Thymocyte Selection-Associated Hmg Box Protein (Tox) Induces Genomic Instability In T-Cell Acute Lymphoblastic Leukemia

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LAMYC and NOTCH are major oncogenic drivers in T-cell Acute Lymphoblastic Leukemia (T-ALL), yet additional collaborating genetic lesions collaborate to induce frank malignancy. To identify these factors, a large-scale transgenic screen was completed where 28 amplified and over-expressed genes found in human T-ALL were assessed for accelerating leukemia onset in the zebrafish transgenic model. From this analysis, Thymocyte selection-associated HMG protein (TOX) synergized with both MYC and NOTCH to induce T-ALL. Here, we show that TOX is highly expressed in 95% of human primary and relapse T-ALL when compared with both normal T cells and B-ALL. TOX is highly and specifically expressed in human T-ALL due to both genomic amplification and transcriptional regulation by the master transcription factors MYB/LMO2. Characterization of zebrafish T-ALLs revealed that TOX promoted genomic instability as assessed by changes in DNA content and Whole Genome Sequencing. Effects on genomic instability were confirmed by metaphase spread following TOX expression in MEF cells, confirming roles for TOX in regulating genomic instability and elevated DNA translocation potential in a wider range of cell types. To identify TOX binding partners, Tandem Mass Spectrometry was performed in human T-ALL cells. TOX was found to interact with KU70/KU80 but not other DNA repair enzymes, a result verified by co-immunoprecipitation studies. Given that TOX elevated genomic instability in the zebrafish model, that Ku70 or Ku80 loss lead to genomic instability and T cell lymphoma in mice, and that TOX bound specifically to KU70/KU80 - the initiating factors required for Non-Homologous End Joining (NHEJ) repair - we hypothesized that TOX is a negative regulator of double-strand break repair. Fluorescent repair assays were completed in 3T3 fibroblasts and confirmed that TOX inhibits NHEJ. Dynamic real-time imaging studies showed that TOX suppresses recruitment of fluorescent-tagged KU80 to DNA breaks. Importantly, TOX loss of function increased NHEJ in human T-ALL cells and reduced time to DNA repair as assessed by fluorescent Traffic Light Reporter assays and quantitative assessment of 53BP1 and γH2A.X foci resolution following irradiation. Our results show that TOX is aberrantly re-activated in 95% of human T-ALL, thereby suppressing KU70/KU80 function to promote genomic instability and ultimately elevating rates at which acquired mutations and rearrangements are amassed in developing pre-malignant T cells. Our work shows that TOX is the major oncogenic driver of genomic instability human T-ALL and locks cells in a constant state of dampened repair.Citation Format: Riadh Lobbardi, Jordan Pinder, Barbara Martinez-Pastor, Jessica Blackburn, Brian J. Abraham, Marc Mansour, Nouran S. Abdelfattah, Aleksey Molodtsov, Gabriela Alexe, Debra Toiber, Manon de Waard, Esha Jain, Deepak Bhere, Khalid Shah, Alejandro Gutierrez, Kimberly Stegmaier, Lewis B. Silverman, Ruslan Sadreyev, John Asara, A Thomas Look, Richard A. Young, Raul Mostoslavsky, Graham Dellaire, David M. Langenau. Thymocyte selection-associated HMG box protein (TOX) induces genomic instability in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3583.