The Target Therapy Hyperbole: "KRAS (p.G12C)"-The Simplification of a Complex Biological Problem.

Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) gene variations are linked to the development of numerous cancers, including non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC). The lack of typical drug-binding sites has long hampered the discovery of therapeutic drugs targeting KRAS. Since "CodeBreaK 100" demonstrated Sotorasib's early safety and efficacy and led to its approval, especially in the treatment of non-small cell lung cancer (NSCLC), the subsequent identification of specific inhibitors for the p.G12C mutation has offered hope. However, the CodeBreaK 200 study found no significant difference in overall survival (OS) between patients treated with Docetaxel and Sotorasib (AMG 510), adding another degree of complexity to this ongoing challenge. The current study compares the three-dimensional structures of the two major KRAS isoforms, KRAS4A and KRAS4B. It also investigates the probable structural changes caused by the three major mutations (p.G12C, p.G12D, and p.G12V) within Sotorasib's pocket domain. The computational analysis demonstrates that the wild-type and mutant isoforms have distinct aggregation propensities, resulting in the creation of alternate oligomeric configurations. This study highlights the increased complexity of the biological issue of using KRAS as a therapeutic target. The present study stresses the need for a better understanding of the structural dynamics of KRAS and its mutations to design more effective therapeutic approaches. It also emphasizes the potential of computational approaches to shed light on the complicated molecular pathways that drive KRAS-mediated oncogenesis. This study adds to the ongoing efforts to address the therapeutic hurdles presented by KRAS in cancer treatment.

Bioinformatics and Experimental Validation for Identifying Biomarkers Associated with AMG510 (Sotorasib) Resistance in KRASG12C-Mutated Lung Adenocarcinoma.

The Kirsten rat sarcoma viral oncogene homolog (KRAS)G12C mutation is prevalent in lung adenocarcinoma (LUAD), driving tumor progression and indicating a poor prognosis. While the FDA-approved AMG510 (Sotorasib) initially demonstrated efficacy in treating KRASG12C-mutated LUAD, resistance emerged within months. Data from AMG510 treatment-resistant LUAD (GSE204753) and single-cell datasets (GSE149655) were analyzed. Gene set variation analysis (GSVA) and gene set enrichment analysis (GSEA) were used to explore enriched signaling pathways, nomogram models were constructed, and transcription factors predicting resistance biomarkers were predicted. CIBERSORT identified immune cell subpopulations, and their association with resistance biomarkers was assessed through single-cell analysis. AMG510-resistant LUAD cells (H358-AR) were constructed, and proliferative changes were evaluated using a CCK-8 assay. Key molecules for AMG510 resistance, including SLC2A1, TLE1, FAM83A, HMGA2, FBXO44, and MTRNR2L12, were recognized. These molecules impacted multiple signaling pathways and the tumor microenvironment and were co-regulated by various transcription factors. Single-cell analysis revealed a dampening effect on immune cell function, with associations with programmed cell death ligand 1 (PDL1) expression, cytokine factors, and failure factors. The findings indicate that these newly identified biomarkers are linked to the abnormal expression of PDL1 and have the potential to induce resistance through immunosuppression. These results highlight the need for further research and therapeutic intervention to address this issue effectively.

First-in-Humans PET Imaging of KRASG12C Mutation Status in Non-Small Cell Lung and Colorectal Cancer Patients Using [18F]PFPMD.

