E1A Expression Research Articles

Benzo[a]pyrene exposure causes exonal switch resulting in reduced surface CD5 expression in an AHR-dependent manner

The function of CD5 protein in T cells is well documented, but regulation of its surface-level expression has yet to be fully understood. However, variation in its surface expression is associated with various immunopathological conditions and haematological malignancies. Briefly, expression of an alternate exon E1B of a human endogenous retroviruses (HERV) origin directly downregulates the conventional transcript variant (E1A), as its expression leads to the retention of the resultant protein at the intracellular level (cCD5). A separate promoter governs the expression of E1B and may be influenced by different transcription factors. Hence, we performed in silico transcription factor binding site (TFBS) analysis of the 3 kb upstream region from TSS of exon E1B and found five putative DREs (Dioxin Response elements) with good similarity scores. Further, we observed the upregulation in E1B expression after the exposure of BaP (a dioxin) and the reduction of E1A expression and their respective protein, i.e. sCD5 and cCD5. The binding of AHR at the predicted DRE sites was confirmed by ChIP qPCR and AHR specific inhibitor and gene silencing studies suggested the involvement of AHR in exonal switch. This study indicates that the polycyclic aromatic hydrocarbon decreases the sCD5 expression by upregulating alternative exon expression, which may adversely affect the overall T cell functions.

HBO1/KAT7/MYST2 HAT complex regulates human adenovirus replicative cycle

Human adenoviruses (HAdV) belong to a small DNA tumor virus family that continues as valuable models in understanding the viral strategies of usurping cell growth regulation. A number of HAdV type 2/5 early viral gene products interact with a variety of cellular proteins to build a conducive environment that promotes viral replication. Here we show that HBO1 (Histone Acetyltransferase Binding to ORC1), a member of the MYST histone acetyltransferase (HAT) complex (also known as KAT7 and MYST2) that acetylates most of the histone H3 lysine 14, is essential for HAdV5 growth. HBO1/MYST2/KAT7 HAT complexes are critical for a variety of cellular processes including control of cell proliferation. In HBO1 downregulated human cells, HAdV5 infection results in reduced expression of E1A and other viral early genes, virus growth is also reduced significantly. Importantly, HBO1 downregulation reduced H3 lysine 14 acetylation at viral promoters during productive infection, likely driving reduced viral gene expression. HBO1 was also associated with viral promoters during infection and co-localized with viral replication centers in the nuclei of infected cells. In transiently transfected cells, overexpression of E1A along with HBO1 stimulated histone acetyltransferase activity of HBO1. E1A also co-immunoprecipitated with HBO1 in transiently transfected cells. In summary, our results demonstrate that HAdV recruits the HBO1 HAT complex to aid in viral replication.

Abstract 7241: Combination of CDK4/6 inhibitor promotes the antitumor effect of oncolytic adenovirus against canine breast cancer cells

Abstract Background: Oncolytic virotherapy has emerged as a novel antitumor platform to induce tumor-specific cytopathic effect against human cancer cells. However, whether oncolytic virotherapy is effective to canine cancer cells remains unclear. We developed telomerase-specific oncolytic adenoviruses OBP-301 and a p53-armed OBP-702 based on human adenovirus serotype 5. OBP-301 and OBP-702 have therapeutic potential against various types of human cancer cells. As recent report has suggested that human adenovirus serotype 5 induces cytopathic effect against canine cancer cells, OBP-301 and OBP-702 are expected to have therapeutic effects against canine cancer cells. Furthermore, it has been reported that CDK4/6 inhibitors clinically used for human breast cancers promote the replication of oncolytic adenoviruses, suggesting that combination of CDK4/6 inhibitors promote the therapeutic potential of oncolytic adenoviruses. In this study, we investigated the therapeutic potential of OBP-301 and OBP-702 against canine breast cancer cells in monotherapy and combination therapy with CDK4/6 inhibitor. Methods: Two canine breast cancer cell lines (CMT-U27, AZA-CB) were used in this study. The antitumor effect of OBP-301 and OBP-702 against canine breast cancer cells was analyzed using the XTT assay. The therapeutic potential of combination therapy with CDK4/6 inhibitor (palibociclib) was further analyzed. Virus-mediated induction of adenovirus E1A and p53 expression was analyzed by Western blotting. The in vivo antitumor effects of OBP-301 and OBP-702 were assessed using CMT-U27 subcutaneous tumor model. Results: OBP-301 and OBP-702 had cytopathic effect against CMT-U27 and AZA-CB cells. The antitumor activity of OBP-702 was superior to that of OBP-301. The combination of palbociclib and OBP-301 showed a more profound antitumor effect compared with monotherapy against CMT-U27 and AZA-CB cells. The expression of E1A and p53 was increased in virus-treated CMT-U27 and AZA-CB cells in a dose-dependent manner. Intratumoral administration of OBP-301 and OBP-702 significantly suppressed the growth of subcutaneous CMT-U27 tumors compared with mock treatment. Conclusion: Our results suggest that telomerase-specific oncolytic adenoviruses have cytopathic effect against canine breast cancer cells via virus infection and replication. Furthermore, combination of CDK4/6 inhibitor may be a promising strategy for promoting the antitumor effect of oncolytic adenovirus against canine breast cancers. Citation Format: Naoyuki Hashimoto, Hiroshi Tazawa, Satoru Kikuchi, Shinji Kuroda, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Combination of CDK4/6 inhibitor promotes the antitumor effect of oncolytic adenovirus against canine breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7241.

