HTLV-1 infected T cells cause bone loss via small extracellular vesicles.

Adult T cell leukemia (ATL), caused by infection with human T cell leukemia virus type 1 (HTLV-1), is often complicated by hypercalcemia and osteolytic lesions. Therefore, we studied the communication between patient-derived ATL cells (ATL-PDX) and HTLV-1 immortalized CD4+ T cell lines (HTLV/T) with osteoclasts and their effects on bone mass in mice. Intratibial inoculation of some HTLV/T lead to a profound local decrease in bone mass similar to marrow-replacing ATL-PDX, despite the fact that few HTLV/T cells persisted in the bone. To study the direct effect of HTLV/T and ATL-PDX on osteoclasts, supernatants were added to murine and human osteoclast precursors. ATL-PDX supernatants from hypercalcemic patients promoted formation of mature osteoclasts, while those from HTLV/T were variably stimulatory, but had largely consistent effects between human and murine cultures. Interestingly, this osteoclastic activity did not correlate with expression of osteoclastogenic cytokine RANKL, suggesting an alternative mechanism. HTLV/T and ATL-PDX produce small extracellular vesicles (sEV), known to facilitate HTLV-1 infection. We hypothesized that these sEV also mediate bone loss by targeting osteoclasts. We isolated sEV from both HTLV/T and ATL-PDX, and found they carried most of the activity found in supernatants. In contrast, sEV from uninfected activated T cells had little effect. Analysis of sEV (both active and inactive) by mass spectrometry and electron microscopy confirmed absence of RANKL and intact virus. Viral proteins Tax and Env were only present in sEV from the active, osteoclast-stimulatory group, along with increased representation of proteins involved in osteoclastogenesis and bone resorption. sEV injected over mouse calvaria in the presence of low dose RANKL caused more osteolysis than RANKL alone. Thus, HTLV-1 infection of T cells can cause release of sEV with strong osteolytic potential, providing a mechanism beyond RANKL production that modifies the bone microenvironment, even in the absence of overt leukemia.

An HTLV-1 envelope mRNA vaccine is immunogenic and protective in New Zealand rabbits.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult T-cell leukemia/lymphoma, a severe and fatal CD4+ T-cell malignancy. Additionally, HTLV-1 can lead to a chronic progressive neurodegenerative disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis. Unfortunately, the prognosis for HTLV-1-related diseases is generally poor, and effective treatment options are limited. In this study, we designed and synthesized a codon optimized HTLV-1 envelope (Env) mRNA encapsulated in a lipid nanoparticle (LNP) and evaluated its efficacy as a vaccine candidate in an established rabbit model of HTLV-1 infection and persistence. Immunization regimens included a prime/boost protocol using Env mRNA-LNP or control green fluorescent protein (GFP) mRNA-LNP. After immunization, rabbits were challenged by intravenous injection with irradiated HTLV-1 producing cells. Three rabbits were partially protected and three rabbits were completely protected against HTLV-1 challenge. These rabbits were then rechallenged 15 weeks later, and two rabbits maintained sterilizing immunity. In Env mRNA-LNP immunized rabbits, proviral load and viral gene expression were significantly lower. After viral challenge in the Env mRNA-LNP vaccinated rabbits, an increase in both CD4+/IFN-γ+ and CD8+/IFN-γ+ T-cells was detected when stimulating with overlapping Env peptides. Env mRNA-LNP elicited a detectable anti-Env antibody response after prime/boost vaccination in all animals and significantly higher levels of neutralizing antibody activity. Neutralizing antibody activity was correlated with a reduction in proviral load. These findings hold promise for the development of preventive strategies and therapeutic interventions against HTLV-1 infection and its associated diseases.IMPORTANCEmRNA vaccine technology has proven to be a viable approach for effectively triggering immune responses that protect against or limit viral infections and disease. In our study, we synthesized a codon optimized human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) mRNA that can be delivered in a lipid nanoparticle (LNP) vaccine approach. The HTLV-1 Env mRNA-LNP produced protective immune responses against viral challenge in a preclinical rabbit model. HTLV-1 is primarily transmitted through direct cell-to-cell contact, and the protection offered by mRNA vaccines in our rabbit model could have significant implications for optimizing the development of other viral vaccine candidates. This is particularly important in addressing the challenge of enhancing protection against infections that rely on cell-to-cell transmission.

