Development Of Adult T-cell Leukemia/lymphoma Research Articles

Clinical Applications of Genomic Alterations in ATLL: Predictive Markers and Therapeutic Targets.

Simple SummaryIn this review paper, we aim to summarize recent findings of genomic alterations found in adult T-cell leukemia/lymphoma (ATLL), which is an incurable disease induced by a virus; human T-cell leukemia virus type 1 (HTLV-1). Genomic alterations of ATLL have been comprehensively analyzed and the identified alterations and HTLV-1 infection synergistically act for ATLL development. As HTLV-1 is an endemic disease, ATLL frequently occurs in the endemic areas. Current clinicogenomic analyses suggest the existence of regional difference in ATLL pathophysiology. From a clinical perspective, several studies identified alterations that act as predictive markers and that a part of the alterations can be targetable in ATLL. The alterations can be leveraged to improve ATLL prognosis.Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell lymphoma (PTCL) caused by human T-cell leukemia virus type 1 (HTLV-1). Recent comprehensive genomic analyses have revealed the genomic landscape. One of the important findings of genomic alterations in ATLL is that almost all alterations are subclonal, suggesting that therapeutic strategies targeting a genomic alteration will result in partial effects. Among the identified alterations, genes involved in T-cell receptor signaling and immune escape mechanisms, such as PLCG1, CARD11, and PD-L1 (also known as CD274), are characteristic of ATLL alterations. From a geographic perspective, ATLL patients in Caribbean islands tend to be younger than those in Japan and the landscape differs between the two areas. Additionally, young Japanese ATLL patients frequently have CD28 fusions, compared with unselected Japanese cases. From a clinical perspective, PD-L1 amplification is an independent prognostic factor among every subtype of ATLL case. Recently, genomic analysis using deep sequencing identified a pre-ATLL clone with ATLL-common mutations in HTLV-1 carriers before development, indicating that genomic analysis can stratify cases based on the risks of development and mortality. In addition to genomic alterations, targetable super-enhancers have been identified in ATLL. These data can be leveraged to improve the prognosis of ATLL.

Autophagy in Human T-Cell Leukemia Virus Type 1 (HTLV-1) Induced Leukemia.

Viruses play an important role in the development of certain human cancers. They are estimated to contribute 16% to all human cancers. Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus to be discovered and is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive T-cell malignancy with poor prognosis. HTLV-1 viral proteins interact with mechanisms and proteins present in host cells for their own benefit, evading the immune system and promoting the establishment of disease. Several viruses manipulate the autophagy pathway to achieve their infective goals, and HTLV-1 is not the exception. HTLV-1 Tax viral protein engages NF-κB and autophagy pathways prone favoring viral replication and T cell transformation. In this review we focus on describing the relationship of HTLV-1 with the autophagy machinery and its implication in the development of ATLL.

Pathophysiological significance of N-myc downstream-regulated gene 2 in cancer development through protein phosphatase 2A phosphorylation regulation.

N‐myc downstream‐regulated gene 2 (NDRG2) is a candidate tumor suppressor in various cancers, including adult T‐cell leukemia/lymphoma (ATLL). NDRG2, as a stress‐responsive protein, is induced by several stress‐related signaling pathways and NDRG2 negatively regulates various signal transduction pathways. Although it has not been found to function alone, NDRG2 binds serine/threonine protein phosphatase 2A (PP2A), generating a complex that is involved in the regulation of various target proteins. The main function of NDRG2 is to maintain cell homeostasis by suppressing stress‐induced signal transduction; however, in cancer, genomic deletions and/or promoter methylation may inhibit the expression of NDRG2, resulting in enhanced tumor development through overactivated signal transduction pathways. A wide variety of tumors develop in Ndrg2‐deficient mice, including T‐cell lymphoma, liver, lung and other tumors, the characteristics of which are similar to those in Pten‐deficient mice. In particular, PTEN is a target molecule of the NDRG2/PP2A complex, which enhances PTEN phosphatase activity by dephosphorylating residues in the PTEN C‐terminal region. In ATLL cells, loss of NDRG2 expression leads to the failed recruitment of PP2A to PTEN, resulting in the inactivation of PTEN phosphatase with phosphorylation, ultimately leading to the activation of PI3K/AKT. Thus, NDRG2, as a PP2A adaptor, regulates the global phosphorylation of important signaling molecules. Moreover, the downregulation of NDRG2 expression by long‐term stress‐induced methylation is directly correlated with the development of ATLL and other cancers. Thus, NDRG2 might be important for the development of stress‐induced leukemia and other cancers and has become an important target for novel molecular therapies.

