Selection-associated High Mobility Group Box Research Articles

Expression pattern, regulation, and clinical significance of TOX in breast cancer.

Thymocyte selection-associated high mobility group box protein (TOX) is a transcription factor implicated in the regulation of T cell exhaustion during chronic infection and cancer. While TOX is being targeted for cancer immunotherapy, limited information is available about its significance in breast cancer and other solid tumors. We performed a comprehensive analysis of TOX gene expression, its epigenetic regulation, protein localization, relation to tumor infiltrating immune cell composition, and prognostic significance in breast cancer using publicly available datasets. Our results suggest an inverse correlation between TOX expression and DNA methylation in tumor cells. However, its expression is elevated in tumor infiltrating immune cells (TIICs), which may compensates for the total TOX levels in the tumor as a whole. Furthermore, higher TOX levels in tumors are associated with T cell exhaustion signatures along with presence of active inflammatory response, including elevated levels of T cell effector cytokines. Survival analysis also confirmed that higher expression of TOX is associated with better prognosis in breast cancer. Therefore, expression of TOX may serve as a novel prognostic marker for this malignancy.

TOX correlates with prognosis, immune infiltration, and T cells exhaustion in lung adenocarcinoma.

BackgroundThymocyte selection‐associated high mobility group box (TOX) plays a crucial role on the development of innate immunity and tumor microenvironment. This study aims to explore the prognostic potential of TOX and comprehensively analyze the correlations between TOX, immune infiltration, and T cells function in diverse cancers particularly lung adenocarcinoma (LUAD).MethodsTIMER was used to analyze TOX expression in different cancers. Potential prognostic value of TOX was evaluated by the PrognoScan, Kaplan‐Meier Plotter, and GEPIA2. The relationships between TOX, immune infiltration, and related gene marker sets were analyzed by TIMER and GEPIA2. Single‐cell RNA‐seq for T cells in LUAD was analyzed to further investigate the correlations between TOX expression and different T cells populations.ResultsTOX downregulates in most of the cancer types and correlates with poor prognosis in LUAD. TOX shows significant impacts on survival of LUAD with early stage, ever‐smoking, or low‐TMB status. Increased TOX expression positively correlates with high immune infiltration levels in most of the immune cells and functional T cells including exhausted T cells. Moreover, multiple key genes of exhausted T cells comprising PD‐1, TIM‐3, TIGHT, and CXCL13 have remarkable interaction with TOX. Specifically, TOX is observed with high enrichment in exhausted CD4+ and CD8+ T cells populations in single‐cell RNA‐seq analysis for LUAD.ConclusionTOX is a prognosis‐related biomarker for multiple cancer types especially LUAD. Increased TOX expression significantly increase immune infiltration levels in most of the immune cells comprising CD8+ T cells, CD4+ T cells, mast cells, and functional T cells. Moreover, we verified that TOX highly correlates with exhausted T cells and is probable a critical regulator promoted T cells exhaustion in LUAD. Detection of TOX expression could help to predict prognosis and regulating TOX expression in exhausted T cells may offer a novel strategy in maximizing immunotherapy efficacy for LUAD.

蕈样肉芽肿皮损中TOX蛋白的表达及其诊断价值评估

Objective To assess the value of thymocyte selection-associated high mobility group box (TOX) protein in the diagnosis of mycosis fungoides (MF) . Methods Totally, 63 paraffin-embedded tissue specimens were collected from Department of Pathology, West China Hospital, Sichuan University between 2003 and 2016, including 30 specimens of suspected MF, 23 specimens of confirmed MF and 10 specimens of benign inflammatory dermatoses. Immunohistochemical staining was performed to compare the expression of TOX among groups. Results Twenty-six (86.7%) suspected MF specimens were positive for TOX, 20 (87%) confirmed MF specimens, and 3 (30%) benign inflammatory dermatosis specimens were positive for TOX. The positive rate of TOX protein was significantly higher in the suspected MF group and MF group than in the benign inflammatory dermatosis group (both P<0.05) . Conclusions TOX protein was expressed to different extents in the cases of suspected or confirmed MF, and its positive rate was markedly higher in the suspected or confirmed MF group than in the benign inflammatory dermatosis group. TOX protein may act as one of molecular markers for MF. Key words: Lymphoma, T-cell; Diagnosis; Mycosis fungoides; Thymocyte selection-associated high mobility group box protein