Kirsten rat sarcoma (KRAS) mutations are an important marker for tumor-targeted therapy. In this study, we sought to develop a KRASG12C oncoprotein-targeted PET tracer and to evaluate its translational potential for noninvasive imaging of the KRASG12C mutation in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) patients. Methods: [18F]PFPMD was synthesized on the basis of AMG510 (sotorasib) by attaching a polyethylene glycol chain to the quinazolinone structure. The binding selectivity and imaging potential of [18F]PFPMD were verified by cellular uptake, internalization, and blocking (H358: KRASG12C mutation; A549: non-KRASG12C mutation) studies, as well as by a small-animal PET/CT imaging study on tumor-bearing mice. Five healthy volunteers were enrolled to assess the safety, biodistribution, and dosimetry of [18F]PFPMD. Subsequently, 14 NSCLC or CRC patients with or without the KRASG12C mutation underwent [18F]PFPMD and [18F]FDG PET/CT imaging. The SUVmax of tumor uptake of [18F]PFPMD was measured and compared between patients with and without the KRASG12C mutation. Results: [18F]PFPMD was obtained with a high radiochemical yield, radiochemical purity, and stability. The protein-binding assay showed that [18F]PFPMD selectively binds to the KRASG12C protein. [18F]PFPMD uptake was significantly higher in H358 than in A549 and was decreased by pretreatment with AMG510 (H358 vs. A549: 3.22% ± 0.28% vs. 2.50% ± 0.25%, P < 0.05; block: 2.06% ± 0.13%, P < 0.01). Similar results were observed in tumor-bearing mice on PET imaging (H358 vs. A549: 3.93% ± 0.24% vs. 2.47% ± 0.26% injected dose/g, P < 0.01; block: 2.89% ± 0.29% injected dose/g; P < 0.05). [18F]PFPMD was safe in humans and was excreted primarily by the gallbladder and intestines. The whole-body effective dose was comparable to that of [18F]FDG. The accumulation of [18F]PFPMD in KRASG12C mutation tumors was significantly higher than that in non-KRASG12C mutation tumors (SUVmax: 3.73 ± 0.58 vs. 2.39 ± 0.22, P < 0.01) in NSCLC and CRC patients. Conclusion: [18F]PFPMD is a safe and promising PET tracer for noninvasive screening of the KRASG12C mutation status in NSCLC and CRC patients.

AMG-510 and cisplatin combination increases antitumor effect in lung adenocarcinoma with mutation of KRAS G12C: a preclinical and translational research.

BACKGROUND: The efficacy of monotherapy of AMG-510 is limited. This study explored whether the AMG-510 and cisplatin combination increases the anti-tumor effect in lung adenocarcinoma with the mutation of Kirsten rat sarcoma viral oncogene (KRAS) G12C. METHODS: Patients' data were used to analyze the proportion of KRAS G12C mutation. Besides, the next-generation sequencing data was used to uncover information about co-mutations. The cell viability assay, the concentration inhibiting 50% of cell viability (IC50) determination, colony formation, and cell-derived xenografts were conducted to explore the anti-tumor effect of AMG-510, Cisplatin, and their combination in vivo. The bioinformatic analysis was conducted to reveal the potential mechanism of drug combination with improved anticancer effect. RESULTS: The proportion of KRAS mutation was 2.2% (11/495). In this cohort with KRAS mutation, the proportion of G12D was higher than others. Besides, KRAS G12A mutated tumors had the likelihood of concurrent serine/threonine kinase 11 (STK11) and kelch-like ECH-associated protein 1 (KEAP1) mutations. KRAS G12C and tumor protein p53 (TP53) mutations could appear at the same time. In addition, KRAS G12D mutations and C-Ros oncogene 1 (ROS1) rearrangement were likely to be present in one tumor simultaneously. When the two drugs were combined, the respective IC50 values were lower than when used alone. In addition, there was a minimum number of clones among all wells in the drug combination. In in vivo experiments, the tumor size reduction in the drug combination group was more than twice that of the single drug group (p < 0.05). The differential expression genes were enriched in the pathways of phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) signaling and extracellular matrix (ECM) proteoglycans compared the combination group to the control group. CONCLUSIONS: The anticancer effect of the drug combination was confirmed to be better than monotherapy in vitro and in vivo. The results of this study may provide some information for the plan of neoadjuvant therapy and the design of clinical trials for lung adenocarcinoma patients with KRAS G12C mutation.

CodeBreak 200: Sotorasib Has Not Broken the KRASG12C Enigma Code.

