Abstract B011: Modeling HER2 heterogeneous breast cancer uncovers therapeutic vulnerabilities and subclonal evolution driving resistance to HER2-targeted therapies

Abstract HER2 heterogenous tumors are defined by the presence of ERBB2 amplified and non-amplified cells accounting for up to 40% of HER2-positive breast cancer cases. HER2 heterogeneity is associated with poor prognosis and resistance to HER2-targeted therapies. Therefore, new therapeutic strategies tailored for HER2 heterogeneous tumors are needed. However, there are no model systems recapitulating HER2 heterogeneity suitable for functional studies and the design of effective treatment strategies. Thus, we established a unique preclinical HER2 heterogeneous breast cancer model composed of ERBB2 amplified (HER2hi) and non-amplified (HER2lo) cell populations derived from the same tumor. DNA barcoding experiments were used to study clonal evolution and resistance during treatment with the antibody-drug conjugate T-DXd and the small molecule inhibitor neratinib. Barcode composition analysis revealed a subclonal cooperation between HER2hi and HER2lo cells, suggesting a functional crosstalk that could lead to progression and resistance specifically in a heterogeneous setting. Consistently, HER2 heterogenous tumors in vivo relapse quicker after T-DXd treatment than HER2 homogenous tumors. As HER2lo cells mainly compose the residual tumors, we aimed at improving T-DXd efficacy in these cells. CRISPR screens in heterogeneous co-cultures uncovered novel sensitizers of HER2lo cells to T-DXd, including the USP9X deubiquitinating enzyme. Targeting USP9X enhances lysosomal trafficking of HER2 to increase ADC payload release, and reduces tumor recurrence after T-DXd treatment, offering a promising strategy to overcome resistance in HER2-heterogeneous breast cancer. In conclusion, our findings reveal that HER2lo cells drive resistance through cooperation with HER2hi cells and highlight novel strategies to sensitize HER2 heterogeneous tumors to T-DXd. These insights provide a mechanistic basis and promising therapeutic avenues to overcome resistance and improve outcomes for patients with HER2 heterogeneous breast cancer. Citation Format: Marie-Anne Goyette, Christopher Graser, Marco Seehawer, Andriana Patmanidis, Ernesto Rojas-Jimenez, Avni Kamat, Pierre Foidart, Zheqi Li, Piotr Sicinski, Franziska Michor, Kornelia Polyak. Modeling HER2 heterogeneous breast cancer uncovers therapeutic vulnerabilities and subclonal evolution driving resistance to HER2-targeted therapies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Cancer Evolution: The Dynamics of Progression and Persistence; 2025 Dec 4-6; Albuquerque, NM. Philadelphia (PA): AACR; Cancer Res 2025;85(23_Suppl):Abstract nr B011.