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ERBB2, the gene encoding HER2, is mutated in 2-4% of breast cancers. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against breast cancers harboring HER2 activating mutations, suggesting these tumors depend on HER2 signaling. Co-occurring HER2 and HER3 (ERBB3) mutations have been reported in patients who respond to neratinib (Hanker et al., Cancer Discov. 2017) suggesting the possibility of cooperativity of both oncogenes. Co-expression of the mutant intracellular domains of HER2 and HER3 in HEK293 cells enhanced phosphorylation of HER3 and ERK compared to expression of either mutant alone, which was blocked by 100 nM neratinib. Interrogation of TCGA, METABRIC, Project GENIE, and Foundation Medicine datasets revealed that gain-of-function mutations in ERBB2 and ERBB3 co-occur with a statistically significant frequency. For example, in GENIE, ERBB2 mutations co-occur with mutations in ERBB3 (8.3% of ERBB2-mutant vs 2.3% of ERBB2 WT; q=1.37x10-10). We hypothesized that co-occurring mutations in HER2 and HER3 cooperate to enhance HER2 signaling and dependence and breast cancer progression. Thirty-four unique breast cancers were found to harbor co-occurring mutations in HER2 and HER3, the most common of which were ERBB2L755S/ERBB3E928G (n=10), ERBB2V777L/ERBB3E928G(n=6), and ERBB2L869R/Q/ERBB3E928G (n=4). Using co-immunoprecipitation assays with HER2 and HER3 antibodies in transfected HEK293 cells, we found that co-expression of HER3E928G with wild type (WT) HER2, or co-expression of HER2L755S or HER2L869R with HER3WT, slightly increased HER2-HER3 dimerization. However, binding was strongest between double mutants. This was accompanied by the highest levels of Y1289 p-HER3 in cells expressing both HER3E928G and each HER2L755S, HER2V777L, or HER2L869R compared to cells expressing each HER2 or HER3 mutant with a respective WT heterodimer partner. Structural modeling of the HER2L869R/HER3E928G double-mutant predicted that the HER3 mutation, located at the dimer interface, may enhance heterodimerization of the kinase domains through decreased bulk and electrostatic repulsion. We also noted that the HER2L755S mutation is predicted to be in close proximity to HER3E928G ( MCF7 “knock-in” cells incorporating HER2L755S, HER2V777L, or HER2L869R (or HER2WT) were stably transduced with HER3E928G or HER3WT. Co-expression of double mutants strongly enhanced estrogen-independent growth in 3D Matrigel over cells expressing either mutant alone. We are currently testing inhibitors of HER2/HER3 signaling, including neratinib ± trastuzumab, trastuzumab + pertuzumab, and the ERBB1-3 antibody mixture Sym013, to determine therapeutic strategies to block the cooperative growth induced by co-occurring HER2 and HER2 mutations. Conclusions: Co-expression of mutant HER2 and mutant HER3 promotes HER2/HER binding, HER3 phosphorylation, and breast tumor cell proliferation. We aim to identify therapeutic vulnerabilities for patients with co-occurring HER2 and HER3 mutations. Citation Format: Hanker AB, Koch JP, Ye D, Sliwoski G, Sheehan J, Kinch LN, Red Brewer M, He J, Miller VA, Lalani AS, Cutler, Jr. RE, Croessmann S, Zabransky DJ, Meiler J, Arteaga CL. Co-occurring gain-of-function mutations in HER2 and HER3 cooperate to enhance HER2/HER3 binding, HER-dependent signaling, and breast cancer growth [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD3-05.

ERBB2, the gene encoding HER2, is mutated in 2-4% of breast cancers. The HER2 irreversible tyrosine kinase inhibitor (TKI) neratinib has shown clinical activity against breast cancer cells harboring HER2 activating mutations. Here, we report for the first time an acquired gatekeeper HER2T798I mutation in a patient with HER2-mutant breast cancer after an initial exceptional response to neratinib. A patient with ER+/PR+/HER2-negative invasive lobular breast cancer progressing on standard therapy was found to harbor a L869R kinase domain mutation in HER2. HER2L869R is homologous to the known activating mutation EGFRL861R/Q. MCF10A breast epithelial cells expressing HER2L869R displayed enhanced HER2-mediated signaling and were resistant to lapatinib and trastuzumab but sensitive to neratinib. The patient was enrolled in the phase II SUMMIT trial (NCT01953926) and treated with neratinib, achieving a partial response lasting 16 months before developing progression. Next gen sequencing of DNA from both a new skin metastasis and plasma cell-free DNA (cfDNA) identified HER2L869R (8.7% cfDNA), whereas a novel HER2T798I mutation was detected only in plasma at 1.3%. Deep sequencing of pre-therapy tumor tissue and plasma did not detect HER2T798I, suggesting that this mutation arose upon resistance. HER2T798I has not been reported in TCGA, COSMIC, or among plasma samples from 17,345 cancer patients subjected to digital DNA sequencing using the Guardant360 assay. HER2T798I is homologous to the EGFRT790M, KITT670I and BCR-ABLT315I gatekeeper mutations known to mediate resistance to erlotinib/gefitinib and imatinib. To examine if HER2T798I mediates resistance to neratinib, we employed biochemical and biological assays and molecular modeling of wild-type (WT) HER2 and HER2T798I. Structural modeling showed the increased bulk of the isoleucine at position 798 would