Thirteen percent of non-small cell lung cancer (NSCLC) patients are estimated to have the KRAS G12C mutation. Sotorasib is a novel KRAS G12C inhibitor that has shown promising results in preclinical and clinical studies, granting its conditional approval by the FDA in May 2021. The phase I clinical trial resulted in a confirmed response of 32% and progression free survival (PFS) of 6.3 months while the phase II trial resulted in a confirmed response of 37.1% and a PFS of 6.8 months. It was also shown to be tolerable with most subjects experiencing grade one or two adverse events, most commonly diarrhea and nausea. The CodeBreaK 200 phase III trial data have recently resulted and showed an improved PFS with the use of sotorasib at 5.6 months compared to that of standard docetaxel of 4.5 months in locally advanced or unresectable metastatic KRAS G12C NSCLC previously treated with at least one platinum-based chemotherapy and checkpoint inhibitor. The lower than expected PFS of sotorasib from the phase III trial opens up opportunities for other G12C inhibitors to join the field. Indeed, adagrasib, another G12C inhibitor just recently gained FDA accelerated approval in NSCLC patients based on the KRYSTAL-1 study where the response rate was 43% with a median duration of response of 8.5 months. With novel agents and combinations, the field of KRAS G12C is quickly evolving. While sotorasib was an exciting start, there is more to do to break the KRAS G12C Enigma code.

Evaluation of KRASG12C inhibitor responses in novel murine KRASG12C lung cancer cell line models.

Introduction: The KRAS(G12C) mutation is the most common genetic mutation in North American lung adenocarcinoma patients. Recently, direct inhibitors of the KRASG12C protein have been developed and demonstrate clinical response rates of 37-43%. Importantly, these agents fail to generate durable therapeutic responses with median progression-free survival of ~6.5 months. Methods: To provide models for further preclinical improvement of these inhibitors, we generated three novel murine KRASG12C-driven lung cancer cell lines. The co-occurring NRASQ61L mutation in KRASG12C-positive LLC cells was deleted and the KRASG12V allele in CMT167 cells was edited to KRASG12C with CRISPR/Cas9 methods. Also, a novel murine KRASG12C line, mKRC.1, was established from a tumor generated in a genetically-engineered mouse model. Results: The three lines exhibit similar in vitro sensitivities to KRASG12C inhibitors (MRTX-1257, MRTX-849, AMG-510), but distinct in vivo responses to MRTX-849 ranging from progressive growth with orthotopic LLC-NRAS KO tumors to modest shrinkage with mKRC.1 tumors. All three cell lines exhibited synergistic in vitro growth inhibition with combinations of MRTX-1257 and the SHP2/PTPN11 inhibitor, RMC-4550. Moreover, treatment with a MRTX-849/RMC-4550 combination yielded transient tumor shrinkage in orthotopic LLC-NRAS KO tumors propagated in syngeneic mice and durable shrinkage of mKRC.1 tumors. Notably, single-agent MRTX-849 activity in mKRC.1 tumors and the combination response in LLC-NRAS KO tumors was lost when the experiments were performed in athymic nu/nu mice, supporting a growing literature demonstrating a role for adaptive immunity in the response to this class of drugs. Discussion: These new models of murine KRASG12C mutant lung cancer should prove valuable for identifying improved therapeutic combination strategies with KRASG12C inhibitors.

Multiple initiatives to conquer KRAS G12C inhibitor resistance from the perspective of clinical therapy.

INTRODUCTION: KRAS G12C targeted covalent inhibitors for cancer therapy are revolutionary. However, resistance to KRAS G12C inhibitors in clinical trials is a proven fact. AREAS COVERED: The authors focus on providing coverage and emphasizing the strategy of conquering KRAS G12C inhibitor resistance from the perspective of clinical therapy. The authors also provide the readers with their expert perspectives for future development. EXPERT OPINION: It is essential to improve the therapeutic effect and achieve long-term disease control through accumulating rapid exploration of drug resistance mechanisms in preclinical trials and developing rational combination dosing approaches from clinical practice. Our presentation of the perspective provides insights into drug resistance in this groundbreaking area of research.

Inside the cracked kernel: establishing the molecular basis of AMG510 and MRTX849 in destabilising KRASG12C mutant switch I and II in cancer treatment.

