RAS is one of the most common oncogenes found in cancer patients, with RAS mutations detected in approximately 21% of all cancers. The RAS family comprises three subtypes: KRAS, NRAS, and HRAS. Due to the unique structural characteristics of RAS proteins, they have long been regarded by the medical community as “undruggable targets”. Tumors driven by RAS mutations are among the most difficult gene-driven cancers to treat. Scientists have spent over 30 years exploring effective RAS inhibitors, and only in recent years have significant breakthroughs been made in treating RAS-mutant tumors. The development of several RAS inhibitors has propelled this field forward substantially. Current therapeutic strategies targeting RAS-driven tumors mainly include direct and indirect targeting approaches.
Biological Characteristics of RAS
RAS proteins are membrane-associated GTPases localized on the inner side of the cell membrane, with a molecular weight of approximately 21 kDa. They possess intrinsic guanosine triphosphatase (GTPase) activity and serve as critical signaling molecules downstream of receptor tyrosine kinases (RTKs). RAS regulates vital physiological processes such as cell growth, differentiation, and apoptosis, playing a pivotal role in tumorigenesis and cancer progression.
Structurally, RAS proteins consist of an effector lobe (amino acids 1-86), an allosteric lobe (amino acids 87-165), and a hypervariable region (HVR, amino acids 167-188/189). The switch I region (aa 30-40) and switch II region (aa 60-76) within the effector lobe are key for effector binding and interactions with guanine nucleotide exchange factors (GEFs) or GTPase-activating proteins (GAPs). The HVR domain facilitates RAS attachment to the cell membrane.
In human cells, three RAS genes encode four isoforms: HRAS, NRAS, and two splice variants of KRAS—KRAS4a and KRAS4b—each including unique exons 4a and 4b, respectively. All four isoforms share identical residues in the first half of the GTPase domain (G-domain) and exhibit 82% sequence identity in the second half. The final 19-20 amino acids at the C-terminus show significant sequence diversity, forming the HVR unique to each isoform.
Under normal conditions, RAS is activated downstream of growth factor receptors, including members of the epidermal growth factor receptor (EGFR) family. RAS functions as a binary molecular switch cycling between an active (ON) and inactive (OFF) state during signal transduction. This cycling is controlled by binding guanosine triphosphate (GTP) or guanosine diphosphate (GDP). Upon receiving extracellular signals, RAS binds GTP and activates, subsequently triggering multiple downstream pathways such as the MAPK and PI3K pathways to regulate diverse cellular activities.
RAS Mutations and Cancer
Mutations in RAS genes are critical drivers in the development of many human malignancies. Since the discovery in 1982 that RAS mutations activate oncogenic signaling in human cancer cells, it has become clear that these mutations disrupt the guanine nucleotide exchange cycle. Mutant RAS proteins are locked in a constitutively active GTP-bound state, leading to continuous downstream signaling activation, promoting uncontrolled cell proliferation and tumorigenesis.
RAS activation predominantly occurs through point mutations at codons 12, 13, and 61. Statistically, RAS mutations are present in about 20-30% of human tumors. Among them, KRAS mutations are the most frequent, especially in pancreatic cancer, colorectal cancer, and lung cancer; NRAS mutations are common in melanoma and leukemia; HRAS mutations appear more often in bladder and head and neck cancers.
Distribution of RAS Mutations in Tumors
The isoform, mutation site, and substitution type of RAS mutations vary clinically by tissue type. These distinct mutation distribution patterns likely reflect the selective environmental pressures during tumor initiation and progression.
The incidence of RAS mutations in different tumor types:
- KRAS mutations account for 14.3% of malignancies, with the highest incidence in pancreatic ductal adenocarcinoma (76.5%), colorectal cancer (42.9%), and non-small cell lung cancer (27.4%).
- NRAS mutations represent 2.5% of malignancies, notably 23.0% in melanoma.
- HRAS mutations are less frequent, at 0.8%, mainly in bladder cancer (4.06%) and head and neck cancers (4.21%).
Regarding mutation hotspots by tumor type:
- KRAS mutations predominantly occur at codon 12 (G12), with a frequency of 78.3%.
- NRAS mutations mainly affect codon 61 (Q61), with a frequency of 58.2%.
- HRAS mutations also cluster at codon 61 (Q61), with a frequency of 29.1%.
