Targeting adaptive EGFR feedback enhances the therapeutic efficacy of RMC-6236 in cholangiocarcinoma

Abstract

Background

Approximately 20% of cholangiocarcinomas harbor KRAS mutations, with codon 12 variants accounting for most of the cases. Among these, G12V is associated with a poor prognosis, whereas G12D is the most frequent subtype. RMC-6236, a novel RAS (ON) multi-selective inhibitor targeting multiple KRAS codon 12 mutations, has shown promise in early clinical studies in KRAS-mutant cholangiocarcinomas.

Methods

We evaluated the therapeutic efficacy of the novel RAS (ON) inhibitor RMC-6236 as a monotherapy and in combination with gemcitabine and the anti-EGFR antibody cetuximab in KRAS-mutant cholangiocarcinoma (CCA) models. Antitumor activity and signaling alterations were assessed using in vitro cell-based assays, western blot analysis, and in vivo xenograft models.

Results

RMC-6236 potently inhibited cell proliferation in KRAS-mutant (G12D and G12V) cell lines at nanomolar concentrations (10–30 nM) and effectively suppressed pERK signaling. Notably, RMC-6236 treatment was accompanied by increased pEGFR expression, indicating adaptive EGFR feedback activation as a potential mechanism of resistance. In vitro, combination treatments significantly enhanced growth inhibition compared with monotherapy. In xenograft models, RMC-6236 treatment induced dose-dependent tumor regression, whereas combination with cetuximab or gemcitabine further enhanced the antitumor efficacy.

Conclusion

EGFR-mediated feedback represents a targetable vulnerability in KRAS-mutant cholangiocarcinomas. Targeting EGFR signaling attenuates adaptive feedback reactivation induced by pan-RAS (ON) inhibition and supports the clinical development of combination strategies with EGFR blockade or cytotoxic chemotherapy.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s40164-026-00835-8.

Background

Cholangiocarcinoma (CCA) is one of the most lethal malignancies of the gastrointestinal tract. Although recent advances in systemic therapy, including immune checkpoint inhibitor-based regimens, have improved outcomes for patients with unresectable disease, gemcitabine-based chemotherapy remains a cornerstone of treatment, and effective molecularly targeted therapies are available only for limited patient subsets [1, 2]. Recently, molecularly targeted agents targeting fibroblast growth factor 2 (FGFR2) fusion genes, present in approximately 10–20% of CCA cases, have demonstrated clinically meaningful efficacy and improved prognosis in selected patients [3]. KRAS mutations are found in 10–20% of CCA cases [4]. Therefore, therapeutic strategies targeting KRAS mutations represent a novel and promising treatment option.

KRAS has long been considered an “undruggable” target; however, this concept has been fundamentally changed with the development of KRAS G12C inhibitors [5–7]. In lung cancer, wherein KRAS G12C mutations are frequent, KRAS G12C inhibitors, such as sotorasib and adagrasib, have been developed and shown favorable clinical efficacy. In contrast, KRAS G12C mutations are relatively rare in gastrointestinal cancers. Furthermore, gastrointestinal cancers exhibit a higher propensity for therapeutic resistance compared with lung cancer, in part because of adaptive feedback activation of upstream signaling pathways, including the epidermal growth factor receptor (EGFR) pathway [8]. In colorectal cancer, adaptive EGFR activation following KRAS inhibition has been extensively reported and has led to clinically feasible combination strategies involving EGFR antibodies [9, 10]. These limitations highlight the need for novel therapeutic strategies that target KRAS-driven gastrointestinal malignancies more broadly and effectively.

In gastrointestinal cancers, including biliary tract, pancreatic, and colorectal cancers, KRAS G12D and G12V are the predominant KRAS mutation subtypes. Among KRAS-mutant CCAs, G12D occurs most frequently (approximately 40–50% of the cases), followed by G12V (approximately 20–30% of the cases). Moreover, codon 12 KRAS mutations have been associated with poor prognosis in CCA [4]. Although KRAS G12D inhibitor MRTX1133 has demonstrated promising antitumor activity in preclinical studies, its clinical efficacy as a monotherapy has not yet been convincingly established [11]. To overcome these limitations, we focused on adaptive feedback response following KRAS inhibition and investigated combination strategies targeting EGFR, a key mediator of adaptive signaling reactivation [12–14].

Sotorasib, adagrasib, and MRTX1133 are the so-called “KRAS (OFF) inhibitors” that bind to the inactive, GDP-bound form of KRAS. RMC-6236, daraxonrasib, is a pan-RAS (ON) inhibitor capable of binding a common active pocket shared by multiple KRAS codon 12 variants, thereby offering a novel therapeutic approach to overcome these challenges. This agent was first reported at the American Association for Cancer Research Annual Meeting in 2022 and has demonstrated promising clinical activity in phase I trials for lung and pancreatic cancers, raising expectations for broader applicability across KRAS mutations [15]. Phase III clinical trials have commenced for this drug in pancreatic, lung, and colorectal cancers [16]. At the molecular level, RMC-6236 functions as a multi-selective RAS (ON) inhibitor that binds to the active GTP-bound form of RAS. Mechanistically, it promotes the formation of a tri-protein complex with cyclophilin A and RAS-GTP, sterically preventing the interaction of RAS with downstream effector proteins, such as RAF and PI3K, thereby suppressing oncogenic RAS signaling [15, 17]. Hence, this agent is different from conventional “OFF inhibitors,” which target GDP-bound KRAS. Moreover, RMC-6236 is active against a broad spectrum of KRAS codon 12 mutations, including the G12D and G12V variants [18]. Among the KRAS alterations in CCA, G12D is the most prevalent subtype, whereas G12V is associated with a poor prognosis [16]. Consequently, therapeutic strategies capable of targeting these clinically relevant KRAS variants, such as the pan-KRAS inhibitor RMC-6236, may have important therapeutic implications for patients with KRAS-mutant CCA. Recent studies have demonstrated the antitumor activity of pan-RAS (ON) inhibition in KRAS-mutant CCA and suggested adaptive feedback reactivation of upstream signaling pathways following KRAS suppression [17, 19]. However, the therapeutic significance of these adaptive feedback responses and the efficacy of clinically applicable combination strategies remain incompletely understood.

In the present study, we evaluated the efficacy of the novel RAS (ON) inhibitor RMC-6236 as a monotherapy, in combination with gemcitabine, a cytotoxic agent widely used in clinical practice, and with the anti-EGFR antibody cetuximab, which targets a key adaptive feedback pathway. By focusing on CCA, a highly aggressive malignancy with limited effective targeted therapies, we aimed to establish a preclinical rationale that can be directly translated into clinical practice and to provide clinically applicable combination strategies incorporating EGFR blockade and cytotoxic chemotherapy with pan-RAS (ON) inhibition.

