T-cell Transformation Research Articles

Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip

Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis.

ETV6 Is An Early T-Cell Progenitor (ETP) Specific Tumor Suppressor Gene in Adult T-ALL

Abstract 406T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease in which genetic lesions coordinately affect cell proliferation, differentiation and survival of thymocytes. Although pediatric T-ALL has been extensively studied on the genomic level during the last decade, large scale genomic studies in adult T-ALL are sparse. Importantly, significant differences in treatment outcome are present between pediatric and adult T-ALL.Here we performed integrative analysis of gene expression, copy number alterations and mutation analysis on 58 adult T-ALL samples treated in the Eastern Cooperative Oncology Group (ECOG) E2993 protocol. Unsupervised and consensus clustering of microarray gene expression data in this series revealed the presence of 2 stable gene expression clusters resembling immature/ETP (n=30) and typical (n=28) T-ALL gene signatures. Indeed, gene set enrichment analysis (GSEA) confirmed that our ETP/immature adult T-ALL signature closely resembled the previously published ETP profiles. In this adult patient cohort, the immature/ETP patient samples were associated with poor outcome (5 year OS 36% for ETP and 58% for non ETP TALL, P=0.088). Additional supervised analyses revealed differential expression of ERAP2, a gene involved in the processing of MHC peptides, between early relapse and long time survivor adult T-ALL patients. Consistently low ERAP2 expression was associated poor prognosis in adult T-ALL (OS 26%, 36% and 78% for 1st 2nd and 3rd terciles respectively, P=0.016). In addition, absence of TCR gamma deletion (p=0.013) as well as the absence of biallelic CDKN2A/CDKN2B deletion (p=0.009) were shown to be poor prognostic markers in adult T-ALL.To gain additional insight in the biology of adult ETP T-ALL, we performed GSEA analysis using the immature/ETP gene expression profiles. This analysis revealed that the adult ETP T-ALL signature is more closely related to acute myeloid leukemia (AML) profiles as compared to ALL signatures. Given this, we hypothesized that myeloid specific genetic alterations might be uniquely present in ETP adult T-ALL. Mutation analysis of AML oncogenes and tumor suppressor genes revealed the presence of myeloid mutations targeting IDH1, IDH2, DNMT3A, FLT3 and N-RAS in 13/30 (43%) of adult ETP T-ALL cases, whereas only 1/28 (3.5%) typical adult T-ALL showed a mutation in N-RAS. Although these ETP adult T-ALL samples seem to have myeloid properties, they still retain T-lymphoid characteristics including activational NOTCH1 mutations in 12/30 (40%) of ETP adult T-ALLs. Of note, the myeloid specific genetic alterations were preferentially present in NOTCH1 wild type ETP adult T-ALL samples.Next, we used a systems biology approach to identify master regulators that might be able to drive the ETP signature identified in adult T-ALL. For this, we interrogated a T-ALL interactome that was constructed from gene expression profiles of 228 human T-ALL samples. This master regulator analysis identified MN1 as the key transcription factor driving the ETP signature. The MN1 gene was initially identified as fusion partner of TEL (ETV6) in patients with AML. Notably, MN1-TEL expression in multipotent progenitors induces both AML and T-ALL and mutation analysis of ETV6 in adult T-ALL revealed the presence of 10 ETV6 mutants (9 frameshift and 1 splice site mutation) in adult T-ALL. Analysis of bone marrow remission genomic DNA confirmed the somatic origin of ETV6 mutations (3 frameshift and 1 splice site) in each of the 4 patient samples with available material. Most notably, ETV6 mutations were exclusively present in the ETP subtype of adult T-ALL. In addition, 8/10 (80%) of ETV6 mutant ETP T-ALL cases were characterized mutations in NOTCH1, suggesting a specific interaction between the oncogenic NOTCH1 programs and the mutational loss of ETV6 in early T-cell transformation.In conclusion, our integrative genomic analyses in adult T-ALL unraveled a high prevalence of immature/early T-cell progenitor (ETP) T-ALLs, associates the pathogenesis of ETP-TALL with myeloid mutations and identifies ETV6 truncating alleles as the first ETP specific gene mutation that strongly interact with NOTCH1 in T-cell transformation. Disclosures:No relevant conflicts of interest to declare.