CARD11 Mutation Induces Oligoclonal Expansion of T-Cells, and Accelerates ATL Development in Combination with HBZ

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma that develops in about 5% of human T-cell leukemia/lymphoma virus 1 (HTLV-1) carriers. In addition to viral oncogenes, namely tax and HTLV-1 bZIP factor (HBZ), gene mutations, highly enriched for T-cell receptor (TCR)-NF-kB signaling, should be involved in the development of ATL. Among gene mutations, mutation in CARD11, a cytoplasmic scaffolding protein required for TCR-induced NF-kB activation, was detected in 24% of ATL patients. Here we generated a mouse model for conditional expression of a human ATL-derived CARD11(E626K) gain-of-function mutant, and demonstrated CARD11 activation induced oligoclonal expansion of T-cell and infiltration to many organs. We also showed that expression of HBZ accelerated mutant CARD11-induced lymphoproliferative diseases. We introduced a human Card11(E626K) into the mouse genome at the ROSA26 locus. After crossing with CD4-Cre Tg, CARD11(E626K)CD4-Cre mice was obtained. In CD4+ cells from CARD11(E626K)CD4-Cre, the amount of cleaved BCL-10 and NF-kBp65 increased compared with those in WT CD4+cells, confirming the activation of NF-kB. About half of CARD11(E626K)CD4-Cre mice died on or after 6 months after birth. At 6 months, leukocytosis was observed in CARD11(E626K)CD4-Cre, and accordingly the number of CD4+ cells cells was about 1.43 times greater than those in WT mice. The most affected organ in CARD11(E626K)CD4-Cre mice was lung. Alveolar septum was thickened by infiltrated cells at 6 months, and worsened subsequently. CD3+ T-cell accumulated around capillary blood vessels, and had high proliferation indices (>50%), as assayed by Ki-67 staining. CARD11(E626K)CD4-Cre mice developed lymphadenopathy (4/8 mice (50%) at 6M and 4/6 mice (66.7%) at 12M). Normal lymph node (LN) architecture was barely preserved, and medullary sinus was expanded with CD3+ T-cell. Some of them were positive for FoxP3, and had moderate proliferation indices (25%-50%). Among CD4+ T-cell, the proportion of naive T-cell (Tn) decreased, and that of effector/memory T-cell (Tem) and regulatory T-cell (Treg) increased compared to WT LNs. The proportion of Treg in CD4+ T-cell from LN of 6M- and 12M-old CARD11(E626K)CD4-Cre mice was 25% and 40%, respectively, which values were much larger than the normal range as 10-15%. We next examined the clonality of CD4+ cells in spleen and swollen LNs from CARD11(E626K)CD4-Cre mice. The clonality of the TCR repertoires of 20 individual Vb gene famines from Vb1-20 was assessed by a PCR. The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 5 splenic CD4+ cells, and 1 of 2 LN CD4+ cells from CARD11(E626K)CD4-Cre mice. To assess the effect of HBZ constant expression on CARD11(E626K)CD4-Cre mice, we generated HBZ Tg, in which HBZ cDNA was expressed under the CD4 promoter. Similar to the previous report (by Satou et al.), our HBZ Tg showed increased number of Tem, destroyed architecture of lung such as thickened alveolar septum by infiltrated cells and decreased alveolar space by edema, and the lymphadenopathy after 12M (66.7%). We then crossed CARD11(E626K)CD4-Creand HBZ Tg, and obtained the compound mice. The compound mice caused more aggressive lymphoproliferative diseases compared with CARD11(E626K)CD4-Cre mice. Most of compound mice died within 8M. At 6M, architecture of lung, kidney, spleen, and LN was destroyed. In lung, alveolar space of lung was scarcely observed caused of T-cell invasion. Alveolar septum was thickened with infiltrated cells, and CD3+ cells accumulated around capillary blood vessel. Some of them were positive for FoxP3, and indicated moderate proliferation indices (25-50%). T-cell invasion was also observed in kidney. Lymphadenopathy was detected in 6 of 9 (66.7%) with completely destroyed architecture, increment of the proportion of Fox3+ cells, and moderate proliferation indices (25-50%). The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 4 splenic CD4+ cells, and 2 of 2 LNs CD4+ cells from compound mice. These results suggest that CARD11 mutant-induced NFkB activation and constant HBZ expression may have similar effects, such as T-cell infiltration into organs and LN adenopathy, and that the simultaneous occurrence of both may have additive effects. Disclosures Sugiyama: Chordia Therapeutics, Japan.: Current Employment. Morishita:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Shimoda:Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Celgene: Honoraria; Shire plc: Honoraria; Novartis: Honoraria, Research Funding; Takeda Pharmaceutical Company: Honoraria; Bristol-Myers Squibb: Honoraria.

