Background: Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a standard first-line therapy for activated EGFR-mutated non-small-cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance and is hence a promising target for anticancer treatment. Patritumab deruxtecan (HER3-DXd) is an antibody drug conjugate comprised of a fully human anti-HER3 IgG1 monoclonal antibody, covalently linked to a topoisomerase 1 inhibitor payload, an exatecan derivative, via a tetrapeptide-based cleavable linker. Clinical trials of theHER3-DXd demonstrated its anticancer activity in EGFR-mutated NSCLC. However, the genomic background that regulates HER3 expression is unknown. This study was conducted with the aim to clarify the genomic background for HER3 regulation in EGFR-mutated NSCLC tumors and to explore the strategy for enhancing the anticancer activity of HER3-DXd. Methods: 48 paired samples were obtained before EGFR-TKI treatment and after the acquisition of EGFR-TKI resistance from patients with EGFR-mutated NSCLC. HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pre-treatment to post-EGFR-TKI resistance acquisition. For statistical tests, linear regression analysis was performed to investigate whether a factor, including EGFR-TKI administration, affected HER3 expression. Pearson correlation coefficients were calculated to explore the relationships between HER3 expression level and other genomic expression in the tumors. Results: We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared to paired pretreatment samples (mean H-score 100.8 vs. 155.9, paired t-test p = 0.0007). Although genomic alterations including EGFR secondary T790M mutation did not correlate with HER3 augmentation, RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation (GSEA; normalized enrichment score -1.78, p = 0.004). Multivariable regression analysis of HER3 augmentation revealed that re-biopsy under continuing EGFR-TKI treatment independently effected HER3 augmentation. Indeed, an in-vitro study also showed that EGFR-TKI increased HER3 expression via repressed AKT phosphorylation in multiple EGFR-mutated cancers, and it finally enhanced the anticancer activity of HER3-DXd. Conclusions: Our findings highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC that is currently being evaluated in a clinical trial for patients with EGFR-mutated NSCLC tumors, not only with regard to an acquired resistance to EGFR-TKI but also in EGFR-TKI-naïve tumors (NCT04676477).