Methods

Cell culture

MMNK-1 (human immortalized cholangiocyte cell line, RRID: CVCL_M266) cells were purchased from Okayama University. YSCCC (wild-type KRAS, RRID: CVCL_3629), HuH28 (wild-type KRAS; RRID: CVCL_0325), and HuCC-T1 (KRAS G12D; RRID: CVCL_0324) cells were purchased from the JCRB Cell Bank. RBE (KRAS G12V; RCB1292) cells were purchased from the RIKEN Cell Bank. All cells were maintained in RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 10% fetal bovine serum (Biowest, Nuaille, France) and 1% penicillin/streptomycin (FUJIFILM Wako Pure Chemicals Corporation, Osaka, Japan) at 37 °C in a 5% CO₂ atmosphere. All cells were routinely tested for Mycoplasma contamination using an EZ-PCR™ Mycoplasma Detection Kit (Biological Industries, Beit Haemek, Israel).

Antibodies and reagents

The following antibodies were used: monoclonal mouse FLAG (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan; #014-22383; RRID: AB_10660291), monoclonal rabbit p-Erk1/2 (Cell Signaling Technology, Danvers, MA, USA; #4376; RRID: AB_331772), monoclonal rabbit Erk1/2 (Cell Signaling Technology; #4695; RRID: AB_390779), monoclonal rabbit p-MEK1/2 (Cell Signaling Technology; #9154; RRID: AB_2138017), monoclonal rabbit MEK1/2 (Cell Signaling Technology; #8727; RRID: AB_10829473), monoclonal rabbit p-AKT (Cell Signaling Technology; #4060; RRID: AB_2315049), monoclonal rabbit AKT (Cell Signaling Technology; #4691; RRID: AB_915783), monoclonal rabbit p-EGFR (Cell Signaling Technology; #3777; RRID: AB_2096270), monoclonal rabbit EGFR (Cell Signaling Technology; #4267; RRID: AB_2246311), β-actin (Cell Signaling Technology; #4967; RRID: AB_2223041), and monoclonal rabbit Ki-67 (Cell Signaling Technology; #9129; RRID: AB_ 2687446). The secondary antibodies used were: polyclonal goat anti-mouse antibody (Dako; Agilent Technologies, Santa Clara, CA, USA; #P0447) and polyclonal goat anti-rabbit IgG (Dako; Agilent Technologies; #P0448) conjugated with horseradish peroxidase (HRP). RMC-6236 was purchased from BLD Pharmatech Ltd., (Shanghai, China). Cetuximab and gemcitabine were purchased from Merck KGaA (Darmstadt, Germany) and Selleck (Houston, TX, USA), respectively.

RMC-6236 formulation

For in vitro studies, RMC-6236 (daraxonrasib) was resuspended in dimethyl sulfoxide (DMSO) and used at a stock concentration of 1 mmol/L. For in vivo studies, RMC-6236 was resuspended in DMSO/PEG400 (FUJIFILM Wako Pure Chemicals Corporation)/Solutol HS-15 (Selleck)/pure water at a volume ratio of 10%/10%/20%/60%, respectively.

Construction and retroviral transduction of KRAS mutations

Wild-type and mutant KRAS (G12D and G12V) constructs with a C-terminal FLAG tag were generated using the In-Fusion cloning system, as previously described [12, 14]. Retroviral transduction was performed using Phoenix-AMPHO packaging cells (American Type Culture Collection, Manassas, VA, USA), and transduced YSCCC cells were selected on G418 (geneticin; Thermo Fisher Scientific, Waltham, MA, USA). The transduction efficiency was confirmed via western blotting.

Cell proliferation assay

Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) (#343–07623; Dojindo Laboratories, Kumamoto, Japan). MMNK-1, YSCCC, HuH28, HuCC-T1, and RBE cells were seeded in tissue culture-treated 96-well plates (2.0 × 10³ cells/well) and incubated at 37 °C overnight. After incubation, each agent (RMC-6236, 0.1–10³ nmol/L; cetuximab, 0.05–50 µg/mL; and gemcitabine, 0.01–30 µmol/L) or DMSO was added to the cells. After 72 h, the CCK-8 reagent (10 µL/well) was added and incubated at 37 °C for 1 h. The absorbance was measured using a plate reader at 450 nm, and cell viability was calculated relative to the absorbance of the DMSO-treated cells. Data are presented as the mean ± standard deviation (SD) of values from at least three independent biological experiments. Dose–response curves were plotted against RMC-6236 concentration on a logarithmic scale (mol/L). IC₅₀ values were calculated using nonlinear regression with a four-parameter logistic model, with the upper and lower asymptotes fixed at 100% and 0%, respectively. Additionally, the combined effects of two or three compounds were assessed using Synergy Finder (https://synergyfinder.fimm.fi), and synergy was quantified based on the Zero Interaction Potency (ZIP) score [20].

Protein sample preparation and western blotting

After drug treatment, the cells were washed with cold phosphate-buffered saline (PBS) and lysed on ice for 30 min using a RIPA Lysis Buffer System (Santa Cruz Biotechnology, Dallas, TX, USA; #sc-24948). Cell lysates were clarified via centrifugation at 10,000 × g for 10 min at 4 °C, and the resulting supernatant was collected as the total protein extract. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific; #23227). Equal amounts of protein (10–15 µg) were separated on NuPAGE 4–12% Bis-Tris gels (Thermo Fisher Scientific; #NP0322) and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween-20 for 30 min at room temperature (22–25 °C), followed by overnight incubation at 4 °C with the primary antibodies. After washing with Tris-buffered saline containing Tween 20, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (Immobilon Western HRP; Amersham ECL Prime Western Blotting Detection Reagent; Cytiva; #RPN2236). Band intensities were quantified using Image Lab Software (version 6.0.1; Bio-Rad Laboratories, Hercules, CA, USA) and normalized to β-actin levels to calculate the relative protein expression.

Single-cell RNA sequencing analysis of human intrahepatic CCA datasets

Publicly available single-cell RNA sequencing (scRNA-seq) data from human intrahepatic CCA cases were obtained from the Gene Expression Omnibus database (accession number GSE138709). Raw UMI count matrices from the tumor samples were analyzed using ScanPy in Python. After quality control to remove low-quality cells and genes, the data were normalized, log-transformed, and subjected to highly variable gene selection, principal component analysis, and harmony-based batch correction. Uniform manifold approximation and projection, as well as Leiden clustering, were used for visualization and cell clustering.

Cell types were annotated based on canonical marker genes for tumor epithelial cells, cancer-associated fibroblasts (CAFs), endothelial cells, macrophages, myeloid cells, T/natural killer (T/NK) cells, and B/plasma cells. The expression patterns of EGFR-related ligands and receptors were also examined across cell populations. Module scores for KRAS/MAPK signaling, EGFR/MAPK signaling, and proliferation-associated programs were calculated to evaluate pathway activity. Tumor epithelial cells were further subdivided and reanalyzed to assess the intratumoral heterogeneity.

