Oncolytic varicella-zoster virus engineered with ORF8 deletion and armed with drug-controllable interleukin-12.

BACKGROUND: The varicella-zoster virus (VZV), belonging to the group of human α-herpesviruses, has yet to be developed as a platform for oncolytic virotherapy, despite indications from clinical case reports suggesting a potential association between VZV infection and cancer remission. METHODS: Here, we constructed oncolytic VZV candidates based on the vaccine strain vOka and the laboratory strain Ellen. These newly engineered viruses were subsequently assessed for their oncolytic properties in the human MeWo melanoma xenograft model and the mouse B16-F10-nectin1 melanoma syngeneic model. RESULTS: In the MeWo xenograft model, both vOka and Ellen exhibited potent antitumor efficacy. However, it was observed that introducing a hyperfusogenic mutation into glycoprotein B led to a reduction in VZV's effectiveness. Notably, the deletion of ORF8 (encodes viral deoxyuridine triphosphatase) attenuated the replication of VZV both in vitro and in vivo, but it did not compromise VZV's oncolytic potency. We further armed the VZV Ellen-ΔORF8 vector with a tet-off controlled mouse single-chain IL12 (scIL12) gene cassette. This augmented virus was validated for its oncolytic activity and triggered systemic antitumor immune responses in the immunocompetent B16-F10-nectin1 model. CONCLUSIONS: These findings highlight the potential of using Ellen-ΔORF8-tet-off-scIL12 as a novel VZV-based oncolytic virotherapy.

Surface-modified measles vaccines encoding oligomeric, prefusion-stabilized SARS-CoV-2 spike glycoproteins boost neutralizing antibody responses to Omicron and historical variants, independent of measles seropositivity.

Serum titers of SARS-CoV-2-neutralizing antibodies (nAbs) correlate well with protection from symptomatic COVID-19 but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are lifelong after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We, therefore, sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain, and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated the potent induction of high titer nAbs in measles-immune mice and confirmed the significant contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin display of the SARS-CoV-2 spike glycoprotein. In animals primed and boosted with a measles virus (MeV) vaccine encoding the ancestral SARS-CoV-2 spike, high-titer nAb responses against ancestral virus strains were only weakly cross-reactive with the Omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the Omicron BA.1 spike, antibody titers to both ancestral and Omicron strains were robustly elevated, and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that by engineering the antigen, we can develop potent measles-based vaccine candidates against SARS-CoV-2.IMPORTANCEAlthough the live-attenuated measles virus (MeV) is one of the safest and most efficacious human vaccines, a measles-vectored COVID-19 vaccine candidate expressing the SARS-CoV-2 spike failed to elicit neutralizing antibody (nAb) responses in a phase-1 clinical trial, especially in measles-immune individuals. Here, we constructed a comprehensive panel of MeV-based COVID-19 vaccine candidates using a MeV with extensive modifications on the envelope glycoproteins (MeV-MR). We show that artificial trimerization of the spike is critical for the induction of nAbs and that their magnitude can be significantly augmented when the spike protein is synchronously fused to a dodecahedral scaffold. Furthermore, preexisting measles immunity did not abolish heterologous immunity elicited by our vector. Our results highlight the importance of antigen optimization in the development of spike-based COVID-19 vaccines and therapies.

Oncolytic α-herpesvirus and myeloid-tropic cytomegalovirus cooperatively enhance systemic antitumor responses.

