Overview of RAS GTPases

RAS proteins are small, membrane-bound GTPases that serve as critical molecular switches in intracellular signaling pathways regulating cell proliferation, differentiation, survival, and migration. As central mediators of signals from growth factor receptors (e.g., EGFR, KRAS, FGFR), RAS cycles between an active GTP-bound state and an inactive GDP-bound state-dysregulation of this cycle drives aberrant cell signaling and oncogenesis.

Mutations in RAS genes are among the most common oncogenic drivers across human cancers, occurring in ~30% of all malignancies. RAS mutations are classified as "driver mutations"-they are sufficient to initiate tumorigenesis and promote cancer progression. Beyond cancer, rare germline RAS mutations cause RASopathies, a group of developmental disorders (e.g., neurofibromatosis type 1, Noonan syndrome).

For decades, RAS was considered "undruggable" due to its smooth protein surface and high affinity for GTP/GDP, leaving no obvious binding pockets for small-molecule inhibitors. However, breakthroughs in structure-based drug design have unlocked targeted therapies:

1) KRAS G12C Inhibitors: The first approved RAS-targeted drugs, including Sotorasib (Lumakras™) and Adagrasib (Krazati™), for advanced non-small cell lung cancer (NSCLC) harboring KRAS G12C mutations. 2) Pan-KRAS Inhibitors: Emerging agents (e.g., MRTX1133) targeting multiple KRAS mutations (G12D, G12V, G12C) are in clinical trials for colorectal, pancreatic, and lung cancers. 3) Upstream/Downstream Inhibition: Agents targeting RAS signaling pathways (e.g., MEK inhibitors, EGFR inhibitors) are used in combination therapies to overcome resistance.

The RAS family comprises three closely related isoforms encoded by distinct genes: KRAS (Kirsten rat sarcoma viral oncogene homolog), NRAS (neuroblastoma RAS viral oncogene homolog), and HRAS (Harvey rat sarcoma viral oncogene homolog). While sharing ~85% amino acid sequence homology, each isoform exhibits unique tissue expression patterns, subcellular localization, and signaling preferences, contributing to distinct pathological roles.

KRAS (Kirsten RAS)

KRAS is the most frequently mutated RAS isoform, encoding a 188-amino-acid protein primarily localized to the plasma membrane. It plays a pivotal role in mediating signaling from receptor tyrosine kinases (RTKs) to downstream effectors (e.g., RAF-MEK-ERK, PI3K-AKT-mTOR), governing cell cycle progression and metabolic reprogramming.

KRAS mutations are predominantly missense mutations, with the most common hotspots at codons 12 (G12C, G12D, G12V, G12R), 13 (G13D), and 61 (Q61H/L). Wild-type (WT) KRAS is also functionally relevant in cancers where RAS signaling is hyperactivated via upstream alterations (e.g., EGFR mutations). KRAS mutations are most prevalent in: non-small cell lung cancer (NSCLC, 30-40%, with G12C accounting for ~13%), colorectal cancer (CRC, 40-50%, predominantly G12D/G12V), pancreatic ductal adenocarcinoma (PDAC, 80-90%, mostly G12D) and other cancers (e.g., biliary tract, ovarian, endometrial).

Two drugs have been approved for KRAS G12C - mutant cancers: Sotorasib, which was approved in 2021 for KRAS G12C - mutant NSCLC and further approved in 2023 for KRAS G12C - mutant CRC, as well as Adagrasib, which obtained its approval in 2022 specifically for KRAS G12C - mutant NSCLC. Meanwhile, a number of promising candidates remain in clinical trial stages, expanding treatment options for patients.

One of the key research challenges in KRAS-targeted therapy lies in the limited coverage of KRAS mutations, as most currently available drugs are only capable of targeting the KRAS G12C mutation and fail to address other common KRAS mutation subtypes. Another major challenge is the emergence of resistance mechanisms, which can manifest in multiple forms: for instance, tumors may undergo a mutation switch from KRAS G12C to KRAS G12D (rendering G12C-specific inhibitors ineffective), or they may activate bypass signaling pathways such as the EGFR (Epidermal Growth Factor Receptor) or IGFR (Insulin-like Growth Factor Receptor) pathways to evade the inhibitory effects of KRAS-targeted drugs. Additionally, a critical obstacle in translating KRAS inhibitors into effective clinical outcomes is the poor penetration of these inhibitors into solid tumors; this limited penetration means that even if the inhibitors exhibit potent activity against KRAS mutations in preclinical models, they cannot reach sufficient concentrations within solid tumor tissues to exert their therapeutic effects.

