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Background Human T cell leukemia virus type 1 (HTLV-1)-associated adult T cell leukemia (ATL) has a very poor prognosis with a median survival of 8 months and a 4-year overall survival of 11% for acute ATL. Present treatment options are limited and there is no curative therapy for ATL. Ubiquitin ligase FBXW7 is commonly mutated or functionally inactivated in human cancers. Consistent with the notion that FBXW7 controls the degradation of many oncoproteins, loss of FBXW7 has been linked to increased drug resistance or sensitivity in cancer cells. Method In this study, we have characterized FBXW7 mutants previously identified in HTLV-I-infected ATL patient samples. TET-inducible ATL cells carrying wild type or mutated FBXW7 were analyzed for target degradation and for drug sensitivity. Results Our results demonstrate that mutations in FBXW7 can selectively disrupt ubiquitination and proteasome degradation of target proteins: c-MYC, cyclin E and MCL1. Both c-MYC and MYCN were highly expressed in uncultured ATL patient’s samples and ATL-derived cell lines; and ATL cells demonstrated sensitivity to BET inhibitors in vitro and in vivo. High-throughput reverse phase protein array revealed BRAF as a novel target of FBXW7 and further experiments showed that mutations in FBXW7 preventing degradation of BRAF provided resistance to BET inhibitors. In contrast to R465, hot spot FBXW7 mutations at R505C retained degradation of BRAF but not NOTCH1, c-MYC, cyclin E, or MCL1. Finally, a combination therapy using BET inhibitors along with a BRAF or an ERK inhibitor prevented tumor cell resistance and growth. Conclusion Our results suggest that FBXW7 status may play an important role in drug therapy resistance of cancer cells. Genetic characterization of FBXW7 may be one factor included in future personalized cancer treatment identification.

Background Human T cell Leukemia virus type 1 (HTLV-I) is etiologically linked to adult T cell leukemia/lymphoma (ATL) and an inflammatory neurodegenerative disease called HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). The exact genetic or epigenetic events and/or environmental factors that influence the development of ATL, or HAM/TSP diseases are largely unknown. The tumor suppressor gene, Fragile Histidine Triad Diadenosine Triphosphatase (FHIT), is frequently lost in cancer through epigenetic modifications and/or deletion. FHIT is a tumor suppressor acting as genome caretaker by regulating cellular DNA repair. Indeed, FHIT loss leads to replicative stress and accumulation of double DNA strand breaks. Therefore, loss of FHIT expression plays a key role in cellular transformation. Methods Here, we studied over 400 samples from HTLV-I-infected individuals with ATL, TSP/HAM, or asymptomatic carriers (AC) for FHIT loss and expression. We examined the epigenetic status of FHIT through methylation specific PCR and bisulfite sequencing; and correlated these results to FHIT expression in patient samples. Results We found that epigenetic alteration of FHIT is specifically found in chronic and acute ATL but is absent in asymptomatic HTLV-I carriers and TSP/HAM patients’ samples. Furthermore, the extent of FHIT methylation in ATL patients was quantitatively comparable in virus-infected and virus non-infected cells. We also found that longitudinal HTLV-I carriers that progressed to smoldering ATL and descendants of ATL patients harbor FHIT methylation. Conclusions These results suggest that germinal epigenetic mutation of FHIT represents a preexisting mark predisposing to the development of ATL diseases. These findings have important clinical implications as patients with acute ATL are rarely cured. Our study suggests an alternative strategy to the current “wait and see approach” in that early screening of HTLV-I-infected individuals for germinal epimutation of FHIT and early treatment may offer significant clinical benefits.

Background Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia/lymphoma (ATLL), a lymphoproliferative malignancy with a dismal prognosis and limited therapeutic options. Recent evidence shows that HTLV-1-transformed cells present defects in both DNA replication and DNA repair, suggesting that these cells might be particularly sensitive to treatment with a small helicase inhibitor. Because the “Werner syndrome ATP-dependent helicase” encoded by the WRN gene plays important roles in both cellular proliferation and DNA repair, we hypothesized that inhibition of WRN activity could be used as a new strategy to target ATLL cells. Methods Our analysis demonstrates an apoptotic effect induced by the WRN helicase inhibitor in HTLV-1-transformed cells in vitro and ATL-derived cell lines. Inhibition of cellular proliferation and induction of apoptosis were demonstrated with cell cycle analysis, XTT proliferation assay, clonogenic assay, annexin V staining, and measurement of mitochondrial transmembrane potential. Results Targeted inhibition of the WRN helicase induced cell cycle arrest and apoptosis in HTLV-1-transformed leukemia cells. Treatment with NSC 19630 (WRN inhibitor) induces S-phase cell cycle arrest, disruption of the mitochondrial membrane potential, and decreased expression of anti-apoptotic factor Bcl-2. These events were associated with activation of caspase-3-dependent apoptosis in ATL cells. We identified some ATL cells, ATL-55T and LMY1, less sensitive to NSC 19630 but sensitive to another WRN inhibitor, NSC 617145. Conclusions WRN is essential for survival of ATL cells. Our studies suggest that targeting the WRN helicase with small inhibitors is a novel promising strategy to target HTLV-1-transformed ATL cells.

