, 9]. Other studies showed that KRAS signaling can be maintained after KRAS G12C inhibition and then drug-resistance is acquired by EGFR or AURKA signaling [10], or epithelial-mesenchymal transition (EMT) via PI3K pathway activation [11]. KRAS-mutant cases are heterogeneous. EMT is also one of the key mechanisms for acquired resistance [12, 13]. Different tumorigenesis is suggested between epithelial and mesenchymal types [14]. KRAS inhibition alone can be thwarted by compensatory or parallel pathway activation [15, 16] and monotherapy may be an insufficient approach.

To overcome the resistance or achieve greater efficiency of target therapy, the usefulness of combination therapies targeting several signaling pathways has been suggested [17,18,19,20]. Co-inhibition by KRAS G12C plus SHP2 [21] or KRAS G12C plus mTORC1 kinase has been suggested to be promising [22]. Solanki et al. evaluated combination therapy by dividing KRAS-mutant cases into epithelial and mesenchymal types; the former is activated by IGFRs and ERBB2/3 and responsive to co-inhibition with SHP2 or SOS1, and the latter is activated by IRAS1 or AXL signaling [14]. Combination treatment for tumors harboring KRAS G12C is a promising approach and is not limited to lung cancer [23, 24].

Combination strategies have been attempted to conquer the KRAS-driving tumors [15, 25, 26]. Phase II-III clinical trials of the combination of the KRAS G12C inhibitor with cytotoxic chemotherapy or immune checkpoint inhibitors (ICIs) for non-small cell lung cancer (NSCLC) are ongoing (NCT05920356, NCT04613596, NCT05472623, NCT05609578, NCT05375994, NCT05840510, NCT05578092, NCT06024174, NCT05118854, NCT05054725, NCT05074810) [27].

We aimed to reveal the alternative pathway changes as a potential mechanism of resistance using several types of KRAS-mutant cell lines (adenocarcinoma or squamous cell carcinoma, epithelial or mesenchymal type, or oncogenic mutations in STK11 and/or TP53) and evaluate the efficiency of combination therapy targeting the alternative pathway change.

Methods

Overview of study design

We aimed to evaluate alternative changes in the tumorigenesis pathway induced by KRAS target treatment. Three lung cancer cell lines (including squamous cell carcinoma) harboring KRAS G12C were cultured with three types of KRAS G12C inhibitors, and the frequency of KRAS G12C was explored in clinical adenocarcinoma and squamous cell carcinoma patients. After exposure to each inhibitor, RNA-Seq was performed and changes in the mRNA expression of tumor-related genes were evaluated. The candidates detected by RNA-Seq were further examined to determine whether their inhibition was effective in KRAS-targeted therapy. The efficiency of combination therapy for KRAS G12C was also determined. All the procedures involving human participants were in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent for collecting samples for use in the study and publication of results was obtained from all patients before surgery. This study was approved by the Institutional Review Board of Hiroshima University (E2020-2081-01).

Detection of KRAS G12C in clinical primary lung cancer cases

The frequency of KRAS G12C was evaluated in consecutive patients with pN0M0 adenocarcinoma and squamous cell carcinoma resected at Hiroshima University Hospital between 2007 and 2019 or 2014 and 2018. DNA was extracted from the resected frozen or formalin-fixed paraffin-embedded tissues using the QIAamp DNA Micro Kit or the QIAamp DNA FFPE Tissue Kit (56304, 56404; Qiagen GmbH, Hilden, Germany). KRAS G12C was detected using commercially available primers and probes (1863506; Bio-Rad, Hercules, CA, USA), and QX100 Droplet Digital PCR System (1863111, Bio-Rad, Hercules, CA, USA). The detailed ddPCR conditions have been described previously [28].

