The RAS gene is a common oncogene, named after the Rat Sarcoma virus, which was first discovered in a mouse sarcoma virus. The human genome contains three RAS genes (HRAS, KRAS and NRAS) that encode four RAS proteins (HRAS, KRAS4A, KRAS4B and NRAS) (1). They are involved in important biological processes such as cell growth, differentiation, and apoptosis in normal cells (2). However, when the RAS gene is mutated or abnormally expressed, it may become a carcinogenic driver gene and promote the carcinogenic process of cells. Research has shown that KRAS is the most common mutation in human cancers, accounting for ~16% of all cancer types (3). Nearly 40% of patients with advanced colorectal cancer carry mutations in RAS oncogenes (KRAS, NRAS and HRAS), with KRAS mutations being the most prevalent (4). KRASG12 hotspot missense mutations (a single point variation that replaces glycine with another amino acid at the 12th codon) account for 68% of all KRAS mutations, including G12D (33.6%), G12V (20.9%), and G12C (8.2%) (5). The RAS signaling pathway is complex. Mutant RAS molecules are difficult to be directly inhibited, which has hindered the development of effective treatments for RAS-mutated tumors and prevented a major therapeutic breakthrough for several years.

The carcinogenic KRAS mutation has been extensively studied for its roles in tumorigenesis and tumor maintenance. At the molecular level of signaling, the mutated KRAS protein with activating mutations eliminates guanosine triphosphate (GTP)ase activity and remains locked in a high-activity state bound to GTP. This results in sustained activation of downstream pro-proliferative and pro-survival pathways such as RAF/MEK/ERK and PI3K/AKT (6,7). Related research indicates that through alternative splicing, the KRAS transcript generates two mRNA isoforms, namely KRAS4A and KRAS4B (8), each encoding functionally distinct mutant proteins. Each variant protein can transform cells and promote tumor growth (9).

The carcinogenic KRAS mutation is intricately linked to the tumor immune microenvironment. Firstly, the innate immune system predominantly relies on the phagocytic activity of macrophages for tumor surveillance. In early tumor formation, macrophages actively infiltrate tumor tissue and engulf tumor cells; subsequently, their phagocytic capabilities are gradually suppressed by tumor-derived inhibitory signals (10). KRAS mutations can directly activate cluster of differentiation (CD)47 in cancer cells. The binding of CD47 to its receptor, signal regulatory protein alpha, on macrophages, inhibits macrophage-mediated phagocytosis (11), rendering tumor cells insensitive to macrophage phagocytosis, thereby leading to evasion of innate immunity and aggressive tumor progression. Additionally, KRAS mutations can induce T-cell exhaustion and limit adaptive immune responses by upregulating programmed death ligand 1. Taken together, research suggests that mutated KRAS serves as a core driver of tumor immune evasion, as carcinogenic KRAS can impair both innate and adaptive immune antitumor capabilities (12).

The carcinogenic KRAS mutation intricately interacts with cellular metabolism. The expression of carcinogenic KRAS mutation is dependent on aerobic glycolysis metabolism, known as the Warburg effect (13). The carcinogenic KRAS mutation enhances aerobic glycolysis by upregulating the expression and activity of glucose transporters and glycolytic enzymes, including hexokinase (14). The metabolic intermediates of aerobic glycolysis, in turn, provide substrates for biosynthetic processes, generating proteins, lipids, and nucleotides to support rapid cell proliferation (15). To exert its biological activity, KRAS must be localized to the plasma membrane (PM) and organized spatially into proteinaceous assemblies called nanoclusters (KRAS-PM) (16). A study by Liu et al (13) revealed that acute glucose deprivation leads to the detachment of KRASG12V from the PM, which is independent of cellular ATP consumption, thereby demonstrating that KRAS-PM localization also requires glycolysis (13).

