Mechanisms of Response and Tolerance to Active RAS Inhibition in KRAS-Mutant Non-Small Cell Lung Cancer

Abstract

Resistance to inactive state-selective RASG12C inhibitors frequently entails accumulation of RASGTP, rendering effective inhibition of active RAS potentially desirable. Here, we evaluated the antitumor activity of the RAS(ON) multiselective tricomplex inhibitor RMC-7977 and dissected mechanisms of response and tolerance in KRASG12C-mutant non-small cell lung cancer (NSCLC). Broad-spectrum reversible RASGTP inhibition with or without concurrent covalent targeting of active RASG12C yielded superior and differentiated antitumor activity across diverse comutational KRASG12C-mutant NSCLC mouse models of primary or acquired RASG12C(ON) or RASG12C(OFF) inhibitor resistance. Interrogation of time-resolved single-cell transcriptional responses established an in vivo atlas of multimodal acute and chronic RAS pathway inhibition in the NSCLC ecosystem and uncovered a regenerative mucinous transcriptional program that supports long-term tumor cell persistence. In patients with advanced KRASG12C-mutant NSCLC, the presence of mucinous histologic features portended poor response to sotorasib or adagrasib. Our results have potential implications for personalized medicine and the development of rational RAS inhibitor-anchored therapeutic strategies. Significance: Our work reveals robust and durable antitumor activity of the preclinical RAS(ON) multiselective inhibitor RMC-7977 against difficult-to-treat subsets of KRASG12C-mutant NSCLC with primary or acquired RASG12C inhibitor resistance and identifies a conserved mucinous transcriptional state that supports RAS inhibitor tolerance. See related commentary by Marasco and Misale, p. 2018.

Figure 1.

Antitumor activity of RAS(ON) inhibition in preclinical models of difficult-to-treatKRAS^G12C-mutant NSCLC. A, Experimental strategy to evaluate acute and long-term effects of RAS inhibition in immune-competent co-mutational models of KRAS^G12C and KRAS^G12D-mutated NSCLC. Six to 13 mice were included in each treatment cohort. B, Histopathologic and IHC characterization of subcutaneous allografts established from murine Kras^G12C and Kras^G12D-mutated NSCLC cell lines that were utilized in the in vivo studies. C and D, RMC-7977 monotherapy and RMC-7977/RMC-4998 combination therapy induce deep and sustained tumor regressions in both STK11-deficient (KL2; C) and p53-deficient (KP2; D) co-mutational models of KRAS^G12C-mutant lung adenocarcinoma. Left, tumor volume growth curves in the study treatment arms. Curves are truncated at the time point that the first mouse in each cohort reached tumor volume ≥1,000 mm³. Top right, Kaplan-Meier estimate of TTD. Comparison of TTD between treatment groups is based on the log-rank test. Bottom right, Kaplan-Meier estimate of relapse-free survival (RFS), assessed as the time from treatment withdrawal on day 60 to TV ≥ 500 mm³. Pairwise comparisons of RFS between treatment groups is based on the log-rank test. The dotted red line indicates treatment withdrawal on day 60. E, RMC-7977 monotherapy/combination therapy overcomes primary resistance to inactive and active state-selective RAS^G12C inhibitors in KL5 derivative models with shRNA-mediated knockdown of Smarca4 or Keap1. F, Antitumor activity of RMC-4988, RMC-7977, and their combination in KEAP1 - and STK11 -mutated H2030 (top) and H2122 (bottom) KRAS^G12C-mutated human NSCLC CDX models. Tumor volume growth curves and Kaplan-Meier estimates of TTD are displayed for each model. Tumor volume data is represented as mean ± SEM and analyzed by two-way ANOVA followed by Bonferroni’s multiple comparison test. Comparison of TTD between treatment groups is based on the log-rank test. G, RMC-7977 exhibits robust antitumor activity in Kras^G12C/+;Lkb1^−/− GEMMs. BOR (top left) and PFS (top right) with RMC-4998 or RMC-7977 were determined based on mouse RECIST. Representative μCT images of primary lung tumors at the time of randomization and following 3 and 6 weeks of treatment. Note the emergence of on-treatment disease progression with RMC-4998, whereas treatment with RMC-7977 results in sustained response (bottom). P ≤ 0.05 was considered statistically significant for all comparisons. (A, Created with BioRender.com.)

Figure 2.

