MRTX849 Is a Potent and Selective Inhibitor of KRAS^G12C, KRAS-Dependent Signal Transduction, and Cell Viability In Vitro

A structure-based drug design approach, including optimization for favorable drug-like properties, led to the discovery of MRTX849 as a potent, covalent KRAS^G12C inhibitor (Fig. 1A; Supplementary Table S1). An LC/MS-based KRAS^G12C protein modification assay revealed that MRTX849 demonstrated much greater modification of KRAS^G12C when preloaded with GDP compared with GTP (Supplementary Table S2), supporting that MRTX849 binds to and stabilizes the inactive GDP-bound form of KRAS^G12C. Indeed, introducing a comutation that impairs the GTPase activity of KRAS^G12C (24) attenuated the inhibitory activity of MRTX1257, a close analogue of MRTX849 (Supplementary Fig. S1A). Secondary mutations that modulate the nucleotide exchange function of KRAS^G12C also affected inhibition by MRTX1257, supporting that the MRTX compound series is dependent on KRAS^G12C nucleotide cycling.

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Figure 1.

 

MRTX849 is a potent, covalent KRAS^G12C inhibitor in vitro. A, Structure of MRTX849. B, Immunoblot protein Western blot analyses of KRAS pathway targets in MIA PaCa-2 cells treated from 1 hours to 72 hours with MRTX849 at 100 nmol/L. C, Immunoblot protein Western blot analyses of KRAS pathway targets in MIA PaCa-2 cells treated for 24 hours with MRTX849 over a 13-point dose response. D, Left y-axis shows active RAS ELISA assay to determine the reduction in RAS-GTP abundance following MRTX849 treatment in MIA PaCa-2 cells for 24 hours. The vehicle value was normalized to 1 by dividing all average values by the vehicle value. Right y-axis shows quantitation of KRAS band shift by MRTX849 treatment in MIA PaCa-2 cells for 24 hours as assessed by Western blot and densitometry. E, In-cell Western blot assay to evaluate modulation of pERK in MIA PaCa-2 cells grown in standard tissue-culture conditions treated with MRTX849 over a time course. F, CellTiter-Glo assay to evaluate cell viability performed on seven KRAS^G12C-mutant cell lines and three non–KRAS^G12C-mutant cell lines grown in 2-D tissue-culture conditions in a 3-day assay (left plot) or 3-D conditions using 96-well, ULA plates in a 12-day assay (right plot).

 

We next determined the cellular activity of MRTX849 utilizing the KRAS^G12C-mutant H358 lung and MIA PaCa-2 pancreatic cancer cell lines. In both models, MRTX849 demonstrated an upward electrophoretic mobility shift of KRAS^G12C protein band migration by immunoblot, indicative of covalent modification of KRAS^G12C. A maximal mobility shift was observed by 1 hour, was maintained through 72 hours (Fig 1B; Supplementary Fig. S1B), and was evident at concentrations as low as 2 nmol/L with near-maximal modification observed at 15.6 nmol/L (Fig. 1C; Supplementary Fig. S1C). Comparable inhibition of active RAS was observed as determined by a RAF RAS-binding domain capture ELISA assay (Fig. 1D; 1D). MRTX849 also inhibited KRAS-dependent signaling targets including ERK1/2 phosphorylation (pERK; Thr²⁰²/Tyr²⁰⁴ ERK1), S6 phosphorylation (pS6; RSK-dependent Ser^235/236), and expression of the ERK-regulated DUSP6, each with IC₅₀ values in the single-digit nanomolar range in both cell lines (Fig. 1B and C; Supplementary Fig. S1B and S1C). The evaluation of pERK over a time course of 48 hours indicated maximal inhibition was observed at 24 hours (Fig. 1E; Supplementary Fig. S1E). Treatment with the des-acrylamide version of MRTX849, which is unable to covalently bind to KRAS^G12C, did not demonstrate significant inhibition of ERK phosphorylation (Supplementary Fig. S1F). The H358 cell line was selected for determination of MRTX849 cysteine selectivity utilizing an LC/MS-based proteomics approach able to detect approximately 6,000 cysteine-containing peptides. After treatment for 3 hours, decreased KRAS^G12C Cys12-free peptide was detected with treated-to-control ratios of 0.029 and 0.008 determined at 1 and 10 μmol/L, respectively, indicating near-complete engagement of the intended target (Supplementary Table S3). In contrast, the only other peptides identified were from lysine-tRNA ligase (KARS) at Cys209 near the detection limit, indicating a high degree of selectivity toward KRAS^CYS12.

