Abstract PS4-07: Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference
Authors: Lee Jangsoon , Pearson Troy , Liu Huey , Fuson Jon A. , Iwase Toshiaki , Diala Irmina , Lalani Alshad S. , Eli Lisa D. , Tripathy Debu , Lim Bora , Ueno Naoto T. 
Published: February 2021

Abstract
Background: Neratinib is a potent, irreversible pan-HER inhibitor that inhibits the ErbB family members EGFR, HER2, and HER4 and downstream signal transduction of these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks druggable levels of receptors for estrogen, progesterone and HER2 and therefore challenging to treat. There is evidence that some cases of TNBC have activated signaling pathways mediated by ErbB family members that may contribute to aggressive behavior. The purpose of this preclinical study was to identify and validate kinases whose targeting may enhance the antitumor activity of neratinib in TNBC cell lines. Methods: In vitro proliferation assays were used to evaluate the efficacy of neratinib in TNBC cell lines. Baseline and post-neratinib-treatment expression of EGFR and phosphorylated EGFR (phospho-EGFR) were assessed via Western blot analysis in 18 TNBC cell lines. Reverse-phase protein array (RPPA) was used to profile and validate the signaling networks induced by neratinib. To identify potential targets or pathways that may synergize with neratinib treatment, we performed high-throughput RNA interference (HT RNAi) screening using a 709-kinome library. CellTiter-Blue, sulforhodamine B, and soft-agar assays were performed to evaluate the antiproliferative effect of neratinib alone and with target inhibitor. Mammary fat pad xenograft models were used to evaluate the efficacy of neratinib alone or with inhibitor in vivo. Results: In vitro proliferation assays showed that the half-maximal inhibitory concentration (IC50) of neratinib in tested TNBC cell lines ranged from 0.16 µM to 1.25 µM. RPPA and Western blot analyses revealed that the efficacy of neratinib correlated with phospho-EGFR expression levels across the TNBC cell lines tested (R2 = 0.3245). Among the tested TNBC cell lines, SUM149 cells (PIK3CA wild-type) were selected for high throughput RNAi screening because this cell line has high EGFR expression and is moderately sensitive to neratinib (IC50 = 0.35 µM). We identified the 40 most relevant kinase targets by the sensitivity index analysis, and further pathway analysis identified PI3K/AKT/mTOR (drug: everolimus) and MAPK (drug: trametinib) as major canonical pathways whose targeting enhanced the cytotoxic effect of neratinib. Everolimus (mTOR inhibitor) produced a strong antiproliferative effect when combined with neratinib in most tested TNBC cell lines (12 of 15 cell lines; combination index [CI] values, 0.1-0.5) and was more effective in PIK3CA-mutated compared to wildtype cell lines. Trametinib (MEK inhibitor) showed a moderate antiproliferative effect (effective in 10 of 15 cell lines; CI values, 0.2-0.9). Synergistic antitumor effects of neratinib combined with everolimus or with trametinib were also observed in anchorage-independent growth conditions (P &lt; 0.05). In vivo experiments demonstrated that neratinib plus everolimus and neratinib plus trametinib combinations inhibited tumor growth in the SUM149 xenograft model for than single drug (neratinib, 42.3% growth inhibition; everolimus, 29.7%; trametinib, 47.1%; neratinib plus everolimus, 69.7%; neratinib plus trametinib, 77.7%; P &lt; 0.0001). Conclusion: Combining neratinib with everolimus or with trametinib enhanced the antitumor effects of these drugs in TNBC regardless of PIK3CA mutation status, and clinical investigations evaluating these combination regimens for the treatment of TNBC are warranted.
Citation Format: Jangsoon Lee, Troy Pearson, Huey Liu, Jon A. Fuson, Toshiaki Iwase, Irmina Diala, Alshad S. Lalani, Lisa D. Eli, Debu Tripathy, Bora Lim, Naoto T. Ueno. Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-07.