The Kirsten rat sarcoma oncoprotein (KRAS) has been punctuated by drug development failures for decades due to frequent mutations that occur mostly at codon 12 and the seemingly intractable targeting of the protein. However, with advances in covalent targeting, the oncoprotein is being expunged from the 'undruggable' list of proteins. This feat has seen some covalent drugs at different stages of clinical trials. The advancement of AMG510 and MRTX849 as inhibitors of cysteine mutated KRAS (KRASG12C) to phase-III clinical trials informed the biased selection of AMG510 and MRTX849 for this study. Despite this advance, the molecular and atomistic modus operandi of these drugs is yet to come to light. In this study, we employed computational tools to unravel the atomistic interactions and subsequent conformational effects of AMG510 and MRTX849 on the mutant KRASG12C. It was revealed that AMG510 and MRTX849 complexes presented similar total free binding energies, (ΔGbind), of -88.15 ± 5.96 kcal/mol and -88.71 ± 7.70 kcal/mol, respectively. Gly10, Lys16, Thr58, Gly60, Glu62, Glu63, Arg68, Asp69, Met72, His95, Tyr96, Gln99, Arg102 and Val103 interacted prominently with AMG510 and MRTX849. These residues interacted with the pharmacophoric moieties of AMG510 and MRTX849 via hydrogen bonds with decreasing bond lengths at various stages of the simulation. These interactions together with pi-pi stacking, pi-sigma and pi-alkyl interactions induced unfolding of switch I whiles compacting switch II, which could interrupt the binding of effector proteins to these interfaces. These insights present useful atomistic perspectives into the success of AMG510 and MRTX849 which could guide the design of more selective and potent KRAS inhibitors.Communicated by Ramaswamy H. Sarma.

Targeting PI3Kα overcomes resistance to KRasG12C inhibitors mediated by activation of EGFR and/or IGF1R.

Although several KRasG12C inhibitors have displayed promising efficacy in clinical settings, acquired resistance developed rapidly and circumvented the activity of KRasG12C inhibitors. To explore the mechanism rendering acquired resistance to KRasG12C inhibitors, we established a series of KRASG12C-mutant cells with acquired resistance to AMG510. We found that differential activation of receptor tyrosine kinases (RTKs) especially EGFR or IGF1R rendered resistance to AMG510 in different cellular contexts by maintaining the activation of MAPK and PI3K signaling. Simultaneous inhibition of EGFR and IGF1R restored sensitivity to AMG510 in resistant cells. PI3K integrates signals from multiple RTKs and the level of phosphorylated AKT was revealed to negatively correlate with the anti-proliferative activity of AMG510 in KRASG12C-mutant cells. Concurrently treatment of a novel PI3Kα inhibitor CYH33 with AMG510 exhibited a synergistic effect against parental and resistant KRASG12C-mutant cells in vitro and in vivo, which was accompanied with concomitant inhibition of AKT and MAPK signaling. Taken together, these findings revealed the potential mechanism rendering acquired resistance to KRasG12C inhibitors and provided a mechanistic rationale to combine PI3Kα inhibitors with KRasG12C inhibitors for therapy of KRASG12C-mutant cancers in future clinical trials.

Evaluation of AMG510 Therapy on KRAS-Mutant Non-Small Cell Lung Cancer and Colorectal Cancer Cell Using a 3D Invasive Tumor Spheroid System under Normoxia and Hypoxia.

A 3D tumor spheroid has been increasingly applied in pharmaceutical development for its simulation of the tumor structure and microenvironment. The embedded-culture of a tumor spheroid within a hydrogel microenvironment could help to improve the mimicking of in vivo cell growth and the development of 3D models for tumor invasiveness evaluation, which could enhance its drug efficiency prediction together with cell viability detection. NCI-H23 spheroids and CT-26 spheroids, from a non-small cell lung cancer and colorectal cancer cell line, respectively, together with extracellular matrix were generated for evaluating their sensitivity to AMG510 (a KRASG12C inhibitor) under normoxia and hypoxia conditions, which were created by an on-stage environmental chamber. Results demonstrated that NCI-H23, the KRASG12C moderate expression cell line, only mildly responded to AMG510 treatment in normal 2D and 3D cultures and could be clearly evaluated by our system in hypoxia conditions, while the negative control CT-26 (G12D-mutant) spheroid exhibited no significant response to AMG510 treatment. In summary, our system, together with a controlled microenvironment and imaging methodology, provided an easily assessable and effective methodology for 3D in vitro drug efficiency testing and screenings.

KRAS in NSCLC: State of the Art and Future Perspectives.

