Epigenetic Mechanisms of Resistance and Biomarkers of Response to EGFR-targeted Therapy in Non-Small Cell Lung Cancer

Doctoranda 
Ester Alemany Cosme

Director de la tesis
Dr. Juan Sandoval del Amor

Unidad de Biomarcadores y Medicina de Precisión (UBMP)

In non-small cell lung cancer (NSCLC) with activating mutations in the EGFR gene, the EGFR tyrosine kinase inhibitor (TKI) osimertinib is the standard of care, yet primary and acquired resistance limit therapeutic benefit. DNA methylation, a reversible epigenetic regulator of gene expression, can modulate cellular programs such as epithelial-mesenchymal transition (EMT), recognized as a mechanism of resistance to osimertinib. This thesis focused on identifying DNA-methylation-driven gene dysregulation underlying EMT-associated resistance (Chapter A) and evaluating circulating cell-free DNA (cfDNA) methylation biomarkers to stratify osimertinib response and progression risk (Chapter B).

In Chapter A, osimertinib-resistant derivatives (OSIR) of H1975, HCC4006, and HCC827 cells were generated by prolonged drug exposure. OSIR models showed EMT-compatible morphology, decreased epithelial and increased mesenchymal markers, and higher motility in two models. Despite sustained p-EGFR inhibition, signaling shifted away from EGFR toward STAT3. Methylome profiling (Illumina MethylationEPIC) revealed 20,213 differentially methylated CpGs annotated to 6,788 genes, enriched for EGFR-TKI resistance, PI3K-AKT/MAPK/RAS signaling, and adhesion/migration. RNA-seq identified 1,338 differentially expressed genes with a dominant EMT signature. Integration of methylome and transcriptome prioritized 15 candidates compatible with methylation-based regulation in resistance: 8 hypermethylated/repressed (CLDN7, CLDN4, TMEM30B, MST1R, CBLC, SPINT1, FUT2, GJB3) and 7 hypomethylated/activated (FGFR1, RIPOR1, TRAF1, ANK1, RNF182, MSRB3, DLC1). Decitabine demethylation validated epigenetic dependence for CLDN7 and GJB3 (re-expression in OSIR) and for MSRB3 and ANK1 (induction in parental lines), whereas other candidates showed model-specific responses. CLDN7 was established as the lead candidate owing to marked mRNA/protein silencing, robust re-expression with locus-specific promoter demethylation after decitabine treatment, and literature supporting tumor-suppressive roles in lung cancer and involvement in EMT. CRISPRi knockdown of CLDN7 in parental cells did not confer resistance or induce EMT, but reduced the number and cohesion of 3D spheroids and tended to increase collective migration. Pharmacologic perturbation of the TGF-β-SMAD2/3 axis altered CLDN7 expression: chronic TGF-β decreased it in parental cells, and the TGFBR1-inhibitor SB-431542 partially restored it in OSIR models.

In Chapter B, a prospective multicenter study enrolled patients with advanced NSCLC harboring activating EGFR mutations treated with first-line osimertinib. For a preliminary cfDNA analysis, 47 valid samples from 28 patients were obtained (24 baseline, 17 first evaluation, 6 progression). Patients were stratified by progression-free survival (PFS) into long-term responders (PFS ≥ 18.9 months) and the short-response group (PFS < 18.9 months). CLDN7 methylation was measurable in cfDNA and variable across CpGs and patients. Two promoter CpGs showed higher median β-values in short-response patients than long-term responders, but the differences were not significant. Longitudinal analysis revealed patient-specific on-treatment changes, with consistent promoter hypermethylation in a short-response patient and hypomethylation in a long-term

responder. Baseline methylome analysis (MethylationEPIC; 575,253 CpGs) identified 591 CpGs differing between groups (FDR < 0.05). From these, a 32-CpG signature derived by penalized Cox (elastic net) was associated with PFS and discriminated between the two groups (C-index = 0.92; AUC = 0.89).

Overall, osimertinib resistance with EMT features is associated with extensive methylome and transcriptome remodeling, and accompanied by marked epigenetic dysregulation of CLDN7, with implications for intercellular cohesion and collective migration. In cfDNA, a preliminary 32-CpG signature can stratify progression risk on first-line osimertinib.

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