Oncolytic virotherapy aims to activate host antitumor immunity. In responsive tumors, intratumorally injected herpes simplex viruses (HSVs) have been shown to lyse tumor cells, resulting in local inflammation, enhanced tumor antigen presentation, and boosting of antitumor cytotoxic lymphocytes. In contrast to HSV, cytomegalovirus (CMV) is nonlytic and reprograms infected myeloid cells, limiting their antigen-presenting functions and protecting them from recognition by natural killer (NK) cells. Here, we show that when co-injected into mouse tumors with an oncolytic HSV, mouse CMV (mCMV) preferentially targeted tumor-associated myeloid cells, promoted the local release of proinflammatory cytokines, and enhanced systemic antitumor immune responses, leading to superior control of both injected and distant contralateral tumors. Deletion of mCMV genes m06, which degrades major histocompatibility complex class I (MHC class I), or m144, a viral MHC class I homolog that inhibits NK activation, was shown to diminish the antitumor activity of the HSV/mCMV combination. However, an mCMV recombinant lacking the m04 gene, which escorts MHC class I to the cell surface, showed superior HSV adjuvanticity. CMV is a potentially promising agent with which to reshape and enhance antitumor immune responses following oncolytic HSV therapy.

A phase I oncolytic virus trial with vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 administered intratumorally and intravenously in uveal melanoma: safety, efficacy, and T cell responses.

Introduction: Metastatic uveal melanoma (MUM) has a poor prognosis and treatment options are limited. These patients do not typically experience durable responses to immune checkpoint inhibitors (ICIs). Oncolytic viruses (OV) represent a novel approach to immunotherapy for patients with MUM. Methods: We developed an OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-ß) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNß-TYRP1), and conducted a Phase 1 clinical trial with a 3 + 3 design in patients with MUM. VSV-IFNß-TYRP1 was injected into a liver metastasis, then administered on the same day as a single intravenous (IV) infusion. The primary objective was safety. Efficacy was a secondary objective. Results: 12 patients with previously treated MUM were enrolled. Median follow up was 19.1 months. 4 dose levels (DLs) were evaluated. One patient at DL4 experienced dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). 4 patients had stable disease (SD) and 8 patients had progressive disease (PD). Interferon gamma (IFNγ) ELIspot data showed that more patients developed a T cell response to virus encoded TYRP1 at higher DLs, and a subset of patients also had a response to other melanoma antigens, including gp100, suggesting epitope spreading. 3 of the patients who responded to additional melanoma antigens were next treated with ICIs, and 2 of these patients experienced durable responses. Discussion: Our study found that VSV-IFNß -TYRP1 can be safely administered via intratumoral (IT) and IV routes in a previously treated population of patients with MUM. Although there were no clear objective radiographic responses to VSV-IFNß-TYRP1, dose-dependent immunogenicity to TYRP1 and other melanoma antigens was seen.

Evidence for Angiotensin II as a Naturally Existing Suppressor for the Guanylyl Cyclase A Receptor and Cyclic GMP Generation.

The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.

EVIDENCE FOR ANGIOTENSIN II AS A NATURALLY EXISTING SUPPRESSOR FOR THE NATRIURETIC PEPTIDE SYSTEM.

Background: Natriuretic peptide system (NPS) and renin angiotensin aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date support this notion. Objectives: This study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro for translational insights. Methods: Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in rat model to determine influence of ANGII on ANP actions. Multiple engineered HEK293 cells and surface plasmon resonance (SPR) technology were leveraged for mechanistic exploration. Results: In humans, ANGII showed inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and interaction term between ANGII and natriuretic peptide increased predicting accuracy of base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed positive association between cGMP with ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at physiological dose attenuated blood pressure reduction and cGMP generation triggered by ANP infusion. In vitro, we showed that the suppression effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), which can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using SPR, we showed ANGII has low affinity for particulate guanylyl cyclase A (GC-A) receptor binding compared to ANP or BNP. Conclusions: Our study reveals ANGII as a natural suppressor for cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular disease.

Surface-modified measles vaccines encoding oligomeric, fusion-stabilized SARS-CoV-2 spike glycoproteins bypass measles seropositivity, boosting neutralizing antibody responses to omicron and historical variants.

Serum titers of SARS-CoV-2 neutralizing antibodies (nAb) correlate well with protection from symptomatic COVID-19, but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are life-long after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We therefore sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated potent induction of high titer nAb in measles-immune mice and confirmed the significant incremental contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin-display of the SARS-CoV-2 spike glycoprotein, and vaccine resurfacing. In animals primed and boosted with a MeV vaccine encoding the ancestral SARS-CoV-2 spike, high titer nAb responses against ancestral virus strains were only weakly cross-reactive with the omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the omicron BA.1 spike, antibody titers to both ancestral and omicron strains were robustly elevated and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that antigen engineering can enable the development of potent measles-based SARS-CoV-2 vaccine candidates.

