Date of Award

January 2017

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Ranjit S. Bindra

Abstract

High-grade gliomas (HGGs) are devastating malignancies of the central nervous system, and few treatment options are available for these tumors. In the most malignant form of the disease, glioblastoma multiforme (GBM), over 90% of patients will succumb to their tumor within 5 years after standard of care treatment, consisting of surgery, radiation therapy, and temozolomide chemotherapy. It is now clear that gliomas are molecularly heterogeneous entities, with mutations in tumor suppressors and oncogenes defining many distinct sub-types with important therapy implications. However, almost all HGGs are treated with a limited array of initial therapies, regardless of these molecular differences. Isocitrate dehydrogenase 1 (IDH1), a gene recently found to be mutated in many gliomas, is involved in the conversion of isocitrate to 2-oxoglutarate in cells. The IDH1 R132H mutant enzyme converts 2-oxoglutarate to the oncometabolite (R)-2- hydroxyglutarate (2HG), which leads to profound metabolic alterations in tumor cells. In addition, recent studies indicate that mutations in IDH1 may also induce altered DSB repair, differential sensitivities to chemo-radiotherapy, and substantial changes in chromatin modifications. Here, we present the creation of a novel HeLa cell line harboring an engineered IDH1 R132H mutation at the endogenous gene locus using CRISPR-Cas9 gene editing. We validated the cell line using a variety of biochemical and

functional assays. In particular, we demonstrated that our mutant cell clones secrete high levels of 2HG, and confirmed that the levels of this oncometabolite can be suppressed with small molecule inhibitors of mutant IDH1. We then performed a focused drug screen using select small molecule inhibitors of DNA repair, leveraging our observation that IDH1 mutant cells are more sensitive to radiation. We report that IDH1-R132h confers increased sensitivity to BMN-673, a PARP inhibitor known to preferentially kill cells with decreased homologous recombination (HR) functionality. We also demonstrate synergy between BMN-673 and the platinating agent, cisplatin, that is enriched by the IDH1-R132H mutation. Finally, preliminary gene expression analysis does not identify any significant decreased expression in a panel of DNA repair-related genes, suggesting that some alternative mechanism may be responsible for the drug sensitivity effect we see. Taken together, these findings suggest that IDH1 mutant tumors may be sensitive to PARP inhibition, representing a new treatment strategy for a devastating disease.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 06/12/2018

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