AbstractDrug insensitivity is arguably one of the biggest challenges in cancer therapeutics. Although effective therapeutic solutions in cancer are limited due to the emergence of drug insensitivity, exploiting evolutionary understanding in this context can provide potential second-line therapeutics sensitizing the drug insensitive populations. Targeted therapeutic agent dabrafenib is used to treat CRC patients with BRAF V600E genotype and insensitivity to dabrafenib is often observed. Understanding underlying clonal architecture of dabrafenib-induced drug insensitivity and identification of potential second-line therapeutics that could sensitize dabrafenib insensitive populations remain to be elucidated. For this purpose, we utilized cellular barcoding technology to decipher dabrafenib-induced clonal evolution in BRAF V600E mutant HT-29 cells. This approach revealed the detection of both pre-existing and de novo barcodes with increased frequencies as a result of dabrafenib insensitivity. Furthermore, our longitudinal monitoring of drug insensitivity based on barcode detection from floating DNA within used medium enabled to identify temporal dynamics of pre-existing and de novo barcodes in relation to dabrafenib insensitivity in HT-29 cells. Moreover, whole-exome sequencing analysis exhibited possible somatic CNVs and SNVs contributing to dabrafenib insensitivity in HT-29 cells. Last, collateral drug sensitivity testing demonstrated oxaliplatin and capecitabine, alone or in combination, as successful second-like therapeutics in inducing collateral sensitivity in dabrafenib-insensitive HT-29 cells. Overall, our findings demonstrate clonal dynamics of dabrafenib-insensitivity in HT-29 cells. In addition, oxaliplatin and capecitabine, alone or in combination, were successful second-line therapeutics in inducing collateral sensitivity in dabrafenib-insensitive HT-29 cells.