Abstract 3720: Utility of oncolytic virus as an immunostimulants and potential for combination therapy with ICIs

Abstract Background: Anti-PD-1 antibodies have been approved for a variety of carcinomas, but their efficacy in gastrointestinal cancers is limited. CD8-positive T cells (CD8) of tumor-infiltrating lymphocytes (TILs) and resident memory T cells (TRMs) with long-term anti-tumor immune function have been reported to be associated with the therapeutic efficacy of anti-PD-1 antibodies. Among our oncolytic adenoviruses (OVs), OBP-301, in which the human telomerase reverse transcriptase (hTERT) promoter element drives the expression of the viral E1A and E1B genes, is currently undergoing clinical trials. In the present study, we investigated the usefulness of OVs as immunostimulants and their potential efficacy in combination with anti-PD-1 antibodies. Methods: The cell lines used were the mouse colon cancer cell line CT26 and the mouse pancreatic cancer cell line PAN02. In vitro, the cytotoxic activity of the OVs and extracellular ATP in the culture supernatant were determined. OBP-502, an OBP-301 variant with the gene cassette of the RGD peptide, and anti-PD-1 antibody were used to examine the antitumor effect on subcutaneous tumors. OBP-702, another OBP-301 variant with the gene cassette of the wild-type p53 tumor suppressor gene, was used to examine the effect of long-term anti-tumor immunity. The effects on memory T-precursor cells (MPEC), CD8 and TRM were immunologically assessed in a rechallenge study. Results: OBP-502 and OBP-702 showed cytotoxic activity against CT26 and PAN02 cells in vitro, and significantly increased ATP in the culture supernatant. In vivo, the anti-PD-1 antibody/OBP-502 combination therapy significantly increased intratumor CD8 in CT26 and PAN02 subcutaneous tumors compared to each monotherapy. Complete response was achieved in 4 out of 12 mice by the combination therapy against CT26 tumors. When CT26 cells was re-seeded into tumor-free mice cured by the combination therapy, 2 out of 4 mice remained tumor-free. In the CT26 and PAN02 subcutaneous tumor model, OBP-702 increased CD44-positive CD62L-negative MPECs that differentiated into TRMs in the spleen, and this effect was blocked by ATP receptor inhibition. In CT26 and PAN02 bilateral subcutaneous tumor models, OBP-702 had a significant antitumor effect on both OBP-702-treated and -non-treated sides, and TIL analysis showed a significant increase in CD8 and TRM. In a rechallenge study, OBP-702-pretreatment showed significant growth inhibition of re-inoculated tumors. Conclusion: The OVs showed potent and long-term immune activation by inducing CD8 and TRM in tumors as well as direct anti-tumor effects, and synergistic effects with ICI. Citation Format: Daisuke Kadowaki, Shinji Kuroda, Masashi Hashimoto, Nobuhiko Kanaya, Yoshihiko Kakiuchi, Satoru Kikuchi, Kazuhiro Noma, Hiroshi Tazawa, Shunsuke Kagawa, Yasuo Urata, Toshiyoshi Fujiwara. Utility of oncolytic virus as an immunostimulants and potential for combination therapy with ICIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3720.

Inhibition of Bromodomain Proteins Enhances Oncolytic HAdVC5 Replication and Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Models.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus.