Abnormalities in RAS Signaling Pathway and Tumorigenesis
RAS plays a crucial role in the EGFR signaling pathway. The proto-oncogene KRAS encodes a GTPase protein that functions as a key downstream effector in the Ras/MAPK pathway activated by growth factors such as EGFR. RAS acts as a molecular switch: once activated, it triggers multiple mitogenic and proliferative factors including c-Raf and PI3-kinase. When active, RAS binds GTP and hydrolyzes its terminal phosphate to convert GTP into GDP, which switches RAS off. Under normal physiological conditions, RAS activation is transient and tightly regulated.
However, mutations in the RAS gene lead to constitutive activation of the RAS protein, which remains bound to GTP independently of upstream signals. This persistent activation causes abnormal hyperactivation of downstream signaling pathways, promoting uncontrolled cell growth and proliferation. As a key node in the EGFR downstream signaling cascade, mutant RAS activates the MAPK pathway directly, bypassing the need for upstream EGFR stimulation. This results in enhanced tumor cell proliferation, metastasis, and poor clinical prognosis. Moreover, tumors harboring constitutively active RAS mutations often show resistance to EGFR inhibitors.
In addition to the MAPK pathway, other RAS-related signaling cascades include the PI3K pathway, RAL signaling, and PLCε pathways. These collectively regulate transcriptional programs controlling cell division, differentiation, migration, adhesion, and apoptosis.
KRAS Mutations
Among RAS mutations, KRAS mutations are the most prevalent and represent a major focus of cancer research. Approximately 17% of solid tumors harbor KRAS mutations and/or amplification of wild-type KRAS. KRAS mutations are particularly common in pancreatic cancer (~90%), colorectal cancer (CRC) (~50%), and lung adenocarcinoma (~25%).
KRAS mutations primarily involve single-base missense mutations, with 98% occurring at codons G12, G13, and Q61. The main KRAS variants include G12C, G12D, G12V, G13D, G12R, and G12A. Mutation distribution varies by cancer type: in non-small cell lung cancer (NSCLC), the G12C variant accounts for 40% of KRAS mutations, followed by G12V (19%) and G12D (15%). In pancreatic ductal adenocarcinoma (PDAC), G12D and G12V are the dominant mutations.
KRAS mutations are a major cause of resistance to EGFR tyrosine kinase inhibitors (TKIs). Receptor tyrosine kinases (RTKs) are a large family of over 60 transmembrane protein subtypes that serve as receptors for cytokines, growth factors, hormones, and other signals. This family includes EGFR, IGFR, TrkR, MCSFR, INSR, NGFR, FGFR, VEGFR, HGFR, among others. Aberrant RTK activation contributes to the development and progression of multiple malignancies such as breast, lung, and colorectal cancers.
Currently, targeted therapies against RTKs mainly consist of monoclonal antibodies and tyrosine kinase inhibitors (TKIs), with EGFR TKIs being the most studied (detailed information on EGFR TKIs is available in related articles). However, resistance to TKIs remains a major limitation in their clinical efficacy. Emerging research indicates that tumor microenvironment, apoptosis resistance, tumor metabolism, epigenetic modifications, and abnormal TKI metabolism are closely linked to tumor progression and TKI resistance.
Moreover, mutations that abnormally activate PTK-associated signaling pathways can render TKIs ineffective. This is because TKIs target PTKs upstream, but cannot inhibit downstream signaling activated by mutated molecules, resulting in therapeutic resistance.
Therapeutic Strategies Targeting RAS-Driven Tumors
RAS mutations are one of the major driving factors in human cancers. However, due to RAS’s unique structural characteristics, biological functions, and the complexity of its role in tumorigenesis, it has long been considered an “undruggable” target. The main challenges in targeting RAS include:
- Dynamic Nature of the Active Site: RAS cycles between an “on” state (GTP-bound) and an “off” state (GDP-bound), each exhibiting distinct structural conformations. Effective inhibitors must selectively modulate this dynamic equilibrium. Additionally, RAS has a very high affinity for GTP, which significantly complicates the development of competitive inhibitors that block GTP binding.
- Lack of Obvious Small-Molecule Binding Pockets: As a small GTP-binding protein, RAS has a relatively smooth three-dimensional surface, lacking deep pockets or clefts suitable for tight binding by traditional small-molecule drugs. Conventional inhibitors typically work by fitting into specific pockets on the target protein to inhibit its activity or function.
- Complex and Extensive Downstream Signaling: Positioned at the core of intricate cellular signaling networks, activated RAS triggers cascades across multiple downstream pathways. Even if RAS activity is inhibited, cells may compensate via alternative signaling routes to sustain growth and survival, limiting the effectiveness of single-agent RAS-targeted therapies.