LncRNA MALAT1 Suppression Protects Endothelium against oxLDL-Induced Inflammation via Inhibiting Expression of MiR-181b Target Gene TOX.

Rare studies have been conducted to investigate the exact interactions between lung adenocarcinoma transcript 1 (MALAT1), thymocyte selection-associated high mobility group box (TOX), and miRNAs in the pathogenesis of atherosclerosis (AS). We aim to investigate the crosstalk between MALAT1 and TOX and evaluate whether the regulatory mechanism was associated with the miRNA network. AS tissues were collected to determine the level of MALAT1 expression in AS patients, together with determination of miR-181b expression. Cultured endothelial cells were utilized to analyze the expressions of MALAT1, miR-181b, and TOX in the presence of oxLDL. Luciferase activity assay was conducted to evaluate the potential target sites of miR-181b on MALAT1 and TOX. In this study, we demonstrated that MALAT1 was upregulated in patients with AS. MALAT1 silencing significantly downregulated the expression of the miR-181b target gene TOX via reversing the effect of miR181b. Importantly, positive modulation of miR181b and inhibition of MALAT1 and TOX significantly attenuated oxLDL-induced endothelial inflammation and oxidative stress. Moreover, the MAPK signal pathways in endothelial cells were also inhibited through regulation of above endogenous RNAs. In summary, MALAT1 suppression protects the endothelium from oxLDL-induced inflammation and oxidative stress in endothelial cells by upregulation of miR-181b and downregulation of TOX.

Sanguinarine inhibits the tumorigenesis of gastric cancer by regulating the TOX/DNA-PKcs/ KU70/80 pathway

Sanguinarine (SAG), a benzophenanthridine alkaloid extracted from Sanguinaria canadensis, exerts antioxidant, anti-inflammatory and antiproliferative activities in a variety of malignancies. However, the underlying mechanisms by which SAG affects the tumorigenesis of gastric cancer (GC) are unclear. The common targets of SAG and GC were identified by network pharmacology, and the association of thymocyte selection-associated high mobility group box (TOX) with the clinicopathological characteristics and prognosis of patients with GC was analyzed by using datasets from The Cancer Genome Atlas (TCGA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays, colony formation assays, flow cytometry analysis, and a xenograft tumor model were conducted to assess the effects of SAG on the growth of GC cells, and Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were used to determine the effects of SAG on the TOX/DNA-PKcs/KU70/80 signaling pathway. We identified 9 collective targets of SAG and GC, of which TOX expression levels were dramatically downregulated in GC tissues compared with adjacent normal tissues, and a low expression of TOX served as an independent prognostic factor of poor survival in patients with GC. SAG suppressed cell viability, colony formation and in vivo tumorigenesis and induced cell apoptosis and cell cycle arrest. Furthermore, SAG increased the expression levels of TOX but decreased those of DNA-PKcs and KU70/80 in GC cells. Our findings indicate that SAG inhibits the tumorigenesis of GC cells by regulating TOX/DNA-PKcs/KU70/80 signaling and may provide therapeutic strategies for the treatment of GC.

From the Editor’s Desk…: October 2019

From the Editor’s Desk…: October 2019

Computer-Aided Discovery of Small Molecule Inhibitors of Thymocyte Selection-Associated High Mobility Group Box Protein (TOX) as Potential Therapeutics for Cutaneous T-Cell Lymphomas.

