Activity and specificity of KRAS variants-directed Revolution Medicines Company (RMC) compounds for pleural effusion-derived non-small cell lung cancer cell lines

Introduction

KRAS family variants are the most frequently occurring mutations in human cancers and are linked to a dismal prognosis (1). KRAS mutations are especially frequent in cancers such as pancreatic adenocarcinoma (88%), lung adenocarcinoma (31%), and colorectal cancers (CRCs) (45–50%) (2-4). Due to the lack of proper small-molecule binding sites and high intracellular GTP levels, KRAS was deemed undruggable for almost four decades (5). In 2013, the Shokat group identified drugs that bound covalently to KRAS G12C-GDP near the cysteine residue (6-8). This novel approach resulted in allele-specific targeting of KRAS G12C in non-small cell lung cancer (NSCLC) by two approved agents, namely sotorasib (AMG-510) and adagrasib (MRTX849) (9,10). These KRAS G12C inhibitors spare KRAS wild-type protein (11). RAS family proteins transmit upstream signals from growth factor receptors via multiple pathways to drive cellular growth, proliferation and survival (12). Mutated or overexpressed RAS was identified as causative oncogene for approximately one in five of all human cancers. RAS cycles between ON (GTP-bound) and OFF (GDP-bound) states with distinct structural features and the increased RAS(ON) state of mutated RAS triggers downstream signaling cascades, resulting in malignant growth and tumor cell survival (5,13). The driving mutations in RAS oncogenes occur mainly at glycine 12 (mainly D, V, C, R, and S), glycine 13 (mainly C and D), and glutamine 61 (mainly R and H) blocking KRAS in the active ON state (14).

Mutations in the RAS gene family (KRAS, NRAS, and HRAS) concern an estimated 3.4 million new patients worldwide per year (15,16). RAS proteins are generally found in almost all cell types, with KRAS representing the most prevalent isotype (17). RAS proteins belong to the GTPase family and guanine nucleotide exchange factors (GEFs) such as SOS1 catalyze nucleotide exchange to promote the GTP-bound ON state, while GTPase activating proteins (GAPs) including NF1 convert RAS to its inactive OFF state (12). Activated RAS proteins modulate the switch I and switch II domains to trigger the downstream activation of the RAF/MEK/ERK pathway, while the ratio of GTP-bound to GDP-bound RAS shift the equilibrium toward the ON state of RAS (12,15). KRAS transmits the signaling cascade triggered by binding of growth factors by receptor tyrosine kinases (RTK) (18).

However, sotorasib and adagrasib entered trials in NSCLC, for the approximately 12% of the patients carrying the KRAS G12C mutation (15,19). Sotorasib demonstrated an objective response rate (ORR) of 41.0% and progression-free survival (PFS) of 6.3 months in patients with G12C mutated NSCLC (20). Similar results were found in a phase 1/1b adagrasib study, revealing an ORR of 42.9% and a PFS of 6.5 months (21,22). Data from the phase 3 CodeBreak 200 study for previously treated metastatic NSCLC showed a modest benefit of sotorasib compared to the chemotherapeutic docetaxel. A favorable ORR was reported (28.1% versus 13.2%) but the OS was not improved (22). In another study, the median OS was 7.6 months, with improved OS of 10.4 months observed for docetaxel plus nintedanib compared to docetaxel monotherapy (6.5 months) (23). The efficacy of docetaxel, administered with or without antiangiogenic agents, was found to be limited in a real-world setting of NSCLC patients with a KRAS G12C mutation, emphasizing the need for novel therapies. For adagrasib, 453 patients were randomly allocated to receive adagrasib (66%) or docetaxel (34%) (24). The adagrasib cohort had a median PFS of 5.5 months, while the docetaxel cohort had 3.8 months. Grade 3 and above treatment-related adverse events (TRAEs) occurred in 47% of 298 patients treated with adagrasib and 46% of 140 patients with docetaxel. The KRAS G12C and G12V mutations are linked to the lowest survival compared to other KRAS mutation subtypes (25). Among the observed KRAS co-mutations, loss of the serine/threonine kinase 11 (STK11) tumor suppressor gene exhibit a dismal outcome, with a median overall survival of only 0.9 years (26). However, KRAS-directed therapies remain subject to rapidly evolving drug resistance. Tumors frequently reactivate effector pathways via secondary genetic mutations and rebound of signaling pathways. Nearly all patients with resistance mutations acquired genetic alterations that reestablish RAS-MAPK signaling (27,28). In the CodeBreaK 100 study of NSCLC, co-occurring mutations in KEAP1, SMARCA4, and CDKN2A showed inferior clinical results following sotorasib therapy (29).