Activation of the STAT6 transcription factor in Jurkat T-cells by the herpesvirus saimiri Tip protein

Herpesvirus saimiri (HVS), a T-lymphotropic monkey herpesvirus, induces fulminant T-cell lymphoma in non-natural primate hosts. In addition, it can immortalize human T-cells in vitro. HVS tyrosine kinase-interacting protein (Tip) is an essential viral gene required for T-cell transformation both in vitro and in vivo. In this study, we found that Tip interacts with the STAT6 transcription factor and induces phosphorylation of STAT6 in T-cells. The interaction with STAT6 requires the Tyr(127) residue and Lck-binding domain of Tip, which are indispensable for interleukin (IL)-2-independent T-cell transformation by HVS. It was also demonstrated that Tip induces nuclear translocation of STAT6, as well as activation of STAT6-dependent transcription in Jurkat T-cells. Interestingly, the phosphorylated STAT6 mainly colocalized with vesicles containing Tip within T-cells, but was barely detectable in the nucleus. However, nuclear translocation of phospho-STAT6 and transcriptional activation of STAT6 by IL-4 stimulation were not affected significantly in T-cells expressing Tip. Collectively, these findings suggest that the constitutive activation of STAT6 by Tip in T-cells may contribute to IL-2-independent T-cell transformation by HVS.

TLX1-Induced T-cell Acute Lymphoblastic Leukemia

The TLX1 transcription factor oncogene is frequently activated by chromosomal translocations in T-cell acute lymphoblastic leukemia (T-ALL) and defines a distinct molecular group of tumors characterized by differentiation arrest at the early cortical stage of thymocyte differentiation and excellent response to therapy. Recent developments from the analysis of genomic data on TLX1-specific transcriptional targets and analysis of the molecular mechanisms of TLX1 transformation in human- and mouse-induced leukemias have shown novel insight into the activity of this transcription factor oncogene. Aberrant expression of TLX1 in T-cell progenitors disrupts normal T-cell development and triggers the development of aneuploidy during T-cell transformation. Importantly, the disruption of the mitotic checkpoint in TLX1-induced tumors may be linked not only to the acquisition of secondary genetic alterations in T-ALL but also to increased sensitivity of these tumors to chemotherapy with drugs targeting the formation of the mitotic spindle.

Activation of the I B kinase complex by HTLV-1 Tax requires cytosolic factors involved in Tax-induced polyubiquitination

Activation of NF-κB by human T cell leukaemia virus type 1 Tax is thought to be crucial in T-cell transformation and the onset of adult T cell leukaemia. Tax activates NF-κB through activation of the IκB kinase (IKK) complex, similar to cytokine-induced NF-κB activation, which involves active signalling complex formation using polyubiquitin chains as a platform. Although polyubiquitination of Tax was reported to be required for IKK activation, most studies have been performed using intact cells, in which secondary NF-κB activation can be induced by various cytokines that are secreted due to Tax-mediated primary NF-κB activation. Therefore, a cell-free assay system, in which IKK can be activated by adding highly purified recombinant Tax to cytosolic extract, was used to analyse Tax-induced IKK activation. In contrast to the cytosolic extract, the purified IKK complex was not activated by Tax, whereas, it was efficiently activated by MEKK1, that does not require polyubiquitination to activate IKK. Moreover, Tax-induced IKK activation was blocked when the cytosolic extract was mixed with either lysine-free, methylated or K63R ubiquitin. These results obtained through our cell-free assay suggest that K63-linked polyubiquitination is critical, but linear polyubiquitination is dispensable or insufficient for Tax-induced IKK activation.