RMC-7977 overcomes acquired resistance to inactive and active state-selective RAS^G12C inhibitors. A, RMC-7977 monotherapy and RMC-7977/RMC-4998 combination therapy elicit deep and sustained tumor regressions in KRAS^G12C-mutated NSCLC models with acquired resistance to sotorasib. B, RMC-7977 is active in NSCLC models with acquired in vivo resistance to the RAS^G12C(ON) inhibitor RMC-4998. C, RMC-7977 overcomes acquired resistance to RMC-4988 driven by secondary alterations in RAS genes. Individual tumors that developed on-treatment resistance to RMC-4988 (KL5shCon RMC-4988 R1-R3 and KP2A RMC-4988 R1-R3) were harvested, processed for WES, and propagated directly in C57BL/6 mice under continuous drug exposure. Mice harboring tumors 450 to 500 mm³ were subsequently treated with RMC-7977 or RMC-7977 plus RMC-4998. Tumor volume growth curves (left) and waterfall plot representation of individual mouse-level best % change in tumor volume (right) are shown. D, OncoPrint depicting somatic genomic alterations (SNVs, indels, and copy number alterations) in the RMC-4988-resistant models and the parental KL5 and KP2 cell lines. Individual tumors from the experiment depicted in D are indicated as R1, R2, and R3.

Figure 3.

Tumor cell-intrinsic and microenvironmental effects of active RAS inhibition in NSCLC. A, Acute phenotypic responses to mutant-selective RAS^G12C and broad-spectrum RAS pathway inhibitors in the KL2 NSCLC model. Representative H&E, histochemical, and IHC images of subcutaneous KL2 tumor allografts following in vivo treatment for 7 days with the indicated inhibitors (N = 6 for all treatment groups). Scale bars, 100 μm. B, Violin plot representation of the Ki67+ TC fraction following 7-day treatment with the indicated RAS pathway inhibitors in the KL2 (top) and KP2 (bottom) allograft models. Ten representative fields in each tumor were selected by two experienced thoracic pathologists blinded to the treatment arm allocation and were analyzed by automated quantitative IHC using Halo software. Only comparisons with significant P values are indicated. C, Western blot analysis of (phospho)-protein abundance reflecting apoptosis (cleaved caspase3 and cleaved PARP), necroptosis (phospho-RIP3 and phospho-MLKL), and pyroptosis (cleaved caspasel and cleaved gasdermin D) in whole-tumor lysates collected at distinct time points following in vivo treatment with the indicated inhibitors. D, FACS-based enumeration of immune cell subsets in KL2 allograft tumors following 7-day treatment with the specified inhibitors. E, Comparison of tumor vascular density following treatment with the indicated inhibitors in the KL2 (left, 7-day treatment) and KP2 (right, 21-day treatment) allograft models. One-way ANOVA with Tukey’s multiple comparison test was used for all pairwise statistical comparisons between treatment groups. P value ≤ 0.05 was considered statistically signifiicant. Asterisks denote statistical signifiicance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Figure 4.

Emergence of a DTP cell population in response to RMC-7977. A, Resurgent tumors following drug withdrawal across diverse Kras-mutant NSCLC allograft models retain long-term sensitivity to rechallenge with RMC-7977 or RMC-7977/RMC4998, even in the context of high baseline tumor burden (≥1,000 mm³). Note that the single KL2 tumor with apparent on-treatment increase in tumor volume (highlighted with an asterisk) displayed prominent treatment-induced cystic degeneration [as illustrated in B (i)]. B, Representative H&E, histochemical, and IHC images of tumor allografts following long-term (6–8 weeks) rechallenge with RMC-7977 or RMC-7977+RMC4998. (i) Low magnification image from an intact KL2 residual tumor following long-term treatment with RMC-7977. (ii-vi) DTP TC populations in KL2 (ii, iii), KLsshCon (iv), KP2 (v), and LKR10 (vi) allograft models following long-term treatment with RMC-7977 (ii, iv, v, vi) or RMC-7977+RMC4998 (iii, KL2 model). (vii-xix) Representative images of Masson’s trichrome (vii, viii) and Alcian Blue (xix) staining in DTPs in the KL2 model. (ix, x) Ki67 immunostaining in KL2 and KP2, respectively. (xii) CD31 staining in DTPs in the KL2 model. Scale bars, 100 μm. C, Representative H&E images of tumor-bearing whole lung samples from Kras^G12C/+;Lkb1^−/− GEMMs following treatment with vehicle or RMC-7977. DTPs following long-term treatment with RMC-7977 (60 days) exhibit mucinous histologic features (high magnification image, right). D, RMC-7977-tolerant persister TCs in KL2 allograft tumors exhibit low Ki67+ proliferative index. E, Sustained inhibition of active RAS suppresses neovascularization in the Kras^G12C-mutant NSCLC tumor microenvironment. Violin plot representation of vascular density in KL2 tumor allografts treated for 7 days with (i) vehicle, (ii) RMC-7977, (iii) following drug release (after 60 days of continuous treatment), and (iv) RMC-7977 rechallenge for a minimum of 6 weeks. One-way ANOVA with Tukey’s multiple comparison test was used for all pairwise statistical comparisons between treatment groups. P value ≤ 0.05 was considered statistically significant. Asterisks denote statistical significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Figure 5.

