Tracing the lineage of replicative RNAs carrying MEK1 in the evolution of drug resistance

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. To explore the accumulation of mutations in various RNAs during the MEK1 evolution experiment, we established a barcoded library and conducted lineage tracing of replicative RNAs carrying MEK1 throughout the drug resistance evolution process. To investigate the process of mutation accumulation in different RNAs in the MEK1 evolution experiment, we generated a library of tens of thousands of barcodes by incorporating a 20-bp random sequence (20 Ns) downstream of the stop codon in MEK1-P2A-PuroR gene. The library, after undergoing transformation, in vitro transcription, and electroporation, was delivered to the BHK-21 host cells. 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,therapeutic drug (2-5 μM cobimetinib) was added into the medium for selection. Puromycin and molnupiravir were also added to the medium simultaneously. One weeks later (i.e., Day 14), survived cells were collected. Cells at day 1, day 5, day 7, and day 14 were collected for total RNA isolation and reverse transcription. The MEK1-T2A-PuroR-barcode region was amplified from both the DNA library plasmid and the aforementioned cDNA libraries using indexed primers. The purified PCR products were mixed and subjected to adapter ligation, fragment selection, primer and polymerase binding, followed by sequencing using a PacBio Sequel IIe instrument for Circular Consensus Sequencing (CCS) reads acquisition. Simultaneously, RNAs from day 1, day 5, day 7, and day 14 were also subjected to FastGNS sequencing.