Targeted approaches reveal epigenetic regulation of lysosomal exocytosis contributing drug sensitivity and transcriptional activation

The limited effectiveness of chemotherapy in many solid tumors often stems from intrinsic cellular mechanisms that reduce intracellular drug accumulation. One such mechanism is lysosomal sequestration followed by exocytosis, wherein chemotherapeutic agents are trapped within acidic vesicles and expelled from the cell, thereby diminishing cytotoxic efficacy. Although this process is increasingly recognized as a contributor to treatment failure, its upstream regulatory control has remained poorly defined. In particular, the role of chromatin-modifying enzymes in governing lysosomal dynamics has not been systematically investigated. This thesis explores the hypothesis that lysosomal exocytosis is subject to epigenetic regulation and that pharmacologic inhibition of specific chromatin-modifying enzymes may suppress vesicle-mediated drug clearance. A focused epidrug library comprising 175 small-molecule inhibitors was conducted to identify compounds that influence lysosomal exocytosis, vesicle accumulation, and cisplatin sensitization in cancer cells. Two inhibitors targeting Type I protein arginine methyltransferases (PRMTs) emerged as potent hits, significantly enhancing cisplatin cytotoxicity without causing baseline toxicity. These inhibitors reduced lysosomal exocytosis and increased lysosomal content independently of canonical transcription factors governing lysosomal biogenesis. Transcriptomic profiling following PRMT inhibition revealed suppression of chromatin maintenance and DNA repair pathways, alongside induction of oxidative stress and apoptosis-related genes. Gene set enrichment analysis further demonstrated enrichment of lysosome-related pathways, including vesicle-mediated transport and regulation of lysosomal enzymes, providing additional support for the impact of PRMT inhibition on lysosomal dynamics. Integration with public ChIP-seq datasets indicated that many differentially expressed genes are direct PRMT targets and the promoter occupancy of PRMTs to these target genes are further verified in ChIP-qPCR experiments. All of these findings were validated across multiple cell lines and extended to other lysosomally sequestered drugs, highlighting broader therapeutic relevance. Moreover, analysis of patient-derived transcriptomes demonstrated that high PRMT1 and PRMT6 expression correlates with poor chemotherapy response, reinforcing the clinical significance of epigenetic control over lysosomal exocytosis. To complement this insight, the second part of the thesis investigates whether epigenetic modifiers can also regulate the transcription of genes involved in lysosome-mediated metal homeostasis and drug resistance. A CRISPR/Cas9-mediated knock-in model was developed by inserting a firefly luciferase cassette into exon 1 of one ATP7B allele, generating a reporter cell line that preserves the endogenous chromatin environment. Screening the same epidrug library in reporter assay identified several histone deacetylase (HDAC) inhibitors as potent inducers of ATP7B transcription. These effects were validated through promoter-reporter assays, RT-qPCR, and chromatin immunoprecipitation, which showed a specific enrichment of H3K18 acetylation mark over H3K9 and H3K27 acetylation at the ATP7B promoter following HDAC inhibition. Importantly, this transcriptional activation was conserved across hepatic and non-hepatic cell types and occurred independently of ROS accumulation. Together, these studies uncover chromatin-level mechanisms that regulate both lysosomal drug efflux and the expression of a clinically relevant copper transporter. By identifying epigenetic targets that influence drug retention and gene activation, this work provides a conceptual and experimental framework for improving therapeutic strategies in cancer and metal storage disorders.