Rapid liposomal formulation for nucleolin targeting to rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. More effective and less toxic therapies are urgently needed for high-risk patients. Peptide-guided targeted drug delivery can increase the therapeutic index of encapsulated drugs and improve patients' well-being. To apply this strategy to RMS, we identified the peptide F3 in a screening for peptides binding to RMS cells surface. F3 binds to nucleolin, which is present on the surface of RMS cells and is abundantly expressed at the mRNA level in RMS patients' biopsies compared to healthy tissues. We developed a rapid microfluidic formulation of F3-decorated PEGylated liposomes and remote loading of the chemotherapeutic drug vincristine. Size, surface charge, drug loading and retention of targeted and control liposomes were studied. Enhanced cellular binding and uptake were observed in three different nucleolin-positive RMS cell lines. Importantly, F3-functionalized liposomes loaded with vincristine were up to 11 times more cytotoxic than non-targeted liposomes for RMS cell lines. These results demonstrate that F3-functionalized liposomes are promising for targeted drug delivery to RMS and warrant further in vivo investigations.

CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, whose prognosis is still poor especially for metastatic, high-grade, and relapsed RMS. New treatments are urgently needed, especially systemic therapies. Chimeric Antigen Receptor T cells (CAR Ts) are very effective against hematological malignancies, but their efficacy against solid tumors needs to be improved. CD276 (B7-H3) is a target upregulated in RMS and detected at low levels in normal tissues. FGFR4 is a very specific target for RMS. Here, we optimized CAR Ts for these two targets, alone or in combination, and tested their anti-tumor activity in vitro and in vivo. METHODS: Four different single-domain antibodies were used to select the most specific FGFR4-CAR construct. RMS cell killing and cytokine production by CD276- and FGFR4-CAR Ts expressing CD8α or CD28 HD/TM domains in combination with 4-1BB and/or CD28 co-stimulatory domains were tested in vitro. The most effective CD276- and FGFR4-CAR Ts were used to generate Dual-CAR Ts. Tumor killing was evaluated in vivo in three orthotopic RMS mouse models. RESULTS: CD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3ζ) showed the strongest killing of RMS cells, and the highest release of IFN-γ and Granzyme B in vitro. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3ζ) showed the most specific killing. CD276-CAR Ts successfully eradicated RD- and Rh4-derived RMS tumors in vivo, achieving complete remission in 3/5 and 5/5 mice, respectively. In CD276low JR-tumors, however, they achieved complete remission in only 1/5 mice. FGFR4 CAR Ts instead delayed Rh4 tumor growth. Dual-CAR Ts promoted Rh4-tumors clearance in 5/5 mice. CONCLUSIONS: CD276- and CD276/FGFR4-directed CAR Ts showed effective RMS cell killing in vitro and eradication of CD276high RMS tumors in vivo. CD276low tumors escaped the therapy highlighting a correlation between antigen density and effectiveness. FGFR4-CAR Ts showed specific killing in vitro but could only delay RMS growth in vivo. Our results demonstrate that combined expression of CD276-CAR with other CAR does not reduce its benefit. Introducing immunotherapy with CD276-CAR Ts in RMS seems to be feasible and promising, although CAR constructs design and target combinations have to be further improved to eradicate tumors with low target expression.

Surfaceome Profiling of Cell Lines and Patient-Derived Xenografts Confirm FGFR4, NCAM1, CD276, and Highlight AGRL2, JAM3, and L1CAM as Surface Targets for Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The prognosis for patients with high-grade and metastatic disease is still very poor, and survivors are burdened with long-lasting side effects. Therefore, more effective and less toxic therapies are needed. Surface proteins are ideal targets for antibody-based therapies, like bispecific antibodies, antibody-drug conjugates, or chimeric antigen receptor (CAR) T-cells. Specific surface targets for RMS are scarce. Here, we performed a surfaceome profiling based on differential centrifugation enrichment of surface/membrane proteins and detection by LC-MS on six fusion-positive (FP) RMS cell lines, five fusion-negative (FN) RMS cell lines, and three RMS patient-derived xenografts (PDXs). A total of 699 proteins were detected in the three RMS groups. Ranking based on expression levels and comparison to expression in normal MRC-5 fibroblasts and myoblasts, followed by statistical analysis, highlighted known RMS targets such as FGFR4, NCAM1, and CD276/B7-H3, and revealed AGRL2, JAM3, MEGF10, GPC4, CADM2, as potential targets for immunotherapies of RMS. L1CAM expression was investigated in RMS tissues, and strong L1CAM expression was observed in more than 80% of alveolar RMS tumors, making it a practicable target for antibody-based therapies of alveolar RMS.

