Abstract
<jats:p><div>Abstract <p>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 <i>ERBB2</i>-amplified (HER2<sup>hi</sup>) and nonamplified (HER2<sup>lo</sup>) cell populations derived from the same tumor. Utilizing these models, together with cellular barcoding, we demonstrate subclonal cooperation between HER2<sup>hi</sup> and HER2<sup>lo</sup> subpopulations. Furthermore, HER2<sup>lo</sup> 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 HER2<sup>lo</sup> 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.</p> Significance:<p>Studies of HER2 HET breast cancer models demonstrated that HER2<sup>lo</sup> cells drive HER2-targeting ADC resistance and accelerate recurrence by cooperating with HER2<sup>hi</sup> cells. We identified novel therapeutic strategies to sensitize HER2<sup>lo</sup> cells to T-DXd, providing mechanistic insight and offering promising avenues to overcome resistance and improve patient outcomes.</p></div></jats:p>