ErbB activation signatures as potential biomarkers for anti-ErbB3 treatment in HNSCC

Abstract

Head and neck squamous cell carcinoma (HNSCC) accounts for 3-5% of all tumor types and remains an unmet medical need with only two targeted therapies approved to date. ErbB3 (HER3), the kinase-impaired member of the EGFR/ErbB family, has been implicated as a disease driver in a number of solid tumors, including a subset of HNSCC. Here we show that the molecular components required for ErbB3 activation, including its ligand neuregulin-1 (NRG1), are highly prevalent in HNSCC and that HER2, but not EGFR, is the major activating ErbB3 kinase partner. We demonstrate that cetuximab treatment primarily inhibits the ERK signaling pathway and KTN3379, an anti-ErbB3 monoclonal antibody, inhibits the AKT signaling pathway, and that dual ErbB receptor inhibition results in enhanced anti-tumor activity in HNSCC models. Surprisingly, we found that while NRG1 is required for ErbB3 activation, it was not sufficient to fully predict for KTN3379 activity. An evaluation of HNSCC patient samples demonstrated that NRG1 expression was significantly associated with expression of the EGFR ligands amphiregulin (AREG) and transforming growth factor α (TGFα). Furthermore, NRG1-positive HNSCC cell lines that secreted high levels of AREG and TGFα or contained high levels of EGFR homodimers (H11D) demonstrated a better response to KTN3379. Although ErbB3 and EGFR activation are uncoupled at the receptor level, their respective signaling pathways are linked through co-expression of their respective ligands. We propose that NRG1 expression and EGFR activation signatures may enrich for improved efficacy of anti-ErbB3 therapeutic mAb approaches when combined with EGFR-targeting therapies in HNSCC.

Fig 1. Prevalence of NRG1 expression in HNSCC and other tumor types.

(A) NRG1 RNA overexpression was found to be most prevalent in HNSCC compared to other major tumor types, and overexpressed in nearly half of all head and neck tumors examined. RNA overexpression is defined as > 4-fold expression over the median expression across all samples (> 29,000). By contrast, NRG1 was overexpressed only in ~ 1% of colorectal cancer. TCGA data were used for this analysis. (B and C) A quantitative ISH-based assay demonstrates detectable NRG1 expression in the majority of human HNSCC tumors (B) and is independent of the histological site (C). Values reflect the ratio of the NRG1 probe signal over that of a control probe.

Fig 2. Prevalence of EGFR, HER2, and ErbB3 expression in HNSCC.

(A) ErbB3 is widely expressed, but not overexpressed, in HNSCC. RNA overexpression was defined as > 4-fold expression over the median expression across all tumor types. TCGA data were used for this analysis (B) ErbB receptors were expressed in the majority of HNSCC patient tumor samples (n = 46), as determined by VeraTag proximity-based immunoassays. Total EGFR (H1T), HER2 (H2T), and ErbB3 (H3T) levels are shown (C) The same pattern of ErbB protein expression was observed in a panel of 8 HNSCC cell lines.

Fig 3. KTN3379 demonstrates single agent in vivo anti-tumor activity in some, but not all NRG1-positive HNSCC models.

Three patient derived xenograft (PDX) models of HNSCC and one cell line (Cal27) were evaluated for expression of NRG1 and EGFR homodimer (H11D) levels. Single agent KTN3379 dosing resulted in significant anti-tumor efficacy in two NRG1 positive models (Cal27 and CTG-0434), but had no effect in NRG1-positive model (CTG-0776) and one NRG-negative model (CTG-0840). Evaluation of EGFR homodimer (H11D) levels indicated that KTN3379 was efficacious in models that are both NRG1-positive and express high H11D levels, but not in models where H11D levels are low.

Fig 4. KTN3379 enhances cetuximab anti-proliferative activity in vitro and in tumor xenograft models of HNSCC.

(A) Titration of KTN3379 (red) enhanced cetuximab (blue) anti-proliferative activity in 4 of 8 HNSCC cell lines (top row) and showed modest enhancement of cetuximab-treated activity in a subset of cell lines (Detroit562 and SCC35). In contrast, neither cetuximab nor KTN3379 showed anti-proliferative activity in other HNSCC lines (UNC10 and SCC9). Combination of KTN3379 and cetuximab is shown in purple. (B) KTN3379 demonstrated significant single agent activity in 2 HNSCC xenograft models (FaDu and OE21), and enhanced the anti-tumor activity of cetuximab. Animals were dosed intraperitoneally twice weekly at 10 mg/kg. Asterisks denote statistical significance; * p-value <0.05; ** p-value <0.01; *** p-value <0.001; **** p-value <0.0001; ns = not significant.

Fig 5. ErbB3 activation depends on HER2.

(A) Cal27 cells were treated with 100 nM anti-ErbB antibodies and cell lysates were analyzed for ErbB3, AKT and ERK phosphorylation. Addition of KTN3379 (KTN) or pertuzumab (Per) inhibited ErbB3 phosphorylation implicating the role of HER2 as the activating co-receptor for ErbB3. In contrast, cetuximab (Cet) did not inhibit ErbB3 phosphorylation on its own. (B) Cal27 cells were treated as described in (A) and phospho-ErbB levels were quantified by RPMA (Reverse-Phase Microarray Assay), yielding results consistent with (A). In addition, cetuximab treatment promoted mild stimulation of EGFR phosphorylation, consistent with previously published results.

Fig 6. Association of NRG1 expression with AREG and TGFα correlate with KTN3379 activity.

(A and B) A search of 303 HNSCC patient samples revealed a significant association between NRG1 expression and expression of EGFR ligands AREG (R² = 0.33) and TGFα (R² = 0.25). However, no association was found between NRG1 expression and any of the EGFR ligands in colorectal cancer patient samples (S7 Fig). (C and D) High levels of secreted AREG or TGFα protein were found associated with improved KTN3379 activity in HNSCC cell lines. Levels of secreted AREG and TGFα derived from a panel of 8 serum-starved HNSCC cell lines were measured after 48 hours. Supernatant-derived ligand concentrations (in pg/mL) are plotted as a function of the combined anti-proliferative activity of KTN3379 with cetuximab. A linear regression fit of the data gave R² values of 0.50 and 0.24 for AREG and TGFα, respectively. A similar result was observed when ligand concentration data were plotted against KTN3379-mediated phospho-AKT inhibition (S8 Fig). (E and F) High levels of EGFR homodimers (H11D) are associated with KTN33379 activity. EGFR homodimer (H11D) levels were evaluated in 8 HNSCC cell lines using a VeraTag immunoassay. H11D values were log-transformed and plotted against the combined anti-proliferative activity of KTN3379 with cetuximab (E), or KTN3379-mediated phospho-AKT inhibition in serum-grown cells (F). Linear regression analysis demonstrates a significant correlation between H11D levels and anti-proliferative activity (R² = 0.48) or phospho-AKT inhibition (R² = 0.76).