Potential cell–cell communication networks were inferred using the CellChat package in R, focusing on EGF pathway interactions involving amphiregulin (AREG), epiregulin (EREG), heparin-binding EGF-like growth factor (HBEGF), EGFR, and Erb-B2 receptor tyrosine kinase 2 (ERBB2).

Establishment of RMC-6236-resistant cells

RMC-6236-resistant HuCC-T1 (HuCC-T1-R) cells were established by continuous exposure to gradually increasing concentrations of RMC-6236. Parental HuCC-T1 cells were initially treated with 0.1 nM RMC-6236, and the drug concentration was increased stepwise at weekly intervals, reaching a final concentration of 30 nM over a total exposure period of approximately 1.5 months. Following long-term drug exposure, the cells were cultured in drug-free medium to allow recovery before subsequent analyses. The RMC-6236-resistant cells were then evaluated for their sensitivity to RMC-6236 using the CCK-8 assay and for changes in EGFR and MAPK signaling via western blotting.

In vivo experiments

Xenograft model establishment

All animal experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals (8th edition) and following the guidelines of the Shinshu University Institutional Animal Care and Use Committee, under an approved protocol (No. 025011). Briefly, 6–8-week-old male BALB/c nude mice weighing 21–29 g, were purchased from CLEA Japan, Inc. (Tokyo, Japan). They were housed in an environmentally controlled room (23 ± 3°C, 55 ± 10% relative humidity) with a 12-hour light/dark cycle (09:00–21:00 light) and provided with standard food and water ad libitum. For the reagent experiment, xenograft tumors were generated via the subcutaneous injection of RBE and HuCC-T1 cells (6–8 × 10⁶ cells), mutant KRAS G12V and G12D, in 200 µL of a solution containing 50% PBS + 50% Matrigel (Corning, NY, USA) into the flanks of mice. Treatment was initiated when the average tumor volume reached 150–200 mm³. The maximal tumor burden was defined as an estimated tumor weight exceeding 10% of the animal’s total body weight. The tumor diameter was measured in two dimensions using a digital caliper, and the tumor volume was calculated using the following formula:

Tumor size was measured twice weekly.

Treatment

RMC-6236 or vehicle was orally administered to mice once daily for 21 consecutive days (n = 4 mice per group). For the single-dose study, mice were randomly assigned to groups (control, 5, 10, and 25 mg/kg). Cetuximab (50 mg/kg) was administered intraperitoneally once per week, whereas gemcitabine (10 mg/kg) was administered intraperitoneally twice per week. All mice were sacrificed via cervical dislocation under 3% isoflurane anesthesia on day 22. Tumor growth was monitored twice weekly, and mice were euthanized before tumors reached the institutional humane endpoint criteria (maximum volume < 1,500 mm³), with no evidence of ulceration or systemic distress. The animals were also euthanized early if adverse effects were observed, and body weight loss was used as a surrogate. The xenograft tumors were fixed in 4% paraformaldehyde solution (FUJIFILM Wako Pure Chemical) for further analysis.

Immunohistochemistry

The resected xenograft tumors were fixed in a 4% paraformaldehyde solution for 16 h at 4 °C and embedded in paraffin. Next, 4 μm-thick sections were prepared for immunostaining. Briefly, antigen retrieval was performed using microwaves and endogenous peroxidase activity was blocked using 3% H₂O₂. Anti-Ki-67 rabbit monoclonal antibody (Cell Signaling Technology; #9129; RRID: AB_ 2687446) and anti-phospho-EGFR (Tyr1068) rabbit monoclonal antibody (Cell Signaling Technology; #3777; RRID: AB_2096270) were used at a dilution of 1:50 and 1:100, respectively, in TBS-T supplemented with 1% bovine serum albumin. The slides were subsequently incubated with primary antibodies for 16 h at 4 °C in a humidified chamber. Histofine Simplestain MAX PO (Nichirei Bioscience, Tokyo, Japan) was used as the secondary antibody according to the manufacturer’s instructions. TUNEL staining was performed according to the manufacturer’s protocol using the in situ Apoptosis Detection Kit (Takara Bio, Shiga, Japan; #MK500). 3,3′-Diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan) was used for visualization, and hematoxylin was used for counterstaining. All slides were visualized and imaged under a CKX53 optical microscope (Olympus, Tokyo, Japan). The cell numbers and H-scores were quantified using the ImageJ software [21].

Statistical analysis

All statistical analyses were performed using R software (version 4.2.2) in the RStudio environment (RStudio, PBC, Boston, MA, USA). Statistical significance was evaluated using unpaired Student’s t-test or one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. Data are presented as means ± SD. Statistical significance was defined as p < 0.05.

Results

KRAS mutations are associated with poor prognosis in CCA

Kaplan–Meier survival analysis using GSEA CCA data revealed that patients with KRAS G12D or G12V mutations exhibited significantly worse overall survival compared with those with wild-type KRAS (p < 0.001) (Fig. 1A), highlighting the clinical relevance of targeting KRAS-driven signaling in CCA.

Fig. 1.

Impact of KRAS mutation status on prognosis and molecular effects of RMC-6236 in KRAS-mutant cholangiocarcinoma. A Kaplan–Meier survival curves from the GSEA CCA dataset showing overall survival in patients with KRAS G12D and G12V mutations or wild-type KRAS. B CCA cell lines with different KRAS status were treated with increasing concentrations of RMC-6236 for 72 h, and cell proliferation was measured using the CCK-8 assay. YSCCC and HuH-28 cells have wild-type KRAS, HuCC-T1 cells have KRAS G12D mutations, and RBE cells have KRAS G12V mutations. MMNK-1 are non-tumor cholangiocytes. IC50 values for each cell line are indicated in the figure. C CCA cell lines with KRAS G12D and G12V mutations or wild-type KRAS were treated with 1–100 nmol/L RMC-6236 for 4 h. Western blot analysis was performed to analyze the expression of proteins related to the EGFR, RAS/MAPK, and PI3K pathways. β-actin was used as a loading control. D Cell lines with KRAS G12D- and G12V-mutations or wild-type KRAS were treated with 10 nmol/L RMC-6236 for 0, 4, 24, and 48 h. Western blot analysis was performed to analyze protein levels related to the EGFR, RAS/MAPK, and PI3K pathways. β-actin was used as a loading control. The expected molecular weights of the detected proteins are as follows: EGFR and phospho-EGFR, ~170 kDa; ERK1/2 and phospho-ERK1/2, 44/42 kDa; MEK1/2 and phospho-MEK1/2, 45/44 kDa; AKT and phospho-AKT, ~60 kDa; β-actin, ~42 kDa. CCA Cholangiocarcinoma, CCK-8 Cell Counting Kit-8, CTR control, EGFR Epidermal growth factor receptor, KRAS Kirsten rat sarcoma viral oncogene homolog, RMC RMC-6236

