Christiane Pinatel

HTLV-1 propels untransformed CD4+ lymphocytes into the cell cycle while protecting CD8+ cells from death

Human T cell leukemia virus type 1 (HTLV-1) infects both CD4+ and CD8+ lymphocytes, yet it induces adult T cell leukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype. Here we show that in vivo infected CD4+ and CD8+ T cells displayed similar patterns of clonal expansion in carriers without malignancy. Cloned infected cells from individuals without malignancy had a dramatic increase in spontaneous proliferation, which predominated in CD8+ lymphocytes and depended on the amount of tax mRNA. In fact, the clonal expansion of HTLV-1-positive CD8+ and CD4+ lymphocytes relied on 2 distinct mechanisms--infection prevented cell death in the former while recruiting the latter into the cell cycle. Cell cycling, but not apoptosis, depended on the level of viral-encoded tax expression. Infected tax-expressing CD4+ lymphocytes accumulated cellular defects characteristic of genetic instability. Therefore, HTLV-1 infection establishes a preleukemic phenotype that is restricted to CD4+ infected clones.

HTLV-1 bZIP Factor HBZ Promotes Cell Proliferation and Genetic Instability by Activating OncomiRs

Viruses disrupt the host cell microRNA (miRNA) network to facilitate their replication. Human T-cell leukemia virus type I (HTLV-1) replication relies on the clonal expansion of its host CD4(+) and CD8(+) T cells, yet this virus causes adult T-cell leukemia/lymphoma (ATLL) that typically has a CD4(+) phenotype. The viral oncoprotein Tax, which is rarely expressed in ATLL cells, has long been recognized for its involvement in tumor initiation by promoting cell proliferation, genetic instability, and miRNA dysregulation. Meanwhile, HBZ is expressed in both untransformed infected cells and ATLL cells and is involved in sustaining cell proliferation and silencing virus expression. Here, we show that an HBZ-miRNA axis promotes cell proliferation and genetic instability, as indicated by comet assays that showed increased numbers of DNA-strand breaks. Expression profiling of miRNA revealed that infected CD4(+) cells, but not CD8(+) T cells, overexpressed oncogenic miRNAs, including miR17 and miR21. HBZ activated these miRNAs via a posttranscriptional mechanism. These effects were alleviated by knocking down miR21 or miR17 and by ectopic expression of OBFC2A, a DNA-damage factor that is downregulated by miR17 and miR21 in HTLV-1-infected CD4(+) T cells. These findings extend the oncogenic potential of HBZ and suggest that viral expression might be involved in the remarkable genetic instability of ATLL cells.

Oncogene- and drug resistance-associated alternative exon usage in acute myeloid leukemia (AML)

In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro, 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents.

HTLV-1-infected CD4+ T-cells display alternative exon usages that culminate in adult T-cell leukemia.

BackgroundReprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects, HTLV-1 qualitatively modifies the pattern of cellular gene expression.ResultsExon expression analysis shows that patients’ untransformed and malignant HTLV-1+ CD4+ T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involved in cancer and cell cycle. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patterns of 3977 exons that discriminate uninfected, infected, and transformed CD4+ T-cells. Furthermore, untransformed infected CD4+ clones and ATLL samples shared 486 exon modifications distributed in 320 genes, thereby indicating a role of AEUs in HTLV-1 leukemogenesis. Exposing cells to splicing modulators revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provide news targets for treating ATLL.ConclusionsTaken together, these data reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets.

Alternative splicing signatures discriminate ATL cells from untransformed CD4+ counterparts deriving from HTLV-1 infected individuals

