Systems Biology Approaches Reveal a Specific Interferon-Inducible Signature in HTLV-1 Associated Myelopathy

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that persists lifelong in the host. In ∼4% of infected people, HTLV-1 causes a chronic disabling neuroinflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is unknown and treatment remains ineffective. We used gene expression microarrays followed by flow cytometric and functional assays to investigate global changes in blood transcriptional profiles of HTLV-1-infected and seronegative individuals. We found that perturbations of the p53 signaling pathway were a hallmark of HTLV-1 infection. In contrast, a subset of interferon (IFN)-stimulated genes was over-expressed in patients with HAM/TSP but not in asymptomatic HTLV-1 carriers or patients with the clinically similar disease multiple sclerosis. The IFN-inducible signature was present in all circulating leukocytes and its intensity correlated with the clinical severity of HAM/TSP. Leukocytes from patients with HAM/TSP were primed to respond strongly to stimulation with exogenous IFN. However, while type I IFN suppressed expression of the HTLV-1 structural protein Gag it failed to suppress the highly immunogenic viral transcriptional transactivator Tax. We conclude that over-expression of a subset of IFN-stimulated genes in chronic HTLV-1 infection does not constitute an efficient host response but instead contributes to the development of HAM/TSP

Modulation of the innate immune response during human T-cell leukemia virus infection implications for development of an oncolytic virotherapy for ATL

Infection with human T cell Leukemia virus (HTLV-1) can cause Adult T-cell Leukemia (ATL) or the neurological disorder HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Although the majority of HTLV-1--infected individuals remain asymptomatic carriers (AC) during their lifetime, 2-5% will develop either ATL or HAM/TSP. The factors that determine HTLV-1 pathogenesis remain elusive, and therefore represent a serious obstacle in the establishment of effective therapies for HTLV-1-associated diseases.Using gene expression profiling of CD4+ T-lymphocytes isolated from HTLV-1-infected individuals, we identified candidate genes differentially regulated in HTLV-1-associated diseases. Of particular interest, SOCS1 was up-regulated in HAM/TSP and AC patients -- but not in ATL. SOCS1 positively correlated with HTLV-1 mRNA in HAM/TSP patient samples. SOCS1-mediated degradation of IRF3 - inhibited antiviral signaling during HTLV-1 infection. Our study reveals a novel evasion mechanism utilized by HTLV-1 which leads to increased retroviral replication, without triggering an IRF3-dependent interferon response. Thus, targeting SOCS1 could represent a potential new approach to enhance the therapeutic potency of IFN-alpha/beta treatment in HAM/TSP disease.Although treatment of hematological malignancies has improved considerably, this has not benefited ATL patients, as they are completely refractory to conventional chemotherapeutic regimens. Oncolytic viruses, such as Vesicular stomatitis virus (VSV), have emerged as a potential treatment for cancer. Here we show that in vitro VSV infection induced significant oncolysis in highly proliferating primary ATL cells, but not in primary Chronic Lymphocytic Leukemia (CLL) cells which are arrested in the G0 phase. As chronic activation and proliferation is characteristic of ATL cells, we examined the effect of T-cell activation on VSV permissiveness and lysis. Activation of primary CD4+ T-lymphocytes was sufficient to induce VSV replication and VSV-triggered cell death, suggesting that cellular signaling pathways - ERK, JNK or AKT - that promote VSV replication are engaged during T-cell activation. Similarly, mitogenic activation of primary CLL promotes the exit from G0 and entrance into the cell cycle, rendering them susceptible to VSV-mediated oncolysis. Moreover, a global increase in protein translation mediated by the activation of mTOR and eIF4E was crucial for VSV replication in primary lymphocytes. These findings provide novel molecular targets for ATL and CLL therapeutics

Modulation of the innate immune response during Human T-cell Leukemia Virus infection: implication for development of an oncolytic vector