The goal of newer KRAS-directed agents is the targeting of additional non-G12C KRAS alleles with increased efficacy, reduced toxicity and less resistance. The so-called Tri-complex inhibitors are a novel class of allele-specific and pan-allele targeting drugs that function as molecular glues for RAS and cyclophilin A (CYPA), inhibiting mutant KRAS by steric hindrance. CYPA is an 18 kDa cytoplasmic immunophilin that functions as a receptor for immunosuppressive drugs (30,31). Additionally, CYPA also has a peptidyl prolyl cis-trans isomerase (PPIase) activity, which is necessary for protein folding in vivo. Previous studies have shown that CYPA levels are increased in carcinoma cell lines, including human NSCLC (31,32). The development of the multi-selective tri-complex RAS(ON) inhibitors Revolution Medicines Company (RMC)-7977 and RMC-6236 provided completely new approaches to target RAS (33). RMC-7977 and RMC-6236 effectively suppress tumor growth, especially in NSCLC and pancreatic ductal adenocarcinoma (PDAC), with minimal effects on healthy tissue. Tri-complex inhibitors bind the KRAS ON state (34). RMC-6236 is a tri-complex RAS ON state inhibitor with activity against mutant and wild-type KRAS, NRAS and HRAS, including cancer cells resistant to first-generation KRAS G12C inhibitors (35,36). For NSCLC, the ORR was 38% and the disease control rate (DCR) was 85%, while the ORR for PDAC was 20% and the DCR was 87% (37,38). Responses were observed in tumors with G12D, G12V and G12R mutations and several prior lines of therapy. The RAS(ON) multiselective inhibitor daraxonrasib (RMC-6236) (39,40) is in a multitude of clinical trials for the treatment of various RAS-driven cancers. RMC-6236 was administered at doses ranging from 160 to 300 mg and led to early antitumor activity with an acceptable safety profile in patients with previously treated PDAC, according to the phase 1 RMS-6236-001 trial (NCT05379985) (41). Dose levels ≥80 mg achieved exposures that induced tumor regressions in human xenograft models with KRAS G12X mutations in mice (37,42).

The specificity of the RMC drugs directed at KRAS variants is mediated by modified chemical fragments derived from Sanglifehrin. These distinct chemical moieties generate a neomorphic interphase between KRAS and CYPA binding with good affinity to mutated KRAS variants. Furthermore, two of the variant-specific RMC compounds are armed with covalent warheads, namely RMC-9805 directed at KRAS G12D with an aziridine group and RMC-6291 targeting KRAS G12C with an ynamide group. Published dose-response curves of RMC compounds on cancer cell lines in vitro showed that even for the highest concentrations used, 10–50% of the cells survived (43). RMC-6291 (elironrasib) which is currently undergoing clinical evaluation (NCT05462717), constitutes a new kind of a KRAS G12C inhibitor. RMC-6291 uses a tri-complex, covalent inhibitor that specifically targets the G12C via a reactive ynamide group (34). The selective RAS(ON) G12C inhibitor RMC-6291 has demonstrated promising early clinical efficacy exhibiting partial response (PR) of 43% in patients with CRC (44). RMC-9805 (zoldonrasib) is a potent, oral, RAS(ON) G12D-selective, covalent, tricomplex inhibitor targeting the active, GTP-bound state of oncogenic RAS G12D isoforms (44-46). In a phase 1 study (NCT06040541), patients with KRASG12D tumors received escalating RMC-9805 doses (150–1200 mg once daily). No dose-limiting toxicities (DLTs) or Grade 4 or 5 TRAEs were reported. In patients with PDAC receiving RMC-9805 the ORR was 30%, and the DCR was 80% (47). RMC-9805 elicited objective responses in 7 of 9 PDAC models (48).

RMC-5127 is a first-in-class, orally bioavailable inhibitor that selectively targets the active form of KRAS G12V, a common oncogenic mutation of NSCLC, PDAC, and CRC. Repeated oral dosing of RMC-5127 resulted in profound and durable anti-tumor activity in CDX and PDX models of KRAS G12V mutant cancers (49). Moreover, RMC-5127 exhibited anti-tumor activity in the intracranial cancer model. The activity of RMC compounds directed at KRAS G12C cancers is roughly comparable to the approved inhibitors sotorasib and adagrasib. However, for KRAS G12D cancers, prominent in PDAC and CRC, RMC-9805 represents the only active drug for this KRAS variant of proven clinical activity.

The activity of the different RMC compounds was tested against recombinant KRAS variants, historical NSCLC and PDAC cell lines and patient-derived xenografts (PDX). However, the established cell lines have poor predictive power and exaggerate the cytotoxicity of the drugs due to adaptation to high proliferative characteristics. Freshly established real-world cell lines more closely reflect the in vivo characteristics of cancer cells (50). We have established a series of KRAS mutated NSCLC cell lines from pleural effusions, comprising KRAS G12C, G12D, G12V, G13D and G13V variants and one cell line with amplified KRAS. To assess potential differences in the cytotoxic efficacy and specificity of the RMC compounds, cell survival assays, phosphoproteome assays and KRAS Western blots were performed in newly established malignant pleura-derived NSCLC cell lines expressing different KRAS variants. We present this article in accordance with the MDAR reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0798/rc).

Methods

Cell lines

Thoracocentesis is routinely carried out in patients with NSCLC for pain relief and to ease breathing. These malignant pleural effusions (MPEs) frequently contain a high number of aggressive cancer cells and can therefore be used to establish cancer cell lines closely resembling real world phenotypes. All pleural effusion samples were sourced in Hospital Floridsdorf, 1210 Vienna, Austria by Dr. Maximilian Hochmair. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Medical University of Vienna, Vienna, Austria (No. 366/2003), and written informed consent was obtained from all participants. This approval also covered the generation of all NSCLC cell lines used in this study.

