Date of Award

6-3-2025

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell Biology, Neurobiology and Anatomy

First Advisor

Nancy Zeleznik-Le

Abstract

MLL-rearranged (MLL-r) leukemia is an aggressive subset of acute leukemia, accounting for up to 10% of adult and 70% of infant AML and ALL cases. These leukemias are driven by MLL fusion proteins, which recruit epigenetic cofactors to dysregulate gene expression, leading to poor prognosis and resistance to conventional chemotherapy. Previous lab work identified EYA1’s role in leukemogenesis, establishing it as a therapeutic target in MLL-r leukemia. EYA1, is a transcriptional coactivator and protein tyrosine phosphatase (PTP). Originally a gout medication, benzbromarone (BBR), was identified in an NCI Diversity Set screen as an EYA1-specific PTP inhibitor. BBR provides a tool to investigate the role of EYA1 PTP activity in MLL-r leukemia and its potential as a therapeutic target. The effects of EYA1 PTP inhibition were evaluated using viability assays, morphological assessments, surface marker and intracellular expression analyses, senescence measurements, RNA Pol II CTD Tyr1 phosphorylation studies, leukemia transplantation models, and combination treatments with Menin-MLL and DOT1L inhibitors. BBR treatment significantly reduced cell viability in responsive MLL-r and MLL-nr leukemia cell lines while sparing normal hCD34⁺ hematopoietic cells. BBR responsiveness required a threshold-level of EYA1 expression, as nonresponsive MLL-ENL cell lines lacked detectable EYA1 expression. BBR induced cellular senescence, promoted myeloid differentiation, impaired cell cycle progression, and triggered cell death. In vivo, BBR treatment delayed disease onset, prolonged survival, and reduced leukemic burden. Additionally, BBR increased RNA Pol II CTD Tyr1 phosphorylation, implicating unregulated EYA1 PTP activity in transcriptional dysregulation. RNA sequencing and gene set enrichment analysis (GSEA) revealed that BBR treatment modulated pathways related to cell cycle regulation, senescence, and apoptosis, validated by functional assays. Combination treatment with BBR and either VTP50469 or EPZ5676 demonstrated additive or synergistic cytotoxic effects, supporting the rationale for combinatorial therapy. These findings establish EYA1 PTP activity as a critical driver of leukemia progression and provide evidence that BBR, a repurposed gout medication, exerts potent anti-leukemic effects in vitro and in vivo. These results support BBR's potential as a targeted therapy for leukemia and provide a foundation for future preclinical and clinical investigations.

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