Oncogenic KRAS blockade therapy: renewed enthusiasm and persistent challenges

RAS genes are mutated at different prevalence rates in human cancers (Fig. 1a) [19]. Elucidating similarities and differences among these RAS mutations from a developmental or evolutionary perspective remains a challenge [20, 21]. Some RAS gene mutations are innocuous, but others cause cancer by producing oncoproteins. For example, three amino acid residues (G12, G13, and Q61) in HRAS, KRAS, and NRAS are mutational hot spots, though with distinct frequencies in different human tumor types (Fig. 1b) [14]. A KRAS-G12 mutation is a common event in pancreatic (91%), colorectal (68%), and lung adenocarcinoma (85%; a subtype of NSCLC) [14]. Among these, KRAS-G12D is the leading mutation in pancreatic (45%) and colorectal adenocarcinoma (45%), while KRAS-G12C mainly occurs in lung adenocarcinoma (46%) (Fig. 1c) [14]. In contrast, 85% of human melanomas have KRAS-Q61 mutations, especially Q61R (46%) [14]. Whole-exome sequencing of human PDAC or NSCLC tumors shows that, despite the genetic heterogeneity within the tumor, KRAS is mutated in different regions [22, 23]. Consistent with this, the co-mutational interactions between each KRAS allele and other unrelated genes are highly tissue-specific, emphasizing the complexity of cell type-specific oncogenesis [24]. KRAS mutations usually co-occur with mutations in tumor protein p53 (TP53) and cyclin-dependent kinase inhibitor 2A (CDKN2A) in PDAC, Kelch-like ECH-associated protein 1 (KEAP1) and/or serine/threonine kinase 11 (STK11) in NSCLC, or APC regulator of WNT signaling pathway (APC) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) in CRC. While mutations in exon 2 of KRAS are the most common, it is important to note that they affect the differential use of exon 4, giving rise to two splice variants, KRAS4A and KRAS4B (Fig. 1b) [25]. These KRAS variants differ in their C-terminal membrane targeting region, posttranslational modifications, and interactomes, thus exhibiting different signal behaviors in development, metabolism, and proliferation [26‐28]. Therefore, to target KRAS, investigators must consider changes in the protein structure caused by point mutations, but also isoform-specific properties [29].

Depending on the clinical settings, the prognostic and predictive values of KRAS mutations are variable and even conflicting. For example, an early study of lung adenocarcinoma patients showed that the KRAS mutation in code 12 is an unfavorable prognostic factor [30]. Similarly, a meta-analysis found that KRAS mutations are associated with poor survival in patients with early resectable NSCLC [31]. In contrast, a pooled analysis of NSCLC patients treated with cisplatin-based chemotherapy revealed that a KRAS mutation was not a prognostic factor [32]. Of note, a KRAS mutation is a negative predictor of response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs; e.g., cetuximab, gefitinib, or erlotinib) (Table 2) [33‐36], but a positive predictor of response to immune checkpoint inhibitors (ICIs; including anti–PD-1 and anti–PD-L1) (Table 3) [37‐39]. However, response rates are variable and dissenting reports suggest that KRAS mutations cannot guide the therapeutic choice between TKIs and ICIs in NSCLC patients [40, 41].

In PDAC, patients with KRAS-G12D mutations (but not total KRAS mutations) have a worse prognosis than patients with wild-type KRAS [42‐44]. Other studies suggest that the prognosis of KRAS-G12V is poorer than that for other mutations [45, 46], commensurate to a KRAS-G12V–associated increase in circulating regulatory T cells that most likely limits antitumor immunity [47]. In advanced PDAC, KRAS mutation status is predictive for the efficacy of erlotinib rather than prognostic [48]. This contradicts other studies reporting that KRAS wild-type patients with PDAC have a significant advantage after treatment with gemcitabine/nimotuzumab [49] or gemcitabine/erlotinib [50] with respect to overall survival. Circulating tumor DNA (ctDNA) has recently become a minimally invasive tool used in precision oncology to evaluate genetic alterations. Mutant KRAS in ctDNA might be a more sensitive predictor of survival than the ELISA-based detection of cancer antigen 19-9 (CA 19-9) [51, 52]. Before a clear conclusion can be drawn regarding the impact of KRAS mutations on overall survival in PDAC [53, 54], additional data from analyses of other genetic alterations are needed.

