Eric Wattel

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.

Telomere deregulations possess cytogenetic, phenotype, and prognostic specificities in acute leukemias

OBJECTIVE Telomeres are protected by tightly regulated factors and elongated by telomerase. Short and/or deprotected chromosomes are recombinogenic and thereby cancer prone. MATERIALS AND METHODS Together with the quantification of telomerase activity (TA), measuring telomere length (TL) and expression of the genes that govern telomere protection and elongation are useful for assessing telomere homeostasis. RESULTS By these means we demonstrate that TL, hTERT, and TA are in the order acute myelogenous leukemia (AML) > T-cell acute lymphoblastic leukemia (T-ALL) > B-cell acute lymphoblastic leukemia (B-ALL) > T-ALL > AML, and B-ALL > AML > T-ALL. AML0 and AML3 display the lowest amounts of hTERT transcripts, and ALL and AML cells with cytogenetic abnormalities possess the shortest telomeres. hTERT expression includes phenotype-specific RNA maturation and correlates with TA but not with TL. A wide ratio of TA to hTERT expression between leukemia subtypes suggests phenotype-specific hTERT post-transcriptional deregulations. B- and T-ALL overexpress Ku70 and Pinx1, T-ALL PTOP and RAP1, and B-ALL TRF2, the expression of which is significantly higher in cases with abnormal karyotype. hTERT transcription and TL correlate with response to intensive chemotherapy, and hTERT and RAD50 are independent prognostic factors for survival. CONCLUSIONS Each leukemia subtype possesses specific telomere dysregulations that rely on phenotype, karyotype, response to treatment, and survival.

Cause-specific telomere factors deregulation in hepatocellular carcinoma

BackgroundAmong the numerous genetic defects associated with hepatocarcinogenesis, telomere abnormalities appear to play a role both in tumor promotion and maintenance. Telomeres, the chromosome extremities, are protected by specific proteins, the shelterin complex and by additional factors. Besides telomerase dysregulation, expression changes of these telomere factors have been observed in cancers.MethodsHere, we tested the hypothesis that such dysregulation might occur in hepatocellular carcinoma (HCC) with specific patterns depending on the cause of HCC. We compared telomere length, telomerase activity (TA), hTERT and telomere genes expression using PCR and Western-blot analyses between non-cirrhotic liver, peritumoral cirrhotic tissue (40 samples) and cancerous tissue (40 samples) derived from 40 patients with HBV-, HCV-, or alcohol-related HCC.ResultsAlterations in TA, hTERT expression and telomere length between non-cirrhotic, cirrhotic, and tumor samples were not significantly influenced by the cause of HCC. In contrast, the expression pattern of hTR, shelterin, and non-shelterin telomere protective factors clearly distinguished the 3 causes of cirrhosis and HCC. For patients with HBV diseased liver, when compared with non-cirrhotic liver, the cirrhotic tissue underexpressed all shelterin and all but HMRE11A and RAD50 non-shelterin telomere factors. For HCV the expression level of POT1, RAP1, Ku80, and RAD50 was higher in cirrhotic than in non-cirrhotic liver samples without evidence for significant transcriptional change for the remaining genes. For alcohol-related liver diseases, the expression level of POT1, RAP1, TIN2, hMRE11A, hMRE11B, Ku70, Ku80, RAD50, TANK1, and PINX1 was higher in cirrhotic than in non-cirrhotic liver samples. For the 3 causes of HCC, there was no significant change in shelterin and non-shelterin gene expression between cirrhosis and HCC samples.ConclusionsThese results validate our hypotheses and demonstrate that cirrhosis and HCC add-up numerous telomere dysfunctions including numerous cause-specific changes that appear to occur early during the course of the disease.

Clonal expansion of HTLV-1 positive CD8+ cells relies on cIAP-2 but not on c-FLIP expression.

Here we investigate the mechanisms by which HTLV-1 infection prevents the cell death of CD8(+) T cells in vivo. We show that upon natural infection, cloned CD8(+) but not CD4(+) cells from patients without malignancy become resistant to Fas-mediated cell death and acquire an antiapoptotic transcriptome that includes the overexpression of cIAP-2 and c-FLIP(L). CD8(+) lymphocyte-restricted cIAP-2 overexpression correlates with resistance to Fas-mediated apoptosis and depends on tax expression via NF-KappaB. In contrast, in the same CD8(+) cells, the HTLV-1-dependent overexpression of c-FLIP(L) does not correlate with resistance to Fas-mediated cell death nor with tax expression. In the present model, infected CD8(+) clones are the only cell subtype in which cIAP-2 expression correlates with resistance to cell death. These results support a role for Tax-dependent cIAP-2 expression in preventing the death of naturally infected CD8(+) cells and thereby in their clonal expansion in vivo.

HTLV-1 positive and negative T cells cloned from infected individuals display telomerase and telomere genes deregulation that predominate in activated but untransformed CD4+ T cells.

Untransformed HTLV‐1 positive CD4+ cells from infected individuals are selected for expressing tax and displaying morphological features consistent with telomere dysfunctions. We show that in resting HTLV‐1 positive CD4+ cells cloned from patients, hTERT expression parallels tax expression and cell cycling. Upon activation, these cells dramatically augment tax expression, whereas their increase in telomerase activity is about 20 times lower than that of their uninfected counterpart. Activated HTLV‐1 positive CD4+ but not uninfected CD4+ or CD8+ clones also repress the transcription of TRF1, TPP1, TANK1, POT1, DNA‐PKc and Ku80. Both infected and uninfected lymphocytes from infected individuals shared common telomere gene deregulations toward a pattern consistent with premature senescence. ATLL cells displayed the highest telomerase activity (TA) whereas recovered a telomere gene transcriptome close to that of normal CD4+ cells. In conclusion HTLV‐1‐dependent telomere modulations seem involved in clonal expansion, immunosuppression, tumor initiation and progression.

