Abstract GS6-04: Co-occurring gain-of-function mutations in HER2 and HER3 modulate HER2/HER3 activation, breast cancer progression, and HER2 inhibitor sensitivity
Authors: Hanker Ariella B. , Jayanthan Harikrishna Sekar , Ye Dan , Lin Chang-Ching , Akamatsu Hiroaki , Sheehan Jonathan H. , Koch James P. , Sudhan Dhivya R. , Brewer Monica Red , Servetto Alberto , He Jie , Miller Vincent A. , Lalani Alshad S. , Meiler Jens , Arteaga Carlos L. 
Published: March 2020

Abstract
Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast cancers. The HER2 tyrosine kinase inhibitor (TKI) neratinib has shown clinical activity against cancers harboring HER2 activating mutations. Co-occurring HER2 and HER3 (ERBB3) mutations have been reported to co-occur in patients with breast cancer, suggesting the possibility of cooperativity of both oncogenes.
Interrogation of the Project GENIE database revealed that gain-of-function missense mutations in HER2 and HER3 exhibit statistically significant co-occurence [q value=0.006 (breast cancer); q=1.01x10−26 (all cancer types)]. On the other hand, HER3 mutations were nearly absent in cancers harboring HER2 in-frame insertions and cancers with HER2 amplification. In breast cancer, co-occurring HER3 mutations were mutually exclusive with PIK3CA mutations, suggestive of potential functional redundancy.
Sixty-eight unique breast cancers from cBioPortal, GENIE, and Foundation Medicine were found to harbor co-occurring mutations in HER2 and HER3, the most common of which were L755S, V777L, L869R/Q, and S310F, each with HER3E928G. Computational structural modeling suggested that the E928G substitution in HER3 enhances the interface energy between HER2 and HER3 kinase domains. This was confirmed by co-immunoprecipitation assays in HEK293 cells transfected with wild-type (WT) or mutant HER2 and HER3. This binding was further enhanced between HER3E928G and HER2 missense mutants. Further, structural modeling of L755S, V777L, and L869R/Q in HER2 revealed that each of these mutations increase HER2 kinase activation. Accordingly, each of these HER2 mutants, as well has HER2S310F in the extracellular domain, increased ligand-independent P-HER2 and P-HER3 in HEK293 cells. P-HER3 levels were the highest HEK293 in cells co-expressing HER2 and HER3 mutations. Similar results were observed in MCF10A breast epithelial cells stably expressing mutant HER2 and mutant HER3. Enhanced activation of the PI3K pathway (P-AKT and P-S6) was observed in cells expressing both mutations compared to cells expressing mutant HER2 with HER3WT or vice-versa.
MCF10A HER2WT/HER3E982G cells did not form colonies in ligand-free 3D Matrigel assays, whereas cells expressing HER2L755S/HER3WT formed organized acini. In contrast, HER2L755S/HER3E928G acini were highly invasive. Addition of the HER3 ligand neuregulin (NRG) to HER2L755S/HER3WT cells phenocopied the effect of HER3E928G. Interestingly, MCF10A cells expressing the HER2Y772_A775 (YVMA) insertion together with HER3WT also formed invasive acini in the absence of NRG, suggesting that this HER2 insertion mutation is more transforming than HER2 missense mutations. Similar results were obtained with invasion assays using Matrigel-coated chambers.
Finally, we examined the effects of HER2/HER3 co-mutations on sensitivity to HER2 inhibitors. Structural analysis was performed of the HER3/HER2 kinase domain complex docked with neratinib. HER3E928G, when bound to HER2, is located close to the neratinib-binding pocket of HER2 and was predicted to reduce HER2/neratinib binding affinity. Similarly, co-expression of HER3E928G reduced the ability of neratinib to inhibit P-HER3, P-AKT, P-S6, and growth of MCF10A cells expressing HER2/HER3 co-mutations. The PI3Kα inhibitor alpelisib restored neratinib sensitivity.
Conclusions: Co-expression of mutant HER2 and mutant HER3 promotes ligand-independent HER2/HER association, HER3 phosphorylation, and cancer cell invasion via enhanced activation of the PI3K pathway; this enhanced signaling output is incompletely blocked by neratinib. Therefore, breast cancers expressing co-occurring HER2 and HER3 mutations may require the addition of a PI3Kα inhibitor to a HER2 TKI.
Citation Format: Ariella B. Hanker, Harikrishna Sekar Jayanthan, Dan Ye, Chang-Ching Lin, Hiroaki Akamatsu, Jonathan H. Sheehan, James P. Koch, Dhivya R. Sudhan, Monica Red Brewer, Alberto Servetto, Jie He, Vincent A. Miller, Alshad S. Lalani, Jens Meiler, Carlos L. Arteaga. Co-occurring gain-of-function mutations in HER2 and HER3 modulate HER2/HER3 activation, breast cancer progression, and HER2 inhibitor sensitivity [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS6-04.