Major Histocompatibility Complex Class II Transactivator CIITA Is a Viral Restriction Factor That Targets Human T-Cell Lymphotropic Virus Type 1 Tax-1 Function and Inhibits Viral Replication

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of an aggressive malignancy of CD4+ T lymphocytes. Since the viral transactivator Tax-1 is a major player in T-cell transformation, targeting Tax-1 protein is regarded as a possible strategy to arrest viral replication and to counteract neoplastic transformation. We demonstrate that CIITA, the master regulator of major histocompatibility complex class II gene transcription, inhibits HTLV-1 replication by blocking the transactivating function of Tax-1 both when exogenously transfected in 293T cells and when endogenously expressed by a subset of U937 promonocytic cells. Tax-1 and CIITA physically interact in vivo via the first 108 amino acids of Tax-1 and two CIITA adjacent regions (amino acids 1 to 252 and 253 to 410). Interestingly, only CIITA 1-252 mediated Tax-1 inhibition, in agreement with the fact that CIITA residues from positions 64 to 124 were required to block Tax-1 transactivation. CIITA inhibitory action on Tax-1 correlated with the nuclear localization of CIITA and was independent of the transcription factor NF-YB, previously involved in CIITA-mediated inhibition of Tax-2 of HTLV-2. Instead, CIITA severely impaired the physical and functional interaction of Tax-1 with the cellular coactivators p300/CBP-associated factor (PCAF), cyclic AMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1), which are required for the optimal activation of HTLV-1 promoter. Accordingly, the overexpression of PCAF, CREB, and ATF1 restored Tax-1-dependent transactivation of the viral long-terminal-repeat promoter inhibited by CIITA. These findings strongly support our original observation that CIITA, beside increasing the antigen-presenting function for pathogen antigens, acts as an endogenous restriction factor against human retroviruses by blocking virus replication and spreading.

Mutual regulation between deubiquitinase CYLD and retroviral oncoprotein Tax

BackgroundOncoprotein Tax, encoded by the human T-cell leukemia virus type 1 (HTLV1), persistently induces NF-κB activation, which contributes to HTLV1-mediated T-cell transformation. Recent studies suggest that the signaling function of Tax requires its ubiquitination, although how the Tax ubiquitination is regulated remains unclear.ResultsWe show here that the deubiquitinase CYLD physically interacts with Tax and negatively regulates the ubiquitination of this viral protein. This function of CYLD is associated with inhibition of Tax-mediated activation of IKK although not that of Tak1. Interestingly, CYLD undergoes constitutive phosphorylation in HTLV1-transformed T cells, a mechanism known to inactivate the catalytic activity of CYLD. Consistently, a phospho-mimetic CYLD mutant fails to inhibit Tax ubiquitination.ConclusionThese findings suggest that CYLD negatively regulates the signaling function of Tax through inhibition of Tax ubiquitination. Conversely, induction of CYLD phosphorylation may serve as a mechanism by which HTLV1 overrides the inhibitory function of CYLD, leading to the persistent activation of NF-κB.

The human T-cell leukemia virus type I (HTLV-I) oncoprotein Tax promotes persistent NF-κB activation by blocking the phosphorylation of the adaptor molecule TAX1BP

Persistent activation of the transcription factor NF-κB by HTLV-1 Tax plays a critical role in T-cell transformation and the onset of adult T-cell leukemia (ATL). Tax activates NF-κB by binding to the NEMO/IKKgamma subunit of the IKK complex, thus leading to persistent IKK activation and nuclear translocation of NF-κB dimers. In addition, Tax counteracts negative regulators of NF-κB such as A20 although the underlying mechanisms remain unclear. We have previously reported that the Tax interacting protein TAX1BP1 serves as a scaffold molecule for the A20 ubiquitin-editing complex, consisting of A20, TAX1BP1, Itch and RNF11, that is critical for the negative feedback of NF-κB. Here we demonstrate that the IKKalpha subunit of IKK phosphorylated TAX1BP1 at Ser593 and Ser624 in response to proinflammatory cytokine stimulation. TAX1BP1 phosphorylation by IKKalpha is essential for the inducible interactions between TAX1BP1, A20, Itch and RNF11 and the subsequent NF-κB inhibitory function of A20. Interestingly, HTLV-I Tax potently blocks TAX1BP1 phosphorylation by preventing the recruitment of IKKalpha to TAX1BP1. Therefore, Tax promotes constitutive NF-κB signaling by inhibiting TAX1BP1 phosphorylation, thus blocking the assembly of the A20 ubiquitin-editing complex and impairing the function of A20.

