Epithelial-mesenchymal transition status is a remarkable biomarker for the combination treatment with avutometinib and defactinib in KRAS-mutated non-small cell lung cancer

Cell cultures and reagents

Nine human NSCLC cell lines with KRAS mutations were used. The cell lines NCI-H358, NCI-H2122, NCI-H441, A549, SW1573, Calu-1, and Calu-6 were purchased from the American Type Culture Collection (Manassas, VA, USA), while the NCI-H23 and HOP-62 cell lines were obtained from the NCI-60 cancer cell line panel of the National Cancer Institute Developmental Therapeutics Programme. We obtained information on the type of point mutations in the KRAS oncogene from the Catalogue of Somatic Mutations in Cancer (COSMIC) database (https://cancer.sanger.ac.uk/cosmic/).

All cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% foetal bovine serum (FBS), 2 mmol/L glutamine, 50 U/mL penicillin, and 100 µg/mL streptomycin in a 5% CO₂ incubator at 37 °C. All cells were passaged for less than 3 months before being renewed with frozen early passage stocks.

Avutometinib was provided by Chugai Pharmaceuticals (Tokyo, Japan). Defactinib was obtained from Selleck Chemicals (Houston, TX, USA). For in vitro experiments, the inhibitors were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. Recombinant human transforming growth factor (TGF)-β1 protein was purchased from R&D Systems (Abingdon, UK).

RTK phosphorylation antibody array

Cell lysates (500 μg/mL) were prepared from NCI-H358 cells treated with DMSO or avutometinib at the indicated concentrations for 72 h. We analysed the phosphorylation status of receptor tyrosine kinases (RTK) in the relevant pathways using RayBio® C-Series Human RTK Phosphorylation Antibody Array C1 (RayBiotech Inc., Norcross, GA, USA) following the manufacturer’s instructions. Arrays were imaged and quantified using the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).

Antibodies and western blotting

Cells treated with each agent at the indicated concentrations were lysed in RIPA buffer (50 mmol/L Tris-HCl, pH 8.0; 150 mmol/L NaCl; 1% NP-40; 0.5% deoxycholic acid; 0.1% SDS; 1 mmol/L dithiothreitol; and 0.5 mmol/L phenylmethylsulfonyl fluoride) supplemented with phosphatase inhibitors (#07574; Nacalai Tesque, Kyoto, Japan) for 30 min at 4 °C and centrifuged. The total protein concentration was measured using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Protein aliquots of 10 µg each were separated using SDS-PAGE. The proteins were subsequently transferred to Immobilon-P membranes (Millipore; Merck KGaA, Darmstadt, Germany), which were then blocked in Tris-buffered saline (TBS) containing 5% skim milk for 1 h at room temperature. After washing three times with TBS with tween-20 (TBS-T), the membranes were incubated overnight at 4 °C with primary antibodies against pFAK (Tyr397, #8556), FAK (#3285), pAKT (Ser473, #4060), AKT (#4691), pERK (#4370), ERK (#4696), Bim (#2933), pFoxO3a (Ser253, #9466), FoxO3a (#2497), Cleaved PARP (#5625), Cleaved Caspase 3 (#9661), pIGF-1R (#3021), IGF-1R (#9750), E-Cadherin (#3195 S), Vimentin (#5741 S) and ZEB1 (#3396 S) (1:1000 dilution; Cell Signalling Technology, Danvers, MA, USA), β-actin (#A5441) (1:1000 dilution; Sigma-Aldrich, Burlington, MA, USA), and cMyc (#C2012) (1:1000 dilution; Abcam, Cambridge MA, UK).

After being washed three times with TBS-T, the membranes were incubated for 1 h at room temperature with HRP-conjugated species-specific secondary antibodies. Immunoreactive bands were visualised using an ECL chemiluminescent detection reagent (Immobilon Western Chemiluminescent HRP Substrate; Merck‐Millipore, Darmstadt, Germany), imaged, and quantified using the ChemiDoc Imaging System. All immunoblotting experiments were performed independently at least three times.

Cell viability assay

Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells (2–3 × 10³ cells/well) were seeded in 96-well plates and incubated with the indicated concentrations of various compounds for 72 h. The CCK-8 solution was added to each well, and the medium’s absorbance (450 nm) was determined using SpectraMax® iD3 (Molecular Devices, LLC., San Jose, CA, USA) after another 4 h of incubation. The percentage growth was determined relative to the DMSO treatment. Experiments were repeated at least three times with triplicate samples.