KRAS-mutant CCA shows preferential sensitivity toward RMC-6236 in vitro

To evaluate the mechanistic basis for the differential clinical responses to treatment with RMC-6236 in CCA, we analyzed the effect of this drug in a panel of cell lines with wild-type KRAS (YSCCC and HuH-28), KRAS G12D (HuCC-T1) and G12V (RBE) mutations, and in non-tumor cells (MMNK-1) (Fig. 1B). Dose-response cell proliferation assay revealed that RMC-6236 exhibited antitumor effects at 3–10 nmol/L in CCA cell lines harboring KRAS G12D or G12V mutations. In contrast, wild-type KRAS cells responded only to higher concentrations, with tumor-suppressive effects observed at approximately 100 nM. Furthermore, at 1000 nmol/L, RMC-6236 inhibited the proliferation of the immortalized normal cholangiocyte cell line, MMNK-1, which indicated potential cytotoxicity at this concentration. Figure 1C shows western blotting of the change in dose-response signal using RMC-6236 in RBE (G12V), HuCC-T1 (G12D), and YSCCC (wild-type) cells. In RBE and HuCC-T1 cell lines, pERK and pAKT were suppressed by RMC-6236 over a concentration range of 3–10 nmol/L.

RMC-6236 induces EGFR-mediated feedback reactivation in KRAS-mutant CCA

To explore the potential role of feedback reactivation in RMC-6236 treatment, CCA cell lines with the KRAS G12X mutation were treated with RMC-6236, and the drug response of each model was assessed from 4 to 48 h. Western blotting revealed decreased pERK and pAKT levels within 4 h in RBE (G12V) and HuCC-T1 (G12D) cells, and the effect was sustained for 24–48 h, accompanied by increased pEGFR levels, suggesting EGFR feedback activation (Fig. 1D, S1A and B). These data suggest that adaptive EGFR feedback reactivation occurs following RMC-6236 in CCA cells.

KRAS mutations drive feedback reactivation following RMC-6236 treatment

To identify the impact of KRAS mutations on feedback reactivation following KRAS G12X inhibition in CCA, we assessed the effects of RMC-6236 using KRAS-transduced CCA cells. Wild-type and mutant KRAS genes (G12V and G12D) carrying a C-terminal FLAG tag were inserted into pDON-5Neo DNA vectors and retrovirally transduced into YSCCC human CCA cells expressing wild-type KRAS (Fig. 2A). Comparable baseline proliferation was observed among the transduced cell lines (Fig. S2A). Consistent with the parental cell lines, RMC-6236 significantly suppressed the proliferation of KRAS G12V- and G12D-transduced cells, whereas minimal effects were observed in wild-type KRAS cells (Fig. 2B).

Fig. 2.

Feedback reactivation of the RAS/MAPK pathway following RMC-6236 treatment in KRAS gene–transduced cholangiocarcinoma cells. Wild-type and mutant KRAS (G12V and G12D) were transduced into YSCCC. A Transduction was confirmed via immunoblotting with an anti-FLAG antibody. B KRAS-transduced cells were treated with 10 nmol/L RMC-6236 for 72 h, and cell proliferation was measured using the CCK-8 assay. C KRAS-transduced cells were treated with 10 nmol/L RMC-6236 for 0, 4, and 24 h. Western blot analysis was performed for proteins related to the EGFR, RAS/MAPK, and PI3K pathways. β-actin was used as a loading control. D Single-cell RNA sequencing data from human intrahepatic CCA samples. Cell-type annotation identified the major cellular populations. E Dot plot showing the sites of EGFR expression and EGFR ligands. F Module score analysis demonstrating the activation of the RAS/MAPK and EGFR/MAPK signaling programs within tumor epithelial cells. G, H Cell–cell communication analysis predicted potential ligand–receptor interactions.The expected molecular weights of the detected proteins are as follows: FLAG-tagged KRAS, ~21 kDa; phospho-EGFR and EGFR, ~170 kDa; phospho-ERK1/2 and ERK1/2, 44/42 kDa; MEK1/2 and phospho-MEK1/2, 45/44 kDa; AKT and phospho-AKT, ~60 kDa; β-actin, ~42 kDa. Data in B are presented as mean ± SD from four independent experiments, each performed with three technical replicates per condition. *p < 0.05, **p < 0.01. CCA Cholangiocarcinoma, EGFR Epidermal growth factor receptor, KRAS Kirsten rat sarcoma viral oncogene homolog, SD Standard deviation

Because EGFR functions upstream of the RAS-MAPK pathway, we next examined whether pharmacological inhibition of KRAS signaling induced adaptive changes in EGFR signaling. In KRAS G12V- and G12D-transduced cells, RMC-6236 rapidly suppressed ERK phosphorylation within 4 h, and this suppression was maintained at 24 h (Fig. 2C). In contrast, phosphorylation of EGFR progressively increased after treatment, resulting in an increased pEGFR/EGFR ratio (Fig. S2B, C). These adaptive changes were not observed in wild-type KRAS-transduced cells. Together, these findings suggest that inhibition of RAS-MAPK signaling by RMC-6236 induces adaptive activation of EGFR signaling specifically in KRAS-mutant CCA cells.

Cell–cell communication analysis supports ligand-driven EGFR activation

To obtain supporting evidence for EGFR reactivation in human tumors, as observed in our in vitro models, we analyzed publicly available scRNA-seq data from human intrahepatic CCA samples. Cell type annotation identified major cellular populations, including tumor epithelial cells, CAFs, endothelial cells, macrophages, myeloid cells, T/NK cells, and B/plasma cells (Fig. 2D). EGFR expression was predominantly detected in tumor epithelial cells, whereas several EGFR ligands, including AREG, EREG, and HBEGF, were expressed in multiple stromal and immune cell populations (Fig. 2E). Consistent with this expression pattern, module score analysis revealed the activation of both KRAS/MAPK and EGFR/MAPK signaling programs within tumor epithelial cells (Fig. 2F).

Furthermore, cell–cell communication analysis predicted ligand–receptor interactions involving HBEGF–EGFR, EREG–EGFR, and AREG–EGFR from the surrounding stromal and immune cell populations toward tumor epithelial cells (Fig. 2G and H). These findings suggest that the tumor microenvironment in human intrahepatic CCA is a potential source of EGFR ligands capable of stimulating EGFR signaling in tumor cells.