Cell lines and inhibitors

Three lung cancer cell lines harboring KRAS G12C were used: H23 (adenocarcinoma), HOP-62 (adenocarcinoma), and Calu-1 (squamous cell carcinoma). As a control, H520 cells (KRAS wild type, lung squamous cell carcinoma) were used in the clonogenic assay. RPMI 1640 Media (11875093, Thermo Fisher Scientific, MA, USA) and McCoy’s 5 A (Modified) Medium (16600082, Thermo Fisher Scientific, MA, USA) medium supplemented with 10% fetal bovine serum and 50 µg/mL penicillin-streptomycin were used for H23/HOP-62/H520 and Calu-1, respectively. Cells were maintained in a humidified atmosphere with 5% CO2 at 37ºC. AMG 510, MRTX849, and ARS-1620 were used as KRAS G12C inhibitors, while RAD001 was used as a mammalian target of rapamycin (mTOR) inhibitor. The profiles of each cell line are shown in Additional file 1: Supplementary Table 1. Information on the inhibitors is provided in Additional file 2: Supplementary Table 2.

RNA sequencing (RNA-Seq) with next-generation sequencing (NGS)

To evaluate changes in mRNA expression levels, we performed RNA-Seq using cell lines treated with KRAS G12C inhibitors. Cells (50,000) were disseminated in a 60 mm dish (83.3901; SARSTEDT, Nümbrecht, Germany). The cells were cultured for 24 h to attach, and the medium was replaced with a fresh medium containing a KRAS G12C inhibitor or dimethyl sulfoxide (DMSO). The number of seeded cells and the concentration of inhibitors were the same regardless of the cell line. The medium containing the inhibitor or DMSO was changed every 3 d for approximately 14 d before reaching cell confluency. RNA was extracted using the Tri Reagent (TR118, COSMO BIO. Tokyo, Japan).

For cDNA synthesis, we used PrimeScript™ Double Strand cDNA Synthesis Kit (6111 A, TAKARA BIO INC., Kusatsu, Siga, Japan) to make cDNA from the mRNA purified with Oligotex™-dT30 < Super > mRNA Purification Kit (9086, TAKARA BIO INC., Kusatsu, Siga, Japan). For library preparation, 14 ng of covaris sheared cDNA was used as a direct input for the xGen Prism DNA Library Prep Kit (10006202, Integrated DNA Technologies Inc., Coralville, IA, USA) and indexed by xGen UDI Primer Pairs, Index 1–96 (Integrated DNA Technologies Inc., Coralville, IA, USA). Libraries were pooled at 500 ng each and subsequently hybridized with the xGen Hybridization and Wash Kit (1080577, Integrated DNA Technologies Inc., Coralville, IA, USA) and xGen Customs Hyb Panels (Integrated DNA Technologies Inc., Coralville, IA, USA) according to the manufacturer’s instructions. The final captured libraries were quantified using a Qubit dsDNA HS Assay Kit (Q32854, Thermo Fisher Scientific, MA, USA) and a Bioanalyzer High Sensitivity DNA Analysis Kit (5067 − 4626, Agilent Technologies, Santa Clara, CA USA). The expression levels of 48 oncogenes and beta-actin were assessed. The genes evaluated are listed in Additional file 3: Supplementary Table 3. We pooled six capture pools together at 4 nM and sequenced them on a HiSeq 2500 (Illumina, PE Rapid v2 workflow) with 2 × 67 bp paired-end reads.

Real-time PCR

mRNA expression levels were examined using real-time PCR and performed independently of RNA sequencing. Predesigned primers and probes (TaqMan Gene Expression Assays, Thermo Fisher Scientific, MA, USA) were used for mTOR (Hs00234508_m1) and β-actin was used as an internal control (Hs01060665_g1). RNA was extracted as described above and purified using the SuperScript IV VIL Master Mix with ezDNase (11766050, Thermo Fisher Scientific, MA, USA). Real-time PCR was performed using StepOnePlus (Thermo Fisher Scientific, MA, USA). Delta-delta cycle threshold (Ct) was employed to quantify expression level using the following formula; 2−(DCt sample), in which delta Ct (DCt) values were determined by subtracting the Ct value of the house-keeping gene (β-actin) from the Ct value of the target gene.

