Extrachromosomal DNA-Driven Oncogene Dosage Heterogeneity Promotes Rapid Adaptation to Therapy in MYCN-Amplified Cancers

Abstract

Extrachromosomal DNA (ecDNA) amplification enhances intercellular oncogene dosage variability and accelerates tumor evolution by violating foundational principles of genetic inheritance through its asymmetric mitotic segregation. Spotlighting high-risk neuroblastoma, we demonstrate how ecDNA amplification undermines the clinical efficacy of current therapies in cancers with extrachromosomal MYCN amplification. Integrating theoretical models of oncogene copy number-dependent fitness with single-cell ecDNA quantification and phenotype analyses, we reveal that ecDNA copy-number heterogeneity drives phenotypic diversity and determines treatment sensitivity through mechanisms unattainable by chromosomal oncogene amplification. We demonstrate that ecDNA copy number directly influences cell fate decisions in cancer cell lines, patient-derived xenografts, and primary neuroblastomas, illustrating how extrachromosomal oncogene dosage-driven phenotypic diversity offers a strong evolutionary advantage under therapeutic pressure. Furthermore, we identify senescent cells with reduced ecDNA copy numbers as a source of treatment resistance in neuroblastomas and outline a strategy for their targeted elimination to improve the treatment of MYCN-amplified cancers.

Significance: ecDNA-driven tumor genome evolution provides a major challenge to curative cancer therapies. We demonstrate that ecDNA copy-number dynamics drives treatment resistance by promoting oncogene dosage-dependent phenotypic heterogeneity in MYCN-amplified cancers. Exploiting phenotype-specific vulnerabilities of ecDNA cells, therefore, presents a powerful strategy to overcome treatment resistance. See related article by Korsah, p. XX.

Figure 1.

Extrachromosomal MNA impacts on oncogene copy-number and phenotypic heterogeneity in neuroblastoma. A, Schematic illustration of the experimental procedure to quantify ecDNA in MYCN-amplified neuroblastoma samples. B, Representative interphase FISH images of ecDNA and HSR neuroblastoma cell lines, PDX, and patient samples. For all pictures, FISH signals are presented in the following color scheme: MYCN (green) and CEP 2 as a reference probe for chromosome 2 (red) with Hoechst as a nuclear counterstain in blue. C, Box plot showing MYCN foci in MYCN-amplified neuroblastoma cell lines, PDX, and patient samples according to their amplification status (ecDNA vs. HSR). n = 10 cell lines, 8 PDX, and 52 patients (see Supplementary Fig. S1A for details; only untreated PDX and patient samples were considered). For all groups, mean absolute deviation values are presented in boxes. P values were calculated using a Fligner–Killeen test. *, P < 0.05 for cell lines and PDX; *, P < 0.0001 for patients. D, CV for all ecDNA samples as shown in A and C. For samples with the highest and lowest CV, MYCN copy number quantifications and distributions are highlighted in square boxes. $\overline{\text{x}}$ indicates the mean CV of the sample copy number over all samples. E, CV for all HSR samples shown in A and C. Square boxes demonstrate MYCN copy-number quantification and distribution for the samples with the highest and lowest coefficients of variation as in D. $\overline{\text{x}}$ indicates the mean CV of the sample copy number over all samples. F, Schematic illustration of the colony formation experiment. ecDNA and HSR cells were seeded as single cells and allowed to grow for 4 days. MYCN copy number was determined by FISH for several groups of colonies defined by cell number, as indicated. G, MYCN copy-number quantification and distribution in colonies with different cell numbers generated as illustrated in F using the near-isogenic cell line STA-NB-10 DM (left) and STA-NB-10 HSR (right). H, Schematic illustration of the computational model to assess the effect of ecDNA on cell fitness. The simulation starts from a random selection of n single cells harboring varying initial ecDNA copy numbers. These cells undergo clonal expansion for a short, defined period. At the end, the population size and ecDNA copy-number distribution within each clone are quantified. I, Representative simulation results of the ecDNA copy-number distribution across clonal populations of varying sizes under ecDNA copy number–dependent fitness (left) and ecDNA copy number–independent fitness (right) from the computational model described in H. J, Schematic illustration of ecDNA and HSR colony formation dynamics and the associated tumor heterogeneity. Cells with fewer ecDNA copies generate smaller colonies compared with cells with a higher initial number of ecDNA, whereas HSR cells with a stable MYCN copy number produce colonies that are more uniform in size.