The ubiquitin-proteasome system (UPS) is involved in multiple aspects of cellular processes, such as cell cycle progression, cellular differentiation, and survival (Davis RJ et al., Cancer Cell 26:455-64, 2014; Skaar JR et al., Nat Rev Drug Discov 13:889-903, 2014; Nakayama KI and Nakayama K, Nat Rev Cancer 6:369-81, 2006). F-box and WD repeat domain containing 7 (FBXW7), also known as Sel10, hCDC4 or hAgo, is a member of the F-box protein family, which functions as the substrate recognition component of the SCF E3 ubiquitin ligase. FBXW7 is a critical tumor suppressor and one of the most commonly deregulated ubiquitin-proteasome system proteins in human cancer. FBXW7 controls proteasome-mediated degradation of oncoproteins such as cyclin E, c-Myc, Mcl-1, mTOR, Jun, Notch and AURKA. Consistent with the tumor suppressor role of FBXW7, it is located at chromosome 4q32, a genomic region deleted in more than 30% of all human cancers (Spruck CH et al., Cancer Res 62:4535-9, 2002). Genetic profiles of human cancers based on high-throughput sequencing have revealed that FBXW7 is frequently mutated in human cancers. In addition to genetic mutations, other mechanisms involving microRNA, long non-coding RNA, and specific oncogenic signaling pathways can inactivate FBXW7 functions in cancer cells. In the following sections, we will discuss the regulation of FBXW7, its role in oncogenesis, and the clinical implications and prognostic value of loss of function of FBXW7 in human cancers.

We have previously reported on the deregulation of cellular microRNAs involved in hematopoiesis and inflammation in human T-cell lymphotropic virus type 1 (HTLV-I)–transformed cells. In this study, we demonstrate that miR-150 and miR-223 specifically target the signal transducer and activator of transcription 1 (STAT1) 3′ untranslated region, reducing STAT1 expression and dampening STAT1-dependent signaling in human T cells. The effects of miR-150 and miR-223 on endogenous STAT1 were confirmed using inducible cell lines. Our studies also showed that miR-150 expression is upregulated by interleukin-2 signaling in adult T cell leukemia/lymphoma (ATL) cells. HTLV-I–transformed and ATL-derived cells have reduced levels of miR150 and miR223 expression, which coincide with increased STAT1 expression and STAT1-dependent signaling. Knockdown of STAT1 by short hairpin RNA demonstrated that the constitutive activation of STAT1 is required for the continuous proliferation of HTLV-I–transformed cells. Our studies further demonstrate that increased expression of STAT1 in ATL cells is associated with higher levels of major histocompatibility complex class I expression. Previous studies have demonstrated that the pressure exerted by natural killer (NK) cells in vivo can edit leukemic tumor cells by forcing an increased expression of major histocompatibility complex class I to escape immune clearance. STAT1-expressing tumor cells produce more aggressive tumors because they cannot be eliminated by NK cells. Our results suggest that therapeutic approaches using combined targeting of STAT1 and MHC class I may be an effective approach to activate NK cell–mediated clearance of ATL tumor cells.

Human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia and T-cell lymphoma (ATL) are aggressive diseases with poor prognoses, limited therapeutic options, and no curative treatment. In this study, we used a mouse model of ATL and restored expression of the microRNA, miR-124a, to identify in vivo downstream effectors responsible for its tumor-suppressive functions in ATL cells. Our results revealed that STAT3, a direct target of miR-124a, is constitutively activated in HTLV-I-transformed cells and ATL cells, and activating STAT3 mutations were detected in 25.5% of primary ATL patients. Interestingly, we found that the STAT3 downstream kinase effector, Pim1, is constitutively activated in ATL cells. The dependence of ATL cells to Pim1 activity was demonstrated using 2 Pim1 small inhibitors, SMI-4a and AZD1208. These studies indicated that HTLV-I-transformed and ATL cells, but not normal peripheral blood mononuclear cells, are highly sensitive to AZD1208, and the inhibition of Pim1 signaling triggers an apoptotic signal in leukemic cells. Finally, preclinical testing of AZD1208 in a mouse model of ATL resulted in significant prevention of tumor growth in vivo. In conclusion, our studies suggest that constitutive activation of the STAT3-Pim1 pathway represents a novel therapeutic target for the treatment of ATL.

Epigenetic regulators play a critical role in the maintenance of specific chromatin domains in an active or repressed state. Disruption of epigenetic regulatory mechanisms is widespread in cancer cells and largely contributes to the transformation process through active repression of tumor suppressor genes. While mutations of epigenetic regulators have been reported in various lymphoid malignancies and solid cancers, mutation of these genes in HTLV-I-associated T-cell leukemia has not been investigated.Here we used whole genome next generation sequencing (NGS) of uncultured freshly isolated ATL samples and identified the presence of mutations in SUZ12, DNMT1, DNMT3A, DNMT3B, TET1, TET2, IDH1, IDH2, MLL, MLL2, MLL3 and MLL4.TET2 was the most frequently mutated gene, occurring in 32 % (10/31) of ATL samples analyzed. Interestingly, NGS revealed nonsense mutations accompanied by loss of heterozygosity (LOH) in TET2 and MLL3, which was further confirmed by cloning and direct sequencing of DNA from uncultured cells. Finally, direct sequencing of matched control and tumor samples revealed that TET2 mutation was present only in ATL tumor cells.Our results suggest that inactivation of MLL3 and TET2 may play an important role in the tumorigenesis process of HTLV-I-induced ATL.