Currently, treatment strategies for RAS mutation-driven tumors mainly fall into direct and indirect targeting approaches. Additionally, immunotherapy is applied under certain conditions to treat RAS-mutant cancers.
(1) Direct Targeting Strategies Include:
Allele-specific inhibitors, such as KRASG12C and KRASG12D allele-specific inhibitors
Targeted protein degradation approaches, including PROTACs
Gene therapies such as antisense oligonucleotides (ASOs) and RAS-specific small interfering RNA (siRNA)
Cell-penetrating antibodies
(2) Indirect Targeting Strategies Include:
Inhibitors that disrupt RAS nucleotide exchange, including SOS1 and SHP2 inhibitors
Inhibitors targeting upstream RAS signaling, such as EGFR inhibitors
Inhibitors targeting downstream RAS signaling pathways, including:
MAPK pathway inhibitors (RAF, MEK, ERK inhibitors)
PI3K pathway inhibitors (PI3K and AKT inhibitors)
(3) Immunotherapy Approaches Include:
Immune checkpoint inhibitors (ICIs)
Adoptive cell therapies and cancer vaccines
Clinical Development Progress of Drugs Targeting RAS Mutations
Currently, there are four approved RAS-targeting drugs worldwide, all of which are small-molecule inhibitors specifically targeting the KRASG12C mutation. These include Amgen’s Sotorasib, Bristol Myers Squibb’s Adagrasib, Jingfang/Xinda Pharmaceuticals’ Fluorzeresib, and Yifang Biotech’s Goseresib. These drugs bind to the inactive (OFF) state of KRAS-G12C, thereby inhibiting downstream signaling pathways.
Sotorasib, Fluorzeresib, and Goseresib are approved for second-line treatment of non-small cell lung cancer (NSCLC), while Adagrasib is approved for second-line and beyond treatment of both NSCLC and colorectal cancer. The majority of drugs progressing rapidly through clinical phases—already marketed or in Phase III trials—are KRASG12C-selective inhibitors primarily developed for NSCLC. Some candidates, such as MK-1084, HJ891, and Olomorasib, are also being tested in combination with PD-1 monoclonal antibodies or chemotherapy as first-line treatments for NSCLC.
| Catalog | Product Name | CAS Number |
| 2296729-00-3 | AMG510 racemate | 2296729-00-3 |
| B2693-291051 | AMG510 | 2252403-56-6 |
| B2693-342068 | MRTX849 | 2326521-71-3 |
Resistance to RAS Inhibitors
Although KRAS is no longer considered undruggable due to the approval of targeted inhibitors, monotherapy with KRAS inhibitors often leads to acquired resistance. The main resistance mechanisms include:
- Bypass Activation: Alternative oncogenic changes that activate the RTK-RAS signaling pathway without directly altering KRAS itself. These involve mutations in genes such as BRAF, MET, ALK, RET, and MAP2K1 beyond KRASG12C.
- Acquired Resistance Mutations: Tumor cells may develop secondary mutations during prolonged KRAS inhibitor treatment, altering the KRAS protein structure so that inhibitors can no longer effectively bind, resulting in loss of drug efficacy. For example, new mutation sites may emerge on the KRASG12C backbone that modify the inhibitor binding pocket.
- Histologic Transformation: NSCLC adenocarcinoma may transform into squamous cell carcinoma, indicating potential non-genetic mechanisms of resistance to KRASG12C inhibition.
Summary
RAS is among the most frequently mutated oncogenes in cancer but has long been regarded as an “undruggable” target, with significant unmet clinical needs. Currently, four KRASG12C inhibitors have been approved, all acting by binding to the inactive state of KRAS-G12C to block downstream signaling through an indirect approach. However, resistance to KRASG12C inhibitors may limit their clinical effectiveness.
Combination therapies are now widely recognized as a strategy to overcome resistance. Several Phase III clinical candidates combine KRASG12C inhibitors with PD-1 monoclonal antibodies or chemotherapy as first-line treatments for NSCLC, showing promising clinical outcomes. Yet, combining drugs is not simply additive—it may improve efficacy while also introducing potential risks.
Due to the diverse mechanisms driving resistance to KRASG12C inhibitors, the variety of emerging KRAS mutations during treatment poses new challenges for developing next-generation, more effective KRASG12C inhibitors. Continued exploration of novel therapeutic strategies is urgently needed to overcome this resistance.