Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Glazer, Peter

Abstract

The identification and exploitation of DNA repair defects has enabled major advances in treating specific cancers. One such defect was discovered in cancers with isocitrate dehydrogenase 1/2 (IDH1/2) mutations, which confer neomorphic production of the oncometabolite 2-hydroxyglutarate (2-HG). Specifically, 2-HG production disrupts homology-directed repair (HDR) by inducing global histone hypermethylation, via histone demethylase inhibition, masking dynamic epigenetic signaling required for HDR. This HDR deficiency underlies subsequent findings of increased IDH mutant sensitivity to radiation, chemotherapy and DNA damage repair inhibitors, initially focusing on poly(ADP)-ribose polymerase (PARP) inhibitors. The discovery of IDH mutant HDR deficiency, and resulting vulnerabilities, has sparked new clinical investigations in cancers with frequent IDH1/2 mutations, specifically the testing of PARP inhibitors. Therefore, further study of strategies targeting IDH mutant DNA repair is warranted to best direct these efforts, and anticipate pathways of resistance to these new therapies. The overall goal of this thesis was to identify strategies to best exploit IDH mutant cancers’ HDR deficiency and inform ongoing trials testing the use of PARP inhibitors, through recommendations for treatment combination as well as second-line options. To evaluate candidates for PARP inhibitor combination, we considered therapeutics currently under investigation in low-grade gliomas, approximately 80% of which are reported to carry IDH1/2 mutations. Among existing therapeutics, we focused on the small molecule histone deacetylase (HDAC) inhibitor vorinostat due to its established ability to down-regulate important HDR factors. We characterized the effects of vorinostat on IDH mutant DNA repair, and assessed its utility alone and in combination with PARP inhibitors. To evaluate second-line approaches to PARP inhibitors, we established resistant IDH mutant models via serial mouse xenograft studies, generating bulk tumor populations and assessing expression differences along resistance progression. Analysis of bulk populations and derived clonal lines highlighted a PARP inhibitor resistance mechanism centered on end resection and HDR overactivation, established in BRCA1/2-mutant cancers. To study the effects of vorinostat on IDH mutant DNA repair, we utilized a panel of engineered IDH1 mutant and wild-type matched cell lines, a patient-derived IDH1 mutant line and a GFP-based HDR reporter cell line. Vorinostat robustly suppressed HDR factors BRCA1 and RAD51 in a dose-dependent fashion independent of IDH1 mutation status, as established in other cancer models. BRCA1 and RAD51 suppression was mediated via disrupted E2F-factor transcriptional regulation, resulting in a marked decrease in factor expression at the mRNA and protein level. As a result, HDAC inhibitor treatment increased DNA double strand breaks, reduced DNA repair factor foci, and conferred functional HDR deficiency even beyond 2-HG’s effects, resulting in a greater repair crisis in IDH1 mutant cells than their wild-type counterparts. This greater net effect on global DNA damage results in the profound sensitivity of IDH1 mutant cells to vorinostat, and underlies the synthetic lethal relationship of histone deacetylase inhibition and 2-HG production. We demonstrated this IDH mutant sensitivity within engineered matched pairs via short-term cell viability assays, long-term clonogenic survival assays and apoptotic marker induction via Western blot analysis. We also found that vorinostat exhibits strong synergy in combination with radiation and the alkylating-agent temozolomide, the current standard of care for glioma, as well as with PARP inhibitors. The efficacy of PARP inhibitor and HDAC inhibitor combination was demonstrated via robust tumor growth delay in two orthotopic xenograft mouse tumor models, presenting this strategy for future clinical investigation. To characterize PARP inhibitor resistance in IDH mutant cancer, we analyzed bulk populations and clonal lines isolated from PARP inhibitor-treated xenograft tumors of a patient-derived, IDH1 mutant fibrosarcoma cell line. An initial survey of factors implicated in BRCA1/2-mutant cancers via Western blot analysis identified down-regulation of negative HDR regulators REV7, RIF1 and 53BP1 among bulk and clonal models. REV7-knockout conferred PARP inhibitor resistance in IDH1 mutant cells in cell culture and, importantly, in xenograft tumors. Concurrently, REV7 loss mitigates 2-HG’s DNA repair suppression, observed via reduced DSB levels in PARP inhibitor resistant clones and the REV7-knockout line. We completed preliminary investigations into the gene expression profiles of our resistant models via bulk RNA-sequencing analysis, and evaluated top candidate therapeutics for targeting PARP inhibitor resistance. These final experiments present avenues for continued research, detailed in this thesis’s concluding chapter. Overall, these projects collectively present new translational implications for IDH mutant cancers’ HDR deficiency, and identify new strategies for targeting these cancers via DNA damage repair.

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