Abstract 3248: Acquired resistance to targeted inhibitors in EGFR-driven glioblastoma: Identification of dual kinase targets
Authors: Shelton Abigail K. , Smithberger Erin , Butler Madison , Stamper Allie , Bash Ryan E. , Angus Steve P. , East Michael P. , Johnson Gary L. , Berens Michael E. , Furnari Frank B. , Miller Ryan 
Published: June 2022

Abstract
Glioblastoma (GBM) is a devastating primary brain tumor with &lt;5% 5-year survival. CDKN2A deletion (~60%) and EGFR amplification (~55%) mutations frequently co-occur in these tumors. EGFR is an attractive therapeutic target due to its mutational frequency and availability of brain-penetrant tyrosine kinase inhibitors (TKI). Several EGFR TKI have failed clinically, due in part to acquired resistance. To mechanistically examine this type of resistance, we used a panel of ten genetically engineered mouse astrocyte lines harboring Cdkn2a deletion and EGFRvIII, a common (~30%) activating mutation. Resistant cells were generated via long-term exposure to gefitinib or erlotinib, either in vitro or in vivo. Both transcriptomic (RNAseq) and proteomic (multiplexed inhibitor beads with mass spectrometry, MIB-MS) experiments showed that cell lines clustered primarily by resistance phenotype and secondarily by method of resistance development when analyzed using principal component analysis and unsupervised hierarchical clustering. Kinases involved in proliferation and differentiation signaling pathways (ex: Pdgfrb, Pdk2, Tnik, Mapk3, Fgfr2) were upregulated in both RNAseq and MIB-MS datasets and thus represent putative druggable targets for dual kinase inhibition. Analysis of commonly upregulated kinases and their commercially available inhibitors revealed dovitinib and dasatinib, two brain-penetrant drugs approved for other cancer indications, as candidates for dual inhibition with an EGFR TKI. Resistant cell lines were all more sensitive to dovitinib than their drug-naïve parents; however, sensitivity to dasatinib varied. BLISS analysis of dual treatment with EGFR TKI neratinib and dasatinib or dovitinib revealed synergistic drug interactions in most lines. Additionally, drug-naïve cells displayed a robust, acute proteomic response to EGFR TKI afatinib over 48h, while the response of resistant lines was significantly blunted. This model system can also be used to examine acute vs. long-term kinome response to EGFR TKI. Acute response was examined by treating drug-naïve cells with afatinib over 48h, and long-term kinome rewiring was observed by comparing untreated cells to gefitinib- and erlotinib-resistant cell lines. Combing both RNAseq datasets for kinases upregulated in both drug-naïve cells over a 48h EGFR TKI treatment course and in resistant cell lines compared to their sensitive parents reveals 21 and 13 common kinases, respectively, at p&lt;0.001. Eight of these kinases (Cdk19, Ddr1, Kalrn, Khk, Mapk3, Pink1, Tnik, Ulk2) appear in both the in vitro and in vivo datasets, indicating a conserved kinome response regardless of method of resistance generation. Overall, integrated kinome profiling in GBM models with defined mutational profiles provides a powerful framework to identify novel therapeutic targets that could significantly alter current treatment paradigms.
Citation Format: Abigail K. Shelton, Erin Smithberger, Madison Butler, Allie Stamper, Ryan E. Bash, Steve P. Angus, Michael P. East, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, Ryan Miller. Acquired resistance to targeted inhibitors in EGFR-driven glioblastoma: Identification of dual kinase targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3248.