Elucidating the Mechanism of Ews-Fli1 Induced Oncogenesis

ELUCIDATING THE MECHANISM OF EWS-FLI1 INDUCED ONCOGENESIS

A DISSERTATION

SUBMITTED TO THE PROGRAM OF CANCER BIOLOGY

AND THE COMMITTEE ON GRADUATE STUDIES

OF STANFORD UNIVERSITY

IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Michelle Renee Marques

September 2012

This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/

This dissertation is online at: http://purl.stanford.edu/dk158wf1278

ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Eric Sweet-Cordero, Primary Adviser

I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Michael Cleary

I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Calvin Kuo

I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Michael Longaker

Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education

This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives.

iii ABSTRACT

Ewing’s sarcoma is the second most common malignant bone cancer in children. The prominent defining feature of Ewing’s sarcoma is a translocation event between a member of the FET family of RNA binding proteins and a member of the Ets transcription factor family. The majority of patients have a translocation event between the EWSR1 gene and the FLI1 gene. The EWS-FLI1 translocation was first discovered in 1992 and to date, the mechanism by which EWS-FLI1 induces the formation of Ewing’s sarcomas remains unclear. Understanding the role of EWS-FLI1 in oncogenesis is critical for Ewing’s sarcoma and would have broad implications for other cancers as well. Translocations involving members of the FET or Ets families are also found in leukemia, prostate cancer and other sarcomas. A primary goal of my graduate work has been to develop tools to express EWS-FLI1 in primary human cells as well as in genetically engineered mice to understand how EWS-FLI1 induces oncogenesis and determine the cell of origin in Ewing’s sarcoma.

As recent work suggested that Ewing’s sarcomas arise from a mesenchymal stem/progenitor cell (MSC), we examined the effects of EWS-FLI1 expression in primary human MSCs. We isolated MSCs from pediatric patients at Lucile Packard Children’s Hospital to establish human bone marrow derived MSC lines (which we call HBMs). Through a series of experiments, we learned that the precise expression levels of EWS-FLI1 were critical in determining the effect of this oncogene on primary cells. High expression of EWS-FLI1 was not tolerated in HBMs. In contrast, when expressed at lower levels, stable EWS-FLI1 expression was maintained in HBMs. To elucidate transcriptional targets of EWS-FLI1 in HBMs, we used next-generation sequencing (RNAseq) to identify genes dysregulated by EWS-FLI1. Using this approach we identified 170 targets that constitute an EWS-FLI1 expression signature, including novel target genes. Expression of a subset of these genes was dependent on EWS-FLI1 expression in Ewing’s sarcoma cell lines, validating their regula