In NSCLC, KRAS mutations occur in up to 30% of all cases, most frequently at codon 12 and 13. KRAS mutations have been linked to adenocarcinoma histology, positive smoking history, and Caucasian ethnicity, although differences have been described across KRAS mutational variants subtypes. KRAS mutations often concur with other molecular alterations, notably TP53, STK11, and KEAP1, which could play an important role in treatment efficacy and patient outcomes. For many years, KRAS mutations have been considered undruggable mainly due to a high toxicity profile and low specificity of compounds. Sotorasib and adagrasib are novel KRAS inhibitors that recently gained FDA approval for pre-treated KRAS mutant NSCLC patients, and other molecules such as GDC-6036 are currently being investigated with promising results. Despite their approval, the efficacy of these drugs is lower than expected and progression among responders has been reported. Mechanisms of acquired resistance to anti-KRAS molecules typically involves either on target secondary mutations (e.g., G12, G13, Q61H, R68S, H95, Y96C, V8L) or off-target alterations. Ongoing trials are currently evaluating strategies for implementing efficacy and overcoming acquired resistance to these compounds. Finally, the efficacy of immune-checkpoint inhibitors still needs to be completely assessed and responses to anti-PD-1/PD-L1 agents may strongly depend on concomitant mutations.

Mechanistic insights into the clinical Y96D mutation with acquired resistance to AMG510 in the KRASG12C.

Special oncogenic mutations in the RAS proteins lead to the aberrant activation of RAS and its downstream signaling pathways. AMG510, the first approval drug for KRAS, covalently binds to the mutated cysteine 12 of KRASG12C protein and has shown promising antitumor activity in clinical trials. Recent studies have reported that the clinically acquired Y96D mutation could severely affect the effectiveness of AMG510. However, the underlying mechanism of the drug-resistance remains unclear. To address this, we performed multiple microsecond molecular dynamics simulations on the KRASG12C-AMG510 and KRASG12C/Y96D-AMG510 complexes at the atomic level. The direct interaction between the residue 96 and AMG510 was impaired owing to the Y96D mutation. Moreover, the mutation yielded higher flexibility and more coupled motion of the switch II and α3-helix, which led to the departing motion of the switch II and α3-helix. The resulting departing motion impaired the interaction between the switch II and α3-helix and subsequently induced the opening and loosening of the AMG510 binding pocket, which further disrupted the interaction between the key residues in the pocket and AMG510 and induced an increased solvent exposure of AMG510. These findings reveal the resistance mechanism of AMG510 to KRASG12C/Y96D, which will help to offer guidance for the development of KRAS targeted drugs to overcome acquired resistance.

Understanding the influence of AMG 510 on the structure of KRASG12C empowered by molecular dynamics simulation.

The KRASG12C mutant is often associated with human cancers, and AMG 510 as a promising covalent inhibitor of KRASG12C has achieved surprising efficacy in clinical trials. However, the interaction mechanism between KRASG12C and AMG 510 is not completely understood. Here, we performed all-atom molecular dynamics simulations on the complex of KRASG12C-AMG 510 to explore the influence of this covalent inhibitor on the conformational change of KRASG12C. A PCA (Principal Component Analysis) model was constructed based on known KRAS crystal structures to distinguish different conformations (active, inactive, and other). By mapping simulation trajectories onto the PCA model, we observed that the conformations of KRASG12C bound with AMG 510 were mainly concentrated in the inactive conformation. Further analysis demonstrated that AMG 510 reduced the flexibility of two switch regions to make the complex of KRASG12C-AMG 510 restricted in the inactive conformation. In the meantime, we also identified key interacting residues between KRASG12C and AMG 510 through the calculation of binding energy. Finally, we built a series of KRAS second-site mutation systems (i.e. KRASG12C/mutations) to conduct large-scale screening of potential resistance mutations. By further combining MD simulations and the PCA model, we not only recapitulated the currently known resistance mutations of AMG 510 successfully but also proposed some novel potential resistant mutations. Taken together, these results broaden our insight into the influence of AMG 510 on the conformational change of the KRASG12C mutant at the atomic level, thereby providing crucial hints for the improvement and optimization of drug candidates.

Characterization of false positive, contaminant-driven mutagenicity in impurities associated with the sotorasib drug substance.