Background Epigenetic regulators play a critical role in the maintenance of specific chromatin domains in an active or repressed state. Disruption of epigenetic regulatory mechanisms is widespread in cancer cells and largely contributes to the transformation process through active repression of tumor suppressor genes. While mutations of epigenetic regulators have been reported in various lymphoid malignancies and solid cancers, mutation of these genes in HTLV-I-associated T-cell leukemia has not been investigated. Method Here we used whole genome next generation sequencing (NGS) of uncultured freshly isolated ATL samples and identified the presence of mutations in SUZ12, DNMT1, DNMT3A, DNMT3B, TET1, TET2, IDH1, IDH2, MLL, MLL2, MLL3 and MLL4. Results TET2 was the most frequently mutated gene, occurring in 32 % (10/31) of ATL samples analyzed. Interestingly, NGS revealed nonsense mutations accompanied by loss of heterozygosity (LOH) in TET2 and MLL3, which was further confirmed by cloning and direct sequencing of DNA from uncultured cells. Finally, direct sequencing of matched control and tumor samples revealed that TET2 mutation was present only in ATL tumor cells. Conclusions Our results suggest that inactivation of MLL3 and TET2 may play an important role in the tumorigenesis process of HTLV-I-induced ATL. Electronic supplementary material The online version of this article (doi:10.1186/s12943-016-0500-z) contains supplementary material, which is available to authorized users.

Contributing reviewers The editors of Molecular Cancer would like to thank all the reviewers who contributed to the journal in Volume 13 (2014) by participating in the review process. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0308-2) contains supplementary material, which is available to authorized users.

Human T cell leukemia virus, type 1 (HTLV-1) replication and spread are controlled by different viral and cellular factors. Although several anti-HIV cellular microRNAs have been described, such a regulation for HTLV-1 has not been reported. In this study, we found that miR-28-3p inhibits HTLV-1 virus expression and its replication by targeting a specific site within the genomic gag/pol viral mRNA. Because miR-28-3p is highly expressed in resting T cells, which are resistant to HTLV-1 infection, we investigated a potential protective role of miR-28-3p against de novo HTLV-1 infection. To this end, we developed a new sensitive and quantitative assay on the basis of the detection of products of reverse transcription. We demonstrate that miR-28-3p does not prevent virus receptor interaction or virus entry but, instead, induces a post-entry block at the reverse transcription level. In addition, we found that HTLV-1, subtype 1A isolates corresponding to the Japanese strain ATK-1 present a natural, single-nucleotide polymorphism within the miR-28-3p target site. As a result of this polymorphism, the ATK-1 virus sequence was not inhibited by miR-28. Interestingly, genetic studies on the transmission of the virus has shown that the ATK-1 strain, which carries a Thr-to-Cys transition mutation, is transmitted efficiently between spouses, suggesting that miR-28 may play an important role in HTLV-1 transmission.

Tumor suppressor functions are essential to control cellular proliferation, to activate the apoptosis or senescence pathway to eliminate unwanted cells, to link DNA damage signals to cell cycle arrest checkpoints, to activate appropriate DNA repair pathways, and to prevent the loss of adhesion to inhibit initiation of metastases. Therefore, tumor suppressor genes are indispensable to maintaining genetic and genomic integrity. Consequently, inactivation of tumor suppressors by somatic mutations or epigenetic mechanisms is frequently associated with tumor initiation and development. In contrast, reactivation of tumor suppressor functions can effectively reverse the transformed phenotype and lead to cell cycle arrest or death of cancerous cells and be used as a therapeutic strategy. Adult T-cell leukemia/lymphoma (ATLL) is an aggressive lymphoproliferative disease associated with infection of CD4 T cells by the Human T-cell Leukemia Virus Type 1 (HTLV-I). HTLV-I-associated T-cell transformation is the result of a multistep oncogenic process in which the virus initially induces chronic T-cell proliferation and alters cellular pathways resulting in the accumulation of genetic defects and the deregulated growth of virally infected cells. This review will focus on the current knowledge of the genetic and epigenetic mechanisms regulating the inactivation of tumor suppressors in the pathogenesis of HTLV-I.

The establishment of a latent reservoir by human tumor viruses is a vital step in initiating cellular transformation and represents a major shortcoming to current therapeutic strategies and the ability to eradicate virus-infected cells. Human T-cell leukemia virus type 1 (HTLV-1) establishes a lifelong infection and is linked to adult T-cell leukemia lymphoma (ATLL). Here, we demonstrate that HTLV-1 p30 recruits the cellular proteasome activator PA28γ onto the viral tax/rex mRNA to prevent its nuclear export and suppress virus replication. Interaction of p30 with a PA28γ retaining fully functional proteasome activity is required for p30's ability to repress HTLV-1. Consistently, HTLV-1 molecular clones replicate better and produce more virus particles in PA28γ-deficient cells. These results define a unique and novel role for the cellular factor PA28γ in the control of nuclear RNA trafficking and HTLV-1–induced latency. Importantly, knockdown of PA28γ expression in ATLL cells latently infected with HTLV-1 reactivates expression of viral tax/rex RNA and the Tax protein. Because Tax is the most immunogenic viral antigen and triggers strong CTL responses, our results suggest that PA28γ-targeted therapy may reactivate virus expression from latently infected cells and allow their eradication from the host.PA28γ acts as a co-repressor of HTLV-1 p30 to suppress virus replication and is required for the maintenance of viral latency. HTLV-1 has evolved a unique function mediated by its posttranscriptional repressor p30, which is not found in HTLV-2.