Mechanisms of Oncogenesis by HTLV-1 Tax.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), a neoplasm of CD4+CD25+ T cells that occurs in 2–5% of infected individuals after decades of asymptomatic latent infection. Multiple HTLV-1-encoded regulatory proteins, including Tax and HTLV-1 basic leucine zipper factor (HBZ), play key roles in viral persistence and latency. The HTLV-1 Tax oncoprotein interacts with a plethora of host cellular proteins to regulate viral gene expression and also promote the aberrant activation of signaling pathways such as NF-κB to drive clonal proliferation and survival of T cells bearing the HTLV-1 provirus. Tax undergoes various post-translational modifications such as phosphorylation and ubiquitination that regulate its function and subcellular localization. Tax shuttles in different subcellular compartments for the activation of anti-apoptotic genes and deregulates the cell cycle with the induction of DNA damage for the accumulation of genomic instability that can result in cellular immortalization and malignant transformation. However, Tax is highly immunogenic and therefore HTLV-1 has evolved numerous strategies to tightly regulate Tax expression while maintaining the pool of anti-apoptotic genes through HBZ. In this review, we summarize the key findings on the oncogenic mechanisms used by Tax that set the stage for the development of ATLL, and the strategies used by HTLV-1 to tightly regulate Tax expression for immune evasion and viral persistence.

Diagnostic Approaches and Established Treatments for Adult T Cell Leukemia Lymphoma.

Adult T-cell leukemia-lymphoma (ATL) is a distinct mature T-cell malignancy caused by human T-cell leukemia/lymphotropic virus type I (HTLV-1) endemic in some areas in the world. HTLV-1 transmits through mother-to-child infection via breastfeeding, sexual intercourses, and blood transfusions. Early HTLV-1 infection, presumably through mother’s milk, is crucial in developing ATL. The estimated cumulative risk of the development of ATL in HTLV-1 carriers is a few percent after transmission from their mothers. The median age of ATL onset is about 70 in Japan and is now rising, whereas an overall mean age in the mid-forties is reported in other parts of the world. ATL is classified into four clinical subtypes (acute, lymphoma, chronic, and smoldering) defined by organ lesions and LDH/calcium values. In aggressive ATL (acute, lymphoma or unfavorable chronic types) and indolent ATL (favorable chronic or smoldering types), intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation and watchful waiting until disease progression has been recommended, respectively, in Japan. Based on a worldwide meta-analysis and multiple other retrospective studies, the antiviral combination of interferon alpha (IFN) and zidovudine (AZT) is recommended in many parts of the world in acute, chronic, and smoldering ATL whereas patients with the lymphoma subtype are treated with chemotherapy, either alone or combined with AZT/IFN. Several new agents have been approved for ATL by the Pharmaceutical and Medical Devices Agency (PMDA) after clinical trials, including an anti-CC chemokine receptor 4 monoclonal antibody, mogamulizumab; an immunomodulatory agent, lenalidomide; and an anti-CD30 antibody/drug conjugate, brentuximab vedotin.

