Abstract 4038: Exploring optimal targeted combination therapies with neratinib for HER2+ breast cancer
Authors: Zhao Ming , Scott Stephen , Evans Kurt , Yuca Erkan , Murthy Rashmi , Avogadri-Connors Francesca , Cutler Richard , Lalani Alshad S. , Piha-Paul Sarina , Meric-Bernstam Funda 
Published: July 2017

Abstract
Activation by amplification or overexpression of the proto-oncogene HER2 (also known as ERBB2) is associated with the development and progression of breast cancer. Neratinib is a novel, irreversible, pan-HER tyrosine kinase inhibitor which selectively inhibits EGFR, HER2 and HER4. In this preclinical study, we explored the efficacy of neratinib in combination with other clinically relevant targeted agents for optimal treatment of HER2-positive breast cancer in in vitro and in vivo models. Western blot (WB) analysis of a panel of breast cancer cell lines showed that BT-474, SKBR-3, and HCC-1954 cells displayed elevated HER2 expression levels (here denoted as HER2+), while MDA-MB-361, MDA-MB-453, and CAMA-1 breast cancer cells expressed relatively lower HER2 expression levels. Interestingly, neratinib inhibition positively correlated with HER2 expression levels as assessed by cell survival assays. The IC50 of neratinib in the HER2+ cell lines was in the 3.6 - 113nM range. Consistent with the in vitro data, treatment of a HER2+ breast cancer PDX model with neratinib single agent (p.o. 5 days/week) led to a significant dose-dependent tumor growth inhibition with tumor regressions observed at a low dose of 10 mg/kg (p&lt;0.0001). The activity of neratinib in combination with other targeted agents was then evaluated in vitro in the HER2+ breast tumor cell lines using a Chou-Talalay method. Neratinib showed synergistic anti-proliferative activity in combination with the PI3Kα inhibitor BYL719 in all the HER2+ cell lines tested. By contrast, synergy with mTOR inhibitors (everolimus and TAK228) and with the MEK inhibitor trametinib was only observed in BT-474 and HCC-1954 cells. HCC-1954 cells had the best response to neratinib combinations among all HER2+ cell lines tested, with combination index values &lt;0.5. Immunoblot analysis demonstrated that neratinib treatment displayed complete inhibition of HER2 phosphorylation in these cells. Substantial inhibition of downstream signaling pathways following single agent neratinib treatment was evident by reduced phosphorylation levels of AKT, S6K1, S6, 4EBP1, ERK1/2 and MEK1/2. Consistently with the cell viability data, the combination of neratinib with PI3K/Akt/mTOR and MAPK inhibitors showed a synergy on signaling blockage in these two downstream pathways respectively. Taken together this data shows that neratinib has efficacy as single agent in preclinical models of HER2+ breast tumors and its anti-proliferative activity in vitro was enhanced when combined with PI3K/Akt/mTOR and MAPK pathway inhibitors. Further studies of neratinib-based combination treatments are underway in HER2+ breast cancer xenograft and PDX models to further corroborate these findings.
Citation Format: Ming Zhao, Stephen Scott, Kurt Evans, Erkan Yuca, Rashmi Murthy, Francesca Avogadri-Connors, Richard Cutler, Alshad S. Lalani, Sarina Piha-Paul, Funda Meric-Bernstam. Exploring optimal targeted combination therapies with neratinib for HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4038. doi:10.1158/1538-7445.AM2017-4038