KRAS was the first described oncogene in human cancer (1) and one of the most frequently mutated genes (2). It encodes a GTPase superfamily protein that mediates the intracellular signaling of activated tyrosine kinase receptors by switching between inactive GDP-bound and active GTP-bound states. This activation induces phosphorylation of effectors of the mitogen-activated protein kinase (MAPK) pathway like RAF, MEK, and ERK leading to apoptosis inhibition and activation of transcription factors that promote cancer cell survival and metastasis (3, 4). Guanine-nucleotide-exchange factors (GEFs) catalyze the exchange of GDP for GTP, hence turning KRAS into its active state; on the other hand, GTPase activating proteins (GAPs) take part in the GTP hydrolysis reaction, inactivating KRAS (5). Single aminoacidic mutations at codons 12,13, and 61 are responsible for structural changes that prevent GAPs from hydrolyzing GTP, and therefore blocking KRAS in its GTP-bound active state (6). The hotspot transversion mutation KRAS^G12C is the most frequently found in lung adenocarcinoma accounting for 13% of cases in the western population (7, 8). Despite its high prevalence and importance in the development of cancer, efforts to effectively target KRAS have long been futile as a consequence of micromolar GTP concentrations in cells (~0.5 mmol/L) combined with a picomolar affinity (dissociation constant at ~10^-10 mol/L) of KRAS for GTP (9). Moreover, the lack of allosteric regulatory sites needed for drug binding coined KRAS as an “undruggable” target (10). This has been modified with the discovery in the KRAS^G12C mutant protein of a pocket that resides beneath de switch II region, adjacent to the mutant cysteine that allows the binding of small inhibitory molecules and, as a consequence favoring the affinity of KRAS for GDP, blocking KRAS^G12C in its inactive state (11). This unique feature of the KRAS G12C mutation has led to the development of specific KRAS^G12C irreversible inhibitors such us adagrasib and sotorasib which is currently FDA approved to treat patients with KRAS^G12C lung cancer.

Unfortunately, primary and acquired resistance invariably occur. This review focuses on the intrinsic and acquired mechanisms of resistance to KRAS^G12C inhibitors, mainly in KRAS^G12C mutant non-small cell lung cancer, and potential clinical strategies to overcome or prevent resistance.

Particular RAS mutations may modify RAS proteins’ biochemical behavior, including their ability to bind GTP and GDP. Further studies continue reporting differences in GTP binding and intrinsic or GAP-mediated GTP hydrolysis (12). Smith et al. detected KRAS^G12V in the GTP-bound conformation, which was consistent with its high transforming potential. In addition, experiments in MCF10A cells transduced with different KRAS mutations revealed that KRAS wild-type (WT) and KRAS^G12D and KRAS^G13D could bind GTP with similar affinity to control cells, which only express endogenous KRAS, after EGF stimulation. In contrast, KRAS^G12C showed an increase in GTP-binding up to 2-fold mutant compared to control cells (13). Another study also reported that KRAS^G12A, ^G12R, ^Q61H, and ^Q61L decreased GTP hydrolysis speed approximately by 40- to 80-fold compared to KRAS wild-type, the ^G12C mutation had a minimal impact in this respect. Regarding this endpoint, KRAS^G12D, ^G12V, and KRAS^G13D mutant proteins displayed an intermediate effect (14).

Anchorage-independent growth is the ability of transformed cells to grow in suspension or unattached to any matrix (13) an associated characteristic for tumor metastasis regulated by the RAS/RAF/MAPK signaling pathway (15). Seeburg et al. reported that except for HRAS WT and HRAS ^G12P, all the HRAS codon 12 mutants could grow in soft agar (16), results paralleling their data on transforming potential of these mutant proteins. Immortalized human bronchial epithelial cells with specific shRNA knockdown of p53 mRNA expressing KRAS^G12C were able to form colonies in soft agar compared to KRAS^G12D easily- and KRAS WT-transfected cells (17), suggesting that the genetic background could also affect the phenotypical manifestation of mutant RAS variants.

The RAF1 serine/threonine kinase is one of the best characterized RAS effector proteins, located directly downstream of RAS in the MAPK pathway (14). Considering that point mutations at codons 12 and 61 of HRAS differ in their phenotypical properties as previously reported (18), Voice et al. hypothesized that mutant RAS proteins might activate RAF1 differentially (19) and found that KRAS^G12A, ^G12C, ^G13D, ^Q61L, and ^Q61H showed 1.2- to 2.3-fold decrease in relative affinity compared to WT KRAS and KRAS^G12D, ^G12R and ^G12V displayed an even more pronounced reduction in affinity for RAF1 (4.8-, 6.2-, and 7.3-fold, respectively).