Epidemiology of HTLV-1 Infection and ATL in Japan: An Update.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection that often occurs in HTLV-1-endemic areas, such as Japan, the Caribbean islands, Central and South America, Intertropical Africa, and the Middle East. In Japan, the nationwide estimation of the number of HTLV-1 carriers was at least 1.08 million in 2006–2007. Furthermore, in 2016, the nationwide annual incidence of newly infected with HTLV-1 was first estimated to be 3.8 per 100,000 person-years based on the age-specific seroconversion rates of blood donors in almost all areas of Japan. The incidence rate was three times higher in women than in men, and it was estimated that at least 4,000 new HTLV-1 infections occur yearly among adolescents and adults in Japan. As well known that HTLV-1 infection alone is not a sufficient condition for ATL to develop. To date, a variety of molecular abnormalities and host susceptibilities have been reported as candidate progression factors for the development of ATL in HTLV-1-carriers. In particular, quite recently in Japan, a variety of immunosuppressive conditions have been recognized as the most important host susceptibilities associated with the development of ATL from HTLV-1-carrier status. Furthermore, in 2013–2016 in Japan, a new nationwide epidemiological study of ATL was conducted targeting patients newly diagnosed with ATL in 2010–2011, from which the most current knowledge about the epidemiological characteristics of Japanese patients with ATL was updated as follows: (1) continuing regional unevenness of the distribution of people with HTLV-1, (2) further aging, with the mean age at diagnosis being 67.5 years, (3) declining M/F ratio, (4) increase of the lymphoma subtype, (5) sex differences in subtype distribution, (6) age differences in subtype distribution, and (7) comorbidity condition. In particular, 32.2% of ATL patients had comorbid malignancies other than ATL. However, the number of deaths due to ATL in Japan has been relatively stable, at around 1,000 patients annually, without significant decline from 1999 to 2017. Because the current epidemiological evidence about HTLV-1 and ATL is insufficient, further epidemiological studies are required.

Mortality and risk of progression to adult T cell leukemia/lymphoma in HTLV-1–associated myelopathy/tropical spastic paraparesis

Human T cell leukemia virus 1 (HTLV-1) causes the functionally debilitating disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) as well as adult T cell leukemia lymphoma (ATLL). Although there were concerns that the mortality of HAM/TSP could be affected by the development of ATLL, prospective evidence was lacking in this area. In this 5-y prospective cohort study, we determined the mortality, prevalence, and incidence of ATLL in 527 HAM/TSP patients. The standard mortality ratio of HAM/TSP patients was 2.25, and ATLL was one of the major causes of death (5/33 deaths). ATLL prevalence and incidence in these patients were 3.0% and 3.81 per 1,000 person-y, respectively. To identify patients at a high risk of developing ATLL, flow cytometry, Southern blotting, and targeted sequencing data were analyzed in a separate cohort of 218 HAM/TSP patients. In 17% of the HAM/TSP patients, we identified an increase in T cells positive for cell adhesion molecule 1 (CADM1), a marker for ATLL and HTLV-1-infected cells. Genomic analysis revealed that somatic mutations of HTLV-1-infected cells were seen in 90% of these cases and 11% of them had dominant clone and developed ATLL in the longitudinal observation. In this study, we were able to demonstrate the increased mortality in patients with HAM/TSP and a significant effect of ATLL on their prognosis. Having dominant clonal expansion of HTLV-1-infected cells with ATLL-associated somatic mutations may be important characteristics of patients with HAM/TSP who are at an increased risk of developing ATLL.

The regulation of NDRG2 expression during ATLL development after HTLV-1 infection

N-myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor that is frequently downregulated in adult T-cell leukemia/lymphoma (ATLL) and functions to negatively regulate several cellular signaling pathways as PP2A recruiter. To clarify the molecular mechanisms of suppression of NDRG2 expression, we initially determined the expression pattern of NDRG2 in various types of T-cells and ATLL cells. NDRG2 expression was significantly upregulated in HTLV-1/Tax-immortalized T-cells, which was mediated by NF-κB activation through Tax expression. On the other hand, NDRG2 expression was suppressed in HTLV-1-infected cell lines and various types of ATLL cells, which was dependent on the DNA methylation of the NDRG2 promoter. We found that the expression of enhancer of zeste homolog 2 (EZH2), a member of the polycomb family, is increased in ATLL, and that EZH2 directly binds to the NDRG2 promoter and induces DNA methylation of the NDRG2 promoter. Since the expression of EZH2 were anti-parallelly regulated with the NDRG2 expression, EZH2 might be one of the most important regulators of the downregulation of NDRG2, contributing to enhanced activation of signaling pathways during ATLL development.

