Extrachromosomal MDM2 amplifications define an ecDNA-permissive tumor cell state that impacts intercellular tumor heterogeneity and treatment response across cancers

Extrachromosomal DNA (ecDNA) amplifies oncogenes with high copy-number variability, promoting heterogeneity, rapid tumor evolution and treatment failure. MDM2, a key negative regulator of TP53, is frequently amplified on ecDNA, yet its impact on ecDNA dynamics and ecDNA dosage-driven treatment responses remains unclear. We integrated pan-cancer ecDNA reconstructions (TCGA + PCAWG), single-cell DNA FISH and ImmunoFISH, scRNA-seq, scG&T-seq from cell lines and PDX models with acute drug perturbations to analyze the impact of extrachromosomal MDM2 amplifications on ecDNA maintenance, TP53 function and treatment response. Consistent with prior work, our analysis of pan-cancer ecDNA reconstructions (TCGA + PCAWG) revealed that MDM2 is the most frequently amplified oncogene on ecDNA and is predominantly amplified extrachromosomally rather than on homogeneously staining regions (HSRs). Among over 100 recurrently amplified oncogenes, we found MDM2 to be one of the strongest ecDNA-biased drivers and to be associated with increased amplicon size and complexity. In contrast to MDM2 amplifications on HSR, MDM2 ecDNA almost exclusively arose in TP53 -wildtype tumors and frequently co-occurred with additional ecDNA oncogenes, suggesting that MDM2 ecDNA creates a phenotypically TP53 -deficient state that supports ecDNA maintenance and diversification. In neuroblastoma and astrocytoma models, MDM2 ecDNA copy number tightly correlated with protein and transcript abundance, generating pronounced single-cell heterogeneity. In this way MDM2 copy number determined TP53 activity and differentially affected treatment outcome in response to cytotoxic or targeted therapies at single-cell level as determined by combined fluorescence-in-situ hybridization and immunofluorescence (FISH/IF) and G&T sequencing data. Whereas chemotherapy rapidly enriched for ecDNA-high cells with reduced TP53 signaling, pharmacologic MDM2 inhibition (Nutlin-3a/Idasanutlin) selectively eliminated ecDNA-high subclones through TP53-dependent apoptosis. Similarly, MDM2 inhibition also reduced MYCN copy numbers in neuroblastoma cell lines and PDX with MYCN amplification, indicating that TP53 activation disrupts ecDNA-permissive states across different oncogenes. Our study demonstrates that MDM2 ecDNA is the predominant ecDNA oncogene in human cancers and defines an ecDNA-permissive state in TP53 -wildtype tumors which facilitates rapid adaptation to different therapeutic pressures. These results identify MDM2 ecDNA copy number variation as a functional determinant of acute treatment responses and reveal MDM2-regulated TP53 function as a vulnerability in ecDNA-carrying cancers that may be exploited through rational treatment sequencing.