Promoter-centric gene regulation in drug-resistant cancer

In eukaryotic cells, gene-distal regulatory elements (REs) facilitate long-range gene regulation, ensuring cell type-specific transcriptional programs. This mechanism is frequently disrupted in cancer, often driven by transcription factors (TFs) that serve as targets for cancer therapy. However, targeting these TFs can lead to acquired resistance mechanisms that are not fully understood. We show that mesothelioma cancer cells, dependent on the oncogenic driver TF TEAD, develop resistance to a pan-TEAD inhibitor and revert to an evolutionarily ancient, promoter-centric gene regulatory mechanism to recover gene expression. Base-pair-resolution 3D chromatin conformation mapping reveals that RE-promoter interactions are disrupted in resistant cells, despite epigenetic and transcriptomic recovery. Mechanistically, in resistant cells, TF complexes, including resistance-specific FOSL1 and KLF4, preferentially bind and enhance promoter activity to recover gene expression, rendering distal REs dispensable. Our findings highlight promoter elements and promoter-specific TFs as potential therapeutic targets using a model of drug-resistant cancer. Overall design: Micro-Capture C: Examination of local chromatin folding in parental and drug resistant mesothelioma cancer cells (NCI-H226). ATACseq: For each sample, ~100k viable cells were processed with the Active Motif ATAC-seq kit according to the manufacturer?s instructions. Nuclei were isolated, tagmentation was performed with Tn5 transposase for 30-minutes at 37C, and libraries were PCR-amplified (12 cycles) using indexing primers. Libraries were size-selected, quality-checked by Tapestation, quantified by Qubit, and sequenced as paired-end (2 × 50 bp). CUT&Run: Epigenetic mapping in parental and drug resistant mesothelioma cancer cells. ChIPseq: NF2-null H226 and MSTO-211H cells were generated by long-term, stepwise dose adaptation to the pan-TEAD inhibitor GNE-7883. H226 cells were gradually exposed from 0.25 µM to 2.5 µM over ~55 days, and MSTO-211H cells from 0.25 µM to 3.0 µM, with incremental increases every 1?2 weeks while maintaining at least 25% cell confluence. During escalation, transient cytostasis was followed by the emergence of proliferating persister populations under sustained drug pressure. Resistant pools were expanded from these outgrowths and maintained in medium containing 5 µM GNE-7883. Characterization of these resistant models followed previously described procedures. For acute treatment conditions, H226 and MSTO cells were treated with 2.5 µM GNE-7883 for 48-hours before collection. RNAseq: NF2-null H226 and MSTO-211H cells were generated by long-term, stepwise dose adaptation to the pan-TEAD inhibitor GNE-7883. H226 cells were gradually exposed from 0.25 µM to 2.5 µM over ~55 days, and MSTO-211H cells from 0.25 µM to 3.0 µM, with incremental increases every 1?2 weeks while maintaining at least 25% cell confluence. During escalation, transient cytostasis was followed by the emergence of proliferating persister populations under sustained drug pressure. Resistant pools were expanded from these outgrowths and maintained in medium containing 5 µM GNE-7883. Characterization of these resistant models followed previously described procedures. For acute treatment conditions, H226 and MSTO cells were treated with 2.5 µM GNE-7883 for 48-hours before collection.