Clinical features and human T-cell leukemia virus type-1 (HTLV-1) proviral load in HTLV-1-positive patients with rheumatoid arthritis: Baseline data in a single center cohort study

Objective: Recently, Human T-cell leukemia virus type-1 proviral load (HTLV-1 PVL) has been evaluated as an important predictor of adult T-cell leukemia/lymphoma (ATL) in HTLV-1 carriers. We aimed to evaluate whether HTLV-1 PVL is also important for the development of ATL among HTLV-1-positive patients with rheumatoid arthritis (RA).Methods: We established a cohort of 82 HTLV-1-positive RA patients between 2017 and 2018. Of those, 27 (32.9%) were treated with biological disease-modifying anti-rheumatic drugs (bDMARDs) with/without methotrexate. We measured HTLV-1 PVL in peripheral blood mononuclear cells (PBMCs) at study entry and compared the value by clinical status and treatment options.Results: The median PVL for all was 9.6 copies per 1000 PBMCs without sex difference (male 17.2 and female 8.6; p = .24). The median PVL was significantly higher for patient’s comorbid bronchiectasis, malignancies, and opportunistic infectious diseases, compared with patients without comorbidity. There were no significant differences in PVL levels among types of bDMARDs, although the level was tended to be higher for patients treated with JAK inhibitor.Conclusions: HTLV-1 seropositive RA patients comorbid for any diseases having higher HTLV-1 PVLs will be a higher risk for developing ATL. Careful follow-up of these patients is necessary to detect ATL development.

Induction of APOBEC3B cytidine deaminase in HTLV-1-infected humanized mice.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL). Following viral infection with HTLV-1, certain infected cells exhibit clonal proliferation. Additional genetic and epigenetic changes in these clonally proliferating cells provide them with the selective advantage of growth, which eventually results in ATL. The precise mechanism, however, has yet to be completely elucidated. It has previously been established that APOBEC3 enzymes are potent host-antiviral restriction factors. Conversely, previous studies have reported that the A3B level is increased in tumor virus infections, such as those caused by HBV and HPV, suggesting that A3B exerts a function as a mutagen. Therefore, the present study analyzed the expression of APOBEC3 family members in various HTLV-1 infection states. No significant differences were observed in the expression between healthy donors and patients with HTLV-1-associated myelopathy. Although no significant changes in the expressions of A3C, A3D, A3F and A3G between uninfected and HTLV-1-infected mice were observed, an increased A3B expression was observed in a short-term humanized mouse model following HTLV-1 infection. In a long-term humanized mouse model following HTLV-1 infection, the gene expression array data exhibited an apparent increase in A3B and CADM1, which are indicators of ATL. Collectively, the results of the present study suggest that A3B is likely involved in the development of ATL in HTLV-1-infected humanized mice.

Cells of adult T-cell leukemia evade HTLV-1 Tax/NF-κB hyperactivation–induced senescence

AbstractHuman T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATL). The HTLV-1 viral trans-activator/oncoprotein Tax is a major driver of ATL, yet it induces rapid p21Cip1/Waf1 (p21)- and p27Kip1-mediated cellular senescence through constitutive activation (hyperactivation) of NF-κB. Although constitutive NF-κB activation is a common feature of T/B-cell leukemia/lymphoma, including ATL, it is not known how ATL cells maintain chronic NF-κB activation without undergoing senescence. Here, we demonstrate that, in contrast to HTLV-1− T-cell lines, ATL cell lines no longer undergo Tax-induced senescence. Although Tax+ and Tax− ATL cell lines showed signatures of constitutive NF-κB activation, their ability to progress through the cell cycle was unaffected. In some cases, ATL cell lines continued to proliferate despite significant upregulation of p21; additionally, many cell lines displayed altered expression of G1 and G1/S cyclins, particularly overexpression of cyclin D2. We propose that, during the course of ATL development, leukemia cells acquire genetic/epigenetic changes that can mitigate the senescence response triggered by NF-κB hyperactivation. Restoring the NF-κB–induced senescence response would likely help to control the development and progression of ATL and similar lymphoid malignancies.