Following collection, pleural effusion samples were subjected to centrifugation, after which the supernatants were divided into aliquots and stored at −80 ℃. The cellular fraction was washed using RPMI-1640 culture medium (R8758, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Eximus, Catus Biotech, Tutzing, Germany) and antibiotics (Sigma-Aldrich). For further evaluation we included several commercially available PDAC cell lines, as well as other KRAS mutated cell lines (Table 1).

All cell lines were cultured in 75 cm² flasks (658170, Greiner Bio-One GmbH, Kremsmünster, Austria) containing RPMI-1640 medium (R8758, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (SFBS-AU, Eximus, Catus Biotech, Tutzing, Germany) and antibiotics (P4458, Sigma-Aldrich, Darmstadt, Germany). Cells were maintained at 37 ℃ in a humidified atmosphere with 5% CO₂ and were passaged regularly using trypsinization. Cell numbers were determined with a LUNA automated cell counter (Biozym, Vienna, Austria). The cultivation of BH1194, BH1440 and BH1467 cells with 2 µM RMC-6236 over seven consecutive days was used to generate RMC-6236 resistant phenotypes.

Cytotoxicity assays

The KRAS(ON) targeting compounds RMC-6236 (pan-KRAS), RMC-6291 (KRAS G12C), RMC-9805 (KRAS G12D) and RMC-5127 (KRAS G12V) were acquired from Targetmol (T74698, T75131, T78212, T204085, Wellesley Hills, MA, USA).

NSCLC and PDAC cells were seeded into 96-well flat-bottom microplates (92096, Techno Plastic Products AG, TPP, Trasadingen, Switzerland) at a density of 1×10⁴ cells per well in 100 µL RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics. To assess half-maximal inhibitory concentration (IC₅₀) values of the tested compounds, seven serial two-fold dilutions of each inhibitor (ranging from 0.004 to 2 µM) were applied, with each condition set up at least in duplicate and experiments conducted in a minimum of three independent replicates.

Following a 4-day incubation period under standard culture conditions, cell viability was quantified by measuring absorbance at 450 nm using a modified MTT-based assay (EZ4U, Biomedica, Vienna, Austria).

Proteome profiler arrays

Relative levels of protein phosphorylation in untreated BH1194 and BH1467 cells and in cells treated with RMC-6236, RMC-6291 and RMC-9805 were assessed using the Proteome Profiler Human Phospho-Kinase Array Kit (ARY003C, R&D Systems, Minneapolis, MN, USA) following the manufacturer’s protocol. Normalization of chemiluminescent signals was performed using the six internal reference spots present on each membrane. Signal quantification and analysis were conducted with Quickspot software (Ideal Eyes System, Bountiful, UT, USA) and further analysis done in Origin 9.1 software (OriginLab, Northampton, MA, USA).

Western blot

To analyze the protein expression of KRAS in BH1194, BH1362, BH1440, BH1467 untreated, as well as RMC-6236 treated cells, cells were lysed in Radioimmunoprecipitation assay (RIPA) buffer consisting of 50 mM Tris-HCl/pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP-40, 10 mM glycerol phosphate, 1 mM aprotinin, 1 mM leupeptin, 1 mM Pefabloc (R0278, Sigma-Aldrich), 1 mM NaVO3, 5 mM NaF and subsequently subjected to protein analysis. Total protein concentration was determined using the bicinchoninic acid (BCA) assay (A55861, ThermoFisher, Waltham, MA, USA); 25 μg of protein of the lysates per well were resolved by SDS-PAGE and run on 12% polyacrylamide gels. To determine protein size, molecular weight markers (RPN755E, Amersham, Buckinghamshire, UK) and Spectra™ Multicolor Broad Range Protein Ladder (#26634, ThermoFisher, Waltham, MA, USA) were used.

Following separation, proteins were transferred (Trans-Blot Turbo Transfer System, BIO-RAD, Hercules, CA, USA) to nitrocellulose membranes (Catalog No. GE10600002, Amersham) and incubated overnight at 4 ℃ with primary monoclonal antibody targeting KRAS (Rabbit #2862, Cell Signaling Technology, Dunvers, MA, USA) and GAPDH (Rabbit #5174, Cell Signaling Technology). The membranes were then incubated for 1 h at 25 ℃ with horseradish peroxidase (HRP)-conjugated secondary antibody (Anti-rabbit IgG, HRP-linked Antibody #7074, Cell Signaling Technology). Finally, specific detection was achieved using WesternBright Sirius chemoluminescence substrate (K-12043-D20, Advansta Inc., San Jose, CA, USA). Western Blots were analyzed using the C-DiGit Blot Scanner (LI-COR, Biosciences GMBH, Bad Homburg, Germany) and the Image Studio Software v6.0 (LI-COR, Biosciences).