Clonogenic assay

Clonogenic assays were performed as previously described [15]. Briefly, 300 cells were seeded in 6-well plates (83.3920, SARSTEDT, Nümbrecht, Germany). The cells were cultured for 24 h and the medium was replaced with inhibitor(s) or DMSO. The number of seeded cells and the concentration of inhibitors were the same regardless of the cell line. The medium containing inhibitor(s) or DMSO was changed every 3 d for 11–14 d before reaching cell confluency. The medium was removed, and the cells were washed with phosphate-buffered saline (PBS). The colonies were fixed with methanol for 25 min on ice and stained with 0.5% crystal violet (031-04852, Wako, Japan) for 15 min at room temperature. The colonies were then washed and photographed. For semiquantitative measurements, the crystal violet was reabsorbed using 2% SDS, and the absorbance was measured at 570 nm using a TriStar LB941 (BERTHOLD, Tokyo, Japan).

MTS assay

The antitumor effect of the drug combination was assessed by cell viability using the Thiazolyl Blue Tetrazolium Bromide assay and the drug concentration required for 50% growth inhibition (IC50) was determined using GraphPad Prism Version 7.05 (GraphPad Software, San Diego, CA, USA). Tumor cells were seeded at a density of 2000 (H23) or 3000 (Calu-1) per well in 96-well plates (83.3924; SARSTEDT, Nümbrecht, Germany) and allowed to attach for 24 h. Subsequently, the cells were treated with a medium containing several inhibitor(s) for 3 d. After treatment, the cells were incubated with a medium containing reagent for MTS assay (G3581, Promega) for 2 h at 37ºC. Cell viability was determined by measuring the absorbance at 450 nm using Multiskan GO (N10588, Thermo Fisher Scientific, MA, USA). To evaluate the combination index (CI), the concentration of each inhibitor ranged from 2− 6 and 22 IC50. The average of the CIs from each concentration (from 2− 6 to 21 IC50) was used as the final CI.

Western blotting

Cells (35 × 104) were seeded in 35 mm dishes (83.3900, SARSTEDT, Nümbrecht, Germany) with medium and allowed to attach for 24 h. The medium was replaced with fresh medium with or without the inhibitor(s). After 24 h of incubation, the cells were washed with PBS and lysed in RIPA buffer (9806, Cell Signaling Technology, Danvers, MA, USA) containing protease inhibitor (04080-24, Nacalai Tesque, Kyoto, Japan) and PMSF (8553, Cell Signaling Technology, Danvers, MA, USA). Following centrifugation at 14,000 × g for 10 min at 4ºC, the supernatant was collected as the total cell lysate and then diluted using the Laemmli Sample Buffer (1610737, BIO-RAD, Hercules, CA, USA) and 2-mercaptoethanol (161–0710, BIO-RAD, Hercules, CA, USA). After heating for 5 min at 95ºC, the lysates containing proteins were electrophoresed on 5–20% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (1704156, BIO-RAD, Hercules, CA, USA). All target proteins were immunoblotted with the appropriate primary and secondary antibodies using the Can Get Signal/PVDF Blocking Reagent (NKB101/NYPBR; TOYOBO LIFE SCIENCE, Tokyo, Japan). β-actin was used as an internal control. Chemiluminescent bands were detected, and expression levels were measured using an Amersham Imager 680 (General Electric, CT, USA). The protein expression levels were quantified using ImageQuant TL (Cytiva, Tokyo, Japan). The antibodies and their concentrations are listed in Additional file 2: Supplementary Table 2. The GTP-binding RAS was extracted and detected using an Active RAS Pull-Down and Detection Kit (16117, Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol. To detect another protein in the same membrane, the attached antibody was removed using the Stripping Solution (193-16375, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

Generation and transfection of mTOR-targeting SiRNA

siRNA targeting mTOR was generated using Silencer Select Validated siRNA (4427038, Thermo Fisher Scientific, MA, USA). siRNAs designed to block mTOR expression (4390824, siRNA ID s603/s604, Thermo Fisher Scientific, MA, USA) or Silencer Select Negative Control (4390843, Thermo Fisher Scientific, MA, USA) was transfected with Lipofectamine RNAiMAX Reagent (3778-030, Thermo Fisher Scientific, MA, USA). After incubation of cell lines with siRNA for 72 h, the cells were seeded in 96-well plates (83.3924; SARSTEDT, Nümbrecht, Germany). The MTS assay was performed as described above, and the effect of silencing mTOR in KRAS G12C cell lines was evaluated.