NRAS (Neuroblastoma RAS)

NRAS is widely expressed in neural tissues, hematopoietic cells, and epithelia. It regulates cell survival, differentiation, and cytoskeletal remodeling, with key roles in hematopoietic stem cell maintenance and neuronal development.

NRAS mutations are most common at codons 12 (G12D/V), 13 (G13D), and 61 (Q61K/R/L)-codon 61 mutations are unique to NRAS and disrupt GTPase activity, leading to constitutive activation. NRAS mutations are hallmark drivers of several types of cancers, including melanoma, where they occur in 20-30% of cases and are predominantly of the Q61K/R variants; hematological malignancies such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), accounting for approximately 15-20% of these disorders; thyroid cancer, specifically the follicular and papillary subtypes, with an incidence rate of 10-20%; and rare solid tumors like ovarian and colorectal cancers.

To date, there are no approved NRAS-specific inhibitors. Current therapeutic strategies primarily focus on two key approaches: first, targeting downstream pathways-such as utilizing MEK inhibitors like Trametinib and Cobimetinib in combination with BRAF inhibitors-for the treatment of NRAS-mutant melanoma; and second, developing novel agents that are currently in preclinical or clinical stages, including NRAS G12D inhibitors and farnesyltransferase inhibitors.

Key research challenges in targeting NRAS-mutant cancers include the lack of selective NRAS inhibitors, which stems from NRAS's high homology with KRAS and HRAS; a high rate of resistance to MEK inhibitors observed in NRAS-mutant cancers; and a limited understanding of the signaling networks that are specific to NRAS.

HRAS (Harvey RAS)

HRAS is primarily expressed in epithelial tissues, skin, and skeletal muscle. It regulates cell adhesion, migration, and angiogenesis, with critical roles in skin development and wound healing.

HRAS mutations occur most frequently at codons 12 (G12V/D) and 61 (Q61L/R), leading to constitutive GTP binding and signaling activation. While HRAS mutations are less common in cancers compared to other RAS family members, they serve as key drivers of several malignancies, including bladder cancer (where they occur in 3-5% of cases), head and neck squamous cell carcinoma (HNSCC, accounting for 2-5% of cases), and skin cancers such as squamous cell carcinoma and melanoma. Additionally, germline HRAS mutations are the cause of Costello syndrome, a type of RASopathy.

Currently, there are no approved therapeutics specifically targeting HRAS, meaning that the treatment of HRAS-mutant cancers depends on chemotherapy, immunotherapy, or non-selective pathway inhibitors-such as MEK inhibitors. At the same time, preclinical research efforts are centered on the development of HRAS-directed therapies, including small-molecule agents and PROTACs (proteolysis-targeting chimeras).

Major research hurdles in investigating and targeting HRAS-mutant cancers encompass the low incidence of HRAS mutations, which restricts recruitment to clinical trials; the overlapping signaling cascades between HRAS and other RAS isoforms, which hinders the development of selective targeting approaches; and the absence of validated preclinical models that are specifically designed for HRAS-mutant cancers.

The research challenges associated with HRAS-mutant cancers include the low frequency of HRAS mutations, which acts as a barrier to enrolling sufficient patients in clinical trials; the overlap in signaling pathways between HRAS and other RAS isoforms, which makes the development of selective targeting strategies more complex; and the absence of validated preclinical models that are well-suited for studying HRAS-mutant cancers.

Our Expertise & Flexible Service Model

With over a decade of expertise in stable cell line development and high-throughput compound screening, we are a leading partner for biotech companies, pharmaceutical firms, academic institutions, and research hospitals worldwide. Our team of molecular biologists, cell biologists, and pharmacologists specializes in RAS family biology-ensuring that our products and services are tailored to the unique needs of RAS-targeted drug discovery. We have supported thousands of clients, from early-stage startups to pharmaceutical companies, in advancing their RAS research programs-from target validation to preclinical candidate selection.

Products & Services

We understand that every research project has unique requirements-so we offer two flexible service models to maximize efficiency and minimize costs:

1) Stable Cell Line Supply

For clients with large compound libraries or in-house screening capabilities, we provide high-quality BaF3 stable cell lines overexpressing WT or mutant RAS (KRAS/NRAS/HRAS). All cell lines are rigorously validated and delivered with comprehensive documentation, enabling you to conduct drug screening, mechanism-of-action (MOA) studies, and resistance profiling in your own laboratory.