Colony formation assay

The cells were seeded at a density of 5 × 10³ cells/well in 6-well plates. After incubation for 24 h, the cells were treated with each agent at the indicated concentrations for 15 days, and the drugs were replenished every 72 h. The plates were stained with 0.1% crystal violet, fixed in formalin, and visually examined. Representative plates from three independent experiments are shown.

Transfection experiments

Duplexed Silencer Select siRNAs against MYC (s9129) and focal adhesion kinase (FAK) (#1: PTK2HSS108800 and #2: PTK2HSS183870) were purchased from Invitrogen (Carlsbad, CA, USA). Cells (1–2 × 10⁵ cells/well) were seeded into 6-well plates. After incubation for 24 h, the cells were transfected using Lipofectamine RNAiMAX (Invitrogen) with these siRNAs (20 nmol/L) or Stealth RNAi as a negative control (Invitrogen) as a scrambled control, according to the manufacturer’s instructions. After incubation for 6 h, the medium containing Lipofectamine was replaced with the medium. Each sample was tested in triplicate in three independent experiments.

Quantitative real-time RT-PCR

The total cellular RNA was extracted from the NCI-H358 and NCI-H2122 cells treated with DMSO or avutometinib (150 nmol/L) for 24 h using Sepasol-RNA I Super G (Nacalai Tesque). Complementary DNA (cDNA) was synthesised from total RNA using ReverTra Ace^® qPCR RT Master Mix kit (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) with cDNA and TaqMan probes (Applied Biosystems) for IGF-1 (Hs01547656_m1) and GAPDH (Hs02758991_g1). The expression level of every gene was determined from standard curves to obtain a relative IGF-1/GAPDH ratio using the 2−ΔΔCT method. Each sample was tested in triplicate in three independent experiments.

Gene set variation analysis (GSVA) of cell lines using the epithelial and mesenchymal gene signature set

RNA-seq gene expression data for the nine KRAS-mutated NSCLC cell lines were retrieved from the Cell Model Passports database (https://cellmodelpassports.Sanger.Ac.uk). These data were log2-transformed from TMM-normalised count data. We calculated the epithelial and mesenchymal scores using GSVA [21]. Scoring was performed using the R package, GSVA. For each cell line (one expression data point), we computed two scores using epithelial (288 genes) and mesenchymal (188 genes) gene lists [22]. Briefly, GSVA estimates a cumulative density function for each gene using all samples, ranks genes across samples, and calculates a score between −1 and 1 for each set of genes using the Kolmogorov–Smirnov random walk statistic. The difference between the two scores was calculated and used as the epithelial-mesenchymal transition (EMT) score. Cell lines with positive (high) EMT scores were mostly mesenchymal, whereas those with negative (low) EMT scores were mostly epithelial. We conducted a hierarchical cluster analysis of the expression of signature genes in all cell lines. The distance between clusters was measured using the Ward method and visualised as a heatmap using Python and Python libraries (Pandas, Matplotlib, and Seaborn).

Quantification of cell cycle and apoptosis

Cells (1–2 × 10⁵ cells/well) were seeded into 6-well plates. After incubation for 24 h, the cells treated with each agent for 48 or 72 h were harvested via trypsinization. After washing twice with phosphate buffered saline (PBS), the cells were suspended in PBS containing 0.1% Triton X-100 and 50 g/mL propidium iodide to stain the nuclei. The cells were analysed using a BD Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA), and the number of cells in the G0-G1, S, G2-M, and sub G1 phases was quantified.

Scratch assay

NCI-H358 and NCI-H2122 cells were seeded into a 6-well plate at a concentration of 1 × 10⁵ cells/mL and cultured for 24 h at 37 °C. After nearly confluent cell monolayers were formed, a linear scratch wound was generated on each plate using a plastic pipette tip. Images of the scratched wound before treatment and 24 h after treatment were photographed to evaluate the relative migration of the cells. Representative images from three independent experiments are shown.