Genome sequencing

KRAS mutation status of the lung tumors and cell lines was assessed by genomic sequencing using the diagnostic Oncomine Next Generation Sequencing (NGS) and for most cell lines also by either whole-exome sequencing (WES) or whole-genome sequencing (WGS). For WES, next-generation sequencing libraries were prepared using SureSelect Human All Exon V8 kit (Agilent, Santa Clara, CA, USA) and sequenced on Illumina NovaSeq6000 (Illumina, San Diego, CA, USA) according to the manufacturers’ protocols. Data was analyzed using open-source software, which included alignment to the reference genome (HG38), deduplication and variant calling using Genome Analysis Toolkit (GATK) (Broad Institute, Cambridge, MA, USA) package (Version 4.4.0.0, Apache 2.0 license) (https://github.com/broadinstitute/gatk).

For WGS, samples were prepared using a DNeasy Blood & Tissue Kit (69504, Quiagen, Hilden, Germany), followed by a SureSelect XT HS2 DNA Reagent Kit (Agilent, Santa Clara, CA, USA). Samples were processed using a DNBSEQ-T7 (MGI Tech Co., Ltd., Shenzhen, China).

Statistical analysis

Origin 9.1 software was used to obtain dose-response curves. MTT experiments were performed at least in triplicate and data was then presented as mean ± standard deviation (SD), if not otherwise stated. For Proteome Profiler arrays pixel values were normalized to ensure comparability. For each spot, differences between groups were assessed using a one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) post hoc test for multiple comparisons. P<0.05 was considered significant. The Chou-Talay combination index (CI) method was used to calculate drug interaction, where CI <1 indicates synergism, CI =1 indicates additivity, and CI >1 indicates antagonism (51).

Results

To illustrate the range of responses observed in the cell line panel, representative dose-response curves are shown first. RMC-6236 and KRAS G12D(ON) inhibitor RMC-9805 on a KRAS wildtype cell line (BH1440), a KRAS G12D cell line (BH1194) and a KRAS G13C cell line (BH1450) (Figure 1). RMC-6236 and RMC-9805 reduced cell viability across all tested cell lines, including the KRAS wildtype line BH1440 as well as the KRAS-mutant lines BH1194 and BH1450. Although some differences in sensitivity were observed, the overall response was not restricted to the KRAS-mutant cell lines. In particular, the wild-type line BH1440 also showed a marked decrease in viability at higher inhibitor concentrations, suggesting that the observed effects were not mutation-specific.

Figure 1 MTT cytotoxicity assays of one KRAS wildtype (BH1440), one KRAS G12D (BH1194) and one KRAS G13C (BH1450) NSCLC cell line. Cells were treated with 9 two-fold dilutions starting at 2 µM of (A) pan-KRAS(ON) inhibitor RMC-6236 and (B) KRAS(ON) G12D inhibitor RMC-9805. Data are presented as mean ± standard deviation.

As additional examples, three KRAS G12C-mutated cell lines, BH1362, BH1467, and BH1658, were treated with the pan-KRAS(ON) inhibitor RMC-6236 and the KRAS G12C(ON) inhibitor RMC-6291 to evaluate the cytotoxic effect of these compounds (Figure 2). RMC-6236 showed only a moderate effect on cell viability. In all three cell lines, viability was reduced with increasing inhibitor concentration, but the survival did not decrease below 50% (Figure 2A). RMC-6291 showed a similar inhibition profile in BH1362 and BH1658 (Figure 2B). However, BH1467 showed a stronger response to RMC-6291, especially at higher concentrations, where viability was clearly reduced. These results show that RMC-6291 does not have the same effect in all KRAS G12C-mutated cell lines. Therefore, the sensitivity to the inhibitor seems to depend not only on the KRAS G12C mutation, but also on other cell line specific factors. These data therefore do not support a mutation-specific effect of the respective inhibitors in selected cases.

Figure 2 MTT cytotoxicity assays of three KRAS G12C-mutant NSCLC cell lines. Cell lines were treated with 9 two-fold serial dilutions, starting at 2 µM, of (A) the pan-KRAS(ON) inhibitor RMC-6236 and (B) the KRAS G12C(ON) inhibitor RMC-6291. Data are presented as mean ± standard deviation.

RMC-6236 lead to variable results across cell lines, only managing to decrease cell viability moderately in most KRAS mutated lines, while it showed more pronounced effects in wildtype BH1440 and BxPC-3, as well as KRAS G12D mutated BH1194 and ASPC-1 (Figure 3).

Figure 3 Viable cells (% of control) of cell lines treated with 2 µM RMC-6236. Data are presented as mean ± standard deviation.

Overall, sixteen NSCLC cell lines established from pleural effusions, four PDAC, one cholangiocarcinoma, and one rhabdomyosarcoma cell line were treated with four RMC compounds. Although one inhibitor is designed as pan-KRAS and three inhibitors are designed to target different KRAS mutations, the minimum percentage of surviving cells at 2 µM did not show a distinct pattern for each inhibitor (Figure 4, Table S1, Figure S1). In general, the pancreatic cancer cell lines and the cholangiocarcinoma cell line EGI-1 exhibited high sensitivity to the RMC compounds and the rhabdomyosarcoma cell line BH1522 was included as a KRAS mutation negative control.