Quantum Dot-Based Screening Identifies F3 Peptide and Reveals Cell Surface Nucleolin as a Therapeutic Target for Rhabdomyosarcoma.

Active drug delivery by tumor-targeting peptides is a promising approach to improve existing therapies for rhabdomyosarcoma (RMS), by increasing the therapeutic effect and decreasing the systemic toxicity, e.g., by drug-loaded peptide-targeted nanoparticles. Here, we tested 20 different tumor-targeting peptides for their ability to bind to two RMS cell lines, Rh30 and RD, using quantum dots Streptavidin and biotin-peptides conjugates as a model for nanoparticles. Four peptides revealed a very strong binding to RMS cells: NCAM-1-targeting NTP peptide, nucleolin-targeting F3 peptide, and two Furin-targeting peptides, TmR and shTmR. F3 peptide showed the strongest binding to all RMS cell lines tested, low binding to normal control myoblasts and fibroblasts, and efficient internalization into RMS cells demonstrated by the cytoplasmic delivery of the Saporin toxin. The expression of the nucleophosphoprotein nucleolin, the target of F3, on the surface of RMS cell lines was validated by competition with the natural ligand lactoferrin, by colocalization with the nucleolin-binding aptamer AS1411, and by the marked sensitivity of RMS cell lines to the growth inhibitory nucleolin-binding N6L pseudopeptide. Taken together, our results indicate that nucleolin-targeting by F3 peptide represents a potential therapeutic approach for RMS.

CD4 + T cells are found within endemic Burkitt lymphoma and modulate Burkitt lymphoma precursor cell viability and expression of pathogenically relevant Epstein-Barr virus genes.

Endemic Burkitt lymphoma (eBL) is an aggressive B cell cancer characterized by an IgH/c-myc translocation and the harboring of Epstein-Barr virus (EBV). Evidence accumulates that CD4 + T cells might contribute to eBL pathogenesis. Here, we investigate the presence of CD4 + T cells in primary eBL tissue and their potential dichotomous impact on an EBV-infected pre-eBL cell model using ex vivo material and in vitro co-cultures. In addition, we establish a novel method to study the effect of IgH/c-myc translocation in primary B cells by employing a CRISPR/Cas9 knock-in approach to introduce and tag de novo translocation. We unprecedently document that CD4 + T cells are present in primary eBL tumor tissue. Furthermore, we demonstrate that CD4 + T cells on the one hand suppress eBL development by killing pre-eBL cells lacking IgH/c-myc translocation in vitro and on the other hand indirectly promote eBL development by inducing crucial EBV Latency III to Latency I switching in pre-eBL cells. Finally, we show that while the mere presence of an IgH/c-myc translocation does not suffice to escape CD4 + T-cell-mediated killing in vitro, the CD4 + T-cell-mediated suppression of EBV's Latency III program in vivo may allow cells harboring an IgH/c-myc translocation and additional mutations to evade immune control and proliferate by means of deregulated c-myc activity, resulting in neoplasia. Thus, our study highlights the dichotomous effects of CD4 + T cells and the mechanisms involved in eBL pathogenesis, suggests mechanisms of their impact on eBL progression, and provides a novel in vitro model for further investigation of IgH/c-myc translocation.

Novel FGFR4-Targeting Single-Domain Antibodies for Multiple Targeted Therapies against Rhabdomyosarcoma.

The fibroblast growth factor receptor 4 (FGFR4) is overexpressed in rhabdomyosarcoma (RMS) and represents a promising target for treatments based on specific and efficient antibodies. Despite progress, there is an urgent need for targeted treatment options to improve survival rates, and to limit long-term side effects. From phage display libraries we selected FGFR4-specific single-domain antibodies (sdAb) binding to recombinant FGFR4 and validated them by flow cytometry, surface plasmon resonance, and fluorescence microscopy. The specificity of the selected sdAb was verified on FGFR4-wild type and FGFR4-knock out cells. FGFR4-sdAb were used to decorate vincristine-loaded liposomes and to generate chimeric antigen receptor (CAR) T cells. First, incubation of RMS cells with FGFR4-sdAb revealed that FGFR4-sdAb can block FGF19-FGFR4 signaling via the MAPK pathway and could therefore serve as therapeutics for FGFR4-dependent cancers. Second, FGFR4-targeted vincristine-loaded liposomes bound specifically to RMS cells and were internalized by the receptor, demonstrating the potential for active drug delivery to the tumor. Third, FGFR4-CAR T cells, generated with one sdAb candidate, demonstrated strong and specific cytotoxicity against FGFR4 expressing RMS cells. We selected novel FGFR4-sdAb with high specificity and nano- to picomolar affinities for FGFR4 which have the potential to enable multiple FGFR4-targeted cancer therapy approaches.