Cell-line-derived xenograft mouse study

Suspensions of NCI-H358 cells (4 × 10⁶ cells/mouse) were injected subcutaneously into the right flank of 6-week-old female BALB/c nu/nu mice obtained from Charles River Laboratories (Yokohama, Japan) and maintained under pathogen-free conditions. Tumours were measured twice a week using calipers, and tumour volumes were calculated using the following formula: 0.5 × lengths × width². The mice were randomly divided into four groups (n = 6 in each group) when the tumour volume reached approximately 70–120 mm³: 1) vehicle, 2) defactinib (10.0 mg/kg), 3) avutometinib (0.3 mg/kg), and 4) avutometinib + defactinib. Avutometinib and defactinib was dissolved in 20% hydroxypropyl-β-cyclodextrin/saline. Drugs were administered once daily via oral gavage up to day 28, and the mice’s body weight and general condition were monitored daily. Approval was obtained from the Institutional Review Board of the Kyoto Prefectural University of Medicine for the mice study (approval no. M2021-38) following the ARRIVE guidelines.

Quantification of immunohistochemistry staining

Formalin-fixed, paraffin-embedded tissue blocks were cut into serial 4-µm thick sections, deparaffinized, and immunostained with Ki-67 (Clone MIB-1; Dako, Agilent, Santa Clara, CA, USA). Apoptosis was quantified via the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) method using a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA), according to the manufacturer’s instructions. Hematoxylin and eosin staining was performed to identify tumour cells. Five randomly selected areas (0.1 mm²/section) containing the most positively stained cells in each section were selected using light microscopy at 400 × magnification. The percentage of cells positive for each immunohistochemical stain was assessed using the ImageJ software.

Statistical analysis

The statistical significance of differences was analysed using student’s t test and one-way ANOVA. The combination index (CI) values were calculated using CompuSyn® software (version 2.0; Biosoft, Cambridge, UK) [21] with cell viability representing the effect when exposed to the drugs at 72 h; CI < 0.7, 0.7 ≤ CI ≤ 1.2, and CI > 1.2 were judged as having synergistic, additive, and antagonistic effects, respectively. All statistical analyses were performed using GraphPad Prism Ver. 9.0 (GraphPad Software, Inc., San Diego, CA, USA). A two-sided p < 0.05 was considered statistically significant.

Dual RAF/MEK inhibitor avutometinib activates FAK and AKT and reactivates ERK

When NCI-H358 cells were treated with avutometinib at concentrations above 1.5 µmol/L for 72 h, ~45% of the cells survived (Fig. S1). To elucidate the mechanism underlying the inhibitory effect of avutometinib, we used an RTK phosphorylation array kit to evaluate the differences in the phosphorylation states of molecules in NCI-H358 cells treated with or without avutometinib for 72 h. The phosphorylation of FAK (pFAK) increased after 72 h of treatment with avutometinib (Fig. 1a). This result suggests that the FAK pathway may function as a bypass signalling pathway against RAF/MEK inhibition by avutometinib. Kinetic analysis revealed that ERK phosphorylation was remarkably inhibited by avutometinib at 3 h but reactivated at 72 h in NCI-H2122 and NCI-H358 cells (Fig. 1b). Conversely, phosphorylation of FAK and AKT was steadily increased after 72 h of avutometinib treatment in these two cell lines (Fig. 1b).

Next, we used siRNAs to evaluate the effects of FAK knockdown on the viability of NCI-H358 and NCI-H2122 cells. The knockdown of FAK expression alone did not affect cell viability. Meanwhile, the knockdown of FAK expression following treatment with avutometinib significantly decreased the viability of both cancer cell lines (Fig. 1c). These results suggest that FAK contributes to the survival of several types of NSCLC cells with KRAS mutations when the MAPK pathway is inhibited by avutometinib. Furthermore, FAK knockdown decreased the avutometinib-induced phosphorylation of AKT but not of ERK (Fig. 1d).

Avutometinib also significantly increased the IGF-1 mRNA levels in NCI-H358 and NCI-H2122 cells (Fig. 1e). Avutometinib treatment decreased c-MYC expression and enhanced IGF-1R phosphorylation over time (Fig. 1f). The knockdown of MYC expression increased the levels of IGF-1 mRNA and the phosphorylation of IGF-1R, FAK, and AKT in both NCI-H358 and NCI-H2122 cells (Fig. S2A, B).

EMT status correlates with synergistic effects of defactinib in combination with avutometinib

To characterise the EMT status in the nine KRAS-mutated lung cancer cell lines, we performed western blot analysis for E-cadherin and vimentin. Additionally, we acquired information on the type of point mutations in the KRAS oncogene status from the COSMIC database (Fig. 2a). We investigated the effects of avutometinib monotherapy on cell viability on KRAS-mutated NSCLC cells (Fig. S3A). There was no significant difference in the efficacy of avutometinib monotherapy in cell lines with epithelial and mesenchymal phenotypes (p = 0.456) or between KRAS G12C and non-G12C point mutations (p = 0.926) (Figs. 2a and S3B–D).

