Abstract P6-12-06: Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study
Authors: Koirala Nischal , Aske Jennifer , Lin Xiaoqian , Dey Nandini , De Pradip 
Published: March 2023

Abstract
Background: Despite the wide-ranging clinical success of human epidermal growth factor receptor 2 (HER2)-directed therapies, many HER2-positive breast cancer patients eventually progress because of the development of primary or acquired resistance. PIK3CA is found often mutated in breast cancer (&gt;30%, TCGA dataset) and responsible for HER2-directed treatment failure. Purpose: Inhibition of pan-tyrosine kinases of HER-receptors along with the blockade of estrogen receptor (ER) prevents the activation of downstream effectors and crosstalk between HER2 and ER, leading to tumor cell death. Similarly, the combined inhibition of HER2-receptor signaling with cell cycle arrest leads to efficient tumor cell apoptosis. Here, we evaluate the mechanistic efficacy and comparability of neratinib (N) (an irreversible pan-ERBB tyrosine kinase inhibitor) in combination with fulvestrant (F) against ribociclib (R) plus trastuzumab (T) in HER2-amplified breast cancer cells. Methods: HER2+ breast cancer cell lines with Rb- wild-type- BT474 (ER+), SKBR3 (ER-), and MDA-MB453 (ER-, PIK3CA mutated [H1047R]) were used for the study. Cells were treated with N+F (F added in ER+ cell line only) or R or T as a single-agent or combination and assessed for real-time proliferation, 3D ON-TOP assay, changes in mitochondrial potential, and apoptosis. Cells treated with R and/or T were additionally examined for cell cycle arrest and changes in CDK4 mRNA transcription. Baseline CCND1 mRNA transcripts were quantified by RT-qPCR. Immunohistochemistry was used to assess baseline BCL2 expression in HER2+ tumor microarrays (TMA). Western blot was used to evaluate the effects on key downstream signaling proteins in response to the above treatment or combination. Results: High CCND1 expression was found in HER2+ cell lines that show promise for CDK4/6 inhibition. High BCL2 expression was found in HER2+ TMA that confirmed the natural resistance to programmed cell death. BT474 and MDA-MB453 were highly sensitive to N (+F), and high growth inhibition was evident at lower doses (&lt; 20 nM). SKBR3 was comparatively less sensitive to N monotherapy and required dosing &gt;160 nM to induce marked cell death. BT474 and MDA-MB453 had pronounced cytostatic responses to R or T+R, while a moderate response was observed in SKBR3. Substantial reduction in CDK4 transcription was noted following R or R+T treatment in MDA-MB453/BT474 but not in SKBR3. Apoptosis assays confirmed enhanced cell death with the R+T combination in BT474 and MDA-MB453 but not in SKBR3. Inhibition of key downstream oncogenic signaling (p-AKT/p-S6RP/p-ERK) was more evident with N compared to R, T, or R+T combination. Attenuated p-Rb expression (Ser 780 & Ser 807/811) was detected in all cell lines from R administration, indicating interruption in cell cycle progression. Conclusion: Mechanistically, N+F was superior to T+R in terms of inhibition of HER2 and its downstream signaling in the HER2+/ER+ BC model. N+F induced significantly more apoptosis and inhibited cell proliferation compared to T+R. Additionally, N monotherapy was highly effective in HER2-amplified/ER-negative breast cancer cells with PIK3CA mutation.
Citation Format: Nischal Koirala, Jennifer Aske, Xiaoqian Lin, Nandini Dey, Pradip De. Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study [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 P6-12-06.