Collin Gilbreath

Extrachromosomal DNAs (ecDNAs) attach to chromosomes during mitosis for random segregation and promote cancer heterogeneity. However, the mechanism governing ecDNA–chromosome mitotic interactions remains poorly understood. Here we show that ecDNAs tether to histone H3 lysine 27 acetylation (H3K27ac)-marked chromatin during mitosis. Depleting H3K27ac disrupts this interaction. Diverse bromodomain proteins, as H3K27ac readers, stabilize ecDNA–chromosome binding in a context-dependent and complementary manner. Although disrupting the Mediator complex in asynchronous cells detaches ecDNAs from mitotic chromosomes, Mediator and active Pol II are absent from ecDNAs during mitosis, suggesting that ecDNAs are transcriptionally silent during mitosis. Instead, inactive Pol II mediates ecDNA attachment. Furthermore, CRISPR interference targeting transcriptional regulatory elements on ecDNA impairs ecDNA segregation. Mis-segregated ecDNAs were expelled into the cytosol, leading to diminished oncogene expression and a reversal of therapy resistance. Our research provides universal cis and trans regulatory mechanisms of ecDNA segregation, offering deeper insight into ecDNA-driven oncogenesis.

EcDNA contributes to cancer genetic heterogeneity through random segregation during mitosis. Emerging evidence links ecDNA to immune evasion, but the mechanism remains elusive. Using genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDAC), we show that Kras and Myc oncogenes are amplified either on ecDNAs or as homogeneously staining regions (HSRs) on chromosomes. ecDNA-driven tumors are more aggressive in immunocompetent mice. Single-cell transcriptomic and histological analyses reveal that ecDNA-driven tumors rapidly establish an immunoevasive tumor microenvironment (TME), marked by increased myofibroblastic cancer-associated fibroblasts (myCAFs) and reduced T cell infiltration. Mechanistically, ecDNA heterogeneity generates a subset of cancer cells with extremely high Kras expression, termed super-expressors, which secrete amphiregulin to promote myCAF expansion and suppress T cell infiltration. Clonally organized super-expressors establish an immunoevasive niche in the TME from patients with PDAC. Our findings demonstrate a causal role of ecDNA in TME remodeling, offering insights into cancer heterogeneity and immune evasion.

Extrachromosomal DNAs (ecDNAs) attach to chromosomes during mitosis for random segregation and promote cancer heterogeneity. However, the mechanism governing ecDNA-chromosome mitotic interactions remains poorly understood. This study shows that ecDNAs tether to histone H3 lysine 27 acetylation (H3K27ac)-marked chromatin during mitosis. Depleting H3K27ac disrupts this interaction. Diverse bromodomain proteins, as H3K27ac readers, stabilise ecDNA-chromosome binding in a context-dependent and complementary manner. Although disrupting the Mediator complex in asynchronous cells detaches ecDNAs from mitotic chromosomes, Mediator and active Pol II are absent from ecDNAs during mitosis, suggesting that ecDNAs are transcriptionally silent during mitosis. Instead, inactive Pol II mediates ecDNA attachment. Furthermore, CRISPR interference targeting transcriptional regulatory elements on ecDNA impairs ecDNA segregation. Mis-segregated ecDNAs were expelled into the cytosol, leading to diminished oncogene expression and a reversal of therapy resistance. Our research provides universal cis and trans regulatory mechanisms of ecDNA segregation, offering deeper insight into ecDNA-driven oncogenesis.

Comparison gene expression and H3K27ac between MGH1518 serial models

Frequency of amplified oncogenes across PDX panel, similarity of ecMYC/L/N structures, and regression analysis of average drug response versus MYC paralog copy number, expression and amplicon type.

Acquired ecMYCN amplification in MGH1578 serial models and lineage oncogene expression compared with ecDNA status across the PDX panel.

Calculation of delta-AUC and comparison of EC with EP in PDX models.

Frequency of amplified oncogenes across PDX panel, similarity of ecMYC/L/N structures, and regression analysis of average drug response versus MYC paralog copy number, expression and amplicon type.

Reconstruction of ecDNAs containing MYC paralogs and MYCL FISH in MGH1501-1A metaphase chromosomes.