The proliferation, survival, differentiation and movement of cancer cells are regulated by different intracellular signaling pathways. Among them, the RAS/RASF/MEK/ERK (MAPK) pathway and the PI3K/AKT pathway have long been revealed to play roles in the pathogenesis of human cancers (17). The RAS molecule is at the core of these tumor signaling pathways and can respond to extracellular signals, such as the activation of epidermal growth factor receptor (EGFR), through effectors such as Src homology-2 protein tyrosine phosphatase (SHP2)/Son of Sevenless (SOS) molecules, leading to the activation of the MAPK, PI3K/AKT and other pathways (18,19). The continuous activation of the MAPK and PI3K pathways leads to uncontrolled cell growth and survival, ultimately resulting in carcinogenic transformation and progression.

Due to the numerous regulatory and regulated molecules, it is difficult to precisely locate a certain link in clinical treatment (19-21), and the pathways such as PI3K and MAPK interact with each other in multiple ways and mutually influence each other by jointly regulating their functions. Therefore, blocking one pathway often induces compensatory activation of another cascade reaction, thereby blocking the effect of KRAS selective inhibitors (22,23).

By contrast, the normal RAS protein cycles between the activated state bound to GTP (ON) and the inactive state bound to guanosine diphosphate (OFF). The cycling disorder of mutant RAS is likely to be in the activated state (24), and its affinity for GTP is extremely high. It is difficult to design small molecule drugs that can directly block its activation (21).

Colorectal cancer carrying the KRAS mutation gene has a poor prognosis (25), and breakthrough treatments have been elusive for numerous years (19).

Currently, both domestic and international guidelines (26-28) recommend a combination of the anti-angiogenic agent bevacizumab with chemotherapy as the first-line standard treatment for advanced KRAS-mutant colorectal cancer, rather than the use of upstream KRAS molecular EGFR-targeted therapy such as cetuximab. In recent years, clinical research has focused on targeted therapies against other molecules associated with the RAS pathway (20,29). Among these, there has been significant progress in drugs targeting the MAPK pathway, notably MEK small molecule inhibitors. However, despite their use as monotherapy [trametinib (30), cobimetinib (31)], in combination with anti-human epidermal growth factor receptor (HER)2 or EGFR therapy [trametinib + lapatinib (32), selumetinib + cetuximab (33)], in combination with chemotherapy [selumetinib + irinotecan (34)], in combination with PI3K/mTOR inhibitors [trametinib + buparlisib (35), trametinib + GSK2126458(36), efametinib + copanlisib (37)], or in combination with AKT inhibitors [cobimetinib + ipatasertib (38), selumetinib + MK-2206(39)], none have shown significant clinical efficacy in advanced colorectal cancer, with minimal objective response rates (ORRs) and almost no objective remissions. Inhibitors targeting downstream molecules of MEK, such as ERK (40) and cyclin dependent kinase (CDK) (41), have also shown suboptimal efficacy as monotherapy.

Forty years after the first discovery of KRAS, in 2013, scientists (18) revealed a novel approach to drug development for this target: A binding pocket exists below the switch II region of the KRAS G12C mutant, where small molecule drugs can be designed to covalently bind, altering the conformation of the KRAS molecule and locking it in an inactive (OFF) state (24,29), without directly targeting GTP. These small molecules that covalently bind to the KRAS G12C mutant are termed ‘allosteric inhibitors’. Following this approach, a series of KRAS G12C allosteric inhibitors have been developed, with the earliest and most representative being sotorasib and adagrasib. Sotorasib, based on the excellent efficacy demonstrated in Phase I/II CodeBreaK100 lung cancer trial, has become the first globally approved KRAS inhibitor (42,43); however it performed poorly in colorectal cancer, with an ORR of only 9.7% for patients with treated KRAS G12C-mutant advanced colorectal cancer (44). Adagrasib exhibited an ORR of 19% and a median duration of response of 4.3 months in the same patient population (44). Subsequent development of KRAS G12C inhibitors GFH925 and JAB-21822 revealed monotherapy ORRs of up to 31% (45) and 33% (46) in patients with colorectal cancer, respectively.