Single-cell transcriptional atlas of in vivo RAS inhibition in KRAS^G12C NSCLC. A, Uniform manifold approximation and projection (UMAP) plot of recovered individual cells, colored by cell-type identity. B, Stacked bar plot representation of absolute (top) and relative (bottom) abundance of distinct cell clusters in each tumor sample. Clusters are color-coded as in A. Note that part of the tumor in mice that were rechallenged with RMC-7977 was stored for IHC studies; therefore, only comparisons of relative cell cluster abundance is applicable. Whole treated tumors were collected and processed in all other treatment arms. C and D, Box plot representation of the scRNA-seq-derived NK cell fraction (C) and TIL to TC ratio (D) in each treatment arm. Dots indicate individual tumor samples. The Wilcoxon rank-sum test was used for statistical comparisons. E, UMAP plot of lymphoid cell subclusters (left) and projection of immune exhaustion marker gene expression (Pdcdl, Tox, Tigit, and Lag3; right). F, Violin plot representation of immune exhaustion signature scores in CD8⁺ T-cell subclusters. G, Relative abundance of CD8⁺ T-cell subclusters in individual treated tumor samples. H, UMAP view of neutrophils colored by cluster membership (left); absolute (center) and relative (right) abundances of neutrophil subclusters in each tumor sample.

Figure 5.

Single-cell transcriptional atlas of in vivo RAS inhibition in KRAS^G12C NSCLC. A, Uniform manifold approximation and projection (UMAP) plot of recovered individual cells, colored by cell-type identity. B, Stacked bar plot representation of absolute (top) and relative (bottom) abundance of distinct cell clusters in each tumor sample. Clusters are color-coded as in A. Note that part of the tumor in mice that were rechallenged with RMC-7977 was stored for IHC studies; therefore, only comparisons of relative cell cluster abundance is applicable. Whole treated tumors were collected and processed in all other treatment arms. C and D, Box plot representation of the scRNA-seq-derived NK cell fraction (C) and TIL to TC ratio (D) in each treatment arm. Dots indicate individual tumor samples. The Wilcoxon rank-sum test was used for statistical comparisons. E, UMAP plot of lymphoid cell subclusters (left) and projection of immune exhaustion marker gene expression (Pdcdl, Tox, Tigit, and Lag3; right). F, Violin plot representation of immune exhaustion signature scores in CD8⁺ T-cell subclusters. G, Relative abundance of CD8⁺ T-cell subclusters in individual treated tumor samples. H, UMAP view of neutrophils colored by cluster membership (left); absolute (center) and relative (right) abundances of neutrophil subclusters in each tumor sample.

Figure 6.

A mucinous regenerative program supports RAS^GTP inhibitor tolerance. A, UMAP plot of cancer cells from KL2 allograft tumor samples, colored by cluster. B, Absolute (top) and relative (bottom) abundance of tumor cell clusters in individual tumor samples. C, Proportions and average expression levels of top differentially expressed TC marker genes. D and E, PROGENy pathway activity scores for cells pooled by tumor cluster (D) or tumor sample (E). F, Volcano plot representation of the top differentially expressed genes and selected mucinous/gastrointestinal marker genes in tumors from mice rechallenged with RMC-7977 or treated with vehicle. G, UMAP visualization of mRNA expression of select GI-related genes (Tff1, Tff2, Tff3, Spink4, Muc1, Mucl3, Gkn2, and Agr2). H, Representative IHC images showing enrichment of TFF1, TFF2, and MUC1-expressing tumor cells following long-term rechallenge with RMC-7977 in the KL2, KP2, KL5shCon, and LKR10 models. I, Representative IHC images showing TFF1 expression in mucinous tumor cells in lung specimens obtained from Kras^G12C/+;Lkb1^−/− GEMMs following long-term treatment with RMC-4998 (top) or RMC-7977 (bottom). Scale bars, 100 μm.