Figure 4 Heatmap of RMC compound efficacy sorted by cell line origin and KRAS mutation. This heatmap shows the minimum percentage of surviving cells after treatment with 2 µM RMC-6236, RMC-6291, RMC-9805, or RMC-5127. This heatmap was sorted cell line origins and respective KRAS mutations. The BHxx cell lines represent NSCLC cell lines established from malignant pleural effusions. BH1522 is a control cell line of a rhabdomyosarcoma in the lung, which was also established via pleural effusion.

RMC treatment led to broad but cell line-dependent changes in phosphorylation patterns (Figures 5,6, Figures S2,S3). ERK1/2 and PRAS40 phosphorylation were consistently reduced by all three compounds in both BH1194 and BH1467 cells. Additionally, the KRAS G12C-mutated BH1467 showed increased HSP27 phosphorylation, particularly after treatment with RMC-6291 (Figure 6).

Figure 5 Relative phosphorylation of selected proteins in BH1194 cells after treatment with RMC-6236, RMC-6291, or RMC-9805. Data are presented as mean ± standard deviation. Asterisks indicate significant differences versus untreated control (*, P<0.05; **, P<0.01; ***, P<0.001).

Figure 6 Relative phosphorylation of selected proteins in BH1467 cells after treatment with RMC-6236, RMC-6291, or RMC-9805. Data are presented as mean ± standard deviation. Asterisks indicate significant differences versus untreated control (* P<0.05, ** P<0.01, *** P<0.001).

In Western Blots, the KRAS wildtype cell line BH1440 and KRAS G12C mutated BH1467 showed an increase in KRAS signal intensity. For the wildtype BH1440 cell line KRAS intensity increased by 621%, while the increase in the KRAS G12C-mutated BH1467 constituted an increase of 325%. Western blots of BH1194 and BH1362 treated with RMC-6236 revealed a shift in KRAS molecular weight (canonical KRAS: 20.20±1.06 kDa vs. shifted KRAS: 27.08±1.03 kDa) (Figures 7,8, Figures S4-S8).

Figure 7 KRAS Western blots. Western blots showing KRAS protein expression in untreated and RMC-6236 treated BH1440 and BH1467 cells (upper). KRAS shift of BH1194 and BH1362 treated with RMC-6236, showing an increase in KRAS molecular weight of approximately 7 kDa (lower).

Figure 8 SDS-PAGE-based molecular weight calibration for KRAS band assignment. A calibration curve was generated using molecular weight ladder bands by plotting the logarithm of molecular weight [log10(kDa)] against relative migration distance. The dashed line represents the linear calibration fit. The apparent molecular weights of KRAS and shifted KRAS bands were estimated from their migration distances relative to the calibration curve. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

There were several significant differences in RMC compound sensitivity between untreated cells and those treated with 2 µM RMC-6236 over seven consecutive days (Figures 9-11). In KRAS G12D BH1194 and KRAS wild-type BH1440, cells were rendered more resistant to the compound, while BH1467 cells were significantly less affected by low doses, than previously untreated control cells.

Figure 9 Cytotoxicity tests of naïve BH1194 cells and those rendered resistant to RMC-6236. Dose-response curves of KRAS G12D-mutated BH1194 and RMC-6236-resistant BH1194R cells after treatment with RMC-6236 two-fold dilutions starting at 2 µM. Data are presented as mean ± standard deviation. Significant differences between the cell lines are noted by an asterisk (*).

Figure 10 Cytotoxicity tests of naïve BH1467 cells and those rendered resistant to RMC-6236. Dose-response curves of KRAS G12C-mutated BH1467 and RMC-6236-resistant BH1467R cells after treatment with RMC-6236 two-fold dilutions starting at 2 µM. Data are presented as mean ± standard deviation. Significant differences between the cell lines are noted by an asterisk (*).

Figure 11 Cytotoxicity tests of naïve BH1440 cells and those rendered resistant to RMC-6236. Dose-response curves of KRAS wild-type cell line BH1440 and RMC-6236-resistant BH1440R cells after treatment with RMC-6236 two-fold dilutions starting at 2 µM. Data are presented as mean ± standard deviation. Significant differences between the cell lines are noted by an asterisk (*).

Combination experiments revealed synergism at high concentrations in combinations of RMC-6236 with mutation specific inhibitors. A combination of RMC-6236 and RMC-9805 in a KRAS G12D-mutated cell line revealed synergism at the two highest concentrations 1 and 2 µM (Figure 12A). Experiments with combinations of RMC-6236 and RMC-6291 in a KRAS G12D-mutated cell line further revealed synergism at 2 µM–0.25 µM of the drugs (Figure 12B).

Figure 12 MTT cytotoxicity assays of combinations of (A) RMC-6236 and RMC-9805 for KRAS G12D-mutated BH1194 and (B) RMC-6236 and RMC-6291 for KRAS G12C-mutated BH1658. Combinations start at 2 µM with 9 two-fold dilutions. Data are presented as mean ± standard deviation. Synergistic values are depicted by an asterisk (*).