Data analysis

The reads resulting from the capture of 49 genes were subjected to RNA-seq analysis using the StrandNGS software, version 2.7 (Tomy Digital Biology). The reads were aligned against a human genome reference (hg19) using RefSeq transcript annotations. Low-quality read removal and QC were then performed, and quantification was performed using the DEseq algorithm. The impact of combination therapy was evaluated using the CI calculated using the Chou–Talalay method [29]. Data for CI calculation was referred to in quantified cell viability in the MTS assay or concentration of crystal violet in the Clonogenic assay. A combination index of less than 0.8 was regarded as statistically synergistic. The significance of the frequency was evaluated using the chi-squared or Yates chi-squared test. Protein and mRNA expression levels were compared using the multiple T test or Mann–Whitney U test. Statistical significance was set at p < 0.05. Statistical analyses and figure creation were performed using SPSS Version 29.0.1.0 (IBM Corp., Armonk, NY, USA), StatMate V (ATMS Co., Ltd., Tokyo, Japan), and GraphPad Prism Version 7.05 (GraphPad Software, San Diego, CA, USA).

Results

Distribution of KRAS G12C in lung adenocarcinoma and squamous cell carcinoma

We tested KRAS mutations using ddPCR in 877 resected adenocarcinoma and 113 squamous cell carcinoma samples. KRAS G12C was detected in 2.9% (25/877) and 1.8% (2/113) of adenocarcinoma and squamous cell carcinoma, respectively. Among KRAS G12C cases, 85.2% (23/27), including two squamous cell carcinoma cases, were current or ex-smokers.

mRNA expression change induced by KRAS G12C Inhibition

RNA-Seq NGS revealed increasing or decreasing changes in mRNA expression. The results are presented as volcano plots in Fig. 1A. The expression of four, 17, and 11 genes was significantly increased by AMG 510, MRTX849, and ARS-1620 treatment, respectively. The expression level of mTOR was significantly increased in all three cell lines by two of the three inhibitors (H23 treated with MRTX849 and ARS-1620, HOP-62 treated with AMG 510 and MRTX849, and Calu-1 treated with AMG 510 and ARS-1620) (Fig. 1B). Although the expression of mTOR in H23 cells after AMG 510 treatment did not significantly increase by NGS, a significant increase in mTOR RNA expression after AMG 510 treatment was confirmed by real-time PCR (Fig. 1C). RNA-Seq revealed that the increased expression of mTOR was induced by several KRAS G12C inhibitors.

Fig. 1
figure 1

Expression of mRNA after KRAS G12C inhibition in three cell lines. (A) Comprehensive RNA-Seq results are shown as volcanic plots. Each dot represents mRNA expression and its statistical significance compared to intact cell lines. Red and green dots indicate mTOR expression levels with and without statistical significance, respectively. (B) Summary of mTOR expression as determined by RNA-Seq. The expression level of mTOR is significantly elevated in all three cell lines by two different KRAS G12C inhibitors. The red circles denote significantly increased mTOR expression levels. (C) Comparison of mRNA expression levels in intact and H23 cell lines treated with AMG 510. Real-time PCR analysis confirmed a significantly greater increase in mTOR mRNA levels in the H23 cell line compared to the intact cell lines

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Synergism of KRAS G12C inhibitors and mTOR inhibitor

A clonogenic assay revealed synergism between each KRAS G12C and mTOR inhibitor (RAD001). The colony formation was suppressed in a dose-dependent manner. CIs were less than 0.8 in all three cell lines and three KRAS G12C inhibitors (Fig. 2). Synergism or dose-dependent suppression by monotherapy has not yet been confirmed in KRAS wild type squamous cell carcinoma cell lines (H520). The MTS assay also showed synergism between KRAS G12C and mTOR inhibition in adenocarcinoma (H23) and squamous cell carcinoma (Calu-1) cell lines (Fig. 3).

