Date of Award

6-2019

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Rosamaria Ruggieri, PhD

Second Advisor

Marc Symons, PhD

Abstract

Glioblastoma Multiforme (GBM) is the most common primary brain tumor with a median survival of less than two years from the time of diagnosis. Survival trends for GBM have not improved significantly over the past decade. The blood-brain barrier (BBB), limited diffusion through the brain parenchyma, “undruggable” molecular targets and intra-tumoral heterogeneity represent significant challenges to the development of new effective therapeutics and inevitably result in tumor recurrence and death. Through extensive genomic profiling, it is known that GBM has a median of three driver mutations which are essential for cancer cell proliferation, survival and invasiveness. Different combinations of these driver mutations are localized to the receptor tyrosine kinase (RTK), p53 and Rb networks. Based on this, the central hypothesis of this thesis is that the simultaneous targeting of the three signaling networks, which are de-regulated in GBM, will optimize treatment.

MicroRNAs (miRNAs) play pivotal roles in eukaryotic post transcriptional gene regulation. Importantly, a single miRNA can modulate the expression of multiple proteins, and hence it was hypothesized in this thesis that an appropriately selected miRNA can target multiple aberrations in deregulated GBM networks and counteract heterogeneity. An in silico analysis identified miR-34a as a potential candidate miRNA to modulate the three signaling networks in GBM. The potential therapeutic effects of miR-34a in GBM were studied in the framework of three specific aims: 1) Characterizing the effects of miR-34a on the GBM deregulated networks and its effects on cell proliferation in 4 different subtypes of GBM; 2) Determining if miR-34a can synergize with temozolomide (TMZ) and if it can sensitize cells with acquired and primary resistance to TMZ; and 3) Implementing nanoparticles which can be loaded with miR for delivery via systemic administration to orthotopically-implanted GBM.

A series of in vitro and in vivo experiments are used to demonstrate that miR-34a strongly inhibits the proliferation of a wide spectrum of glioblastoma cells, including primary patient derived xenograft cultures belonging to the three subtypes of GBM. It is also established in this dissertation that transfection of miR-34a strongly sensitizes GBM cell cultures to TMZ, including cells that show either primary or acquired resistance to TMZ. Importantly, evidence is provided that miR-34a can be effectively delivered to an orthotopic PDX tumor using nanocells derived from bacteria, leading to long term survival in combination with TMZ. These findings have been submitted for publication and are described in detail in chapter 2 of this dissertation.

Additionally, multiple miR-34a regulated genes are identified that are likely to contribute to GBM therapeutic resistance. The expression of these genes is spatially heterogeneous, which strongly suggests that their simultaneous targeting will be beneficial for achieving therapeutic benefit. A preliminary analysis indicates that miR-34a can down-regulate the expression of these genes, suggesting that it can sensitize all the different tumor compartments to TMZ. Furthermore, it is demonstrated that miR-34a can sensitize to radiation (RT) and combination of RT and TMZ. These findings are described in the third chapter of this dissertation.

The data from this dissertation strengthen the preclinical basis for the development of miRNA therapeutics for GBM and suggest that delivery of miR-34a may be a powerful new adjuvant for the treatment of glioblastoma in combination with temozolomide and radiation, mitigating both inter- and intra-tumor heterogeneity. Importantly, miR-34a nanocells can be potentially used in the treatment of both primary and recurrent GBM.

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