In localized CRC, KRAS mutations usually suggest a poor prognosis [55‐57]. Some KRAS mutations, including KRAS-G12V, are more aggressive than others [58]. In contrast to BRAF mutations, KRAS mutations have no major prognostic value in advanced CRC patients [59]. The association between KRAS mutations and poor clinical outcomes from TKI treatment has been confirmed in several independent studies [60‐63]. Compared with KRAS-G12V mutations or wild-type tumor groups, CRC patients with KRAS-G13D mutations are insensitive to cetuximab therapy [64, 65]. Nevertheless, the impact of KRAS mutations on cetuximab treatment needs to be further evaluated in prospective randomized trials. Reportedly, KRAS-G12D–mediated inhibition of interferon regulatory factor 2 (IRF2) drives CRC resistance to anti–PD-1 therapy in preclinical models [66]. However, the clinical implications of these findings remain elusive.

In conclusion, the prognostic and predictive values of KRAS mutations are affected by many factors, such as tumor type, stage, patient age, sex, the coexistence of mutations affecting other oncogenes or tumor suppressors, and treatment regimens. For this reason, the clinical utility of detecting KRAS mutations may be over- or underestimated. Careful meta-analyses that avoid nonrandom systematic errors due to variations between trials [67] are needed to clarify this issue.

Wild-type KRAS protein mostly resides on the cytoplasmic side of the plasma membrane, as well as at membranes of intracellular organelles, and is guided by protein localization signals (such as lipid moieties added to the carboxyl terminus) [68]. The RAS family of proteins belongs to a class of enzymes called small GTPases, which play a central role in cell signal transduction [69]. The functions and activities of RAS protein depend on the transition from an inactive guanosine diphosphate (GDP)-bound state to an active guanosine triphosphate (GTP)-bound state (Fig. 2a) [69]. Normally, this conversion process of RAS status is reversible and is maintained in a balanced manner by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). GAPs, such as neurofibromin 1 (NF1), accelerate the GTP hydrolysis of RAS, leading RAS to an inactive state [70, 71]. GEFs, including the most universally expressed SOS Ras/Rac guanine nucleotide exchange factor 1 (SOS1), are responsible for producing active GTP-bound RAS [72‐74]. The balance between hydrolysis and exchange determines the level of activated KRAS in the cell [75]. Oncogenic mutations of RAS disrupt GAP-mediated GTP hydrolysis, allowing these oncoproteins to accumulate in a continuous GTP-binding active state (Fig. 2b) [13]. Among distinct KRAS mutants, the KRAS-G12C protein exhibits the highest intrinsic GTP hydrolysis rate [76].

The upstream regulators of the RAS pathway involve receptor tyrosine kinases (RTKs), which are cell surface receptors for many growth factors, cytokines, and hormones (Fig. 3). One particular RTK subclass, the EGFR family, is composed of four closely related members, EGFR (also known as ERBB1 or HER1), ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4. EGFR/ERBB1, the best characterized activator of RAS signaling, acts through binding to an adaptor protein, namely growth factor receptor bound protein 2 (GRB2) [77, 78]. GRB2 further mediates the recruitment and activation of SOS1- and SH2-containing protein tyrosine phosphatase 2 (SHP2) to activate GTP-bound RAS [79‐84]. Activated EGFR mutations are often found in human cancers, especially NSCLC, and lead to the constitutive activation of downstream signals, including RAS [85]. G-protein–coupled receptors (GPCRs), the largest group of membrane receptors, also participate in RAS activation [86, 87]. Thus, the interplay between RTKs and GPCRs may increase the plasticity of RAS activation.

