Date of Award

Spring 1-1-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

James D. Orth

Second Advisor

Rui Yi

Third Advisor

Tin Tin Su

Fourth Advisor

Ding Xue

Fifth Advisor

Sabrina L. Spencer

Abstract

Exportin-1 is a promising new anti-cancer target for selective inhibitors of nuclear export (SINE) molecules. Selinexor is a first-in-class SINE molecule in clinical trials for a variety of different cancers. Selinexor and other SINE molecules covalently bind exportin-1 and prevent nuclear export of cargo proteins. Over 200 Exportin-1 protein cargoes have been identified, including p53, pRB, IκB, and BRCA1. While early clinical success with inhibitors of Exportin-1 has been observed, the molecular mechanisms of response are still being examined. Previous studies have shown a variety of cell cycle effects and cell death. Here we show that inhibition of Exportin-1 causes DNA damage concurrent with nucleolar dysfunction. DNA damage occurs in about 35-45% of fibrosacrcoma derived cells, and to varying extent in other cancer derived cell lines. DNA damage occurs in G1-phase in 74% of the cells that acquire damage and 26% in S/G2-phase. In addition, more death is observed in cells that acquire DNA damage in G1-phase versus S/G2-phase. DNA damage occurs in large distinct foci, and are believed to be composed of clusters of double strand break sites. The proximity of DNA damage to nucleoli led to the investigation of nucleolar function. When Exportin-1 is inhibited, transcription of ribosomal RNA is reduced and proteins associated with ribosome biogenesis are redistributed within the nucleolus. Both these effects are indicative of nucleolar stress. When RNA polymerase I is acutely inhibited in combination with selinexor, DNA damage formation is no longer increased over control treated cells. In addition, DNA damage formation is blocked when cell cycle progression is inhibited. These data suggest that DNA damage is dependent both on nucleolar function and cell cycle progression. DNA damage formation is enhanced both in cells that have a loss of function for p53 or overexpression of the RNA polymerase I transcription factor, UBF, indicating potential sensitivity in cancer where these two processes are often disrupted. In all, this thesis explores a variety of different pathways in the complex response to Exportin-1 inhibition. It presents a novel anti-cancer mechanism of selinexor, leading to a more complete understanding for therapeutically targeting Exportin-1.

Share

COinS