Assessment of the safety of nivolumab in people living with HIV with advanced cancer on antiretroviral therapy: the AIDS Malignancy Consortium 095 Study.

BACKGROUND: Although immunotherapy has emerged as a therapeutic strategy for many cancers, there are limited studies establishing the safety and efficacy in people living with HIV (PLWH) and cancer. METHODS: PLWH and solid tumors or Kaposi sarcoma (KS) receiving antiretroviral therapy and a suppressed HIV viral load received nivolumab at 3 mg/kg every 2 weeks, in two dose deescalation cohorts stratified by CD4 count (stratum 1: CD4 count > 200/µL and stratum 2: CD4 count 100-199/µL). An expansion cohort of 24 participants with a CD4 count > 200/µL was then enrolled. RESULTS: A total of 36 PLWH received nivolumab, including 15 with KS and 21 with a variety of other solid tumors. None of the first 12 participants had dose-limiting toxicity in both CD4 strata, and five patients (14%) overall had grade 3 or higher immune related adverse events. Objective partial response occurred in nine PLWH and cancer (25%), including in six of 15 with KS (40%; 95% CI, 16.3-64.7). The median duration of response was 9.0 months overall and 12.5 months in KS. Responses were observed regardless of PDL1 expression. There were no significant changes in CD4 count or HIV viral load. CONCLUSIONS: Nivolumab has a safety profile in PLWH similar to HIV-negative subjects with cancer, and also efficacy in KS. Plasma HIV remained suppressed and CD4 counts remained stable during treatment and antiretroviral therapy, indicating no adverse impact on immune function. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02408861.

Brentuximab vedotin with AVD for stage II-IV HIV-related Hodgkin lymphoma (AMC 085): phase 2 results from an open-label, single arm, multicentre phase 1/2 trial.

BACKGROUND: Brentuximab vedotin in combination with doxorubicin, vinblastine, and dacarbazine (AVD) is approved in the upfront setting for advanced stage classical Hodgkin lymphoma (cHL). People living with HIV have been excluded from these studies. We aimed to understand the activity and safety of brentuximab vedotin-AVD in people living with HIV diagnosed with Hodgkin lymphoma, while focusing on HIV disease parameters and antiretroviral therapy (ART) interactions. METHODS: We present the phase 2 portion of a multicentre phase 1/2 study. Eligible patients were 18 years or older, had untreated stage II-IV HIV-associated cHL (HIV-cHL), a Karnofsky performance status of more than 30%, a CD4+ T-cell count of 50 cells per µL or more, were required to take ART, and were not on strong CYP3A4 or P-glycoprotein inhibitors. Patients were treated intravenously with 1·2 mg/kg of brentuximab vedotin (recommended phase 2 dose) with standard doses of AVD for six cycles on days 1 and 15 of a 28-day cycle. The primary endpoint of the phase 2 portion was 2-year progression-free survival (PFS), assessed in all eligible participants who began treatment. Accrual has been completed. This trial is registered at ClinicalTrials.gov, NCT01771107. FINDINGS: Between March 8, 2013, and March 7, 2019, 41 patients received study therapy with a median follow up of 29 months (IQR 16-38). 34 (83%) of 41 patients presented with stage III-IV and seven (17%) with stage II unfavourable HIV-cHL. 37 (90%) of 41 patients completed therapy, all 37 of whom achieved complete response. The 2-year PFS was 87% (95% CI 71-94) and the overall survival was 92% (78-97). The most common grade 3 or worse adverse events were peripheral sensory neuropathy (four [10%] of 41 patients), neutropenia (18 [44%]), and febrile neutropenia (five [12%]). One treatment-related death was reported, due to infection. INTERPRETATION: Brentuximab vedotin-AVD was highly active and had a tolerable adverse event rate in HIV-cHL and is an important therapeutic option for people with HIV-cHL. The complete reponse rate is encouraging and is possibly related to a unique aspect of HIV-cHL biology. Upcoming 5-year data will evaluate the sustainability of the outcomes obtained. FUNDING: National Institutes of Health and National Cancer Institute.

Clinical characteristics and outcomes of infection with human T-lymphotropic virus in a non-endemic area: a single institution study.

