bioRxiv2026
Activating HER2 mutations are significantly enriched in both primary and metastatic invasive lobular breast cancer (ILC), with large public datasets of primary breast tumors linking them to a worse prognosis in ILC. Despite their oncogenic role, no FDA-approved therapies currently target HER2-mutant breast cancers. While the HER2-directed antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd) has shown efficacy in HER2-mutant non-small cell lung cancer, its activity in HER2-mutant ILC remains unknown. Using the Caris real-world database, one of the largest cohorts with survival data in advanced breast cancers, we confirmed that HER2 mutations are more prevalent in advanced ILC than in invasive breast cancer of no special type (NST) tumors, are associated with worse survival in both histologies, yet predict improved response to T-DXd across subtypes, highlighting the need for mutation-directed, histology-informed therapies. Using endogenous HER2-mutant ILC cell lines (UACC3133-S310F, BCK4-L755S) and CRISPR-engineered isogenic ILC models with clinically relevant HER2 mutations (S310F, V777L), we found these mutations drive HER2/HER3 hyperactivation and downstream signaling, conferring increased sensitivity to HER2 tyrosine kinase inhibitors (TKIs) and T-DXd. Mechanistically, HER2 mutants showed enhanced receptor ubiquitination, internalization, and lysosomal degradation upon T-DXd treatment, explaining the observed drug sensitivity. While combining T-DXd with neratinib or the HSP90 inhibitor ganetespib yielded synergistic effects in long-term growth assays, accompanied by increased HER2 ubiquitination, the concurrent hyperactivation of HER3 in HER2-mutant cells suggested that co-targeting HER3 could provide an effective alternative strategy. Accordingly, HER2-mutant ILC exhibited enhanced sensitivity to the HER3-directed ADC patritumab deruxtecan (P-DXd) or LJM716, a HER3-targeting antibody. We further uncovered a previously unrecognized mechanism of P-DXd beyond HER3 ligand blockade and payload delivery: P-DXd promotes HER2/HER3 association, increases HER2 ubiquitination, and enhances T-DXd internalization, resulting in potent synergy with T-DXd. Mechanistically, we identified HER3 extracellular domains I and II as essential for P-DXd binding and for mediating P-DXd-induced HER2/HER3 association, establishing a structural basis for this activity. In vivo, both T-DXd and P-DXd suppressed UACC3133 and BCK4 xenograft growth, with combination therapy trending toward greater efficacy and prevented regrowth of tumors. Extending these findings beyond HER2-mutant ILC, combination treatment with T-DXd and P-DXd demonstrated synergistic activity across multiple breast cancer models, including (i) HER2-amplified NST patient-derived organoids (PDOs) harboring hotspot HER2 mutations, (ii) HER2-wild-type NST PDOs with clinically intrinsic or acquired T-DXd resistance, and (iii) isogenic HER2-mutant ILC PDOs with experimentally induced resistance after prolonged T-DXd exposure. Collectively, these findings support HER2 as an actionable target in HER2-mutant ILC and position T-DXd-based regimens, particularly in combination with HER3 inhibition, as a promising therapeutic strategy for this underserved patient population.