Epidermal growth factor receptor dimerization status determines response to HER-kinase targeted therapies in keratinocytes

Abstract

3062 Background: Acne-like skin rash is common among cancer patients treated with EGFR tyrosine kinase inhibitors (Gefitinib, Erlotinib) or anti-EGFR antibodies (C225). Interestingly, these cutaneous side effects are rarely observed in patients treated with 2C4, a humanized monoclonal antibody which abrogates HER2-mediated signaling. Our molecular hypothesis for this observation is that in skin EGFR homodimers are preferred to EGFR/HER2 heterodimers which lead to strong cutaneous side-effects in response to gefitinib but not to 2C4. Methods: This hypothesis was tested in primary human epidermal keratinocytes (NHEK) by evaluating absolute mRNA and protein expression levels of the HER-kinase axis members and the effect of HER-kinase directed therapies by phospho-MAPK western blot analysis. The preference status of HER2-specific heterodimers was determined using semi-quantitative co-immunoprecipitation analysis. The results were compared to an ovarian cancer cell line (OVCA433) which is responsive to both gefitinib and 2C4. Results: Gefitinib inhibited ligand-mediated EGFR phosphorylation and MAPK activation in both OVCA433 and NHEK cells in a dose-dependent manner. In contrast, 2C4 blocked EGFR phosphorylation and downstream signaling only in OVCA433 cells but did not demonstrate any effect in keratinocytes. Co-immunoprecipitation studies showed that significant levels of HER2 interact with EGFR in OVCA433 cells while HER2 is absent in EGFR-immunoprecipitations performed with NHEK lysates. These results were not driven by the level of receptor expression. Studies are underway to validate our observations using a colorimetric heterodimerization assay to assess the EGFR dimerization status in these cell lines and in skin biopsies from patients who developed a skin rash in response to gefitinib treatment. Conclusions: Our results suggest a preference for EGFR homodimerization in primary keratinocytes and indicate the inability of HER2 to engage in active EGFR/HER2 heterodimers. This is of particular interest as the cutaneous adverse effects of gefitinib result from direct interference of EGFR signaling in skin. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Genentech