Cutaneous T-cell lymphomas (CTCL) are the most common primary lymphomas of the skin. We have previously identified thymocyte selection-associated high mobility group (HMG) box protein (TOX) as a promising drug target in CTCL; however, there are currently no small molecules able to directly inhibit TOX. We aimed to address this unmet opportunity by developing anti-TOX therapeutics with the use of computer-aided drug discovery methods. The available NMR-resolved structure of the TOX protein was used to model its DNA-binding HMG-box domain. To investigate the druggability of the corresponding protein–DNA interface on TOX, we performed a pilot virtual screening of 200,000 small molecules using in silico docking and identified ‘hot spots’ for drug-binding on the HMG-box domain. We then performed a large-scale virtual screening of 7.6 million drug-like compounds that were available from the ZINC15 database. As a result, a total of 140 top candidate compounds were selected for subsequent in vitro validation. Of those, 18 small molecules have been characterized as selective TOX inhibitors.

TOX3 is a favorable prognostic indicator and potential immunomodulatory factor in lung adenocarcinoma.

Thymocyte selection-associated high mobility group box (TOX) genes represent a novel family of genes. Deregulated expression of TOXs has been reported in a variety of cancer types, including lung cancer. It has also been reported that TOXs are crucial regulators of the immune system. The present study systematically evaluated the prognostic values of TOX family members using a set of publicly accessible databases, including Oncomine, Kaplan-Meier plotter and cBioPortal. It was revealed that TOX expression profiles differed between lung cancer and normal tissues, and high expression of TOX mRNAs generally predicted improved survival outcomes. Notably, TOX3 expression was significantly increased in lung adenocarcinoma, compared with other pathological subtypes of lung cancer. Survival analysis demonstrated that elevated TOX3 expression was significantly associated with improved progression-free and overall survival in patients with lung adenocarcinoma. Furthermore, correlation analysis indicated that TOX3 expression was negatively correlated with the expression of programmed death-1 receptor (PD-1), PD-ligand 1 and Hepatitis A virus cellular receptor 2 in lung adenocarcinoma. These results indicated that TOX3 is a prognostic indicator and promising immunomodulatory factor in lung adenocarcinoma. Future studies investigating the role of TOX3 in lung cancer immunity are warranted.

TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential1-4. This process, known as T cell exhaustion or dysfunction1, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1)5-8. The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood9-12. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein13-15. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in miceand hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1+ self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.

Single-cell RNA-seq reveals TOX as a key regulator of CD8+ T cell persistence in chronic infection.

Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including TCF1, but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursors before the peak of the T-cell response. We discovered a co-expression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursors. Moreover, TOX promoted persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T-cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.

TOX promotes the exhaustion of antitumor CD8+ T cells by preventing PD1 degradation in hepatocellular carcinoma

The thymocyte selection-associated high mobility group box protein (TOX) plays a vital role in T cell development and differentiation, however, its role in T cell exhaustion was unexplored. Here, we aim to investigate the role of TOX in regulating the antitumor effect of CD8+ T cells in hepatocellular carcinoma. Fully functional, partially and severely exhausted tumor-infiltrating CD8+ T cells were sorted by flow cytometry and subjected to transcriptome sequencing analysis. Upregulated TOX expression was validated by flow cytometry. The antitumor function of CD8+ T cells with TOX downregulation or overexpression was studied in a mouse HCC model and HCC patient-derived xenograft mouse model. Transcriptome sequencing analysis was performed in TOX-overexpressing and control CD8+ T cells. The mechanism underlying the TOX-mediated regulation of PD1 expression was studied by laser confocal detection, immune co-precipitation and flow cytometer. TOX was upregulated in exhausted CD8+ T cells in hepatocellular carcinoma. TOX downregulation in CD8+ T cells inhibited tumor growth, increased CD8+ T cell infiltration, alleviated CD8+ T cell exhaustion and improved the anti-PD1 response of CD8+ T cells. The mechanism behind this involved the binding of TOX to PD1 in the cytoplasm, which facilitated the endocytic recycling of PD1, thus maintaining abundant PD1 expression at the cell surface. High expression of TOX in peripheral CD8+ T cells correlated with poorer anti-PD1 responses and prognosis. TOX promotes CD8+ T cell exhaustion in hepatocellular carcinoma by regulating endocytic recycling of PD1. Downregulating TOX expression in CD8+ T cells exerts synergistic effects with anti-PD1 therapy, highlighting a promising strategy for cancer immunotherapy. Abundant TOX expression in CD8+ T cells impairs their antitumor function in hepatocellular carcinoma. Mechanically, TOX reduces PD1 degradation and promotes PD1 translocation to the cell surface in CD8+ T cells, thus maintaining high PD1 expression at the cell surface. Downregulating TOX expression improves the antitumor function of CD8+ T cells, which shows the synergetic role of anti-PD1 therapy, highlighting a promising strategy for enhancement of cancer immunotherapy.

