Abstract 5227: Extrachromosomal MYC-paralogs amplification shapes suppressive tumor microenvironment in small cell lung cancer

Abstract Introduction: Although immunotherapy has demonstrated promise in small cell lung cancer (SCLC), certain patients encounter limited benefits, highlighting the need for identifying immunosuppressive biomarkers. Extrachromosomal circular DNA (ecDNA) promotes amplification of MYC-paralogs, which drive cross-resistance in SCLC. Here, we aim to investigate whether ecDNA-mediated MYC-paralogs amplification (ecMYC+) represents immunosuppressive features in SCLC. Methods: Bulk RNA sequencing (RNA-seq) data were retrieved from public database and paraffin-embedded samples (n = 40). The copy number of MYC-paralogs was investigated using real-time polymerase chain reaction. RNA-seq, flow cytometry and metaphase fluorescence in situ hybridization (FISH) were performed in SCLC cell lines. The overexpression and amplification of MYC-paralogs in SCLC clinical samples (n = 42) were identified using immunohistochemistry (IHC) and FISH. The spatial distribution of tumor immune microenvironment (TIME) was characterized by multiplex immunofluorescence and imaging mass cytometry (IMC). Results: The mean copy number of ecDNAs was higher in SCLC than in the other cell lines (15.3 vs 8.4, p = 4.0×10-3), and the frequency of ecMYC+ cell lines was higher in SCLC than that in the other lineages (SCLC 22/47 vs others 15/282, p = 3.9×10 -12). In ecMYC+ SCLC, multiple immune-related pathways were down-regulated, while nucleotide metabolism processes were up-regulated. Inhibition of nucleotide metabolism by hydroxyurea and gemcitabine resulted in the reduction of ecDNA, along with the activation of antigen presenting pathways. Immunohistochemical analysis revealed that overexpression of MYC-paralogs was observed in 19 out of 42 resected SCLC samples. FISH identified 8 samples as MYC-paralogs copy number gain or amplification, and 3 samples exhibited a scattered signals distribution resembling ecDNA amplification. Through the resolution of 103, 341 cells from 24 pathological regions, we observed higher expression of MKI67, VEGFA, FAP and FOXP3 and reduced T cell infiltration in ecMYC+ samples. Moreover, ecMYC+ samples exhibited elevated cellular neighborhoods dominated by Ki67+ tumors, with reduced spatial interaction with immune cells. Conclusions: Extrachromosomal amplification of MYC-paralogs shapes suppressive tumor microenvironment, identifying potential subgroup of immunotherapy resistant patients. Citation Format: Jingwei Zhang, Yueqi Jin, Ence Yang, Xiao Li. Extrachromosomal MYC-paralogs amplification shapes suppressive tumor microenvironment in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5227.