Glucose Starvation Causes ptauS409 Increase in N2a Cells Through ATF3/PKAcα Signaling Pathway.

In this work, we report that glucose starvation (GS) causes ptauS409 increase, which may participate in GS-induced neurites retraction in neuro-2a (N2a) cells. Upon GS treatment, PKAcα was stimulated at mRNA and protein levels. Luciferase reporter gene assays indicated that GS regulated PKAcα expression through a core promoter (-345 to -95 bp upstream the transcription starting site) consisting of a cis-acting element of Activating Transcription Factor 3 (ATF3). Knockdown and over-expression experiments demonstrate that ATF3 transcriptionally regulated PKAcα expression. Moreover, GS stimulated ATF3 expression in a time-dependent manner. These findings reveal that glucose starvation induces ptauS409 increase in N2a cells through an ATF3- PKAcα axis, which shed some light on the relationship between brain glucose metabolism and neurodegenerative diseases.

HERC5/IFI16/p53 signaling mediates breast cancer cell proliferation and migration.

AIMS: This study aims to find differentially expressed ubiquitination-related gene(s) and elucidates their biological significance in breast cancer. MAIN METHODS: Differentially expressed genes were profiled in MCF-7 and MDA-MB-231 cells by using PCR array method. Abnormal expression of HERC5 was studied in the cells and in breast cancer specimens via Quantitative Real-time PCR and western blot. Cell proliferation and cell migration abilities were evaluated by using cell counting kits, or through colony formation, wound healing and trans-well assays. HERC5 target proteins were investigated via proteomic, co-immunoprecipitation and western blot methods. Down-stream signaling pathways were investigated through gene expression/knockdown methods. KEY FINDINGS: Huge increase of HERC5 expression was found in MCF-7 and MDA-MB-231 cells, knockdown of which repressed the cell proliferation and migration. HERC5 interacted with IFI16, mediated IFI16 ISGylation at K274 and facilitated IFI16 proteasomal degradation. IFI16 acted as a tumor suppressor and to some extent mediated the HERC5 function in the breast cancer (BC) cells. HERC5 was negatively correlated with IFI16 protein, while IFI16 was positively correlated to p53 expression at mRNA and protein levels, which indicates a novel signaling pathway - HERC5/IFI16/p53. HERC5 expression was increased in glucose-starved BC cells and in human breast cancer tissues, accompanied with the decrease of IFI16 and P53. SIGNIFICANCE: Our work reveals the abnormal expression of HERC5 and its carcinogenic role in breast cancer cells, which is probably mediated by an HERC5/IFI16/p53 signaling pathway. This work also provides potential diagnostic/therapeutic biomarkers for breast cancer diagnosis and treatment.

Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine.

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

Clinical activity of single-dose systemic oncolytic VSV virotherapy in patients with relapsed refractory T-cell lymphoma.