The clonal expansion and malignant transformation of HTLV-1 infected CD4+ T-cells have been linked to the reprogramming effects of HTLV-1 on host transcriptional profile. Coupled to transcription, alternative splicing (AS) is a post-transcriptional process that plays critical role in the complexity of transcriptome and splicing abnormalities frequently occur in cancer. To examine whether AS modifications associate with HTLV-1-associated leukemogenesis, we compared the exon expression profiles of ATL cells with that of CD4+ T-cell clones obtained by limited-dilution cloning of PBMC deriving from HTLV-1 carriers. 3 ATL cells and 12 untransformed infected clones clustering in infected, uninfected, PHA-stimulated or unstimulated CD4+ T cells were compared for exon RNA content using Exon Chip Human microarray. Hierarchical clustering analysis identified 12516 alternative spliced events (3642 genes) that clearly separated ATL samples from the 4 untransformed phenotypes mentioned above. In contrast, the exon content of 1539 genes differed between untransformed infected and uninfected T-CD4+ cells. Overall, less than 5% alternatively spliced genes were found differentially expressed at the transcriptional level. Microarray data were confirmed for 18 AS events using exon specific RT-PCR analysis. Pathway analysis of alternatively spliced genes (3642) in ATL cells revealed new AS-based pathways for p53 signaling, cell cycle and DNA replication while those of untransformed infected CD4+ T-cells were enriched in pathways for cellular movement and DNA repair. These findings unveil a new layer of complexity in the interplay between HTLV-1 and host cell gene expression machinery in which AS might play a central role in tumor initiation and promotion.

TET2 exon 2 skipping is an independent favorable prognostic factor for cytogenetically normal acute myelogenous leukemia (AML)

In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2Shigh patients were found to be younger than TET2low patients without a difference in the rate of complete remission. However, TET2E2Shigh patients exhibited a significantly lower CIR (p<10-4). TET2E2S and FLT3-ITD, but not age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both the intermediate-1 and favorable ELN genetic categories, TET2E2S remained significantly associated with prolonged survival. There was no correlation between TET2E2S status and outcomes in 34 additional AML patients who were unfit for IC. Therefore our results suggest that assessments of TET2 exon 2 splicing status might improve risk stratification in CN-AML patients treated with IC.

Tax promotes the expression of the alternative spliced isoform CD44v10 by slowing down the elongating RNA polymerase II

Deregulation of gene expression is the hallmark of HTLV-1 infection, involving a complex interplay between Tax and host-cell transcription machinery. It has been recently shown that changes in promoter structure and occupation impinge on both transcription and alternative splicing (AS) by modulating the RNApolII elongation rate. These data prompted us to examine whether Tax could induce changes in alternative splicing of cellular genes. The exon expression profiling of 293T-cells expressing or not Tax were investigated using Exon Chip Human microarray. The exon content of 857 genes differed between the two cell-categories, among which only 309 genes were found differentially expressed at the whole transcriptional level. AS events were validated by exon-specific RT-PCR experiments. Of these, we evidenced that Tax-induced NF-kB-dependent transcription of CD44 resulted in the inclusion of variable exon v10 (CD44v10) in mature transcripts. CD44 variants are described in a wide variety of cellular functions including lymphocyte activation, migration, and tumor metastasis. CD44v10 mRNA expression was significantly higher in HTLV-1-infected than uninfected clones deriving from HTLV-1 carriers. The protein expression of CD44v10 at the plasma membrane of Tax+293T-cells was detected by FACS analysis. At the mechanistic level, qChIP analysis of CD44 gene occupancy showed that Tax-induced splicing of CD44v10 coincides with a significant enrichment of both RNApolII and Tax at the region encoding variable exons, indicating that Tax interferes with the elongation rate of RNApolII. Together, these results unmask new mechanisms that connect Tax, transcription and alternative splicing with the HTLV-1-induced transcriptome reprogramming of CD4+ T cells.

HTLV-1 modulates alternative splicing in CD4+ T cells in vivo

Transcriptional dysregulation is the hallmark of HTLV-1 infected cells yet the impact of HTLV-1 on alternative splicing (AS) remains unknown. Given that about 95% of genes are spliced, we investigated the exon expression profiling of CD4+ T-cell clones obtained by limited-dilution cloning of PBMC deriving from HTLV-1 carriers. Overall, 12 infected clones clustering in infected, uninfected, PHA-stimulated or unstimulated CD4+ T cells were compared for exon RNA content using Exon Chip Human microarray. The splicing index method was used to identify differentially expressed exons in paired comparisons. The exon content of 558 genes differed between infected and uninfected CD4+ T cells. Of these, only 76 genes were found differentially expressed at the whole transcriptional level. 360 exon events (185/558 genes) could be manually annotated in FASTDB database and corresponded to exon skipping (59 genes), mutually exclusive exons (151), 5’ splice site (5’SS) (4), 3’SS (3), internal exon deletion (23), and intron retention (15). Some of these are already validated by exon specific RT-PCR experiments that are in progress. Exon-based hierarchical clustering analysis identified alternative exons associated with HTLV-1 infection and/or PHA stimulation. Ingenuity Pathway Analysis revealed new AS-based pathways of gene deregulation irrespective of whole transcriptional deregulation. These corresponded to hitherto unknown deregulation pattern of cell cycling and DNA damage response. Thus, HTLV-1 infection possesses a transcription-independent but AS-based signature that unmasks numerous new putative leukemogenic pathways.

