HER2-positive/oestrogen receptor-positive (HER2+/ER+) represents a unique breast cancer subtype. The use of individual HER2- or ER-targeting agents can lead to the acquisition of therapeutic resistance due to compensatory receptor crosstalk. New drug combinations targeting HER2 and ER could improve outcomes for patients with HER2+/ER+ breast cancer. In this study, the pre-clinical rationale is explored for combining amcenestrant (Amc), a selective oestrogen receptor degrader (SERD), with HER2-targeted therapies including trastuzumab, trastuzumab-emtansine (T-DM1) and tyrosine kinase inhibitors (TKIs). The combination of Amc and anti-HER2 therapies was investigated in a panel of four HER2+/ER+ cell lines: BT-474, MDA-MB-361, EFM-192a and a trastuzumab-resistant variant BT-474-T. Proliferation (IC₅₀ and matrix combination assays) was determined using acid phosphatase assays. HER2/ER and intracellular signalling pathway protein levels/activity were investigated by western blot. Apoptosis was assessed using caspase 3/7 assays. Additivity and synergy were observed between Amc and the TKIs neratinib, lapatinib and tucatinib in all cell lines. Amc increased the anti-proliferative effect of trastuzumab in MDA-MB-361 and BT-474-T. Addition of Amc also increased anti-proliferative efficacy of T-DM1 in BT-474-T. TKI/Amc combinations reduced p-HER2 and ER levels and resulted in increased apoptosis. Higher ER expression in MDA-MB-361 and BT-474-T was associated with greater potential for synergy. In conclusion, the combination of Amc- and HER2-targeted treatments has potential as a therapeutic strategy for the treatment of HER2+/ER+ breast cancer and warrants further clinical investigation to validate safety and efficacy in patients.

Background: Targeting the human epidermal growth factor receptor (HER) family of tyrosine kinase receptors with small molecule TKIs lapatinib, neratinib and tucatinib, monoclonal antibodies like trastuzumab and more recently with ADCs like T-DM1 and T-DXd has resulted in improved survival rates for patients with HER2+ BC. TKIs are primarily utilised in trastuzumab-refractory disease. Neratinib, and the pan-HER TKI afatinib, bind irreversibly to all members of the HER family, while lapatinib binds reversibly to HER2 and EGFR, and tucatinib binds reversibly to HER2 alone. Previous work in our lab has shown that TKIs like lapatinib are capable of modulating tumour surface HER2 levels and increasing trastuzumab load on tumours. Thus, there is a rationale for the use of TKI/ADC combinations in HER2+ BC. Using innately trastuzumab-resistant HER2+ BC cell lines, this in vitro study aims to assess the anti-proliferative potential of HER2-targeting ADCs T-DM1 or T-DXd in combination with the TKIs afatinib, lapatinib, neratinib or tucatinib. Methods: HCC1569 and HCC1954 cells (HER2+, estrogen receptor (ER)-negative; innately trastuzumab-resistant) were grown in RPMI1640/10% FBS at 37°C and 5% CO2. The anti-proliferative effects of afatinib, lapatinib, neratinib and tucatinib; of T-DM1 and T-DXd; and of each TKI/ADC combination thereof were assessed in these cells via 5-day acid phosphatase. ADCs were obtained from Saint Vincent’s University Hospital, Dublin, and TKIs were purchased from commercial sources. Calcusyn software was used to generate IC50 values and combination index (CI) values at ED50. CI values > 1 represent an antagonistic combination, CI values = 1 are additive, and CI values < 1 are synergistic. All assays were carried out in triplicate. Results: The HCC1569 cell line was more sensitive to all four TKIs (IC50 values were 12.8 ± 0.3, 453.8 ± 47.1 nM, 4.7 ± 1.4 and 381.5 ± 37.3 nM for afatinib, lapatinib, neratinib and tucatinib respectively) compared to the HCC1954 cell line (IC50 22.3 ± 2.8, 652.4 ± 36.0, 57.0 ± 6.3 and 2365.1 ± 185.3 nM for afatinib, lapatinib, neratinib and tucatinib respectively). Despite their innate trastuzumab resistance, both cell lines displayed high sensitivity to T-DM1 (IC50 25.2 ± 8.7 ng/mL for HCC1569 and IC50 30.4 ± 3.2 ng/mL for HCC1954) and to T-DXd (IC50 16.0 ± 3.2 ng/mL for HCC1569 and IC50 36.8 ± 8.7 ng/mL for HCC1954). In both cell lines, co-treatment with ADCs and the irreversible TKIs afatinib and neratinib resulted in additive or synergistic responses, while the combination of the ADCs with the reversible TKIs lapatinib and tucatinib resulted in mild to moderate antagonism (Table 1). Conclusions: In this pre-clinical study, T-DM1 and T-DXd consistently exhibited antagonistic interactions with reversible HER2-targeting TKIs, and synergy/additivity in combination with the irreversible TKIs tested. Future work will explore the underlying mechanisms of this observed synergy and antagonism, including impacts of TKIs on HER2 expression and real-time ADC internalisation rates. Table 1. CI values +/- Std Dev for each TKI/ADC treatment combination in HCC1569 and HCC1954 cells Citation Format: Niall Ashfield, Amira F. Mahdi, Neil T. Conlon, John Crown, Denis M. Collins. Reversible versus irreversible tyrosine kinase inhibitors (TKIs) combined with antibody-drug conjugates (ADCs) in HER2-positive (HER2+) breast cancer (BC) cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-10.

