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Bay846, a new irreversible small molecule inhibitor of EGFR and Her2, is highly effective against malignant brain tumor models

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Summary

The epidermal growth factor receptor (EGFR) pathway is aberrantly activated in tumors and plays a key role in promoting tumor growth. Small molecule inhibitors which bind reversibly to EGFR have demonstrated limited clinical activity. Thus, there is a continued need to develop novel EGFR inhibitors with improved anti-tumor activity. Bay846 is a newly developed small molecule inhibitor that binds irreversibly to the tyrosine kinase domains of EGFR and Her2. The in vitro and in vivo efficacy of Bay846 was tested using a panel of nine human malignant brain tumor (glioma) models. Lapatinib, a reversible inhibitor of EGFR and Her2, was included for comparison. Six glioma cell lines were sensitive to Bay846 treatment. Bay846 strongly suppressed tumor cell growth in vitro by inducing cell lysis/death rather than cell cycle arrest. Consistent with this, Bay846 had potent anti-tumor activity which led to regressions in tumor size. The active, phosphorylated form of EGFR was reduced by Bay846 treatment in vitro and in tumors. Importantly, the efficacy of Bay846 was significantly greater than lapatinib in all assays. Bay846-sensitivity was associated with expression of a wild-type PTEN in conjunction with high levels of an oncogenic EGFR variant (A289V or EGFRvIII). These studies demonstrate that targeting the EGFR pathway with the irreversible inhibitor Bay846 has great potential to increase the efficacy of this cancer therapy.

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Acknowledgements

Financial support was provided by Bayer Healthcare (DEP and GWC), an American Cancer Society Institutional Research grant from the Upstate Cancer Institute at SUNY Upstate Medical University (DEP), and the Dept. of Neurosurgery at SUNY Upstate Medical University. We thank Bayer Healthcare for providing Bay846, Rebecca Sager for assistance with the LDH studies, and Ed Shillitoe for critical reading of the manuscript.

Financial support

Bayer Healthcare (DEP and GWC), an American Cancer Society Institutional Research grant from the Upstate Cancer Institute at SUNY Upstate Medical University (DEP), and Dept. of Neurosurgery, SUNY Upstate Medical University.

Conflict of Interest

The authors declare that financial support and the Bay846 compound was provided by Bayer Healthcare. KP, HS, and OP are employees of Bayer Healthcare. The commercial funder had no role in the analysis or interpretation of data.

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Authors and Affiliations

  1. Department of Neurosurgery, State University of New York (SUNY), Upstate Medical University, 750 East Adams St., Rm 4117 IHP, Syracuse, NY, 13210, USA

    Sharon L. Longo, David J. Padalino, Sandra McGillis, Gregory W. Canute & Dawn E. Post

  2. Microbiology & Immunology, State University of New York (SUNY), Upstate Medical University, Syracuse, NY, USA

    Dawn E. Post

  3. Bayer Healthcare, Berlin, Germany

    Kirstin Petersen, Hartmut Schirok & Oliver Politz

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  1. Sharon L. Longo
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Supplemental Fig. 1

Cell surface EGFR expression levels. Cell surface EGFR levels were quantified by flow cytometry using an anti-EGFR specific antibody (open curves). An isotype matched IgG antibody was used as a negative control (red shaded curves). The relative EGFR level (SFI) was calculated as the ratio of the mean fluorescence value obtained with the specific EGFR antibody versus the control antibody. (PPT 87 kb)

Supplemental Fig. 2

Cell surface Her2 expression levels. Cell surface Her2 levels were quantified by flow cytometry using an anti-Her2 specific antibody (open curves). An isotype matched IgG antibody was used as a negative control (red shaded curves). The relative Her2 level (SFI) was calculated as the ratio of the mean fluorescence value obtained with the specific Her2 antibody versus the control antibody. The study was performed twice for each cell line, except for GBM39 and U87 which were measured one time, and the resulting SFI was averaged. Representative data for each cell line is shown. (PPT 86 kb)

Supplemental Fig. 3

Cell cycle profile of cell lines which were resistant to Bay846 and lapatinib treatment. Cells were treated with Bay846, lapatinib, or untreated at the indicated drug concentrations for 48 h. The relative DNA content per cell was obtained by staining cells with propidium iodide and then measuring fluorescence using a LSRII flow cytometer. The cell cycle profile was analyzed using FACSDiva software (Becton-Dickinson). See Fig. 2 for cells lines which were sensitive to Bay846 and lapatinib. (PPT 120 kb)

Supplemental Fig. 4

Total and phospho-EGFR levels in cells lines which were resistant to Bay846 and lapatinib treatment. Cells were treated with Bay846, lapatinib, or untreated. Bay846 and lapatinib were used at a concentration of 1 μM. Total cell lysates were examined by western blotting for total and phospho-(p1086 and p1173) EGFR, PTEN, and actin (loading control) at Day 2 (Bai, U87, Arn) or Day 3 [Mor(lo)]. Asterisks (*) indicate non-specific bands that are cross-reactive with the anti-PTEN antibody. See Fig. 4 for cells lines which were sensitive to Bay846 and lapatinib. (PPT 166 kb)

Supplemental Table 1

Primers used for sequencing PTEN and EGFR cDNAs. The PCR product size represents the expected size in base pairs of a wild-type cDNA sequence. (DOC 30 kb)

Supplemental Table 2

Inhibition of GBM cell growth by Bay846 and lapatinib measured by MTT assay at 3 days post-treatment. Data is the average percent cell growth inhibition ± SD and is shown graphically in Fig. 1. Asterisks indicate a significant increase in cell growth inhibition by Bay846 compared to lapatinib (p < 0.002, t-test). (DOC 32 kb)

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Longo, S.L., Padalino, D.J., McGillis, S. et al. Bay846, a new irreversible small molecule inhibitor of EGFR and Her2, is highly effective against malignant brain tumor models. Invest New Drugs 30, 2161–2172 (2012). https://doi.org/10.1007/s10637-011-9784-4

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