Targeting Braf And Nf1 Mutations In Glioma

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Abstract
Gliomas are responsible for a disproportionate share of cancer-related morbidity and mortality, despite being less common than other cancers, so treatments for glioma patients are a serious unmet need. About 3% of gliomas harbor either constitutively activating point mutations or fusions in the gene BRAF which activate the BRAF MEK-ERK pathway. Because there are specific inhibitors for this pathway available, in this work we interrogated the underling biology of the sensitivity of BRAF-mutant gliomas to these inhibitors. We identified cell lines with BRAF mutations and showed that BRAF-V600E gliomas are dependent on BRAF signaling and MEK kinase activity for tumor maintenance. Next we showed that the monomeric BRAF inhibitor vemurafenib is able to inhibit mutant-BRAF signaling in glioma, However, this inhibition is transient, and ERK phosphorylation quickly rebounds. This ERK rebound is associated with incomplete inhibition of Cyclin D1 and lack of growth suppression. We then sought to identify possible candidate RTKs that were upregulated by relief of ERK-dependent feedback and might reactivate ERK signaling. We found that ERBB family members and their activity were upregulated. However, we also found that EGFR and HER2 kinase activity does not appear to be necessary in order to induce ERK rebound. We then looked into the role of negative feedback on RAS signaling and showed that vemurafenib relieves negative feedback on RAS signaling, leading to increases in RAS-GTP and the induction of a vemurafenib insensitive state. However, drugs that target the RAF dimers that are a product of increased RAS-GTP are able potently suppress ERK rebound, Cyclin D1 expression, and achieve greater growth suppression. Finally, we tried to understand the role of the RAS-GAP NF1 in GBM. We established and biochemically validated an inducible NF1 expression system and examined its biological role. Interestingly, NF1 expression is not growth suppressive in all NF1-deficient cell lines. Despite this, NF1-deficient cell lines appear to maintain a MEK dependency that can be therapeutically targeted with a MEK inhibitor, even though NF1 reconstitution has little direct effect on MEK and ERK signaling. Together, this works describes alterations at two different nodes of the MAP-Kinase pathway and their therapeutic relevance.
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2016
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BRAF; ERK; glioblastoma; glioma; RAS; vemurafenib
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Pharmacology
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Doctor of Philosophy
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Government Document
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Attribution-NonCommercial-NoDerivatives 4.0 International
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dissertation or thesis
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