Human adenovirus oncolytic properties and the inhibitory role of E4 orf4 and E4 orf6/7 on endogenously activated NF-κB

Human adenovirus is a promising tool for cancer therapy as an oncolytic virus. To predict which region of the oncolytic adenovirus E4 gene could be deleted, we investigated the relationship between the E4 proteins and NF-κB. Here, we report that TLR2-dependent NF-κB activation in Ad5-infected cells was significantly inhibited 24 h post-infection. Among the six E4 proteins, E4 orf4 and E4 orf6/7 exhibited notable suppressive effects on NF-κB activation. However, only E4 orf4 was co-immunoprecipitated with the RelA protein, also known as p65. It appears likely that E4 orf6/7 represses NF-κB activation via E2F-dependent pathways. Our results suggest that both E4 orf4 and E4 orf6/7 are novel inhibitors of NF-κB activation. The inhibition of endogenous NF-κB activation by E4 proteins during the late phase of infection also appears to elucidate the previously reported suppression of E1A expression in the late phase of infection. These redundant suppressive effects of E4 orf4 and E4 orf6/7 on NF-κB suggest that these proteins may play a major role in the anticancer properties of oncolytic adenovirus.

Inhibition of human adenovirus replication by TRIM35-mediated degradation of E1A.

Human adenovirus (HAdV) is ubiquitous in the human population, constituting a significant burden of global respiratory diseases. Children and individuals with low immunity are at risk of developing severe infections without approved antiviral treatment for HAdV. Our study demonstrated that TRIM35 inhibited HAdV-C5 early gene transcription, early protein expression, genome replication, and infectious virus progeny production. Furthermore, TRIM35 was found to inhibit HAdV replication by attenuating E1A expression. Mechanistically, TRIM35 interacts with and degrades E1A by promoting its K48-linked ubiquitination. Additionally, K253 and K285 are the key sites necessary for TRIM35 degradation. Moreover, an oncolytic adenovirus carrying shTRIM35 was constructed and observed to exhibit improved oncolysis in vivo, providing new ideas for clinical tumor treatment. Our results expand the broad antiviral activity of TRIM35 and mechanically support its application as a HAdV replication inhibitor. IMPORTANCE E1A is an essential human adenovirus (HAdV) protein responsible for the early replication of adenovirus while interacting with multiple host proteins. Understanding the interaction between HAdV E1A and TRIM35 helps identify effective antiviral therapeutic targets. The viral E1A protein is a crucial activator and regulator of viral transcription during the early infection stages. We first reported that TRIM35 interacts with E1A to resist adenovirus infection. Our study demonstrated that TRIM35 targets E1A to resist adenovirus, indicating the applicability of targeting virus-dependent host factors as a suitable antiviral strategy.

Inducible HEK293 AAV packaging cell lines expressing Rep proteins

Packaging or producer cell lines for scalable recombinant adeno-associated virus (rAAV) production have been notoriously difficult to create due in part to the cytostatic nature of the Rep proteins required for AAV production. The most difficult challenge being creating AAV packaging cell lines using HEK293 parental cells, currently the best mammalian platform for rAAV production due to the constitutive expression of E1A in HEK293 cells, a key REP transcription activator. Using suspension and serum-free media adapted HEK293SF carrying a gene expression regulation system induced by addition of cumate and coumermycin, we were able to create REP-expressing AAV packaging cells. This was achieved by carefully choosing two of the AAV Rep proteins (Rep 40 and 68), using two inducible promoters with different expression levels and integrating into the cells through lentiviral vector transduction. Three of our best clones produced rAAV titers comparable to titers obtained by standard triple plasmid transfection of their parental cells. These clones were stable for up to 7weeks under continuous cultures condition. rAAV production from one clone was also validated at scale of 1L in a wave bioreactor using serum-free suspension culture.

Characterizing the tumor selectivity of oncolytic adenovirus ICVB-1042: Analysis of S-phase induction, viral replication, and cytolytic activity in human primary normal cell types.

