Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive human disease-causing thousands of deaths each year both in the United States and in the word. Late detection of the disease, failure of standard chemotherapies, and the lack of effective new treatments make PDAC largely incurable. It is one of the major RAS-driven cancers, representing over 90% of KRAS mutations. The MAPK pathway is the most important downstream effector of mutant RAS. Efforts to target the RAS-MAPK pathway using MEK inhibitors (MEKi) in PDAC have remained unsuccessful, mainly due to the rapid development of resistance. This highlights the need for a deeper understanding of the molecular mechanisms underlying resistant tumor progression, particularly for the successful application of recently developed RAS inhibitors.
Mitochondria are highly dynamic organelles that can exhibit a range of phenotypes from highly fragmented to highly networked. In response to internal and external stimuli, coordination between the mitochondrial fission GTPase DRP1 and fusion GTPases MFN1/2 and OPA1 maintains a balanced dynamic morphology of mitochondrial continuum in healthy cells. Interestingly, many cancer cells exhibit fragmented mitochondrial networks, which supports metabolic reprogramming that promotes tumor growth. Previously, we demonstrated that in RAS mutant tumors, ERK phosphorylates the mitochondrial fission GTPase DRP1 to promote fission. DRP1-mediated mitochondrial fission plays a tumor-promoting role in pancreatic and other RAS-driven cancers, but its role in therapeutic resistance is unknown. In the present study, we aimed to understand if DRP1-mediated mitochondrial fission contributes to the resistance to MEK inhibitor trametinib in PDAC.
We have generated a panel of patient-derived pancreatic cancer cell lines resistant to trametinib and analyzed their mitochondrial phenotypes. We found that trametinib-resistant cells exhibit increased expression and phosphorylation of DRP1 compared to sensitive counterparts. Furthermore, quantitative analysis of mitochondrial structure reveals that mitochondria in resistant cells are distinct from sensitive cells on trametinib. c-Myc activation is required for DRP1 S616 phosphorylation in trametinib-resistant pancreatic cancer cells. Furthermore, inhibition of CDK4/6 activity, either pharmacologically or genetically, is sufficient to block DRP1 phosphorylation in resistant cells, suggesting that activation of these kinases drives reactivation of mitochondrial fission in the absence of MAPK signaling. We have confirmed that CDK6 is a kinase of DRP1 S616 phosphorylation in an in vitro kinase assay. Importantly, deletion of DRP1 leads to either growth inhibition or re-sensitization to trametinib in resistant lines. In addition, we observed decreased tumor growth in DRP1 knock-out mice compared to DRP1 wild-type mice on trametinib.
These findings suggest that CDK4/6 plays a central role in reactivating DRP1- one of the ERK effectors downstream of RAS in MEKi-resistant tumors. Targeting mitochondrial fission, hence, might be a promising therapeutic strategy to combat resistance to MAPK and RAS inhibitors.