The mannose 6-phosphate receptor targeted with porphyrin-based periodic mesoporous organosilica nanoparticles for rhabdomyosarcoma theranostics.

Porphyrin-based periodic mesoporous organosilica nanoparticles (PMO) synthesized from a large functional octatriethoxysilylated porphyrin precursor and allowing two-photon excitation photodynamic therapy (TPE-PDT) and NIR imaging were synthesized. These PMO were grafted with polyethylene glycol (PEG) moieties and an analogue of mannose 6-phosphate functionalized at the anomeric position (AMFA). AMFAs are known to efficiently target mannose 6-phosphate receptors (M6PRs) which are over-expressed in various cancers. Here, we demonstrated for the first time that M6PRs were over-expressed in rhabdomyosarcoma (RMS) cells and could be efficiently targeted with PMO-AMFA allowing TPE imaging and TPE-PDT of RMS cells. The comparison with healthy myoblasts demonstrated an absence of biological effects, suggesting a cancer cell specificity in the biomedical action observed.

Cyclin-dependent kinase 1 and survivin as potential therapeutic targets against nasal natural killer/T-cell lymphoma.

Nasal natural killer/T-cell lymphoma (NNKTL) is closely associated with Epstein-Barr virus (EBV) and is characterized by poor prognosis, resulting from rapid progression of lesions in the affected organs. Recent data have shown that NNKTL is associated with the aberrant expression of cyclin-dependent kinase 1 (CDK1) and its downstream target survivin, but little is known about the functional roles of CDK1 and survivin in NNKTL. In the current study, we show that knockdown of the EBV-encoded oncoprotein latent membrane protein 1 (LMP1) induces downregulation of CDK1 and survivin in NNKTL cells. Immunohistochemistry detected CDK1 and survivin expression in LMP1-positive cells of NNKTL biopsy specimens. Inhibition of CDK1 and survivin in NNKTL cells with several inhibitors led to a dose-dependent decrease in cell proliferation. In addition, the Sp1 inhibitor mithramycin, which can downregulate both CDK1 and survivin, significantly suppressed the growth of established NNKTL in a murine xenograft model. Our results suggest that LMP1 upregulation of CDK1 and survivin may be essential for NNKTL progression. Furthermore, targeting CDK1 and survivin with Sp1 inhibitors such as mithramycin may be an effective approach to treat NNKTL, which is considered to be a treatment-refractory lymphoma.

IRAK4 is essential for TLR9-induced suppression of Epstein-Barr virus BZLF1 transcription in Akata Burkitt's lymphoma cells.

Burkitt's lymphoma (BL) is the most common childhood cancer in equatorial Africa, and is endemic to areas where people are chronically co-infected with Epstein-Barr virus (EBV) and the malaria pathogen Plasmodium falciparum. The contribution of these pathogens in the oncogenic process remains poorly understood. We showed earlier that the activation of Toll-like receptor (TLR) 9 by hemozoin, a disposal product formed from the digestion of blood by P. falciparum, suppresses the lytic reactivation of EBV in BL cells. EBV lytic reactivation is regulated by the expression of transcription factor Zta (ZEBRA), encoded by the EBV gene BZLF1. Here, we explore in the BL cell line Akata, the mechanism involved in repression by TLR9 of expression of BZLF1. We show that BZLF1 repression is mediated upon TLR9 engagement by a mechanism that is largely independent of de novo protein synthesis. By CRISPR/Cas9-induced inactivation of TLR9, MyD88, IRAK4 and IRAK1 we confirm that BZLF1 repression is dependent on functional TLR9 and MyD88 signaling, and identify IRAK4 as an essential element for TLR9-induced repression of BZLF1 expression upon BCR cross-linking. Our results unprecedentedly show that TLR9-mediated inhibition of lytic EBV is largely independent of new protein synthesis and demonstrate the central roles of MyD88 and IRAK4 in this process contributing to EBV's persistence in the host's B-cell pool.

The proprotein convertase furin in tumour progression.

