Visualizing the Dynamic Locus-specific DNA Repair and Cancer Resistance in Single Cells

Introduction: : DNA damage is one of the most critical challenges faced by cells, as it can lead to genomic instability, cell death, and various pathological conditions, including cancer. The repair of DNA damage involves complex mechanisms that must balance both speed and accuracy to maintain genomic integrity. Traditional methods for studying DNA repair have largely relied on fixed-cell imaging techniques. While these approaches have provided valuable insights, they lack the temporal and spatial resolution necessary to fully understand the dynamics of repair processes. In this study, we introduce the GLIDRE (Genomic Locus Imaging and DSB Repair Monitoring by Elastin-like Proteins) system for the concurrent imaging of genomic loci and monitoring of DNA repair. Using GLIDRE, we examined the mechanisms underlying the enhanced DNA repair capacity in H3.3K27M mutant tumor cells and its role in CAR-T therapy resistance.

Materials and

Methods: : To visualize non-repetitive loci in live cells, we adopted the liquid-liquid phase separation property of elastin-like polypeptides (ELP), which enables the automatic recruitment of signals hundreds and thousands of times higher than simple repeats. In our GLIDRE system, a catalytically dead Cas9 fused to an eight repeats SunTag scaffold (dCas9-8×SunTag) recruits ScFv (anti-SunTag)-nVenus-ELP, forming a high-concentration ELP microdomain at the target locus. This microdomain can further recruit additional ELP fusion proteins, forming a visible droplet at the target non-repetitive site. To induce and monitor DNA damage, we generated double-strand breaks using a heat-inducible AsCas12a nuclease and tracked repair foci via 53BP1 fluorescence (Panel A). To investigate the impact of H3.3K27M mutation on DNA repair, we analyzed public H3K27me3 ChIP-Seq and ATAC-Seq data for DIPG patients’ samples and applied GLIDRE to follow repair at the PLK1 oncogene and CDKN2A tumor suppressor loci. Lastly, we treated H3.3K27M mutant cells with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) to induce global chromatin acetylation and quantified its impact on repair kinetics. We also performed CAR-T cell cytotoxicity assays to assess how the H3.3K27M mutation and SAHA treatment modulate tumor cell resistance.

Results, Conclusions, and Discussions:: We utilized the GLIDRE system to dynamically track DNA repair at non-repetitive loci. Using time-lapse imaging, we captured multiple rounds of DSB repair at the target site (Panel B). The dwelling time of 53BP1-Halotag at the DSB site provided a quantitative measure of repair kinetics, reflecting the speed and efficiency of DSB repair. H3K27me3 ChIP-seq analysis showed that while most genes lose H3K27me3 marks in H3.3K27M mutant cells including oncogenes, tumor suppressor genes, such as CDKN2A, generally remain heavily trimethylated at the H3K27 site. Consequently, chromatin accessibility is also enhanced in these regions with H3.3K27M according to the ATAC-Seq. To investigate the impact of H3.3K27M mutations on DNA repair, we utilized our GLIDRE system to analyze the repair dynamics of the PLK1 (oncogene) and CDKN2A (tumor suppressor) genes. Interestingly, we observed faster DNA repair at the PLK1 locus in H3.3K27M-expressing cells, while repair at the CDKN2A locus was significantly slower in these mutant cells (Panel C). We measured DNA repair at the CDKN2A locus before and after SAHA treatment in both H3.3K27WT and H3.3K27M mutant cells. As hypothesized, SAHA treatment significantly accelerated DNA repair at the CDKN2A locus in mutant cells. Encouragingly, SAHA pretreatment sensitized H3.3K27M mutant tumor cells, resulting in killing rates comparable to those observed in WT cells (Panel D). By circumventing many of the limitations inherent to traditional fixed-cell imaging approaches, our method offers unique opportunities to dissect the spatial and temporal dynamics of DNA repair, particularly as they relate to chromatin accessibility and transcriptional status. These results not only confirm that targeted epigenetic therapies can enhance the efficacy of immunotherapeutic strategies but also provide a rationale for broader applications in oncology. For example, future studies could explore how modulation of chromatin accessibility might potentiate the effects of genotoxic agents in various cancer models, including those driven by other histone mutants or epigenetic deregulations.

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