Introduction: Understanding of human T-lymphotropic virus (HTLV) remains largely based on epidemiologic and clinical data from endemic areas. Globalization has resulted in migration of persons living with HTLV (PLHTLV) from endemic to non-endemic areas, and a rise of HTLV infection in the United States. Yet, due to the historical rarity of this disease, affected patients are often under- and mis-diagnosed. Thus, we sought to characterize the epidemiology, clinical features, comorbidities, and survival of HTLV-1- or HTLV-2-positive individuals identified in a non-endemic area. Methods: Our study was a single institution, retrospective case-control analysis of HTLV-1 or HTLV-2 patients between 1998 and 2020. We utilized two HTLV-negative controls, matched for age, sex, and ethnicity, for each HTLV-positive case. We evaluated associations between HTLV infection and several hematologic, neurologic, infectious, and rheumatologic covariates. Finally, clinical factors predictive of overall survival (OS) were assessed. Results: We identified 38 cases of HTLV infection, of whom 23 were HTLV-1 and 15 were HTLV-2 positive. The majority (~54%) of patients in our control group received HTLV testing for transplant evaluation, compared to ~24% of HTLV-seropositive patients. Co-morbidities associated with HTLV, hepatitis C seropositivity were higher in HTLV-seropositive patients compared to controls (OR 10.7, 95% CI = 3.2-59.0, p < 0.001). Hepatitis C and HTLV co-infection resulted in decreased OS, compared to no infection, hepatitis C infection alone, or HTLV infection alone. Patients with any cancer diagnosis and HTLV infection had worse OS compared to patients with cancer or HTLV alone. HTLV-1 positive patients had lower median OS compared to HTLV-2 patients (47.7 months vs. 77.4 months). In univariate analysis, the hazard for 1-year all-cause mortality was increased among patients with HTLV-seropositivity, adult T-cell leukemia, acute myelogenous leukemia, and hepatitis C infection. When corrected, multivariate analysis showed that HTLV seropositivity was no longer associated with 1 year all-cause mortality; however association with AML and hepatitis C infection remained significant. Conclusion: HTLV-seropositivity was not associated with increased 1 year mortality in multivariate analysis. However, our study is limited by our small patient sample size, as well as the biased patient control population due to selection factors for HTLV testing.

Phase I Trial of the Multi-kinase Inhibitor Cabozantinib, a CYP3A4 Substrate, plus CYP3A4-Interacting Antiretroviral Therapy in People Living with HIV and Cancer (AMC-087).

PURPOSE: To evaluate the safety, pharmacokinetics, and pharmacodynamic effects of cabozantinib, a CYP3A4 substrate, in people living with human immunodeficiency virus and cancer receiving antiretrovirals (ARV). PATIENTS AND METHODS: Patients received a reduced dose of cabozantinib (20 mg orally daily) with strong CYP3A4 inhibitors (ARV ritonavir or non-ARV cobicistat, stratum A), or a standard 60 mg dose with ARVs that are CYP3A4 inducers (efavirenz or etravirine, stratum B) or noninteracting ARVs (stratum C). Initial dose escalation in stratum A and stratum B was performed on the basis of tolerability. RESULTS: 36 patients received cabozantinib plus ARVs, including 20 in stratum A, 9 in B, and 7 in C. The recommended initial cabozantinib doses for stratum A, B, and C were 20, 60, and 60 mg, respectively. Doses of 40 or 60 mg plus CYP3A4 inhibitors in stratum A and 100 mg plus CYP3A4 inducers in stratum B were associated with excessive toxicity, whereas 60 mg with noninteracting ARVs was not. The steady state minimal concentrations were lower at 20 mg in stratum A or 60 mg in stratum B compared with 60 mg in stratum C, while total exposure was only lower in 60 mg in stratum B compared with 60 mg in stratum C. Activity was observed in Kaposi sarcoma and an AXL-amplified sarcoma. CONCLUSIONS: Cabozantinib as a single agent should be initiated at 20 mg daily and 60 mg daily when taken concurrently with ARVs that are strong CYP3A4 inhibitors and inducers, respectively, with consideration for subsequent escalation per current cabozantinib guidelines. See related commentary by Eisenmann and Sparreboom, p. 4999.

Defactinib, Pembrolizumab, and Gemcitabine in Patients with Advanced Treatment Refractory Pancreatic Cancer: A Phase I Dose Escalation and Expansion Study.