To evaluate the breadth of MRTX849 activity, its effect on cell viability was determined across a panel of 17 KRAS^G12C-mutant and 3 non–KRAS^G12C-mutant cancer cell lines using 2-D (3-day, adherent cells) and 3-D (12-day, spheroids) cell-growth conditions. MRTX849 potently inhibited cell growth in the vast majority of KRAS^G12C-mutant cell lines with IC₅₀ values ranging between 10 and 973 nmol/L in the 2-D format and between 0.2 and 1,042 nmol/L in the 3-D format (Supplementary Table S4; Fig. 1F). In agreement with prior KRAS^G12C inhibitor studies (5), MRTX849 demonstrated improved potency in the 3-D assay format, as all but one KRAS^G12C-mutant cell line exhibited an IC₅₀ value below 100 nmol/L. Although MRTX849 was broadly effective in inhibiting viability of KRAS^G12C-mutant cell lines, IC₅₀ values varied across the cell panel by 100-fold, suggesting a differential degree of sensitivity to treatment. All three non–KRAS^G12C-mutant cell lines tested demonstrated IC₅₀ values greater than 1 μmol/L in 2-D conditions and greater than 3 μmol/L in 3-D conditions, suggesting the effect of MRTX849 on cell viability was dependent on the presence of KRAS^G12C.

To determine whether the difference in sensitivity across the cell panel correlated with the ability of MRTX849 to bind to KRAS or inhibit KRAS-dependent signal transduction, seven KRAS^G12C-mutant cancer cell lines were selected from the panel for further evaluation. In each cell line, MRTX849 demonstrated a very similar concentration-dependent electrophoretic mobility shift (IC₅₀) for KRAS^G12C protein migration, suggesting that the ability to bind to and modify KRAS^G12C does not readily account for differences in response in viability studies (Fig. 1B and C; Supplementary Figs. S1B and S1C and S2A and S2B). The effect of MRTX849 on selected phosphoproteins implicated in mediating KRAS-dependent signaling was also evaluated across the cell panel by immunoblot and/or reverse-phase protein array (RPPA) following treatment for 6 or 24 hours. Notably, the concentration–response relationship and maximal effect of MRTX849 on inhibition of ERK and S6^S235/236 phosphorylation varied across the cell panel (Supplementary Fig. S2A and S2C; Supplementary Table S7). MRTX849 demonstrated only partial inhibition of pERK in KYSE-410 and SW1573 cells and a minimal effect on pS6^S235/236 in SW1573, H2030, and KYSE-410 cells (Supplementary Fig. S2A and S2C). Each of these cell lines were among those that exhibited a submaximal response to MRTX849 in both 2-D and 3-D viability assays (Fig. 1F). Although KRAS is implicated in mediating signal transduction through the PI3K and mTOR pathways, there was minimal evidence of a significant and/or durable effect of MRTX849 on AKT (S473, T308) or 4E-BP1 (T37/T46, S65, T70) phosphorylation at any time point in any cell lines evaluated (Supplementary Fig. S2D). However, MRTX849 demonstrated concentration-dependent partial inhibition of the mTOR-dependent signaling targets p70 S6 kinase (T412) and/or pS6 (S240/44) in the H358, MIA PaCa-2,H2122, and H1373 cell lines, each of which exhibited a maximal response to treatment. Together, these data suggest that maximizing inhibition of KRAS-dependent ERK and S6 signaling may be required to elicit a robust response in tumor-cell viability assays.