Figure 2.

MYCN copy-number differences diversify cellular phenotypes in neuroblastoma. A, Schematic illustration of the FISH-guided proteomics workflow using image-guided laser microdissection followed by label-free quantitation of proteins with LC/MS in MYCN-high vs. MYCN-low copy-number cells in the neuroblastoma ecDNA cell line CHP-212. B, Volcano plot of differentially expressed proteins (P value ≤ 0.05) in pooled MYCN-high (>40 spots) vs. MYCN-low (<5 spots) cells by FISH-guided proteomics. Representative proteins of significantly enriched gene sets and pathways are indicated as follows: INTS13 and CTDSPL2 (DNA replication stress), STAG1 and PRDX2 (DNA damage), HLA-C (inflammation), CD44 and ANXA2 (mesenchymal phenotype), IGF2R and PIK3C3 (autophagy), and MMP2, MMP14, IGF2BP1, and MAP4K3 (senescence). C, Gene set and pathway enrichment analysis in MYCN-high vs. MYCN-low cells as in B. Tested gene sets and pathways were derived from the HALLMARK gene set of the human Molecular Signatures Database, WikiPathways, or reference 21 for the MES.signature.genes (van Groningen) and reference 85 for the senescence (Casella_up) signatures. D, Box plots comparing gene module enrichment scores for selected gene sets in MYCN-high vs. MYCN-low cells from scG&T-seq of a neuroblastoma PDX with extrachromosomal MNA. Enrichment scores were calculated for each cell, and box plots representing the IQR (25th–75th percentiles), with the median indicated by a central line, were plotted for cells with the highest (top 25%; red) vs. lowest (bottom 25%; blue) MYCN copy numbers. FDR-adjusted P values were calculated using a Wilcoxon rank-sum test. P < 0.05 for WP_DNA_DAMAGE_RESPONSE and senescence (Casella_up); P < 0.0001 for HALLMARK_MYC_TARGETS_V1, WP_CELL_CYCLE, REACTOME_Activation of ATR in response to replication stress, and the gene expression signature of cellular senescence (CellAge; ref. 28). E, Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) visualization of multiome (RNA + ATAC) single-cell data obtained from an SHH MB tumor (7316-178) containing an extrachromosomal MNA. Individual cells with high (n = 1225) and low (n = 1225) MYCN ecDNA amplifications are highlighted in blue and red, respectively, oligodendrocyte precursor cells (OPC) in purple and hematopoietic cells in green. F, Violin plots showing gene module enrichment scores for selected gene sets in single cells with high (red) and low (blue) extrachromosomal MNA. P values were calculated using the Student t test. Inset box plots represent the IQR (25th–75th percentiles), with the median indicated by a central line; outliers are displayed as individual points. P < 0.05 for senescence (Casella_up) comparison; P < 0.0001 for all the other comparisons. G, Relative frequency distribution of MYCN protein levels, γH2AX as a marker of DNA damage, and trimethylation of H3K9me3 as a marker of senescence in the near-isogenic neuroblastoma cell line pair STA-NB-10 HSR (red) and STA-NB-10 DM (blue). Mean (dashed lines) and median absolute deviation values are indicated in corresponding colors. P values were calculated using a Fligner–Killeen test. *, P < 0.05. H, Relative frequency distribution of MYCN, γH2AX, and H3K9me3 in 3 ecDNA PDX and 3 HSR neuroblastoma PDX. Mean (dashed lines) and MAD values are indicated in corresponding colors. P values were calculated using a Fligner–Killeen test. *, P < 0.05. BH, Benjamini–Hochberg.

Figure 2.

