Abstract The clinical challenges associated with T-cell acute lymphoblastic leukemia (T-ALL) are two-fold: 1) the recently improved cure rate for primary T-ALL is largely attributed to highly toxic chemotherapy regimens that have both short- and long-term adverse effects in patients, and 2) chemotherapy is often ineffective against relapsed T-ALL, which has a dismal 5-year survival rate of <30% in children and <10% in adults. The development of new and better chemotherapies requires a detailed understanding of the genes and pathways that drive T-ALL malignancy. We have completed an unbiased cell transplantation screen using a zebrafish model of T-ALL and in excess of 6,000 adult recipient animals to identify molecular targets that promote T-ALL progression and relapse. Single leukemic cells were grown in syngeneic recipient fish and animals were assessed for differences in growth, leukemia propagating cell frequency, and therapy resistance. Serial transplantation experiments then followed evolution within 48 single-cell clones, identifying 6 clones that evolved increased LPC frequency and/or elevated growth potential. Comparative genomic hybridization arrays were used to identify recurrent amplifications associated with clonal evolution, and identified Protein Tyrosine Phosphatase 4A3 (PRL3) as being genomically amplified in 30% of clones with elevated LPC frequency and growth. Real-time quantitative PCR showed that 90% of clones with high LPC frequency and growth expressed high levels of PRL3, suggesting additional genetic pathways likely activate PRL3. PRL-3 was also genomically amplified in a subset of human T-ALL and highly expressed in 58% of primary T-ALL patient samples, suggesting that this phosphatase is an important and previously undefined driver of human T-ALL. PRL3 has not been previously associated with leukemia progression or survival, although it is expressed in Multiple Myeloma and Acute Myelogenous Leukemia. PRL3 knock-down in human T-ALL significantly reduced the viability of cell lines in vitro and in xenograft models (p<0.0001). Additionally, a specific PRL3 inhibitor strongly induced apoptosis of PRL3-expressing T-ALL cells in a dose-dependent manner. PRL3 inhibition also killed T-ALL cell lines that were resistant to dexamethasone, the standard chemotherapy for T-ALL, suggesting that therapies that inactivate PRL3 will be useful for the treatment of refractory and relapse disease. We have also identified several FDA-approved, general phosphatase inhibitors that have potent anti-PRL3 activity and are capable of killing T-ALL cells in vitro. Current work is focused on moving these inhibitors into pre-clinical testing using patient-derived xenografts. Phospho-profiling approaches are also being used to identify the substrates of PRL3 tyrosine phosphatase activity that are critical to T-ALL survival and may represent new, tractable drug targets for the treatment of T-ALL and other types of leukemias. Note: This abstract was not presented at the meeting. Citation Format: Jessica S. Blackburn, Aleksey Molodstov, Riadh Lobbardi, Finola Moore, David Langeau. The tyrosine phosphatase PRL3 as a novel drug target in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1725. doi:10.1158/1538-7445.AM2015-1725