Abstract 4439: Adaptive rewiring after prolonged KRAS inhibition sensitizes PDAC cells to sequential targeting with ferroptosis inducers

Abstract Recent advances in direct targeting of oncogenic KRAS necessitate the need to understand and overcome intrinsic and extrinsic resistance mechanisms of cancer cells, especially in pancreatic ductal adenocarcinoma (PDAC) with KRAS as the main oncogenic driver. To investigate time-dependent KRAS resistance, we created a dual-recombinase PDAC mouse model that allows for inducible genetic deletion of KrasG12D in vivo and in vitro. A drug screen on primary PDAC cell lines derived from the model that were resistant to KRASG12D inhibition identified a strong RSL3-induced ferroptosis sensitivity of KrasG12D-depleted cell lines compared to their KrasG12D-proficient counterpart. Inhibitors MRTX1133 and RMC-6236 also conferred ferroptosis sensitization in various mouse and human PDAC cell lines. Interestingly, ferroptosis sensitization was stronger after 7-day treatment with KRAS inhibitor, compared to acute inhibition of 1-3 days, indicating that exhaustive rewiring of cellular pathways is needed for complete sensitization. This was also evident in the transcriptome analysis of cells at different time-points after KRASG12D depletion revealing temporary and chronic adaptations spanning various metabolic, proliferative and stress-associated pathways, several of which were previously linked to ferroptosis. KrasG12D-depleted cells exhibited higher ROS, higher iron ion content, and lower capacity to reduce ROS based on redox ratio of intracellular glutathione. Intriguingly, the sensitized phenotype was maintained in resistant cells treated with MRTX1133 over a period of 30-45 days, suggesting a stable phenotype that could be exploited in a clinical setting using sequential therapy approach. To better understand which genes are essential for viability of KrasG12D-depleted cells, we performed a whole-genome CRISPR knock-out screen on cell line with KrasG12D-depletion (genomic and inhibitor-based), as well as with additional RSL3 treatment. Kras-depletion screen identified several ferroptosis-associated regulators among the top depleted and enriched genes, including redox balance regulators (Slc7a11, Txnrd1, Keap1, Nadk), iron homeostasis (Ttc7, Glrx5) and KRAS-resistance-associated pathways (Taz, Map3k7). Machine learning algorithm applied on RNAseq of differentially sensitized cell lines revealed a signature of genes whose expression is altered based on sensitivity to RSL3. These, together with global changes in transcriptome, suggest that multiple pathways likely contribute to ferroptosis sensitization, including KEAP-NRF2 redox balance pathway, iron and lipid metabolism, and also hint at involvement of TGFβ-EMT signaling axis. Our results suggest that a sequential two-hit therapeutic approach with KRAS inhibition followed by subsequent ferroptosis induction could efficiently mitigate resistance to KRAS-inhibitor based therapies in PDAC. Citation Format: Tina Kos, Christian Schneeweis, Shinjini Bandopadhyay, Birgit Prinker, Chen Zhao, Dieter Saur. Adaptive rewiring after prolonged KRAS inhibition sensitizes PDAC cells to sequential targeting with ferroptosis inducers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4439.