Clinical success with intravenous (IV) oncolytic virotherapy (OV) has to-date been anecdotal. We conducted a phase 1 clinical trial of systemic OV and investigated the mechanisms of action in responding patients. A single IV dose of vesicular stomatitis virus (VSV) interferon-ß (IFN-ß) with sodium iodide symporter (NIS) was administered to patients with relapsed/refractory hematologic malignancies to determine safety and efficacy across 4 dose levels (DLs). Correlative studies were undertaken to evaluate viremia, virus shedding, virus replication, and immune responses. Fifteen patients received VSV-IFNß-NIS. Three patients were treated at DL1 through DL3 (0.05, 0.17, and 0.5 × 1011 TCID50), and 6 were treated at DL4 (1.7 × 1011 TCID50) with no dose-limiting toxicities. Three of 7 patients with T-cell lymphoma (TCL) had responses: a 3-month partial response (PR) at DL2, a 6-month PR, and a complete response (CR) ongoing at 20 months at DL4. Viremia peaked at the end of infusion, g was detected. Plasma IFN-ß, a biomarker of VSV-IFNß-NIS replication, peaked between 4 hours and 48 hours after infusion. The patient with CR had robust viral replication with increased plasma cell-free DNA, high peak IFN-ß of 18 213 pg/mL, a strong anti-VSV neutralizing antibody response, and increased numbers of tumor reactive T-cells. VSV-IFNß-NIS as a single agent was effective in patients with TCL, resulting in durable disease remissions in heavily pretreated patients. Correlative analyses suggest that responses may be due to a combination of direct oncolytic tumor destruction and immune-mediated tumor control. This trial is registered at www.clinicaltrials.gov as #NCT03017820.

Serotypic evolution of measles virus is constrained by multiple co-dominant B cell epitopes on its surface glycoproteins.

After centuries of pestilence and decades of global vaccination, measles virus (MeV) genotypes capable of evading vaccine-induced immunity have not emerged. Here, by systematically building mutations into the hemagglutinin (H) glycoprotein of an attenuated measles virus strain and assaying for serum neutralization, we show that virus evolution is severely constrained by the existence of numerous co-dominant H glycoprotein antigenic sites, some critical for binding to the pathogenicity receptors SLAMF1 and nectin-4. We further demonstrate the existence in serum of protective neutralizing antibodies targeting co-dominant fusion (F) glycoprotein epitopes. Lack of a substantial reduction in serum neutralization of mutant measles viruses that retain even one of the co-dominant antigenic sites makes evolution of pathogenic measles viruses capable of escaping serum neutralization in vaccinated individuals extremely unlikely.

MeV-Stealth: A CD46-specific oncolytic measles virus resistant to neutralization by measles-immune human serum.

The frequent overexpression of CD46 in malignant tumors has provided a basis to use vaccine-lineage measles virus (MeV) as an oncolytic virotherapy platform. However, widespread measles seropositivity limits the systemic deployment of oncolytic MeV for the treatment of metastatic neoplasia. Here, we report the development of MeV-Stealth, a modified vaccine MeV strain that exhibits oncolytic properties and escapes antimeasles antibodies in vivo. We engineered this virus using homologous envelope glycoproteins from the closely-related but serologically non-cross reactive canine distemper virus (CDV). By fusing a high-affinity CD46 specific single-chain antibody fragment (scFv) to the CDV-Hemagglutinin (H), ablating its tropism for human nectin-4 and modifying the CDV-Fusion (F) signal peptide we achieved efficient retargeting to CD46. A receptor binding affinity of ~20 nM was required to trigger CD46-dependent intercellular fusion at levels comparable to the original MeV H/F complex and to achieve similar antitumor efficacy in myeloma and ovarian tumor-bearing mice models. In mice passively immunized with measles-immune serum, treatment of ovarian tumors with MeV-Stealth significantly increased overall survival compared with treatment with vaccine-lineage MeV. Our results show that MeV-Stealth effectively targets and lyses CD46-expressing cancer cells in mouse models of ovarian cancer and myeloma, and evades inhibition by human measles-immune serum. MeV-Stealth could therefore represent a strong alternative to current oncolytic MeV strains for treatment of measles-immune cancer patients.

A living cell-based fluorescent reporter for high-throughput screening of anti-tumor drugs.

Suppression of cellular O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) can repress proliferation and migration of various cancer cells, which opens a new avenue for cancer therapy. Based on the regulation of insulin gene transcription, we designed a cell-based fluorescent reporter capable of sensing cellular O-GlcNAcylation in HEK293T cells. The fluorescent reporter mainly consists of a reporter (green fluorescent protein (GFP)), an internal reference (red fluorescent protein), and an operator (neuronal differentiation 1), which serves as a "sweet switch" to control GFP expression in response to cellular O-GlcNAcylation changes. The fluorescent reporter can efficiently sense reduced levels of cellular O-GlcNAcylation in several cell lines. Using the fluorescent reporter, we screened 120 natural products and obtained one compound, sesamin, which could markedly inhibit protein O-GlcNAcylation in HeLa and human colorectal carcinoma-116 cells and repress their migration in vitro. Altogether, the present study demonstrated the development of a novel strategy for anti-tumor drug screening, as well as for conducting gene transcription studies.

