Sensitizing K-Ras G12D mutant colorectal patient-derived organoids via ferroptosis pathway.

211 Background: Colorectal cancer remains the third most fatal form of cancer for males and females combined, with K-Ras mutations present in approximately 40% of all cases. These mutations drive cancer progression and confer resistance to conventional therapies, underscoring the need for novel treatment strategies. Ferroptosis, a regulated non-apoptotic form of cell death characterized as iron-dependent lipid peroxidation, has emerged as a target for cancer therapy. A study published in 2023 found that K-Ras mutant pancreatic ductal adenocarcinoma and non-small cell lung cancer cells lack ferroptosis induced lipid peroxidation and concurrently upregulated ferroptosis suppressing protein (FSP-1). It is unknown if K-Ras mutant colorectal tumors also evade ferroptosis-induced cell death as a method of cancer progression and therapy resistance. This study aims to measure the effects of inhibiting K-Ras G12D, the most common K-Ras mutation found in CRC, on ferroptosis through a patient derived colorectal cancer organoid (PDO) model. We hypothesize that blocking K-Ras G12D using a MRTX-1133, a K-Ras G12D specific inhibitor, can induce ferroptosis and synergistically work with ferroptosis inducers. Methods: As a pilot study, two patient derived organoid lines were established. Surveillance screening and whole exome sequencing was performed to verify K-Ras mutation. CRC9T has wild-type and CRC30T has mutant K-Ras. Additionally, Columbia Pathology Core confirmed tissues used to generate organoids were moderately differentiated colorectal adenocarcinoma. CTG-3D assays were performed to measure IC50 using ferroptosis inducers RSL3, iFSP, and K-Ras inhibitor MRTX-1133. Organoids were cultured in standard gut media for 72 hours, treated, and collected after 72 hours. After treatment, organoids were imaged and stained with BODIPY-C11, a lipid peroxidation fluorescence dye. Flow cytometry was performed, and results were analyzed using FlowJo to show ferroptosis-induced lipid peroxidation in stained organoids. Results: Inhibiting K-Ras G12D and inducing ferroptosis showed a synergistic effect on ferroptosis-induced lipid peroxidation. CRC9T was more sensitive (IC50 RLS3: 2.96 uM, iFSP 3.46 uM) to ferroptosis inducers than CRC30T (IC50: RSL3: 5.68 uM, iFSP: 8.6 uM). Blocking K-Ras in was only efficacious in CRC30T (IC50: MRTX1133: 9.73 uM). Cells from the mutant line showed greatest lipid peroxidation when combining ferroptosis inducers with K-Ras G12D inhibitors in flow cytometry analysis. Conclusions: These findings demonstrate a synergistic effect when blocking K-Ras G12D and inducing ferroptosis and align with previous studies that showed a connection between ferroptosis suppression and oncogenic KRAS expression. We plan to validate these findings in other patient derived organoid model systems and to assess the effects of pan-KRAS inhibition and ferroptosis inducer on non-K-Ras G12D PDOs.