Abstract 1828: Hyperactivation of mTORC1 drives acquired resistance to the pan HER tyrosine kinase inhibitor neratinib in HER2 mutant cancers

Abstract

Abstract Background: Tumor genomic profiling has identified patients with cancers harboring activating ERBB2 (HER2) mutations that are sensitive to HER2 targeted therapies. In the SUMMIT phase II ‘basket' trial, a subset of patients with ERBB2 mutant cancers have exhibited significant clinical benefit from treatment with the pan-HER irreversible tyrosine kinase inhibitor (TKI) neratinib. However, durable responses to neratinib are few, suggesting mechanisms of de novo and acquired drug resistance. Thus, we sought to identify druggable mechanisms of resistance to neratinib. Methods: We utilized 5637 bladder cancer (with HER2S310F) and OVCAR8 ovarian cancer (with HER2G776V) cells. Drug resistant cells were developed by exposing cells to increasing concentrations of neratinib over 6 months (5637, 600 nM; OVCAR8, 2 µM). Neratinib resistant H1781 lung cancer cells (with HER2G776>VC) and MCF7 breast cancer cells (with L755S or V777L) knock-in mutations are currently being developed. For immunoblot and drug sensitivity assays, neratinib resistant cells were maintained drug-free for 96 hours and then retreated with neratinib and other inhibitors. Candidate pathways/genes driving neratinib resistance were identified by performing RNA sequencing and whole exome sequencing in drug-resistant and -sensitive cells. Results: Neratinib-resistant 5637 and OVCAR8 cells were cross-resistant to the HER2 TKIs afatinib and lapatinib. Immunoblot analysis of both cells treated with neratinib showed effective suppression of HER2, EGFR and HER3 phosphorylation. However, they exhibited a striking increase in S6 kinase (S6K) activity and S6 phosphorylation compared to drug-sensitive parental cells, which was maintained in the presence of supra-pharmacological levels of neratinib (1 µM). S6 phosphorylation and viability of drug resistant cells was completely ablated by the combination of neratinib and the mTORC1 inhibitor everolimus, but not with the PI3Kα inhibitor alpelisib, the pan-PI3K inhibitor buparlisib, or the AKT inhibitor MK-2206, suggesting PI3K- and AKT-independent activation of mTORC1. Gene set enrichment analysis (GSEA) of RNA seq data from the drug-resistant cells revealed significant enrichment of K-Ras pathway components in addition to mTORC1 pathway. Consistent with these results, whole exome sequencing revealed activating alterations of the Ras pathway including a truncating mutation in RASA2 and a P200L mutation in PIK3CA Ras binding domain; thereby suggesting potential Ras mediated mTOR activation driving neratinib resistance. Studies are underway to confirm the contribution of Ras pathway in mTOR mediated neratinib resistance. Conclusions: These data suggest that hyperactivation of mTORC1 promotes acquired resistance to neratinib across histologically distinct ERBB2-mutant cancers. Citation Format: Dhivya R. Sudhan, Ariella B. Hanker, Angel Guerrero-Zotano, Luigi Formisano, Yan Guo, Qi Liu, Francesca Avogadri-Connors, Richard E. Cutler, Alshad S. Lalani, Richard Bryce, Alan Auerbach, Carlos L. Arteaga. Hyperactivation of mTORC1 drives acquired resistance to the pan HER tyrosine kinase inhibitor neratinib in HER2 mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1828.