Abstract PO4-14-12: Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids
Authors: Liu Jie Bin , Tseng Danielle , Hooda Jagmohan , Brown Daniel , Lee Adrian , Oesterreich Steffi 
Published: May 2024

Abstract
Background: Activating mutations in ERBB2 (HER2) are enriched &gt;4-fold in invasive lobular carcinoma (ILC) with a rate of up to 19% in metastatic ILC. ILC is a histologic subtype of breast cancer characterized by loss of E-cadherin (CDH1), suggesting a potential interaction between loss of CDH1 and mutations in ERBB2. Recent trials have demonstrated promising single agent efficacy using the irreversible pan-HER tyrosine kinase inhibitor (TKI), neratinib, in patients with metastatic ERBB2 mutant ILC. However, further studies on combination therapies with other anticancer agents are needed to increase response rate and progression free survival for these patients. HER2-targeted antibody drug conjugates (ADC), particularly trastuzumab deruxtecan (T-DXd), have shown great promise in HER2-low metastatic breast cancer. Yet, their efficacy as a single agent or with HER2 TKIs in HER2-low and -mutant ILC is unknown and warrants investigation.
Methods: Past studies analyzing ERBB2 mutations used overexpression of ERBB2 mutant cDNA, but this approach does not faithfully recapitulate the human disease. To model ERBB2 missense mutations as found in human breast cancers, we used CRISPR-based prime editing to generate a panel of isogenic ILC cell lines and patient-derived organoids (PDO) harboring ERBB2 wild-type (WT) or ERBB2 mutations (S310F or V777L). Both of these ERBB2 mutations have been previously characterized and are known to be activating mutations. We then used them to test neratinib and other TKIs with ADCs, including T-DXd and trastuzumab emtansine (T-DM1).
Results: We successfully introduced single copy, heterozygous activating ERBB2 mutations (S310F or V777L) into two ERBB2-nonamplified metastatic ILC cell lines (MDA-MB-134 and SUM44PE) and one ERBB2-nonamplified metastatic ILC PDO (IPM-BO-053). Positive clones carrying the mutations were verified by Sanger sequencing and droplet digital PCR and subsequently pooled together. We further demonstrated that these mutations hyperactivated HER2 and downstream signaling pathways. ILC cell lines harboring these mutations showed enhanced sensitivity to HER2 TKIs but not ADCs. In contrary, ERBB2 mutations did not alter responses of IPM-BO-053 PDOs to HER2 TKIs but significantly increased responses to ADCs. Interestingly, we also observed accelerated HER2 protein degradation upon heregulin stimulation in ERBB2 mutant ILC PDOs, suggesting activating ERBB2 mutations may enhance ADC/HER2 complex internalization, degradation, and release of payloads. Lastly, we explored drug synergy between HER2 TKIs and ADCs in isogenic IPM-BO-053 PDOs and found that combination of T-DXd and neratinib or afatinib showed synergy (Combination Index &lt; 1).
Conclusions: Although the reason for the discrepancies in drug response between ILC cell lines and PDOs is not clear, we hypothesize that response in 3D PDOs might be more faithfully representing response seen in patients. We will generate additional ILC PDOs with knock-in ERBB2 mutations to validate our findings. Irreversible HER2 TKIs, such as neratinib and afatinib, showed synergy with T-DXd in ERBB2 mutant ILC PDOs. This holds important therapeutic implications in light of current treatment options for ILC. In future experiments, we will test if neratinib or afatinib increases endocytic uptake of T-DXd using a fluorescently labeled endocytosis tracker. Further, an in-depth molecular characterization of our isogenic cell lines and PDOs models is ongoing to gain mechanistic insights into how activating ERBB2 mutations increase HER2 internalization and degradation. Our in vitro studies provide a strong foundation for in vivo testing of neratinib or afatinib with T-DXd for ERBB2 mutant ILC.
Citation Format: Jie Bin Liu, Danielle Tseng, Jagmohan Hooda, Daniel Brown, Adrian Lee, Steffi Oesterreich. Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-12.