Acquired Resistance to KRASG12C Inhibition in Cancer

Abstract

Background: Clinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRAS^G12C). The mechanisms of acquired resistance to these therapies are currently unknown.

Methods: Among patients with KRAS^G12C -mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRAS^G12C inhibitors.

Results: A total of 38 patients were included in this study: 27 with non-small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRAS^G12C allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN. In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of KRAS mutations that confer resistance to KRAS^G12C inhibitors.

Conclusions: Diverse genomic and histologic mechanisms impart resistance to covalent KRAS^G12C inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.).

Figure 1.. Resistance to Adagrasib Conferred by Acquired KRAS Mutations.

Serial axial computed tomographic scans are shown for a patient with KRAS^G12C-mutant lung adenocarcinoma (Panel A) who had a partial response to adagrasib followed by disease progression. A lung tumor biopsy at the time of acquired resistance showed an acquired KRAS Y96C mutation. A patient with colorectal cancer (Panel B) showed multiple mechanisms of adagrasib resistance, including mutations in KRAS H95Q, H95R, G12R/D/V, G13D, and Q61H (listed twice owing to distinct c.183A→C and c.183A→T mutations leading to the same amino acid substitution); MAP2K1 K57N/T mutations; and a CCDC6-RET fusion. Pretreatment sequencing of RIT1 and PTEN was not performed. In this patient, the pretreatment variant allele frequency (VAF) for KRAS^G12C was from tissue, whereas the VAF at resistance was from circulating tumor DNA. A patient with lung adenocarcinoma (Panel C) was found to have KRAS R68S, H95D, and G12V/W along with BRAF V600E mutations at the time of acquired resistance to adagrasib. A patient with colorectal cancer (Panel D) showed KRAS H95R and G12D mutations as well as an activating MAP2K1 deletion. In Panels B through D, red coloring indicates KRAS alleles. ND denotes not detected.

Figure 2.. Genetic and Nongenetic Mechanisms of Resistance to Adagrasib.

A patient with colorectal cancer (Panel A) was found to have multiple acquired mutations in KRAS, EGFR, and MAP2K1 and an EML4-ALK rearrangement. Multiple oncogenic fusions (Panel B) involving FGFR3, BRAF, and RAF1, along with activating mutations of KRAS, NRAS, and MAP2K1, were detected in a patient with colorectal cancer. A patient with colorectal cancer (Panel C) was observed to have focal amplification of the KRAS^G12C allele at the time of adagrasib resistance. In Panels A through C, red coloring indicates KRAS alleles. Acquired focal MET amplification (Panel D) is shown for a patient with lung adenocarcinoma. Focal MYC amplification was also observed in both the pretreatment and postresistance biopsies. The y axis shows the log₂ ratio of copy number for tumor to paired normal tissue. Histologic transformation from lung adenocarcinoma to squamous-cell carcinoma (Panel E) was observed at the time of adagrasib resistance in a patient for whom a genetic mechanism of resistance could not be identified.

Figure 3.. Summary of Putative Mechanisms of Acquired Resistance to Adagrasib Treatment.

Of 38 patients with KRAS^G12C-mutant cancers, 17 had at least one putative resistance mechanism. A summary of the genomic and histologic mechanisms of resistance among these 17 patients is shown in the comutation plot, with each row indicating a patient and each column indicating a specific acquired alteration. Seven patients had more than one putative resistance mechanism identified. AC denotes appendiceal cancer, CRC colorectal cancer, ctDNA circulating tumor DNA, and NSCLC non–small-cell lung cancer.

Figure 4.. Resistance to Adagrasib or Sotorasib Conferred by Acquired Missense Mutations in the KRAS^G12C Drug-Binding Site.

Putative second-site resistance mutations were mapped onto crystal structures of KRAS^G12C bound to adagrasib (Panel A) or sotorasib (Panel B). Shown are representative dose–response curves for Ba/F3 cells that were grown in the absence of interleukin-3, that expressed the indicated KRAS^G12C variants, and that were treated for 5 days with adagrasib (Panel C) or sotorasib (Panel D). The curves represent means ±SE from at least three independent experiments. Immunoblots (Panel E) are shown for extracellular signal-regulated kinase (ERK) phosphorylation levels (pERK1/2 T202/Y204) and KRAS total protein levels from Ba/F3 cells that expressed KRAS^G12C or three clinically observed resistance mutations: KRAS^G12C/H95D, KRAS^G12C/H95R, and KRAS^G12C/Y96C. An immunoblot for vinculin is shown as a loading control. Six-hour adagrasib or sotorasib treatment of cells that expressed sensitive but not insensitive alleles conferred suppression of pERK and upward band shift of KRAS due to covalent binding of the inhibitors. GDP denotes guanosine-5′-diphosphate, and DMSO dimethyl sulfoxide.

Figure 5.. Landscape of On-Target Resistance Mutations to KRAS^G12C Inhibitors Revealed by Deep Mutational Scanning.

A heatmap depicts the results of a positive-selection screen in Ba/F3 cells for second-site missense mutations in KRAS^G12C that confer resistance to the KRAS^G12C inhibitors MRTX1257 (a close analogue of adagrasib) or sotorasib. Relative allele abundance is shown as a z score comparing the end point of the screen for each treatment group to the pretreatment allelic representation in the library, with red indicating enrichment of putative resistance variants. Columns represent the amino acid position within the KRAS^G12C allele, and each row represents 1 of 20 possible amino acids or the stop codon (X). The structural motifs within the KRAS protein are indicated in the tracks above the heatmap. Gray coloration indicates alleles that were not included in the screening library. HVR denotes hypervariable region.