HTLV-1 Tax1 represses the proapoptotic protein Bim, which is crucial for T-cell transformation

Human T cell leukemia virus type 1 (HTLV-1) is an etiological agent of adult T-cell leukemia (ATL) and its viral oncoprotein Tax1 plays critical roles in T cell transformation and ATL development. Tax1 converts a T-cell line CTLL-2 from IL-2 dependent growth into IL-2-independent one, and the conversion requires inhibition of apoptosis. In this study, we investigated the involvement of Bim, an apoptosis inducer after cytokine deprivation, in Tax1-induced transformation. Bim expression is strongly induced in CTLL-2 cells after IL-2 starvation, whereas such induction is markedly reduced in CTLL-2 cells transformed by Tax1. A Tax1 mutant defective of NF-kB2 activation transformed CTLL-2 less efficiently than wild type Tax, but this low transforming activity was rescued by knockdown of Bim in CTLL-2, suggesting that activation of noncanonical NF-kB2 pathway is involved in Tax1-mediated Bim repression. Furthermore, the expression of Bim was lower in HTLV-1 transformed and ATL cell lines than HTLV-1-negative T-cell lines. These results suggest that the Bim repression by Tax1 is crucial for the survival of HTLV-1 infected cells and may play a role in ATL development.

Physical and functional interaction between the HTLV-1 Tax oncoprotein and the cellular LIM domain protein FHL3

HTLV-1 is the ethiologic agent of adult T-cell leukemia (ATL). The viral regulatory protein Tax1 plays a pivotal role in T-cell transformation by deregulating several signalling pathways including CREB, NF-κB and AP-1 leading to the abnormal expression of several cellular proteins involved in cell survival and proliferation. Previous studies in the lab, using the yeast-2-hybrid approach to screen a T-cell library for Tax1 interacting partners identified the cellular Four and a Half Lim domain protein 3 (FHL3) as a possible Tax1 interacting candidate. FHL3 can regulate the actin-cytoskeleton and functions as a transcriptional activator. The aim of this study is to investigate the physical and functional interaction between Tax1 and FHL3. We have found that Tax1 and FHL3 interact both in vitro and in vivo and deletion analysis of FHL3 revealed that each LIM domain of FHL3 can interact with Tax1. Deletion analysis of Tax1 determined that the interaction with FHL3 occurs at both the N and C terminus, namely amino acids 1-50 and a fragment which encompasses the predicted LIM binding domain 207-353aa. We have demonstrated that FHL3 enhances Tax1 mediated LTR transcriptional activation without affecting basal activity. In contrast to this we show that FHL3 down regulates NF-κB activation by Tax1. Using confocal microscopy, we show that FHL3 co localizes with Tax in the nucleus and at the periphery of co transfected cells. In addition we demonstrate that FHL3 induces nanotubules in Cos7 cells and may be capable of transporting Tax1 from one cell to another. Overall our results so far suggest that the interaction between Tax1 and FHL3 alters both the transactivating activity and the sub cellular localization of Tax1 and provide new insights into molecular mechanisms that underlie the oncogenic nature of this HTLV-1 protein.

The MHC-II transactivator CIITA, a viral restriction factor inhibiting the replication of Human T-Cell Lymphotropic Virus Type 1