RAS proteins also activate the PI3K/AKT/mTOR pathway to promote cell survival by activating survival factors and inhibiting apoptotic proteins (20). Therefore, different in vitro (13) and in vivo (18, 21) studies also assessed the activation of this pathway by the interaction of various RAS mutated variants with PI3K and different downstream proteins, such as AKT, 4EBP, or RPS6. The comparison of KRAS^G12C and ^G12V with WT KRAS in a panel of 67 NSCLC cell lines showed that these mutations decreased AKT activation compared to WT KRAS (17). Despite this low activation of AKT, cells expressing KRAS^G12C or ^G12V showed the same phosphorylation levels of 70S6K and 4EBP proteins compared to WT KRAS in the absence of serum. In contrast, the addition of serum to the media enabled KRAS^G12C and ^G12V to strongly phosphorylate 70S6K compared to WT KRAS (17).

RAS proteins can also interact and activate effectors that do not belong to the MAPK and the PI3K canonical cascades. For example, RAC, a subfamily of small GTPases of the RHO family, can interact with RAS via the RacGEF called Tiam1. The RAS/RAC signaling pathway controls several cellular functions by regulating actin cytoskeleton, including cell morphology, locomotion, and polarity (22). Another RAS downstream effector subfamily is the RAL group of proteins involved in membrane trafficking, proliferation, survival, and metastasis in many types of cancer (23). In this sense, WT KRAS and KRAS^G12C, but not KRAS^G12D, activated RALA and RALB effector proteins (17).

Brunelli et al. characterized the metabolic profile of the isogenic NCI-H1299 NSCLC cell line overexpressing WT KRAS or KRAS^G12C, ^G12D, or ^{G12V (} 24). Most metabolites identified were common to all three KRAS-mutated lines (G12C, G12D, and G12V), although these mutants harbored 74, 58, and 48 unique metabolites, respectively, compared to WT. Moreover, the deregulated metabolites between WT and mutant KRAS variants were classified into biochemical groups. The two most abundant classes for KRAS^G12C, ^G12D, and ^G12V were glycerophospholipids and amino acids. KRAS^G12C and KRAS^G12D mainly affected phosphatidylcholines (PC) and phosphatidylinositol (PI), whereas KRAS^G12V influenced PI and phosphatidylserine. In addition, the report by Brunelli et al. provided further insights into the biology of the deregulated metabolites. KRAS^G12C, ^G12D, and ^G12V variants showed an increase of metabolites related to protein biosynthesis, glutathione, glutamate metabolism and ammonia recycling (24). Moreover, KRAS^G12C, ^G12D, and ^G12V had lower levels of glutamate, glutamine, asparagine, and proline, amino acids interconnected in the glutamate synthase cycle, and lower levels of NAD+, an essential coenzyme involved in many cellular metabolic pathways.

Following on these results, the group of Pastorelli continued studying the metabolic profile of KRAS^G12C, as it is the most representative KRAS mutation in NSCLC patients. In this work, the NCI-H1299 NSCLC cell line expressing WT or KRAS^G12C and xenograft tumors generated from this cell line were analyzed (25). They identified 26 and 23 deregulated metabolites in vitro and in vivo, respectively, between WT KRAS and KRAS^G12C. The enriched pathway analysis of these deregulated metabolites showed that KRAS^G12C alters the same metabolic pathways in vitro and in vivo, including pathways involved in protein biosynthesis, ammonia recycling, and urea cycle (25). Focusing on the deregulated metabolites whose abundance changed significantly in vitro and in vivo between WT KRAS and KRAS^G12C, 11 and 16 metabolites were significantly altered, respectively. Moreover, in both in vitro and in vivo models, KRAS^G12C, decreased glutamine and glutamate levels, two amino acids involved in nitrogen balance maintenance, supporting the central role of glutaminolysis and nitrogen anabolism to provide energy for cancer cell growth and proliferation. This indicates that cells expressing the KRAS^G12C variant use glutaminolysis as a source of energy (25). In addition, KRAS^G12C, mutation induced a significant increase in the levels of carnitine, acetyl-carnitine, and butyryl-carnitine, which are involved in the oxidation of fatty acids. This increase could be associated with the mitochondrial fatty acid beta-oxidation to respond to the increasing energy demand triggered by KRAS^G12C, to fuel cell or tumor growth and proliferation (25).