PURPOSE: Targeting focal adhesion kinase (FAK) renders checkpoint immunotherapy effective in pancreatic ductal adenocarcinoma (PDAC) mouse model. Defactinib is a highly potent oral FAK inhibitor that has a tolerable safety profile. PATIENTS AND METHODS: We conducted a multicenter, open-label, phase I study with dose escalation and expansion phases. In dose escalation, patients with refractory solid tumors were treated at five escalating dose levels of defactinib and gemcitabine to identify a recommended phase II dose (RP2D). In expansion phase, patients with metastatic PDAC who progressed on frontline treatment (refractory cohort) or had stable disease (SD) after at least 4 months of standard gemcitabine/nab-paclitaxel (maintenance cohort) were treated at RP2D. Pre- and posttreatment tumor biopsies were performed to evaluate tumor immunity. RESULTS: The triple drug combination was well-tolerated, with no dose-limiting toxicities. Among 20 treated patients with refractory PDAC, the disease control rate (DCR) was 80%, with one partial response (PR) and 15 SDs, and the median progression-free survival (PFS) and overall survival (OS) were 3.6 and 7.8 months, respectively. Among 10 evaluable patients in the maintenance cohort, DCR was 70% with one PR and six SDs. Three patients with SD came off study due to treatment- or disease-related complications. The median PFS and OS on study treatment were 5.0 and 8.3 months, respectively. CONCLUSIONS: The combination of defactinib, pembrolizumab, and gemcitabine was well-tolerated and safe, had promising preliminary efficacy, and showed biomarker activity in infiltrative T lymphocytes. Efficacy of this strategy may require incorporation of more potent chemotherapy in future studies.

A role for an HTLV-1 vaccine?

HTLV-1 is a global infection with 5-20 million infected individuals. Although only a minority of infected individuals develop myelopathy, lymphoproliferative malignancy, or inflammatory disorders, infection is associated with immunosuppression and shorter survival. Transmission of HTLV-1 is through contaminated blood or needles, mother-to-child exposure through breast-feeding, and sexual intercourse. HTLV-1 is a delta retrovirus that expresses immunogenic Gag, Envelope, TAX, and Hbz proteins. Neutralizing antibodies have been identified directed against the surface envelope protein, and cytotoxic T-cell epitopes within TAX have been characterized. Thus far, there have been few investigations of vaccines directed against each of these proteins, with limited responses, thus far. However, with new technologies developed in the last few years, a renewed investigation is warranted in search for a safe and effective HTLV-1 vaccine.

Evolution of Multiple Domains of the HIV-1 Envelope Glycoprotein during Coreceptor Switch with CCR5 Antagonist Therapy.

HIV-1 uses CD4 as a receptor and chemokine receptors CCR5 and/or CXCR4 as coreceptors. CCR5 antagonists are a class of antiretrovirals used to inhibit viral entry. Phenotypic prediction algorithms such as Geno2Pheno are used to assess CCR5 antagonist eligibility, for which the V3 region is screened. However, there exist scenarios where the algorithm cannot give an accurate prediction of tropism. The current study examined coreceptor shift of HIV-1 from CCR5-tropic strains to CXCR4-tropic or dual-tropic strains among five subjects in a clinical trial of the CCR5 antagonist vicriviroc. Envelope gene amplicon libraries were constructed and subjected to next-generation sequencing, as well as single-clone sequencing and functional analyses. Approximately half of the amplified full-length single envelope-encoding clones had no significant activity for infection of cells expressing high levels of CD4 and CCR5 or CXCR4. Functional analysis of 9 to 21 individual infectious clones at baseline and at the time of VF were used to construct phylogenetic trees and sequence alignments. These studies confirmed that specific residues and the overall charge of the V3 loop were the major determinants of coreceptor use, in addition to specific residues in other domains of the envelope protein in V1/V2, V4, C3, and C4 domains that may be important for coreceptor shift. These results provide greater insight into the viral genetic determinants of coreceptor shift. IMPORTANCE This study is novel in combining single-genome sequence analysis and next-generation sequencing to characterize HIV-1 quasispecies. The work highlights the importance of mutants present at frequencies of 1% or less in development of drug resistance. This study highlights a critical role of specific amino acid substitutions outside V3 that contribute to coreceptor shift as well as important roles of the V1/V2, V4, C3, and C4 domain residues.