O-GlcNAcylation of Thr12/Ser56 in short-form O-GlcNAc transferase (sOGT) regulates its substrate selectivity.

O-GlcNAcylation is a ubiquitous protein glycosylation playing different roles on variant proteins. O-GlcNAc transferase (OGT) is the unique enzyme responsible for the sugar addition to nucleocytoplasmic proteins. Recently, multiple O-GlcNAc sites have been observed on short-form OGT (sOGT) and nucleocytoplasmic OGT (ncOGT), both of which locate in the nucleus and cytoplasm in cell. Moreover, O-GlcNAcylation of Ser389 in ncOGT (1036 amino acids) affects its nuclear translocation in HeLa cells. To date, the major O-GlcNAcylation sites and their roles in sOGT remain unknown. Here, we performed LC-MS/MS and mutational analyses to seek the major O-GlcNAcylation site on sOGT. We identified six O-GlcNAc sites in the tetratricopeptide repeat domain in sOGT, with Thr12 and Ser56 being two "key" sites. Thr12 is a dominant O-GlcNAcylation site, whereas the modification of Ser56 plays a role in regulating sOGT O-GlcNAcylation, partly through Thr12In vitro activity and pulldown assays demonstrated that O-GlcNAcylation does not affect sOGT activity but does affect sOGT-interacting proteins. In HEK293T cells, S56A bound to and hence glycosylated more proteins in contrast to T12A and WT sOGT. By proteomic and bioinformatics analyses, we found that T12A and S56A differed in substrate proteins (e.g. HNRNPU and PDCD6IP), which eventually affected cell cycle progression and/or cell proliferation. These findings demonstrate that O-GlcNAcylation modulates sOGT substrate selectivity and affects its role in the cell. The data also highlight the regulatory role of O-GlcNAcylation at Thr12 and Ser56.

Targeting glycogen synthase kinase 3 for therapeutic benefit in lymphoma.

Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and ß are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3ß compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3ß genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3ß binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3ß is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3ß correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

Hepatocyte nuclear factor 1 alpha (HNF1A) regulates transcription of O-GlcNAc transferase in a negative feedback mechanism.

O-GlcNAc transferase (OGT)-catalyzed protein O-GlcNAcylation is implicated in diverse cellular events. In the present study, we report the regulation of ogt transcription by the hepatocyte nuclear factor 1 homologue A (HNF1A) in HEK293T cells. We first identified a core ogt promoter (-150 to +200 bp) and confirmed its binding to the transcription factor HNF1A. We found that HNF1A regulates ogt transcription in a time-dependent manner and that O-GlcNAcylation of HNF1A represses ogt transcription. Electron-transfer dissociation based tandem mass spectrometry analysis revealed 14 O-GlcNAc sites on HNF1A, six of which are predominantly modified, including Ser303/304 , Ser471 , Ser560 and Thr563/564 . We further found that loss of O-GlcNAcylation at Ser303/304 or Thr563/564 significantly elevates ogt transcription. These findings highlight a negative feedback mechanism for ogt transcription, which partially explains the homeostasis of cellular O-GlcNAcylation.

Dual-Isotope SPECT Imaging with NIS Reporter Gene and Duramycin to Visualize Tumor Susceptibility to Oncolytic Virus Infection.

