EGFR feedback blockade boosts pan-RAS inhibitor RMC-6236 in cholangiocarcinoma models

A cholangiocarcinoma is among the most lethal malignancies of the gastrointestinal tract, and for most patients with unresectable disease, gemcitabine-based chemotherapy remains a cornerstone of treatment even as immune checkpoint inhibitor regimens have improved outcomes. Molecularly targeted therapies exist only for limited subsets, notably FGFR2 fusion genes, which are present in roughly 10 to 20 percent of cases. KRAS mutations occur in 10 to 20 percent of cholangiocarcinomas, and codon 12 variants account for most of them. Within this group, G12D is the most frequent subtype, making up approximately 40 to 50 percent of KRAS-mutant cases, while G12V accounts for roughly 20 to 30 percent and is associated with a poor prognosis. A new preclinical study now reports that the pan-RAS inhibitor RMC-6236, also known as daraxonrasib, potently suppresses KRAS-mutant cholangiocarcinoma cells but simultaneously triggers an adaptive feedback loop through the epidermal growth factor receptor, and that blocking this loop with the anti-EGFR antibody cetuximab or pairing the drug with gemcitabine substantially strengthens the antitumor effect in cell and mouse models.

The research, published in Experimental Hematology & Oncology, was motivated by a long-standing therapeutic gap. KRAS was long considered an undruggable target, a view overturned by the development of KRAS G12C inhibitors such as sotorasib and adagrasib, which have shown favorable clinical efficacy in lung cancer where G12C mutations are frequent. In gastrointestinal cancers, however, KRAS G12C mutations are relatively rare, and G12D and G12V predominate across biliary tract, pancreatic, and colorectal tumors. These cancers also show a higher propensity for therapeutic resistance, in part because adaptive feedback activates upstream signaling pathways, including the EGFR pathway. In colorectal cancer, adaptive EGFR activation following KRAS inhibition has been extensively reported and has led to clinically feasible combination strategies using EGFR antibodies. The G12D-specific inhibitor MRTX1133 has shown promising antitumor activity in preclinical studies, but its clinical efficacy as a monotherapy has not been convincingly established.

RMC-6236 belongs to a mechanistically distinct class. Sotorasib, adagrasib, and MRTX1133 are so-called KRAS OFF inhibitors that bind the inactive, GDP-bound form of the protein. RMC-6236 is a pan-RAS ON inhibitor that binds the active, GTP-bound state shared by multiple KRAS codon 12 variants. At the molecular level, it promotes the formation of a tri-protein complex with cyclophilin A and RAS-GTP, sterically preventing RAS from interacting with downstream effector proteins such as RAF and PI3K, thereby suppressing oncogenic RAS signaling. First reported at the American Association for Cancer Research Annual Meeting in 2022, the drug has demonstrated promising clinical activity in phase I trials for lung and pancreatic cancers, and phase III trials have commenced in pancreatic, lung, and colorectal cancers. The authors of the new work set out to test whether RMC-6236 could serve as a backbone for clinically applicable combinations in cholangiocarcinoma, a disease with limited effective targeted options.

The experimental system combined cell-line assays, western blotting, single-cell transcriptomics of human tumors, and xenograft experiments. The panel included YSCCC and HuH28 cells with wild-type KRAS, HuCC-T1 cells carrying KRAS G12D, RBE cells carrying KRAS G12V, and MMNK-1, an immortalized non-tumor cholangiocyte line. In dose-response proliferation assays, RMC-6236 inhibited proliferation of the KRAS-mutant lines at nanomolar concentrations, with antitumor effects at 3 to 10 nanomoles per liter, while wild-type KRAS cells responded only at approximately 100 nanomoles per liter. At 1000 nanomoles per liter, the drug also inhibited the non-tumor cholangiocyte line, indicating potential cytotoxicity at that concentration. Western blotting showed that in RBE and HuCC-T1 cells, phosphorylated ERK and phosphorylated AKT were suppressed over the 3 to 10 nanomole per liter range, consistent with direct shutdown of RAS pathway output.

The pivotal observation came from time-course experiments. When KRAS-mutant cells were treated with RMC-6236, western blotting revealed decreased pERK and pAKT within 4 hours, an effect sustained at 24 to 48 hours, accompanied by increased phosphorylated EGFR, indicating EGFR feedback activation. To confirm that this response depended on the oncogene, the team transduced wild-type KRAS YSCCC cells with FLAG-tagged wild-type, G12V, or G12D KRAS constructs. RMC-6236 significantly suppressed proliferation of the G12V- and G12D-transduced cells while having minimal effects on wild-type KRAS cells. In the mutant-transduced cells, ERK phosphorylation fell within 4 hours and remained suppressed at 24 hours, while EGFR phosphorylation progressively increased, raising the pEGFR-to-EGFR ratio. These adaptive changes were absent in wild-type KRAS-transduced cells, indicating that inhibition of RAS-MAPK signaling induces adaptive EGFR activation specifically in KRAS-mutant cholangiocarcinoma cells.

To connect the cell-culture findings to human disease, the researchers analyzed publicly available single-cell RNA sequencing data from human intrahepatic cholangiocarcinoma samples from the Gene Expression Omnibus database. Cell-type annotation identified tumor epithelial cells, cancer-associated fibroblasts, endothelial cells, macrophages, myeloid cells, T and natural killer cells, and B and plasma cells. EGFR expression was predominantly detected in tumor epithelial cells, whereas several EGFR ligands, including amphiregulin, epiregulin, and heparin-binding EGF-like growth factor, were expressed in multiple stromal and immune cell populations. Module score analysis revealed activation of both KRAS/MAPK and EGFR/MAPK signaling programs within tumor epithelial cells. Cell-cell communication analysis predicted ligand-receptor interactions involving HBEGF-EGFR, EREG-EGFR, and AREG-EGFR from surrounding stromal and immune cells toward tumor cells, suggesting that the tumor microenvironment provides a paracrine source of EGFR ligands capable of stimulating EGFR signaling in tumor cells.

