Tiek, D. M. | Search bioRxiv

bioRxiv2019

Targeting destabilized DNA G-quadruplexes and aberrant splicing in drug-resistant glioblastoma

Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Though initially beneficial, TMZ- resistance is universal and second-line interventions are an unmet clinical need. Here we took advantage the mechanism of action of TMZ to target guanines (G) and investigated G-rich g- quadruplex (G4) and splice site changes that occur upon TMZ-resistance. We report TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulated alternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4 stabilizing drug TMPyP4 or a novel splicing kinase inhibitor of cdc2- like kinase. Finally, we show that the G4 and RNA-binding protein EWSR1 aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples. Together, our findings provide insight into targetable vulnerabilities of TMZ-resistant GBM and present cytoplasmic EWSR1 as a putative biomarker. TeaserTargeting temozolomide mutations in drug resistant glioma via g-quadruplex and splicing modulators with a putative biomarker.

cancer biology↗

bioRxiv2019

Estrogen-related receptor beta activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma

Glioblastoma (GBM; grade 4 glioma) is a highly aggressive and incurable tumor. GBM has recently been characterized as highly dependent on alternative splicing, a critical driver of tumor heterogeneity and plasticity. Estrogen-related receptor beta (ERR{beta}, ESRRB, NR3B2) is an orphan nuclear receptor expressed in the brain, where alternative splicing of the 3 end of the pre-mRNA leads to the production of three validated ERR{beta} protein products - ERR{beta} short form (ERR{beta}sf), ERR{beta}2, and ERR{beta} exon 10-deleted (ERR{beta}-{Delta}10). Our prior studies have shown the ERR{beta}2 isoform to play a role in G2/M cell cycle arrest and induction of apoptosis, in contrast to the function of the shorter ERR{beta}sf isoform in senescence and G1 cell cycle arrest. In this study, we sought to better define the role of the pro-apoptotic ERR{beta}2 isoform in GBM. We show that the ERR{beta}2 isoform is located in the nucleus, but also the cytoplasm. ERR{beta}2 suppresses GBM cell migration, interacts with the actin nucleation-promoting factor cortactin, and an ERR{beta} agonist is able to remodel the actin cytoskeleton and similarly suppress GBM cell migration. We further show that inhibition of the splicing regulatory cdc2-like kinases (CLKs) in combination with an ERR{beta} agonist shifts isoform expression in favor of ERR{beta}2 and potentiates inhibition of growth and migration in GBM cells and intracranial tumors. Financial SupportThis work was supported by R21 CA191444 (RBR), a Georgetown University Medical Center (GUMC) Dean for Researchs Toulmin Pilot Project Award (RBR), and a Partners in Research Breakthrough Award (RBR), as well as F99 CA234799 (DMT) and a student research grant from the Medical Center Graduate Student Organization (MCGSO, to DMT). Additional fellowship funding for DMT and MMH was provided by the Tumor Biology Training Grant (T32 CA009686, PI: Dr. Anna T. Riegel). Technical services were provided by the GUMC Animal Models, Flow Cytometry & Cell Sorting, Histopathology & Tissue, Microscopy & Imaging, and Tissue Culture Shared Resources, which are supported in part by Cancer Center Support Grant P30 CA051008 (PI: Dr. Louis M. Weiner). The Mayo Clinic Brain Tumor Patient-Derived Xenograft National Resource is supported in part by P50 CA108961 (JNS, PI: Dr. Patrick ONeill). The content of this article is the sole responsibility of the authors and does not represent the official views of the National Institutes of Health. Conflict of Interest and Author Contribution StatementThe authors declare no potential conflict of interest. DMT contributed to study design, performed experiments, analyzed data, and wrote the paper. SAK performed experiments, analyzed data, and wrote the paper. CJT performed experiments and analyzed data. MMH performed experiments. SDD performed experiments. JNS provided patient-derived xenograft samples. EG contributed to study design, and performed experiments. RBR contributed to study design, performed experiments, analyzed data, and wrote the paper. All authors reviewed, edited, and approved the manuscript.

cancer biology↗

bioRxiv2017

Alterations in Cell Motility, Proliferation, and Metabolism in Novel Models of Acquired Temozolomide Resistant Glioblastoma

Glioblastoma (GBM) is an aggressive and incurable tumor of the brain with limited treatment options. Current first-line standard of care is the DNA alkylating agent temozolomide (TMZ), but this treatment strategy adds only ~4 months to median survival due to the rapid development of resistance. While some mechanisms of TMZ resistance have been identified, they are not fully understood. There are few effective strategies to manage therapy resistant GBM, and we lack diverse preclinical models of acquired TMZ resistance in which to test therapeutic strategies on TMZ resistant GBM. In this study, we create and characterize two new GBM cell lines resistant to TMZ, based on the 8MGBA and 42MGBA cell lines. Analysis of the TMZ resistant (TMZres) variants in conjunction with their parental, sensitive cell lines shows that acquisition of TMZ resistance is accompanied by broad phenotypic changes, including increased proliferation, migration, chromosomal aberrations and secretion of cytosolic lipids. Importantly, each TMZ resistant model captures a different facet of the \"go\" (8MGBA-TMZres) or \"grow\" (42MGBA-TMZres) hypothesis of GBM behavior. These model systems will be important additions to the available tools for investigators seeking to define molecular mechanisms of acquired TMZ resistance.

cancer biology↗