EGFR blockade enhances the efficacy of RMC-6236 in KRAS-mutant CCA

We next investigated EGFR feedback reactivation induced by RMC-6236 and evaluated the efficacy of the combined use of anti-EGFR antibodies and RMC-6236 in KRAS G12X cells. KRAS G12V and G12D cells were treated with RMC-6236 and/or cetuximab for 72 h, and their proliferation was measured using the CCK-8 assay (Fig. 3A). The combined efficacy of RMC-6236 and cetuximab was significantly higher in both cell lines, but not in cells with wild-type KRAS. Synergy analysis revealed that the combination exhibited the highest efficacy at concentrations of 1–10 nmol/L for RMC-6236 and 0.1–10 µg/mL for cetuximab (Fig. 3B). Changes in EGFR and MAPK signaling were assessed using western blot analysis (Fig. 3C). pERK rebound was observed following treatment with RMC-6236 alone for 24 h, accompanied by the upregulation of pEGFR. The addition of cetuximab suppressed the reactivation of EGFR, and pERK expression was reduced upon combined treatment with RMC-6236 and cetuximab. This was also observed in the KRAS gene-transduced YSCCC cell lines, G12V and G12D (Fig. S3A–C).

Fig. 3.

Synergistic anti-tumor effects of combined RMC-6236 and cetuximab treatment on KRAS-mutant cholangiocarcinoma cells. A RBE (KRAS G12V), HuCC-T1 (KRAS G12D), and YSCCC (wild-type KRAS) cells were treated for 72 h with RMC-6236 (10 nmol/L) and/or cetuximab (5 µg/mL), and cell proliferation was assessed using the CCK-8 assay. B Synergy landscape map for the effect of the RMC-6236 and cetuximab combination on RBE and HuCC-T1 cells. The concentration of each drug is shown on its respective axis. The color scale represents the ZIP score, where red indicates strong synergy and green indicates antagonism. C RBE, HuCC-T1, and YSCCC cells were treated with RMC-6236 (10 nmol/L) and/or cetuximab (5 µg/mL) for 24 h, and western blot analysis of the EGFR and RAS/MAPK pathway proteins was performed. β-actin was used as a loading control. D Effects of RMC-6236, with or without cetuximab, on the proliferation of RMC-6236-resistant HuCC-T1 cells (HuCC-T1-R). HuCC-T1-R cells were treated with the indicated concentrations of RMC-6236 in the presence or absence of cetuximab (5 μg/mL) for 72 h, and cell proliferation was assessed using the CCK-8 assay. E Effects of RMC-6236 and cetuximab on EGFR and MAPK signaling in HuCC-T1-R cells. HuCC-T1-R cells were treated with RMC-6236 and/or cetuximab for 24 h, and the expression of EGFR and MAPK pathway proteins was analyzed via western blotting. The expected molecular weights of the detected proteins are as follows: EGFR and phospho-EGFR, ~170 kDa; ERK1/2 and phospho-ERK1/2, 44/42 kDa; MEK1/2 and phospho-MEK1/2, 45/44 kDa; AKT and phospho-AKT, ~60 kDa; β-actin, ~42 kDa. Data are presented as mean ± SD from three independent experiments, each performed with three technical replicates per condition. *p < 0.05, **p < 0.01. CTR control, Cet cetuximab, CCK-8 Cell Counting Kit-8, EGFR Epidermal growth factor receptor, KRAS Kirsten rat sarcoma viral oncogene homolog, RMC RMC-6236, ZIP score Zero Interaction Potency score, SD Standard deviation

Cetuximab restores sensitivity to RMC-6236 in acquired-resistant HuCC-T1 cells

To further investigate whether EGFR blockade could enhance the activity of RMC-6236 in an acquired-resistant setting, we established an RMC-6236-resistant derivative of HuCC-T1 cells (HuCC-T1-R) through prolonged exposure to gradually increasing concentrations of RMC-6236. We then evaluated the effects of RMC-6236, with or without cetuximab, on HuCC-T1-R cells. The addition of cetuximab (5 µg/mL) significantly enhanced the growth-inhibitory effect of RMC-6236 across the concentration range of 1–100 nM compared with RMC-6236 monotherapy (Fig. 3D). We next examined EGFR and MAPK signaling via western blotting. Combined treatment with RMC-6236 and cetuximab reduced both EGFR and ERK phosphorylation compared with RMC-6236 monotherapy (Fig. 3E). These findings suggest that EGFR blockade may enhance the activity of RMC-6236 in acquired-resistant CCA cells, potentially through the suppression of EGFR–MAPK signaling.

EGFR blockade enhances the antitumor efficacy of RMC-6236 in KRAS-mutant CCA in vivo

We generated murine xenografts of KRAS G12V and G12D cells and evaluated the efficacy of RMC-6236 in vivo (Fig. 4A). First, we examined the antitumor effects of RMC-6236 at different concentrations: 5, 10, and 25 mg/kg/day. RMC-6236 showed dose-dependent antitumor effects (Fig. 4B). We then evaluated whether there was a synergistic effect of RMC-6236 and cetuximab. Mice were treated with RMC-6236 (5 mg/kg/day) and/or cetuximab (50 mg/kg/week). The combination of RMC-6236 and cetuximab showed significant growth inhibition compared with RMC-6236 or cetuximab alone (Fig. 4C and D, S4A). Immunohistochemical analysis of subcutaneous tumor tissues showed that the Ki-67 positivity index was significantly reduced by the RMC-6236 and cetuximab combination compared with that in the case of RMC-6236 monotherapy. Furthermore, pEGFR immunohistochemical staining demonstrated that the pEGFR H-score was significantly lower in the RMC-6236 plus cetuximab treatment than in the RMC-6236 monotherapy, indicating that cetuximab effectively suppressed the adaptive EGFR activation induced by RMC-6236 in vivo (Fig. 4E and F, S4B and C). Similarly, TUNEL staining revealed increased apoptosis in tumors treated with a combination of RMC-6236 and cetuximab (Fig. 4E and G). Fluctuations in body weight were within 10% for all mice throughout the experiment (Fig. S4D).

Fig. 4.