Time dependent expression of the blood biomarkers EIF2D and TOX in patients with schizophrenia

BackgroundDuring last years, there has been an intensive search for blood biomarkers in schizophrenia to assist in diagnosis, prognosis and clinical management of the disease. MethodsIn this study, we first conducted a weighted gene coexpression network analysis to address differentially expressed genes in peripheral blood from patients with chronic schizophrenia (n = 30) and healthy controls (n = 15). The discriminating performance of the candidate genes was further tested in an independent cohort of patients with first-episode schizophrenia (n = 124) and healthy controls (n = 54), and in postmortem brain samples (cingulate and prefrontal cortices) from patients with schizophrenia (n = 34) and healthy controls (n = 35). ResultsThe expression of the Eukaryotic Translation Initiation Factor 2D (EIF2D) gene, which is involved in protein synthesis regulation, was increased in the chronic patients of schizophrenia. On the contrary, the expression of the Thymocyte Selection-Associated High Mobility Group Box (TOX) gene, involved in immune function, was reduced. EIF2D expression was also altered in first-episode schizophrenia patients, but showing reduced levels. Any of the postmortem brain areas studied did not show differences of expression of both genes. ConclusionsEIF2D and TOX are putative blood markers of chronic patients of schizophrenia, which expression change from the onset to the chronic disease, unraveling new biological pathways that can be used for the development of new intervention strategies in the diagnosis and prognosis of schizophrenia disease.

143 Molecular targeting of the DNA binding domain of TOX protein results in selective inhibition of cutaneous T cell lymphoma cells

Thymocyte selection-associated high mobility group box protein (TOX) is highly enriched in the malignant T cells of cutaneous T cell lymphoma (CTCL). Knocking down its expression resulted in efficient eradication of CTCL cells in culture and in mouse models, suggesting TOX is a desirable target for therapeutic development. Here we describe identification of a novel class of small molecules targeting the DNA binding domain of TOX and evaluation of their ability to inhibit growth and survival of CTCL cells. The 3D structural model of TOX was used for in silico screening of more than 500 million small molecules using computer aided molecular docking algorithms designed for selection of chemical structures with predicted affinity to the DNA binding groove of TOX protein. One hundred and fifty three structures were identified and synthesized. Their activities were evaluated using TOX-dependent and TOX-independent cell lines, revealing 12 molecules with selective inhibitory effects on TOX-dependent CTCL cells. These molecules will serve as lead compounds for structure-activity optimization steps. In conclusion, we showed that small molecules targeting the DNA binding groove of TOX can lead to specific inhibition of CTCL cells, demonstrating the feasibility of this approach for developing therapies for diseases that are dependent on TOX functions, including CTCL.