MRTX849 Treatment In Vivo Leads to Dose-Dependent KRAS^G12C Modification, KRAS Pathway Inhibition, and Antitumor Efficacy

Studies were conducted to evaluate MRTX849 antitumor activity along with its pharmacokinetic and pharmacodynamic properties in vivo, both to understand the clinical utility of this agent and to provide insight toward response to treatment. MRTX849 demonstrated moderate plasma clearance and prolonged half-life following oral administration (Supplementary Table S1; Supplementary Fig. S3). To evaluate the pharmacodynamic response to MRTX849 and to correlate drug exposure with target inhibition, MRTX849 was administered via oral gavage over a range of dose levels to H358 xenograft–bearing mice, and plasma and tumors were collected at defined time points. The fraction of covalently modified KRAS^G12C protein was proportional to the plasma concentration of MRTX849 (Fig. 2A). When evaluated over time after a single oral dose at 30 mg/kg, the modified fraction of KRAS^G12C was 74% at 6 hours after dose and gradually decreased to 47% by 72 hours (Fig. 2B). This extended pharmacodynamic effect, despite declining levels of MRTX849 in plasma, was consistent with the irreversible inhibition of KRAS^G12C by MRTX849 and the relatively long half-life for the KRAS^G12C protein (∼24–48 hours; Supplementary Table S5). The modification of KRAS^G12C was maximized after repeated daily dosing for 3 days at 30 mg/kg (Fig. 2B), and higher dose levels did not demonstrate additional KRAS^G12C modification in multiple tumor models (data not shown). The maximum level of modification of approximately 80%, despite increasing dose and plasma levels of MRTX849, suggests that accurate measurement of complete inhibition of KRAS^G12C utilizing LC/MS may not be attainable, potentially due to a pool of active, noncycling, or unfolded KRAS^G12C protein in tumors. Together, these studies demonstrated a dose-dependent increase in covalent modification of KRAS^G12C by MRTX849 and that the majority of targetable KRAS was covalently modified by MRTX849 over a repeated administration schedule at dose levels at or exceeding 30 mg/kg.

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Figure 2.

 

MRTX849 modifies KRAS^G12C and inhibits KRAS signaling and tumor growth in vivo. A, MRTX849 was administered orally as a single dose to mice bearing established H358 xenografts (average tumor volume ∼350 mm³) at 10, 30, and 100 mg/kg. KRAS modification and MRTX849 plasma concentration data from n = 3 mice are shown as mean ± SD. KRAS^G12C modification was statistically significant versus vehicle control using the two-tailed Student t test. **, P < 0.01. B, MRTX849 was administered orally as a single dose or daily (QD) for 3 days to mice bearing established H358 xenografts (average tumor volume ∼350 mm³) at 30 mg/kg. Plasma was collected at 0.5, 2, 6, 24, 48, and 72 hours after administration of the last dose, and tumors were collected at 6, 24, 48, and 72 hours after dose. KRAS^G12C modification and MRTX849 plasma concentration data are shown from n = 3 mice as mean ± SD. Induction of modified KRAS^G12C protein at all time points was determined to be statistically significant versus vehicle control using two-way ANOVA. In addition, induction of modified KRAS^G12C protein at 72 hours in day 1 samples and 48 and 72 hours in day 3 samples was statistically significant versus the 6-hour time point. Brackets indicate P < 0.05 as compared with left-most sample. C, MRTX849 was administered as in A. Tumors were collected 6 hours after dose, and total and phosphorylated ERK1/2 and total and phosphorylated S6 were analyzed by immunoblot and quantified by densitometric analysis. Relative fluorescence intensity of pERK1/2 and pS6 was normalized by dividing pERK1/2 and pS6 by total ERK1/2 and total S6, respectively. Vehicle-treated tumors were normalized to 1 by dividing all average values by the vehicle value. Average pERK1/2 and pS6 values were divided by the average value in vehicle-treated tumors. Data shown represent the average of 2 to 3 tumors per treatment group plus SD. Reduction of pS6 relative fluorescence intensity was determined to be statistically significant versus vehicle control using the two-tailed Student t test. Brackets indicate P < 0.05 compared with left-most sample. D, MRTX849 was administered as in B. Tumors were collected at 6, 24, 48, or 72 hours after administration of the last dose, and total and phosphorylated ERK1/2 and total and phosphorylated S6 were analyzed as in C. Data shown represent the average of 3 to 4 tumors per treatment group plus SD. Reduction of pS6 relative fluorescence intensity on day 3 was determined to be statistically significant versus vehicle control using two-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample. E, MRTX849 was administered via daily oral gavage at the doses indicated to mice bearing established MIA PaCa-2 xenografts. Dosing was initiated when tumors were approximately 350 to 400 mm³.MRTX849 was administered to mice daily until day 16. Data are shown as mean tumor volume ± SEM. Tumor volumes at day 16 were determined to be statistically significant versus vehicle control via two-tailed Student t test. **, P < 0.01; *, P < 0.05.