MYCN copy-number differences diversify cellular phenotypes in neuroblastoma. A, Schematic illustration of the FISH-guided proteomics workflow using image-guided laser microdissection followed by label-free quantitation of proteins with LC/MS in MYCN-high vs. MYCN-low copy-number cells in the neuroblastoma ecDNA cell line CHP-212. B, Volcano plot of differentially expressed proteins (P value ≤ 0.05) in pooled MYCN-high (>40 spots) vs. MYCN-low (<5 spots) cells by FISH-guided proteomics. Representative proteins of significantly enriched gene sets and pathways are indicated as follows: INTS13 and CTDSPL2 (DNA replication stress), STAG1 and PRDX2 (DNA damage), HLA-C (inflammation), CD44 and ANXA2 (mesenchymal phenotype), IGF2R and PIK3C3 (autophagy), and MMP2, MMP14, IGF2BP1, and MAP4K3 (senescence). C, Gene set and pathway enrichment analysis in MYCN-high vs. MYCN-low cells as in B. Tested gene sets and pathways were derived from the HALLMARK gene set of the human Molecular Signatures Database, WikiPathways, or reference 21 for the MES.signature.genes (van Groningen) and reference 85 for the senescence (Casella_up) signatures. D, Box plots comparing gene module enrichment scores for selected gene sets in MYCN-high vs. MYCN-low cells from scG&T-seq of a neuroblastoma PDX with extrachromosomal MNA. Enrichment scores were calculated for each cell, and box plots representing the IQR (25th–75th percentiles), with the median indicated by a central line, were plotted for cells with the highest (top 25%; red) vs. lowest (bottom 25%; blue) MYCN copy numbers. FDR-adjusted P values were calculated using a Wilcoxon rank-sum test. P < 0.05 for WP_DNA_DAMAGE_RESPONSE and senescence (Casella_up); P < 0.0001 for HALLMARK_MYC_TARGETS_V1, WP_CELL_CYCLE, REACTOME_Activation of ATR in response to replication stress, and the gene expression signature of cellular senescence (CellAge; ref. 28). E, Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) visualization of multiome (RNA + ATAC) single-cell data obtained from an SHH MB tumor (7316-178) containing an extrachromosomal MNA. Individual cells with high (n = 1225) and low (n = 1225) MYCN ecDNA amplifications are highlighted in blue and red, respectively, oligodendrocyte precursor cells (OPC) in purple and hematopoietic cells in green. F, Violin plots showing gene module enrichment scores for selected gene sets in single cells with high (red) and low (blue) extrachromosomal MNA. P values were calculated using the Student t test. Inset box plots represent the IQR (25th–75th percentiles), with the median indicated by a central line; outliers are displayed as individual points. P < 0.05 for senescence (Casella_up) comparison; P < 0.0001 for all the other comparisons. G, Relative frequency distribution of MYCN protein levels, γH2AX as a marker of DNA damage, and trimethylation of H3K9me3 as a marker of senescence in the near-isogenic neuroblastoma cell line pair STA-NB-10 HSR (red) and STA-NB-10 DM (blue). Mean (dashed lines) and median absolute deviation values are indicated in corresponding colors. P values were calculated using a Fligner–Killeen test. *, P < 0.05. H, Relative frequency distribution of MYCN, γH2AX, and H3K9me3 in 3 ecDNA PDX and 3 HSR neuroblastoma PDX. Mean (dashed lines) and MAD values are indicated in corresponding colors. P values were calculated using a Fligner–Killeen test. *, P < 0.05. BH, Benjamini–Hochberg.

Figure 3.

Fitness under chemotherapeutic selection reveals ecDNA-driven treatment response heterogeneity. A, Schematic representation of model B (ecDNA-dependent cytotoxicity). B, Growth rate, apoptosis rate, and net growth rate of model B, in which cell apoptosis is dependent on ecDNA copy numbers. C, Box plot showing mean ecDNA copy numbers before and after treatment in model B across all colony sizes (150 repetitions). The P value was calculated using the Mann–Whitney U test. *, P ≤ 0.05. D, Paired cell numbers before and after treatment in model B for colonies of different sizes (2–5 cells, 6–9 cells, and ≥15 cells) as defined in Fig. 1 (50 repetitions). E, Schematic overview of the experimental setup to test the theoretical models. ecDNA or HSR cell lines were seeded at low density and allowed to proliferate for 4 days before treatment with doxorubicin for 3 days. Colony size was defined by cell number as described in Fig. 1G and measured for 50 colonies prior to and after exposure to doxorubicin. F, Colony sizes of STA-NB-10 DM cells corresponding to cell numbers from 2 to 5 (left), from 6 to 9 (middle), and of ≥15 (right) in untreated and doxorubicin-treated colonies as described in E. G, Colony size of STA-NB-10 HSR cells corresponding to cell numbers of ≥15 in untreated and doxorubicin-treated colonies as described in E.