Human T-cell Lymphotropic Virus type-1 (HTLV-1) is the causative agent of an aggressive malignancy of CD4+ T lymphocytes. It is believed that the viral transactivator Tax-1 is a major player in T-cell transformation. Thus, targeting Tax-1 protein is regarded as a possible strategy to arrest viral replication and ultimately to counteract neoplastic transformation. Here, we demonstrated that CIITA, the master regulator of MHC class II gene transcription, inhibits HTLV-1 replication by blocking the transactivating function of Tax-1. Co-immunoprecipitation experiments have shown that CIITA and Tax-1 physically interact in vivo and that the first 108 amino acids of Tax-1 were necessary for this binding. Two adjacent regions (1-252 and 253-410) of CIITA bound independently to Tax-1, but only region 1-252 mediated Tax-1 inhibition, in agreement with the fact that CIITA residues from positions 64-124 were required to block Tax-1 transactivation. CIITA inhibitory action on Tax-1 function correlated with the nuclear localization of CIITA and was independent of the transcription factor NF-YB, previously involved in CIITA-mediated inhibition of Tax-2 of HTLV-2, a virus with still elusive pathogenic action. Furthermore, CIITA severely impaired the physical and functional interaction of Tax-1 with the cellular co-activator PCAF, which is required for the optimal activation of HTLV-1 promoter. Accordingly, the over-expression of PCAF restored Tax-1-dependent transactivation of the viral LTR promoter inhibited by CIITA. These findings strongly support our original observation that CIITA, beside increasing the antigen-presenting function for pathogen antigens, acts as an endogenous restriction factor against human retroviruses by blocking virus replication and spreading.

Cytoplasmic and nuclear events controlling Tax-mediated activation of the NF-κB pathway: involvement of TAB2, IKKgamma/NEMO and calreticulin

The Tax oncoprotein of HTLV-1 initiates T-cell transformation by dysregulating cell cycle progression and inhibiting DNA damage responses. The subsequent genomic instability might result in constitutive activation of the NF-κB pathway observed in HTLV-1-transformed T lymphocytes. Our previous results indicated that differential modifications of Tax by ubiquitination or sumoylation controlled its retention either in the cytoplasm or in the nucleus, respectively. Here we show that Tax is targeted to pre-existing punctate cytoplasmic structures which contain the TNF-receptor associated protein 2 (TAB2). Colocalization of Tax with TAB2 in these cytoplasmic structures induced the recruitment of additional components involved in the NF-κB activation cascade, including the regulatory subunit of the IKK complex, IKKgamma/NEMO, the RelA subunit of NF-κB and TAX1-BP1, which are Tax interacting partners. Overexpression of TAB2 strongly stimulated Tax-mediated activation of the NF-κB pathway indicating that the concentration of these NF-κB factors in Tax-containing cytoplasmic punctate structures is a step important for activation of the NF-κB pathway by Tax. Interestingly, calreticulin, a multifunctional calcium buffering chaperone previously involved in the nucleocytoplasmic transport of nuclear receptors such as glucocorticoid and thyroid receptors, was also recruited by Tax in these cytoplasmic structures. The fact that overexpressed IKKgamma concentrated in nuclear foci that included calreticulin and that co-expression of Tax and IKKgamma led to the redistribution of both IKKgamma and calreticulin from the nucleus to the cytoplasm suggested that calreticulin might be involved in the nuclear export of these factors and their recruitment in TAB2-containing cytoplasmic punctate structures. The possibility that Tax activates the NF-κB pathway via initiation of events in the nucleus leading to the transport of IKKgamma from the nucleus to the cytoplasm will be discussed.

Tax ubiquitylation and SUMOylation control the dynamic shuttling of Tax and NEMO between Ubc9 nuclear bodies and the centrosome

The human T-lymphotropic virus type I oncoprotein Tax is critical for T-cell transformation, acting mainly through nuclear factor kappa B essential modulator (NEMO) binding and subsequent nuclear factor-κB activation. Tax localizes to Tax nuclear bodies and to the centrosome and is subjected to ubiquitylation and small ubiquitin-like modifier (SUMO)ylation, which are both necessary for complete transcriptional activation. Using the photoconvertible fluorophore Dendra-2 coupled with live video confocal microscopy, we show for the first time that the same Tax molecule shuttles among Tax nuclear bodies and between these nuclear bodies and the centrosome, depending on its posttranslational modifications. Ubiquitylation targets Tax to nuclear bodies to which NEMO is recruited and subsequently SUMOylated. We also demonstrate that Tax nuclear bodies contain the SUMOylation machinery including SUMO and the SUMO conjugating enzyme Ubc9, strongly suggesting that these nuclear bodies represent sites of active SUMOylation. Finally, both ubiquitylation and SUMOylation of Tax control NEMO targeting to the centrosome. Altogether, we are proposing a model where both ubiquitylation and SUMOylation of Tax control the shuttling of Tax and NEMO between the cytoplasmic and nuclear compartments.