 

To evaluate the effect of MRTX849 on KRAS-dependent signal transduction in vivo, a single dose of MRTX849 at 10, 30, or 100 mg/kg was administered to H358 tumor–bearing mice. Dose-dependent inhibition of ERK1/2 and pS6^S235/36 phosphorylation was observed at 6 hours after dose based on immunoblot and densitometric analysis (Fig. 2C). MRTX849 also demonstrated marked inhibition of ERK1/2 and S6^S235/36 phosphorylation after one or three daily doses at 6 or 24 hours, and levels gradually recovered by 72 hours after the final dose (Fig. 2D). pERK1/2 and pS6^S235/36 were further evaluated in formalin-fixed, paraffin-embedded sections from vehicle-treated and MRTX849-treated xenografts in four tumor models utilizing IHC methods coupled with image analysis algorithms. These studies demonstrated increased pERK1/2 and pS6 in nontumor/stromal cells following MRTX849 administration, indicating that immunoblotting studies with bulk tumor lysate likely underrepresent the degree of pathway inhibition in tumor cells, whereas IHC-based evaluation may more accurately reflect both the degree and spatial impact of pathway inhibition. Maximal inhibition was observed for both ERK and S6^S235/36 phosphorylation after a single dose at the 6-hour time point, with a rebound in signaling evident 24 hours after single dose in each model (Supplementary Fig. S4). Marked inhibition of ERK phosphorylation was observed at 6 hours after administration, with 89%, 94%, and 94% inhibition observed compared with vehicle controls in MIA PaCa-2, H1373, and H2122 tumors, respectively (H358 pERK not quantifiable). This indicates that dose levels at or exceeding 30 mg/kg dose maximized inhibition of ERK phosphorylation in multiple models (Supplementary Fig. S4A and S4B). Inhibition of S6 phosphorylation at 6 hours was more variable, with percent inhibition values of 76%, 50%, 86%, and 56% observed in MIA PaCa-2, H1373, H358, and H2122 tumors, respectively (Supplementary Fig. S4B). Together, these data indicate that consistent acute (6 hours) inhibition of KRAS-dependent ERK phosphorylation was maximized in all evaluated models, whereas inhibition of S6^S235/36 was more variable, presumably due to varying degrees of KRAS-independent activation of this pathway in different tumor models.

MIA PaCa-2 and H358 were selected as MRTX849-responsive tumor models, thereby enabling a high-resolution understanding of dose–response relationships. Significant, dose-dependent, antitumor activity was observed at the 3, 10, 30, and 100 mg/kg dose levels in the MIA PaCa-2 model (Fig. 2E). Evidence of rapid tumor regression was observed at the earliest post-treatment tumor measurement, and animals in the 30 and 100 mg/kg cohorts exhibited evidence of a complete response at study day 15. Dosing was stopped at study day 16, and all 4 mice in the 100 mg/kg cohort and 2 of 7 mice in the 30 mg/kg cohort remained tumor-free through study day 70 (Supplementary Fig. S5A). In a second MIA PaCa-2 study, dose-dependent antitumor efficacy was observed at the 5, 10, and 20 mg/kg dose levels, and 2 of 5 mice at the 20 mg/kg dose level exhibited complete tumor regression (Supplementary Fig. S5B). Significant dose-dependent antitumor efficacy was also observed in the H358 model, including 61% and 79% tumor regression at the 30 and 100 mg/kg dose levels, respectively, at day 22 (Supplementary Fig. S5C). MRTX849 was well tolerated, and no effect on body weight was observed at all dose levels evaluated (Supplementary Fig. S5D). These studies indicated that MRTX849 demonstrated dose-dependent antitumor efficacy over a well-tolerated dose range and that the maximally efficacious dose of MRTX849 is between 30 and 100 mg/kg/day.