EGFR blockade enhances the antitumor efficacy of RMC-6236 in KRAS-mutant cholangiocarcinoma in vivo. A Murine xenografts from HuCC-T1 (KRAS G12D) cells and the course of treatment. B Antitumor effects of RMC-6236 at different concentrations: 5, 10, and 25 mg/kg/day, in G12D cells. C In vivo treatment with the RMC-6236 and cetuximab combination. Mice were treated with RMC-6236 (5 mg/kg/day) and/or cetuximab (50 mg/kg/week). D RMC-6236 plus cetuximab combination showed significant growth inhibition compared to RMC-6236 or cetuximab alone. E Representative images of hematoxylin and eosin staining, Ki-67 and pEGFR immunohistochemistry, and TUNEL staining in subcutaneous tumor tissues. F Ki-67 positivity index was significantly reduced by the RMC-6236 and cetuximab combination compared with that in the case of RMC-6236 monotherapy. G TUNEL-positive cells were significantly increased by the RMC-6236 and cetuximab combination compared with that in the case of RMC-6236 monotherapy. Data are presented as mean ± SD. Data in (F) and G for each tumor, positively stained cells were quantified in five randomly selected high-power fields. *p < 0.05, **p < 0.01. CTR control, EGFR Epidermal growth factor receptor, KRAS Kirsten rat sarcoma viral oncogene homolog, RMC RMC-6236, SD Standard deviation, TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling

Combination therapy of RMC-6236 and gemcitabine for CCA

Based on the clinical guidelines for CCA therapy, we evaluated the synergistic effects of RMC-6236 and gemcitabine on KRAS G12V and G12D cells. A synergy landscape map for the effect of the RMC-6236 and gemcitabine combination on KRAS G12V and G12D cells is shown in Fig. 5. The combination was effective in both cells when the concentration of gemcitabine was 0.01–0.3 µmol/L and that of RMC-6236 was 1–30 nmol/L (Fig. 5A and B, S5A and B), with significantly higher tumor-suppressive effects compared with those of 3 nmol/L RMC-6236 alone or 0.1 µmol/L gemcitabine alone (Fig. 5C and D). Changes in the EGFR and MAPK pathways after 24 h of treatment were determined via western blot analysis (Fig. 5E). The pEGFR expression was reduced more with the combination therapy than with gemcitabine alone.

Fig. 5.

Synergistic anti-tumor effects of RMC-6236 and gemcitabine combination in KRAS-mutant cholangiocarcinoma cells. A, B Synergy landscape map for the effects of the RMC-6236 and gemcitabine combination on RBE (KRAS G12V) (A) and HuCC-T1 (KRAS G12D) (B) cells. The concentration of each drug is shown on its respective axis. The color scale represents the ZIP score, where red indicates strong synergy and green indicates antagonism. C, D CCK-8 assay confirming significantly greater growth inhibition in the most synergistic region identified by the ZIP model of RBE (C) and HuCC-T1 (D) cells. E CCA cell lines were treated with RMC-6236 and/or gemcitabine for 24 h. Results of the western blot analysis for the EGFR and RAS/MAPK pathway are shown, with β-actin as a loading control. Expected molecular weights of the detected proteins are as follows: EGFR and phospho-EGFR, ~170 kDa; ERK1/2 and phospho-ERK1/2, 44/42 kDa; MEK1/2 and phospho-MEK1/2, 45/44 kDa; AKT and phospho-AKT, ~60 kDa; β-actin, ~42 kDa. Data are presented as mean ± SD from three independent experiments, each performed with three technical replicates per condition. **p < 0.01. CCA Cholangiocarcinoma, CCK-8 Cell Counting Kit-8, CTR control, EGFR Epidermal growth factor receptor, Gem gemcitabine, KRAS Kirsten rat sarcoma viral oncogene homolog, RMC RMC-6236, SD Standard deviation, ZIP Zero Interaction Potency

Combination of RMC-6236 and gemcitabine enhances antitumor efficacy in KRAS-mutant CCA xenograft models

We also generated murine xenografts of KRAS G12V and G12D cells and tested the efficacy of RMC-6236 and gemcitabine in vivo (Fig. 6A). Furthermore, we evaluated whether RMC-6236 and gemcitabine exerted a synergistic effect. The dose of RMC-6236 was 5 mg/kg/day, whereas that of gemcitabine was 10 mg/kg twice a week. The RMC-6236 and gemcitabine combination significantly inhibited the growth compared with RMC-6236 monotherapy (Fig. 6B and C, and S5C). Immunohistochemical analyses of the subcutaneous tumor tissues showed that the Ki-67 positivity index was significantly reduced by the RMC-6236 and gemcitabine combination compared with that in the case of RMC-6236 monotherapy (Fig. 6D and E). TUNEL staining demonstrated a significant increase in the number of apoptotic cells in the combination treatment group compared with that in the gemcitabine monotherapy group (Fig. 6F). The fluctuation in body weight was within 10% for all mice throughout the experiment (Fig. S5D).

Fig. 6.

Combination of RMC-6236 and gemcitabine enhances antitumor efficacy in KRAS-mutant cholangiocarcinoma xenograft models. A RMC-6236 and gemcitabine efficacy was assessed using murine xenografts from KRAS G12D cells in vivo. B Time-course analysis of tumor volume in response to treatment with RMC-6236 and gemcitabine. C RMC-6236 plus gemcitabine combination showed significant growth inhibition compared to that achieved with RMC-6236 treatment alone. D Representative images of subcutaneous tumor tissue sections subjected to hematoxylin and eosin staining, Ki-67 immunohistochemistry, and TUNEL staining. E Ki-67-positive index was significantly reduced by the RMC-6236 and gemcitabine combination compared with that in the case of RMC-6236 monotherapy. F TUNEL staining demonstrated a significant increase in the number of apoptotic cells in the combination treatment group compared with that in the gemcitabine monotherapy group. Data are presented as mean ± SD. For each tumor, positively stained cells were quantified in five randomly selected high-power fields. *p < 0.05. CTR control, KRAS Kirsten rat sarcoma viral oncogene homolog, RMC RMC-6236, SD Standard deviation, TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling

Triplet therapy with RMC-6236, cetuximab, and gemcitabine shows limited efficacy and incomplete MAPK pathway suppression in KRAS-mutant CCA

Next, we evaluated the efficacy of a triplet therapy with RMC-6236, cetuximab, and gemcitabine in CCA cells with KRAS G12X mutation. Cell proliferation was measured using the CCK-8 assay. The three-drug combination significantly inhibited the growth of KRAS G12V and G12D cells compared with RMC-6236 alone or in combination with cetuximab or gemcitabine (Fig. 7A and B). In contrast, combination therapy with gemcitabine and cetuximab showed antagonism, and the efficacy of the three-drug therapy was observed only at limited concentrations (Fig. 7C and D, S6A and B). Next, KRAS G12X cells were treated for 24 h, followed by analysis of the EGFR and MAPK pathways using western blotting (Fig. 7E, S6C). Rebinding to pMEK and pERK was observed after a single treatment with RMC-6236 for 24 h. pERK expression was suppressed when the cells were treated with RMC-6236, cetuximab, or gemcitabine, and the expression of pEGFR was downregulated. However, combination therapy with gemcitabine and cetuximab restored pMEK and pERK expression. In the triplet therapy, the upregulation of pEGFR and rebound of pMEK and pERK continued.

Fig. 7.