The Past, Present, and Future of a Human T-Cell Leukemia Virus Type 1 Vaccine.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic human retrovirus which causes a lifelong infection. An estimated 5-10 million persons are infected with HTLV-1 worldwide - a number which is likely higher due to lack of reliable epidemiological data. Most infected individuals remain asymptomatic; however, a portion of HTLV-1-positive individuals will develop an aggressive CD4+ T-cell malignancy called adult T-cell leukemia/lymphoma (ATL), or a progressive neurodegenerative disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Few treatment options exist for HAM/TSP outside of palliative care and ATL carries an especially poor prognosis given the heterogeneity of the disease and lack of effective long-term treatments. In addition, the risk of HTLV-1 disease development increases substantially if the virus is acquired early in life. Currently, there is no realistic cure for HTLV-1 infection nor any reliable measure to prevent HTLV-1-mediated disease development. The severity of HTLV-1-associated diseases (ATL, HAM/TSP) and limited treatment options highlights the need for development of a preventative vaccine or new therapeutic interventions. This review will highlight past HTLV-1 vaccine development efforts, the current molecular tools and animal models which might be useful in vaccine development, and the future possibilities of an effective HTLV-1 vaccine.

Safety and Tolerability of Carboplatin and Paclitaxel in Cancer Patients with HIV (AMC-078), an AIDS Malignancy Consortium (AMC) Study.

BACKGROUND: Persons living with human immunodeficiency virus are an underserved population for evidence-based cancer treatment. Paclitaxel and carboplatin (PCb) is an active regimen against a variety of solid tumors, including several seen in excess in patients with HIV infection. We performed a pilot trial to evaluate the safety of full-dose PCb in people living with human immunodeficiency virus and cancer. METHODS: Eligible patients, stratified by concurrent antiretroviral therapy (ART) that included CYP3A4 inhibitors or not, received paclitaxel (175 mg/m2) in combination with carboplatin (target AUC 6) intravenously every 3 weeks for up to 6 cycles. RESULTS: Sixteen evaluable patients received 64 cycles of PCb, including 6 patients treated with CYP3A4 inhibiting ART (ritonavir). The adverse event profile was consistent with the known toxicity profile of PCb, with no differences between the 2 strata. There were 4 partial responses (25%, 95% CI: 7%-52%), and overall, CD4+ lymphocyte count was similar after completion of therapy (median: 310/µL) compared with baseline values (median: 389/µL). Pharmacokinetic studies in 6 patients revealed no significant differences in Cmax or AUCinf for paclitaxel between the 2 cohorts. CONCLUSION: Full doses of PCb chemotherapy are tolerable when given concurrently with ART in people living with human immunodeficiency virus with cancer, including patients receiving CYP3A4 inhibitors. CLINICALTRIALS.GOV IDENTIFIER: NCT01249443.

Heparanase Blockade as a Novel Dual-Targeting Therapy for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused over 5 million deaths worldwide. Pneumonia and systemic inflammation contribute to its high mortality. Many viruses use heparan sulfate proteoglycans as coreceptors for viral entry, and heparanase (HPSE) is a known regulator of both viral entry and inflammatory cytokines. We evaluated the heparanase inhibitor Roneparstat, a modified heparin with minimum anticoagulant activity, in pathophysiology and therapy for COVID-19. We found that Roneparstat significantly decreased the infectivity of SARS-CoV-2, SARS-CoV-1, and retroviruses (human T-lymphotropic virus 1 [HTLV-1] and HIV-1) in vitro. Single-cell RNA sequencing (scRNA-seq) analysis of cells from the bronchoalveolar lavage fluid of COVID-19 patients revealed a marked increase in HPSE gene expression in CD68+ macrophages compared to healthy controls. Elevated levels of HPSE expression in macrophages correlated with the severity of COVID-19 and the expression of inflammatory cytokine genes, including IL6, TNF, IL1B, and CCL2. In line with this finding, we found a marked induction of HPSE and numerous inflammatory cytokines in human macrophages challenged with SARS-CoV-2 S1 protein. Treatment with Roneparstat significantly attenuated SARS-CoV-2 S1 protein-mediated inflammatory cytokine release from human macrophages, through disruption of NF-κB signaling. HPSE knockdown in a macrophage cell line also showed diminished inflammatory cytokine production during S1 protein challenge. Taken together, this study provides a proof of concept that heparanase is a target for SARS-CoV-2-mediated pathogenesis and that Roneparstat may serve as a dual-targeted therapy to reduce viral infection and inflammation in COVID-19. IMPORTANCE The complex pathogenesis of COVID-19 consists of two major pathological phases: an initial infection phase elicited by SARS-CoV-2 entry and replication and an inflammation phase that could lead to tissue damage, which can evolve into acute respiratory failure or even death. While the development and deployment of vaccines are ongoing, effective therapy for COVID-19 is still urgently needed. In this study, we explored HPSE blockade with Roneparstat, a phase I clinically tested HPSE inhibitor, in the context of COVID-19 pathogenesis. Treatment with Roneparstat showed wide-spectrum anti-infection activities against SARS-CoV-2, HTLV-1, and HIV-1 in vitro. In addition, HPSE blockade with Roneparstat significantly attenuated SARS-CoV-2 S1 protein-induced inflammatory cytokine release from human macrophages through disruption of NF-κB signaling. Together, this study provides a proof of principle for the use of Roneparstat as a dual-targeting therapy for COVID-19 to decrease viral infection and dampen the proinflammatory immune response mediated by macrophages.