MRTX849 Demonstrates Broad-Spectrum Tumor Regression in KRAS^G12C Cell Line and Patient-Derived Xenograft Models

To evaluate the breadth of antitumor activity across genetically and histologically heterogeneous KRAS^G12C-mutant cancer models, MRTX849 was evaluated at a fixed dose of 100 mg/kg/day (a dose projected to demonstrate near-maximal target inhibition in most models) in a panel of human KRAS^G12C-mutant cell line–derived xenograft (CDX) and patient-derived xenograft (PDX) models. MRTX849 demonstrated tumor regression exceeding 30% volume reduction from baseline in 17 of 26 models (65%) at approximately 3 weeks of treatment (Fig. 3A; Supplementary Table S6). By comparison, MRTX849 did not exhibit significant antitumor activity at 100 mg/kg in three non–KRAS^G12C-mutant models (Fig. 3A; Supplementary Table S6). Together, these results indicate that KRAS^G12C-mutant tumors are broadly dependent upon mutant KRAS for tumor-cell growth and survival and that MRTX849 elicits antitumor activity through a KRAS^G12C-dependent mechanism.

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Figure 3.

 

Antitumor activity of MRTX849 in KRAS^G12C-mutant and non–KRAS^G12C-mutant human tumor xenograft models. A, MRTX849 was administered via oral gavage at 100 mg/kg every day to mice bearing the CDX or PDX model indicated. Dosing was initiated when tumors were, on average, approximately 250 to 400 mm³. MRTX849 was formulated as a free base and resuspended as a solution in 10% Captisol and 50 mmol/L citrate buffer, pH 5.0. The percent change from baseline control was calculated at days 19 to 22 for most models. Statistical significance was determined for each model and is shown in Supplementary Table S6. Status of mutations and alterations in key genes is shown below each model. MAF (%), percent KRAS^G12C-mutant allele fraction by RNA-seq; CNV, copy-number variation; * denotes very high CDK4 expression by RNA-seq and possible amplification. HER family status was determined by averaging EGFR, ERBB2, and ERBB3 RNA-seq expression for CDX (CCLE) or PDX (Crown huBase) models. Positive HER family calls denote greater than the median expression of the models tested. CDX and PDX model HER family calls were determined independently. B, Tumor-growth inhibition plots from representative xenograft models that were categorized as sensitive, partially sensitive, or treatment refractory.

 

Although MRTX849 exhibited marked antitumor responses in the majority of models tested, a response pattern ranging from delayed tumor growth to complete regression was observed across the xenograft panel. The response to treatment was categorized as sensitive, partially sensitive, or treatment refractory (Fig. 3B). Rank order and Pearson statistical analyses were performed to evaluate the correlation between in vitro potency (IC₅₀ in 2-D or 3-D viability assays) and antitumor response in vivo (% regression or progression on day 22), and a significant correlation between response in cell lines compared with tumor models was not observed (Supplementary Fig. S6A and S6B). Thus, we focused on a comprehensive analysis of correlates with MRTX849 tumor response in vivo, including tumor histology, co-occurring genetic alterations, as well as baseline or drug-induced changes in expression of KRAS-related genes [RNA sequencing (RNA-seq)] and/or protein signaling networks (RPPA in 18 models, ref. 28; Supplementary Fig. S7). No individual genetic alteration, including but not limited to KRAS-mutant allele frequency, TP53, STK11, or CDKN2A, predicted the antitumor activity of MRTX849. Interestingly, baseline gene and/or protein expression of selected members of the HER family of RTKs and of regulators of early cell-cycle transition did exhibit a trend with the degree of antitumor response, suggesting these pathways may influence the response to KRAS inhibitors (Supplementary Fig. S7A). Together, these data indicate that there are no individual binary biomarkers that clearly predict therapeutic response and that the molecular complexity and heterogeneity present in distinct KRAS-mutated tumors may contribute to the response to target blockade.

MRTX849 Antitumor Activity Translates to RECIST Responses in Patients with Cancer

A 45-year-old female former smoker diagnosed with stage IV lung adenocarcinoma and refractory to multiple lines of therapy including carboplatin/pemetrexed/pembrolizumab, docetaxel, and investigational treatment with binimetinib and palbociclib was enrolled onto the MRTX849-001 phase Ib clinical trial with two bilateral lung lesions and mediastinal lymph node as target lesions. Targeted next-generation sequencing (NGS) demonstrated a KRAS^G12C mutation (c.34G>T). In addition, loss-of-function KEAP1 (K97M) and STK11 (E223*) mutations were detected and are predicted to be deleterious to their respective proteins. The patient was administered MRTX849 (600 mg twice a day) and had marked clinical improvement within 2 weeks, including complete resolution of baseline cough and oxygen dependency. A RECIST-defined partial response of 33% reduction of target lesions was observed at cycle 3 day 1 (45 days), and the patient continues on study (Fig. 4A).