Proprotein convertases are proteases that have been implicated in the activation of a wide variety of proteins. These proteins are generally synthesised as precursor proteins and require limited proteolysis for conversion into their mature bioactive counterparts. Many of these proteins, including metalloproteases, growth factors and their receptors or adhesion molecules, have been shown to facilitate tumour formation and progression. Hence, this review will focus on the proprotein convertase furin and its role in cancer. The expression of furin has been confirmed in a large spectrum of cancers such as head and neck squamous cell carcinoma, breast cancer and rhabdomyosarcoma. Functional studies modulating furin activity uncovered its importance for the processing of many cancer-related substrates and strongly indicate that high furin activity promotes the malignant phenotype of cancer cells. In this review, we summarise the expression and function of furin in different cancer types, discuss its role in processing cancer-related proproteins and give examples of potential therapeutic approaches that take advantage of the proteolytic activity of furin in cancer cells.

Prolonged circulation and increased tumor accumulation of liposomal vincristine in a mouse model of rhabdomyosarcoma.

AIM: Our goal was to improve vincristine (VCR) based rhabdomyosarcoma (RMS) therapy by encapsulating the drug into liposomes. A targeting strategy was attempted to enhance tumor accumulation. MATERIALS & METHODS: VCR was loaded in control and peptide-decorated liposomes via an active method. The interaction of an RMS-specific peptide with the presumed target furin and the cellular uptake of both liposomal groups were studied in vitro. Pharmacokinetics and biodistribution of VCR-containing liposomes were assessed in an RMS xenograft mouse model. RESULTS: Liposomes ensured high VCR concentration in plasma and in the tumor. Peptide-decorated liposomes showed modest uptake in RMS cells. CONCLUSION: The investigated peptide-modified liposomal formulation may not be optimal for furin-mediated RMS targeting. Nevertheless, VCR-loaded liposomes could serve as a delivery platform for experimental RMS.

Peptides for tumor-specific drug targeting: state of the art and beyond.

Important elements for an efficient tumor targeted delivery are cancer-specific de novo- or over-expression of target receptors and their availability on the tumor cell surface. Peptides can be designed to selectively bind to such receptors and act as tumor-targeting ligands, as has been revealed in several preclinical studies. Notably, the amino acid sequences of these peptides can be chemically modified to prevent enzymatic degradation and improve the stability of the peptide. Furthermore, tumor-targeting peptides can be conjugated to the surface of nanosized drug carriers for the selective delivery of anticancer drugs to tumors. In this review, tumor receptors for which targeting peptides have been identified are described, and their targeting potential is considered. Various chemical modifications of peptides are thoroughly described, and targeting peptides as well as peptide-functionalized nanocarriers are critically discussed. The limitations of active targeting nanocarriers are evaluated in detail, and an outlook on the potential of tumor-targeting peptides and their clinical applications is provided.

Activation of ATR-Chk1 pathway facilitates EBV-mediated transformation of primary tonsillar B-cells.

Primary infection of the immunocompromised host with the oncovirus Epstein-Barr virus (EBV) that targets mainly B-cells is associated with an increased risk for EBV-associated tumors. The early events subsequent to primary infection with potential for B-cell transformation are poorly studied. Here, we modeled in vitro the primary infection by using B-cells isolated from tonsils, the portal of entry of EBV, since species specificity of EBV hampers modeling in experimental animals. Increasing evidence indicates that the host DNA damage response (DDR) can influence and be influenced by EBV infection. Thus, we inoculated tonsillar B-cells (TBCs) with EBV-B95.8 and investigated cell proliferation and the DDR during the first 96 hours thereafter. We identified for the first time that EBV infection of TBCs induces a period of hyperproliferation 48-96 hours post infection characterized by the activation of ataxia telangiectasia and Rad3-releated (ATR) and checkpoint kinase-1 (Chk1). Whereas inhibition of Chk1 did not affect B-cell transformation, the specific inhibition of ATR robustly decreased the transformation efficiency of EBV. Our results suggest that activation of ATR is key for EBV-induced B-cell transformation. Thus, targeting the interaction between ATR/Chk1 and EBV could offer new options for the treatment of EBV-associated malignancies.

