Mechanisms of Resistance to Carcinogenic KRAS Inhibition in Pancreatic Cancer

Background

Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal disease. Most patients are diagnosed at an advanced stage and typically die within 12 months, primarily due to limited treatment options and poor response to standard chemotherapy. KRAS is the most critical oncogene in this cancer, altered in over 90% of tumors. The G12D, G12V, and G12R mutations in KRAS are most common. KRAS gene mutations typically increase the stability of the protein in its active GTP-bound state, driving tumor signaling through downstream effector pathways such as the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways. Previous studies have shown that genetic ablation of KRAS gene expression leads to cell cycle arrest and cell death, resulting in potent tumor regression in PDAC animal models. Therefore, KRAS is a high-priority therapeutic target for PDAC.

Research Aim and Motivation

This study aims to explore the mechanisms of resistance to KRAS oncogene inhibition. Despite showing clinical efficacy in PDAC patients, resistance to KRAS inhibitors is common. By performing multimodal analyses of clinical samples and various PDAC preclinical models, this study aims to define both genetic and non-genetic mechanisms of resistance, providing a basis for designing combination therapy strategies in the future.

Research Source

This paper was authored by Professor Andrew J. Aguirre and his team, with members from the Dana-Farber Cancer Institute, The Broad Institute of Harvard and MIT, Harvard Medical School, The University of Texas MD Anderson Cancer Center, and the Perelman School of Medicine at the University of Pennsylvania, among other institutions. The study was published in the journal “Cancer Discovery.”

Research Methods and Processes

Research Processes

The research includes multiple steps, conducted in different models and patient samples:

1. Clinical Sample Collection and Processing: Peripheral blood samples were collected from PDAC patients participating in the KRYSTAL-1 and CodeBreaK100 clinical trials, obtaining resistance mechanisms through circulating tumor DNA (ctDNA) analysis.
2. Preclinical Model Analysis:
   • Cell Line and Organoid Models: Applying the KRASG12D inhibitor MRTX1133 to cell line and organoid models to study in vitro resistance mechanisms.
   • Syngeneic KPC Mouse Model: Evaluating the efficacy and long-term resistance mechanisms of the KRASG12D inhibitor in immunocompetent syngeneic KPC monkey models.
   • Xenotransplant PDX Model: Using PDX models to validate the efficacy of the KRASG12D inhibitor in different cell states.

Data Analysis

Comprehensive genomic and transcriptomic analyses were conducted using tools such as whole genome sequencing (WGS), RNA sequencing (RNA-seq), and reverse phase protein array (RPPA) to capture differences and commonalities between models. Further validation of resistance mechanisms was performed using immunohistochemistry (IHC) and multiplex immunofluorescence (MIF).

Research Results

Genetic Mechanisms

In 22 PDAC patients with KRASG12C mutations, ctDNA analysis detected several genetic alterations during the resistance stage, including mutations in PIK3CA and KRAS, as well as amplifications of KRASG12C, MYC, MET, EGFR, and CDK6. Similar gene amplifications and alterations were observed in various preclinical models treated with MRTX1133, suggesting these mutations may be common resistance mechanisms.

Non-Genetic Mechanisms

The study found that epithelial-mesenchymal transition (EMT) and the activated PI3K–AKT–mTOR signaling pathway were highly associated with resistance to KRAS inhibition across various models. Further confirmation using RNA-seq and RPPA data identified significant upregulation of EMT features and RTK signaling in resistance models. In the MRTX1133-treated KPC mouse model, rapid and significant tumor regression was observed in the early stages of treatment, but tumors eventually developed resistance and regrew.

Evolution of Cell States

Single-cell RNA sequencing (snRNA-seq) revealed that early MRTX1133 treatment induced a transition of tumor cells to a classical (epithelial) state, whereas, during the acquired resistance stage, tumor cells exhibited an enrichment of partial EMT (pEMT) and mesenchymal states. These findings were consistent with studies in PDX models, indicating that different cell states have varying sensitivities and resistance mechanisms to KRAS inhibition.

Efficacy of Combination Treatments

Studies of various PDAC models showed that co-treatment with KRASG12D inhibitors and chemotherapy significantly improved efficacy compared to monotherapy. Specific combinations, such as MRTX1133 with gemcitabine/nab-paclitaxel, significantly delayed tumor recurrence and markedly reduced metastatic burden in metastatic models.

Research Conclusions

Through comprehensive analysis of multiple models, this study defined genetic and non-genetic mechanisms of resistance to KRAS inhibition, suggesting potential multiple combination treatment strategies. Future clinical trial designs can leverage these resistance mechanisms by employing combination therapies targeting RTK signaling or cellular states.

Research Significance

This study is the first to comprehensively elucidate the mechanisms of resistance to KRAS inhibitors in PDAC, offering various possible combination therapy strategies aimed at improving the clinical efficacy and durability of KRAS inhibitors, providing more effective treatment options for PDAC patients.