Abstract 4025: STEAP4 ISH and IHC diagnostics for Tarloxotinib activation in EGFR/HER2 mutant cancers

Background: Tarloxotinib is a clinical-stage prodrug that releases a potent, irreversible EGFR/HER2 inhibitor (Tarloxotinib-E) selectively in severely hypoxic regions of tumors. One-electron reduction leads to loss of the trigger moiety under oxygen-deficient conditions, releasing the neutral, diffusible ‘warhead’ Tarloxotinib-E. CRISPR/cas9-mediated disruption of the metalloreductase STEAP4 has demonstrated its role as the dominant plasma membrane reductase that facilitates one-electron reduction of Tarloxotinib. Methods: Cancer cell lines, PDX and resected tumor tissues processed in Formalin Fixed Paraffin Embedded (FFPE) blocks were evaluated. RNAScope technology was used to develop a method to assess STEAP4 mRNA expression by in situ hybridization using specific probes for human STEAP4. mRNA ISH technology for STEAP4 was optimized on a Leica Bond RX autostainer for detection of STEAP4 in FFPE specimens. STEAP4 antibodies were evaluated for Immunohistochemistry (IHC) analysis. STEAP4 IHC was employed as cross-reference, using an anti-STEAP4 antibody validated against STEAP4 knockout and over-expressing cell line and tumor models. Staining data was compared to quantitative PCR results for mRNA ISH and Western blot analysis for IHC. STEAP4 mRNA expression was assessed in FFPE sections of various EGFR/HER2 mutant tumor xenografts and EGFR/HER2 mutant patient tumor specimens. Results: The STEAP4 mRNA ISH assay was optimized and validated using FFPE sections from cell lines and STEAP4 overexpressing tumor blocks and compared to wild type tumors with minimal expression. High level of STEAP4 mRNA was observed in STEAP4 overexpressing tumors. STEAP4 mRNA ISH analysis revealed variable levels of expression in EGFR/HER2 mutant xenograft tumors. STEAP4 mRNA and protein expression level correlated with Tarloxotinib conversion to Tarloxotinib-E. STEAP4 mRNA expression in EGFR/HER2 mutant patient tumor specimens will be presented. Conclusion: STEAP4 reduces Tarloxotinib and leads to the conversion to the active drug, Tarloxotinib-E, in hypoxic tumors. Assessment of STEAP4 expression in individual patients is important to guide patient stratification in Tarloxotinib clinical trials and represents an attractive biomarker to enroll tarloxotinib-responsive patients in clinical trials. Citation Format: Hui Yu, Shevan Silva, Adriana Estrada-Bernal, Chris Rivard, Fred Hirsch, Maria Abbatista, Jeff Smaill, Robert C. Doebele, Adam Patterson, Avanish Vellanki, Vijaya G. Tirunagaru. STEAP4 ISH and IHC diagnostics for Tarloxotinib activation in EGFR/HER2 mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4025.