MCT1 causes resistance to SMAD3 inhibition in cancer and involves dysregulation of the cholesterol synthesis pathway (CRISPR-SAM gene activation screen)
Non-genetic resistance remains a critical challenge in cancer therapy. Using CRISPR-based activation and knockout screens, this study identifies MCT1 as a pivotal mediator of resistance to the SMAD3 inhibitor SIS3 in BRAF-mutated melanoma and KRAS-mutated lung adenocarcinoma. Mechanistically, MCT1 drives metabolic reprogramming by promoting oxidative phosphorylation (OXPHOS) and supporting critical anabolic pathways such as lipid and cholesterol biosynthesis. Also, our data highlight epigenetic regulation and WNT/ß-catenin pathway as important modulators of drug resistance. Integrated CRISPR screening and long-read direct transcriptomic analyses reveal that enzymes involved in OXPHOS, fatty acid synthesis, and cholesterol production are vital for MCT1-driven drug tolerance. Moreover, MCT1 overexpression prevents decreased cholesterol synthesis in response to SMAD3 inhibitor. We show that pharmacological inhibition or genetic depletion of MCT1 severely impairs tumor cell viability under SIS3 pressure. Furthermore, combining SMAD3 inhibition with MCT1 and cholesterol synthesis blockade reduces abundance of persister cells and produces a more durable anti-tumor effect in BRAF inhibitor resistant melanoma cells. This study reinforces the potential of targeting MCT1-dependent metabolic pathways to overcome drug resistance in cancer therapy.