2) Custom Compound Screening & Validation

For clients with a small number of compounds or limited in-house resources, we offer end-to-end compound screening services. Our team handles cell culture, compound dosing, assay execution, and data analysis-delivering actionable results (e.g., IC50 values, inhibition curves). This model eliminates the need for upfront investment in cell line validation and screening infrastructure, accelerating your research timeline by a few months.

Advantages

1) Diverse Mutation Coverage: We offer BaF3 cell lines for key RAS hotspots (e.g., KRAS G12C/D/V/R, NRAS Q61K/R, HRAS G12V) and WT RAS-covering the multiple clinically relevant mutations across cancers.

2) High-Throughput Compatibility: Our cell lines are optimized for 96-well/384-well plate formats, supporting high-throughput screening (HTS) of large compound libraries with minimal variability.

3) Exceptional Sensitivity: BaF3 cells are RAS-dependent for proliferation, ensuring robust detection of RAS inhibitor activity-even for low-potency compounds.

4) Customization: We provide personalized cell line development (e.g., rare RAS mutations, dual RAS-pathway alterations) and tailored screening assays (e.g., combination therapy screening, MOA-focused assays) to meet your specific research goals.

Key Applications

Our BaF3 RAS cell lines and screening services support two core research stages:

1) In Vitro Compound Screening (Cellular Level)

• Assay Type: CellTiter-Glo (CTG) Luminescent Cell Viability Assay-quantifies compound-induced inhibition of RAS-driven cell proliferation and viability.
• Output: IC50 values, dose-response curves, and inhibition percentages at multiple concentrations.
• Purpose: Rapidly identify lead compounds with potential RAS-targeted activity, prioritize candidates for further development, and eliminate inactive compounds early in the pipeline.

2) In Vivo Target Validation & Efficacy Screening (Animal Level)

• Model Type: BaF3 cell-derived xenograft (CDX) models in immunocompromised mice (NSG, BALB/c nude).
• Assays: Tumor growth inhibition (TGI), tumor volume measurement, survival analysis, and pharmacodynamic (PD) marker assessment (e.g., p-ERK, Ki67).
• Purpose: Validate in vitro hits in a physiological setting, evaluate the efficacy of small-molecule inhibitors, antibodies, or combination therapies against RAS-mutant tumors, and generate preclinical data to support IND filings.

Product List

We currently have established over 50 BaF3 stable cell lines expressing RAS family WT/mutant genes. All these cell lines have been validated for high-level gene expression via quantitative real-time PCR (qPCR). Most of the cell lines exhibit interleukin-3 (IL-3)-independent growth, and their IC₅₀ values have been determined using known inhibitors (e.g., adagrasib, Lonafarnib, Trametinib). We are actively expanding this panel of stable cell lines and also offer custom development services for stable cell lines overexpressing user-specified RAS mutant genes.

Table 1. List of KRAS cell lines

Table 2. List of NRAS cell lines

Table 3. List of HRAS cell lines

Product Data

Our cell lines undergo rigorous quality control (QC) to ensure consistency, purity, and functionality.

1. Cell Morphology

BaF3 cells (RAS WT and mutant-overexpressing) exhibit characteristic round, suspension-cell morphology.

2. QPCR Overexpression Validation

RAS mRNA expression levels in BaF3 stable cell lines compared to parental BaF3 cells are measured by quantitative real-time PCR (QPCR) using RAS isoform-specific primers and GAPDH as internal control.

3. IC₅₀ Validation (Positive Control)

We screened a panel of well-characterized RAS inhibitors and performed the CellTiter-Glo® (CTG) Luminescent Cell Viability Assay on our complete repertoire of IL-3-independent BaF3-RAS-expressing cell lines. The resultant data demonstrated that these engineered cell models are robust and reliable for high-throughput in vitro RAS inhibitor screening and drug sensitivity evaluation.

4. Mycoplasma Detection

All of our cell lines are mycoplasma-negative tested by PCR-based method (no amplification of mycoplasma-specific bands) and MycoAlert Mycoplasma Detection Kit.

Contact Us

Ready to accelerate your RAS-targeted research? Our team is here to support your project-whether you need stable cell lines, custom screening services, or technical guidance. Please contact us at info@creative-biogene.com or 1-631-626-9181 (USA), 44-208-123-7131 (Europe). Our team will respond within 24 hours.