HER2 Heterogeneous Breast Cancer Models Reveal Novel Therapeutic Targets and Subclonal Dynamics during Evolution to Resistance to HER2-Targeted Therapies

Intratumor heterogeneity for human epidermal growth factor receptor 2 (HER2) in HER2-positive breast cancer is a driver of resistance to HER2-targeted therapies. The advancement of treatments for HER2 heterogeneous (HET) tumors has been hindered by the lack of preclinical models that accurately mimic the human disease. In this study, we describe human HER2 HET breast cancer models composed of ERBB2-amplified (HER2hi) and nonamplified (HER2lo) cell populations derived from the same tumor. Utilizing these models, together with cellular barcoding, we demonstrate subclonal cooperation between HER2hi and HER2lo subpopulations. Furthermore, HER2lo cells drive resistance to HER2-targeting antibody-drug conjugates (ADC) like trastuzumab deruxtecan (T-DXd) but are sensitive to HER2 kinase inhibitors. CRISPR screens in HET cocultures identified sensitizers of HER2lo cells to T-DXd, including ATP-binding cassette subfamily C member 1 and ubiquitin-specific peptidase 9 X (USP9X). USP9X inhibition enhances the lysosomal targeting of HER2, thereby potentiating ADC payload release and reducing tumor recurrence after T-DXd treatment. Our results elucidate the functional relevance of HER2 heterogeneity and propose improved therapies for these tumors. SIGNIFICANCE: Studies of HER2 HET breast cancer models demonstrated that HER2lo cells drive HER2-targeting ADC resistance and accelerate recurrence by cooperating with HER2hi cells. We identified novel therapeutic strategies to sensitize HER2lo cells to T-DXd, providing mechanistic insight and offering promising avenues to overcome resistance and improve patient outcomes.