Tax alters telomerase activity through both quantitative and qualitative hTERT transcriptional dysregulations

In untransformed cells impaired for DNA damage repair such as Tax positive cells, insufficient telomerase activity (TA) triggers tumorigenic telomere dysfunctions. Here we show that in quiescent HTLV-1+ T cells deriving from carriers, hTERT expression paralleled tax expression, suggesting that Tax promotes an immortalized phenotype in quiescent T cells. Interestingly, PHA-stimulation significantly augmented tax expression in CD4+ (2.6 times, p= 0.022) but not in CD8+ T cells (1.3, ns). 48 hours after PHA stimulation, uninfected and HTLV-1+CD8+ cells augmented their TA and hTERT expression (p<0.03 for each). In contrast PHA possessed no significant effect on TA and hTERT in HTLV-1+CD4+ cells. The PHA-induced fold changes in TA and hTERT expression were respectively 22 and 6.9-folds lower in infected than in uninfected CD4+ T cells, indicating that PHA decreased the level of TA more than 3 folds that of hTERT expression in the sole HTLV-1+CD4+ T cells subset. This hiatus between hTERT expression and TA was reproduced in vitro upon ectopic Tax expression in epithelial cells. We thus supposed that upon PHA-dependent Tax expression, a negative post-transcriptional element targeted hTERT for decreasing TA in HTLV-1+CD4+ T cells. Accordingly we next demonstrated that, beside its repressive effect on the hTERT promoter, ectopic Tax expression strongly redistributed hTERT RNA isoforms towards a significantly increased proportion in inactive isoforms and a significantly decreased proportion in the active A+B+ isoform. Thus via Tax expression, naturally HTLV-1 infected CD4+ T cells undergo both transcriptional and post-transcriptional hTERT modifications that significantly decrease TA in vivo.

Cloned HTLV-1+CD4+, but not CD8+, T-cells display an oncogenic miRNome

HTLV-1 persistence in vivo relies on the persistent clonal expansion of its host cells. These are CD4+ and CD8+ T cells, yet ATL is regularly CD4+. Accordingly, untransformed HTLV-1+CD4+ but not CD8+ T cells cloned from carriers cumulate the features of preleukemic cells, including multinuclearity, chromatin bridges, increased cell cycling and inappropriate telomerase activity. MicroRNAs (miR) modify the maturation of a plethora of T-cells RNA and their deregulation would therefore constitute an appropriate explanation for the Tax-dependent or -independent pleiotropic changes in the phenotype of HTLV-1+CD4+ T cells. As the miRNome of naturally infected untransformed cells has not been investigated to date, we assessed the miR expression profiling of T cells cloned from carriers. Microarray results, confirmed by quantitative RTPCR, showed that, upon infection, CD4+ and CD8+ clones yielded aberrant expression of 15 distinct miRs including miR-34b and miR-494 that were respectively over- and underexpressed in both compartments. The more prominent effect of the infection consisted in the CD4+-restricted overexpression of the cancer-related miRs miR-21, -27b and -23b associated with the CD4+-restricted downregulation of the proapoptotic miR-15 and -16. Data were extended by the analysis of 40 additional CD4+ clones (20 infected). Crossing the miRNome against the whole transcriptome data identified putative miR-targeted genes. In silico, those targeted by miR-23b and -27b defined 2 hitherto unknown pathways involving the cell cycle and genetic disorders. Therefore HTLV-1 triggers a phenotype-specific miR signature consistent with the preleukemic HTLV-1+CD4+ phenotype.