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Figure 4.

 

Activity of MRTX849 in patients with lung and colon cancers. A, Pretreatment and 6-week scans of a heavily pretreated patient with a KRAS^G12C mutation–positive lung adenocarcinoma indicating 33% reduction of target lesions. Patient continues on study. The top plots show a coronal view, and bottom plots an axial view of CT chest images prior to MRTX849 treatment (left) and after two cycles of MRTX849 treatment (right). B, Baseline, 6-week (Cycle 2), and 12-week (Cycle 4) scans of a patient with a KRAS^G12C mutation–positive colon adenocarcinoma. Partial response (PR) was confirmed at Cycle 4, and patient continues on study. Four lesions (TL1–4) are shown with axial views of CT images prior to MRTX849 treatment (top), after two cycles of MRTX849 treatment (center), and after four cycles of MRTX849 treatment (bottom).

 

A 47-year-old female never-smoker with metastatic adenocarcinoma of the left colon who exhibited progressive disease after receiving multiple lines of systemic therapy, including FOLFOX plus bevacizumab, single-agent capecitabine, FOLFIRI plus bevacizumab, and an investigational antibody–drug conjugate, was enrolled into the MRTX849-001 phase Ib clinical trial. This patient had extensive metastases involving the liver, peritoneum, ovaries, and lymph nodes. Targeted NGS identified a KRAS^G12C mutation. The patient was administered MRTX849 (600 mg twice a day) and demonstrated marked clinical improvement within 3 weeks and a visible decrease in size of her umbilical Sister Mary Joseph nodule. Her carcinoembryonic antigen levels decreased from 77 ng/mLat baseline to 11 ng/mL at cycle 2 day 1 and 3 ng/mL by cycle 3 day 1 (normal range, 0–5 ng/mL). A RECIST-defined partial response with a 37% reduction of target lesions and complete response of a nontarget lesion was observed at cycle 3 day 1(day 42). Confirmatory CT scans were conducted at cycle 5, day 1 (day 84) and indicated a confirmed RECIST partial response with further reduction of target lesions at −47% from baseline (Fig. 4B). The patient remains on treatment through Cycle 6.

Temporal Effects of MRTX849 on KRAS-Dependent Signaling and Feedback Pathways and Relationship to Antitumor Activity Following Repeat Dosing in Xenograft Models

A comprehensive analysis was conducted to evaluate MRTX849-induced temporal molecular changes to further interrogate mechanisms of drug response across sensitive and partially sensitive models. To evaluate temporal changes in global gene expression, xenograft-bearing mice were administered vehicle or 100 mg/kg MRTX849, and RNA-seq was performed on tumors at 6 and 24 hours after treatment. Gene expression was evaluated at day 1 and day 5 for the sensitive models MIA PaCa-2 and H1373 to ensure sufficient tissue availability from regressing tumors, or at day 7 in the partially sensitive models H358, H2122, and H2030 to coordinate with tumor stasis plateau. The top differentially expressed gene set enrichment analysis (GSEA) hallmark gene sets, regardless of tumor response, in all five models were several KRAS-annotated gene sets confirming MRTX849 selectively inhibits multiple genes directly related to KRAS signaling. MYC, mTOR, cell cycle, and apoptosis/BCL2 pathway gene sets were also strongly differentially expressed, confirming MRTX849 broadly affected multiple well-established, KRAS-regulated pathways, several of which have proved difficult to directly inhibit with previous targeted therapies (Fig. 5A and B; Supplementary Fig. S8A—S8D). The marked impact of MRTX849 on a large number of genes that regulate cell cycle and apoptosis provides further insight into molecular mechanisms which mediate its antitumor activity.

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Figure 5.

 

MRTX849 treatment in vivo regulates KRAS-dependent oncogenic signaling and feedback-inhibitory pathways. A, Volcano plots displaying differentially expressed genes in xenograft tumors 24 hours after oral administration of vehicle or 100 mg/kg MRTX849 in a representative MRTX849-sensitive (H1373) and MRTX849-partially sensitive (H358) model. Significance denoted in the legend (P_adj < 0.01). B, GSEA heat maps depicting hallmark signature pathways differentially regulated in at least one model 24 hours following oral administration of a single 100 mg/kg MRTX849 dose compared with vehicle. Normalized enrichment score shown in all models 6 or 24 hours after a single dose (QD × 1) or 5 (QD × 5) or 7 (QD × 7) days dosing. C, Genes that feedback-inhibit MAP kinase signaling are downregulated following MRTX849 treatment in all five cell line xenografts assessed by RNA-seq. TPM, Transcripts Per Kilobase Million.

