Abstract B12: A large-scale transgenic screen in zebrafish identifies TOX as a novel oncogene in T-cell acute lymphoblastic leukemia

Abstract

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric malignancy of thymocytes. To identify molecular pathways underlying T-ALL progression, a large-scale zebrafish transgenic screen was performed in which 38 amplified and over-expressed genes found in human relapsed and refractory T-ALL were assessed for accelerating leukemia onset in the zebrafish T-ALL model. From this analysis, Thymocyte Selection-associated high mobility group box (TOX) was identified as a potent collaborating oncogene that synergized with both MYC and NOTCH to enhance T-ALL aggression. TOX exerts important roles in the development of CD4SP T cells and in myeloid cell development; however, a role for TOX in regulating T-ALL has not been reported. Importantly, TOX is genomically amplified in both human and mouse T-ALL and is highly expressed in a majority of human T-ALL. Early onset T-ALL was associated with increased numbers of transformed clones and elevated proliferation, suggesting that TOX both expands the pool of progenitor cells capable of initiating disease and alters the phenotype of established T-ALL. shRNA knock down studies in human T-ALL cells resulted in potent cell killing associated with elevated apoptosis and late S- phase cell cycle arrest, confirming a critical role for TOX in T-ALL maintenance and continued growth. TOX binding partners were identified by antibody immunoprecipitation followed by Tandem Mass Spectrometry. From this analysis, Ku70 and Ku80 were identified as key binding factors with TOX. This interaction was confirmed by reciprocal pull downs performed in the presence of DNAse. Intriguingly, Ku70-deficient mice have severely impaired double-strand break repair and are predisposed to T-cell lymphoma, suggesting that TOX would be a negative regulator of Ku70/Ku80 function. In support of this, knockdown of TOX in human T-ALL cell lines accelerates double strand break repair as assessed by comet assay and by quantitative assessment of 53BP1 and γH2A.X foci formation following irradiation. Moreover, gain-of-function experiments show that full-length TOX efficiently inhibits non-homologous end-joining (NHEJ), while mutants that lack either the nuclear localization signal or the HMG-box that binds DNA fail to alter the double-strand break repair. Finally, Ku80 recruitment to sites of DNA damage is reduced in TOX-overexpressing cells as assessed by laser-induced DNA damage and real-time imaging analysis. Our data support a role for TOX in accelerating T-ALL onset by suppressing double-strand break repair and subsequent accumulation of DNA alterations resulting in genomic instability. Citation Format: Riadh Lobbardi, Nouran Abdelfattah, Barbara Martinez, Jordan Pinder, Deborah Toiber, Jessica S. Blackburn, Manon De Waard, Graham Dellaire, Raul Mostoslavsky, David M. Langenau. A large-scale transgenic screen in zebrafish identifies TOX as a novel oncogene in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B12.