Triplet-therapy exhibits limited efficacy and incomplete MAPK pathway suppression in KRAS-mutant cholangiocarcinoma. A, B Effect of triplet therapy with RMC-6236, cetuximab, and gemcitabine on RBE (KRAS G12V) and HuCC-T1 (KRAS G12D) cells was measured using the CCK-8 assay. C, D The efficacy of the three-drug therapy was observed only at limited concentrations in both cell lines. E KRAS G12V cells were treated with RMC-6236, cetuximab, and/or gemcitabine for 24 h, followed by analysis of EGFR and MAPK pathways using western blot analysis, with β-actin as a loading control. F Antitumor effects of RMC-6236 plus cetuximab, with or without gemcitabine, in a xenograft model. Mice bearing KRAS-mutant CCA xenografts were treated with RMC-6236 plus cetuximab or with the triplet combination of RMC-6236, cetuximab, and gemcitabine. The expected molecular weights of the detected proteins are as follows: EGFR and phospho-EGFR, ~170 kDa; ERK1/2 and phospho-ERK1/2, 44/42 kDa; MEK1/2 and phospho-MEK1/2, 45/44 kDa; AKT and phospho-AKT, ~60 kDa; β-actin, ~42 kDa. Data are presented as mean ± SD from three independent experiments, each performed with three technical replicates per condition. *p < 0.05, **p < 0.01. Gem gemcitabine, Cet cetuximab, EGFR Epidermal growth factor receptor, KRAS Kirsten rat sarcoma viral oncogene homolog, RMC RMC-6236, SD Standard deviation

To further evaluate whether the addition of gemcitabine provides additional therapeutic benefit to RMC-6236 plus cetuximab in vivo, we compared the antitumor effects of the two- and three-drug regimens in a xenograft model. Both the therapies effectively suppressed tumor growth; however, no significant difference in tumor growth inhibition was observed between RMC-6236 plus cetuximab and the triplet therapy (Fig. 7F, S6D). These findings indicate that the addition of gemcitabine does not provide any significant additional antitumor benefit over RMC-6236 plus cetuximab in this model.

Discussion

Although recent advances in KRAS-targeted therapies have enabled effective inhibition of oncogenic KRAS, accumulating evidence suggests that adaptive feedback reactivation of upstream signaling pathways can attenuate therapeutic responses and limit their clinical benefit. Consistent with this, feedback-mediated resistance to KRAS inhibitors has been reported in other gastrointestinal tumors, such as colorectal and pancreatic cancers [8, 12]. KRAS (OFF) inhibitors often lead to treatment resistance through mutations in the switch II pocket, secondary mutations in RAS isoforms, and activation of upstream receptor tyrosine kinases. To address these limitations, we used RMC-6236 (daraxonrasib), a multi-selective RAS (ON) tricomplex inhibitor that binds to cyclophilin A and inhibits the active GTP-bound state of RAS, in this study [15]. RMC-6236 exhibits potent inhibitory effects and antitumor activity against KRAS G12X mutant variants (i.e., codon 12 substitutions) and has demonstrated antitumor efficacy in a phase III clinical trial in pancreatic cancer [16]. With the use of RMC-6236, substantial improvements in therapeutic outcomes have been anticipated in patients with gastrointestinal cancers harboring KRAS G12X mutations. In our study, RMC-6236 demonstrated anti-tumor effects in CCA cells, but it also induced reactivation of EGFR and the MAPK pathway. These findings are consistent with those of a recent Cancer Cell study demonstrating the antitumor activity of pan-RAS (ON) inhibition in KRAS-mutant CCA while simultaneously highlighting adaptive reactivation of upstream receptor tyrosine kinase signaling following suppression of the KRAS pathway [17]. This adaptive reactivation of receptor tyrosine kinase signaling is a recognized escape mechanism in which the loss of ERK-dependent negative feedback promotes upstream signaling, thereby restoring the MAPK pathway [22, 23] and driving adaptive signaling response following KRAS inhibition [22, 24].

Our single-cell transcriptomic analysis of human CCA provides an additional mechanistic framework for understanding adaptive signaling within the tumor microenvironment. EGFR expression is localized in tumor epithelial cells, whereas ligands, such as AREG, EREG, and HBEGF, are expressed in multiple stromal and immune cell populations. This suggests that the tumor microenvironment (TME) provides a paracrine source of EGFR ligands capable of stimulating tumor cells. Furthermore, we identified significant transcriptional heterogeneity within the epithelial compartment, specifically in the expression of the EGFR/MAPK pathway genes. This heterogeneity indicates that certain tumor cell subpopulations may possess differential capacities to engage adaptive EGFR-mediated feedback signaling following MAPK pathway inhibition. Together, these TME interactions and intratumoral heterogeneity likely cooperate to facilitate EGFR-mediated feedback observed in our experimental models. Consequently, concomitant medication is essential for maximizing the therapeutic potential of RMC-6236. The efficacy of an EGFR inhibitor and RMC-7977 combination has been reported for lung cancer [25]. Moreover, recent clinical studies have demonstrated promising activity of daraxonrasib (RMC-6236) across KRAS G12X-mutant solid tumors. Early clinical data presented at the AACR Annual Meeting 2026 showed encouraging results for the efficacy and manageable safety profile in patients with metastatic pancreatic ductal adenocarcinoma (PDAC). Favorable antitumor activity and acceptable tolerability were also reported for a combination of daraxonrasib and gemcitabine plus nab-paclitaxel (GnP) in metastatic PDAC [26, 27]. Based on these encouraging findings, randomized phase III studies evaluating daraxonrasib support the translational relevance of pan-RAS (ON) inhibition and provide a strong rationale for exploring daraxonrasib-based combination approaches in other KRAS-driven malignancies [28, 29], including CCA.

Recent studies have established the therapeutic potential of pan-RAS (ON) inhibition in KRAS-mutant CCA and suggested adaptive reactivation of upstream receptor tyrosine kinase signaling following the KRAS pathway suppression [17]. Building upon these findings, we further demonstrate that RMC-6236 induces adaptive EGFR feedback reactivation in KRAS-mutant CCA cells and that pharmacologic EGFR blockade with cetuximab effectively suppresses this signaling response and enhances the antitumor activity of RMC-6236. These findings provide additional mechanistic insights into adaptive feedback signaling following pan-RAS (ON) inhibition and support EGFR-directed combination strategies as a potential therapeutic approach in KRAS-mutant CCA.

Activating EGFR mutations are uncommon in gastrointestinal malignancies. In pancreatic adenocarcinoma, EGFR mutation was detected in 4.3% of KRAS wild-type tumors and in 0.35% of KRAS-mutated tumors [30]. Given that activating EGFR mutations are uncommon in CCA, the rationale for combining cetuximab with RMC-6236 is distinct from that of EGFR-targeted therapy in EGFR-mutant cancers. Rather than targeting constitutive EGFR oncogenic signaling, cetuximab is intended to suppress adaptive EGFR activation induced by KRAS inhibition.

