Long Interspersed Element-1 (LINE-1; L1) is an autonomous retrotransposon whose expression is frequently dysregulated in cancer. L1 encodes two proteins, ORF1p and ORF2p, which are essential for retrotransposition. Reverse transcriptase inhibitors (RTIs), including nucleoside (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), have been reported to exert anti-cancer effects; however, the molecular mechanisms underlying these effects remain incompletely understood. This project investigates the cellular response to RTI treatment and its relationship with LINE-1 expression and activity. RTI treatment also promoted the association of ORF1p with proteins involved in transcriptional regulation, DNA damage, and nuclear architecture.