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Patients with breast cancer brain metastases have extremely limited survival and no approved systemic therapeutics. Triple-negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis. TNBC frequently harbors BRCA mutations translating to platinum sensitivity potentially augmented by additional suppression of DNA repair mechanisms through PARP inhibition. We evaluated brain penetrance and efficacy of carboplatin ± the PARP inhibitor ABT888, and investigated gene-expression changes in murine intracranial TNBC models stratified by BRCA and molecular subtype status. Athymic mice were inoculated intracerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low); or BRCA–wild-type (wt): MDA-MB-468 (basal), MDA-MB-231BR (claudin-low). TNBC cells were treated with PBS control [intraperitoneal (IP), weekly], carboplatin (50 mg/kg/wk, IP), ABT888 (25 mg/kg/d, oral gavage), or their combination. DNA damage (γ-H2AX), apoptosis (cleaved caspase-3, cC3), and gene expression were measured in intracranial tumors. Carboplatin ± ABT888 significantly improved survival in BRCA-mutant intracranial models compared with control, but did not improve survival in BRCA-wt intracranial models. Carboplatin + ABT888 revealed a modest survival advantage versus carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436, but not in the SUM149 model. BRCA-mutant SUM149 expression of γ-H2AX and cC3 proteins was elevated in all treatment groups compared with control, whereas BRCA-wt MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene-expression changes in BRCA-mutant models. Carboplatin ± ABT888 penetrates the brain and improves survival in BRCA-mutant intracranial TNBC models with corresponding DNA damage and gene-expression changes. Combination therapy represents a potential promising treatment strategy for patients with TNBC brain metastases warranting further clinical investigation. Mol Cancer Ther; 14(4); 920–30. ©2015 AACR.

This file contains additional details on the microarray analysis and normalization.

This file contains the compiled Supplemental Figures S1-S9. Figure S1 is a flowchart describing the process of normalizing the microarray data for the SUM149 model for the different scanners used. Figure S2 depicts the scanner differences in the microarray data for the SUM149 model before and after normalization. Figure S3 is a flowchart depicting the analytical process used to analyze and compare the SUM149 and MDA-MB-436 models microarray results, and Figure S4 shows the difference in scale between models before and after the scaling process for visual comparison of the SUM149 and MDA-MB-436 models. Figure S5 demonstrates the lack of survival benefit of Carboplatin +/- ABT88 in the two BRCA-wt models, MDA-MB-468 and MDA-MB-231BR. Figure S6 contains representative images of IHC cC3 staining for the BRCA-wt model, MDA-MB-468. Figure S7 shows the unsupervised clustering of the BRCA-mut SUM149 and MDA-MB-436 models with treatment. Figure S8 contains the supervised clustering based on carboplatin treatment in these two models. Figure S9 demonstrates the expression levels of the 38 commonly regulated genes in both models with carboplatin treatment.

This file contains the differentially expressed genes and enriched pathways lists from the gene expression microarray analyses in Supplemental Table S1, with sample information in S1a. Lists are for the following comparisons: SUM149 Control vs. ABT888 (S1b), MDA-MB-436 Control vs. ABT888 (S1c), SUM149 Control vs. Carboplatin (S1d), MDA-MB-436 Control vs. Carboplatin (S1e), SUM149 Control vs Carboplatin+ABT888 (S1f), MDA-MB-436 Control vs Carboplatin+ABT888 (S1g), SUM149 Carboplatin vs Carboplatin+ABT888 (S1h), MDA-MB-436 Carboplatin vs Carboplatin+ABT888 (S1i). DAVID pathway enrichment analysis results are shown for the significantly up-regulated (S1j) and down-regulated (S1k) genes with Carboplatin treatment for both models.

This file contains the figure legends for Supplemental Figures S1-S9.

