Kinome profiling of non-Hodgkin lymphoma identifies Tyro3 as a therapeutic target in primary effusion lymphoma.

Non-Hodgkin lymphomas (NHLs) make up the majority of lymphoma diagnoses and represent a very diverse set of malignancies. We sought to identify kinases uniquely up-regulated in different NHL subtypes. Using multiplexed inhibitor bead-mass spectrometry (MIB/MS), we found Tyro3 was uniquely up-regulated and important for cell survival in primary effusion lymphoma (PEL), which is a viral lymphoma infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Tyro3 was also highly expressed in PEL cell lines as well as in primary PEL exudates. Based on this discovery, we developed an inhibitor against Tyro3 named UNC3810A, which hindered cell growth in PEL, but not in other NHL subtypes where Tyro3 was not highly expressed. UNC3810A also significantly inhibited tumor progression in a PEL xenograft mouse model that was not seen in a non-PEL NHL model. Taken together, our data suggest Tyro3 is a therapeutic target for PEL.

In Vivo Models of Oncoproteins Encoded by Kaposi's Sarcoma-Associated Herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus. KSHV utilizes its proteins to modify the cellular environment to promote viral replication and persistence. Some of these proteins are oncogenic, modulating cell proliferation, apoptosis, angiogenesis, genome stability, and immune responses, among other cancer hallmarks. These changes can lead to the development of KSHV-associated malignancies. In this Gem, we focus on animal models of oncogenic KSHV proteins that were developed to enable better understanding of KSHV tumorigenesis.

Deletion of the K1L Gene Results in a Vaccinia Virus That Is Less Pathogenic Due to Muted Innate Immune Responses, yet Still Elicits Protective Immunity.

All viruses strategically alter the antiviral immune response to their benefit. The vaccinia virus (VACV) K1 protein has multiple immunomodulatory effects in tissue culture models of infection, including NF-κB antagonism. However, the effect of K1 during animal infection is poorly understood. We determined that a K1L-less vaccinia virus (vΔK1L) was less pathogenic than wild-type VACV in intranasal and intradermal models of infection. Decreased pathogenicity was correlated with diminished virus replication in intranasally infected mice. However, in intradermally inoculated ears, vΔK1L replicated to levels nearly identical to those of VACV, implying that the decreased immune response to vΔK1L infection, not virus replication, dictated lesion size. Several lines of evidence support this theory. First, vΔK1L induced slightly less edema than vK1L, as revealed by histopathology and noninvasive quantitative ultrasound technology (QUS). Second, infiltrating immune cell populations were decreased in vΔK1L-infected ears. Third, cytokine and chemokine gene expression was decreased in vΔK1L-infected ears. While these results identified the biological basis for smaller lesions, they remained puzzling; because K1 antagonizes NF-κB in vitro, antiviral gene expression was expected to be higher during vΔK1L infection. Despite these diminished innate immune responses, vΔK1L vaccination induced a protective VACV-specific CD8+ T cell response and protected against a lethal VACV challenge. Thus, vΔK1L is the first vaccinia virus construct reported that caused a muted innate immune gene expression profile and decreased immune cell infiltration in an intradermal model of infection yet still elicited protective immunity.IMPORTANCE The vaccinia virus (VACV) K1 protein inhibits NF-κB activation among its other antagonistic functions. A virus lacking K1 (vΔK1L) was predicted to be less pathogenic because it would trigger a more robust antiviral immune response than VACV. Indeed, vΔK1L was less pathogenic in intradermally infected mouse ear pinnae. However, vΔK1L infection unexpectedly elicited dramatically reduced infiltration of innate immune cells into ears. This was likely due to decreased expression of cytokine and chemokine genes in vΔK1L-infected ears. As such, our finding contradicted observations from cell culture systems. Interestingly, vΔK1L conferred protective immunity against lethal VACV challenge. This suggests that the muted immune response triggered during vΔK1L infection remained sufficient to mount an effective protective response. Our results highlight the complexity and unpredictable nature of virus-host interactions, a relationship that must be understood to better comprehend virus pathogenesis or to manipulate viruses for use as vaccines.