The proprotein convertase furin is required to maintain viability of alveolar rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Success of current therapies is still limited and outcome is particularly poor for metastatic alveolar rhabdomyosarcoma (aRMS). We previously identified the proprotein convertase furin as potential target for specific drug delivery with RMS-homing peptides. Furin is a protease that converts inactive precursor proteins into bioactive proteins and peptides. In this study, we investigate the biological role of furin in aRMS progression in vitro and in vivo. Furin expression was confirmed in over 86% RMS biopsies in a tissue microarray (n=89). Inducible furin silencing in vitro led to significant impairment of cell viability and proliferation in all investigated aRMS cell lines, but not in MRC5 fibroblasts. Furthermore, the aRMS cell lines Rh3 and Rh4 revealed to be very sensitive to furin silencing, undergoing caspase-dependent cell death. Notably, furin silencing in vivo led to complete remission of established Rh4 tumors and to delayed growth in Rh30 tumors. Taken together, these findings identify furin as an important factor for aRMS progression and survival. Thus, we propose furin as a novel therapeutic target for treatment of aRMS.

The Proprotein Convertase Furin Contributes to Rhabdomyosarcoma Malignancy by Promoting Vascularization, Migration and Invasion.

The proprotein convertase (PC) furin cleaves precursor proteins, an important step in the activation of many cancer-associated proteins. Substrates of furin and furin-like PCs play a role in proliferation, metastasis and invasion. Some of them are involved in the progression of the pediatric soft tissue sarcoma rhabdomyosarcoma (RMS). In this study, we show that PCs, and in particular furin, are expressed in RMS cell lines. To investigate the functional role of furin, we generated RMS cell lines with modulated furin activity. Silencing or stable inhibition of furin delayed tumor growth in Rh30 and RD xenografts in vivo, and was correlated with lower microvessel density. Reduced furin activity also decreased migration and invasion abilities in vitro, and inhibition of furin in RMS cells diminished processing of IGF1R, VEGF-C, PDGF-B and MT1-MMP, leading to lower levels of mature proteins. Furthermore, we found that furin activity is required for proper IGF signaling in RMS cells, as furin silencing resulted in reduced phosphorylation of Akt upon IGF1 stimulation. Taken together, our results suggest that furin plays an important role in the malignant phenotype of RMS cells by activating proteins involved in tumor growth and vascularization, metastasis and invasion.

Telomerase activity impacts on Epstein-Barr virus infection of AGS cells.

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva.

Prolonged survival upon ultrasound-enhanced doxorubicin delivery in two syngenic glioblastoma mouse models.

Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans with a very poor prognosis. Chemotherapeutical treatment of GBMs is limited by the blood-brain barrier (BBB). This physical and metabolic barrier separates the blood from the brain parenchyma and prevents the entry of toxins but also of potentially useful chemotherapeutics from the blood into the brain. Microbubble-enhanced focused ultrasound (MB-FUS) has been proposed to disrupt locally and reversibly the BBB to facilitate diffusion of drugs from the micro vasculature into brain tissue. The present study investigates the feasibility and the safety of such an approach in two syngenic mouse models of GBM (GL261 and SMA-560). Local doxorubicin (DOX) concentration in MB-FUS sonicated normal brain tissue as well as in brain tumor tissue was increased as compared to the unsonicated control tissue in the contralateral hemisphere. Moreover, ultrasound mediated BBB disruption, in combination with DOX therapy, resulted in a significant increase of survival and in a slower disease progression in the two syngenic GBM mouse models. In conclusion, our results confirm that MB-ultrasound might ultimately be an effective technology to improve the therapy of GBM, and they provide for the first time evidence that combining MB-FUS with DOX treatment is effective in syngenic mouse models for GBM which can serve as preclinical models to study the impact of immune system on the therapeutic application of MB-FUS chemotherapy.

Activation of NF-κB via endosomal Toll-like receptor 7 (TLR7) or TLR9 suppresses murine herpesvirus 68 reactivation.