Noninvasive dual-imaging methods that provide an early readout on tumor permissiveness to virus infection and tumor cell death could be valuable in optimizing development of oncolytic virotherapies. Here, we have used the sodium iodide symporter (NIS) and 125I radiotracer to detect infection and replicative spread of an oncolytic vesicular stomatitis virus (VSV) in VSV-susceptible (MPC-11 tumor) versus VSV-resistant (CT26 tumor) tumors in BALB/c mice. In conjunction, tumor cell death was imaged simultaneously using technetium (99mTc)-duramycin that binds phosphatidylethanolamine in apoptotic and necrotic cells. Dual-isotope single-photon emission computed tomography/computed tomography (SPECT/CT) imaging showed areas of virus infection (NIS and 125I), which overlapped well with areas of tumor cell death (99mTc-duramycin imaging) in susceptible tumors. Multiple infectious foci arose early in MPC-11 tumors, which rapidly expanded throughout the tumor parenchyma over time. There was a dose-dependent increase in numbers of infectious centers and 99mTc-duramycin-positive areas with viral dose. In contrast, NIS or duramycin signals were minimal in VSV-resistant CT26 tumors. Combinatorial use of NIS and 99mTc-duramycin SPECT imaging for simultaneous monitoring of oncolytic virotherapy (OV) spread and the presence or absence of treatment-associated cell death could be useful to guide development of combination treatment strategies to enhance therapeutic outcome.

New transgenic NIS reporter rats for longitudinal tracking of fibrogenesis by high-resolution imaging.

Fibrogenesis is the underlying mechanism of wound healing and repair. Animal models that enable longitudinal monitoring of fibrogenesis are needed to improve traditional tissue analysis post-mortem. Here, we generated transgenic reporter rats expressing the sodium iodide symporter (NIS) driven by the rat collagen type-1 alpha-1 (Col1α1) promoter and demonstrated that fibrogenesis can be visualized over time using SPECT or PET imaging following activation of NIS expression by rotator cuff (RC) injury. Radiotracer uptake was first detected in and around the injury site day 3 following surgery, increasing through day 7-14, and declining by day 21, revealing for the first time, the kinetics of Col1α1 promoter activity in situ. Differences in the intensity and duration of NIS expression/collagen promoter activation between individual RC injured Col1α1-hNIS rats were evident. Dexamethasone treatment delayed time to peak NIS signals, showing that modulation of fibrogenesis by a steroid can be imaged with exquisite sensitivity and resolution in living animals. NIS reporter rats would facilitate studies in physiological wound repair and pathological processes such as fibrosis and the development of anti-fibrotic drugs.

Sialic acid linkage-specific permethylation for improved profiling of protein glycosylation by MALDI-TOF MS.

Protein glycosylation mediates a wide range of cellular processes, affecting development and disease in mammals. Deciphering the "glycocodes" requires rapid, sensitive and in-depth characterization of diverse glycan structures derived from biological samples. In this study, we described a two-step derivatization strategy termed linkage-specific sialic acid permethylation (SSAP) consisting of dimethylamination and permethylation for the improved profiling of glycosylation by matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS). High linkage-specificity (∼99%) of SSAP to both the two most common forms of sialic acid, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), permitted direct discrimination of α2,3- and α2,6-linked sialic acids in MALDI-TOF MS. The enhanced intensity (>10-fold) and increased detection limit (>10-fold) of derivatized glycans were valued for sensitive glycomics. Moreover, the good compatibility and reaction efficiency of the two steps of SSAP allowed rapid sample preparation (<2 h), benefiting robust analysis of glycans in a high-throughput manner. The SSAP strategy was further applied to investigate the protein glycosylation of human serum associated with rheumatoid arthritis (RA). It was demonstrated that the relative abundances of individual glycans were different in RA negative and RA positive samples, and meanwhile the RA patient/control ratios of both α2,3- and α2,6-sialylated glycans tended to elevate accompanied with the increase of sialylation. Those findings of the glycosylation changes occurred in human serum protein may contribute to the diagnosis of RA. Herein, SSAP derivatization combined with MALDI-TOF MS exhibits unique advantages for glycomic analysis and shows potential in glycosylation profiling of therapeutic proteins and clinical glycan biomarker discovery.