Evolution of drug resistance enabled by replicative RNAs carrying MEK1

Drug resistance poses a significant clinical challenge, and comprehending the mechanisms underlying this resistance can facilitate the design of novel inhibitors and advance cancer treatment. The REPLACE system was employed to examine resistance mutations in MEK1 during the administration of 3 allosteric inhibitors (i.e., cobimetinib, trametinib, and selumetinib). These experiments represent a category of experiments that utilize REPLACE to achieve continuous evolution (or adaptation) by linking the activity of evolutionary targets with the survival of mammalian cells. MEK1 cDNA from H. sapiens was cloned into the repRNA-v4-derived vector. The vector was in vitro transcribed into RNA and the resulting RNAs were electroporated into aforementioned host cells expressing nsP4. Medium was replaced with fresh medium containing 5 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 days of selection by puromycin (i.e., on Day 5), approximately 2 million of cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using a concentration of 2 μM molnupiravir. On Day 7, the cells in each dish were approaching 90% confluence (~ 107 cells per dish). Therapeutic drug (i.e., 2-5 μM cobimetinib, 5-10 μM trametinib, or 50-100 μM selumetinib) was added into the medium for selection. Puromycin and molnupiravir were also added to the medium simultaneously. One week later (i.e., Day 14), approximately 105 cells successfully survived and adapted to the environment with inhibitor. The cells were collected for total RNA extraction, sequencing, and mutation analysis.