The development of specific KRAS^G12C inhibitors started with ARS-1620, a small covalent specific inhibitor, that showed in vitro and in vivo activity in KRAS ^G12C mutant models (26). This was followed by the development of two KRAS^G12C inhibitors, sotorasib (AMG 510) and adagrasib (MRTX849), supported by robust preclinical evidence and reaching clinical development in phase I/II trials (27, 28). Both compounds have a common mechanism of action by inhibiting KRAS in its GDP-bound state, however, the binding properties of both inhibitors differ slightly. The KRAS^G12C cryptic pocket is formed by residues H95/Y96/Q99 is exploited by the hydrogen mediated bonding of the hydroxyl group of Y96 with the pyrimidine ring of adagrasib (29) and by the water bridges between Y96 and a carboxyl group in sotorasib (30).

The phase I trial of sotorasib CodeBreak100 included pretreated 125 patients with metastatic KRAS^G12C mutant solid tumors; 45.7% had non-small cell lung cancer (NSCLC) (31). The objective response was 32.2% in patients with lung cancer, and the disease control rate was 88.1%. No dose-limiting toxicities or treatment-related deaths were observed at the established phase II dose of 960mg (31). These results were recently confirmed by the phase II trial, which specifically included 122 patients with NSCLC. The objective response rate was 37.1%, with a median duration of response of 11 months and an 80.6% disease control rate. Median progression-free survival was 6.8 months, and median overall survival was 12.5 months (32). In the explorative analysis, assessing the association between response to sotorasib and the presence of previously described co-occurring mutations (STK11, KEAP1, and TP53), which define three subgroups of KRAS mutant cancers with different biology and response to treatment (33), sotorasib showed efficacy in all subgroups, with a lower percentage of response in patients with KEAP1 mutated tumors, although this was not statistically significant. Also, similar response rates were observed in PDL-1 positive or negative (score <1%) patients as well as in tumors with high (≥10 mutations per megabase) or low (<10 mutations per megabase) mutational burden. These findings led to the FDA’s accelerated approval in May 2021 of sotorasib in previously treated patients with mutant KRASG12C NSCLC. Efficacy of sotorasib versus docetaxel in previously treated patients KRAS^G12C mutant NSCLC is currently being tested on a phase III clinical trial (NCT04303780). Moreover, sotorasib as first-line treatment is under investigation on a phase II trial (NCT04933695).

In a similar manner, data from the phase I/II KRYSTAL-I clinical trial, that included 79 previously treated patients with advanced stage KRAS^G12C mutant NSCLC treated with adagrasib, showed an objective response rate of 45% and a 96% diseases control rate (34). This trial is still ongoing (NCT03785249).

Interestingly, response rates for KRAS^G12C mutant colorectal cancer cohorts included in both trials were considerably lower, with 7% and 17% for sotorasib and adagrasib, respectively (31, 34). This primary resistance is driven by upstream activation of EGFR, which can be overcome by combining KRAS^G12C inhibitors with anti EGFR monoclonal antibodies, like sotorasib and cetuximab, respectively (35).

Given the clinical evidence of the efficacy of KRAS^G12C inhibitors in lung cancer, a considerable proportion of patients do not experience a tumor response or have primary progression, for which treatment intensification may be required. Also, compared to other targeted therapies like EGFR, ALK, and ROS1, patients with KRAS mutant tumors that initially achieve a partial response have a shorter duration of responses, driven by acquired resistance mechanisms (36).

Despite the encouraging clinical results, about half of the patients included in clinical trials with sotorasib and adagrasib do not experience significant tumor shrinkage with specific KRAS^G12C inhibitors. Moreover, nearly 10% of patients will experience primary disease progression. On the other hand, as with other target therapies, all patients who initially experience an objective response or stable disease will eventually progress with KRAS^G12C inhibitors, given the emergence of resistance mechanisms in cancer cells. In general, biologic mechanisms of resistance can be categorized into two main groups: on-target resistance, driven by mutations or amplification in KRAS that halters drug binding, and off-target or bypass mechanisms of resistance, when KRAS is inhibited, but another effector commands oncogenic signaling. In addition, other resistance mechanisms imply histologic transformation and other events affecting epigenetic modifications and apoptosis. A summary of biologic resistance mechanisms to KRAS^G12C inhibitors is shown in Figure 1 .

