Atypical teratoid rhabdoid tumor (AT/RT) is among the most fatal of all pediatric brain tumors. Aside from loss of function mutations in the SMARCB1 (BAF47/INI1/SNF5) chromatin remodeling gene, ...little is known of other molecular drivers of AT/RT. LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. We identified high-levels of LIN28A and LIN28B in AT/RT primary tumors and cell lines, with corresponding low levels of the LIN28-regulated microRNAs of the let-7 family. Knockdown of LIN28A by lentiviral shRNA in the AT/RT cell lines CHLA-06-ATRT and BT37 inhibited growth, cell proliferation and colony formation and induced apoptosis. Suppression of LIN28A in orthotopic xenograft models led to a more than doubling of median survival compared to empty vector controls (48 vs 115 days). LIN28A knockdown led to increased expression of let-7b and let-7g microRNAs and a down-regulation of KRAS mRNA. AT/RT primary tumors expressed increased mitogen activated protein (MAP) kinase pathway activity, and the MEK inhibitor selumetinib (AZD6244) decreased AT/RT growth and increased apoptosis. These data implicate LIN28/RAS/MAP kinase as key drivers of AT/RT tumorigenesis and indicate that targeting this pathway may be a therapeutic option in this aggressive pediatric malignancy.
The latency-associated transcript (LAT) of herpes simplex virus 1 (HSV-1), CD8α(+) dendritic cells (DCs), and programmed death 1 (PD-1) have all been implicated in the HSV-1 latency-reactivation ...cycle. It is not known, however, whether an interaction between LAT and CD8α(+) DCs regulates latency and T-cell exhaustion. To address this question, we used LAT-expressing LAT(+) and LAT-negative LAT(-) viruses. Depletion of DCs in mice ocularly infected with LAT(+) virus resulted in a reduction in the number of T cells expressing PD-1 in the trigeminal ganglia (TG), whereas depletion of DCs in mice similarly infected with LAT(-) virus did not alter PD-1 expression. CD8α(+) DCs, but not CD4(+) DCs, infected with LAT(+) virus had higher levels of ICP0, ICP4, thymidine kinase (TK), and PD-1 ligand 1 (PD-L1) transcripts than those infected with LAT(-) virus. Coculture of infected bone marrow (BM)-derived DCs from wild-type (WT) mice, but not infected DCs from CD8α(-/-) mice, with WT naive T cells contributed to an increase in PD-1 expression. Transfer of bone marrow from WT mice but not CD8α(-/-) mice to recipient Rag1(-/-) mice increased the number of latent viral genomes in reconstituted mice infected with the LAT(+) virus. Collectively, these data indicated that a reduction in latency correlated with a decline in the levels of CD8α(+) DCs and PD-1 expression. In summary, our results demonstrate an interaction among LAT, PD-1, and CD11c CD8α(+) cells that regulates latency in the TG of HSV-1-infected mice.
Very little is known regarding the interrelationship of LAT, PD-1, and CD8α(+) DCs and how such interactions might contribute to relative numbers of latent viral genomes. We show here that (i) in both in vivo and in vitro studies, deficiency of CD8α(+) DCs significantly reduced T-cell exhaustion in the presence of LAT(+) virus but not LAT(-) virus; (ii) HSV-1 infectivity was significantly lower in LAT(-)-infected DCs than in their LAT(+)-infected counterparts; and (iii) adoptive transfer of bone marrow (BM) from WT but not CD8α(-/-) mice to recipient Rag1(-/-) mice restored latency to the level in WT mice following infection with LAT(+) virus. These studies point to a key role for CD8α(+) DCs in T-cell exhaustion in the presence of LAT, which leads to larger numbers of latent viral genomes. Thus, altering this negative function of CD8α(+) DCs can potentially be used to generate a more effective vaccine against HSV infection.
We have shown previously that HSV-1 glycoprotein K (gK) exacerbates corneal scarring (CS) in mice and rabbits. Here, we investigated the relative impact of gK overexpression on host responses during ...primary corneal infection and latency in trigeminal ganglia (TG) of infected mice.
Mice were infected ocularly with HSV-gK(3) (expressing two extra copies of gK replacing latency associated transcript LAT), HSV-gK(3) revertant (HSV-gK(3)R), or wild-type HSV-1 strain McKrae. Individual corneas on day 5 post infection (PI) and TG on day 28 PI were isolated and used for detection of gB DNA in the TG, HSV-1 receptors in the cornea and TG, and inflammatory infiltrates in TG.