The downstream effectors of the RAS pathway are mainly involved in the activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways, which favor anabolic processes including cell growth, protein translation, and proliferation (Fig. 3) [88, 89]. Thus, the constitutive activation of membrane RAS-dependent signal pathways favors oncogenesis [90]. The aggregation of mitochondrial KRAS-G12V protein also favors tumor cell growth through metabolic effects [91]. Another distinctive feature of KRAS-G12D protein is that it can be released by PDAC cells into the tumor microenvironment and then mediates the polarization of pro-tumor macrophages (Fig. 4) [92]. Indeed, PDAC patients with KRAS-G12D–positive macrophages exhibit low survival rates [92]. There is also strong preclinical evidence that mutated KRAS requires additional factor (such as chronic inflammation or a high-fat or high-iron diet) to deploy its full carcinogenic activity [93‐95]. Further work is needed to elucidate the likely complex cell-autonomous and non-autonomous effects of mutated and unmutated KRAS protein on the tumor microenvironment [21].

The most successful way to inhibit oncogenic kinases is to develop inhibitors that compete with ATP to bind to the kinase domain. However, KRAS uses GTP instead of ATP as a phosphate donor for signal transmission. Attempts to directly enzymatically inhibit KRAS function have been largely frustrated, leading to the development of indirect methods for KRAS inhibition. Below, we highlight some representative drugs that exemplify the main strategies for indirectly targeting mutant KRAS (Table 1 and Fig. 3).

Different G12Ci compounds exhibit distinct activities and toxicities. For example, adagrasib was found to bind to KRAS-G12C equally in a series of different cell lines, despite the major variability in downstream signals [176]. The main risk of covalent inhibitors is the possibility of nonspecific reactions with off-target proteins containing cysteine residues [173]. Thus, the molecular identification of proteins accounting for off-target effects of G12Ci may improve structure-based drug design [203]. In addition to organ-mediated drug metabolism, tumor-resident microorganisms can directly decompose chemotherapeutic drugs to cause drug resistance [207]. Hence, optimizing the in vivo pharmacokinetic properties of KRAS inhibitors and evaluating the (in)activity of their metabolites is still an important area for examination. Finally, it remains to be seen whether it is possible to develop additional mutation-specific inhibitors or pan-mutant KRAS inhibitors [208].

The design and implementation of strategies to minimize or overcome drug resistance is an important goal of clinical oncology in order to achieve complete and durable clinical responses [209]. The observed tumor heterogeneity and the extensive feedback between RAS and other tumor-related signals may promote drug resistance. The combination of several drugs intercepting different signaling pathways (e.g., upstream signaling, downstream signaling, parallel signaling, cell cycle processes, or immune checkpoints) may prevent or delay the development of therapy resistance, but usually at the cost of increased toxicity [14, 17]. A number of clinical trials combining G12Ci with other established agents (including TKIs and ICIs) are being launched. The design of such trials should avoid random combinations and follow a rationale based on the genetic, metabolic, and immune mechanisms of drug resistance.

Predictive biomarkers can guide treatment decisions by indicating the likely impact of a particular therapy on a patient. Some genetic alterations, especially secondary gene mutation, are associated with the development of adagrasib resistance in patients. In addition to using endoscopic ultrasound-guided fine needle aspiration, the analysis of circulating tumor DNA combined with next-generation sequencing technologies provides a way for examining the genetic characteristics of tumors. Despite these obvious technological advances, circulating metabolites or proteins should not be neglected as potential biomarkers, given that their quantitation would be much more convenient.

In summary, the development of ever more efficient and specific drugs blocking oncogenic RAS must be in parallel with major efforts to avoid and overcome therapeutic resistance. Given the continued efforts of industry, academia, and health care providers, as well as the latest breakthroughs in basic, clinical, and translational research on G12Ci, the prospects look bright.