The promoter of the telomerase catalytic subunit (TERT) is subject to tight regulation and remains repressed in somatic cells to ensure their limited life span and to prevent tumor initiation. Here we report that the hTERT promoter is strongly repressed by p53 and the related family members p63 and p73. We found that p53-mediated repression was different in human and mouse cells and occurred through p53-dependent transcription inhibition of c-Myc or through E-box/E2F pathways, respectively. Although p63TAα-mediated repression occurred through SP1, p63TAy-mediated repression occurred through E2F signaling. Finally, p73α- and p73β-mediated repression occurred through NF-YB2. Our results show a complex multifactorial mechanism used by p53 and its family members to keep hTERT expression under tight control.

Whereas oncogenic retroviruses are common in animals, human T-lymphotropic virus 1 (HTLV-1) is the only transmissible retrovirus associated with cancer in humans and is etiologically linked to adult T-cell leukemia. The leukemogenesis process is still largely unknown, but relies on extended survival and clonal expansion of infected cells, which in turn accumulate genetic defects. A common feature of human tumor viruses is their ability to stimulate proliferation and survival of infected pretumoral cells and then hide by establishing latency in cells that have acquired a transformed phenotype. Whereas disruption of the DNA repair is one of the major processes responsible for the accumulation of genomic abnormalities and carcinogenesis, the absence of DNA repair also poses the threat of cell-cycle arrest or apoptosis of virus-infected cells. This study describes how the HTLV-1 p30 viral protein inhibits conservative homologous recombination (HR) DNA repair by targeting the MRE11/RAD50/NBS1 complex and favors the error-prone nonhomologous-end–joining (NHEJ) DNA-repair pathway instead. As a result, HTLV-1 p30 may facilitate the accumulation of mutations in the host genome and the cumulative risk of transformation. Our results provide new insights into how human tumor viruses may manipulate cellular DNA-damage responses to promote cancer.

The identification of the genes necessary for human T-cell leukemia virus (HTLV-1) persistence in humans may provide targets for therapeutic approaches. We demonstrate that ablation of the HTLV-1 genes encoding p12, p30, or the HBZ protein, does not affect viral infectivity in rabbits and in this species, only the absence of HBZ is associated with a consistent reduction in virus levels. We observed reversion of the HTLV-1 mutants to the HTLV-1 wild-type genotype in none of the inoculated rabbits. In contrast, in macaques, the absence of HBZ was associated with reversion of the mutant virus to the wild-type genotype in 3 of the 4 animals within weeks from infection. Similarly, reversion to the wild type was observed in 2 of the 4 macaque inoculated with the p30 mutant. The 4 macaques exposed to the p12 knock remained seronegative, and only 2 animals were positive at a single time point for viral DNA in tissues. Interestingly, we found that the p12 and the p30 mutants were also severely impaired in their ability to replicate in human dendritic cells. These data suggest that infection of dendritic cells may be required for the establishment and maintenance of HTLV-1 infection in primate species.

Background Human T-cell leukemia virus type I (HTLV-I) has efficiently adapted to its host and establishes a persistent infection characterized by low levels of viral gene expression and slow proliferation of HTLV-I infected cells over decades. We have previously found that HTLV-I p30 is a negative regulator of virus expression. Results In this study we show that p30 targets multiple cell cycle checkpoints resulting in a delayed entry into S phase. We found that p30 binds to cyclin E and CDK2 and prevents the formation of active cyclin E-CDK2 complexes. In turn, this decreases the phosphorylation levels of Rb and prevents the release of E2F and its transcriptional activation of genes required for G1/S transition. Our studies also show that HTLV-II p28 does not bind cyclin E and does not affect cell cycle progression. Conclusions In contrast to HTLV-I, the HTLV-II-related retrovirus is not oncogenic in humans. Here we report that the HTLV-I p30 delays cell cycle progression while its homologue, HTLV-II p28, does not, providing evidence for important differences between these two related retrovirus proteins.

The Notch signaling pathway plays an important role in cellular proliferation, differentiation, and apoptosis. Unregulated activation of Notch signaling can result in excessive cellular proliferation and cancer. Human T-cell leukemia virus type 1 (HTLV-I) is the etiological agent of adult T-cell leukemia (ATL). The disease has a dismal prognosis and is invariably fatal. In this study, we report a high frequency of constitutively activated Notch in ATL patients. We found activating mutations in Notch in more than 30% of ATL patients. These activating mutations are phenotypically different from those previously reported in T-ALL leukemias and may represent polymorphisms for activated Notch in human cancers. Compared with the exclusive activating frameshift mutations in the proline, glutamic acid, serine, and threonine (PEST) domain in T-ALLs, those in ATLs have, in addition, single-substitution mutations in this domain leading to reduced CDC4/Fbw7-mediated degradation and stabilization of the intracellular cleaved form of Notch1 (ICN1). Finally, we demonstrated that inhibition of Notch signaling by γ-secretase inhibitors reduced tumor cell proliferation and tumor formation in ATL-engrafted mice. These data suggest that activated Notch may be important to ATL pathogenesis and reveal Notch1 as a target for therapeutic intervention in ATL patients.