Glioblastoma (GBM) is the most common malignant brain tumor in adults with a dismal 15-month median survival. Standard therapy consisting of surgical resection, radiation, and temozolomide has been unsuccessful in meaningfully extending survival and preventing recurrence; thus, novel therapeutics are urgently needed. One proposed targeted treatment strategy for GBM involves using small molecule inhibitors against common genetic mutations. Epidermal growth factor receptor (EGFR) is the most commonly overexpressed oncogene in GBM (~56%). While EGFR tyrosine kinase inhibitors (TKI) have shown promise in other cancers, GBM clinical trials with EGFR TKI have failed. One reason for this failure is the development of adaptive therapeutic resistance. Understanding the mechanisms behind drug resistance is essential for the development of novel, effective therapeutics for GBM. To better understand adaptive resistance in GBM, we utilized two genetically engineered mouse astrocyte lines harboring common GBM mutations: Cdkn2a-/-, EGFRvIII (CEv3) and Cdkn2a-/-, Pten-/-, EGFRvIII (CEV3P). CDKN2A and PTEN are commonly deleted or otherwise inactivated tumor suppressor genes in GBM while the vIII variant of EGFR is the single most common oncogene mutation, making it an attractive therapeutic target. Cell lines CEv3 and CEv3P are both sensitive to neratinib, an irreversible second-generation EGFR TKI, at IC50 of 0.24μM and 0.13µM, respectively. To better understand adaptive response to neratinib treatment, we profiled the transcriptome with RNA sequencing at 0, 4, 24, and 48 hours. Our data shows that kinome rewiring is detectable after just 4 hours of treatment and sustained through 48 hours, with differential expression of 70% or more of the expressed kinome. We propose that differentially expressed kinases in response to neratinib can potentially activate alternative signaling pathways that bypass EGFR inhibition, which ultimately confers resistance to EGFR targeted therapy. Furthermore, we hypothesize that the epigenome is directly responsible for this adaptive kinome response through BRD4 dependent enhancer remodeling. Because dual therapy against EGFR and BRD4 has shown promising results in other cancers, targeting the epigenome through BRD4 represents a potential combination therapy with EGFR TKI in GBM. To profile BRD4-associated epigenomic changes, we used Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to interrogate several regulatory marks (H3K4me1, K3K4me3, H3K27ac) in addition to BRD4. We seek to integrate RNA sequencing and CUT&RUN data to determine if kinases differentially expressed following neratinib treatment correlate with epigenetic marks for their respective enhancer(s). This work will provide insight into the adaptive resistance mechanism of EGFR driven GBM. Citation Format: Benjamin Lin, Julia Ziebro, Kasey R. Skinner, Abigail Shelton, Erin Smithberger, Ryan Bash, Frank B. Furnari, Ryan Miller. Elucidating the transcriptomic response to EGFR-targeted therapy in EGFR-driven glioblastoma [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 1125.

Glioblastoma (GBM) is an aggressive primary brain tumor with poor survival and limited treatment options. However, it is an attractive candidate for precision therapeutic approaches due to the frequency of amplification and/or activating mutations in the epidermal growth factor receptor (EGFR) gene and the availability of several brain penetrant second- and third-generation EGFR tyrosine kinase inhibitors (TKI). We used comprehensive molecular profiling of a panel of genetically engineered mouse astrocyte models to examine whether mutational profiles, particularly EGFR and PTEN status, could be used to identify kinases upregulated in specific mutational backgrounds. Using RNA-seq and multiplex inhibitor bead/mass spectrometry (MIB-MS) to analyze the kinase transcriptomes and proteomes, respectively, we have identified several potential targets for combination therapy. Overexpression of wild type EGFR in immortalized, Cdkn2a-/- astrocytes resulted in mild rewiring of the GBM kinome. Only 5 kinases aside from EGFR itself were overexpressed on either the transcript or protein levels. One overexpressed kinase, Hck, has been shown to be involved in cell survival, proliferation, adhesion, and migration. In contrast, overexpression of EGFRvIII, a constitutively active, extracellular domain truncation mutant of EGFR, resulted in significant alteration of the GBM kinome – 81 kinases showed differential expression, with 27 upregulated. One potentially attractive target among these was Cdk6, a drug-targetable, prognostically significant cyclin-dependent kinase implicated in proliferation, migration, and invasion. Finally, overexpression of EGFRvIII in cells lacking Pten dysregulated 46 kinases, including 15 upregulated. One particularly interesting target in these cells was Ddr2, a tyrosine kinase involved in migration, invasion, and extracellular matrix remodeling. We conclude that Hck, Cdk6, and Ddr2 represent attractive targets for therapeutic intervention in their relevant genetic contexts. These findings also suggest that molecular diagnostics for EGFR and PTEN status may be useful in guiding development of rational, EGFR TKI-centric drug combinations.

