Date of Award

2016

Document Type

Open Access Dissertation

Department

College of Pharmacy

Sub-Department

Pharmaceutical Sciences

First Advisor

Michael Shtutman

Second Advisor

Igor Roninson

Abstract

Effective cancer treatment depends on the development of therapeutics that efficiently eliminate the disease without significant reduction to quality of life. Targeted therapeutics achieve these goals, but their long-term efficacy is limited by the ability of cancers to become resistant to these therapies over time. The identification of new therapeutic targets is crucial for the future of cancer treatment. In this work we present functional identification and characterization of novel targets for cancer therapy.

Loss-of-function screens are a well-founded approach for the functional identification of new therapeutic targets, and, in combination with high-throughput sequencing technology, can now be performed at a whole genome scale. However, whole genome LOF screens often produce unmanageably large data sets from which a few genes are often arbitrarily selected for further functional studies. We developed a new statistical analysis and applied it to a focused shRNA library to bridge the gap between a whole genome LOF study and the identification of promising targets. This approach revealed a highly promising target gene, DEAD-box helicase 24 (DDX24).

Previously we used a functional screening approach to identify the z-1 subunit of coatomer complex I (COPZ1) as a target that causes cancer-specific cell death. This subunit is encoded by the COPZ1 gene, which is part of a complex system including the homologous gene COPZ2 and intronically encoded microRNAs, miR-148b (COPZ1) and miR-152 (COPZ2). To extend the understanding of how inhibition of COPZ1 causes tumor specific cell death, we probed the cellular signaling pathways which promote cell death and found that depletion of COPZ1 activates the unfolded protein response (UPR), but ultimately promotes cell death by a UPR-independent mechanism.

Finally, we used cross-linking, ligation, and sequencing of hybrids (CLASH), a cutting edge approach to identify direct microRNA targets, to study the microRNA- 148/152 family. MiR-152 has been proposed to play a causal role in the silencing of the COPZ2 gene and the subsequent efficacy of COPZ1 as a therapeutic target. We integrated this target dataset with RNA sequencing analysis to identify the functional target network for the microRNA-148/152 family including genes that computational target prediction algorithms failed to identify.

This work advances the development of new cancer therapeutics at multiple points along the therapeutic development pipeline including identification of novel therapeutic targets, elucidation of pathways responsible for cancer specific sensitivity to therapeutic targets, and functional analysis of cellular consequences of inhibiting a known target.

Available for download on Wednesday, December 12, 2018

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