Abstract 331: Dynamic kinome targeting reveals kinases involved in acquired resistance to tyrosine kinase inhibitors in EGFR-driven glioblastomas
Authors: Shelton Abby , Smithberger Erin , Butler Madison , Flores Alex , Bash Ryan , Angus Steve , Sciaky Noah , Dhruv Harshil , Johnson Gary L. , Berens Michael E. , Furnari Frank , Miller C. Ryan 
Published: August 2019

Abstract
Glioblastoma (GBM) is a devastating primary brain tumor with limited treatment options. Extensive molecular characterization has revealed two particularly frequent mutations: CDKN2A deletion (50-60%) and EGFR (40-50%). EGFRvIII (~35%) is a constitutively active truncation mutant with exons 2-7 deleted. EGFR is a particularly attractive therapeutic target due to frequent activating mutations, such as EGFRvIII, and ready availability of multiple targeted inhibitors. Several EGFR tyrosine kinase inhibitors (TKI) have failed clinically, due in part to acquired resistance. To mechanistically examine this type of resistance, we used genetically-engineered mouse astrocytes harboring homozygous deletions of Cdkn2a, as well as EGFRvIII (CEv3). CEv3 astrocytes were made intrinsically resistant to the EGFR TKI gefitinib or erlotinib via long-term exposure, both in vitro and in vivo. We found that long-term gefitinib or erlotinib exposure conferred variable levels of cross resistance to a panel of second- and third-generation EGFR TKI (ΔIC50 1.12-36.1-fold), relative to non-resistant parent lines. We have previously shown that dynamic kinome reprogramming may be responsible for TKI resistance in glioblastoma. Therefore, we used a chemical proteomics method, multiplexed inhibitor beads and mass spectrometry (MIB-MS), to examine changes in the expressed and functional kinome, in both the presence or absence of one of several EGFR TKI known to penetrate the blood-brain barrier. Additionally, we performed RNA sequencing (RNA-seq) to inspect transcriptomic alterations in response to these drugs. RNA-seq showed that resistant CEv3 mouse astrocytes clustered separately from their non-resistant in vitro and in vivo counterparts. Acquired resistance also induced transcriptome alterations governing cellular metabolism, including upregulation of metabolic pathways and downregulation of RNA processing genes. Importantly, the kinase transcriptome was rewired, as 67 kinases were differentially expressed across parental and resistant cell lines (Q&lt;0.001). Probing the dynamic kinome response to afatinib, an EGFR TKI, using RNA-seq identified two potential kinases involved in acute, adaptive resistance to afatinib, Bmx and Ntrk3. Integrated kinome profiling using RNA-seq and MIB-MS in murine models of GBM with defined mutational profiles provides a powerful framework to define novel therapeutic targets that could significantly alter current treatment paradigms.
Citation Format: Abby Shelton, Erin Smithberger, Madison Butler, Alex Flores, Ryan Bash, Steve Angus, Noah Sciaky, Harshil Dhruv, Gary L. Johnson, Michael E. Berens, Frank Furnari, C. Ryan Miller. Dynamic kinome targeting reveals kinases involved in acquired resistance to tyrosine kinase inhibitors in EGFR-driven glioblastomas [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 331.