KRAS G12C allosteric inhibitors indeed have the potential to alter the treatment landscape of advanced colorectal cancer with KRAS mutations. However, the two following major issues have been noted: i) Monotherapy for colorectal cancer has shown inferior efficacy compared with lung cancer, with a short duration of response, indicating both primary and acquired resistance. This may be related to the different degrees of reliance on the RAS pathway in different cancer types, activation of bypass signaling pathways, and secondary mutations in the KRAS molecule itself (47-49) KRAS G12C mutations represent only a small fraction of molecular abnormalities in advanced colorectal cancer, and the most common mutations (G12D, G12V and G13D) are unresponsive to G12C inhibitors (29).

To explore the latest trends in KRAS-targeted therapy for colorectal cancer and to address the aforementioned issues, the Trialtrove database was utilized (https://www.citeline.com/en/products-services/clinical/trialtrove) to extract data on ongoing clinical trials worldwide as of June 1st, 2024, involving direct targeting of the KRAS protein or encoding nucleic acids, with study subjects including patients with colorectal cancer, resulting in a total of 85 trials. The start year, drug name, mechanism, combination strategies, study population, and preliminary efficacy results are displayed for each project (Table I).

It was found that combination therapy is a potential strategy to improve the response rate of KRAS-targeted therapy in colorectal cancer. Among the aforementioned 85 clinical trials, 53 (62.4%) involved combination therapy, which presented the following three main directions: i) Blocking upstream KRAS signaling, using combination therapies with EGFR monoclonal antibodies (such as cetuximab and panitumumab), or inhibitors of effectors such as SHP2 and SOS1; ii) blocking downstream KRAS signaling, using combination therapies with MEK, PI3K, or CDK inhibitors; and iii) the use of combination of therapies with immunotherapy or chemotherapy. The first approach has shown some efficacy, with both sotorasib combined with panitumumab (50) and adagrasib (51), divarasib (52), or JAB-21822(47) combined with cetuximab achieving a doubling of ORR compared with monotherapy, along with a prolonged duration of response. Although the combination with SHP2 inhibitors has not yet been reported in clinical data, it also holds great potential based on mechanism and preclinical research results (53,54).

Unfortunately, since the ‘backbone’ drugs for combination therapy are all KRAS G12C inhibitors, the aforementioned strategies are only effective for this rare subtype of colorectal cancer, making it difficult to overcome other types of KRAS mutations. The activation/inactivation cycling rate of other KRAS mutants is different from G12C, and there is no clear allosteric binding pocket, making it difficult for them to be locked in an inactive state (55). Certain studies have examined the use of targeted drugs directly to GTP-bound KRAS molecules, forming a ternary complex with farnesyl (22,56). These drugs, termed RAS (ON) inhibitors, not only inhibit G12C mutations but may also be effective against other types of mutations, laying the foundation for the development of pan-KRAS targeted drugs (22). In addition to conventional small molecule targeted drugs, tumor vaccines and protein degraders targeting other KRAS, or even RAS mutants, are also in early development stages. The aforementioned drugs hold promise in changing the current situation where the common KRAS mutation subtypes G12D, G12V, and G13D in colorectal cancer are considered ‘intractable’.

Collectively, the findings indicate that the KRAS mutation is the most common molecular abnormality in colorectal cancer. Drug therapy targeting this mutation has also undergone arduous exploration. Despite the advent of KRAS G12C inhibitors, monotherapy demonstrates suboptimal clinical efficacy in colorectal cancer, attributed to primary resistance and limited coverage of prevalent KRAS mutations (such as G12D and G12V). Combination therapy with KRAS G12C targeted drugs, notably in combination with EGFR monoclonal antibodies, has effectively increased the ORR. Novel drugs that exploit the mechanim of action of KRAS and target other KRAS mutation types are also in early clinical development. Colorectal cancer KRAS-targeted therapy is a major scientific topic for novel drug development, and the future looks promising.