The complex and diverse resistance mechanisms to KRAS^G12C inhibitors pose a challenge to design clinical strategies to overcome or prevent resistance. A main mechanism of primary resistance is feedback reactivation mediated by multiple RTKs, leading to preclinical research aiming to combine the inhibition of KRAS^G12C and other RTKs such as EGFR, FGFR, and MET (56). In this setting the combination of sotorasib and MET inhibitors such as crizotinib and capmatinib has shown to overcome resistance to KRAS^G12C inhibitors in vitro in cancer cells with MET amplification (46). One of the main focuses of current development is targeting transduction molecules situated downstream from RTKs such as SHP2 and SOS1 which after phosphorylation favor de GTP-bound state thus activating the ERK pathway (57). Consequently, targeting SHP2 and SOS1 can synergize with KRAS^G12C inhibitors given in combination. Preclinical research showed that the combination of a KRAS^G12C inhibition (ARS-1620) with a SHP2 inhibitor (SHP099) conveyed a more profound suppression of KRAS-GTP and total RAS-GTP in vitro and enhance tumor regression in xenograft models compared to ARS-1620 and SHP099 alone (39). In the same manner the combination of sotorasib and BI-3406, a SOS1 inhibitor, resulted in diminished activity of the ERK pathway in KRAS^G12C mutant cells (46). Multiple clinical trials are ongoing to test the efficacy of the combination of KRAS^G12C and SHP2 inhibitors including: the phase 1/2 KRYSTAL 2 trial evaluating the combination of MRTX849 and TNO155 in patients with solid tumor with KRAS^G12C mutation (NCT04330664) and a phase Ib/II evaluating JDQ443 (KRAS^G12C inhibitor) and TNO155 (NCT04699188). Other strategies to overcome primary resistance are being evaluated in preclinical models including combination of KRAS^G12C inhibitors with: PI3K/AKT inhibitors, chemotherapy, CDK4/6 inhibitors targeting alterative pathways, cell cycle and DNA damage (56). RM-108 is a specific KRAS^G12C inhibitor which binds to a chaperone protein forming a “tri-complex” that prevents the association to the downstream effector proteins (58). This has shown to be active in KRAS^G12C/Y96D resistant BA/F3 models (29). These strategies represent multiple opportunities to overcome primary and acquired resistance, which need validation in clinical trials.

In addition to the efficacy of single targeted therapies and combinations, cell death induced by KRAS^G12C inhibitors induce a pro-inflammatory microenvironment that set the bases for the observed synergism between KRAS^G12C and anti-PD1 immune checkpoint inhibitors in murine models (27, 59); this combination strategy is being evaluated in the multi-arm CodeBreak 101 trial (NCT04185883) with an estimated enrollment of 1273 participants. This trial includes both an experimental arm of sotorasib plus pembrolizumab in dose exploration and dose expansion cohorts and a sotorasib plus atezolizumab cohort. Other arms in this trial include the combination of sotorasib plus: afatinib, palbociclib, chemotherapy, everolimus, among others. In a similar manner the phase 2 KRYSTAL-7 trial is designed to include 250 patients with treatment naïve advanced KRAS^G12C mutant NSCLC to evaluate the clinical activity of adagrasib in combination with pembrolizumab in (NCT04613596).

Given the recent clinical approval of specific KRAS^G12C inhibitors and the ongoing development of other strategies to target other KRAS mutations, understanding the biological mechanisms that confer primary and acquired resistance is necessary to improve patients’ clinical outcomes to the response rates and progression free survival.

Resistance to KRAS^G12C is still not fully understood. However initial reports show that these mechanisms are complex, heterogeneous, and can emerge concomitantly during treatment. In contrast with other targets like EGFR-mutant NSCLC in which a single the T790M mutation is responsible for approximately half of the progressions after first- or second-line specific inhibitors (60), progressions after treatment with KRAS^G12C inhibitors are caused by both single and combined mechanisms, including: on-target mutations affecting the docking site of this inhibitors; other activating KRAS mutations; activation of the canonical pathways by mutations or activation of upstream and downstream effectors and also activation of non-mutant RAS isoforms. These early reports may be selecting patients with tumors that have more complex biology; hence there is a need to further explore resistance mechanisms in patients that experience a more extended benefit with KRAS^G12C inhibitors. In the clinical setting, liquid biopsy NGS has proven to be a useful tool in identifying the heterogeneity of resistance mechanisms occurring in a single patient (61). The identification and understanding of acquired resistance mutations to test and develop new therapies to target them.

JB, AC, and GR: drafted and critically reviewed the work. All authors contributed to the article and approved the submitted version.

JB reports educational grants from Amgen, GR reports grants from Amgen, personal fees from Roche, Bayer, BMS, MSD, Pfizer, Takeda, and Amgen. AC disclosed financial research support from MSD, Boehringer Ingelheim, Roche, BMS, and The Foundation for Clinical and Applied Cancer Research - FICMAC. Additionally, he was linked and received honoraria as an advisor, participated in speakers’ bureau, and gave expert testimony to MSD, Boehringer Ingelheim, Roche, BMS, Pfizer, Novartis, Celldex Therapeutics, Foundation Medicine, Eli Lilly, and Foundation for Clinical and Applied Cancer Research - FICMAC.

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