Figure 6.

A mucinous regenerative program supports RAS^GTP inhibitor tolerance. A, UMAP plot of cancer cells from KL2 allograft tumor samples, colored by cluster. B, Absolute (top) and relative (bottom) abundance of tumor cell clusters in individual tumor samples. C, Proportions and average expression levels of top differentially expressed TC marker genes. D and E, PROGENy pathway activity scores for cells pooled by tumor cluster (D) or tumor sample (E). F, Volcano plot representation of the top differentially expressed genes and selected mucinous/gastrointestinal marker genes in tumors from mice rechallenged with RMC-7977 or treated with vehicle. G, UMAP visualization of mRNA expression of select GI-related genes (Tff1, Tff2, Tff3, Spink4, Muc1, Mucl3, Gkn2, and Agr2). H, Representative IHC images showing enrichment of TFF1, TFF2, and MUC1-expressing tumor cells following long-term rechallenge with RMC-7977 in the KL2, KP2, KL5shCon, and LKR10 models. I, Representative IHC images showing TFF1 expression in mucinous tumor cells in lung specimens obtained from Kras^G12C/+;Lkb1^−/− GEMMs following long-term treatment with RMC-4998 (top) or RMC-7977 (bottom). Scale bars, 100 μm.

Figure 7.

Mucinous differentiation is associated with RAS inhibitor tolerance in human KRAS-mutant NSCLC. A, Flow cytometry-based assessment of the fraction of MUC1^High-expressing tumor cells following treatment with vehicle or RMC-7977 (40 nmol/L) for 6 days in a panel of nine KRAS^G12C (H1792, H23, H727, HCC44, H358, H1373, H441) and KRAS^non-G12C-mutant (H460, A549) NSCLC cell lines. B, Long-term (>4 weeks) in vitro exposure to RMC-7977 promotes accumulation of MUC1^High tumor cells. Error bars represent SEM from technical triplicates; a paired t test was used for comparisons between treatment arms in both A and B. Asterisks denote statistical significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P≤ 0.001; ****, P ≤ 0.0001. C, Western blot analysis of MUC1 protein abundance in whole cell lysates following in vitro treatment with vehicle or RMC-7977 (40 nmol/L) for 6 days. Similar results were observed with a second well-characterized antibody [MUC1-C (D5K9I) XP Rabbit mAb #16564, Cell Signaling Technology] against the C-terminal subunit of MUC1 (Supplementary Fig. S13B). D, Representative IF images depicting increased MUC1 expression (green fluorescence) in the H460 (top row) and HCC44 cell lines following long-term (>4 weeks) in vitro treatment with RMC-7977. DNA is stained with DAPI. Scale bars, 50 μm. E, IHC expression of TFF1, MUC1, AGR2, and CDX2 in the H460 and H1373 CDX models in response to treatment with vehicle (top) or RMC-7977 for 21 days (bottom). Scale bars, 100 μm. F, Flow cytometry-based assessment of the proportion of MUC1^High tumor cells following treatment for 6 days with (i) vehicle, (ii) sotorasib (1 μmol/L), (iii) RMC-4998 (100 nmol/L), and (iv) RMC-7977 (40 nmol/L). The A549 and H460 cell lines represent negative controls for RAS^G12C-selective inhibitors. G and H, RECIST v1.1-based overall response rate (G) and Kaplan-Meier estimates of PFS and OS (H) in patients with advanced KRAS^G12C-mutant NSCLC treated with sotorasib or adagrasib monotherapy (MD Anderson clinical cohort), according to the presence or absence of mucinous features in baseline biopsy reports. The log-rank test was used for comparison of PFS and OS. HRs and corresponding CIs were estimated with the use of a stratified Cox proportional hazards model with adjustment for clinical variables [age, history of brain metastasis, prior lines of therapy for metastatic disease (0 vs. ≥1), PS (0–1 vs. 2)] and histologic subtype (adenocarcinoma vs. other histologic subtypes). P ≤ 0.05 was considered statistically significant.