Functional testing then asked whether blocking EGFR could translate the feedback observation into a therapeutic advantage. In KRAS G12V and G12D cells treated for 72 hours, the combination of RMC-6236 and cetuximab produced significantly greater growth inhibition than either agent alone, an effect not seen in wild-type KRAS cells. Synergy analysis using the Zero Interaction Potency score showed the strongest combination effect at 1 to 10 nanomoles per liter RMC-6236 with 0.1 to 10 micrograms per milliliter cetuximab. Western blotting explained the mechanism: RMC-6236 alone caused pERK rebound at 24 hours alongside pEGFR upregulation, but adding cetuximab suppressed EGFR reactivation and kept pERK reduced. The same pattern appeared in the KRAS gene-transduced YSCCC lines. The team also established an RMC-6236-resistant HuCC-T1 derivative through roughly 1.5 months of stepwise exposure up to 30 nanomoles per liter; in these acquired-resistant cells, cetuximab significantly enhanced growth inhibition across 1 to 100 nanomoles per liter of RMC-6236 and reduced both EGFR and ERK phosphorylation, suggesting EGFR blockade may restore sensitivity through suppression of EGFR-MAPK signaling.

In vivo, the combination held up. In xenografts of KRAS G12D and G12V cells in mice, RMC-6236 given orally once daily for 21 days produced dose-dependent antitumor effects at 5, 10, and 25 milligrams per kilogram per day. Adding cetuximab at 50 milligrams per kilogram weekly to 5 milligrams per kilogram per day of RMC-6236 yielded significantly greater growth inhibition than either drug alone. Immunohistochemistry of subcutaneous tumors showed a significantly reduced Ki-67 positivity index with the combination versus RMC-6236 monotherapy, a significantly lower pEGFR H-score indicating that cetuximab suppressed adaptive EGFR activation in vivo, and increased apoptosis on TUNEL staining. Body weight fluctuations stayed within 10 percent for all mice. A parallel gemcitabine program showed synergy in vitro at 0.01 to 0.3 micromoles per liter gemcitabine with 1 to 30 nanomoles per liter RMC-6236, and in xenografts the combination of 5 milligrams per kilogram per day RMC-6236 with 10 milligrams per kilogram gemcitabine twice weekly significantly inhibited growth versus RMC-6236 alone, with reduced Ki-67 staining and increased apoptotic cells.

The triplet of RMC-6236, cetuximab, and gemcitabine told a more nuanced story. In cell proliferation assays, the three-drug combination significantly inhibited growth of KRAS G12V and G12D cells compared with RMC-6236 alone or with either doublet, yet gemcitabine plus cetuximab alone showed antagonism, and triplet efficacy appeared only at limited concentrations. Western blotting revealed that gemcitabine plus cetuximab restored pMEK and pERK expression, and in triplet therapy the upregulation of pEGFR and rebound of pMEK and pERK persisted. In xenografts, both the RMC-6236 plus cetuximab doublet and the triplet effectively suppressed tumor growth, but no significant difference separated them, indicating that adding gemcitabine provided no significant additional antitumor benefit in this model. The authors note that the RMC-6236 and cetuximab pair elicited a more reproducible and concentration-dependent effect than the triple regimen.

Several caveats frame the conclusions. The analyses used a limited number of KRAS-mutant cholangiocarcinoma cell lines, which may not represent the molecular diversity of the disease; the mechanistic basis of how RMC-6236 modifies gemcitabine-cetuximab interactions requires further study; the molecular mechanisms underlying acquired resistance and the role of sustained EGFR signaling during its development remain unresolved; and expanded in vivo models are needed to confirm generalizability. The authors also emphasize that because activating EGFR mutations are uncommon in cholangiocarcinoma, the rationale for cetuximab here is distinct from 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. Within those limits, the study supports EGFR-directed combination strategies as a potential therapeutic approach in KRAS-mutant cholangiocarcinoma and argues that additional preclinical studies and well-designed clinical trials are essential to determine the safety, feasibility, and clinical applicability of RMC-6236-based regimens.

Subject of Research: Preclinical evaluation of the pan-RAS inhibitor RMC-6236 and adaptive EGFR feedback targeting in KRAS-mutant cholangiocarcinoma

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

Article References: Yamazaki, S., Hondo, N., Yasukawa, K., Kitazawa, M., Nakamura, S., Yamamoto, Y., Miyazaki, S., Tanaka, H., Kuroiwa, M., Ishizaka, N., Shimizu, T., Shimizu, A., & Soejima, Y. (2026). Targeting adaptive EGFR feedback enhances the therapeutic efficacy of RMC-6236 in cholangiocarcinoma. Experimental Hematology & Oncology, 15(1), Article 102. https://doi.org/10.1186/s40164-026-00835-8

Image Credits: AI Generated

DOI: 10.1186/s40164-026-00835-8

Keywords: cholangiocarcinoma, RMC-6236, daraxonrasib, KRAS G12D, KRAS G12V, EGFR feedback, cetuximab, gemcitabine, RAS ON inhibitor, xenograft models, MAPK signaling, single-cell RNA sequencing