 

Targeted RNA-seq analysis was performed on genes implicated in the temporal regulation of external signaling inputs and feedback pathways which collectively temper signaling flux through the RAS–RAF–MEK–ERK MAP kinase (MAPK) pathway including DUSP, SPRY, and PHLDA family genes (13, 18). These MAPK pathway–negative regulators were each ranked among the most strongly decreased genes following MRTX849 treatment, providing evidence that ERK-dependent transcriptional output is blocked and that pathways involved in reactivation of RTK- and ERK-dependent signaling were activated (Fig. 5C; Supplementary Fig. S4A).

On the basis of the observation of dynamic changes in transcriptional programs linked to KRAS pathway reactivation, IHC plus quantitative imaging of tumor cell–specific pERK and pS6 was evaluated over a range of time points. In the sensitive MIA PaCa-2 and H1373 tumor models, treatment with MRTX849 (100 mg/kg) demonstrated ≥90% inhibition of ERK phosphorylation at 6 and 24 hours on both days 1 and 5 (Supplementary Fig. S4). In contrast, in the partially sensitive H358 and H2122 models, robust inhibition of ERK phosphorylation was observed at 6 hours after a single dose; however, marked recovery of ERK phosphorylation was observed at 24 hours after single dose and at both 6 and 24 hours following 7 days of repeat-dose administration. Because DUSP, SPRY, and ETV family transcripts remain downregulated through 5 to 7 days in all models, it is evident that other independent factors contribute to temporal reactivation of ERK (Fig. 5C). Similar to what was observed with single-dose administration, the effect of MRTX849 on pS6 was variable over time and did not track with the antitumor activity of MRTX849. Together, these results suggest that the extent and duration of inhibition of pERK may track with the magnitude of antitumor efficacy of KRAS^G12C inhibitors and that further evaluation of the role of S6 is required to understand if it plays a role in drug sensitivity.

The effect of MRTX849 on cell proliferation and apoptosis was characterized by IHC analysis of Ki-67 or cleaved caspase-3 after a single dose or repeat administration. The fraction of Ki-67–positive cells was significantly reduced in tumors after repeat administration in all four models tested, further supporting a broadly operative antiproliferative mechanism, independent of the magnitude of MRTX849 antitumor response (Supplementary Fig. S4). Induction of apoptosis as determined by cleaved caspase-3 immunostaining was also evident on day 1 of treatment (6 and/or 24 hours after treatment) in the sensitive H358, MIA PaCa-2, and H1373 models (79%–100% maximal regression) but not in the partially sensitive H2122 model (Supplementary Fig. S4). An expanded RPPA-based pathway analysis of several models also indicated a correlation between antitumor activity of MRTX849 and decreased survivin (statistically significant at days 5/7 in 7 models evaluated; Supplementary Fig. S7B) and a trend toward increased cleaved caspase-3 induction (day 1, P = 0.08, 16 models), supporting the induction of apoptosis as a key mediator of a cytoreductive antitumor response (Supplementary Fig. S7C). Interestingly, the magnitude of reduction of MYC and cyclin B1 protein levels at days 5/7 also closely correlated with MRTX849 antitumor activity, consistent with their roles as critical regulators of KRAS-mediated cell growth and survival pathways (Supplementary Fig. S7B). Collectively, these data support that durable inhibition of ERK activity and maximal inhibition of ERK-regulated outputs including MYC and E2F-mediated transcription are associated with induction of apoptosis and maximal response to MRTX849 treatment.