The HTLV-1 Tax protein inhibits formation of stress granules by interacting with histone deacetylase 6

Human T cell leukemia virus type-1 (HTLV-1) is the causative agent of a fatal adult T-cell leukemia. Through deregulation of multiple cellular signaling pathways the viral Tax protein has a pivotal role in T-cell transformation. In response to stressful stimuli, cells mount a cellular stress response to limit the damage that environmental forces inflict on DNA or proteins. During stress response, cells postpone the translation of most cellular mRNAs, which are gathered into cytoplasmic mRNA-silencing foci called stress granules (SGs) and allocate their available resources towards the production of dedicated stress-management proteins. Here we demonstrate that Tax controls the formation of SGs and interferes with the cellular stress response pathway. In agreement with previous reports, we observed that Tax relocates from the nucleus to the cytoplasm in response to environmental stress. We found that the presence of Tax in the cytoplasm of stressed cells prevents the formation of SGs and counteracts the shutoff of specific host proteins. Unexpectedly, nuclear localization of Tax promotes spontaneous aggregation of SGs, even in the absence of stress. Mutant analysis revealed that the SG inhibitory capacity of Tax is independent of its transcriptional abilities but relies on its interaction with histone deacetylase 6, a critical component of SGs. Importantly, the stress-protective effect of Tax was also observed in the context of HTLV-1 infected cells, which were shown to be less prone to form SGs and undergo apoptosis under arsenite exposure. These observations identify Tax as the first virally encoded inhibitory component of SGs and unravel a new strategy developed by HTLV-1 to deregulate normal cell processes. We postulate that inhibition of the stress response pathway by Tax would favor cell survival under stressful conditions and may have an important role in HTLV-1-induced cellular transformation.

HTLV-1 HBZ Protein Deregulates Interactions between Cellular Factors and the KIX Domain of p300/CBP

The complex retrovirus human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. Deregulation of cellular transcription is thought to be an important step for T-cell transformation caused by viral infection. HTLV-1 basic leucine zipper factor (HBZ) is one of the viral proteins believed to be involved in this process, as it deregulates the expression of numerous cellular genes. In the context of the provirus, HBZ represses HTLV-1 transcription, in part, by binding to the homologous cellular coactivators p300 and CBP. These coactivators play a central role in transcriptional regulation. In this study, we determined that HBZ binds with high affinity to the KIX domain of p300/CBP. This domain contains two binding surfaces that are differentially targeted by multiple cellular factors. We show that two φXXφφ motifs in the activation domain of HBZ mediate binding to a single surface of the KIX domain, the mixed-lineage leukemia (MLL) binding surface. Formation of this interaction inhibits binding of MLL to the KIX domain while enhancing the binding of the transcription factor c-Myb to the opposite surface of KIX. Consequently, HBZ inhibits transcriptional activation mediated by MLL and enhances activation mediated by c-Myb. CREB, which binds the same surface of KIX as c-Myb, also exhibited an increase in activity through HBZ. These results indicate that HBZ is able to alter gene expression by competing with transcription factors for the occupancy of one surface of KIX while enhancing the binding of factors to the other surface.