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.

HBx triggers either cellular senescence or cell proliferation depending on cellular phenotype.

Replicative senescence is a hallmark of chronic liver diseases including chronic hepatitis B virus (HBV) infection, whereas HBV‐encoded oncoproteins HBx and preS2 have been found to overcome senescence. HBx possesses a C‐terminal truncation mainly in hepatocellular carcinomas but also in noncancerous liver tissues. Here, by cell counting, BrdU incorporation, MTT proliferation assay, cell cycle analysis, SA‐βgal staining and Western blotting in primary and malignant cells, we investigated the effect of HBx C‐terminal mutants on cellular senescence. HBx C‐terminal mutants were found to trigger cellular senescence in primary MRC5 cells, and malignant liver cells Huh7, and SK‐Hep1. In contrast, these mutants promoted the proliferation of HepG2 malignant liver cells. The pro‐senescent effect of HBx relied on an increased p16INK4a and p21Waf1/Cip1 expression, and a decreased phosphorylation of Rb. Together, these results suggest that the two main variants of HBx present in HBV‐infected liver possess opposite effects on cellular senescence that depend on the phenotype of infected cells.

Effect of Initial Body Mass Index on Survival Outcome of Patients With Acute Leukemia: A Single-Center Retrospective Study

BACKGROUND Obesity is associated with an increased risk of mortality from cardiovascular causes and occurrence of malignancies. However, the effect of body mass index (BMI) on survival outcome remains controversial in acute leukemia (AL) patients. PATIENTS AND METHODS A total of 531 adults with AL who entered clinical trials in our institution between 1994 and 2012 were analyzed retrospectively for the effect of BMI at diagnosis on outcome. The median follow-up was 4.7 years (95% confidence interval [CI], 4.0-5.1). RESULTS BMI had no significant effect on complete response rate, disease-free survival (DFS), or overall survival (OS) in patients from the whole cohort when considering a cutoff value for BMI of 25, and when analyzed according to age, or initial cytogenetics. In T-acute lymphoblastic leukemia (T-ALL) patients with BMI > 25, median DFS was not reached with a 3-year DFS at 76%, and median DFS was 16.1 months with 3-year DFS at 13% for those with BMI ≤ 25 (P = .005). Median OS was not reached in T-ALL patients with BMI > 25 versus 28.3 months in those with BMI ≤ 25 (3-year OS: 78% vs. 41%; P = .04). Multivariate analyses confirmed the prognostic value of BMI (> 25 vs. < 25) in T-ALL, but only in terms of DFS (hazard ratio, 0.25; 95% CI, 0.05-0.87; P = .037). However, in a validation cohort of 211 T-ALL patients, these results were not confirmed. CONCLUSION Results from the literature are very heterogeneous and contradictory regarding the effect of BMI on leukemia outcome. Even if nutritional status during chemotherapy courses is critical, these findings provide further evidence that initial body size does not have a major prognostic effect on survival in AL patients.

A Dose-Effect Relationship For Deltaretrovirus-Dependent Leukemogenesis In Sheep

BackgroundRetrovirus-induced tumors develop in a broad range of frequencies and after extremely variable periods of time, from only a few days to several decades, depending mainly on virus type. For hitherto unexplained reasons, deltaretroviruses cause hematological malignancies only in a minority of naturally infected organisms and after a very prolonged period of clinical latency.ResultsHere we demonstrate that the development of malignancies in sheep experimentally infected with the deltaretrovirus bovine leukemia virus (BLV) depends only on the level of BLV replication. Animals were experimentally infected with leukemogenic or attenuated, but infectious, BLV molecular clones and monitored prospectively through 8 months for viral replication. As early as 2 weeks after infection and subsequently at any time during follow-up, leukemogenic viruses produced significantly higher absolute levels of reverse transcription (RT), clonal expansion of infected cells, and circulating proviruses with RT- and somatic-dependent mutations than attenuated viruses. These differences were only quantitative, and both kinds of viruses triggered parallel temporal fluctuations of host lymphoid cells, viral loads, infected cell clonality and proliferation.ConclusionDeltaretrovirus-associated leukemogenesis in sheep appears to be a two-hit process over time depending on the amounts of first horizontally and then vertically expanded viruses.

The HTLV-1 encoded bZIP factor promotes cell proliferation and genetic instability through activation of oncogenic microRNAs

Viruses disrupt their host cells microRNAs (miRNAs) network for facilitating their replication. That of HTLV-1 relies on the clonal expansion of its host CD4+ and CD8+ T-cells yet the virus causes adult T-cell leukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype. Infected cells express Tax and HBZ viral oncoproteins. Tax is expressed in untransformed cells where it promotes cell proliferation, genetic instability and miRNAs deregulation whereas in contrast, HBZ is expressed by untransformed and malignant T-cells where hitherto, it is considered to promote cell proliferation and to silence virus expression. Here we show that an HBZ/miRNAs axis promotes cell proliferation and genetic instability. Infected CD4+ but not CD8+ T-cells were found to overexpress oncogenic miRNAs such as miR-17 and miR-21. HBZ activated these miRNAs via a posttranscriptional mechanism while in addition to promoting cellular growth; HBZ decreased DNA stability. These effects were alleviated by either miR-21/miR-17 knockdown or by the ectopic expression of OBFC2A, a factor that protects genome stability and that we found targeted by miR-17 and miR-21 in HTLV-1 infected CD4+ T-cells. This considerably extends the oncogenic potential of HBZ and suggests that viral expression might be involved in the remarkable genetic instability of ATLL cells.