Moreover, we calculated the CI values of the FAK inhibitor defactinib in combination with avutometinib using CompuSyn. Among the nine KRAS-mutated NSCLC cell lines, NCI-H358, NCI-H2122, and NCI-H441 had values < 0.7. Meanwhile, the other cell lines had values between 0.7 and 1.2 (Fig. 2b). We found a strong combined effect of avutometinib and defactinib in cancer cells with an epithelial phenotype, but not in those with a mesenchymal phenotype, regardless of the presence or absence of phosphorylated FAK expression, or KRAS point mutation (G12C or not) (Fig. 2c). Therefore, the EMT status might be a biomarker for combination therapy with avutometinib and defactinib.

Next, we evaluated the EMT status in the nine KRAS-mutated NSCLC cell lines based on the mRNA expression levels of the 416 signature EMT genes. RNA-seq data were obtained from the Cell Model Passports database. Hierarchical cluster analysis revealed that the transcriptomes of cell lines were broadly classified into two groups, each corresponding to epithelial or mesenchymal cell lines, based on western blotting results (Fig. 2d). To examine the degree of EMT in these cells, we calculated an epithelial and a mesenchymal score that summarised the overall transcriptional activities of epithelial and mesenchymal genes for each cell line. These scores were based on GSVA using a list of 228 epithelial genes and 188 mesenchymal genes as signature gene sets [22]. A positive/high value for each score indicates more epithelial or mesenchymal cells. The transcriptional state of the cell lines was unevenly distributed in the epithelial and mesenchymal regions in the epithelial and mesenchymal score spaces (Fig. 2e). Therefore, the western blot result-based classification of epithelial or mesenchymal cell lines was validated via a transcriptome analysis of 416 genes related to EMT.

Furthermore, the relationship between the CI values and degree of EMT in these cell lines was examined. EMT scores were calculated as a single score representing the degree of EMT and evaluated for correlation with the CI value, as described in the Methods section. Cell lines with positive/high EMT scores are highly mesenchymal, whereas those with negative/low scores are highly epithelial. A strong positive correlation was observed between CI value and EMT score (R² = 0.7937; Fig. 2f). Therefore, cell lines with CI values above 0.7 were confirmed to be in a stronger mesenchymal state. These results indicate that the EMT status, not the FAK phosphorylation status or KRAS point mutation type, is a predictive biomarker for the efficacy of combination therapy with avutometinib and defactinib.

Defactinib sensitises epithelial phenotype cancer cells to avutometinib

We evaluated the combined effects of avutometinib and defactinib on KRAS-mutated NSCLC cells. Although avutometinib alone did not adequately reduce cell viability in both the epithelial and mesenchymal phenotypes of KRAS-mutated NSCLC cells (Fig. S3A), the combined use of avutometinib and defactinib significantly reduced the viability of epithelial cells (Figs. 2b and S4A). Moreover, continuous co-treatment for 15 days decreased the viability of epithelial-phenotype cells, but not of mesenchymal-phenotype cells (Fig. S4B).

Next, we evaluated the effects of these drugs on the expression of various proteins using western blotting. Combination treatment for 6 h inhibited the activation of FAK and AKT induced by avutometinib alone in epithelial-phenotype cells but not in mesenchymal-phenotype cells (Fig. 3a). Subsequently, we examined the alterations in the cell cycle and sub G1 populations with a combination of avutometinib and defactinib. The sub G1 population was induced by the combination of epithelial phenotype cells but not in mesenchymal phenotype cells (Fig. 3b). Conversely, the cell cycle was not obviously affected by the combination (Fig. S5). Regarding cell migration, no additional effects of defactinib were observed in cells with an epithelial phenotype (Fig. S6). Given the increase in sub G1 with combination therapy, we evaluated the apoptosis-associated Bim protein. The combination treatment for 24 h increased Bim protein levels more than avutometinib alone in NCI-H358 and NCI-H441 cells (Fig. 3c). We then assessed the effects of the combination treatment on the Bim-associated protein, FoxO3a, and found a decrease in the phosphorylation of FoxO3a induced by avutometinib alone in epithelial cells (Fig. 3c). The combination treatment for 72 h inhibited the activation of FAK and AKT induced by avutometinib alone in epithelial cells (Fig. 3d). Consistent with the flow cytometry results, combination treatment with avutometinib and defactinib for 72 h increased the cleavage of caspase 3 and PARP, markers of apoptotic cells, compared with avutometinib alone in epithelial phenotype cells (Fig. 3d). Hence, the use of defactinib in combination with avutometinib may induce apoptosis in epithelial-phenotype KRAS-mutated NSCLC cells via the FoxO3a-Bim pathway, regardless of the type of KRAS point mutation.

