Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

Dr. James Koropatnick

Abstract

Genomic instability and a high mutation rate lead to heterogeneity in human tumors. Mathematical modelling predicts that these characteristics promote acquired resistance to cytotoxic and targeted therapies, by increasing the likelihood that resistant subpopulations exist at the start of treatment (and promoting the accumulation of de novo resistance mutations during treatment). As a result, genome plasticity promotes increased fitness on the population level, but individual tumor cells must nonetheless maintain a level of DNA integrity that allows for continued survival, particularly in the context of DNA-damaging therapy (which DNA repair counteracts). Thus, DNA repair proteins are a source of innate resistance to many common anti-cancer drugs, and represent intriguing targets for therapeutic attack.

One way to forestall treatment resistance is to sensitize tumor cells to DNA-damaging therapy by inhibiting DNA repair and decreasing survival following drug treatment. I developed the concept of complementary lethality’, defined as “potentiation of drug therapy by the inhibition of DNA repair factors responsible for resistance to that specific drug”. BRCA2 is involved in homologous recombination repair (HRR) of double stranded breaks (DSBs) in DNA, and mutations in the BRCA2 gene predispose to various cancers. However, patients with BRCA2-mutated tumors respond more favourably to therapy. I found that inhibition of BRCA2 with siRNA sensitized tumor cells to DNA-damaging drugs and thus overcame innate resistance to their action. Combined inhibition of BRCA2 and thymidylate synthase (TS), the enzyme responsible for de novo synthesis of thymidylate (and the source of innate resistance to several treatments), rendered tumor cells responsive to a broader range of drugs and created a state of multi-drug sensitivity.

Based on these results, I created a novel BRCA2-targeting antisense oligodeoxynucleotide (ASO) and tested it in the context of cisplatin treatment. ASOs exhibit several advantages over siRNAs in vivo, and some ASO-based drugs have been approved by the FDA. I found that BRCA2 downregulation (with the BRCA2 ASO) enhanced the ability of cisplatin (a cytotoxic, DNA-damaging drug) to control tumor cell proliferation in vitro and metastasis in vivo, and also induced alterations in cellular metabolism.

Further studies using the PARP-1 inhibitor olaparib, which is selectively lethal in cells with HRR deficiency, led me to formulate the concept of “reciprocal positive selection for weakness”: in a population heterogeneous for HRR-proficiency, olaparib selects for HRR-proficient cells, while BRCA2 inhibition selects for HRR-deficient cells. Each individual treatment thus selects for cells ‘weak’ to the other in a reciprocal manner, and combined inhibition of both targets should prevent selection-mediated escape. This is a strategy that aims to prevent acquired resistance in a heterogeneous tumor population by nullifying enrichment of specific cell subpopulations.

I found that BRCA2 inhibition can render HRR-positive cancer cells (with innate resistance to olaparib) sensitive to PARP inhibition. Furthermore, olaparib monotherapy in a primarily HRR-deficient mixed cell population (3:1 ratio of HRR-deficient:HRR-proficient cells) induced resistance to further olaparib treatment after just one dose. This parallels clinical reports which show that patients with BRCA2-mutated tumors can present with tumors harboring functional BRCA2 protein following olaparib therapy, presumably due to treatment-mediated selection of subclones present at the start of therapy. In my experiments, co-treatment of this same mixed population with BRCA2 ASO and olaparib prevented enrichment based on HRR-proficiency and eliminated the tumor cell population. In addition, treatment of ovarian tumor-bearing mice with BRCA2 siRNA and olaparib in vivo decreased both the number and weight of tumor nodules when compared with each treatment individually. These studies highlight the important role that DNA repair mediators such as BRCA2 play in innate and acquired resistance to treatment, and provide rationale for therapeutic targeting of DNA repair in human tumors.

Available for download on Sunday, October 01, 2017


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