Figure 4.

Chemotherapy induces dynamic copy-number changes of MYCN and MYC in ecDNA-containing cancers. A, Dot plot analysis and representative pictures of MYCN copy numbers in three neuroblastoma ecDNA cell lines (CHP-212, SIMA, and UKF-NB-6) exposed to doxorubicin for 5 days or left untreated. Mean copy numbers are presented as horizontal lines. P values were calculated using a Mann–Whitney U test. *, P ≤ 0.05. For all pictures, FISH signals are presented in the following color scheme: MYCN (green) and CEP 2 as a reference probe for chromosome 2 (red) with Hoechst as a nuclear counterstain in blue. B, Dot plot analysis and representative pictures of MYC copy numbers in the colorectal carcinoma cell line COLO320 DM, the mesothelioma cell line MSTO, and the gastric adenocarcinoma cell line SNU-16 exposed to doxorubicin for 5 days or left untreated. Mean copy numbers are presented as horizontal lines. P values were calculated using a Mann–Whitney U test. *, P ≤ 0.05. For all pictures, FISH signals are presented in the following color scheme: MYC (green) and CEN 8 as a reference probe for chromosome 8 (red) with Hoechst as a nuclear counterstain in blue. C, Dot plot analysis of MYCN copy numbers in two ecDNA PDX before and at different time points after doxorubicin treatment. P values were calculated using a Mann–Whitney U test. *, P ≤ 0.05. D, Dot plot analysis of MYCN copy numbers from untreated (n = 36) or treated (n = 14) ecDNA neuroblastomas from the German High Risk Neuroblastoma Trial Protocol NB-2004-HR. P values were calculated using a Mann–Whitney U test. *, P ≤ 0.05. E, (Left) Dot plot analysis of MYCN copy numbers from four patient-matched untreated and treated ecDNA neuroblastomas as in D. P values were calculated using a Mann–Whitney U test. *, P ≤ 0.05. (Right) Representative FISH images of matched untreated vs. treated tumor samples from one patient with MYCN signal in green, CEP 2 as a reference probe for chromosome 2 in red, and Hoechst as a nuclear counterstain in blue.

Figure 5.