Sotorasib (Lumakras™) is a first-in-class, non-genotoxic, small molecule inhibitor of KRAS G12C developed as an anticancer therapeutic for treatment of patients that have a high unmet medical need. Anticancer therapeutics are considered out of scope of ICH M7 guidance for control of mutagenic impurities; however, based on ICH S9 Q&A, mutagenicity assessments are needed for impurities that exceed the qualification threshold, consistent with ICH Q3A/B, and non-mutagenic drugs. Here, we carried out hybrid-based mutagenicity assessment of sotorasib drug substance (DS) impurities using in silico quantitative structure-activity relationship (QSAR) modelling and Ames tests (for in silico positive mutagens). We encountered contradictive mutagenicity results for 2 impurities (Beta-Chloride and PAC). PAC was negative initially by QSAR but positive in a GLP full plate Ames test and Beta-Chloride was positive by QSAR, negative in a non-GLP micro-Ames but positive in a GLP full plate Ames assay. Root cause analyses identified and characterized mutagenic contaminants, 3-chloropropionic acid in batches of Beta-Chloride and 3-chloropropionic acid and Chloro-PAC in batches of PAC, used in initial GLP full-plate Ames tests. Significant reduction of these contaminants in re-purified batches resulted in no induction of mutagenicity in follow-up GLP micro-Ames tests. In summary, root-cause analyses led to accurate mutagenicity assessment for sotorasib DS-associated impurities.

Spotlight on Sotorasib (AMG 510) for KRAS G12C Positive Non-Small Cell Lung Cancer.

Mutations in codon 12 of KRAS have been identified in 13% of non-small cell lung cancer patients. Developing targeted therapies against KRASG12C mutation has proven to be challenging due to the abundance of GTP in the cytoplasm, rapid hydrolysis of GTP, and difficulty designing small molecules to achieve sufficient concentration for KRAS inhibition. Based on promising results in both preclinical and clinical trials, sotorasib, a novel KRASG12C inhibitor, was given conditional approval by the FDA in May 2021. The Phase I portion of the clinical trial produced 32% confirmed response with 56% of patients with stable disease. About 91.2% of patients who received the highest dose of 960mg daily achieved disease control. The Phase II portion, which used 960mg daily dosing resulted in 37.1% of patients with confirmed response and 80.6% of patients with disease control. Both phase I and phase II had similar progression-free survival, in 6.3 months and 6.8 months, respectively. In both phases, grade 4 adverse events occurred in only one patient. The most common adverse events were elevations in LFTs, which down-trended upon dose reduction and steroid treatment. While the conditional approval of sotorasib was a major breakthrough for those patients harboring KRASG12C mutations, resistance mutations to sotorasib are increasingly common. Many proposals have been made to address this, such as the use of combination therapy for synthetic lethality, which are producing encouraging results. Here, we explore in further detail the development of sotorasib, its efficacy, mechanism of resistance, and strategies to overcome these resistances.

Cytotoxic activity of KRAS inhibitors in combination with chemotherapeutics.

INTRODUCTION: KRAS is the most frequently mutated oncogenic driver in pancreatic, lung, and colon cancer. Recently, KRAS inhibitors in clinical use show promising activity but most responses are partial and drug resistance develops. The use of therapeutics in combination with KRAS inhibitors are expected to improve outcomes. AREAS COVERED: This review describes the KRAS G12C mutation-specific inhibitors and the SOS1-targeting inhibitors that reduce the GTP-loading of wildtype and mutated KRAS. Both types of compounds reduce tumor cell proliferation in vitro and in vivo. The combinations of the various KRAS inhibitors with downstream signaling effectors, modulators of KRAS-associated metabolic alterations and chemotherapeutics are summarized. EXPERT OPINION: The clinical potency of mutated KRAS-specific inhibitors needs to be improved by suitable drug combinations. Inhibition of downstream signaling cascades increases toxicity and other combinations exploited comprise G12C-directed inhibitors with SOS1 inhibitors, glucose/glutamine metabolic modulators, classical chemotherapeutics, and others. The most suitable inhibitor combinations corroborated in preclinical development await clinical verification.

Drug sensitivity profile of minor KRAS mutations in colorectal cancer using mix culture assay: The effect of AMG-510, a novel KRAS G12C selective inhibitor, on colon cancer cells is markedly enhanced by the combined inhibition of MEK and BCL-XL.

