Abstract 5039: EGFR regulates Mcl-1 dependence in chemoresistant and acquired ABT-737-resistant high-risk neuroblastoma (HR NB).
Authors: Peirce Susan K. , Nalluri Srilatha , Abdella Haneen , Goldsmith Kelly 
Published: August 2014

Patients with HR NB initially respond to treatment yet &gt; 60% die of chemoresistant disease due to inhibited apoptosis. We have shown that HR NBs depend on anti-apoptotic Bcl-2 or Mcl-1 to sequester the pro-death protein Bim for survival. Mice carrying Bcl-2 dependent NB xenografts were cured with the Bcl-2/Bcl-xL antagonist ABT-737 plus cytotoxics, but Mcl-1 dependent NBs were resistant, as ABT-737 does not target Mcl-1. We also showed a Bcl-2 dependent NB cell line (SMS-SAN) selected in vitro to resist high dose ABT-737 (SAN-ABTR) altered Bim binding from Bcl-2 to Mcl-1, despite persistent Bcl-2 expression. SAN-ABTR is also resistant to common cytotoxic drugs compared to SMS-SAN, likely through acquired Mcl-1 dependence. As no current molecules inhibit Mcl-1 directly, we hypothesized that targeting pathways that regulate Mcl-1 expression will restore chemotherapy and Bcl-2 antagonist sensitivity in HR NB. Real time cell sensing (xCELLigence) showed that SAN-ABTR at steady state had a &gt; 2 log increase in proliferation compared to SMS-SAN. Tyrosine kinases (TK) enhance NB proliferation and regulate Mcl-1 in adult cancers, but how they regulate Bcl-2 proteins in NB is unknown. Using phospho-protein microarrays, we found that EGFR and its downstream effectors, AKT, ERK 1/2, and CREB were phosphorylated in SAN-ABTR but not in SMS-SAN. Co-immunoprecipitation (co-IP) showed that inhibition of EGFR by shRNA in SAN-ABTR reverted Bim from Mcl-1 back to Bcl-2 sequestration, despite persistent Mcl-1 expression and restored sensitivity to ABT-737 in vitro. EGFR inhibition also re-sensitized SAN-ABTR to cytotoxic agents. Immunoblots of a panel of 12 NB cell lines characterized for Bcl-2 dependence patterns showed EGFR to be highly expressed in Mcl-1 dependent NBs but not in Bcl-2 dependent NBs. We inhibited EGFR by shRNA in Mcl-1 dependent SKNBE(2) and again Bim moved from Mcl-1 sequestration over to Bcl-2, suggesting a role for EGFR in de novo Mcl-1 dependence. Inhibition of ERK 1/2 (using UO126) resulted in enhanced sensitivity to ABT-737 in Mcl-1 dependent, EGFR expressing NLF cells, suggesting EGFR is regulating Mcl-1:Bim binding through ERK. Work is ongoing to evaluate post-translational modifications causing EGFR mediated Bim:Mcl-1 binding in NB. This data supports a role for EGFR in both pre-existing chemoresistance and emergent targeted therapy resistance in HR NB through modulation of Bcl-2 family interactions. Earlier studies suggest that EGFR inhibitors alone have little utility in NB treatment. Here, we demonstrate that Bcl-2 sequesters Bim upon EGFR inhibitor displacement of Bim from Mcl-1. Therefore, EGFR inhibitors will be more effective if the Bcl-2 survival bias is subverted using Bcl-2 antagonists. The novel targeted combination of EGFR and Bcl-2 blockade has the potential to impact the survival of patients with HR NB as well as other EGFR overexpressing and Mcl-1 dependent cancers.

Citation Format: Susan K. Peirce, Srilatha Nalluri, Haneen Abdella, Kelly Goldsmith. EGFR regulates Mcl-1 dependence in chemoresistant and acquired ABT-737-resistant high-risk neuroblastoma (HR NB). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5039. doi:10.1158/1538-7445.AM2013-5039