Novel Relationships of Splicing Factors and Regulation in TMZ-Resistant Glioblastoma

Abstract

Glioblastoma (GBM) is a devastating cancer, due to both our narrow understanding of its molecular drivers and limited therapeutic strategies. The blood-brain barrier (BBB) limits the chemotherapeutic options for the treatment of GBM, but the DNA-damaging agent temozolomide (TMZ) can penetrate the BBB and is the first-line standard of care treatment together with surgery and radiation for this tumor. However, most patients’ tumors rapidly become resistant to TMZ, and TMZ-resistant GBM is uniformly fatal. The overall premise of my thesis research was to identify strategies for the second-line treatment of GBM through the study of models and mechanisms of TMZ resistance. To this end, I established two TMZ-resistant GBM cell line pairs. Molecular and phenotypic characterization these GBM TMZ- resistant cell lines indicated that one, 42MGBA-TMZ, captured the “grow” of the “go or grow” model for GBM-resistance. By contrast, the second, 8MBGA-TMZ, exhibited increased in migration, encompassing the “go” aspect of TMZ resistance. One potential mechanistic driver of TMZ resistance is alternative splicing, as the brain contains the most alternatively spliced transcripts of any organ, and the GBM tissue exhibits the deregulated expression of some splicing factors compared with healthy brain tissue. The orphan nuclear receptor - estrogen-related receptor beta (ERRβ) expresses multiple isoforms, and using the 42MGBA-TMZ and 8MBGA-TMZ, I assessed the expression and function of two ERRβ isoforms - ERRβ short form (ERRβ-sf) and ERRβ-2 in GBM. Although ERRβ is an orphan nuclear receptor with no known endogenous ligand, the synthetic agonist DY131 can potently activate the receptor. Previous studies have shown ERRβ-2 to be pro-apoptotic when activated by DY131. I therefore used in silico and in vitro methods to determine proteins required for the expression of the pro-apoptotic ERRβ-2 isoform. I found that the serine/arginine (SR) rich splicing factor SRSF6may function in the generation of the ERRβ-2 isoform, and that inhibition of cdc2-like kinases (CLKs, which phosphorylate SR proteins) with TG-003 in combination with the ERRβ synthetic agonist DY-131 potently inhibits GBM cell migration and growth in vitro. Overall, these studies add two new cell lines to the limited repertoire of TMZ-resistant GBM cell culture models and begin to define the importance of studying ERRβ isoform function and switching as a potential

strategy to target TMZ-resistant GBM cells.