CRISPR/Cas9 Screen Identifies Vulnerabilities and Modifiers of Response to MRTX849 in KRAS^G12C-Mutant Cancer Cell Lines In Vitro and In Vivo

The correlative analysis of genomic or proteomic markers with response to MRTX849 in the defined panel of models provided only limited insight toward mechanism of therapeutic response or resistance. Therefore, we directly interrogated the role of selected genes in mediating therapeutic response utilizing a focused CRISPR/Cas9 knockout screen targeting approximately 400 genes including many genes involved in KRAS signaling. This was conducted in H358 and H2122 cells in vitro and in H2122 xenografts in vivo in the presence and absence of MRTX849 treatment (Supplementary Fig. S9A–S9F). In MRTX849-anchored screens in vitro, single guide RNAs (sgRNA) that target RAS signaling pathway genes including MYC, SHP2 (H2122), mTOR pathway (MTOR and RPS6), and cell-cycle genes (CDK1, CDK2, CDK4/6, and RB1) were identified to affect cell fitness. sgRNAs that target KEAP1 and CBL were enriched in the H2122 model, demonstrating cell-specific genetic routes toward improved fitness through loss of classic tumor-suppressor genes, including in the context of MRTX849 treatment. KRAS sgRNA dropout was less pronounced in the MRTX849-treated cells compared with DMSO control–treated cells, as would be expected with redundant depletion of the drug target (Supplementary Fig. S9C and S9D). To evaluate whether a distinct KRAS dependence or modulation of MRTX849 therapeutic response was observed in vitro versus in vivo, xenograft-bearing mice bearing H2122 cells (∼250 mm³) transduced with the sgRNA library were orally administered vehicle or MRTX849 for 2 weeks (Supplementary Fig. S9A, S9E, and S9F). In MRTX849-treated xenografts, sgRNAs targeting cell cycle, SHP2, MYC, and mTOR pathway genes remained among the top depleted sgRNAs, demonstrating that inhibition of these targets in vivo, in the context of KRAS inhibition, leads to further tumor-growth inhibition over and above the effects of KRAS inhibition alone (Supplementary Fig. S9E and S9F). sgRNAs targeting the tumor suppressor KEAP1 were enriched in MRTX849-treated xenografts, suggesting loss of KEAP1 may represent a mechanism of intrinsic or acquired resistance. Interestingly, NRAS was one of the top enriched genes in the vehicle-treated xenografts, suggesting NRAS functions as a tumor suppressor in this context; however, enrichment was not as pronounced in the MRTX849-treated xenografts, suggesting NRAS may compensate for KRAS in the context of KRAS inhibition (Supplementary Fig. S9F). Collectively, these data demonstrate the importance of selected proteins that regulate RTK- and RAS-dependent signaling and cell-cycle transition in mediating the oncogenic effects of mutant KRAS, and also provide a catalog of potentially druggable vulnerabilities that complement KRAS blockade.

Cancer Therapeutic Combination Screen to Identify Rational and Clinically Tractable Strategies to Address Feedback and Resistance Pathways

To further interrogate pathways that mediate the antitumor response to MRTX849 and to identify combinations capable of enhancing response to MRTX849, a combination screen was conducted in vitro using a focused library of small-molecule inhibitors across a panel of cell lines (Supplementary Fig. S10A and S10B; Supplementary Table S8). Approximately 70 compounds targeting relevant pathways (RTKs, MAPK/ERK, PI3K, mTOR, cell cycle) were tested in a 3- or 7-day viability assay, and synergistic combinations were identified and ranked. Multiple hits from this screen were then identified for additional evaluation in combination studies with MRTX849, including the HER family inhibitor afatinib, the CDK4/6 inhibitor palbociclib, the SHP2 inhibitor RMC-4550, and mTOR pathway inhibitors.

Combination Strategies That Target Upstream Signaling Pathways Implicated in Extrinsic Regulation of KRAS Nucleotide Cycling and Feedback/Bypass Pathways

MRTX849 in combination with HER family inhibitors synergistically inhibited tumor-cell viability in the majority of cell lines evaluated and were the top hit in the combination screen in vitro (Supplementary Fig. S10). Cell lines with the highest (top 50th percentile) average composite baseline RNA expression values of selected HER family members exhibited the highest synergy scores to these combinations (Supplementary Fig. S11A). Afatinib was selected as a prototype HER family inhibitor based on its broad in vitro combination activity. Combination studies were conducted with MRTX849 and afatinib in five tumor models that were partially sensitive or treatment refractory to single-agent MRTX849. The MRTX849 and afatinib combination demonstrated significantly greater antitumor efficacy compared with either single agent in all five models evaluated, including multiple models exhibiting complete or near-complete responses to the combination (Fig. 6A; Supplementary Fig. S11B).