Cancer drug resistance is the leading cause of cancer related deaths. The development of drug resistance can be partially contributed to tumor heterogeneity and epigenetic plasticity. However, the detailed molecular mechanism underlying epigenetic modulated drug resistance remains elusive. In this work, we systematically analyzed epigenetic changes in tamoxifen (Tam) responsive and resistant breast cancer cell line MCF7, and adopted a data-driven approach to identify key epigenetic features distinguishing between these two cell types. Significantly, we revealed that DNA methylation and H3K9me3 marks that constitute the heterochromatin are distinctively different between Tam-resistant and -responsive cells. We then performed time-lapse imaging of 5mC and H3K9me3 features using engineered probes. After Tam treatment, we observed a slow transition of MCF7 cells from a drug-responsive to -resistant population based on DNA methylation features. A similar trend was not observed using H3K9me3 probes. Collectively, our results suggest that DNA methylation changes partake in the establishment of Tam-resistant breast cancer cell lines. Instead of global changes in the DNA methylation level, the distribution of DNA methylation features inside the nucleus can be one of the drivers that facilitates the establishment of a drug resistant phenotype in MCF7.

Background: Alzheimer's disease and related dementias (ADRD) affect 6.2 million people in the U.S., a number that is rapidly increasing due to an aging population, yet no effective cure or treatment exists. TDP‐43 pathology, initially identified as a biomarker for ALS and FTD, is now observed in the majority of ADRD patients and is often associated with accelerated cognitive decline. However, the molecular mechanisms by which TDP‐43 contributes to ADRD progression remain unclear, largely due to the lack of tools to monitor its aggregation dynamics in living cells. Methods: To address this gap, we engineered a FRET‐based biosensor to detect TDP‐43 aggregation. The biosensor was designed using various TDP‐43 isoforms (full‐length, C‐terminal domain, and ΔNLS) fused to different linker constructs. We evaluated biosensor performance under proteostasis stress conditions known to induce TDP‐43 aggregation and validated its sensitivity using preformed TDP‐43 fibrils. To further explore the progression of TDP‐43 pathology, we transduced the biosensor into human iPSC‐derived cortical neurons. These neurons were then treated with cellular stressors that mimic aging‐related proteostasis dysfunction, cellular stressors that mimic aging‐related proteostasis dysfunction, i.e. preformed fibrils and pharmacological drugs. FRET signal growth was monitored over time, and neurons exhibiting high FRET signals were isolated using FACS for downstream RNA‐seq analysis. Results: The engineered FRET biosensor successfully detected TDP‐43 aggregation in response to proteostasis stressors. Neurons treated with aging‐mimicking compounds exhibited an increase in FRET signal over time, indicating progressive TDP‐43 aggregation. Conclusion: This study presents a novel FRET‐based biosensor for tracking TDP‐43 aggregation in living neurons. Our approach enables real‐time monitoring of TDP‐43 pathology and facilitates the identification of molecular pathways disrupted by its accumulation.