Graduation Date

Fall 12-15-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Interdisciplinary Graduate Program in Biomedical Sciences

First Advisor

Gargi Ghosal, PhD

Abstract

Ewing sarcoma (EWS) is a malignant pediatric bone cancer. EWS express a fusion oncogene resulting from the reciprocal translocation of EWSR1 an ETS transcription factor. In most EWS tumors, this occurs with FLI1, creating the EWS::FLI1 fusion oncogene which encodes an aberrant transcription factor. Independent of its transcriptional activity, EWS::FLI1 deregulates other cellular processes driving oncogenesis. For cases with a single, localized tumor, roughly 80% of patients remain event-free after five years. Disease dissemination is a problem – about 25% of patients have metastatic disease at diagnosis. Long-term survival for these patients is less than 40%. Presently, no second line standard of care regimen exists for patients with metastatic or relapsed EWS. Identification of novel therapeutic targets is needed to improve patient outcomes, especially for metastatic or recurrent disease. Oncogene expression causes replication stress (RS), which occurs whenever replisome progression is impeded. RS underlies early oncogenesis by creating an environment promoting transformation of precancerous cells. RS is deleterious if unresolved. Consequently, the RS response is important for cell survival in the presence of both endogenous and chemotherapeutic- induced RS. Cancer cells rely on the RS response for survival and evolution toward drug resistance. EWS cells experience elevated endogenous RS, but the mechanism by which EWS cells tolerate it has not been described fully. We observed elevated USP1 levels in EWS cell lines and v patient tumors from transcriptional upregulation by EWS::FLI1. USP1 is a key regulator of both the RS and DDR. USP1 downregulation or inhibition in EWS cell lines decreases cell proliferation and induces apoptotic cell death. Mechanistically, this results from destabilization of Survivin after loss of USP1. Survivin, a pro-survival protein, ensures proper chromosomal segregation during mitosis and suppresses apoptosis throughout the cell cycle. We propose a model wherein USP1 dually promotes EWS cell survival by mitigating the RS response, allowing continuous proliferation despite high levels of RS, in addition to suppressing apoptosis via Survivin. Our work identifies the USP1-Survivin axis as a critical facilitator of EWS cell survival. Importantly, USP1 inhibition sensitizes EWS cells to RS-inducing standard of care agents. By identifying key players in the EWS RS response, targeted therapies can be designed to exploit inherent vulnerabilities of this disease with the goal of improving patient outcomes.

Comments

2023 Copyright, the authors

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