Abstract P6-12-09: Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in HER2+ breast cancer (BC) cell lines
Authors: O’Reilly Debbie , Eli Lisa D. , Wong Alvin , Crown John , Collins Denis M. 
Published: March 2023

Introduction: NER is a pan-HER tyrosine kinase inhibitor (TKI) approved for the treatment of HER2+ BC in the adjuvant setting following trastuzumab and in combination with capecitabine for advanced disease. Resistance to small molecule TKIs like NER can develop in the clinic. Pre-clinical studies have highlighted that retinoic acid can inhibit BC growth and modulate HER2 signalling pathways. The RAR family of nuclear transcription factors consists of RARα, RARβ and RARγ. The synthetic retinoic acid fenretinide (FN) acts as a pan-RAR agonist, while AGN194310 (AG) acts as a pan-RAR antagonist. In order to investigate the anti-proliferative potential of co-targeting RAR and HER2 pathways in sensitive and resistant BC cell line models, we examined the effect of FN and AG in combination with NER in two HER2+, estrogen receptor-negative, trastuzumab-resistant cell lines HCC1569 and HCC1954, and their NER-resistant (NR) sub-lines HCC1569-NR and HCC1954-NR. Methods: HCC1569 and HCC1954 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. NR cell lines were generated by continuous exposure to 150nM NER for 6 months. 10 mM stock solutions of FN (H7779-Sigma), AG (SML2665-Sigma) and NER (supplied by Puma Biotechnology, Inc) were made in DMSO. Proliferation was measured as percentage growth versus DMSO control using an acid phosphatase based assay after 5 days drug exposure. The half-maximal inhibitory concentration (IC50) was calculated for each drug using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50) using CalcuSyn. Values &lt; 1 represent a synergistic effect, a value of 1 is additive and values &gt; 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research found that the NR cell lines were &gt;10-fold resistant to NER (HCC1569-NR IC50 0.44 ± 0.1 μM, HCC1954-NR IC50 0.198 ± 0.019 μM) compared to the parental HCC1569 (IC50 0.018 ± 0.015 μM) and HCC1954 (IC50 0.017 ± 0.001 μM) cell lines. Pan-RAR agonism by FN had a potent anti-proliferative effect in the HCC1569 (FN IC50 0.22 ± 0.02 μM) and the HCC1569-NR cell lines (FN IC50 0.28 ± 0.13 μM), with the HCC1954 and HCC1954-NR cell lines proving less sensitive (IC50 6.47 ± 1.3 μM and 1.9 ± 0.2 μM, respectively). When combined with NER, FN produced a strong antagonistic effect in the HCC1569 cell line (CI value: 15.63 ± 9.5) and a strong synergistic effect in the HCC1954 cell line (CI value: 0.42 ± 0.06). For the NR cell line models, the NER/FN combination proved synergistic (HCC1569-NR, CI value: 0.84 ± 0.46) or additive (HCC1954-NR, CI value: 0.97 ± 0.15). Next, we wanted to assess the impact of antagonising rather than activating RAR activity in our cell line models. All four cell lines were less sensitive to antagonist AG (IC50 &gt;8μM for all cell lines) compared to FN. The addition of AG to NER resulted in responses diametrically opposed to the FN/NER combination. The AG/NER combination produced a strong synergistic effect in the HCC1569 cell line (CI value: 0.52 ± 0.17), an antagonistic effect in the HCC1954 cell line (CI value: 2.1 ± 0.4) and an antagonistic effect in both NR cell lines (HCC1954-NR, CI value: 2.69 ± 0.6 and HCC1569-NR, CI value: 1.58 ± 0.12). Conclusions: This pre-clinical study suggests involvement of the RAR signalling pathway in response to NER and the development of NR. Results also suggest pan-RAR agonism, rather than pan-RAR antagonism, as a potential therapeutic strategy to overcome NR. Further investigation is warranted to determine how targeting the RAR signalling pathway may assist in the treatment of HER2+ BC.

Citation Format: Debbie O’Reilly, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in 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 P6-12-09.