Discussion

The KRAS G12C-directed covalent inhibitors sotorasib and adagrasib, which lock mutant KRAS in its OFF configuration, allowed for targeting of KRAS in patients (6,8,21). However, the clinical benefit for the KRAS G12C mutant NSCLC patients is of relatively short duration with a median duration of response (DOR) reaching roughly 10.9 months for sotorasib (36). The modest efficacy of the KRAS targeting drugs is due to primary resistance, as well as through adaptive and acquired refractoriness (21,22,36). Mechanisms of escape include secondary RAS mutations, amplification of the KRAS G12C allele and reactivation of upstream RTK (36). Although sotorasib and adagrasib may replace cytotoxic treatments using docetaxel or other chemotherapeutics, they offer no higher response rates or prolongation of survival but a similar rate of adverse events (AEs) (22).

Therefore, the search for next-generation KRAS inhibitors with higher potency is ongoing and inhibitors able to target mutations beyond G12C, such as G12D and G12V, are required urgently. The novel multi-selective tri-complex RAS(ON) inhibitors RMC-7977 and RMC-6236 block KRAS by steric hindrance through glueing CYPA to the KRAS switch regions (33). These RMC drugs contain CYPA-binding moieties of sanglifehrin, an immunosuppressive compound. CYPA is a chaperone highly abundant in cells (12.3 µM), especially in tumor cells, and RMC compounds model a neomorphic surface on CYPA with high binding specificity for RAS. Among the RMC compounds, RMC-6236 (daraxonrasib) was the first developed as a clinical candidate that blocks the activity of all KRAS variants and wildtype. The RMC ON strategy blocks RAS signaling in its active state rather than by impairing activation, which results in durable signaling inhibition and extended isoform coverage.

In detail, the preclinical tool compound RMC-7977 showed a reversible binding to CYPA (K_d1 =195 nM) and of the binary complex to RAS (K_d2 =85 nM), suppressing the phosphorylation of ERK (EC₅₀ =0.421 nM) and cell proliferation (EC₅₀ =2.20 nM) (36). It binds all KRAS G12X mutants with threefold higher affinity compared to wild-type KRAS. Cell lines with mutationally activated RTKs also responded to RMC-7977 inhibition, including those with mutations or fusions of ERBBs, FGFRs, ROS1, RET, NTRK1 and ALK (median EC₅₀ =6.14 nM). The RMC drugs were shown to accumulate specifically in tumor tissues and to spare healthy tissues (52). RMC-7977 causes minimal toxicity at doses that produce high tumor regression in murine models (36,52).

RMC-6236 (daraxonrasib), the clinical counterpart to RMC-7977, is a potent, noncovalent, and multi-selective molecule glue inhibitor of the active state of both mutant variants of RAS and likewise the wild-type of RAS (35,53,54). The affinity of RMC-6236 for CYPA protein (K_D1) was 55.3 nmol/L and the affinity of the RMC-6236:CypA binary complex for KRAS G12V, G12D, and WT (K_D2), amounts to 131, 364, and 154 nM, respectively. RMC-6236 potently inhibited cell growth in KRAS G12X with a median EC₅₀ of 8 nM (35). A trial comprising 42 patients with PDAC harboring KRAS G12X mutations treated with daraxonrasib reported a median PFS of 8.5 months and ORR of 29% (55). However, the initial ORRs observed with daraxonrasib in NSCLC, PDAC, and CRC are lower than those observed with the previously used mutant-selective inhibitors (4,55). Early clinical data for RMC-6236 report DCRs of 85% in NSCLC and PDAC, with manageable toxicity. Normal cells are spared due to low levels of RAS-GTP and tumor-specific drug accumulation (56,57). Discontinuation of treatment was observed in 5% of patients, with grade 3 rash, nausea, diarrhea, and vomiting as AEs (37,56). However, gastro-intestinal effects are likewise known to limit the administration of KRAS/SOS1-directed compounds. Intestinal epithelial cells (IECs) regulate the absorption of water, nutrients, and electrolytes from the external environment and act as a barrier to protect the host tissue (56). The RAS superfamily of small GTPases are critical regulators of intestinal epithelial homeostasis and the barrier function of epithelial junctions (58,59).

The mechanisms of resistance in patients treated with these RMC inhibitors remains largely unknown. A group of RMC resistant patients had acquired alterations in BRAF, RAF1, MAP2K1/2 and PIK3CA signaling mediators (60). Acquired resistance to RMC-6236 correlated with KRAS amplifications in 35% of patients and KRAS Y64X mutations conferred on-target resistance to RMC-6236 through impairment of tri-complex formation (60,61).

