Abstract 3865: Thymocyte selection-associated HMG box protein (TOX) induces genomic instability in T-cell acute lymphoblastic leukemia

Abstract

Abstract MYC and NOTCH are major oncogenic drivers in T-cell Acute Lymphoblastic Leukemia (T-ALL), yet additional collaborating genetic lesions likely 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 a zebrafish transgenic model. From this analysis, Thymocyte selection-associated HMG protein (TOX) synergized with both MYC and NOTCH to induce T-ALL. TOX is dynamically regulated in T cell development with peak expression occurring when thymocytes are actively undergoing T cell receptor (TCR) recombination. Despite TOX being genomically amplified in a subset of human and mouse T-ALL and being overexpressed near all human T-ALL, a role for TOX in inducing T-ALL has not been reported. Characterization of zebrafish T-ALLs revealed that TOX expands the overall number of malignant T-ALL clones and promoted genomic instability as assessed by changes in DNA content and Whole Genome Sequencing. To identify TOX binding partners, TOX immunoprecipitation-Tandem Mass Spectrometry has been 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 and 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 KU70 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. Given the prominent role TOX has in T cell development and its coordinated regulation during active TCRβ and TCRα rearrangement, it is likely that the normal function of TOX is to transiently suppress the NHEJ pathway during RAG-mediated recombination. Prolonging the time to DNA repair would likely facilitate long-range repair across VDJ segments. In the setting of T-ALL, TOX is aberrantly re-activated, 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. Citation Format: Riadh Lobbardi, Jordan Pinder, Barbara Martinez, Jessica Blackburn, Nouran Abdelfattah, Debra Toiber, Manon De Waard, Esha Jain, Ruslan Sadreyev, John Asara, 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 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 3865. doi:10.1158/1538-7445.AM2015-3865