MYCN copy-number status determines treatment response to cytotoxic therapies. A, Correlation plot and representative images showing the association of MYCN copy numbers with γH2AX intensity as a marker of DNA damage in single cells of the ecDNA cell line CHP-212 by ImmunoFISH. Each dot represents a cell before (red) or 4 hours after (blue) doxorubicin treatment. Correlation for the untreated and treated conditions is assessed using Pearson’s correlation coefficient and its associated P value. B, Correlation plot and representative images showing the association of MYCN copy number and cleaved PARP1 as a marker of apoptosis for single cells as in A. Correlation for the untreated and treated conditions is assessed using Pearson’s correlation coefficient and its associated P value. C, Box plot showing MYCN copy numbers in cells that stain either negative or positive for the senescence marker H3K9me3, as determined by ImmunoFISH, before and 5, 10, or 20 days after doxorubicin treatment. P values were calculated using a Mann–Whitney U test. *, P ≤ 0.05. All comparisons between H3K9me3-positive cells are not significant. D, Gene set and pathway enrichment analysis comparing MYCN-high and MYCN-low cells 5 days after doxorubicin treatment using FISH-guided proteomics. Tested gene sets and pathways were derived from the Hallmark gene set of the human Molecular Signatures Database, WikiPathways, or reference 21 for the MES.signature.genes (van Groningen) and reference 85 for the senescence (Casella_up) signatures. E, Mean senescence score for individual neuroblastoma cells in untreated (n = 32) and chemotherapy-exposed (n = 38) MYCN-amplified tumors. Boxes from first to third quartile; whiskers indicate data at a maximum of 1.5-fold IQR from the box. P value were calculated using Welch’s two-sample t test (two-sided; mean score before chemotherapy −0.065, mean score after chemotherapy −0.035; t = −3.27, d_f = 65.35, P = 0.0017). F, Mean MYCN expression and senescence score for neuroblastoma cells in MYCN-amplified samples (n = 70). Correlation is assessed using Pearson’s correlation coefficient and its associated P value. G, Box plot showing MYCN copy number and the senescence marker H3K9me3, as determined by ImmunoFISH, in cells before treatment, treated with doxorubicin for 5 days or sequentially with doxorubicin for 5 days followed either by vehicle (DMSO) or tranylcypromine (2-PCPA) every 3 days until day 20. P values were calculated using a Mann–Whitney U test. *, P ≤ 0.05. H, Detection of MYCN copy numbers by FISH in untreated vs. doxorubicin-treated CHP-212 cells stably transduced with either BCL2 or an empty vector as control. Boxes from the first to third quartile; whiskers indicate data at a maximum of 1.5-fold IQR from the box. P values were calculated using a Mann–Whitney U test. *, P ≤ 0.05. I, Detection of MYCN copy numbers by FISH in untreated vs. doxorubicin-treated CHP-212 cells stably transduced either with a short hairpin against TP53 or against GFP as a control. Quantification as in H. For all pictures, FISH and immunofluorescence signals are presented in the following color scheme: MYCN (green); CEP 2 as a reference probe for chromosome 2 (red); immunofluorescence signal for γH2AX, cleaved PARP1, H3K9me3, or PCNA (purple); and Hoechst as a nuclear counterstain (blue). Scale bar, 10 μm. Simple linear regression was performed to analyze the relationship between MYCN copy number and γH2AX, cleaved PARP1, or H3K9me3. BH, Benjamini–Hochberg.

Figure 6.

Targeted elimination of chemotherapy-resistant low MYCN copy-number cells improves treatment outcome. A, Bar plot showing cell viability as assessed by CellTiter-Glo in the ecDNA cell line CHP-212 without treatment, exposed to one of the indicated senolytic drugs (navitoclax, nintedanib, bafilomycin A1, digoxin, or fisetin) at their respective IC₅₀ or IC₁₀ individually for 3 days, to doxorubicin for 5 days, or to the sequential treatment with doxorubicin for 5 days followed by the indicated senolytic drugs at their respective IC₅₀ or IC₁₀. Values are presented as mean ± SD, n = 3 technical replicates. P values were calculated using the Student t test. *, P ≤ 0.05. B, Zero interaction potency (ZIP) synergy score from cell viability measurements with CellTiter-Glo in the near-isogenic neuroblastoma cells STA-NB-10 DM (left) and STA-NB-10 HSR (right) exposed sequentially to doxorubicin and navitoclax as in A, n = 3 technical replicates. C, Correlation plot and representative image showing the association of MYCN copy number and BCL-xL in single cells of the ecDNA cell line CHP-212 by ImmunoFISH. Each dot represents a cell before doxorubicin treatment. Correlation is assessed using Pearson’s correlation coefficient and its associated P value. For imaging, cells were stained with MYCN FISH (green), CEP 2 as a reference for chromosome 2 abundance (red), BCL-xL by IF (purple), and Hoechst as a nuclear counterstain (blue). D, Dot plot indicating MYCN copy numbers in CHP-212 cells left untreated or exposed to navitoclax for 3 days. Mean copy numbers are presented as horizontal lines [x = 23.5 (untreated) and x = 36 (navitoclax)]. *, P ≤ 0.05. E, Correlation plot showing the association of MYCN copy numbers with cleaved PARP1 as a marker of apoptosis for single cells of the ecDNA cell line CHP-212 by ImmunoFISH. Each dot represents a cell 16 hours after navitoclax treatment. Correlation is assessed using Pearson’s correlation coefficient and its associated P value. F, Tumor volumes in neuroblastoma PDX with extrachromosomal MNA treated with doxorubicin, navitoclax, or both drugs in sequential combination. Changes in tumor volume were monitored for 20 days and compared with mice receiving vehicle treatment (n = at least 3 independent mice per treatment group). *, P < 0.05 for all comparisons.