Abstract LB283: Reactivation of Drp1S616 phosphorylation by c-MYC-CDK4/6 signaling axis plays a functional role in resistance to MEK inhibition in pancreatic cancer cells

Abstract Mitochondria are highly dynamic organelles that can exhibit a range of phenotypes from highly fragmented to highly networked. Many cancer cells exhibit fragmented mitochondrial networks, and this phenotype supports metabolic reprogramming to promote tumor growth. We previously demonstrated that in Ras mutant tumors, ERK mediated phosphorylation of the mitochondrial fission GTPase Drp1 at S616 leads to an increase in mitochondrial fission activity that is responsible for the fragmented phenotype. We and others have demonstrated that Drp1-mediated mitochondrial fission plays a tumor-promoting role in pancreatic and other Ras-driven cancers, but its role in therapeutic resistance is unknown. The MAPK pathway is the most critical effector pathway downstream of mutant Ras. Despite this, targeting this pathway directly in Ras-driven pancreatic cancer has been unsuccessful due to the development of resistance. In the present study we have generated a panel of patient-derived pancreatic cancer cell lines resistant to the MEK inhibitor trametinib and analyzed their mitochondrial phenotypes. We find that trametinib-resistant pancreatic cancer cells exhibit increased expression and phosphorylation of Drp1 compared to sensitive counterparts, even in the presence of trametinib. Furthermore, quantitative analysis of mitochondrial structure reveals that trametinib-induced mitochondrial restructuring is inhibited in resistant cells. Intriguingly, we have identified CDK4/6 as novel kinase of Drp1. Inhibition of CDK4/6 activity is sufficient to block Drp1 phosphorylation in resistant cells, suggesting that activation of these kinases is driving the reactivation of mitochondrial fission in the absence of MAPK signaling. Importantly, CRISPR-mediated deletion of Drp1 from resistant cells either partially re-sensitizes the cells to trametinib (1 line) or leads to growth inhibition independent of drug treatment (3 lines). These data highlight the importance of Drp1 reactivation in maintaining the growth of trametinib resistant tumor cells. Furthermore, we have identified c-MYC oncoprotein is reactivated in trametinib resistant pancreatic cancer cells and mediates Drp1S616 phosphorylation through CDK4/6 kinases. Together, these findings point to the importance of mitochondrial structure in drug resistance and suggest that inhibition of mitochondrial fission might be a promising therapeutic strategy to combat resistance to MAPK and RAS inhibitors. Citation Format: Salma Sharmin, Jennifer A. Kashatus, Sara J Adair, Todd W Bauer, David F Kashatus. Reactivation of Drp1S616 phosphorylation by c-MYC-CDK4/6 signaling axis plays a functional role in resistance to MEK inhibition in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB283.