Additional data_RAF1 extrachromosomal DNA amplification confers acquired erlotinib resistance in non-small cell lung cancer cell model

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Fig. S1. EGFR gene status and Structural variant cluster and ecDNA-associated oncogene amplification and expression in ER-R (a) ER-R cells retained the sensitizing EGFR exon 19 deletion, with only additional silent mutations detected.(b) Reduced EGFR copy number and expression reflect the loss of EGFR dependency, consistent with an EGFR-independent bypass mechanism. (c) Copy number (CN) and structural variation (SV) profiles across the focally amplified region on chromosome 3. ER-R cells showed markedly elevated CN, allelic imbalance, and clustered SV breakpoints. (d–e) Copy number analysis demonstrated concordant circular amplicons on chromosome 3, in which VHL, FANCD2, PPARG, and RAF1 were co-amplified.Fig. S2. RAF1-containing ecDNA promotes MAPK pathway activation (a-b) mRNA expression analyses show focal high-level amplification and marked overexpression of these oncogenes in ER-R compared with ER-S. (c) Gene set enrichment analysis (GSEA) demonstrated significant enrichment of the MAPK signaling cascade in ER-R cells relative to ER-S cells (NES = 1.39, p < 0.01). Fig. S3. RAF1 upregulation is required for ER-R cell survival (a) Cell lysates prepared from ER-S and ER-R cells were subjected to immunoblotting analysis with antibodies against total EGFR, p-EGFR (Y1068), total ERK, p-ERK (T202/Y204), RAF1 and p-RAF1 (S289/296/301), with vinculin as a loading control. (b) Colony formation assays were performed by seeding si-RAF1- or si-GFP-transfected cells at low density and culturing for 7 days. Colonies were fixed and stained with crystal violet, and representative images from one of three independent experiments are shown.Fig. S4. RAF1-mediated resistance confers therapeutic sensitivity (a) Cell lysates prepared from cells treated with erlotinib (1 µM), AZD6244 (1 µM), or their combination for 24 hours were subjected to immunoblotting analysis with antibodies against total EGFR, p-EGFR (Y1068), total ERK, p-ERK (T202/Y204), RAF1, p-RAF1 (S289/296/301), and c-PARP with vinculin. (b) Cells expressing either the vector control or RAF1-overexpression construct were treated with erlotinib (1 µM) and imaged under bright-field conditions every 7 days for up to 21 days using the Cytation 5 (BioTek) imaging system. ER-S cells, which stably expressed either a RAF1 cDNA plasmid (RAF1 OE) or an empty vector, underwent 40 days of treatment with 1 μM erlotinib. Cell quantification was performed using Hoechst 33342 staining, followed by automated nuclei counting via the Cytation 5 imaging system. Data were normalized to untreated controls and are presented as the mean ± SD (n = 4). Cell viability was measured using CCK-8, and dose–response curves were generated. The results are presented as the mean ± SD from quadruplicate experimentsFig. S5. ecDNA perturbation reduces RAF1 expression and resistance phenotypes (a) Cell proliferation was monitored in real time using the Incucyte system, with confluency quantified at 4-hour intervals using Incucyte® AI Confluence Analysis. ER-S cells received single treatments of erlotinib (1 μM) or HU (250 μM), while ER-R cells were treated with a combination of erlotinib (1 μM) and HU (250 μM). Statistical significance was determined using an unpaired Welch’s t-test.Fig. S6. Pan-cancer analysis of RAF1-carrying ecDNA formation propensity. A genomic survey across TCGA, PCAWG, and HMF cohorts was conducted, totaling 3,427 amplicon-positive samples. While the baseline ecDNA formation rate for oncogene-containing amplicons is 32.1% (1,805/5,618), the RAF1 locus exhibits a significantly higher frequency of circularization at 58.3% (14/24). This enrichment is statistically significant (Odds Ratio = 2.96, p = 0.0084, Fisher’s exact test), suggesting a heightened structural predisposition for ecDNA formation at this locus compared to the pan-cancer average.Fig. S7. Proposed model of ecDNA-mediated RAF1-driven resistance and therapeutic dependency. Created using BioRender.com.

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