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 anti-proliferative 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 anti-proliferative and pro-apoptotic 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.
Inhibitors of BRAFV600E kinase are currently under investigations in preclinical and clinical studies involving BRAFV600E glioma. Studies demonstrated clinical response to such individualized therapy in the majority of patients whereas in some patients tumors continue to grow despite treatment. To study resistance mechanisms, which include feedback activation of mitogen-activated protein kinase (MAPK) signaling in melanoma, we developed a luciferase-modified cell line (2341luc) from a BrafV600E mutant and Cdkn2a- deficient murine high-grade glioma, and analyzed its molecular responses to BRAFV600E- and MAPK kinase (MEK)-targeted inhibition. Immunocompetent, syngeneic FVB/N mice with intracranial grafts of 2341luc were tested for effects of BRAFV600E and MEK inhibitor treatments, with bioluminescence imaging up to 14-days after start of treatment and survival analysis as primary indicators of inhibitor activity. Intracranial injected tumor cells consistently generated high-grade glioma-like tumors in syngeneic mice. Intraperitoneal daily delivery of BRAFV600E inhibitor dabrafenib only transiently suppressed MAPK signaling, and rather increased Akt signaling and failed to extend survival for mice with intracranial 2341luc tumor. MEK inhibitor trametinib delivered by oral gavage daily suppressed MAPK pathway more effectively and had a more durable anti-growth effect than dabrafenib as well as a significant survival benefit. Compared with either agent alone, combined BRAFV600E and MEK inhibitor treatment was more effective in reducing tumor growth and extending animal subject survival, as corresponding to sustained MAPK pathway inhibition. Results derived from the 2341luc engraftment model application have clinical implications for the management of BRAFV600E glioma.
BRAF and MEK inhibitor combinations have shown promising results against several types of BRAF V600E mutated cancers. Patients with high-grade BRAF V600E mutated gliomas frequently experience therapy failure with concurrent BRAF V600E and MEK inhibition (BRAFi+MEKi). A step toward overcoming therapy resistance includes improving the understanding how these inhibitors affect tumor cells and (immune) microenvironment. In novel syngeneic murine models and patient-derived cell lines of BRAF V600E-mutated high-grade astrocytomas we analyzed effects of BRAF V600E expression and BRAF V600E inhibitor Dabrafenib and MEK inhibitor Trametinib. BRAFi+MEKi promoted glial differentiation as determined by immunostaining and RNA expression profiling. In addition, increased interferon alpha and gamma signatures and pro-inflammatory cytokines were detected in response to BRAFi+MEKi. Quantitative rtPCR validated upregulation of known downstream targets of interferon gamma, the major histocompatibility genes, and programmed death (PD-1) receptor signaling. Cytometry analyses showed heightened frequency of tumor-infiltrating T cells positive for PD-1 and tumor cells co-expressing programmed death ligand-1 (PD-L1). Combining BRAFi+MEKi with dual immune checkpoint inhibition by anti-PD-L1 and anti-CTLA-4 treatment decreased T cell deactivation and resulted in a T cell-dependent survival benefit of mice with orthotopic BRAF V600E-mutated high-grade glioma. These data showed that clinically relevant BRAFi+MEKi sensitizes BRAF V600E-mutated gliomas to the anti-tumor activity of concurrent dual immune checkpoint blockade. Immunofluorescence staining identified an active T cell infiltrate in human MAPK pathway altered gliomas. Collectively, our data suggest that an improved therapeutic benefit could be derived from combining BRAFi+MEKi with immune checkpoint inhibitors in patients with BRAF V600E high-grade gliomas.
In the last decade evidence has accumulated that suggests that the cerebellum is involved not only in motor but also in behavioral and cognitive functions. A myriad of anatomical, clinical and imaging studies support that assumption. The lengthened survival of patients with cerebellar tumors has also brought an increased awareness of neurocognitive deficits to the neurooncological community. Although evidence from neurosurgical case series exists that clearly demonstrates that patients afflicted from posterior fossa tumors are at high risk for long-term cognitive or adaptive deficits, there is still a lack of systematic translational review on this issue. Accordingly a systematic review was conducted to summarize the impact of cerebellar lesions on behavior and cognition. The findings and clinical implications are discussed in the light of the recent advances in neuroimaging techniques.
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