The pan-RAS inhibitor RMC-6236 was the starting point to develop further KRAS variant-selective RMC compounds (16). RMC-6291 (elironrasib) is a first-in-class covalent tri-complex ON state inhibitor for KRAS G12C armed with a reactive thiol group (34,62). RMC-6291 exhibited tolerability and efficacy in patients revealing an ORR of 50% in patients which progressed previously during treatment with KRAS G12C OFF inhibitors (46). The absence of a reactive cysteine residue in KRAS G12D presents a significant challenge for the development of covalent inhibitors. MRTX1133 was the first potent and selective noncovalent inhibitor targeting KRAS G12D that positioned a positively charged amine moiety adjacent to the acidic side chain of Asp12 for inhibition (63). However, its phase 1/2 clinical trial was terminated due to highly variable and suboptimal pharmacokinetics. RMC-9805 (zoldonrasib) is a first-in-class covalent tri-complex inhibitor that selectively targets the ON state of KRAS G12D. RMC-9805 covalently interacts with the KRAS G12D Asp12 residue through a reactive aziridine warhead (64). A phase I trial of RMC-9805 in patients with KRAS G12D-mutant tumors reported ORRs of 61% and 30% for NSCLC and PDAC, respectively (47,65). KRAS G12V replaces glycine with valine that causes high steric hindrance and favors the active ON state (47). RMC-5127 is a potent, orally bioavailable, and selective noncovalent inhibitor that targets the active, GTP-bound form of KRAS G12V (66). For PDAC, no effective targeted therapies have been approved and the standard of care remains surgery and chemotherapy (67,68). For unresectable PDAC the first-line therapy is FOLFIRINOX, a combination of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin or gemcitabine combined with nab-paclitaxel (69,70). PDAC is dominated by mutations in KRAS (99%) with G12D as the most frequent substitution (41%), followed by G12V (32%), G12R (16%) and G12C (2%) (68,71-74).

In preclinical drug development, the success rate of promoting drugs from phase I trials to a final approval is of a minor rate of approximately 10%. Early in this process, compounds reacting with specific molecular targets are selected via testing on permanent cell lines, xenotransplants, and animal experimentation. However, a host of failed clinical trials indicate that these models lack enough predictive power (50). In contrast to established cell lines kept for long periods in tissue culture, freshly established new cancer cell lines may better preserve the original tumor characteristics. For typical solid tumors, access to representative tissue by operations or biopsies is difficult, but pleura-derived tumor cells of advanced lung cancer patients is a notable exception (50). In case of MRTX1133, the cytotoxicity of the compound differed markedly from highly sensitive permanent cell lines to less sensitive newly established cancer cell lines and this KRAS G12D-directed agent ultimately failed. However, the high cytotoxic activity of RMC compounds reported during the preclinical development needs verification in real-world cell lines, both with respect to cell killing and in regard to their specificity for the distinct KRAS variants.

The RMC compounds were preclinically developed and tested for activity against recombinant KRAS variant cell lines, commercial cancer cell lines and xenografts. For this study, we employed a panel of KRAS-mutated NSCLC and control cell lines that were established from MPEs. The KRAS genotype of these cell lines was either determined by diagnostic NGS sequencing, whole exome sequencing (WES) or whole genome sequencing (WGS). The cytotoxic activity of the RMC compounds was assessed in MTT viability tests for a 2 µM–5 nM concentration range. The fraction of surviving cancer cells at the highest RMC concentration was regarded as a measure of their cytotoxic activity, as IC₅₀ values could not be reliably calculated due to limited cytotoxicity. Dose-response curves of RMC-6236 showed a fraction of 50–70% surviving cells at up to 2 µM for KRAS G12C mutated NSCLC. The KRAS G12C-specific RMC compound RMC-6291 resulted in a survival of 60% in two G12C mutated cell lines and good activity against the sotorasib-relapsed BH1467 cell line. RMC-6236 exhibited good activity against wildtype-KRAS and BH1194 KRAS G12D cells but low activity against BH1450 KRAS G13C cells. However, the KRAS G12D-directed specific RMC-9805 compound revealed no better cytotoxicity compared to the pan-RAS inhibitor RMC-6236. RMC-6291, which contains a KRAS G12C-specific warhead, exhibited high activity against G12C and G13C cell lines, good activity against most G12X lines, and low activity against the BH1362 G12C cell line. It also showed good activity against a range of non-G12C control lines. Thus, with exception of BH1658 KRAS G12C, the warhead of RMC6291 lacked to confer superior cytotoxic activity. Peculiarly, the RMC-5127 compound directed to G12V exhibited good activity against a G12C line and pleural breast cancer cells, besides two KRAS G12V cell lines. In summary, the KRAS variant-specific RMC compounds did not fare better than pan-KRAS RMC-6236 and revealed a poor specificity pattern for the corresponding KRAS mutations. The heatmap relating the cytotoxicity of the RMC compounds to the KRAS mutation pattern depicted no clear interrelationship between these two variables.

Of course, the clinical activity of the RMC compounds is subject to chemoresistance that limits responses and survival. Exposure of two KRAS mutant cell lines, namely BH1467 KRAS G12C and BH1194 KRAS G12D, to RMC-6236 for 7 days killed a fraction of the tumor cells which detached and could be removed by aspiration. The intact remaining cells exhibited increased resistance to RMC-6236 and KRAS Western blots revealed either an increased expression of this oncogene or a molecular size shift indicating an attachment of one ubiquitin chain to KRAS. Monoubiquitiniylation of KRAS has been reported as an activating KRAS modification (75). Thus, KRAS amplification or activation interferes with RMC-6236 cytotoxic activity. Accordingly, aquired KRAS amplification in response to RMC compounds has been reported in a group of patients (61).

