HER2-positive breast cancer accounts for 15–20% of all breast cancer cases. Second-generation antibody-drug conjugates (ADC) trastuzumab deruxtecan (T-DXd; Enhertu®) and trastuzumab duocarmazine (SYD985) have shown effectiveness in HER2-positive and HER2-low metastatic breast cancer. However, not all patients respond due to intrinsic or acquired resistance, as observed with other HER2-targeting therapies. Efforts to understand the mechanisms involved in resistance are essential for identifying strategies to overcome it.

Cas9 expressing HER2-positive HCC1954 and MDA-MB-361 cells were transduced with the MinLibCas9 single guide RNA (sgRNA) library and exposed to T-DXd and a SYD985 biosimilar as well as their respective cytotoxic payloads (DXd, a topoisomerase 1 inhibitor and DUBA, a DNA alkylator) for 30–45 days. High-throughput sequencing identified gene knockouts enriched or depleted in response to treatment. BT-474, HCC1954, and MDA-MB-361 cells were continuously cultured in T-DXd for 30–45 days to establish resistant models. RNA was extracted and subjected to RNA sequencing (RNASeq) to identify transcriptional changes associated with resistance. Antiproliferative activity of treatments was evaluated by thymidine incorporation assay.

Whole-genome CRISPR/Cas9 screens revealed a high correlation of gene hits between the ADCs and their payload, particularly for T-DXd and DXd. Gene hits showed limited overlap between T-DXd and the SYD985 biosimilar, suggesting distinct resistance mechanisms for different ADCs. The top 500 candidate genes were identified for further validation using a focused sgRNA library. T-DXd resistance emerged after continuous culture in all three cell lines. HER2 expression was retained in BT-474 and MDA-MB-361 but decreased in HCC1954 cells. BT-474 and MDA-MB-361 resistant cells retained sensitivity to the ADC trastuzumab emtansine and the tyrosine kinase inhibitors neratinib and lapatinib. All resistant models also exhibited cross-resistance to the DXd payload. RNASeq analysis revealed upregulation of interferon-alpha and interferon-gamma signalling pathways across all resistant cell lines, while RET emerged as the most significantly downregulated gene. Combination therapy with T-DXd and the RET inhibitor selpercatinib demonstrated synergy in all three treatment-naïve cell lines.

Our study provides insight into the molecular basis of ADC resistance, identifying potential targets to overcome resistance and enhance ADC efficacy in HER2-positive breast cancer. The identification of RET downregulation in resistant cells and the observed synergy between T-DXd and selpercatinib highlight a potential strategy for limiting T-DXd resistance.