Tax ubiquitylation and SUMOylation control the dynamic shuttling of Tax and NEMO between Ubc9 nuclear bodies and the centrosome

AbstractThe human T-lymphotropic virus type I oncoprotein Tax is critical for T-cell transformation, acting mainly through nuclear factor kappa B essential modulator (NEMO) binding and subsequent nuclear factor-κB activation. Tax localizes to Tax nuclear bodies and to the centrosome and is subjected to ubiquitylation and small ubiquitin-like modifier (SUMO)ylation, which are both necessary for complete transcriptional activation. Using the photoconvertible fluorophore Dendra-2 coupled with live video confocal microscopy, we show for the first time that the same Tax molecule shuttles among Tax nuclear bodies and between these nuclear bodies and the centrosome, depending on its posttranslational modifications. Ubiquitylation targets Tax to nuclear bodies to which NEMO is recruited and subsequently SUMOylated. We also demonstrate that Tax nuclear bodies contain the SUMOylation machinery including SUMO and the SUMO conjugating enzyme Ubc9, strongly suggesting that these nuclear bodies represent sites of active SUMOylation. Finally, both ubiquitylation and SUMOylation of Tax control NEMO targeting to the centrosome. Altogether, we are proposing a model where both ubiquitylation and SUMOylation of Tax control the shuttling of Tax and NEMO between the cytoplasmic and nuclear compartments.

Oncogenic activation of the Notch1 gene by deletion of its promoter in Ikaros-deficient T-ALL

AbstractThe Notch pathway is frequently activated in T-cell acute lymphoblastic leukemias (T-ALLs). Of the Notch receptors, Notch1 is a recurrent target of gain-of-function mutations and Notch3 is expressed in all T-ALLs, but it is currently unclear how these receptors contribute to T-cell transformation in vivo. We investigated the role of Notch1 and Notch3 in T-ALL progression by a genetic approach, in mice bearing a knockdown mutation in the Ikaros gene that spontaneously develop Notch-dependent T-ALL. While deletion of Notch3 has little effect, T cell–specific deletion of floxed Notch1 promoter/exon 1 sequences significantly accelerates leukemogenesis. Notch1-deleted tumors lack surface Notch1 but express γ-secretase–cleaved intracellular Notch1 proteins. In addition, these tumors accumulate high levels of truncated Notch1 transcripts that are caused by aberrant transcription from cryptic initiation sites in the 3′ part of the gene. Deletion of the floxed sequences directly reprograms the Notch1 locus to begin transcription from these 3′ promoters and is accompanied by an epigenetic reorganization of the Notch1 locus that is consistent with transcriptional activation. Further, spontaneous deletion of 5′ Notch1 sequences occurs in approximately 75% of Ikaros-deficient T-ALLs. These results reveal a novel mechanism for the oncogenic activation of the Notch1 gene after deletion of its main promoter.

A role for proapoptotic Bax and Bak in T-cell differentiation and transformation

AbstractProapoptotic Bax and Bak are the key B-cell lymphoma-2 family members mediating apoptosis through the intrinsic pathway. Cells doubly deficient for Bax and Bak are profoundly resistant to apoptotic stimuli originating from multiple stimuli. Here we describe mice in which Bax and Bak have been deleted specifically in T-cells using Lck-Cre. In these T cell–specific BaxBak-deficient mice, early T-cell progenitors accumulate in the thymus, with relative depletion of more mature T cells. In addition, bone marrow progenitor cells fail to progress to the double positive stage when cultured on OP9 stromal cells expressing the Notch ligand Delta-like 1, consistent with a critical role for Bax and Bak in early T-cell development. Over time, T cell–specific BaxBak-deficient mice progress to an aggressive T-cell lymphoblastic leukemia/lymphoma. Interestingly, quantitative real-time polymerase chain reaction analysis of BaxBak-deficient T-cell lymphomas does not display amplification of the Notch signal transduction pathway, commonly activated in T-cell leukemia in both mouse and man. Bax and Bak, key regulators of the intrinsic pathway of apoptosis, are thus required to prevent T-cell malignancy, and for normal T-cell differentiation, regulating early T-cell development at the stage of early T-lineage progenitor cells.