Background: Up to 70% of HER2 over-expressing (HER2+) breast cancers also express estrogen receptor (ER). Dual targeting of HER2 and ER has shown pre-clinical and clinical benefit in HER2+/ER+ breast cancer. Rationale for these combinations is based on circumvention of resistance mediated by crosstalk between HER2 and ER signalling pathways. Fulvestrant is the only selective estrogen receptor degrader (SERD) approved by the FDA for the treatment of ER+ breast cancer. Development of alternative orally available SERDs with improved bioavailability, such as amcenestrant, is underway. The HER2-targeted tyrosine kinase inhibitors (TKIs) lapatinib, neratinib and tucatinib and the antibody drug conjugate T-DM1 are approved for the treatment of HER2+ breast cancer. This pre-clinical study aims to investigate the potential of combining the oral SERD amcenestrant with HER2-targeted therapies for the treatment of HER2+/ER+ breast cancer through identification of synergistic combinations. Methods: ER and HER2 expression levels were assessed in the HER2+/ER+ BT-474 and MDA-MB-361 cell lines by western blot. 5-day acid phosphatase-based assays were used to assess cell viability following treatment with neratinib, lapatinib, tucatinib, and T-DM1; alone and in combination with amcenestrant. All TKIs were commercially sourced, T-DM1 was obtained from Saint Vincent’s University Hospital. Amcenestrant was supplied by Sanofi. IC50 values were determined using Calcusyn, while matrix combination assays of the HER2-targeted therapies combined with amcenestrant were analysed using Combenefit software employing the Loewe model (LM) to determine synergy (Score > 0) or antagonism (Score < 0). All data are the result of a minimum of three independent experiments. Results: HER2 and ER expression was confirmed in the MDA-MB-361 and BT-474 cell lines. MDA-MB-361 cells express higher levels of ER and lower levels of HER2 and phosphorylated HER2 than BT474 cells. MDA-MB-361 exhibited a higher sensitivity to 1 µM amcenestrant alone (% cell viability = 53.3 ± 2.3 %) than the BT-474 (% viability = 85.3 ± 5%). A comparison of IC50 values for each TKI alone or in combination with 1 µM amcenestrant showed amcenestrant significantly lowered the IC50 values for neratinib and tucatinib in BT-474, and all three TKIs in MDA-MB-361 (Table 1). In matrix combination assays, all TKIs exhibited synergistic combinations, with the combination of neratinib and amcenestrant demonstrating the highest synergy values in MDA-MB-361 (LM max value = 25 ± 7) and in BT-474 (LM max value = 19 ± 7). The combination of amcenestrant and T-DM1 displayed additivity (LM value = 0) rather than synergy in the BT-474 cell line, likely due to the high potency of T-DM1 as a single agent. Conclusions: The combination of amcenestrant and HER2-targeting therapies such as neratinib shows potential as a dual-target therapeutic strategy for the treatment of HER2+/ER+ breast cancer and warrants further investigation. Citation Format: Amira F. Mahdi, Niall Ashfield, Neil T. Conlon, John Crown, Denis M. Collins. Pre-clinical study of amcenestrant and HER2-targeted therapies in HER2+/ER+ breast cancer cell line models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-16.