Maintenance of long remission in adult T-cell leukemia by Tax-targeted vaccine: A hope for disease-preventive therapy.

Adult T‐cell leukemia/lymphoma (ATL) is an aggressive lymphoproliferative disease caused by human T‐cell leukemia virus type 1 (HTLV‐1). Multi‐agent chemotherapy can reduce ATL cells but frequently allows relapses within a short period of time. Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) following chemotherapy is now a standard therapy for ATL in Japan as it can achieve long‐term remission in approximately one‐third of recipient ATL patients; however, it also has a risk of treatment‐related mortality. Allo‐HSCT often induces HTLV‐1 Tax‐specific cytotoxic T cells (CTL) as well as graft‐versus‐host (GVH) response in ATL patients. This observation led to development of a new therapeutic vaccine to activate Tax‐specific CTL, anticipating anti‐ATL effects without GVH response. The newly developed Tax‐DC vaccine consists of autologous dendritic cells pulsed with Tax peptides corresponding to CTL epitopes that have been identified in post‐allo‐HSCT ATL patients. In a pilot study of Tax‐DC therapy in three ATL patients after various initial therapies, two patients survived for more than 4 years after vaccination without severe adverse effects (UMIN000011423). The Tax‐DC vaccine is currently under phase I trial, showing a promising clinical outcome so far. These findings indicate the importance of patients’ own HTLV‐1‐specific T‐cell responses in maintaining remission and provide a new approach to anti‐ATL immunotherapy targeting Tax. Although Tax‐targeted vaccination is ineffective against Tax‐negative ATL cells, it can be a safe alternative maintenance therapy for Tax‐positive ATL and may be further applicable for treatment of indolent ATL or even prophylaxis of ATL development among HTLV‐1‐carriers.

Human T-cell lymphotropic/leukemia virus type 1 (HTLV-1) Tax-specific T-cell exhaustion in HTLV-1-infected individuals.

Adult T‐cell leukemia/lymphoma (ATL) is caused by Human T‐cell lymphotropic/leukemia virus type 1 (HTLV‐1), and a higher HTLV‐1 provirus load in PBMC is a risk factor for ATL development. Here, we document a significant inverse correlation between the function of HTLV‐1 Tax‐specific CTL (Tax‐CTL), as assessed by ex vivo cytokine production in response to cognate peptide, and the HTLV‐1 provirus load in PBMC in both HTLV‐1 asymptomatic carriers (AC) (Spearman rank correlation coefficient [Rs] = −0.494, P = .037, n = 18) and ATL patients (Rs = −0.774, P = .001, n = 15). There was also a significant correlation between the HTLV‐1 provirus load and the percentage of PD‐1‐positive Tax‐CTL in both HTLV‐1 AC (Rs = 0.574, P = .013) and ATL patients (Rs = 0.676, P = .006). Furthermore, the percentage of PD‐1‐positive Tax‐CTL was inversely correlated with their function in HTLV‐1 AC (Rs = −0.542, P = .020), and ATL patients (Rs = −0.639, P = .010). These findings indicate that the function of Tax‐CTL decreased as their programmed cell death protein 1 (PD‐1) levels increased, parallel to the increased HTLV‐1 provirus load in PBMC. We propose that functional Tax‐CTL are crucial for determining the HTLV‐1 provirus load in PBMC, not only in HTLV‐1 AC, but also in ATL, and that PD‐1 expression levels are reliable markers of Tax‐CTL function. Thus, modulating the immunological equilibrium between Tax‐CTL and HTLV‐1‐infected cells to achieve dominance of functional effectors could represent an ideal strategy for controlling HTLV‐1‐associated disease.

A Preclinical Model for the ATLL Lymphoma Subtype With Insights Into the Role of Microenvironment in HTLV-1-Mediated Lymphomagenesis.

