Abstract PD8-06: Acquired resistance to tucatinib is associated with EGFR amplification in HER2+ breast cancer (BC) models and can be overcome by a more complete blockade of HER receptor layer
Authors: Jamunarani Veeraraghavan, Sreyashree Bose, Ragini Mistry, Pier Selenica, Sarmistha Nanda, Lanfang Qin, Andrea Gazzo, Yingjie Zhu, Michael A Mancini, Fabio Stossi, Britta Weigelt, Jorge S Reis-Filho, C. Kent Osborne, Mothaffar F Rimawi, Rachel Schiff
Journal: Cancer Research (2022)

Abstract Background: With recent approval of the irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (N) and the HER2-specific TKI tucatinib (T) in the advanced setting and their edging towards the early setting in HER2+ BC, resistance will likely emerge as a challenge, as illustrated by the HER2CLIMB study, where only one patient with brain metastasis remained progression free after 2 years on T. We set out to define the mechanisms of resistance to T and treatment strategies to overcome it. Materials and Methods: Our previously characterized HER2+ BT474 models with acquired resistance to lapatinib (L; LapR) or N (NrbR) (SABCS20-PD3-09), and our recently developed models of BT474 and SKBR3 with acquired resistance to T (TucaR) developed through long-term exposure to increasing doses of T (up to 200nM) and their naïve parental (P) were used. Genomic (DNA-seq), transcriptomic (RNA-seq), and proteomic (RPPA, western blot) characterization were performed. Drug efficacy studies involved cell growth assays by the imaging-based IncuCyte system. Results: We recently reported that while LapR is associated with acquisition of HER2 L755S mutation, which partially reactivates the HER pathway, NrbR is associated with the additional co-acquisition of a pathogenic PIK3CA mutation. Preliminary analysis of 2 BT474 TucaR models (ATCC and AZ) showed highly elevated levels of phosphorylated (p) and total (t) EGFR, suggesting EGFR signaling activation. Levels of pHER2, pHER3, and downstream pAKT and pS6 were also markedly higher in the TucaR models compared to P or short-term T. The TucaR but not LapR or NrbR models exhibited EGFR amplification, explaining the higher EGFR levels and signaling. Further, the elevated pEGFR, pHER2, pHER3, pAKT, and pS6 levels in TucaR models were substantially suppressed by the EGFR-specific TKI gefitinib (G) (50, 500nM) or even further when combined with T (500 nM G+200nM T). These results suggest that the higher pHER2 levels in TucaR models is probably due to heterodimerization of the amplified EGFR with HER2 and subsequent HER2 phosphorylation. In contrast to the P cells where the apoptotic marker cleaved (c)-PARP was not induced with G alone (50, 500nM), but with T (200nM) or 500nM G+T, in the TucaR model, 500nM G alone was enough to induce c-PARP, which was further enhanced when combined with T, implying the survival dependence of TucaR cells on EGFR signaling. The TucaR models were hypersensitive to G compared to P cells, and this growth inhibition was further enhanced with G+T. Whilst we previously reported that the LapR and NrbR cells were cross-resistant to T, the TucaR cells remained highly sensitive to the pan-HER TKIs N, poziotinib, and pyrotinib. Finally, in a second HER2+ model SKBR3, at 200nM TucaR, we observed elevated pEGFR, pHER2, pHER3, and pAKT levels, the underlying mechanism of which is under investigation by genomic and molecular analysis. In-depth characterization of our TucaR models to determine the differential gene expression and signatures is ongoing to gain additional mechanistic insights. Conclusions: Our findings suggest that whilst complete blockade of the HER layer using N is evaded by acquisition of HER and PIK3CA mutations, resistance to the HER2 TKI T is associated with EGFR amplification, a finding that underscores the HER signaling pathway redundancy and cross-talk between HER receptors to compensate for partial blockade of the pathway. Further, while resistance to L and N confers cross-resistance to T, resistance to T may be overcome using pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Together, our findings hold crucial implications in light of the current treatment landscape of HER2+ BC, with a particular emphasis on the considerations to strategize the treatment sequence of currently available TKIs. Citation Format: Jamunarani Veeraraghavan, Sreyashree Bose, Ragini Mistry, Pier Selenica, Sarmistha Nanda, Lanfang Qin, Andrea Gazzo, Yingjie Zhu, Michael A Mancini, Fabio Stossi, Britta Weigelt, Jorge S Reis-Filho, C. Kent Osborne, Mothaffar F Rimawi, Rachel Schiff. Acquired resistance to tucatinib is associated with EGFR amplification in HER2+ breast cancer (BC) models and can be overcome by a more complete blockade of HER receptor layer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD8-06.