Phase 1b trial of anti-VEGF/PDGFR vorolanib combined with immune checkpoint inhibitors in patients with advanced solid tumors.

PURPOSE: Vorolanib is a multi-target tyrosine kinase inhibitor with anti-angiogenic properties. This study aimed to evaluate the tolerability, safety and efficacy of vorolanib when added to checkpoint inhibitors (CPIs) in patients with advanced solid tumors. METHODS: We conducted a phase 1b study of vorolanib (300 or 400 mg orally once daily) plus pembrolizumab or nivolumab using a standard 3 + 3 design to determine the dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). The endpoints included safety, toxicity and objective response rate, according to Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST 1.1). RESULTS: Sixteen patients (9 in pembrolizumab arm, 7 in nivolumab arm) with gastrointestinal or lung cancers were enrolled. All patients had at least 1 treatment-related adverse event (TRAE). The most common TRAEs across all cohorts were lymphopenia (n = 7), leukopenia (n = 5), fatigue (n = 5), and alanine aminotransferase elevation (n = 5); most toxicities were grade (G) 1-2. DLTs were reported in 3 patients at vorolanib 400 mg dose level, with G3 aspartate aminotransferase elevation, G3 rectal hemorrhage, and G3 rash. Of 13 total response-evaluable patients, 2 patients had confirmed partial responses (1 rectal squamous cell cancer and 1 small cell lung cancer). Two patients achieved prolonged stable disease. Vorolanib 300 mg daily was determined to be the RP2D for either pembrolizumab or nivolumab. CONCLUSION: Combination vorolanib 300 mg orally once daily plus CPI appears to be a feasible regimen with manageable toxicity and promising efficacy in select tumor types. NCT03511222. Date of Registration: April 18, 2018.

Adult T-Cell Leukemia-Lymphoma Presenting Concurrently with Myelopathy.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus. Of the approximate ten to twenty million people currently infected worldwide, 4-9% of infected individuals develop adult T-cell leukemia/lymphoma (ATLL) or HTLV-associated myelopathy/tropical spastic paresis (HAM/TSP) in their lifetime. The current report is based on a patient who presented concurrently with CD30+ lymphoma subtype ATLL and HAM/TSP. The patient's ATLL responded to brentuximab-vedotin-based chemotherapy; however, HAM/TSP did not improve. The patient's peripheral blood mononuclear cells were cultured and injected into immunodeficient mice, and the mice developed massive organ involvement and chronic lymphocytic leukemia-subtype ATLL. This case study is novel in the findings of concurrent development of ATLL and HAM/TSP, the response to brentuximab-vedotin chemotherapy, and the use HTLV-1 helix basic zipper protein-targeted probe for RNAscope for diagnosis.

Targeting NF-κB with Nanotherapy in a Mouse Model of Adult T-Cell Leukemia/Lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive, clonal malignancy of mature T cells caused by human T-cell leukemia virus type 1. Although it is a rare tumor type, it serves as an excellent model of a virus driven process that transforms cells and engenders a highly malignant tumor that is extraordinarily difficult to treat. The viral transcriptional transactivator (Tax) in the HTLV-1 genome directly promotes tumorigenesis, and Tax-induced oncogenesis depends on its ability to constitutively activate NF-κB signaling. Accordingly, we developed and evaluated a nano-delivery system that simultaneously inhibits both canonical (p65) and noncanonical (p100) NF-κB signaling pathways locally in tumors after systemic administration. Our results demonstrate that siRNA is delivered rapidly to ATLL tumors after either i.p. or i.v. injection. The siRNA treatment significantly reduced both p65 and p100 mRNA and protein expression. Anti-NF-κB nanotherapy significantly inhibited tumor growth in two distinct tumor models in mice: a spontaneous Tax-driven tumor model, and a Tax tumor cell transplant model. Moreover, siRNA nanotherapy sensitized late-stage ATLL tumors to the conventional chemotherapeutic agent etoposide, indicating a pleiotropic benefit for localized siRNA nanotherapeutics.

