Abstract P4-01-01: Resistance to next generation tyrosine kinase inhibitors (TKIs) in HER2-positive breast cancer (BC): Role of HER and PIK3CA mutations and development of new treatment strategies and study models
Authors: Bose Sreyashree , Mistry Ragini , Liu Chia Chia , Nanda Sarmistha , Qin Lanfang , Selenica Pier , Gazzo Andrea , Zhu Yingjie , Mancini Michael A. , Stossi Fabio , Diala Irmina , Eli Lisa D. , Weigelt Britta , Reis-Filho Jorge S. , Rimawi Mothaffar F. , Osborne C. Kent , Schiff Rachel , Veeraraghavan Jamunarani 
Published: March 2022

Abstract
Background: We recently reported that acquired resistance to the dual HER1/2 TKI lapatinib (Lap) was mediated by HER2 L755S, while resistance to the pan-HER TKI neratinib (Nrb) was associated with co-acquisition of an additional pathogenic PIK3CA mutation. Though the role of HER2 mutations is gaining attention in HER2-positive (+) BC, less is known about their role and clinical implications in next generation TKI resistance, particularly when co-occurring with PIK3CA mutations in HER2+ BC. Investigating optimal treatment combinations and the development of new clinically relevant 3D models are warranted.Materials and Methods: HER2+ BT474 parental (P) cells and models with acquired resistance to Lap (LapR) and Nrb (NrbR) (SABCS20-PD3-09) were used. Xenografts established in mice using P, LapR, and NrbR cells and 3D organoids derived from these xenografts using the Hans Clevers (HC, PMID 29224780) or Mark Burkard (MB, PMID 31175091) method were characterized by qRT-PCR and western blot. Drug efficacy was assessed by growth changes in 2D and 3D models using the IncuCyte system or by microscopy-based analysis. Results: We previously showed that Lap and Nrb resistance confers cross-resistance to tucatinib (Tuca) and trastuzumab, and that targeting the HER and downstream PI3K pathway, especially using small molecule agents that are key for treatment of CNS lesions, is effective only in combination with Nrb or poziotinib (Pozio), but not Tuca. Our new studies revealed that the MEK inhibitor (i) AZD6244 (selumetinib; Sel), mTORi everolimus (Eve), and selective estrogen receptor degrader fulvestrant (Ful) were not effective as single agents in inhibiting the growth of either LapR or NrbR models. Whilst the LapR cells were highly sensitive to the irreversible HER1/2 TKI afatinib (Afa) and the irreversible dual/pan-HER TKI pyrotinib (Pyro) as single agents, the NrbR models were cross-resistant to both TKIs, highlighting the importance of the co-occurring PIK3CA mutation in resistance. Interestingly, Afa and Pyro were only partly effective when combined with Eve+Ful, Sel+Eve, or Sel+the PIK3CAi alpelisib in inhibiting NrbR growth. Consistent with our previously reported findings for Nrb and Pozio, Pyro was highly effective with TDM1. As opposed to the P xenografts, the LapR and NrbR tumors grew in the presence of the respective TKI, confirming their resistant phenotype in vivo. P and resistant xenograft-derived organoids (XDOs) were successfully established using the HC but not MB method, but the HC-derived XDOs were subsequently grown in MB condition and used for molecular and functional studies. Preliminary characterization showed that the LapR tumors and XDOs harbor HER2 L755S, whereas the NrbR tumors and XDOs also have a concomitant PIK3CA E542V mutation, findings that are in line with our 2D results, suggesting that the xenografts and XDOs retain and recapitulate the molecular profile of their 2D or tumor counterparts. Early drug efficacy studies indicate that, akin to the 2D models, the LapR XDOs are highly sensitive to Nrb, whereas both the LapR and NrbR XDOs exhibit cross-resistance to Tuca but remain sensitive to Pozio.Conclusions: Our data suggest that the potency of next generation irreversible HER TKIs in HER2+ BC may be challenged by the emergence of mutations in HER2, together with other co-occurring downstream mutations, such as PIK3CA. Our findings present a clear roadmap for the development of combinatorial therapies that should be individualized for patients with HER2+ BC. Our newly developed XDO strategy may offer a new platform to confirm and prioritize optimal drug combinations to overcome this resistance and may facilitate the near future development of patient-derived organoids for precision medicine of resistant HER2+ BC.
Citation Format: Sreyashree Bose, Ragini Mistry, Chia Chia Liu, Sarmistha Nanda, Lanfang Qin, Pier Selenica, Andrea Gazzo, Yingjie Zhu, Michael A. Mancini, Fabio Stossi, Irmina Diala, Lisa D. Eli, Britta Weigelt, Jorge S. Reis-Filho, Mothaffar F. Rimawi, C. Kent Osborne, Rachel Schiff, Jamunarani Veeraraghavan. Resistance to next generation tyrosine kinase inhibitors (TKIs) in HER2-positive breast cancer (BC): Role of HER and PIK3CA mutations and development of new treatment strategies and study models [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 P4-01-01.