Abstract 1077: Acquired neratinib resistance is associated with acquisition of HER2 and PIK3CA mutations and can be overcome using potent drug combinations in HER2-positive breast cancer models
Authors: Veeraraghavan Jamunarani , Mistry Ragini , Nanda Sarmistha , Bose Sreyashree , Liu Chia Chia , Sethunath Vidyalakshmi , Shea Martin J. , Mitchell Tamika , Anurag Meenakshi , Mancini Michael A. , Diala Irmina , Lalani Alshad S. , Stossi Fabio , Osborne C. Kent , Rimawi Mothaffar F. , Schiff Rachel 
Published: July 2021

Abstract
The role of HER2 and PIK3CA mutations in anti-HER2 resistance is gaining more importance in HER2-positive (+) breast cancer. We recently reported that acquired resistance to lapatinib (Lap)-containing regimens is mediated by HER2 L755S, which could be overcome using the irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (Nrb). However, less is known about the role of L755S in resistance to next-generation TKIs, particularly when co-occurring with PIK3CA mutations. HER2+ BT474 cell models with primary or sequential acquired resistance (R) to Lap (LapR) or Nrb (NrbR) and their parental (P) counterparts were profiled for alterations in signaling and gene expression by RPPA, western blot, and RNA-seq. For drug efficacy studies, change in cell growth was assessed using imaging-based high-throughput system. Proteomic profiling revealed partial restoration of HER2 phosphorylation and downstream signaling in the LapR and NrbR derivatives. RNA-seq analysis showed that the LapR and NrbR models, but not P cells, harbor HER2 L755S mutation. Importantly, the NrbR but not LapR cells also co-acquire a PIK3CA pathogenic mutation. GSEA analysis of RNA-seq data showed significant downregulation of G2/M checkpoint in the R derivatives compared to P cells, suggesting genetic instability. In line with the presence of HER2 and PIK3CA activating mutations and HER pathway reactivation in the R models, GSEA revealed an enrichment of mTORC1 and KRAS signaling in the R cells. Furthermore, enrichment of epithelial mesenchymal transition signature and downregulation of apical surface genes was observed in the R models compared to P cells, suggestive of their aggressive phenotype. Interestingly, the LapR cells remained sensitive to Nrb, though a higher dose (IC50: ~50nM) was required compared to P cells (IC50: ~2nM). The LapR and NrbR cells were cross-resistant to the HER2-selective TKI tucatinib, and trastuzumab. We recently showed that the NrbR cells were either partially or completely sensitive to poziotinib or TDM1, respectively, suggesting their therapeutic promise against HER2- and PIK3CA-mutant tumors. Of note, our studies using small molecule agents targeting HER and its downstream pathway to facilitate treatment of CNS lesions suggest that AKT or mutant PIK3CA inhibitors are effective only when combined with either neratinib or poziotinib, but not tucatinib, findings which we are currently expanding to xenograft-derived organoids. Overall, our findings suggest a complex disease evolution upon resistance to neratinib but indicate their potentially continued efficacy in overcoming resistance through drug combinations. Ongoing integrative omics analysis to determine the genomic and mutational complexity and landscape will uncover additional mechanistic insights and guide the discovery of other actionable targets.
Citation Format: Jamunarani Veeraraghavan, Ragini Mistry, Sarmistha Nanda, Sreyashree Bose, Chia Chia Liu, Vidyalakshmi Sethunath, Martin J. Shea, Tamika Mitchell, Meenakshi Anurag, Michael A. Mancini, Irmina Diala, Alshad S. Lalani, Fabio Stossi, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. Acquired neratinib resistance is associated with acquisition of HER2 and PIK3CA mutations and can be overcome using potent drug combinations in HER2-positive breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1077.