BCL11B Mutations In T-Cell Acute Lymphoblastic Leukemia

AbstractAbstract 471T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with the activation of oncogenic transcription factors. Thus, 5% to 10% of pediatric and up to 30% of adult T-ALL patients show aberrant expression of the TLX1 transcription factor oncogene. Aberrant expression of TLX1 in Lck-TLX1 transgenics induces transformation of T-cell progenitors and the development of clonal T-cell lymphoblastic tumors with over 80% penetrance and a latency of 29 weeks. The prolonged latency before T-ALL development in TLX1 transgenic mice and the clonal nature of these tumors suggested the presence of cooperating mutations involved in the pathogenesis of these leukemias. Array Comparative Genomic Hybridization (aCGH) analysis identified several recurring chromosomal alterations in these tumors including three heterozygous deletions on chromosome 12, with a common deleted region containing only the Bcl11b gene. Moreover, DNA sequence analysis of Bcl11b showed the presence of Bcl11b mutations in 4/15 (27%) mouse TLX1 induced T-ALLs, which together with the 3 Bcl11b deletions identified in our aCGH analysis brings the prevalence of Bcl11b alterations in mouse TLX1 induced tumors to 7/15 (47%). Notably, mutation analysis of Bcl11b in a panel of 21 non TLX1-transgenic mouse T-ALL tumors failed to detect any Bcl11b mutations (P<0.001).Bcl11b encodes a zinc finger transcription factor with a critical role in the differentiation and survival of T-cell progenitors in the thymus. Given the similarities between our mouse model of TLX1-induced leukemia and human T-ALL, we hypothesized that BCL11B alterations could also be implicated in the pathogenesis of human T-ALL. Most notably, aCGH analysis of human T-ALL samples showed the presence of focal heterozygous deletions encompassing the BCL11B locus in 2/69 (3%) T-ALL cases. In addition, mutation analysis of BCL11B in human T-ALL demonstrated the presence of somatic truncating frameshift mutations and somatic missense point mutations in 9/71 (13%) cases analyzed. Interestingly, all 6 BCL11B missense mutations involved zinc finger domains. One sample showed compound heterozygous BCL11B mutations while the remaining cases harbored heterozygous BCL11B lesions. Finally, expression analysis of TLX1 or TLX3 in 8 T-ALLs harboring mutations or deletions in BCL11B with RNA available showed aberrant expression of these transcription factor oncogenes in 5/8 (63%) [TLX1 (4/8); TLX3 (1/8)] of these samples.The identification of a high prevalence of Bcl11b mutations and deletions in TLX1-induced tumors suggested a specific genetic interaction between these two genes in T-cell transformation. We contemplated two alternative scenarios. First, TLX1 could induce partial inactivation of BCL11B in T-cell transformation, an effect that would be reinforced by secondary genetic alterations in the BCL11B locus during tumor progression. Alternatively, BCL11B mutations and TLX1 overexpression could operate in parallel pathways that contribute to distinct but complementary aspects of tumor development. To test if BCL11B could be a direct transcriptional target of TLX1 in T-ALL, we analyzed the binding of TLX1 to the BCL11B promoter via chromatin immunoprecipitation analysis in ALL-SIL, a T-ALL cell line expressing high levels of TLX1 as result of the t(10;14)(q24;q11.2) translocation. This analysis revealed that indeed TLX1 binds to the BCL11B promoter. Moreover, siRNA knockdown of TLX1 in this cell line resulted in transcriptional upregulation of BCL11B, indicating that BCL11B is a direct transcriptional target downregulated by TLX1 in T-ALL. The model that emerges from these results is that the TLX1 oncogene directly downregulates the BCL11B tumor suppressor gene, and that the oncogenic effects of this transcriptional regulatory axis can be fixed and reinforced by secondary deletions and mutations in the BCL11B locus acquired during tumor progression. Altogether, these results highlight the power of integrated genomic analysis of human and mouse leukemias, identify a TLX1-BCL11B transcriptional regulatory axis in the pathogenesis of T-ALL and define BCL11B as a tumor suppressor gene recurrently deleted and mutated in 16% of human T-ALL patients. Disclosures:No relevant conflicts of interest to declare.

The T-ALL paradox in cancer

Overexpression of T cell leukemia homeobox 1 (TLX1) occurs in some leukemias. New findings in mice and humans show how TLX1 drives chromosomal aberrations and deletions of tumor suppressor genes during T-cell transformation (pages 1321–1327). Surprisingly, its expression does not correlate with bad prognosis.