Adult T cell Leukemia/Lymphoma (ATLL) is a mature T cell malignancy associated with Human T cell Leukemia Virus type 1 (HTLV-1) infection. Among its four main clinical subtypes, the prognosis of acute and lymphoma variants remains poor. The long latency (3–6 decades) and low incidence (3–5%) of ATLL imply the involvement of viral and host factors in full-blown malignancy. Despite multiple preclinical and clinical studies, the contribution of the stromal microenvironment in ATLL development is not yet completely unraveled. The aims of this study were to investigate the role of the host microenvironment, and specifically fibroblasts, in ATLL pathogenesis and to propose a murine model for the lymphoma subtype. Here we present evidence that the oncogenic capacity of HTLV-1-immortalized C91/PL cells is enhanced when they are xenotransplanted together with human foreskin fibroblasts (HFF) in immunocompromised BALB/c Rag2-/-γc-/- mice. Moreover, cell lines derived from a developed lymphoma and their subsequent in vivo passages acquired the stable property to induce aggressive T cell lymphomas. In particular, one of these cell lines, C91/III cells, consistently induced aggressive lymphomas also in NOD/SCID/IL2Rγc KO (NSG) mice. To dissect the mechanisms linked to this enhanced tumorigenic ability, we quantified 45 soluble factors released by these cell lines and found that 21 of them, mainly pro-inflammatory cytokines and chemokines, were significantly increased in C91/III cells compared to the parental C91/PL cells. Moreover, many of the increased factors were also released by human fibroblasts and belonged to the known secretory pattern of ATLL cells. C91/PL cells co-cultured with HFF showed features reminiscent of those observed in C91/III cells, including a similar secretory pattern and a more aggressive behavior in vivo. On the whole, our data provide evidence that fibroblasts, one of the major stromal components, might enhance tumorigenesis of HTLV-1-infected and immortalized T cells, thus throwing light on the role of microenvironment contribution in ATLL pathogenesis. We also propose that the lymphoma induced in NSG mice by injection with C91/III cells represents a new murine preclinical ATLL model that could be adopted to test novel therapeutic interventions for the aggressive lymphoma subtype.

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.

Genetic analysis and its clinical implication in adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm with a dismal prognosis. This disease is caused by human T-cell leukemia virus type-1 (HTLV-1) retrovirus. Not only viral proteins, such as Tax and HBZ, but also additional genetic and epigenetic aberrations, including gain-of-function alterations in the components of T-cell receptor/NF-κB pathway or deteriorating alterations responsible for immune surveillance, are essential for ATL development and progression. Furthermore, recent investigation has demonstrated the effects of genetic and epigenetic alterations prevalently identified in ATL on the disease phenotype and its clinical outcomes. Aggressive ATL is associated with an increased burden of genetic and epigenetic abnormalities. Higher frequencies of TP53 and IRF4 mutations and several copy number alterations, including PD-L1 amplifications and CDKN2A deletions, are more predominant in aggressive than indolent diseases. In contrast, STAT3 mutations are more frequent in indolent ATL. Several genetic alterations, such as PD-L1 amplification, can predict the clinical outcomes independent of established clinical factors. Therefore, ATL subtypes can further be subdivided into genetically distinct subgroups with different prognoses. The genetic profiling of ATL cases has revealed its molecular pathology and should contribute to improved prognostication and management of patients with ATL.

Stability of the HTLV-1 Antisense-Derived Protein, HBZ, Is Regulated by the E3 Ubiquitin-Protein Ligase, UBR5