Fig. 2
figure 2

Clonogenic assay in three cell lines treated using three KRAS G12C inhibitors and mTOR inhibitor. (A) Representative image of the clonogenic assay in H23 cell lines treated with AMG 510 and RAD001. The colony growth is suppressed in a dose-dependent manner by monotherapy and stronger inhibited by combination therapy. The heatmap represents the amount of colony growth by the density of crystal violet. (B, C) Two representative images of the clonogenic assay in HOP-62 (B) and Calu-1 (C) cell lines after treatment with AMG 510 and RAD001. In both cell lines, the colony growth is suppressed in a dose-dependent manner and stronger in combination therapy. (D) Summary of the clonogenic assay in cell lines treated with three different KRAS G12C inhibitors and RAD001. Cell growth is suppressed in a dose-dependent manner. Combination index is below 0.8 (dashed line) in all three cell lines in three different agents. (E) Results of the clonogenic assay in H520, which is lung squamous cell carcinoma cell lines without KRAS mutation. Neither colony growth inhibition in a dose-dependent manner nor higher suppression by combination treatment is observed. Combination index is above 0.8 (dashed line) in all concentrations, being non-synergistic in H520 cell lines

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Fig. 3
figure 3

Results of MTS assay using KRAS G12C and mTOR inhibitor. (A-B) Results of MTS assay in H23 and Calu-1 cell lines using three different KRAS G12C inhibitors with RAD001. Tumor growth is assessed in a range of 2− 6 to 22 times of concentration of IC50. Cell viability is lower in combination therapy in both cell lines. The dashed lines indicate the 50% cell viability and drug concentration for IC50. (C) Summary of the MTS assay representing the combination index. Combination indexes are lower than 0.8 (dashed line) in both cell lines with three different agents

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Impact of mTOR on tumor growth in lung adenocarcinoma and squamous cell carcinoma harboring KRAS G12C

Next, we evaluated the effect of mTOR suppression on tumor growth. Expression of mTOR was suppressed by siRNA and cell growth was compared with that of intact H23 and Calu-1 cell lines. The MTS assay showed suppressed cell growth following mTOR inhibition in both cell lines (Fig. 4A). These results suggest that the activation of mTOR is involved in the tumor growth of KRAS G12C-driving lung cancer.

Fig. 4
figure 4

Impact of mTOR expression on cell growth and western blotting results in H23 and Calu-1 cell lines. (A) Growth curves for intact or mTOR-inhibited H23 or Calu-1 cell lines. Cell growth was assessed using the MTS assay at 24, 48, and 72 h after allowing cells to adhere overnight. Significant suppression of cell growth was observed with mTOR inhibition. (B) Western blot analysis shows elevated levels of pAKT (Ser473), pERK (Thr202/Tyr204), and GTP-RAS following RAD001 monotherapy. In contrast, combination therapy with AMG 510 and RAD001 leads to reduced levels of pmTOR (Ser2448), pAKT (Ser473), pERK (Thr202/Tyr204), and GTP-RAS

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Alternative pathway activation by mono- or combination treatment targeting KRAS G12C and/or mTOR

Western blotting revealed that KRAS G12C inhibition suppressed ERK and PI3K- protein kinase B (AKT)-mTOR pathways. Expression of activated GTP-bound RAS was induced by a single mTOR inhibitor. The expression of phosphorylated ERK (Thr202/Tyr204) and AKT (Ser473) was also elevated, suggesting the activation of the RAS-MEK-ERK and PI3K-AKT-mTOR pathways. The combination treatment inhibited these alterations and significantly suppressed the expression of GTP-bound RAS and phosphorylated mTOR (Ser2448) (Fig. 4B).