Human T-cell leukemia virus type I (HTLV-I)–associated malignancies are seen in a small percentage of infected persons. Although in vitro immortalization by HTLV-I virus is very efficient, we report that Tax has poor oncogenic activity in human primary T cells and that immortalization by Tax is rare. Sustained telomerase activity represents one of the oncogenic steps required for Tax-mediated immortalization. Tax expression was required for the growth of primary T cells, but was not sufficient to propel T cells into cell cycle in the absence of exogenous interleukin-2 (IL-2). Tax was sufficient to activate the phosphoinositide-3 kinase (PI3K)/Akt pathway as shown by down regulation of Src homology phosphatase-1 and increased phosphorylation of Akt. We also found disruption of putative tumor suppressors IL-16 and translocated promoter region (TPR) in Tax-immortalized and HTLV-I–transformed cell lines. Our results confirmed previous observations that Tax activates the anaphase-promoting complex. However, Tax did not affect the mitotic spindle checkpoint, which was also functional in HTLV-I–transformed cells. These data provide a better understanding of Tax functions in human T cells, and highlight the limitations of Tax, suggesting that other viral proteins are key to T-cell transformation and development of adult T-cell leukemia.

BackgroundHuman T-cell leukemia virus type 1 (HTLV-I) is a human retrovirus that is etiologically linked to adult T-cell leukemia (ATL), an aggressive and fatal lymphoproliferative disease. The viral transactivator, Tax, is thought to play an important role during the initial stages of CD4+T-cell immortalization by HTLV-1. Tax has been shown to activate transcription through CREB/ATF and NF-KB, and to alter numerous signaling pathways. These pleiotropic effects of Tax modify the expression of a wide array of cellular genes. Another viral protein encoded by HTLV-I, p30, has been shown to affect virus replication at the transcriptional and posttranscriptional levels. Little is currently known regarding the effect of p30 on the expression and nuclear export of cellular host mRNA transcripts. Identification of these RNA may reveal new targets and increase our understanding of HTLV-I pathogenesis. In this study, using primary peripheral blood mononuclear cells, we report a genome wide analysis of human genes transcriptionally and post-transcriptionally regulated by the HTLV-I protein p30.ResultsUsing microarray analysis, we analyzed total and cytoplasmic cellular mRNA transcript levels isolated from PBMCs to assess the effect of p30 on cellular RNA transcript expression and their nuclear export. We report p30-dependent transcription resulting in the 2.5 fold up-regulation of 15 genes and the down-regulation of 65 human genes. We further tested nuclear export of cellular mRNA and found that p30 expression also resulted in a 2.5 fold post-transcriptional down-regulation of 90 genes and the up-regulation of 33 genes.ConclusionOverall, our study describes that expression of the HTLV-I protein p30 both positively and negatively alters the expression of cellular transcripts. Our study identifies for the first time the cellular genes for which nuclear export is affected by p30. These results suggest that p30 may possess a more global function with respect to mRNA transcription and the nuclear shuttling of cellular mRNA transcripts. In addition, these alterations in gene expression may play a role in cell transformation and the onset of leukemia.

The human T-cell leukemia/lymphoma virus Type 1 (HTLV-1) ORF-I encodes, a ninety-nine amino acid hydrophobic membrane protein, p12I that affects receptors in different cellular compartments. We report here that proteolytic cleavage dictates different cellular localization and functions of p12I. The removal of a non-canonical endoplasmic reticulum (ER) retention/retrieval signal within the amino terminus of p12I is necessary for trafficking to the Golgi apparatus and generation of a completely cleaved 8 kDa protein. The 8 kDa protein in turn traffics to the cell surface, is recruited to the immunological synapse following T-cell receptor (TCR) ligation and down-regulates TCR proximal signaling. The uncleaved 12 kDa form of p12I resides in the ER and interacts with the β and γc chains of the interleukin-2 receptor (IL-2R), the heavy chain of the major histocompatibility complex (MHC) class I, as well as calreticulin and calnexin. Genetic analysis of ORF-I from ex vivo samples of HTLV-1-infected patients reveals predominant amino acid substitutions within ORF-I that inhibits proteolytic cleavage, suggesting that ER associated functions of p12I may contribute to the survival and proliferation of the infected T-cells in the host.

The persistence of human T-cell leukemia/lymphoma virus-I (HTLV-I)–infected cells is dependent upon clonal expansion and up-regulation of telomerase (hTERT). We have previously found that in interleukin (IL)–2–independent transformed HTLV-I cells, Tax strongly activates the hTERT promoter through nuclear factor-κB (NF-κB)–mediated Sp1 and c-Myc activation. In IL-2–dependent cells and adult T-cell leukemia/lymphoma (ATLL) patient samples, however, Tax expression is very low to undetectable, yet these cells retain strong telomerase activity. This suggests the existence of compensatory mechanisms in IL-2–dependent cells and ATLL patients. In this study, we demonstrate that telomerase activity is significantly decreased upon IL-2 withdrawal in immortalized HTLV-I cell lines. Inhibition of PI3K or AKT signaling pathways reduced telomerase activity in HTLV-I cells. We found that IL-2/IL-2R signaling was associated with a PI3K-dependent/AKT-independent transcriptional up-regulation of the endogenous hTERT promoter. We found that activation of the PI3K pathway mediated cytoplasmic retention of the Wilms tumor (WTI) protein, which strongly suppressed the hTERT promoter. The importance of this regulatory pathway for telomerase expression is underscored by findings that the PI3K pathway is commonly found activated in cancer cells.

