The quest for better cancer therapies:Identification of transcription factor involvement in ATP7B regulation via dCas9-Apex2 (Caspex), TOX4 as a potential regulator of Cisplatin resistance

The identification of specific transcription factors and epigenetic modifications that regulate the ATP7B gene could provide new insights into the development of therapeutic strategies targeting cancer drug resistance mechanisms. Here, we used genomic locus proteomics dCas9-Apex2 (CASPEX) to identify proteins that interact with the promoter of ATP7B which can provide a deeper understanding of the role of transcription factors and chromatin modifiers in the regulation of the ATP7B, and ultimately, contribute to the development of new therapies for diseases associated with ATP7B dysfunction and cancer drug resistance. To explore the transcriptional regulators responsible for controlling the expression of the ATP7B gene, we dissected the -3000 bp to +1 bp region at the ATP7B promoter into seven distinct regions, which was carried out to comprehensively cover the proteins that bind to the promoter, using the CASPEX labeling system. The targeting efficiency of the gRNAs on ATP7B promoter was confirmed via chromatin immunoprecipitation (ChIP). Following mass spectrometry, we identified over 150 transcription factors that were enriched at the ATP7B promoter. We selected specific candidates to examine their impact on ATP7B expression through overexpression or knock-out experiments. Additionally, we investigated their ability to bind to the ATP7B promoter using ChIP-qPCR analysis. Our screening suggested that TOX4 acts as a novel regulator of ATP7B in HEK293T and HUH7 cell lines. The activity of TOX4 at the ATP7B promoter was confirmed via both luciferase reporter assays and ChIP-qPCR experiments. Furthermore, while TOX4 Ox conferred cisplatin resistance, its downregulation lead to sensitization by out hypothesis. In conclusion, our discoveries offer new perspectives on the regulatory mechanisms governing the ATP7B gene, which could have significant implications for comprehending and addressing cancer drug resistance involving TOX4. The recognition of these transcription factors, particularly TOX4, as crucial regulators of the ATP7B gene, opens up possibilities for potential targeting in future cancer therapeutic approaches. Keywords: ATP7B, Cisplatin Resistance, TOX4, Proteomics, Molecular Biology