The elevation of phosphorylated ERK (Thr202/Tyr204), AKT (Ser473), and GTP-bound RAS induced by mTOR single inhibition suggested that mTOR activation is crucial for maintaining tumor growth in KRAS G12C-driven lung cancer, and suppression of mTOR was restored by the alternative activation of GTP-bound KRAS, resulting in ERK elevation as a downstream change of the RAS-MEK-ERK pathway. Targeting KRAS G12C and mTOR in combination abrogates GTP-RAS, pmTOR, and pERK expression Although pAKT was elevated even with combined inhibition, this may be due to the steady suppression of mTOR in the PI3K-AKT-mTOR pathway (see Fig. 5).

Fig. 5
figure 5

Illustration summarizing pathway alterations in lung cancer with KRAS G12C under monotherapy and combination treatment. Continuous KRAS G12C inhibition induces mTOR activation, while single-agent mTOR inhibition causes feedback activation of GTP-bound KRAS, resulting in activation of both the RAS-MEK-ERK and PI3K-AKT-mTOR pathways. Simultaneous dual inhibition of KRAS G12C and mTOR abrogates both cascades, leading to tumor suppression

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Discussion

KRAS is one of the most frequently identified onco-driving genes in lung cancer, particularly in Western countries [30]. In oncogenic KRAS-mutant cases, RAS proteins are binary molecular switches that convert active GTP-bound to inactive guanosine diphosphate (GDP)-bound forms. KRAS mutations maintain their active form by compromising GTPase and GTPase-activating protein (GAPs) activity, which hydrolyzes GTP to GDP [16, 31, 32].

Several treatment approaches have been suggested for RAS-mutant tumors: inhibition of RAS processing, inhibition in the upstream or downstream of the RAS pathway, or synthetic lethality strategies [3, 33, 34]. As combination therapy, preclinical studies and clinical trials have been conducted [35].

We used three KRAS G12C inhibitors, including two clinically approved agents with RAD001 (everolimus), which is clinically available for treating breast cancer, kidney cancer, and neuroendocrine tumors.

Sotorasib (AMG 510) and adagrasib (MRTX849) monotherapies have shown therapeutic efficacy in NSCLC in clinical phase I-II trials [36,37,38]. ARS-1620 inhibited RAS-GDP and atropisomerically inhibited KRAS G12C activity [39].

Combination therapy suppressing GTP-bound KRAS and mTOR is classified as “co-targeting a critical downstream node” [35]. A previous study suggested mTOR, IGF1R, and KRAS G12 inhibition by ARS-1620 markedly increased tumor regression in three different KRAS-driven lung cancer mouse models compared to mTOR, IGF1R, and MEK inhibition [40]. The combination of the PI3K inhibitor and ARS-1620 showed efficacy in the KRAS G12C NSCLC model [28]. Phase I-II trials combining sotorasib and several inhibitors, including mTOR inhibitors, are ongoing (NCT05840510 and NCT04185883) [27].

Here, we showed that the activation of mTOR was induced by KRAS G12C inhibition in three different types of cell lines, and a single inhibition of mTOR induced a feedback enhancement in GTP-bound RAS, leading to RAS-MEK-ERK pathway activation. Dual inhibition of KRAS G12C and mTOR abrogated the RAS-MEK-ERK and phosphatidylinositol-3-kinase (PI3K)-AKT-mTOR cascades and suppressed tumor growth more effectively. The PI3K-AKT-mTOR pathway is activated by RAS in KRAS-mutant lung cancers [41, 42] and has been suggested as a promising candidate target for combination therapy.