During primary HSV-1 infection, gK overexpression resulted in altered expression of herpesvirus entry mediator (HVEM), 3-O-sulfated heparin sulfate (3-OS-HS), paired immunoglobulin-like type 2 receptor-α (PILR-α), nectin-1, and nectin-2 in cornea of BALB/c, but not C57BL/6 mice. However, gK overexpression did have an effect on 3-OS-HS, PILR-α, nectin-1, and nectin-2 expression (but not HVEM expression) in TG of C57BL/6 mice during latency. These differences did not affect the level of latency, but instead were correlated with the presence of CS. The presence of LAT increased HVEM expression and this effect was enhanced further by the presence of CS in latently-infected mice. Finally, the presence of LAT, but not overexpression of gK, affected CD4, CD8, TNF-α, Tim-3, PD-1, IL-21, IL-2, and IFN-γ expression in TG.
We demonstrate a novel link between gK exacerbation of CS and HSV-1 receptors, suggesting a gK-induced molecular route for the pathogenesis as well as selective advantage of these entry routes for the pathogen during latency-reactivation cycle.
CD80 plays a critical role in stimulation of T cells and subsequent control of infection. To investigate the effect of CD80 on HSV-1 infection, we constructed a recombinant HSV-1 virus that expresses ...two copies of the CD80 gene in place of the latency associated transcript (LAT). This mutant virus (HSV-CD80) expressed high levels of CD80 and had similar virus replication kinetics as control viruses in rabbit skin cells. In contrast to parental virus, this CD80 expressing recombinant virus replicated efficiently in immature dendritic cells (DCs). Additionally, the susceptibility of immature DCs to HSV-CD80 infection was mediated by CD80 binding to PD-L1 on DCs. This interaction also contributed to a significant increase in T cell activation. Taken together, these results suggest that inclusion of CD80 as a vaccine adjuvant may promote increased vaccine efficacy by enhancing the immune response directly and also indirectly by targeting to DC.
The majority of diffuse midline gliomas, H3 K27-altered (DMG-H3 K27-a), are infiltrating pediatric brain tumors that arise in the pons with no effective treatment. To understand how clonal evolution ...contributes to the tumor's invasive spread, we performed exome sequencing and SNP array profiling on 49 multi-region autopsy samples from 11 patients with pontine DMG-H3 K27-a enrolled in a phase I clinical trial of PDGFR inhibitor crenolanib. For each patient, a phylogenetic tree was constructed by testing multiple possible clonal evolution models to select the one consistent with somatic mutations and copy number variations across all tumor regions. The tree was then used to deconvolute subclonal composition and prevalence at each tumor region to study convergent evolution and invasion patterns. Somatic variants in the PI3K pathway, a late event, are enriched in our cohort, affecting 70% of patients. Convergent evolution of PI3K at distinct phylogenetic branches was detected in 40% of the patients. 24 (~ 50%) of tumor regions were occupied by subclones of mixed lineages with varying molecular ages, indicating multiple waves of invasion across the pons and extrapontine. Subclones harboring a PDGFRA amplicon, including one that amplified a PDGRFA
mutant allele, were detected in four patients; their presence in extrapontine tumor and normal brain samples imply their involvement in extrapontine invasion. Our study expands the current knowledge on tumor invasion patterns in DMG-H3 K27-a, which may inform the design of future clinical trials.
Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. In this study we ...have demonstrated for the first time that inhibitors of SPP, such as L685,458, (Z-LL)2 ketone, aspirin, ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activity via (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, when administered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes of ocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedient approach to the reduction of viral replication and the resulting pathology.
•Five different inhibitors of SPP reduced HSV-1 infectivity in vitro.•The reduced HSV-1 titer was due to blocking viral transcriptions in the nucleus.•One of the tested inhibitor also reduced HSV-1 ocular titers in vivo.
We sought to determine the possibility of an interrelationship between primary virus replication in the eye, the level of viral DNA in the trigeminal ganglia (TG) during latency, and the amount of ...virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS and RE) strains of HSV-1, and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 postinfection (p.i.), and virus reactivation on day 28 p.i. as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1, even after corneal scarification, had lower virus titers in the eye, had less latency in the TG, and took a longer time to reactivate than virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT((-)) McKrae-derived mutant. The viral dose with the McKrae strain of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG and the level of viral DNA in latent TG and time to reactivation.
Very little is known regarding the interrelationship between primary virus replication in the eye, the level of latency in TG, and the time to reactivate in the mouse model. This study was designed to answer these questions. Our results point to the absence of any correlation between the level of primary virus replication and the level of viral DNA during latency, and neither was an indicator of how rapidly the virus reactivated following explant TG-induced reactivation.
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