Germinal epimutation of Fragile Histidine Triad (FHIT) gene is associated with progression to acute and chronic adult T-cell leukemia diseases.

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.

Epigenomic regulation of human T-cell leukemia virus by chromatin-insulator CTCF.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes an aggressive T-cell malignancy and a variety of inflammatory conditions. The integrated provirus includes a single binding site for the epigenomic insulator, CCCTC-binding protein (CTCF), but its function remains unclear. In the current study, a mutant virus was examined that eliminates the CTCF-binding site. The mutation did not disrupt the kinetics and levels of virus gene expression, or establishment of or reactivation from latency. However, the mutation disrupted the epigenetic barrier function, resulting in enhanced DNA CpG methylation downstream of the CTCF binding site on both strands of the integrated provirus and H3K4Me3, H3K36Me3, and H3K27Me3 chromatin modifications both up- and downstream of the site. A majority of clonal cell lines infected with wild type HTLV-1 exhibited increased plus strand gene expression with CTCF knockdown, while expression in mutant HTLV-1 clonal lines was unaffected. These findings indicate that CTCF binding regulates HTLV-1 gene expression, DNA and histone methylation in an integration site dependent fashion.

Epigenetic Regulation of Human T-Cell Leukemia Virus Gene Expression.

Viral and cellular gene expression are regulated by epigenetic alterations, including DNA methylation, histone modifications, nucleosome positioning, and chromatin looping. Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus associated with inflammatory disorders and T-cell lymphoproliferative malignancy. The transforming activity of HTLV-1 is driven by the viral oncoprotein Tax, which acts as a transcriptional activator of the cAMP response element-binding protein (CREB) and nuclear factor kappa B (NFκB) pathways. The epigenetic effects of Tax and the induction of lymphoproliferative malignancy include alterations in DNA methylation and histone modifications. In addition, alterations in nucleosome positioning and DNA looping also occur in HTLV-1-induced malignant cells. A mechanistic definition of these effects will pave the way to new therapies for HTLV-1-associated disorders.

Response-adapted therapy with infusional EPOCH chemotherapy plus rituximab in HIV-associated, B-cell non-Hodgkin's lymphoma.

Four cycles of rituximab plus CHOP chemotherapy is as effective as 6 cycles in low-risk diffuse large B-cell lymphoma (DLBCL). Here we report a post-hoc analysis of a prospective clinical trial in patients with HIV-associated DLBCL and high-grade lymphoma treated with 4-6 cycles of EPOCH plus rituximab based a response-adapted treatment strategy. 106 evaluable patients with HIV-associated DLBCL or high-grade CD20-positive non-Hodgkin's lymphoma were randomized to receive rituximab (375 mg/m2) given either concurrently prior to each infusional EPOCH cycle, or sequentially (weekly for 6 weeks) following completion of EPOCH. EPOCH consisted of a 96-hour IV infusion of etoposide, doxorubicin, and vincristine plus oral prednisone followed by IV bolus cyclophosphamide every 21 days for 4 to 6 cycles. Patients received 2 additional cycles of therapy after documentation of a complete response (CR) by computerized tomography after cycles 2 and 4. 64 of 106 evaluable patients (60%, 95% CI 50%, 70%) had a CR in both treatment arms. The 2-year event-free survival (EFS) rates were similar in the 24 patients with CR who received 4 or fewer EPOCH cycles (78%, 95% confidence intervals [55%, 90%]) due to achieving a CR after 2 cycles, compared with those who received 5-6 cycles of EPOCH (85%, 95% CI 70%, 93%) because a CR was first documented after cycle 4. A response-adapted strategy may permit a shorter treatment duration without compromising therapeutic efficacy in patients with HIV-associated lymphoma treated with EPOCH plus rituximab, which merits further evaluation in additional prospective trials. Clinical Trials.gov identifier NCT00049036.