Glioblastoma (GBM) is a devastating primary brain tumor with 5-year survival < 5%. CDKN2A deletion (~60%) and EGFR amplification (55–60%) mutations frequently co-occur in these tumors. EGFR is an attractive therapeutic target due to its mutational frequency and availability of multiple 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 genetically engineered mouse astrocytes harboring Cdkn2a deletion and EGFRvIII, a common (35%) activating mutation. Resistant cells were generated via chronic exposure to gefitinib or erlotinib, either in vitro or in vivo. Resistance to these first-generation EGFR TKI conferred cross resistance (up to 36-fold ΔIC50) to a panel of second- and third-generation TKI relative to sensitive parental lines. Moreover, integrated RNA sequencing (RNA-seq) and chemical proteomics (multiplexed inhibitor beads and mass spectrometry (MIB-MS)) showed that the kinase transcriptome and proteome were rewired in resistant cells: 113 of ~300 detected kinases were differentially expressed (p< 0.05). We then used these techniques to examine acute (≤ 48 h) kinome changes in both sensitive and resistant cells upon treatment with a CNS-penetrant, second-generation EGFR TKI, afatinib. Whereas exposure of treatment-naïve, sensitive cells to afatinib significantly rewired the kinome (120 differentially expressed kinases), the response of resistant cells to drug re-challenge was significantly blunted (13 differentially expressed kinases). A subset of expressed kinases (35 of 263) dynamically responded to afatinib in both sensitive and resistant cells. Overall, upregulated kinases include those implicated in the biology of gliomas (Bmx, Fgfr2) and of other cancers (Pdgfrb, Mapk3/4, Ddr1/2, Pdk2). These kinases thus represent putative druggable targets for dual inhibition therapy. Integrated kinome profiling using MIB-MS and RNA-seq in GBM models with defined mutational profiles provides a powerful framework to identify novel therapeutic targets that could significantly alter current treatment paradigms.

Glioblastoma (GBM) is an aggressive primary brain tumor with a poor survival rate. One of the most common molecular alterations seen in GBM is amplification and/or mutation of the Epidermal Growth Factor Receptor (EGFR), which has made it an attractive therapeutic target. However, several EGFR tyrosine kinase inhibitors have been tested clinically in GBM with minimal success. One reason for this lack of efficacy could be due to acute, adaptive resistance via alternative pathway activation. To investigate this mechanism of tumor resistance, we used RNA-seq and multiplex inhibitor bead/mass spectrometry (MIB-MS) to analyze the transcriptomes and kinomes of genetically engineered murine astrocytes with common GBM genotypes. We have previously shown that 38% of the expressed kinome varied among a panel of diverse nGEM astrocytes harboring Cdkn2a deletion (C) plus Pten deletion (CP), wild-type human EGFR (CE) or EGFRvIII (CEv3) overexpression or both EGFRvIII overexpression and Pten deletion (CEv3P). Although CE have a similar transcriptional profile to C cells at baseline, when treated with the EGFR inhibitor afatinib, CE respond more similarly to CEv3 cells. When cells containing endogenous murine EGFR (C and CP) are treated with afatinib, fewer than 0.5% of kinases showed differential expression. In cells with EGFR overexpression alone, more than 6% of kinases were differentially expressed upon afatinib treatment, including Ntrk3, Fgfr2 and 3, Lyn, Bmx, Epha2 and 5, Fn3k, a kinase involved in fructosamine processing, and Nrbp2, a kinase involved in regulation of apoptosis. This effect was blunted in cells lacking Pten in addition to having EGFRvIII (CEv3P), resulting in less than 2% of kinases being differentially expressed. The only kinase upregulated in all three EGFR-overexpressing cell types was Coq8a, which is involved in electron transport and response to DNA damage. Given this overlap in response, Coq8a could be a potential dual treatment target for GBM.

Gliomas are diffusely invasive brain tumors with fatal outcomes and few effective treatments. Precision medicine focuses on targeting the genetics of individual tumors, but not host genetics, despite studies that have linked germline polymorphisms with glioma risk. Accordingly, glioma survival studies in mice utilize genetically variable tumors on identical host genetic backgrounds, which fails to differentiate between cancer cell-autonomous (CCA) and tumor microenvironment (TME) effects on glioma progression and host survival. The Collaborative Cross (CC) is a panel of genetically diverse mouse strains derived from both wild- and traditional inbred laboratory strains that facilitates high-resolution genetic mapping in models of complex disease. Here, we implement a novel platform to discover genetic modifiers of both CCA and TME phenotypes using genetically defined orthotopic murine allograft gliomas and CC hosts. We stereotactically injected Nf1;Trp53-/-oligodendrocyte progenitor-derived mouse tumor cells into syngeneic C57BL/6 control mice and 14 different CC strains. Seven strains survived significantly longer than controls (P Citation Format: Kasey Skinner, Martin Ferris, Ryan Bash, Abigail Shelton, Erin Smithberger, Steve Angus, Brian Golitz, Noah Sciaky, Jeremy Simon, Jason Stein, Glenn Matsushima, Quinn Ostrom, Lindsay Stetson, Jill Barnholtz-Sloan, Harshil Dhruv, Michael Berens, Fernando Pardo Manuel de Villena, C. Ryan Miller. Tumor microenvironment and host genetics impact glioma progression in a Collaborative Cross-based orthotopic allograft model [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 2745.