Clinical Acquired Resistance to KRASG12C Inhibition through a Novel KRAS Switch-II Pocket Mutation and Polyclonal Alterations Converging on RAS-MAPK Reactivation

Abstract

Mutant-selective KRAS^G12C inhibitors, such as MRTX849 (adagrasib) and AMG 510 (sotorasib), have demonstrated efficacy in KRAS ^G12C-mutant cancers, including non-small cell lung cancer (NSCLC). However, mechanisms underlying clinical acquired resistance to KRAS^G12C inhibitors remain undetermined. To begin to define the mechanistic spectrum of acquired resistance, we describe a patient with KRAS ^G12C NSCLC who developed polyclonal acquired resistance to MRTX849 with the emergence of 10 heterogeneous resistance alterations in serial cell-free DNA spanning four genes (KRAS, NRAS, BRAF, MAP2K1), all of which converge to reactivate RAS-MAPK signaling. Notably, a novel KRAS ^Y96D mutation affecting the switch-II pocket, to which MRTX849 and other inactive-state inhibitors bind, was identified that interferes with key protein-drug interactions and confers resistance to these inhibitors in engineered and patient-derived KRAS ^G12C cancer models. Interestingly, a novel, functionally distinct tricomplex KRAS^G12C active-state inhibitor RM-018 retained the ability to bind and inhibit KRAS^G12C/Y96D and could overcome resistance. SIGNIFICANCE: In one of the first reports of clinical acquired resistance to KRAS^G12C inhibitors, our data suggest polyclonal RAS-MAPK reactivation as a central resistance mechanism. We also identify a novel KRAS switch-II pocket mutation that impairs binding and drives resistance to inactive-state inhibitors but is surmountable by a functionally distinct KRAS^G12C inhibitor.See related commentary by Pinnelli and Trusolino, p. 1874.This article is highlighted in the In This Issue feature, p. 1861.

Figure 1.. Acquired resistance to KRAS^G12C inhibitor MRTX849 (adagrasib).

A, Computed tomography (CT) images of the patient’s axillary lymph node metastasis at baseline, during response to MRTX849, and at progression on MRTX849. B, Variant allele fractions (VAFs) of mutations detected in the patient’s serial plasma samples. † indicates the mutations were detected by digital droplet PCR but not by plasma NGS. C, Alterations detected in post-MRTX849 cfDNA include acquired mutations in KRAS as well as multiple components of the MAPK signaling cascade. *KRAS^G12F represents a potential resistance mechanism supported by limited sequencing reads, as shown in Supplementary Figure S2.

Figure 2.. Structural basis for resistance to KRAS G12C inhibition conferred by KRAS^Y96D.

Shown are the modeled crystal structures of MRTX849 (6UT0), AMG 510 (6OIM), and ARS-1620 (5V9U) bound to KRAS^G12C (top panels) and KRAS^G12C/Y96D (bottom panels), highlighting the loss of the hydrogen bonds between MRTX849 or AMG 510 and the Y96 residue and the disruption of the switch II pocket dynamics between ARS-1620 and KRAS^G12C/Y96D.

Figure 3.. Cellular characterization of KRAS^Y96D in KRAS^G12C-mutant models.

A, Cell viability assays performed with NCI-H358, MIA PaCa-2 and Ba/F3 cells infected with retrovirus packaging KRAS (G12C or G12C/Y96D). Cell lines were treated with indicated drugs for 72 hours and the viabilities were measured with CellTiter-Glo. B, Western blot analysis was performed after treating MIA PaCa-2 cells stably expressing KRAS^G12C or KRAS^G12C/Y96D with MRTX849 for 4 hours. C, MGH1138-1 cells expressing KRAS^G12C or KRAS^G12C/Y96D were treated with MRTX849 for 4 hours and subjected to western blot analysis (left) and cell viability assay following 72 hours of treatment with the indicated concentrations of MRTX849 (right). D, Western blot analysis of HEK293T cells transiently expressing KRAS mutants after treatment with MRTX849 for 4 hours. E, RAS-GTP pulldown was performed after treating HEK293T stably expressing KRAS mutants with MRTX849.

Figure 4.. Novel KRAS inhibitor RM-018 overcomes KRAS^G12C/Y96D.

A, Mechanism of action of RM-018. B, RM-018 selectively inhibits cell viability in cells harboring KRAS^G12C. C, Cell viability assays performed with NCI-H358, MIA PaCa-2, Ba/F3 and MGH1138-1 cells stably infected with KRAS^G12C or KRAS^G12C/Y96D treated for 72 hours with RM-018. D and E, Western blot analysis performed in MIA PaCa-2 stably expressing KRAS^G12C or KRAS^G12C/Y96D (D) and HEK293T cells transiently expressing KRAS mutant (E) after treatment of RM-018 for 4 hours. F, Western blot analysis of MGH1138-1 cells transiently expressing KRAS^G12C or KRAS^G12C/Y96D) after treatment with RM-018 for 4 hours. G, HEK293T cells transiently expressing KRAS mutant were treated with the indicated drug at 100 nmol/L each for 4 hours and then subjected to western blot analysis.