Colorectal cancer with a Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) gene mutation is considered to be resistant to anti-EGFR agents. G12D is the most common KRAS mutation in colorectal cancer, followed by G12V and G13D. According to clinical and basic research data, patients with colorectal cancer exhibiting G12D and G12V KRAS mutations are resistant to anti-EGFR agents; however, this is not true of G13D and other minor mutations, which are still not well understood. The current study focused on minor KRAS mutations (G12A, G12C, G12S, Q61H and A146T) and evaluated whether these were resistant to anti-EGFR antibodies using a mix culture assay. The results demonstrated that all KRAS mutations, including minor mutations, were resistant to two anti-EGFR agents: Cetuximab and panitumumab. The combined effect of MEK and BCL-XL inhibition on colorectal cancer cells with KRAS minor mutations were subsequently evaluated. The combined effect of MEK and BCL-XL inhibitors was confirmed in all KRAS minor mutations. The sensitivity of AMG510, a novel KRAS G12C selective inhibitor, was also assessed. The mix culture assay revealed that AMG510 selectively exerted an antitumor effect on colon cancer cells with a G12C KRAS mutation. The combination of MEK and BCL-XL inhibition markedly enhanced the effect of AMG510 in colon cancer cells. The current study suggested that AMG510 may have potential clinical use in combination with MEK and BCL-XL inhibitors in the treatment of patients with colorectal cancer exhibiting the G12C KRAS mutation.

Nonclinical Safety Profile of Sotorasib, a KRASG12C-Specific Covalent Inhibitor for the Treatment of KRAS p.G12C-Mutated Cancer.

Sotorasib is a first-in-class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. A comprehensive nonclinical safety assessment package, including secondary/safety pharmacology and toxicology studies, was conducted to support the marketing application for sotorasib. Sotorasib was negative in a battery of genotoxicity assays and negative in an in vitro phototoxicity assay. Based on in vitro assays, sotorasib had no off-target effects against various receptors, enzymes (including numerous kinases), ion channels, or transporters. Consistent with the tumor-specific target distribution (ie, KRASG12C), there were no primary pharmacology-related on-target effects identified. The kidney was identified as a target organ in the rat but not the dog. Renal toxicity in the rat was characterized by tubular degeneration and necrosis restricted to a specific region suggesting that the toxicity was attributed to the local formation of a putative toxic reactive metabolite. In the 3-month dog study, adaptive changes of hepatocellular hypertrophy due to drug metabolizing enzyme induction were observed in the liver that was associated with secondary effects in the pituitary and thyroid gland. Sotorasib was not teratogenic and had no direct effect on embryo-fetal development in the rat or rabbit. Human, dog, and rat circulating metabolites, M24, M10, and M18, raised no clinically relevant safety concerns based on the general toxicology studies, primary/secondary pharmacology screening, an in vitro human ether-à-go-go-related gene assay, or mutagenicity assessment. Overall, the results of the nonclinical safety program support a high benefit/risk ratio of sotorasib for the treatment of patients with KRAS p.G12C-mutated tumors.

Targeting KRAS in Solid Tumors: Current Challenges and Future Opportunities of Novel KRAS Inhibitors.

Activating mutations in RAS family proteins are found in ~25% of all human cancers. Different solid tumors are correlated with mutations in certain isoforms of RAS, with Kirsten RAS (KRAS) being the most frequently mutated isoform. Historically, KRAS has been acknowledged as "undruggable", largely because the RAS proteins do not appear to present suitable pockets to which small inhibitory molecules can bind. However, this scenario has changed over the last years with the advent of novel KRAS inhibitors. In this review, we describe the role of KRAS mutation across different solid tumors, providing data on novel KRAS inhibitors currently under development and an updated overview of ongoing research in this field. A literature search was performed to select papers, abstracts, and oral presentation on KRAS inhibitory strategies in KRAS mutated solid tumors. Overall, the most promising therapeutic results have been obtained with molecules targeting KRAS G12C, thus paving the way for a significant therapeutic improvement in non-small cell lung cancer. Unfortunately, KRAS G12C mutation is rather uncommon in other solid tumors, namely pancreatic ductal adenocarcinoma and colorectal cancer. Several combination strategies are currently under evaluation in clinical trials, in order to bypass the resistance mechanisms responsible for the intrinsic resistance of mutated KRAS to the main therapeutic strategies adopted to date. Results suggest that the therapeutic scenario of KRAS has started to change, and further research will bring therapeutic results in this field.