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Purpose: Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor in adults with a median age of onset of 68 to 70 years old. Although advanced age is often associated with poorer GBM patient survival, the predominant source(s) of maladaptive aging effects remains to be established. Here, we studied intratumoral and extratumoral relationships between adult patients with GBM and mice with brain tumors across the lifespan., Experimental Design: Electronic health records at Northwestern Medicine and the NCI SEER databases were evaluated for GBM patient age and overall survival. The commercial Tempus and Caris databases, as well as The Cancer Genome Atlas were profiled for gene expression, DNA methylation, and mutational changes with varying GBM patient age. In addition, gene expression analysis was performed on the extratumoral brain of younger and older adult mice with or without a brain tumor. The survival of young and old wild-type or transgenic (INK-ATTAC) mice with a brain tumor was evaluated after treatment with or without senolytics and/or immunotherapy., Results: Human patients with GBM ≥65 years of age had a significantly decreased survival compared with their younger counterparts. While the intra-GBM molecular profiles were similar between younger and older patients with GBM, non-tumor brain tissue had a significantly different gene expression profile between young and old mice with a brain tumor and the eradication of senescent cells improved immunotherapy-dependent survival of old but not young mice., Conclusions: This work suggests a potential benefit for combining senolytics with immunotherapy in older patients with GBM., (©2023 The Authors; Published by the American Association for Cancer Research.)

Purpose: Selective RAF-targeted therapy is effective in some patients with BRAF V600E -mutated glioma, though emergent and adaptive resistance occurs through ill-defined mechanisms., Experimental Design: Paired pre-/post- RAF inhibitor (RAFi)-treated glioma samples ( N = 15) were obtained and queried for treatment-emergent genomic alterations using DNA and RNA sequencing (RNA-seq). Functional validation of putative resistance mechanisms was performed using established and patient-derived BRAF V600E -mutant glioma cell lines., Results: Analysis of 15 tissue sample pairs identified 13 alterations conferring putative resistance were identified among nine paired samples (including mutations involving ERRFI1, BAP1, ANKHD1 , and MAP2K1 ). We performed functional validation of mechanisms of resistance, including loss of NF1, PTEN , or CBL , in BRAF V600E -mutant glioma lines, and demonstrate they are capable of conferring resistance in vitro . Knockdown of CBL resulted in increased EGFR expression and phosphorylation, a possible mechanism for maintaining ERK signaling within the cell. Combination therapy with a MEKi or EGFR inhibitor was able to overcome resistance to BRAFi, in NF1 knockdown and CBL knockdown, respectively. Restoration of wild-type PTEN in B76 cells (PTEN -/- ) restored sensitivity to BRAFi. We identified and validated CRAF upregulation as a mechanism of resistance in one resistant sample. RNA-seq analysis identified two emergent expression patterns in resistant samples, consistent with expression patterns of known glioma subtypes., Conclusions: Resistance mechanisms to BRAFi in glioma are varied and may predict effective precision combinations of targeted therapy, highlighting the importance of a personalized approach., (©2021 The Authors; Published by the American Association for Cancer Research.)

The treatment of glioblastoma (GBM) remains challenging in part due to the presence of stem-like tumor-propagating cells that are resistant to standard therapies consisting of radiation and temozolomide. Among the novel and targeted agents under evaluation for the treatment of GBM are BRAF/MAPK inhibitors, but their effects on tumor-propagating cells are unclear. Here, we characterized the behaviors of CD133(+) tumor-propagating cells isolated from primary GBM cell lines. We show that CD133(+) cells exhibited decreased sensitivity to the antiproliferative effects of BRAF/MAPK inhibition compared to CD133(-) cells. Furthermore, CD133(+) cells exhibited an extended G2-M phase and increased polarized asymmetric cell divisions. At the molecular level, we observed that polo-like kinase (PLK) 1 activity was elevated in CD133(+) cells, prompting our investigation of BRAF/PLK1 combination treatment effects in an orthotopic GBM xenograft model. Combined inhibition of BRAF and PLK1 resulted in significantly greater antiproliferative and proapoptotic effects beyond those achieved by monotherapy (P < 0.05). We propose that PLK1 activity controls a polarity checkpoint and compensates for BRAF/MAPK inhibition in CD133(+) cells, suggesting the need for concurrent PLK1 inhibition to improve antitumor activity against a therapy-resistant cell compartment., (©2015 American Association for Cancer Research.)

Loss of neurofibromin 1 (NF1) leads to hyperactivation of RAS, which in turn signals through the RAF/MEK/ERK and phosphoinositide 3-kinase (PI3K)/mTOR pathways to regulate cell growth and survival. Because NF1-deficient acute myeloid leukemias are sensitive to MEK inhibitors, we investigated here whether NF1-deficient glioblastoma multiforme (GBM) would respond to MEK inhibition. In 19 GBM cell lines, we found that treatment with the clinically available MEK inhibitors PD0325901 or AZD6244 decreased levels of phospho-ERK, the downstream effector of MEK, regardless of NF1 status. However, growth inhibition occurred only in a subset of NF1-deficient cells, in association with decreased levels of cyclin D1, increased levels of p27, and G1 arrest. As a single agent, PD0325901 suppressed the growth of NF1-deficient, MEK inhibitor-sensitive cells in vivo as well. Mechanistically, NF1-deficient, MEK inhibitor-sensitive cells were dependent upon the RAF/MEK/ERK pathway for growth and did not activate the PI3K pathway as a mechanism of acquired resistance. Importantly, NF1-deficient cells intrinsically resistant to MEK inhibition were sensitized by the addition of the dual PI3K/mTOR inhibitor PI-103. Taken together, our findings indicate that a subset of NF1-deficient GBMs may respond to MEK inhibitors currently being tested in clinical trials., (©2012 AACR.)

Unlabelled: Although mutational activation of the epidermal growth factor receptor (EGFR) features prominently in glioma and non-small cell lung cancer (NSCLC), inhibitors of EGFR improve survival only in patients with NCSLC. To understand how mutations in EGFR influence response to therapy, we generated glioma cells expressing either glioma- or NSCLC-derived alleles and quantified kinase-site occupancy by clinical inhibitors with the use of a novel affinity probe and kinetic methodology. At equivalent doses, erlotinib achieved lower kinase-site occupancy in glioma-derived EGFRvIII compared with NSCLC-derived EGFR mutants. Kinase-site occupancy correlated directly with cell-cycle arrest. EGFRvIII released erlotinib rapidly compared with wild-type EGFR, whereas NSCLC-derived mutants released erlotinib slowly., Significance: These data suggest that kinase-site occupancy is a biomarker for efficacy of EGFR inhibitors, that rapid binding and release of erlotinib in glioma-derived EGFRvIII opposes the blockade of downstream signaling, and that slower cycling of erlotinib within the active site of NSCLC-derived mutants underlies their improved clinical response., (© 2012 AACR)