Adult T-cell leukemia (ATL) is an aggressive lymphoproliferative disease of poor clinical prognosis associated with infection by the human T-cell leukemia virus type I (HTLV-I). The use of arsenic trioxide (As2O3) has been shown to effectively treat acute promyelocytic leukemia (APL) with greater than 80% of patients achieving complete remission. The combination of arsenic and interferon has also shown promising results in the treatment of ATL. The requirement for slow dosage increases of arsenic and the time required to achieve a pharmacologic active dose in patients is a major obstacle because median survival of patients with ATL is about 6 months. In this study we report a potent synergistic effect of the combination of arsenic trioxide and interferon α (As/IFN-α) with emodin and DHA on cell-cycle arrest and cell death of HTLV-I–infected cells. Importantly, we found that clinically achievable doses of DHA and emodin allowed for reduced arsenic concentrations by 100-fold while still remaining highly toxic to tumor cells. Our data provide a rationale for combined use of As/IFN-α with emodin and DHA in patients with ATL refractory to conventional therapy.

The antiviral thymidine analog azidothymidine (AZT) is used to treat several virus-associated human cancers. However, to date the mechanism of AZT action remains unclear and thus, reasons for treatment failure are unknown. Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy of poor prognosis. Here, we report that enduring AZT treatment of T-cell leukemia virus I–infected cells, in vitro and in vivo in ATL patients, results in inhibition of telomerase activity, progressive telomere shortening, and increased p14ARF expression. In turn, this elicits stabilization and reactivation of the tumor suppressor p53-dependent transcription, increased expression of the cyclin-dependent kinase inhibitor p21Waf1, and accumulation of p27kip1, thereby inducing cellular senescence and tumor cell death. While ATL patients carrying a wild-type p53 enter remission following treatment with AZT, those with a mutated p53 did not respond, and patients' disease relapse was associated with the selection of a tumor clone carrying mutated inactive p53.

The proto-oncogene c-myb is essential for a controlled balance between cell growth and differentiation. Aberrant c-Myb activity has been reported for numerous human cancers, and enforced c-Myb transcription can transform cells of lymphoid origin by stimulating cellular proliferation and inhibiting apoptotic pathways. Here we demonstrate that activation of the NF-kappaB pathway by the HTLV-1 Tax protein leads to transcriptional inactivation of c-Myb. This conclusion was supported by the fact that Tax mutants unable to stimulate the NF-kappaB pathway could not inhibit c-Myb transactivating functions. In addition, inhibition of Tax-mediated NF-kappaB activation by coexpression of IkappaBalpha restored c-Myb transcription, and Tax was unable to block c-Myb transcription in a NEMO knockout cell line. Importantly, physiological stimuli, such as signaling with the cellular cytokines tumor necrosis factor alpha, interleukin 1 beta (IL-1beta), and lipopolysaccharide, also inhibited c-Myb transcription. These results uncover a new link between extracellular signaling and c-Myb-dependent transcription. The mechanism underlying NF-kappaB-mediated repression was identified as sequestration of the coactivators CBP/p300 by RelA. Interestingly, an amino-terminal deletion form of p300 lacking the C/H1 and KIX domains and unable to bind RelA retained the ability to stimulate c-Myb transcription and prevented NF-kappaB-mediated repression.