In case of PDAC cell lines, RMC-6236 showed high activity against one wild-type KRAS and several KRAS G12D-mutated cell lines, with the exception of PANC-1. Similar patterns were observed for RMC-9805 and RMC-5127. However, these cytotoxic activities were found for classical and permanent cell lines that are generally more sensitive to drugs.

Phosphoprotein arrays showed characteristic alterations for BH1194 and BH1467 following exposure to RMC-6236, RMC-6291 and RMC-9805, respectively. In BH1194 cells all RMC compounds tested suppressed the phosphorylation of ERK1/2, lowered phosphorylation of EGFR, GSK-3 and PRAS40 as most consistent alterations. Thus, signaling via EGFR and the MAPK pathway is impaired and PRAS40 once phosphorylated, releases mTORC1 and counteracts growth inhibition. GSK-3 activity is inhibited through phosphorylation of serine 21 in GSK-3α and serine 9 in GSK-3β. GSK-3 is required for the in vitro and in vivo growth and survival of human mutant KRAS-dependent tumors (76). In BH1467 KRAS G12C cells, ERK1/2 is suppressed with RMC-6291 showing the lowest activity, EGFR is slightly activated and CHK2 (checkpoint kinase 2) and PRAS40 are hypophosphorylated. CHK2 is a critical tumor suppressor and serine/threonine kinase and its activation is strictly regulated by phosphorylation. Once activated, CHK2 halts the cell cycle, initiates DNA repair, or triggers apoptosis by phosphorylating key substrates (77).

The present findings suggest that RMC-6236 is a drug suppressing the growth-stimulating activities of a range of KRAS mutated variants. However, the RMC compounds specifically designed for distinct KRAS mutations show varying activities that are poorly correlated with the specific mutations that they were developed for. Our results are derived with the help of pleural effusion-derived newly established NSCLC lines that are a selection of highly metastatic and aggressive lung cancer cells.

Combinations of tricomplex pan-RAS inhibitors with mutant-selective or isoform selective switch II pocket KRAS inhibitors may offer additional mechanistic synergy. Preclinical studies combining the pan-RAS inhibitor daraxonrasib with either elironrasib (KRAS G12C specific; NCT06128551) or zoldonrasib (KRAS G12D specific; NCT06040541) support the feasibility of this concept (78). Further selective ON-state tri-complex inhibitors, KRAS Q61H and G13C, as well as covalent inhibitors KRAS G12S are in preclinical development (79-81).

We tested RMC combinations of RMC-6236 with RMC-6291 in the KRAS G12C mutated cell line BH1658 and RMC-6236 in combination with RMC-9805 in the KRAS G12D mutated BH1194 NSCLC cells, respectively. The pan-RAS inhibitor RMC-6236 in combination with G12C-directed RMC-6291 exhibited synergy for a concentration range of 0.25–2 µM. However, the combination of the pan-RAS RMC with the G12D-targeting RMC-9805 only showed synergistic interactions for the two highest concentrations of 1 and 2 µM that turned to antagonism at lower concentrations of the drugs. Thus, combinations of the pan-RAS RMC-6236 with the RMC drugs equipped with a G12C-or a G12D-directed warhead yielded limited synergy at concentrations within the clinical PPCs of the drugs. Considering the poor drug specificity these combinations may not significantly increase the antitumor activity of RMC-6236/daraxonrasib.

Conclusions

The RMC KRAS-directed compounds have been successfully explored as a new approach to target this oncogene by exploiting their function as intracellular glues interacting with CYPA. Clinically, daraxonrasib (RMC.6236) doubles the survival time in PDAC but is less effective in NSCLC. The RMC compounds kill approximately half of the cells at highest concentrations when tested against newly established KRAS mutant NSCLC cell lines. The cytotoxicity tests described here determined the activity of the RMC compounds after one single dose, whereas in patients the drugs are administered on 21 successive days and may accumulate. ERK signaling is suppressed by all RMC compounds tested such blocking the MAPK pathway. Resistance to RMC-6236 seems to be partially due to simple KRAS amplification and increases in KRAS activity. RMC developed a series of KRAS variant-directed agents against KRAS G12C, G12D and G12V, in RMC-6291 and RMC-9805 equipped with warheads. However, these mutation-specific drugs frequently exhibit poor selectivity for the diverse KRAS-mutated NSCLC cell lines derived from MPEs. Variable cytotoxic responses across distinct tumor types show that mutation alone is not sufficient to predict therapeutic outcomes and KRAS is modulated by tumor type-dependent comutations. Inhibition of wild-type KRAS and lack of KRAS mutant specificity point to problems in optimizing the RMC compounds using cells that poorly represent actual NSCLC cells or that the binding of these compounds to KRAS is modulated by the molecular background of specific cancer cell. In conclusion, the novel RMC KRAS ON-targeting compounds need further characterization and a search for synergistic KRAS agents to increase the clinical efficacy.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0798/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0798/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0798/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0798/coif). G.H. serves as an unpaid editorial board member of Translational Lung Cancer Research from September 2025 to August 2027. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Medical University of Vienna, Vienna, Austria (No. 366/2003), and written informed consent was obtained from all participants. This approval also covered the generation of all NSCLC cell lines used in this study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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