Voluntary Wheel Running Does Not Alter Mortality to or Immunogenicity of Vaccinia Virus in Mice: A Pilot Study.

Exercise has been shown to improve immune responses to viral infections and vaccines in several mouse models. However, previous pathogen studies have primarily used infections limited to the respiratory tract. Additionally, previous studies have utilized forced treadmill exercise paradigms, and voluntary wheel running (VWR) has been shown to have differential effects on the immune system in non-infection models. We examined whether VWR could improve morbidity and mortality to a 50% lethal dose of vaccinia virus (VACV), a systemic pathogen commonly used to examine immune responses. Additionally, we examined whether VWR could improve antibody response to a replication-deficient strain of VACV, mimicking a vaccination. Male C57Bl/6J mice underwent 8 weeks of VWR or remained sedentary, then were infected intranasally with 105 PFU VACV strain WR and followed 14 days for weight loss. Mice in the vaccination study ran or were sedentary for 8 weeks, then were given 106 PFU of replication-deficient VACV strain MVA intraperitoneally. Blood was collected at 1, 2, and 4 weeks post-inoculation, and anti-VACV IgG titer was determined by ELISA. VWR did not improve mortality due to VACV infection (p = 0.26), although fewer VWR mice (4/10) died compared to sedentary (SED, 6/10). VWR did not prevent body weight loss due to infection compared to SED (p = 0.20), although VWR mice loss slightly less weight compared to SED through the first 6 days post-infection. Food intake was significantly reduced in SED post-infection compared to VWR (p = 0.05). VWR mice developed a greater IgG antibody response, although this was not significant (p = 0.22). In summary, VWR did not protect against mortality to VACV or prevent infection-induced weight loss, and VWR did not enhance antibody responses. However, there were non-significant trends toward VWR-related improvements in these outcomes, and post-infection food intake was improved by VWR.

Vaccinia virus K1 ankyrin repeat protein inhibits NF-κB activation by preventing RelA acetylation.

The vaccinia virus (VACV) K1 protein inhibits dsRNA-dependent protein kinase (PKR) activation. A consequence of this function is that K1 inhibits PKR-induced NF-κB activation during VACV infection. However, transient expression of K1 also inhibits Toll-like receptor (TLR)-induced NF-κB activation. This suggests that K1 has a second NF-κB inhibitory mechanism that is PKR-independent. This possibility was explored by expressing K1 independently of infection and stimulating NF-κB under conditions that minimized or excluded PKR activation. K1 inhibited both TNF- and phorbol 12-myristate 13-acetate (PMA)-induced NF-κB activation, as detected by transcription of synthetic (e.g. luciferase) and natural (e.g. CXCL8) genes controlled by NF-κB. K1 also inhibited NF-κB activity in PKRkd cells, cells that have greatly decreased amounts of PKR. K1 no longer prevented IκBα degradation or NF-κB nuclear translocation in the absence of PKR, suggesting that K1 acted on a nuclear event. Indeed, K1 was present in the nucleus and cytoplasm of stimulated and unstimulated cells. K1 inhibited acetylation of the RelA (p65) subunit of NF-κB, a nuclear event known to be required for NF-κB activation. Moreover, p65-CBP (CREB-binding protein) interactions were blocked in the presence of K1. However, K1 did not preclude NF-κB binding to oligonucleotides containing κB-binding sites. The current interpretation of these data is that NF-κB-promoter interactions still occur in the presence of K1, but NF-κB cannot properly trigger transcriptional activation because K1 antagonizes acetylation of RelA. Thus, in comparison to all known VACV NF-κB inhibitory proteins, K1 acts at one of the most downstream events of NF-κB activation.