The dysregulation of cellular apoptosis pathways has emerged as a critical early event associated with the development of many types of human cancers. Numerous viral and cellular oncogenes, aside from their inherent transforming properties, are known to induce programmed cell death, consistent with the hypothesis that genetic defects are required to support tumor survival. Here, we report that nuclear expression of the CREB-binding protein (CBP)/p300-binding domain of the human T-cell lymphotropic virus type 1 (HTLV-1) transactivator, Tax, triggers an apoptotic death-inducing signal during short-term clonal analyses, as well as in transient cell death assays. Coexpression of the antiapoptotic factor Bcl-2 increased serum stimulation; incubation with the chemical caspase inhibitor z-Val-Ala-DL-Asp fluoromethylketone antagonized Tax-induced cell death. The CBP/p300-binding defective Tax mutants K88A and V89A exhibited markedly reduced cytotoxic effects compared to the wild-type Tax protein. Importantly, nuclear expression of the minimal CBP/p300-binding peptide of Tax induced apoptosis in the absence of Tax-dependent transcriptional activities, while its K88A counterpart did not cause cell death. Further, Tax-mediated apoptosis was effectively prevented by ectopic expression of the p300 coactivator. We also report that activation of the NF-kappaB transcription pathway by Tax, under growth arrest conditions, results in apoptosis that occurs independent of direct Tax coactivator effects. Our results allude to a novel pivotal role for the transcriptional coactivator p300 in determining cell fate and raise the possibility that dysregulated coactivator usage may pose an early barrier to transformation that must be selectively overcome as a prerequisite for the initiation of neoplasia.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (40, 55) and a neurodegenerative disorder called tropical spastic paraparesis/HTLV-1-associated myelopathy (19, 37). HTLV-1 replication is dependent on the viral transactivator, Tax, and three 21-bp repeat elements, collectively referred to as the Tax response element (TxRE), localized in the U3 region of the proviral long terminal repeat (LTR). Each 21-bp repeat contains a core cyclic AMP (cAMP) response element (CRE) flanked by 5′ G-rich and 3′ C-rich sequences that confer Tax-dependent transactivation and dictate the assembly of Tax, CREB (CRE-binding protein), and the 21-bp repeat into a ternary (3°) complex (38, 46, 53, 56, 57). Tax interacts with CREB via the latter’s basic domain and amino acid residues in its immediate vicinity (2, 39, 54). A cellular homolog of CREB, ATF-1, can also interact with Tax in the form of a CREB/ATF-1 heterodimer or as an ATF-1 homodimer (11, 44, 57). Due to subtle amino acid sequence differences in the ATF-1 basic domain, Tax binds ATF-1 with reduced affinity (2, 57). Like CREB, Tax functions as a dimer (47). Analyses of Tax mutants have suggested that the NH2-terminal domain of Tax is important for CREB bZip (basic domain-leucine zipper) binding, while the midsection and the COOH terminus of Tax are important for subunit dimerization and possibly interaction with basal transcription factors, respectively (1, 23, 42, 47). Several groups have also reported that Tax enhances the dimerization and DNA binding of bZip proteins in vitro (3, 7, 12, 39, 50, 52). Transcriptional activation via the TxRE, CREB (and/or ATF-1), and Tax is independent of cellular signaling processes. Irrespective of the cell types used, cotransfection of a Tax expression plasmid and a reporter containing the TxRE results in 30- to 100-fold transcriptional induction. This contrasts with cellular somatostatin, proenkephalin, and phosphoenolpyruvate carboxyl kinase genes, whose expression is under CRE control and becomes induced only when the cAMP signaling pathway is activated. Work from many laboratories has indicated that an elevation in the intracellular levels of cAMP, as a consequence of cell surface signaling events, results in activation of protein kinase A, which can phosphorylate a sequence-specific DNA-binding protein, CREB, at amino acid residue Ser-133 (21, 22). Phospho-CREB subsequently recruits a transcriptional coactivator, CBP (CREB-binding protein) (16, 31, 35), or its homolog, p300 (4, 5), to CRE-containing enhancers to augment mRNA transcription (16, 31, 35). CBP and p300 appear to serve as integrators of numerous cellular signaling processes (26). The massive sizes of CBP and p300 allow them to interact with many cellular transcription factors in a signal-dependent and sometimes mutually exclusive fashion. To date, steroid and retinoid hormone receptors, CREB, c-Jun, c-Myb, Sap-1a, c-Fos, MyoD, p53, Stat-1/2, NF-κB, pp90rsk, TATA-binding protein, and TFIIB have been found to interact with CBP and p300 (see reference 26 for a review). This list will probably continue to grow. The oncoproteins of two DNA tumor viruses, adenovirus E1A and simian virus 40 large T antigen, also target and affect CBP/p300 functions (4, 5, 8). P/CAF (p300/CBP-associated factor), a histone acetyltransferase (HAT) that acetylates histones H3 and H4 but not H2A or H2B, has also been shown to bind to the CBP/p300 domain where E1A binds (51). Finally, in addition to their proposed roles as adaptor or coactivator molecules for transcription, CBP and p300 possess intrinsic HAT activity for all four core histones (9, 36). For this reason, they are suggested to play an important role in changing the nucleosomal structure at or around certain promoters (9, 36). The HAT activities of CBP/p300 and P/CAF presumably release promoter sequences from the repressive effect of the nucleosome and render them accessible to basal transcription factors and RNA polymerase II for transcriptional initiation. Using fluorescence anisotropy techniques, Kowk et al. have recently shown that HTLV-1 Tax directly binds CBP and p300 (30), results which have been confirmed as well as extended (20). These observations, combined with previous reports that Tax, CREB bZip, and TxRE specifically form a 3° complex, suggests that both CBP and p300 can be recruited to CREB/TxRE in a manner that bypasses signal-induced CREB phosphorylation by interacting with Tax. Thus, via the action of Tax, viral transcription can proceed constitutively without extracellular stimuli. The protein domain in CBP that is important for binding Tax has been localized to amino acid residues 451 to 682 (30). Interestingly, this domain has also been found to be important for binding phosphorylated CREB, c-Jun, c-Myb, and Sap-1a (6, 10, 17, 27, 30). In this study, we show by electrophoretic mobility shift assay (EMSA) that both the full-length p300 and residues 451 to 682 of CBP (CBP451-682) form stable quaternary (4°) complexes with Tax, CREB bZip, and the HTLV-1 21-bp repeat in vitro. The presence of CBP451-682 or p300 greatly facilitated the formation of a stable, high-order protein-DNA complex on the HTLV-1 21-bp repeat. The 4° complex readily forms on the HTLV-1 21-bp repeat but not on a CRE with nonspecific flanking sequences. While ATF-1 bZip could participate in 4° complex assembly, ATF-2 and ATF-3 failed to do so. Analyses of a series of Tax deletion mutants by glutathione S-transferase (GST) pull-down assays and EMSA, coupled with peptide competition, further revealed Tax amino acid residues 81 to 95 (81QRTSKTLKVLTPPIT95) to be important for CBP/p300 binding. This region lies between the domains for binding CREB bZip and for Tax subunit dimerization (1, 47). Three amino acid residues (88KVL90) in this region constitute sites that are rapidly cleaved by trypsin and chymotrypsin, suggesting that they are highly exposed and accessible to protein-protein interactions. We further report a series of point mutations targeted to this domain in Tax, which result in impairments in LTR transactivation and generate proteins that are defective in the ability to interact with CBP/p300. These data provide direct in vivo evidence in support of the role of CBP/p300 in Tax-mediated transactivation. Importantly, mutations that inactivate the COOH-terminal transactivation domain of Tax did not significantly alter the incorporation of CBP and p300 in vitro, suggesting that 4° complex formation alone might not be sufficient for transcriptional activation. Interactions with additional cellular factors via the COOH-terminal transactivation domain of Tax are likely to be important for efficient LTR-driven gene expression.

