CDK4 or CDK6 upregulation induces DNA replication stress and genomic instability to cause EGFR targeted therapy resistance in lung cancer

Epidermal growth factor receptor (EGFR)–mutant lung adenocarcinomas (LUAD) harbor a complex landscape of genetic co-alterations and potential oncogenic interactions. Among them are recurrent amplifications of the cell cycle regulatory genes CDK4 and CDK6 , which have been clinically implicated in resistance to EGFR tyrosine kinase inhibitors (TKIs). However, the mechanisms by which CDK4/6 upregulation promotes therapy resistance remain poorly defined. Here, we demonstrate that CDK4 or CDK6 overexpression limits EGFR inhibitor-induced proliferative arrest, promoting continued cell cycle progression. This is accompanied by elevated replication stress, increased TPX2 expression, DNA damage leading to ATM activation, and ultimately genomic instability. Integrative transcriptomic and copy number analyses of EGFR-mutant LUAD tumors from both patients and preclinical models revealed that CDK4 or CDK6 amplification is associated with the upregulation of genes linked to tumor progression, including AGR2 , ASNS , and STEAP1 . CDK4 amplification was also highly correlated with gene expression changes associated with epithelial-to-mesenchymal transition (EMT) in a single-cell RNA sequencing dataset from patient biopsies. In preclinical models, co-treatment with CDK4/6 and EGFR inhibitors restored proliferative arrest, induced tumor cell apoptosis, and reduced replication stress, DNA damage, and genomic instability. Our findings uncover a mechanistic basis for EGFR inhibitor resistance of CDK4 and CDK6 amplified EGFR-mutant LUAD. They also provide a rationale for the biomarker-driven clinical development of combination EGFR and CDK4/6-targeted therapies for the treatment of a subset of EGFR-mutant LUAD patients.