We also evaluated the efficacy of combining RMC-6236 with gemcitabine and observed synergistic antitumor effects in KRAS-mutant models. Gemcitabine-based chemotherapy remains a cornerstone of first-line treatment for advanced biliary tract cancer, originally established by the pivotal ABC-02 trial and subsequent guideline updates demonstrating its survival benefits [31, 32]. More recently, the TOPAZ-1 trial demonstrated a survival benefit with the addition of durvalumab to gemcitabine and cisplatin, leading to the adoption of chemoimmunotherapy as a standard first-line treatment for advanced biliary tract cancer [1]. Furthermore, the KEYNOTE-966 trial showed that pembrolizumab combined with gemcitabine and cisplatin significantly improved the overall survival compared with that achieved with chemotherapy alone [2]. Despite these advances, outcomes remain unsatisfactory, particularly in patients harboring KRAS mutations. In this context, evaluating whether novel KRAS-targeted agents can further enhance the therapeutic efficacy of gemcitabine-based treatment remains of considerable clinical relevance. In contrast, we confirmed that gemcitabine combined with cetuximab produced antagonistic effects in line with previous clinical observations in biliary tract cancer [33, 34]. These clinical data underscore the complexity of upstream EGFR blockade in a disease frequently driven by downstream KRAS mutations. Notably, the limited efficacy of anti-EGFR therapies in unselected biliary tract cancer populations suggests that EGFR signaling alone may not represent a dominant oncogenic driver in most cases. Our results indicate that the antagonism observed with the gemcitabine/cetuximab combination can be overcome by introducing RMC-6236 as a third agent. Triple combination therapy restored and further enhanced the antitumor activity, suggesting that the direct suppression of active KRAS mitigates escape signaling and feedback activation induced by EGFR inhibition. This observation highlights a mechanistic distinction: whereas cetuximab alone cannot surmount oncogenic KRAS signaling, RMC-6236 effectively blocks the downstream effector pathways necessary for tumor survival, thereby allowing both gemcitabine and cetuximab to exert their therapeutic effects without mutual interference. These findings are consistent with recent advances in precision oncology demonstrating that adaptive signaling substantially limits the efficacy of targeted therapies. In BRAF V600E-mutant colorectal cancer, feedback activation of EGFR following BRAF inhibition provided the rationale for combined BRAF and EGFR inhibition, which significantly improved clinical outcomes [35]. More recently, addition of cytotoxic chemotherapy to this targeted combination further enhanced the therapeutic efficacy, establishing a new first-line treatment paradigm [36].

We noted that the RMC-6236 and cetuximab combination elicited a more reproducible and concentration-dependent antitumor effect than the triple-agent regimen. A rebound in the MAPK pathway and upregulation of EGFR was also observed with triple therapy. These findings suggest that RAS (ON) inhibition effectively attenuates downstream oncogenic signaling and stabilizes the therapeutic effects of EGFR blockade. Importantly, our in vivo xenograft models confirmed the antitumor efficacy of RMC-6236–based therapy, providing robust preclinical support for this treatment strategy. Gemcitabine is an established therapeutic agent for CCA. We, therefore, propose that the combination of RMC-6236 and cetuximab represents a promising therapeutic approach for KRAS-mutant CCA and warrants further evaluation in well-designed clinical trials to establish its safety and clinical efficacy.

This study had certain limitations. First, analyses were conducted using a limited number of KRAS-mutant CCA cell lines, which may not fully represent the molecular diversity of the disease. Second, although we evaluated major downstream signaling pathways, more comprehensive mechanistic studies are required to clarify how RMC-6236 modifies the interaction between gemcitabine and cetuximab. Third, although we demonstrated that cetuximab enhanced the activity of RMC-6236 in an acquired-resistant HuCC-T1 cell model, the molecular mechanisms underlying the development of acquired resistance and the role of sustained EGFR signaling during resistance acquisition remain to be fully elucidated. Finally, further preclinical studies, including those using expanded in vivo models, will help confirm the generalizability of these observations.

Conclusion

In conclusion, RMC-6236 demonstrated potent antitumor activity against KRAS-mutant CCA cells and its efficacy was further enhanced by EGFR blockade or chemotherapy. These findings support RMC-6236 as a promising therapeutic option for KRAS-mutant CCA; however, additional preclinical studies and well-designed clinical trials are essential to determine its safety, feasibility, and clinical applicability.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AKT

Protein kinase B

ANOVA

Analysis of variance

AREG

Amphiregulin

BCA

Bicinchoninic acid assay

CAF(s)

Cancer–associated fibroblast(s)

CCA

Cholangiocarcinoma

CCK

8–Cell Counting Kit–8

DMSO

Dimethyl sulfoxide

EGFR

Epidermal growth factor receptor

ERBB2

Erb–B2 receptor tyrosine kinase 2

EREG

Epiregulin

ERK

Extracellular signal–regulated kinase

FGFR2

Fibroblast growth factor receptor 2

GDP

Guanosine diphosphate

GnP

Gemcitabine plus nab–paclitaxel

GTP

Guanosine triphosphate

HBEGF

Heparin–binding EGF–like growth factor

HRP

Horseradish peroxidase

KRAS

Kirsten rat sarcoma viral oncogene homolog

MAPK

Mitogen–activated protein kinase

MEK

Mitogen–activated protein kinase kinase

PBS

Phosphate–buffered saline

RIPA

Radioimmunoprecipitation assay buffer

RMC

6236–Pan–RAS (ON) inhibitor (daraxonrasib)

RPMI

Roswell Park Memorial Institute (medium)

scRNA

seq–Single–cell RNA sequencing

SD

Standard deviation

TME

Tumor microenvironment

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labeling

UMI

Unique molecular identifier

ZIP score

Zero interaction potency score

Author contributions

SY and NH conceived and designed the study. SY, KY, MK, SN, YY, and SM performed the experiments and collected the data. SY and NH analyzed and interpreted the data. HT, MK, NI, TS, AS, and YS contributed to data interpretation and provided scientific supervision. SY drafted the manuscript. NH, MK and YS critically revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported in part by the Japan Society for the Promotion of Science KAKENHI (grant no. 24K18534, to N. Hondo). The funders had no role in the design of the study; collection, analysis, and interpretation of data; writing of the manuscript; or the decision to publish the results.

Data availability

Publicly available single-cell RNA sequencing (scRNA-seq) data from human intrahepatic CCA cases were obtained from the Gene Expression Omnibus database (accession number GSE138709).

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Shinshu University Institutional Animal Care and Use Committee (protocol No. 025011) and conducted in accordance with institutional guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.