Plasmacytoid dendritic cells (pDCs) isolated from HTLV-1-infected individuals express viral proteins and can infect CD4+ T cells in vitro [1], and the proviral load in pDCs parallels the PBMC proviral load in infected individuals [2], suggesting a role for these cells in HTLV-1 transmission and persistence. We recently generated immortalized cell lines from primary pDCs infected with HTLV-1. Peripheral blood pDCs can survive in ex vivo culture for a limited time in the presence of IL-3. Following infection with cell-free virus, pDC were cultured with activated CD4+ T cells, and separated by immunomagnetic beads. Cells capable of continuously proliferating in the absence of IL-3 were isolated and characterized. FACS analysis revealed that these cells are phenotypically similar to pDCs (CD123+, BDCA2+, CMKLR1+, CD3-, CD19-, CD14-). Preliminary studies suggest that these cells spontaneously produce cytokines and chemokines expressed by pDCs. These pDC-like cells produce high levels of HTLV-1, as determined by ELISA analyses of culture supernatants and transmission EM and, like freshly isolated pDCs, can efficiently transmit HTLV-1 to T cells. As previously shown for HTLV-1-infected T cells [3], expression of HTLV-1 in these pDC-like cells is markedly decreased following exposure to type 1 interferons. Studies to characterize the viral integration sites and to examine expression levels of proteins previously shown to be altered in HTLV-1-transformed T cells are currently in progress. The in vitro generation of immortalized HTLV-1-infected pDC-like cells capable to transmitting the virus to T cells support the notion that pDCs could be an in vivo reservoir for HTLV-1.

Programmed cell death (PCD) characteristically involves chromatin condensation, membrane blebbing, and DNA oligonucleosomal fragmentation. These events, collectively referred to as apoptosis, represent an active cell suicide mechanism that eliminates cellular threats including potentially cancerous or virus-infected cells. Various types of programmed cell death can be blocked by the proto-oncogene Bcl-2. Levels of this protein are consistently low or undetectable in human endothelial cells (EC), which are important for immunoregulation through their interactions with circulating lymphocytes and are potential targets for infection by virus-bearing T-cells. Accumulating evidence suggests that EC may be infected in vivo to play an important role in HTLV-I-associated neuromyelopathy. In the present study, we report the establishment and characterization of human endothelial cell lines stably transfected with an HTLV-I-derived molecular clone. We observed constitutive expression of HTLV-I genes coinciding with activated Bcl-2 expression. Transient transfection of EC with the viral transactivator Tax and a reporter construct Bcl-2 promoter-CAT did not result in a significant increase in CAT activity and suggests that, in EC, expression of a second viral protein might be required for Bcl-2 activation. Further, Tax-induced apoptosis in rat fibroblasts has been shown to be blocked by Bcl-2 expression. Thus, HTLV-I-mediated induction of Bcl-2 expression in EC may provide protection against viral-induced apoptosis or extend cellular survival and create a reservoir for viral gene expression.

International audience; Background: Human T-cell leukemia virus type 1 (HTLV-I) is a human retrovirus associated with adult T-cell leukemia (ATL), an aggressive CD4 T-cell proliferative disease with dismal prognosis. The long latency preceding the development of the disease and the low incidence suggests that the virus itself is not sufficient for transformation and that genetic defects are required to create a permissive environment for leukemia. In fact, ATL cells are characterized by profound genetic modifications including structural and numerical chromosome alterations. Results: In this study we used molecular combing techniques to study the effect of the oncoprotein Tax on DNA replication. We found that replication forks have difficulties replicating complex DNA, fork progression is slower, and they pause or stall more frequently in the presence of Tax expression. Our results also show that Tax-associated replication defects are partially compensated by an increase in the firing of back-up origins. Consistent with these effects of Tax on DNA replication, an increase in double strand DNA breaks (DDSB) was seen in Tax expressing cells. Tax-mediated increases in DDSBs were associated with the ability of Tax to activate NF-kB and to stimulate intracellular nitric oxide production. We also demonstrated a reduced expression of human translesion synthesis (TLS) DNA polymerases Pol-H and Pol-K in HTLV-I-transformed T cells and ATL cells. This was associated with an increase in DNA breaks induced by Tax at specific genome regions, such as the c-Myc and the Bcl-2 major breakpoints. Consistent with the notion that the non-homologous end joining (NHEJ) pathway is hyperactive in HTLV-I-transformed cells, we found that inhibition of the NHEJ pathway induces significant killing of HTLV-I transformed cells and patient-derived leukemic ATL cells. Conclusion: Our results suggest that, replication problems increase genetic instability in HTLV-I-transformed cells. As a result, abuse of NHEJ and a defective homologous repair (HR) DNA repair pathway can be targeted as a new therapeutic approach for the treatment of adult T-cell leukemia.