TFEB-mediated lysosomal biogenesis and lysosomal drug sequestration confer resistance to MEK inhibition in pancreatic cancer

Ben Zhao,Jens T Siveke,Jiang-Ning Gu,Diana Behrens,Smiths S Lueong,Marija Trajkovic-Arsic,Stephan Hahn,Silvia Vega-Rubin-De-Celis,Konstantinos Savvatakis,Laura Dierichs,Ralf Axel Hilger,Sven-Thorsten Liffers,Samuel Peña-Llopis

doi:10.1038/s41420-020-0246-7

Abstract

Oncogenic KRAS mutations are encountered in more than 90% of pancreatic ductal adenocarcinomas. MEK inhibition has failed to procure any clinical benefits in mutant RAS-driven cancers including pancreatic ductal adenocarcinoma (PDAC). To identify potential resistance mechanisms underlying MEK inhibitor (MEKi) resistance in PDAC, we investigated lysosomal drug accumulation in PDAC models both in vitro and in vivo. Mouse PDAC models and human PDAC cell lines as well as human PDAC xenografts treated with the MEK inhibitor trametinib or refametinib led to an enhanced expression of lysosomal markers and enrichment of lysosomal gene sets. A time-dependent, increase in lysosomal content was observed upon MEK inhibition. Strikingly, there was a strong activation of lysosomal biogenesis in cell lines of the classical compared to the basal-like molecular subtype. Increase in lysosomal content was associated with nuclear translocation of the Transcription Factor EB (TFEB) and upregulation of TFEB target genes. siRNA-mediated depletion of TFEB led to a decreased lysosomal biogenesis upon MEK inhibition and potentiated sensitivity. Using LC-MS, we show accumulation of MEKi in the lysosomes of treated cells. Therefore, MEK inhibition triggers lysosomal biogenesis and subsequent drug sequestration. Combined targeting of MEK and lysosomal function may improve sensitivity to MEK inhibition in PDAC.

Highlights

Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive and lethal malignant human diseases, with a 5-year survival rate of less than 8%1
We show that disruption of lysosomal biogenesis by Transcription Factor EB (TFEB) knockdown partially sensitizes PDAC cells to trametinib treatment and demonstrate the presence of trametinib in lysosomes
Models, we investigated the impact of MEK inhibition on lysosomal biogenesis

Summary

Results

MEKi treatment triggers expression of lysosomeassociated genes in mouse PDAC models Using mouse PDAC-derived cell lines and mouse PDAC models, we investigated the impact of MEK inhibition on lysosomal biogenesis. Fluorescent lysosomal staining of the TFEB-depleted cells after trametinib treatment did not show any significant increase in lysosomal contents as was observed in wildtype cells or cells transfected with a non-targeting siRNA Both transcript and protein levels of lysosomal markers LAMP1 and LAMP2 as well as lysosomal-associated ATPases such as vATP6V0D1 and the hydrolase CTSD remained stable in the MEKi-treated group after TFEB knockdown compared with cell transfected with a non-targeting control (Fig. 4e, f). We assessed whether MEKi-induced lysosomal biogenesis occurs in human PDAC in vivo To test this hypothesis, we first performed gene expression analyses on human PDAC cell lines treated with single dose IC50 of trametinib or vehicle control. The TFEB target gene ATP6V1A was highly expressed under MEKi treatment (Supplementary Fig. 7a)

Discussion

Conclusion

Findings

Materials and methods