Infection with human T cell Leukemia virus (HTLV-1) can cause Adult T-cell Leukemia (ATL) or the neurological disorder HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Although the majority of HTLV-1–infected individuals remain asymptomatic carriers (AC) during their lifetime, 2-5% will develop either ATL or HAM/TSP. The factors that determine HTLV-1 pathogenesis remain elusive, and therefore represent a serious obstacle in the establishment of effective therapies for HTLV-1-associated diseases. Using gene expression profiling of CD4+ T-lymphocytes isolated from HTLV-1-infected individuals, we identified candidate genes differentially regulated in HTLV-1-associated diseases. Of particular interest, SOCS1 was up-regulated in HAM/TSP and AC patients – but not in ATL. SOCS1 positively correlated with HTLV-1 mRNA in HAM/TSP patient samples. SOCS1-mediated degradation of IRF3 - inhibited antiviral signaling during HTLV-1 infection. Our study reveals a novel evasion mechanism utilized by HTLV-1 which leads to increased retroviral replication, without triggering an IRF3-dependent interferon response. Thus, targeting SOCS1 could represent a potential new approach to enhance the therapeutic potency of IFN-α/β treatment in HAM/TSP disease. Although treatment of hematological malignancies has improved considerably, this has not benefited ATL patients, as they are completely refractory to conventional chemotherapeutic regimens. Oncolytic viruses, such as Vesicular stomatitis virus (VSV), have emerged as a potential treatment for cancer. Here we show that in vitro VSV infection induced significant oncolysis in highly proliferating primary ATL cells, but not in primary Chronic Lymphocytic Leukemia (CLL) cells which are arrested in the G0 phase. As chronic activation and proliferation is characteristic of ATL cells, we examined the effect of T-cell activation on VSV permissiveness and lysis. Activation of primary CD4+ T-lymphocytes was sufficient to induce VSV replication and VSV-triggered cell death, suggesting that cellular signaling pathways - ERK, JNK or AKT - that promote VSV replication are engaged during T-cell activation. Similarly, mitogenic activation of primary CLL promotes the exit from G0 and entrance into the cell cycle, rendering them susceptible to VSV-mediated oncolysis. Moreover, a global increase in protein translation mediated by the activation of mTOR and eIF4E was crucial for VSV replication in primary lymphocytes. These findings provide novel molecular targets for ATL and CLL therapeutics.Le rétrovirus T-lymphotropique humain (HTLV-1) est l'agent étiologique de la leucémie à cellule T de l'adulte (ATL) - une leucémie agressive et fatale des lymphocytes T CD4+. HTLV-1 est également associé à une forme de myélopathie chronique appelée Paraparésie Spastique Tropicale ou atteinte neurologique connue sous le nom de HTLV-1 - Associated Myelopathy (HAM/TSP). Bien que la majorité des individus infectés avec HTLV-1 demeurent asymptomatiques (AC) au cours de leur vie, 2 à 5% développent soit une ATL soit une HAM/TSP. Les facteurs qui déterminent la pathogénèse de l'HTLV-1 restent inconnus, et représentent donc un sérieux obstacle à la mise en place de traitements efficaces contre les maladies associées au virus HTLV-1. Les études sur l'expression des gènes des lymphocytes T CD4+ isolés de patients infectés par HTLV-1, ont permis l'identification de gènes d'intérêt exprimés de façon différentielle dans les maladies associées au virus HTLV-1. De façon intéressante, il a été mis en évidence que l'expression de SOCS1 est plus élevé chez les patients asymptomatiques ou HAM/TSP que chez les patients ATL qui expriment généralement très peu d'ARN viral. Chez les patients HAM, il existe une corrélation directe entre le niveau d'expression de SOCS1 et l'activité transcriptionelle provirale. Du point de vue fonctionnel, SOCS1 inhibe la réponse antivirale, entre autre via la dégradation du facteur de transcription IRF3. Cette étude a donc permis d'identifier un nouveau mécanisme utilisé par HTLV-1 afin d'inhiber la réponse antivirale et ainsi d'augmenter sa capacité de réplication.Dans cette étude, nous démontrons également que l'infection in vitro par le VSV induit la lyse oncogénique des cellules ATL, à fort potentiel prolifératif, mais pas celle des cellules primaires de la leucémie lymphocytique chronique (CLL), qui sont quiescentes in vitro. Puisque l'activation et la prolifération chronique est une caractéristique des cellules ATL, nous avons étudié l'effet de l'activation des cellules T sur leur permissivité au VSV ainsi que leur lyse induite par le virus. L'activation des cellules T CD4+ primaires de patients sains est suffisante pour permettre la réplication virale et la mort cellulaire induite par le VSV. L'utilisation d'inhibiteurs de la signalisation cellulaire a montré que l'activation de ERK, JNK ou AKT est suffisante pour permettre la réplication de VSV dans les cellules T CD4+. De façon similaire, la stimulation mitogénique des cellules CLL de patients induisant leur entrée dans le cycle cellulaire, les rend susceptibles à l'oncolyse par le VSV. De plus, une augmentation globale de la traduction protéique induite par l'activation de mTOR et eIF4E est essentielle pour la réplication du VSV dans les lymphocytes primaires.En conclusion, ces résultats permettent d'identifier de nouvelles voies thérapeutiques pour les leucémies de l'ATL et CLL

Human T Cell Leukemia Virus Type 1 Tax Inhibits Innate Antiviral Signaling via NF-κB-Dependent Induction of SOCS1▿

Human T cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-β expression accompanied by reduced HTLV-1 replication and p19Gag levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein

VSV Oncolysis in Combination With the BCL-2 Inhibitor Obatoclax Overcomes Apoptosis Resistance in Chronic Lymphocytic Leukemia

In chronic lymphocytic leukemia (CLL), overexpression of antiapoptotic B-cell leukemia/lymphoma 2 (BCL-2) family members contributes to leukemogenesis by interfering with apoptosis; BCL-2 expression also impairs vesicular stomatitis virus (VSV)-mediated oncolysis of primary CLL cells. In the effort to reverse resistance to VSV-mediated oncolysis, we combined VSV with obatoclax (GX15-070)—a small-molecule BCL-2 inhibitor currently in phase 2 clinical trials—and examined the molecular mechanisms governing the in vitro and in vivo antitumor efficiency of combining the two agents. In combination with VSV, obatoclax synergistically induced cell death in primary CLL samples and reduced tumor growth in severe combined immunodeficient (SCID) mice-bearing A20 lymphoma tumors. Mechanistically, the combination stimulated the mitochondrial apoptotic pathway, as reflected by caspase-3 and -9 cleavage, cytochrome c release and BAX translocation. Combination treatment triggered the release of BAX from BCL-2 and myeloid cell leukemia-1 (MCL-1) from BAK, whereas VSV infection induced NOXA expression and increased the formation of a novel BAX-NOXA heterodimer. Finally, NOXA was identified as an important inducer of VSV-obatoclax driven apoptosis via knockdown and overexpression of NOXA. These studies offer insight into the synergy between small-molecule BCL-2 inhibitors such as obatoclax and VSV as a combination strategy to overcome apoptosis resistance in CLL