Human T-cell leukemia virus type 1 (HTLV-1) encodes a protein derived from the antisense strand of the proviral genome designated HBZ (HTLV-1 basic leucine zipper factor). HBZ is the only viral gene consistently expressed in infected patients and adult T-cell leukemia/lymphoma (ATL) tumor cell lines. It functions to antagonize many activities of the Tax viral transcriptional activator, suppresses apoptosis, and supports proliferation of ATL cells. Factors that regulate the stability of HBZ are thus important to the pathophysiology of ATL development. Using affinity-tagged protein and shotgun proteomics, we identified UBR5 as a novel HBZ-binding partner. UBR5 is an E3 ubiquitin-protein ligase that functions as a key regulator of the ubiquitin proteasome system in both cancer and developmental biology. Herein, we investigated the role of UBR5 in HTLV-1-mediated T-cell transformation and leukemia/lymphoma development. The UBR5/HBZ interaction was verified in vivo using over-expression constructs, as well as endogenously in T-cells. shRNA-mediated knockdown of UBR5 enhanced HBZ steady-state levels by stabilizing the HBZ protein. Interestingly, the related HTLV-2 antisense-derived protein, APH-2, also interacted with UBR5 in vivo. However, knockdown of UBR5 did not affect APH-2 protein stability. Co-immunoprecipitation assays identified ubiquitination of HBZ and knockdown of UBR5 resulted in a decrease in HBZ ubiquitination. MS/MS analysis identified seven ubiquitinated lysines in HBZ. Interestingly, UBR5 expression was upregulated in established T lymphocytic leukemia/lymphoma cell lines and the later stage of T-cell transformation in vitro. Finally, we demonstrated loss of UBR5 decreased cellular proliferation in transformed T-cell lines. Overall, our study provides evidence for UBR5 as a host cell E3 ubiquitin-protein ligase responsible for regulating HBZ protein stability. Additionally, our data suggests UBR5 plays an important role in maintaining the proliferative phenotype of transformed T-cell lines.

Epidemiological and clinical features of adult T-cell leukemia-lymphoma in Japan, 2010-2011: A nationwide survey.

Adult T‐cell leukemia–lymphoma (ATL) is a mature T‐cell malignancy associated with human T‐cell leukemia virus type 1 (HTLV‐1) infection. Japan is the most endemic country for HTLV‐1 and ATL in the world. Recent nationwide studies of Japanese blood donors reported that HTLV‐1 carriers spread from endemic areas to non‐endemic areas. Therefore, the latest information on nationwide epidemiological and clinical data for ATL is necessary to guide clinical practice. We undertook a multicenter, hospital‐based survey of newly diagnosed ATL patients from 2010 to 2011. A total of 996 patients with ATL were registered from 126 hospitals across Japan. Of those, 922 (487 men and 435 women) were included in the analysis. The median age at diagnosis was 68 years (interquartile range, 60–75 years). Overall, 67.2% of ATL was diagnosed in the Kyushu–Okinawa area. The most common subtype was acute (49.5%), followed by lymphoma (25.7%), chronic (14.2%), and smoldering (10.6%). Lymphoma type was more prevalent in men (60%), whereas chronic was more prevalent in women (60%). Half of patients with lymphoma type were aged over 70 years, whereas one‐third of patients with the chronic type were aged under 60 years. All of these characteristics were different from those of the previous nationwide surveys in the 1980s and 1990s. This survey clarified that half of current patients with ATL are aged over 68 years who were unable to receive intensive cytotoxic therapies. New less toxic agents for aged patients and further strategies to prevent the development of ATL from HTLV‐1 carrier status are needed.

HTLV-1 viral oncogene HBZ induces osteolytic bone disease in transgenic mice.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell malignancy that occurs in HTLV-1 infected patients. Most ATL patients develop osteolytic lesions and hypercalcemia of malignancy, causing severe skeletal related complications and reduced overall survival. The HTLV-1 virus encodes 2 viral oncogenes, Tax and HBZ. Tax, a transcriptional activator, is critical to ATL development, and has been implicated in pathologic osteolysis. HBZ, HTLV-1 basic leucine zipper transcription factor, promotes tumor cell proliferation and disrupts Wnt pathway modulators; however, its role in ATL induced osteolytic bone loss is unknown. To determine if HBZ is sufficient for the development of bone loss, we established a transgenic Granzyme B HBZ (Gzmb-HBZ) mouse model. Lymphoproliferative disease including tumors, enlarged spleens and/or abnormal white cell counts developed in two-thirds of Gzmb-HBZ mice at 18 months. HBZ positive cells were detected in tumors, spleen and bone marrow. Importantly, pathologic bone loss and hypercalcemia were present at 18 months. Bone-acting factors were present in serum and RANKL, PTHrP and DKK1, key mediators of hypercalcemia and bone loss, were upregulated in Gzmb-HBZ T cells. These data demonstrate that Gzmb-HBZ mice model ATL bone disease and express factors that are current therapeutic targets for metastatic and bone resident tumors.