Background: HER receptor tyrosine kinases (EGFR/HER1; HER2; HER3; HER4) and their soluble ligands represent a robust system involved in the regulation of a diverse array of cellular processes. Deregulation of HER receptors, notably HER2, has been linked to the initiation and progression of breast cancer and other solid tumors. Relatively less is known about the role of HER receptor soluble ligands in tumorigenesis and responsiveness to HER targeted therapies. Here we will discuss the impact of the HER3 ligand heregulin (HRG) on the sensitivity of breast cancers to HER tyrosine kinase inhibitors (TKIs), and identify TKI strategies to treat HRG-driven breast cancers. Methods: The effects of exogenous HRG (50ng/ml) on the antitumor activity of a panel of TKIs with different enzymatic properties e.g. a reversible, selective inhibitor (lapatinib) and irreversible, pan-HER inhibitors (neratinib; CI-1033) were assessed in HER2+ breast cancer (BC) cell lines. The concentration of TKIs used was in the 1–2.5 uM range, and cells were treated over a 72 hr course. Vehicle treatment alone served as controls. The impact of the above mentioned treatments on total HER receptor and specific EGFR, HER2, and HER3 phosphotyrosine sites, in addition to the phosphorylation state of components of downstream MAPK and PI3K signaling pathways was analyzed through western blot. In addition to studying the effects of exogenous HRG, the impact of TKIs on a model of triple negative BC (TNBC) known to produce HRG in an autocrine manner was also evaluated. QRT-PCR was used to assess the effects of TKIs on HER receptor mRNA levels. Results: Lapatinib inhibited proliferation and phosphorylation of EGFR, HER2, HER3, and downstream MAPK and PI3K signaling pathways in HER2+ SKBR3 and BT474 cell lines. Pre-treatment with HRG abrogated the antitumor effects of a therapeutic concentration of lapatinib (1 uM), and reversed the inhibitory effects of lapatinib on the phosphorylation of HER receptors and components of their downstream signaling pathways e.g. Erk1/2 and Akt. Neratinib also blocked proliferation and phosphorylation in HER2+ BC cells. In contrast to lapatinib, the antitumor effects of neratinib were not reversed by exogenous HRG. Interestingly, treatment with neratinib and similar pan-HER irreversible TKIs, but not reversible TKIs, resulted in loss of HER2 and EGFR protein expression. QRT-PCR was used to evaluate if this effect was at the level of transcription. Furthermore, neratinib inhibited cell proliferation and blocked EGFR signaling in a TNBC model driven by autocrine produced HRG. Conclusions: Our findings suggest that HRG is a mediator of therapeutic resistance to lapatinib in HER2+ and TNBC. Neratinib demonstrates profound effects on HER signaling by markedly reducing HER2 and EGFR protein expression and blocking the pro-tumorigenic effects of autocrine or paracrine expression of HRG. In contrast to HRG, we previously showed that EGF, an EGFR specific ligand did not reverse the antitumor effects of lapatinib in breast cancer cells. Thus, the selection of HER targeted therapies in a given tumor should take into account not only the HER receptor expression profile, but also the presence of autocrine or paracrine derived ligands activating HER receptors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-03.

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Background: Immune-based therapy for metastatic breast cancer has had limited success. Strategies to augment adaptive immunity include vaccines targeting genomic amplifications like Human Epidermal Growth Factor Type 2 (HER2), an established driver of malignancy. Using a novel alphaviral vector, we constructed a vaccine encoding a portion of HER2 (VRP-HER2). Methods: In preclinical studies, mice were immunized before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and anti-tumor function were assessed. We then translated this vaccine into a phase I clinical trial in which subjects with advanced HER2-overexpressing breast cancers received VRP-HER2 every 2 weeks for a total of three doses (cohort 1). In cohort 2, subjects received the same dose of VRP-HER2 along with a standard HER2 targeted therapy. Results: VRP-HER2 induced HER2-specific T cell and antibody responses while controlling tumor growth in murine models. Vaccination with VRP-HER2 was well tolerated in both patient cohorts. PFS was modest, while median OS was 50.2 months in cohort 1 and 32.7 months in cohort 2. In cohort 2, there is one partial response and two patients with continued stable disease. Vaccine induced anti-HER2 antibodies and T cells were identified. Increased perforin expression by memory CD8 T cells post vaccination significantly correlated with improved PFS. Conclusions: VRP-HER2 led to an increase in perforin expressing HER2-specific memory CD8 T cells in preclinical and clinical studies, and had profound antitumor effects in murine models. The generation of HER2-specific memory CD8 T cells was significantly correlated with increased PFS in patients. Subsequent studies will seek to enhance T cell activity by combination with anti-PD-1/PD-L1 antibodies. Citation Format: Crosby EJ, Gwin WR, Chang S, Maecker HT, Lubkov V, Snyder JC, Broadwater G, Hyslop T, Osada T, Hobeika AC, Hartman ZC, Morse MA, Lyerly HK. CD8 T cells induced by novel alphaviral vector predict improved progression free survival in advanced HER2+ breast cancer patients [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 P2-09-16.