Value of TOX Immunoexpression in the Diagnosis of Early Mycosis Fungoides

Background: The lack of a specific marker to differentiate malignant from reactiveT-cells makes a definite diagnosis difficult in patients suspected of having mycosisfungoides. This study has evaluated value of thymocyte selection-associated highmobility group box factor expression in the differentiation of early mycosis fungoidesfrom a benign inflammatory dermatosis.Methods:We selected 22 mycosis fungoides cases, 18 cases suspicious for mycosisfungoides, and 12 cases of eczematous dermatitis from patients who attendedDermatology Clinic, Shiraz University of Medical Sciences (2008-2015). The obtainedskin biopsies were immunostained for thymocyte selection-associated high mobilitygroup box factor antigen in addition to pan T cell markers. The slides were evaluatedfor the percentage of tumor cells and intensity of immunoreactivity.Results: From 22 cases of mycosis fungoides, 40.9% showed massive infiltrationof thymocyte selection-associated high mobility group box factor-positive lymphocytes(>30%) with high (3+) intensity in the epidermis; there was no case negative forthymocyte selection-associated high mobility group box factor expression. Only oneeczematous dermatitis case had expression of thymocyte selection-associated highmobility group box factor-positive lymphocytes (>30%) with high intensity and 4 caseswere negative for thymocyte selection-associated high mobility group box factorexpression. The frequency of thymocyte selection-associated high mobility groupbox factor expressing lymphocytes was higher in biopsies from mycosis fungoidescompared to eczematous dermatitis (P<0.05). CD7- cases expressed more thymocyteselection-associated high mobility group box factor-positive lymphocytes in the dermisand epidermis, which were significantly correlated (P=0.013).Conclusion: Thymocyte selection-associated high mobility group box factor, as apositive marker, in combination with pan T cell markers (especially CD7-) can be usefulto detect mycosis fungoides malignant lymphoid cells.

Long noncoding RNA expression profile reveals lncRNAs signature associated with extracellular matrix degradation in kashin-beck disease

Kashin-Beck disease (KBD) is a deformative, endemic osteochondropathy involving degeneration and necrosis of growth plates and articular cartilage. The pathogenesis of KBD is related to gene expression and regulation mechanisms, but long noncoding RNAs (lncRNAs) in KBD have not been investigated. In this study, we identified 316 up-regulated and 631 down-regulated lncRNAs (≥ 2-fold change) in KBD chondrocytes using microarray analysis, of which more than three-quarters were intergenic lncRNAs and antisense lncRNAs. We also identified 232 up-regulated and 427 down-regulated mRNAs (≥ 2-fold change). A lncRNA-mRNA correlation analysis combined 343 lncRNAs and 292 mRNAs to form 509 coding-noncoding gene co-expression networks (CNC networks). Eleven lncRNAs were predicted to have cis-regulated target genes, including NAV2 (neuron navigator 2), TOX (thymocyte selection-associated high mobility group box), LAMA4 (laminin, alpha 4), and DEPTOR (DEP domain containing mTOR-interacting protein). The differentially expressed mRNAs in KBD significantly contribute to biological events associated with the extracellular matrix. Meanwhile, 34 mRNAs and 55 co-expressed lncRNAs constituted a network that influences the extracellular matrix. In the network, FBLN1 and LAMA 4 were the core genes with the highest significance. These novel findings indicate that lncRNAs may play a role in extracellular matrix destruction in KBD.

Multiple Linkage Disequilibrium Mapping Methods to Validate Additive Quantitative Trait Loci in Korean Native Cattle (Hanwoo).

