Adaptive DNA amplification of synthetic gene circuit opens new way to overcome cancer chemoresistance

Drug resistance remains a formidable barrier to the efficacy of cancer treatments, underscoring the necessity for experimental model systems that are comprehensive yet streamlined to allow the identification of the underlying mechanisms and corresponding countermeasures that can be applied across various cancer types. To address this need, we investigated a collection of engineered mammalian cell lines with synthetic gene circuits integrated into their genome that evolved resistance to Puromycin. Through bulk RNA sequencing and DNA copy number analysis, we identified DNA amplification as the major mechanism underlying drug resistance in 4 out of 6 replicate populations, without any noticeable single-point mutations. To address drug resistance due to DNA amplification, we successfully applied Triplex-Forming Oligonucleotides (TFOs) aimed at directly targeting the amplified DNA regions. A combination of multiple TFOs with the pertinent drug or a Nucleotide Excision Repair (NER) inhibitor further inhibited cell growth. Furthermore, we observed comparable inhibitory effects in human cancer cell lines by combining a mix of TFOs with chemotherapy drugs, suggesting potent combinatorial therapies to counter drug-resistance in cancer.

This work was supported by the National Institutes of Health, NIGMS MIRA Program (R35GM122561) and by the Laufer Center for Physical and Quantitative Biology.