Eric G. Sun

Although EGFR inhibitors are highly effective for treating EGFR- mutant lung adenocarcinomas ( EGFR + LUADs), resistance inevitably emerges, with histologic transformation to small cell lung cancer (SCLC) representing a particularly aggressive mechanism. Our prospective trial enrolled patients with EGFR + LUAD at high risk of transformation ( TP53 / RB1 -deficient) and treated them with first-line osimertinib combined with platinum and etoposide. Despite the addition of SCLC-directed chemotherapy, nearly half of patients (5 of 11) underwent SCLC transformation, with median progression-free survival of 11 months and overall survival of 33 months. Genomic and single-cell transcriptomic profiling of patient samples revealed clinical and subclinical plasticity associated with TP53 / RB1 comutations, including histological transformation to SCLC, as well as mesenchymal and squamous histologies. These changes in plasticity were linked to progressive disruption of the RB pathway, leading to altered chromatin accessibility and induction of neuronal differentiation and epithelial-to-mesenchymal transition transcriptional programs, further intensified by osimertinib treatment. These findings provide insights into the role of plasticity in mediating drug tolerance.

Genomic insults in the form of DNA damage and mitotic errors can result in mislocalization of nuclear DNA into the cytoplasm in the form of micronuclei or as fragmented chromosomal elements. Recent work from the Ly lab has demonstrated that cytoplasmic DNAs can undergo intercellular transfer via nanotube-like connections. Using a variety of cell lines, the authors demonstrate the transfer of DNA through nanotubes and that various sources of genome instability can promote this phenomenon. Crucially, the transferred DNA can be incorporated into the nucleus of recipient cells and intermix with host chromosomes. Additionally, the transferred DNA molecules are functional and can provide a fitness advantage to recipient cells. These findings uncover a novel horizontal gene transfer mechanism in human cells, which could have profound implications in human disease and biology.

High-level copy-number (CN) amplification (HLAMP) is a major mechanism of oncogene activation in human cancer. Despite progress in therapeutically targeting HLAMPs, the processes underlying HLAMP evolution are incompletely understood, leaving critical knowledge gaps in their etiology and mechanisms of therapeutic response. To address this, we analyzed using novel computational approaches, the evolutionary trajectories of HLAMP in single-cell whole-genome sequencing of 86,239 cancer cells from 94 patients and 8 experimental systems. We found that two common etiologies of oncogene amplification - intrachromosomal amplification (ICamp) and extrachromosomal circular DNA (ecDNA) - lead to distinct CN distributions across cells in amplitude, variance and clone specificity. Notably, ICamp events exhibited widespread subclonal specificity, indicating their dynamic evolution through numeric or structural modulation associated with clonal expansions. In contrast, ecDNAs exhibited higher cell-to-cell CN variation linked to structural rearrangements, whereby complex ecDNA architectures were resolved at single-nucleotide resolution in individual cell genomes. Through joint analysis of ecDNA structure and their cellular abundance, we observed both divergent and convergent modes of ecDNA evolution shaped by DNA damage, selection, and chromosomal re-integration. Finally, modeling single-cell distributions of ecDNAs substantially improved bulk genome-graph based predictions, revealing that ecDNA prevalence is tissue-type specific, rather than broadly shared across cancer types, with implications for ecDNA-directed therapy.

In mammalian cells, gene copy number is controlled to maintain gene expression and genome stability. However, a common molecular feature across cancer types is oncogene amplification, increasing the copy number and expression of tumor-promoting genes and thus promoting cancer progression. For example, in tyrosine kinase inhibitor (TKI)-resistant lung adenocarcinoma (LUAD), oncogene amplification is frequent. Despite the prevalence of oncogene amplification in TKI-resistant tumors, the underlying mechanisms are not fully understood. Here, we find that LUADs exhibit a unique chromatin signature demarcated by strong CTCF and cohesin deposition in drug-naive tumors, which correlates with the boundaries of oncogene amplicons in TKI-resistant LUAD cells. We identify a global chromatin-priming effect during the acquisition of TKI resistance, marked by a dynamic increase of H3K27Ac, cohesin loading and inter-TAD interactions, which occur before the onset of oncogene amplification. Furthermore, we show that METTL7A, reported to localize to the endoplasmic reticulum and inner nuclear membrane, has a chromatin regulatory function by binding to amplified loci and regulating cohesin recruitment and inter-TAD interactions. METTL7A appears to remodel the chromatin landscape prior to large-scale copy number gains. Although METTL7A depletion exerts little phenotypical effects on drug-naive cells, its depletion prevents the formation and maintenance of TKI resistant-clones, showcasing its role as cells become resistant. In summary, we unveil a mechanism required for the acquisition of TKI resistance regulated by an unexpected chromatin function of METTL7A.

DNA double-strand breaks (DSBs) disrupt the continuity of the genome, with consequences for malignant transformation. Massive DNA damage can elicit a cellular checkpoint response that prevents cell proliferation^1,2. However, how highly aggressive cancer cells, which can tolerate widespread DNA damage, respond to DSBs alongside continuous chromosome duplication is unknown. Here we show that DSBs induce a local genome maintenance mechanism that inhibits replication initiation in DSB-containing topologically associating domains (TADs) without affecting DNA synthesis at other genomic locations. This process is facilitated by mediators of replication and DSBs (MRDs). In normal and cancer cells, MRDs include the TIMELESS–TIPIN complex and the WEE1 kinase, which actively dislodges the TIMELESS–TIPIN complex from replication origins adjacent to DSBs and prevents initiation of DNA synthesis at DSB-containing TADs. Dysregulation of MRDs, or disruption of 3D chromatin architecture by dissolving TADs, results in inadvertent replication in damaged chromatin and increased DNA damage in cancer cells. We propose that the intact MRD cascade precedes DSB repair to prevent genomic instability, which is otherwise observed when replication is forced, or when genome architecture is challenged, in the presence of DSBs^3,4,5. These observations reveal a previously unknown vulnerability in the DNA replication machinery that may be exploited to therapeutically target cancer cells.

Focal gene amplifications are among the most common cancer-associated mutations¹ but have proven challenging to engineer in primary cells and model organisms. Here we describe a general strategy to engineer large (more than 1 Mbp) focal amplifications mediated by extrachromosomal DNAs (ecDNAs)² in a spatiotemporally controlled manner in cells and in mice. By coupling ecDNA formation with expression of selectable markers, we track the dynamics of ecDNA-containing cells under physiological conditions and in the presence of specific selective pressures. We also apply this approach to generate mice harbouring Cre-inducible Myc- and Mdm2-containing ecDNAs analogous to those occurring in human cancers. We show that the engineered ecDNAs spontaneously accumulate in primary cells derived from these animals, promoting their proliferation, immortalization and transformation. Finally, we demonstrate the ability of Mdm2-containing ecDNAs to promote tumour formation in an autochthonous mouse model of hepatocellular carcinoma. These findings offer insights into the role of ecDNA-mediated gene amplifications in tumorigenesis. We anticipate that this approach will be valuable for investigating further unresolved aspects of ecDNA biology and for developing new preclinical immunocompetent mouse models of human cancers harbouring specific focal gene amplifications.