The efficiency of genome-wide association analysis (GWAS) depends on power of detection for quantitative trait loci (QTL) and precision for QTL mapping. In this study, three different strategies for GWAS were applied to detect QTL for carcass quality traits in the Korean cattle, Hanwoo; a linkage disequilibrium single locus regression method (LDRM), a combined linkage and linkage disequilibrium analysis (LDLA) and a BayesCπ approach. The phenotypes of 486 steers were collected for weaning weight (WWT), yearling weight (YWT), carcass weight (CWT), backfat thickness (BFT), longissimus dorsi muscle area, and marbling score (Marb). Also the genotype data for the steers and their sires were scored with the Illumina bovine 50K single nucleotide polymorphism (SNP) chips. For the two former GWAS methods, threshold values were set at false discovery rate <0.01 on a chromosome-wide level, while a cut-off threshold value was set in the latter model, such that the top five windows, each of which comprised 10 adjacent SNPs, were chosen with significant variation for the phenotype. Four major additive QTL from these three methods had high concordance found in 64.1 to 64.9Mb for Bos taurus autosome (BTA) 7 for WWT, 24.3 to 25.4Mb for BTA14 for CWT, 0.5 to 1.5Mb for BTA6 for BFT and 26.3 to 33.4Mb for BTA29 for BFT. Several candidate genes (i.e. glutamate receptor, ionotropic, ampa 1 [GRIA1], family with sequence similarity 110, member B [FAM110B], and thymocyte selection-associated high mobility group box [TOX]) may be identified close to these QTL. Our result suggests that the use of different linkage disequilibrium mapping approaches can provide more reliable chromosome regions to further pinpoint DNA makers or causative genes in these regions.

Differential expression of TOX by skin-infiltrating T cells in Sézary syndrome and erythrodermic dermatitis.

The histopathologic differentiation between Sézary syndrome (SS) and erythrodermic dermatitis may be extremely difficult. In this immunohistochemical study, it was investigated if thymocyte selection-associated high mobility group box protein (TOX) and C-MYC can be used as additional diagnostic markers to differentiate between SS and erythrodermic dermatitis. Paraffin-embedded skin biopsies from 15 SS patients and 17 erythrodermic dermatitis patients were stained and scored for TOX or C-MYC expression. Strong nuclear staining for TOX in more than 50% of skin-infiltrating T cells was observed in 13 of 15 (87%) SS cases, whereas erythrodermic dermatitis cases showed weak nuclear staining in 11-50% (median: 25%) of the T cells; strong nuclear staining as found in SS was never observed in erythrodermic dermatitis. No significant differences in C-MYC expression between SS and erythrodermic dermatitis were found. In most patients of both groups, percentages of C-MYC positive-cells varied between less than 10 and 25% of skin-infiltrating T cells. Our results suggest that strong expression of TOX in more than 50% of skin-infiltrating T cells in erythrodermic skin is a useful marker in the differentiation between SS and erythrodermic dermatitis, whereas staining for C-MYC does not contribute to differential diagnosis.

TOX2 regulates human natural killer cell development by controlling T-BET expression

AbstractThymocyte selection-associated high mobility group box protein family member 2 (TOX2) is a transcription factor belonging to the TOX family that shares a highly conserved high mobility group DNA-binding domain with the other TOX members. Although TOX1 has been shown to be an essential regulator of T-cell and natural killer (NK) cell differentiation in mice, little is known about the roles of the other TOX family members in lymphocyte development, particularly in humans. In this study, we found that TOX2 was preferentially expressed in mature human NK cells (mNK) and was upregulated during in vitro differentiation of NK cells from human umbilical cord blood (UCB)-derived CD34+ cells. Gene silencing of TOX2 intrinsically hindered the transition between early developmental stages of NK cells, whereas overexpression of TOX2 enhanced the development of mNK cells from UCB CD34+ cells. We subsequently found that TOX2 was independent of ETS-1 but could directly upregulate the transcription of TBX21 (encoding T-BET). Overexpression of T-BET rescued the TOX2 knockdown phenotypes. Given the essential function of T-BET in NK cell differentiation, TOX2 therefore plays a crucial role in controlling normal NK cell development by acting upstream of TBX21 transcriptional regulation.

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

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.

Abstract B30: A large-scale transgenic screen in zebrafish identifies TOX as a novel oncogene in T-cell acute lymphoblastic leukemia

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 on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B30.