result in a steric clash with neratinib, thus reducing drug binding. We stably expressed HER2WT, HER2T798I, HER2L869R and HER2L869R/T798I in MCF10A cells and NR6 mouse fibroblasts. Neratinib (10-100 nM) blocked HER2-mediated signaling in cells expressing HER2WT or HER2L869R but did not in cells expressing HER2T798I. The EGFR irreversible TKI osimertinib (100 nM), which isselective for mutant EGFR (including EGFRT790M) and approved for treatment of NSCLC expressing EGFRT790M, failed to inhibit HER2WT, HER2L869R or HER2T798I. In contrast, either the EGFR/HER2 irreversible TKI afatinib or AZ5104, a metabolite of osimertinib, strongly blocked signaling induced by HER2WT, HER2L869R or HER2T798I. Cells expressing HER2T798M displayed a significantly higher IC50 to neratinib than cells expressing HER2WT, whereas afatinib or AZ5014 were very active against all cells (IC50 Conclusions: The acquisition of a T798I gatekeeper mutation in HER2 upon development of clinical resistance to neratinib in a breast cancer with an initial activating mutation in HER2 strongly suggests that HER2L869R is a driver mutation. We speculate that HER2T798I may arise as a secondary mutation following response to effective HER2 TKIs in other cancers with HER2 activating mutations. Certain irreversible EGFR inhibitors may be effective in patients with HER2-driven breast cancer resistant to neratinib. Citation Format: Hanker AB, Red Brewer M, Sheehan JH, Koch JP, Lanman R, Hyman DM, Cutler, Jr. RE, Lalani AS, Cross D, Lovly CM, Meiler J, Arteaga CL. An acquired HER2 T798I gatekeeper mutation induces resistance to neratinib in a patient with HER2 mutant-driven breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-03.

Background: Estrogen receptor (ER)-positive breast cancers (BC) initially respond to antiestrogens but eventually become hormone-independent and recur. FGFR1 is amplified in ∼10% of ER+ BC and is associated with early recurrence on antiestrogen therapy. Notably, one third of FGFR1-amplified tumors have simultaneous amplification of CCND1, FGF3, FGF4 and FGF19 on chromosome 11q12-14. Herein, we investigated the mechanisms by which FGFR1 amplification confers resistance to antiestrogen therapy in ER+ BC cells. Results: We performed whole exome sequencing in tumor biopsies from 130 patients with an operable ER+/HER2- BC who had received letrozole for 10-21 days prior to surgery. Tumors were categorized by the natural log (ln) of post-letrozole Ki67 as sensitive (ln ≤1 or ≤2.7% Ki67+ cells; n=68) or resistant (ln ≥2 or ≥7.4%; n=18). We found amplifications in FGFR1 and/or 11q12-14 in 6/11 (55%) resistant tumors compared with 5/34 (15%) in sensitive tumors (p=0.006); all cases were confirmed by FGFR1-fluorescence in situ hydridization (FISH). Resistant tumors with FGFR1 and/or 11q12-14-amplification showed a marked increase in nuclear FGFR1 with letrozole. ER+/FGFR1-amplified CAMA1 and MDA134 cell lines also exhibited co-localization of ER and FGFR1 in the nucleus. Cell proliferation was partially reduced by estrogen deprivation, and FGFR1 siRNA further reduced cell growth in hormone-depleted medium. We generated CAMA1 and MDA134 cells resistant to long-term estrogen deprivation (LTED). These cells exhibited overexpression of FGF3/4/19 and ERα with a concomitant increase in ligand-independent ER transcriptional activity and growth. An ER-FGFR1 interaction was observed in the nucleus and cytosol of CAMA1 parental cells with enhanced interaction in CAMA1 LTED cells. Genetic (with siRNA) and pharmacologic (with lucitinib) inhibition of FGFR1 reduced a) nuclear localization of FGFR1; b) ER transcriptional activity; and c) cell proliferation. Nuclear localization and ER-FGFR1 interaction were disrupted by a kinase-deficient FGFR1. Conversely, addition of FGF3 ligand stimulated ER-FGFR1 interaction and ER transcriptional activity, suggesting FGFR activation can regulate ER function. Inhibition of FGF receptor-specific substrate (FRS2), a principal mediator of FGFR1 signal transduction to the MAPK and PI3K pathways, with siRNA or pharmacologic inhibition of PI3K with buparlisib or MEK with GSK1120212 did not reduce ER transcriptional activity suggesting that, in ER+/FGFR1-amplified cancer cells, ER function is not modulated by FGFR signal transducers. Finally, using chromatin immunoprecipitation (ChIP) we showed that FGFR1 binds directly to estrogen response elements (ERE). This association was reduced with lucitanib. We are currently investigating genes modulated by ER/FGFR1 in ER+ BC and the in vivo anti-tumor efficacy of dual inhibition of FGFR1 and ER in ER+/FGFR1-amplified patient-derived breast cancer xenografts. Conclusions: These data support a critical role of ER and FGFR1 interaction in endocrine resistance in ER+/FGFR1-amplified breast cancer. Targeting of FGFR1 in combination with antiestrogens may abrogate resistance to endocrine therapy in these tumors and is worthy of clinical investigation. Citation Format: Formisano L, Young CD, Bhola NE, Bulen B, Estrada VM, Wagle N, Van Allen E, Red Brewer ML, Jansen VM, Guerrero AL, Giltnane JM, Strcker T, Arteaga CL. Nuclear FGFR1 interaction with estrogen receptor (ER) α is associated with resistance to endocrine therapy in ER+/FGFR1-amplified breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S3-03.