EMT induction reduces the activity of the combination treatment of avutometinib and defactinib

EMT plays an important role in cancer progression and is induced by TGF-β [23]. We evaluated the effects of combination therapy with avutometinib and defactinib by inducing the mesenchymal phenotype. First, NCI-H358 cells were incubated with TGF-β1 for 2 weeks to induce EMT. EMT-induced cells (H358-TGF-β cells) showed a spindle-cell shape, loss of polarity, and decreased intercellular adhesion compared to parental cells (Fig. 4a). Moreover, E-cadherin expression was decreased, while those of ZEB1 and vimentin increased in H358-TGF-β cells compared to parental cells (Fig. 4b). Regarding the cell growth and cell cycle, TGF-β-induced EMT slowed down cell growth and increased the percentage of cells in the G0-G1 phase (Fig. 4c, d).

Next, we calculated the CI values for avutometinib and defactinib in NCI-H358 and H358-TGF-β cells. CI values in H358-TGF-β cells were significantly higher than those in parental NCI-H358 cells (p = 0.012; Fig. 4e). Thus, EMT appeared to decrease the effects of avutometinib and defactinib combination therapy. When treated with avutometinib alone, FAK was activated in NCI-H358 cells but not in H358-TGF-β cells (Fig. 4f). Combination therapy with avutometinib and defactinib for 6 h inhibited the phosphorylation of AKT induced by avutometinib alone in NCI-H358 cells, but not in H358-TGF-β cells (Fig. 4f). Moreover, steady-state levels of AKT phosphorylation were higher in H358-TGF-β cells than in NCI-H358 cells. However, there was no difference in the inhibitory effect of avutometinib on phosphorylated ERK between NCI-H358 and H358-TGF-β cells (Fig. 4f). These results suggested that the additive effect of defactinib on epithelial-type KRAS-mutated cell lines treated with avutometinib was due to its contribution to the FAK-AKT-mediated feedback mechanism.

Combination therapy of avutometinib and defactinib maintains tumour regression in vivo

In vivo experiments were conducted to evaluate the effects of avutometinib and defactinib on NCI-H358 cells. Mice were treated with avutometinib alone, defactinib alone, or a combination of these therapies via daily oral gavage up to day 28. Treatment with avutometinib alone and combination therapy with avutometinib plus defactinib caused tumour regression. After treatment cessation, tumours treated with avutometinib alone exhibited rapid regrowth, while those subjected to combination therapy maintained a sustained tumour-suppressive effect. Tumour regression was observed in two mice receiving combination therapy. (Fig. S7A). Defactinib monotherapy only suppressed tumour growth without regression (Fig. 5a). No apparent adverse effects, including weight loss, were observed during the treatment period (Fig. S7B). The size and weight of the resected tumours were measured at the end of the experiment (day 49) to evaluate the effects of avutometinib and defactinib. Tumour-suppressive effect was observed with avutometinib alone and with combination therapy (Fig. 5b). The mean tumour weight in the combination therapy group was significantly lower than that in the avutometinib-treated group (p = 0.029; Fig. 5c).

To further investigate the molecular changes induced by avutometinib and defactinib, we excised tumours from mice treated with these agents for 4 days and performed western blotting and immunohistochemical staining. Similar to the in vitro assays, the in vivo experiments showed that the combination of defactinib and avutometinib inhibited the avutometinib-activated phosphorylation of FAK and AKT, and Bim expression was higher in the combination group than in the other groups (Fig. 5d). The number of Ki-67-positive proliferative tumour cells was significantly lower in the combination group than in the avutometinib alone group (p < 0.001; Fig. 5e, f). Conversely, the number of TUNEL-positive tumour cells, i.e., apoptotic cells, was significantly higher in the combination group than in the avutometinib alone group (p < 0.001; Fig. 5e, f). Thus, these in vivo analyses showed that combination therapy with avutometinib and defactinib inhibited the AKT and MAPK pathways and induced apoptosis, resulting in the regression of the epithelial phenotype in KRAS-mutated NSCLC tumours (Fig. S8).