Abstract 1857: Glioblastoma growth is suppressed dual inhibition of EGFR and CDK6 kinases
Authors: Smithberger Erin , Shelton Abigail K. , Bash Ryan E. , Butler Madison K. , Flores Alex R. , Stamper Allie , Angus Steven P. , East Michael P. , Johnson Gary L. , Berens Michael E. , Furnari Frank B. , Miller Ryan 
Published: June 2022

Abstract
Glioblastoma (GBM) is a malignant brain tumor that has proven difficult to treat, despite expressing promising targets such as EGFRvIII. EGFRvIII, a mutant version of the epidermal growth factor receptor (EGFR), is constitutively active and not present in normal brain cells. The tumor specificity of EGFRvIII and the frequent EGFR amplification seen in GBM make EGFR a potentially attractive therapeutic target; however, clinical studies have shown little to no efficacy for EGFR tyrosine kinase inhibitors (TKI). One reason for this lack of efficacy may be adaptive resistance. We used RNA sequencing and multiplexed inhibitor beads with mass spectrometry (MIB-MS) to study the transcriptomes and kinomes of genetically engineered mouse astrocytes to investigate this resistance and identify potential targets for dual inhibition. Out of 329 kinases detected by MIB-MS, 76 were differentially expressed between cells with Cdkn2a deletion (“C”) and cells that also overexpressed EGFRvIII (“CEv3”). Thirty-four of these kinases were overexpressed in the CEv3 cells relative to the parental C cells (log2 fold change of 5.6, p&lt;1x105). One of these kinases, Cdk6, is also significantly overexpressed in CEv3 cells versus cells that have a further loss of function mutation of Pten (“CEv3P”) (log2 fold change of 5.6, p&lt;1x105). Despite this significant differential expression at the protein level, RNA expression of Cdk6 was similar between cell lines. When these cells were treated with the CDK6 inhibitor abemaciclib, CEv3 cells were found to be significantly more sensitive to inhibition than C and CEv3P cells (IC50 of 0.10 μM vs. 0.18 μM and 0.23 μM, respectively). Similarly, when cells were treated with abemaciclib in combination with the EGFR inhibitor neratinib, there was significantly higher synergy in CEv3 cells than C or CEv3P cells. Genotypically-matched patient-derived xenograft (PDX) cells were assayed for EGFR-CDK6 inhibitor synergy and showed a similar pattern of greater synergy in cells with EGFRvIII overexpression and functional PTEN than cells with EGFRvIII overexpression and PTEN loss. CEv3 and CEv3P cells were orthotopically implanted into mice and treated with neratinib, abemaciclib, or a combination. In CEv3-injected mice, combination treatment led to significantly longer survival than either single agent or control treatment. However, in CEv3P-injected mice, no survival difference was seen between any of the treatment arms. Taken together, these data provide strong evidence that CDK6 is a promising target for combination treatment with EGFR inhibitors in glioblastoma.
Citation Format: Erin Smithberger, Abigail K. Shelton, Ryan E. Bash, Madison K. Butler, Alex R. Flores, Allie Stamper, Steven P. Angus, Michael P. East, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, Ryan Miller. Glioblastoma growth is suppressed dual inhibition of EGFR and CDK6 kinases [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 1857.