Genetically targeted therapies have been developed almost exclusively for adenocarcinomas. We confirmed similar alternate changes by KRAS G12C or mTOR inhibition in different phenotypes of lung cancer cell lines. KRAS G12C can also occur in non-adenocarcinoma lung cancers. The incidence of KRAS mutations in lung squamous cell carcinoma ranged 0–14% (median: 4.5% from 13 studies) [2]. Targeted therapy has been suggested to be effective in non-adenocarcinoma cases harboring targetable genetic variants [43, 44]. We have previously shown that targeting specific gene expression could be effective in both adenocarcinoma and squamous cell carcinoma, even if the target signal does not stem from oncogenic mutations [45]. Skoulidis et al. divided KRAS-mutant lung adenocarcinoma by co-occurring genomic alterations into three subtypes and found that mTORC1 signaling was inactivated in a subtype characterized by the absence of TP53 or STK11/LKB1 mutations [46]. H23 included cancer gene mutations in STK11 and TP53, and HOP-62 harbored mutations in TP53 (Additional file 1: Supplementary Table 1). De novo mTORC1 suppression might not occur in H23 and HOP-62, but no data regarding Calu-1 cell lines exist. We tried to reveal the genetic features, including TP53/STK11/LKB1 mutations and mTOR expression, in non-adenocarcinoma KRAS-mutant cases, and compare the genetic features between KRAS-mutant adenocarcinoma and squamous cell carcinoma cases using clinical samples. Although the incidence of KRAS G12C in squamous cell carcinoma was not significantly different compared with adenocarcinoma (1.8% (2/113) vs. 2.9% (25/877), p-value = 0.721), the number of squamous cell carcinoma cases was too small. Further exploration of a combination strategy targeting mTOR is warranted in the mTORC1-suppressed subtype and more non-adenocarcinoma cases with KRAS G12C mutation. We evaluated the pathway change as a potential mechanism in drug resistance. However, we did not directly confirm cell lines with mTOR activation showed the drug resistance. Further research which validates mTOR activation induced by KRAS G12C inhibition can be a resistant mechanism in several lung cancer phenotypes is warranted.

Conclusions

Activation of mTOR has been suggested as an alternative change induced by KRAS G12C inhibition, and mTOR solo-inhibition induces GTP-bound KRAS activation. A combination strategy inhibiting GTP-bound KRAS G12C and mTOR is promising for primary lung cancers harboring a KRAS G12C mutation. This combination strategy might be effective for lung cancers harboring KRAS G12C mutation regardless of difference in histology, treatment agent, coexisting oncological mutations, or EMT phenotype.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

The work was conducted with the facilities in the Natural Science Center for Basic Research and Development(N-BARD) at Hiroshima University. The authors would like to thank Editage (http://www.editage.jp) for English language editing.

Funding

This study was supported by the Japan Society for the Promotion of Science (JSPS KAKENHI Grant number 22K08975).

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Author notes

  1. Junichi Soh

    Present address: Department of Thoracic Surgery, Osaka Metropolitan University Graduate School of Medicine, Osaka, Japan

Authors and Affiliations

  1. Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan

    Masaoki Ito, Yoshihiro Miyata & Morihito Okada

  2. Division of Thoracic Surgery, Department of Surgery, Kindai University Faculty of Medicine, Osaka-Sayama, Japan

    Masaoki Ito, Kenichi Suda, Junichi Soh & Yasuhiro Tsutani

  3. Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan

    Shoko Hirano, Nagisa Morihara, Misako Takemoto & Fumiko Irisuna

  4. Department of Pathology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan

    Kei Kushitani & Yukio Takeshima

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Contributions

Conceptualization; MI, YM. Data collection and curation; MI, KK, YT, Interpretation; MI, YM, SH, NM, KS, JS, Formal analysis; MI, SH, NM, MT, FI. Investigation; MI, SH. Methodology; MI, YM, SH. Project administration; MI, YM. Supervision; YT, MO, Visualization; MI, Roles/Writing– original draft; MI, Writing– review & editing and approving the final manuscript; all authors.

Corresponding authors

Correspondence to Masaoki Ito or Yoshihiro Miyata.

Ethics declarations

Ethics approval and consent to participate

All the procedures involving human participants were in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent for collecting samples for use in study and publication of results was obtained from all patients before surgery. This study was approved by the Institutional Review Board of Hiroshima University (E2020-2081-01).

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The authors declare no competing interests.

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Ito, M., Miyata, Y., Hirano, S. et al. Dual inhibition of GTP-bound KRAS and mTOR in lung adenocarcinoma and squamous cell carcinoma harboring KRAS G12C. Cell Commun Signal 23, 220 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12964-025-02187-y

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Keywords

Cell Communication and Signaling

ISSN: 1478-811X