Synergistic Effect Of Kras G12C Inhibitors And Regorafenib In The Combined Treatment Of Colorectal Cancer

Objective: Kras is an important driver gene in malignant tumors such as colorectal cancer.As a molecular switch,this protein helps regulate cell growth,differentiation,and survival.Mutations in the Kras gene lead to changes in the amino acid sequence of Kras protein,resulting in a persistently active or "on" state.This leads to continuous cell signaling even in the absence of external stimuli,eventually causing uncontrolled cell growth and proliferation,and promoting cancer development.Kras mutations are found in about 30-50% of colorectal cancer cases,with G12 C mutations accounting for about 11%.They are more common in later stages of the disease and are associated with poorer prognosis.Novel small molecule inhibitors targeting Kras G12 C mutations,such as AMG 510 and MRTX849,have shown promising results in recent preclinical and clinical studies for the treatment of colorectal cancer.However,the emerging issue of drug resistance makes the combination strategy of Kras inhibitors with other drugs an urgent problem to be solved.Regorafenib,as a multi-kinase inhibitor with multiple targets in the Kras upstream and downstream pathways,has potential synergistic effects with Kras G12 C inhibitors.We investigated the effects and mechanisms of Kras G12 C inhibitors combined with regorafenib in the treatment of colorectal cancer.Methods:1.The effects of Kras G12 C inhibitors combined with regorafenib on the proliferation and apoptosis of Kras G12 C wild-type/mutant and G12 C gene knock-in CRC cells were evaluated using MTS,crystal violet staining,colony formation assay,drug combination index calculation,synergy finder score calculation,flow cytometry,and Hoechst staining experiments.2.Kras G12 C wild-type/mutant and Kras G12 C knock-in cells were treated with a combination of AMG510/MRTX849 and regorafenib,and the mechanism of the combined action of the two drugs was investigated using western blot experiments.3.Patient-derived organoid(PDO)and PDX models from Kras G12 C mutant CRC patient tissues were established,and the therapeutic effects of Kras G12 C inhibitors combined with regorafenib in these models were further investigated.Results:1.MTS,crystal violet staining,drug combination index calculation,synergy finder calculation,and colony formation assays experiments showed that,compared to Kras G12 C wild-type cells,the combination of Kras G12 C inhibitors and regorafenib significantly inhibited cell proliferation in Kras G12 C mutation/knock-in cells colorectal cancer cells.2.Flow cytometry and Hoechst staining experiments showed that,compared to Kras G12 C wild-type cells,the combination of Kras G12 C inhibitors and regorafenib promoted apoptosis in Kras G12 C mutation/knock-in colorectal cancer cells.3.Kras G12 C inhibitors combined with regorafenib had a significant synergistic effect in Kras G12 C mutant PDO,as evidenced by a significant reduction in PDO viability.In the PDX model,the combination therapy significantly enhanced the inhibition of tumor growth in Kras G12 C mutant PDXs compared to single-agent treatment.4.The combination of Kras G12 C inhibitors and regorafenib inhibited the ERK rebound effect and activated apoptosis-related proteins.Conclusions:1.Compared with wild-type Kras G12 C cells,the combination of Kras G12 C inhibitor and regorafenib can significantly inhibit the proliferation and promote apoptosis in CRC cells with Kras G12 C mutation.2.Compared with wild-type Kras G12 C cells,the combination of Kras G12 C inhibitor and regorafenib can significantly inhibit the proliferation and promote apoptosis in CRC cells in Kras G12 C knock-in cells.3.The combination of Kras G12 C inhibitor and regorafenib can suppress the ERK feedback rebound effect,upregulate the expression of pro-apoptotic proteins and downregulate the expression of anti-apoptotic proteins in the Bcl-2 family,thereby inducing caspase-mediated cell apoptosis.4.The combination of Kras G12 C inhibitor and regorafenib has a significant synergistic effect in Kras G12 C mutated PDOs,as demonstrated by a significant reduction in PDO activity.In PDX models,compared to single drug groups,combined drug treatment can significantly enhance the inhibition of Kras G12 C mutated PDX tumor proliferation.