UNLABELLED: In order to understand and possibly treat B-cell malignancies associated with latent gammaherpesvirus infection, it is vital to understand the factors that control the balance between the two transcriptional states of gammaherpesviruses: latency and lytic replication. We used murine gammaherpesvirus 68 (MHV 68) as a model system to investigate how engagement of endosomal Toll-like receptors (TLRs) impacts reactivation from latency in vitro and establishment of latent infection in vivo. We found that treatment with TLR7 ligand R848 or TLR9 ligand CpG oligodeoxynucleotide (ODN) suppresses reactivation of MHV 68 in vitro. These suppressive effects correlated with the ability to activate cellular transcription factor NF-κB. Downregulation of TLR9 by RNA interference in vitro led to a reduction of nuclear levels of NF-κB p65 and consequently to an increase of spontaneous reactivation in cells latently infected with MHV 68, indicating that the TLR9 pathway suppresses spontaneous reactivation events. In vivo, sustained stimulation of TLR7 by repeated R848 treatment led to an increased frequency of infected splenocytes compared to mock-treated control results. Frequencies of infected splenic B cells in tlr7-/- or tlr9-/- mice after establishment of latency did not differ from those seen with their wild-type counterparts. Nevertheless, MHV 68-infected B cells from tlr9-/- mice showed a higher frequency of reactivation than B cells from wild-type or tlr7-/- mice in ex vivo reactivation assays. Thus, we show a suppressive effect of TLR7 or TLR9 triggering on MHV 68 reactivation that correlates with NF-κB activation and that the mere presence of a functional TLR9 signaling pathway contributes to dampen lytic gammaherpesvirus reactivation in infected cells. IMPORTANCE: A hallmark of gammaherpesviruses is their establishment of latency in B cells that is reversible through lytic reactivation. Latency can result in B-cell malignancies. Activation of the innate immune system is thought to contribute to controlling the switch between the transcriptional states of latency and reactivation. Nevertheless, the mechanisms involved are not clear. Here, we show that engagement of Toll-like receptor 7 (TLR7) and TLR9 suppresses reactivation of murine gammaherpesvirus MHV 68 in vitro and that stimulation of TLR7 in vivo increases the frequency of infected cells. TLR7 and TLR9 are innate immunity sensors of nucleic acids localized in endosomes. Additionally, we demonstrate that impairment of TLR9 signaling in latently infected B cells leads to increased reactivation. Thus, activated endosomal TLR7 and TLR9 pathways play an important role in promoting establishment of latent gammaherpesvirus infection. Counteracting signaling of these pathways allows reactivation and could represent treatment targets in gammaherpesvirus-associated malignancies.

Oropharyngeal group A streptococcal colonization disrupts latent Epstein-Barr virus infection.

Epstein-Barr virus (EBV) infects >90% of the human population within the first 2 decades of life and establishes reversible latent infection in B cells. The stimuli that lead to switching from latent to lytic EBV infection in vivo are still elusive. Group A streptococci (GAS) are a common cause of bacterial pharyngotonsillitis in children and adolescents and colonize the tonsils and pharynx of up to 20% of healthy children. Thus, concomitant presence of EBV and GAS in the same individual is frequent. Here, we show that EBV carriers who are colonized with GAS shed EBV particles in higher numbers in their saliva, compared with EBV carriers not colonized with GAS. Messenger RNA levels of the master lytic regulatory EBV gene BZLF1 were more frequently detected in tonsils from EBV carriers colonized with GAS than from EBV carriers not colonized. Heat-killed GAS, potentially mimicking GAS colonization, elicited lytic EBV in latently infected lymphoblastoid cell lines (LCLs) partially via Toll-like receptor 2 triggering, as did purified GAS peptidoglycan. Thus, colonization by GAS might benefit EBV by increasing the EBV load in saliva and thereby enhancing the likelihood of EBV spread to other hosts.

Early gene expression changes by Epstein-Barr virus infection of B-cells indicate CDKs and survivin as therapeutic targets for post-transplant lymphoproliferative diseases.

Lymphoproliferative diseases (LPDs) associated with Epstein-Barr virus (EBV) infection cause significant morbidity and mortality in bone marrow and solid organ transplant recipients. To gain insight into LPD pathogenesis and to identify potential effective therapeutic approaches, we investigated early molecular events leading to B-cell transformation by gene expression profiling of EBV-infected B-cells from tonsils by Affymetrix microarray 72 hr postinfection when the B-cells hyperproliferation phase starts. Cell cycle and apoptosis were the most significantly affected pathways and enriched gene sets. In particular, we found significantly increased expression of cyclin-dependent kinase (CDK)1 and CCNB1 (cyclin B1) and of one of their downstream targets BIRC5 (survivin). Importantly, the strong upregulation of the antiapoptotic protein survivin was confirmed in lymphoblastoid cell lines (LCLs) and 71% of EBV-positive post-transplant EBV-LPD lesions scored positive for survivin. The validity of early transforming events for the identification of therapeutic targets for EBV-LPD was confirmed by the marked antiproliferative effect of the CDK inhibitor flavopiridol on LCLs and by the strong induction of apoptosis by survivin inhibition with YM155 or terameprocol. Our results suggest that targeting of CDKs and/or survivin in post-transplant EBV-LPD by specific inhibitors might be an important approach to control and eliminate EBV-transformed B-cells that should be further considered.