Search

Purpose:Activating missense mutations of KRAS are the most frequent oncogenic driver events in lung adenocarcinoma (LUAD). However, KRAS isoforms are highly heterogeneous, and data on the potential isoform-dependent therapeutic vulnerabilities are still lacking.Experimental Design:We developed an isogenic cell-based platform to compare the oncogenic properties and specific therapeutic actionability of KRAS-mutant isoforms. In parallel, we analyzed clinicopathologic and genomic data from 3,560 patients with non–small cell lung cancer (NSCLC) to survey allele-specific features associated with oncogenic KRAS mutations.Results:In isogenic cell lines expressing different mutant KRAS isoforms, we identified isoform-specific biochemical, biological, and oncogenic properties both in vitro and in vivo. These exclusive features correlated with different therapeutic responses to MEK inhibitors, with KRAS G12C and Q61H mutants being more sensitive compared with other isoforms. In vivo, combined KRAS G12C and MEK inhibition was more effective than either drug alone. Among patients with NSCLCs that underwent comprehensive tumor genomic profiling, STK11 and ATM mutations were significantly enriched among tumors harboring KRAS G12C, G12A, and G12V mutations. KEAP1 mutation was significantly enriched among KRAS G12C and KRAS G13X LUADs. KRAS G13X-mutated tumors had the highest frequency of concurrent STK11 and KEAP1 mutations. Transcriptomic profiling revealed unique patterns of gene expression in each KRAS isoform, compared with KRAS wild-type tumors.Conclusions:This study demonstrates that KRAS isoforms are highly heterogeneous in terms of concurrent genomic alterations and gene-expression profiles, and that stratification based on KRAS alleles should be considered in the design of future clinical trials.

Supplementary Data from Comparative Analysis and Isoform-Specific Therapeutic Vulnerabilities of KRAS Mutations in Non–Small Cell Lung Cancer

Assembly of RAS molecules into complexes at the cell membrane is critical for RAS signaling. We previously showed that oncogenic KRAS codon 61 mutations increase its affinity for RAF, raising the possibility that KRASQ61H, the most common KRAS mutation at codon 61, upregulates RAS signaling through mechanisms at the level of RAS assemblies. We show here that KRASQ61H exhibits preferential binding to RAF relative to PI3K in cells, leading to enhanced MAPK signaling in in vitro models and human NSCLC tumors. X-ray crystallography of KRASQ61H:GTP revealed that a hyperdynamic switch 2 allows for a more stable interaction with switch 1, suggesting that enhanced RAF activity arises from a combination of absent intrinsic GTP hydrolysis activity and increased affinity for RAF. Disruption of KRASQ61H assemblies by the RAS oligomer–disrupting D154Q mutation impaired RAF dimerization and altered MAPK signaling but had little effect on PI3K signaling. However, KRASQ61H oligomers but not KRASG12D oligomers were disrupted by RAF mutations that disrupt RAF–RAF interactions. KRASQ61H cells show enhanced sensitivity to RAF and MEK inhibitors individually, whereas combined treatment elicited synergistic growth inhibition. Furthermore, KRASQ61H tumors in mice exhibited high vulnerability to MEK inhibitor, consistent with cooperativity between KRASQ61H and RAF oligomerization and dependence on MAPK signaling. These findings support the notion that KRASQ61H and functionally similar mutations may serve as predictive biomarkers for targeted therapies against the MAPK pathway. Significance: These findings show that oncogenic KRASQ61H forms a cooperative RAS–RAF ternary complex, which renders RAS-driven tumors vulnerable to MEKi and RAFi, thus establishing a framework for evaluating RAS biomarker-driven targeted therapies.

Purpose:Activating missense mutations of KRAS are the most frequent oncogenic driver events in lung adenocarcinoma (LUAD). However, KRAS isoforms are highly heterogeneous, and data on the potential isoform-dependent therapeutic vulnerabilities are still lacking.Experimental Design:We developed an isogenic cell-based platform to compare the oncogenic properties and specific therapeutic actionability of KRAS-mutant isoforms. In parallel, we analyzed clinicopathologic and genomic data from 3,560 patients with non–small cell lung cancer (NSCLC) to survey allele-specific features associated with oncogenic KRAS mutations.Results:In isogenic cell lines expressing different mutant KRAS isoforms, we identified isoform-specific biochemical, biological, and oncogenic properties both in vitro and in vivo. These exclusive features correlated with different therapeutic responses to MEK inhibitors, with KRAS G12C and Q61H mutants being more sensitive compared with other isoforms. In vivo, combined KRAS G12C and MEK inhibition was more effective than either drug alone. Among patients with NSCLCs that underwent comprehensive tumor genomic profiling, STK11 and ATM mutations were significantly enriched among tumors harboring KRAS G12C, G12A, and G12V mutations. KEAP1 mutation was significantly enriched among KRAS G12C and KRAS G13X LUADs. KRAS G13X-mutated tumors had the highest frequency of concurrent STK11 and KEAP1 mutations. Transcriptomic profiling revealed unique patterns of gene expression in each KRAS isoform, compared with KRAS wild-type tumors.Conclusions:This study demonstrates that KRAS isoforms are highly heterogeneous in terms of concurrent genomic alterations and gene-expression profiles, and that stratification based on KRAS alleles should be considered in the design of future clinical trials.

The clinical efficacy of epidermal growth factor receptor (EGFR)–targeted therapy in EGFR-mutant non–small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor–resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.

A subset of drug-resistant EGFR -mutant MET -amplified lung cancer switches oncogenic dependence to MET and is treatable with MET inhibitor monotherapy.

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the MAPK pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras-effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric, GTP-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1, and accessory proteins (Galectin-3 and 14-3-3σ) on the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.

Assembly of RAS molecules into complexes at the cell membrane is critical for RAS signaling. We previously showed that oncogenic KRAS codon 61 mutations increase its affinity for RAF, raising the possibility that KRAS