e14503 Background: Adenoviral-based oncolytic virus (OV) therapeutics include mutations that enable activity in tumor cells and limit toxicity in normal cells. These tumor selectivity approaches often involve mutating viral E1A function or expression; however, such modifications allow viral activity in normal cells or limit anti-tumor potency. ICVB-1042 was engineered with dual modifications in E1A and E4orf6/7, resulting in superior tumor selectivity that does not compromise lytic potency in tumor cells. This improved tumor selective virus minimizes off-tumor activity and promotes robust tumor lysis. Methods: ICVB-1042 was compared to viruses with wildtype E1A and E4orf6/7 and a clinically tested virus with an E1A mutation (ICVB-2006). Viral function and killing were assessed in primary normal human renal (HRE) and bronchial (BrE) epithelial cells in the presence of the inhibitor palbociclib to pharmacologically block S-phase entry via host cell signaling. HRE and BrE were infected with virus and evaluated for indicators of viral toxicity in normal cells. Viral replication was quantified by droplet digital polymerase chain reaction (ddPCR); cell lysis was quantified by WST-1 assay. An S-phase induction assay was developed using an mCherry-Geminin reporter that allows imaging-based quantification of viral-dependent entry to S-phase. Viral activity in cells was compared with and without the CDK4/6 inhibitor using a viral-encoded fluorescent reporter to determine the requirement for host cell proliferation on viral function. Results: ICVB-1042 demonstrated superior protection in normal cells to comparator ICVB-2006 and viruses lacking tumor selectivity modifications. Palbociclib steeply reduced viral reporter gene expression (63-88% reduction), viral replication ( > 10-fold reduction), and cytolytic activity of ICVB-1042, while non-selective viruses were unaffected by this treatment. Non-selective virus infection resulted in 3.3- to 6.1-fold higher levels of S-phase entry compared to ICVB-1042. ICVB-2006 infection resembled non-selective viruses at the level of S-phase induction (3.1- to 3.3-fold above ICVB-1042) and viral reporter gene expression was modestly inhibited by palbociclib (12-31% reduction). Conclusions: The improved tumor selectivity of ICVB-1042 results in protection of normal, non-proliferative cells from viral-mediated toxicity. The conventional approach of ICVB-2006 with a single E1A mutation leaves viral replication and cell death in normal human cells mostly intact. Together, these observations support that ICVB-1042 presents normal cell protection and reduced toxicity risks associated with OV therapy.

Antiviral effect of natural and semisynthetic diterpenoids against adenovirus infection in vitro.

The emergence and re-emergence of viruses has highlighted the need to develop new broad spectrum antivirals to mitigate human infections. Pursuing our search for new bioactive plant-derived molecules, we studied several diterpene derivatives synthesized from jatropholones A and B and carnosic acid isolated from Jatropha isabellei and Rosmarinus officinalis, respectively. Here, we investigated the antiviral effect of the diterpenes against human adenovirus (HAdV-5) that causes several infections for which there is no approved antiviral therapy yet. Ten compounds were evaluated and none of them presented cytotoxicity in A549 cells. Only compounds 2, 5 and 9 inhibited HAdV-5 replication in a concentration-dependent manner, without virucidal activity, whereas the antiviral action took place after virus internalization. The expression of viral proteins E1A and Hexon was strongly inhibited by compounds 2 and 5 and, in a lesser degree, by compound 9. Since compounds 2, 5 and 9 prevent ERK activation, they might exert their antiviral action by interfering host cell functions required for virus replication. Besides, the compounds had an anti-inflammatory profile since they significantly inhibited the levels of IL-6 and IL-8 produced by THP-1 cells infected with HAdV-5 or with an adenoviral vector. In conclusion, diterpenes 2, 5 and 9 not only exerted antiviral activity against adenovirus but also are able to restrain pro-inflammatory cytokines induced by the virus.

Evaluation of Anti-bacterial, Anti-adenoviral, and Apoptosis-inducing Activity of Bacillus clausii Supernatant

Background: Bacillus clausii is being studied as a probiotic candidate. There is insufficient information on the antimicrobial and anticancer effects of B. clausii. Objectives: The present investigation was designed to evaluate the anti-bacterial, anti-adenoviral, and apoptosis-inducing activity of B. clausii cell-free supernatant (CFS). Methods: First, the supernatant of B. clausii was collected after culture for 24 h. Then, its anti-bacterial impact on several genera of bacteria was assessed through the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Adenovirus 5 (Ad5) was exposed to the CFS under four conditions, including pre-treatment: First infecting cells with CFS and then with the virus; pre-incubation: Incubation of the supernatant and virus for 1.5 hours and then adding to the cells; competition: Infection of cells with the simultaneous mixture of the supernatant and virus, and post-treatment: First infecting cells with the virus and then with CFS. The median tissue culture infectious dose (TCID50) technique determined the virus titer. Real-time PCR was performed to assess the E1A expression. After exposure to the CFS, real-time PCR was utilized to measure the expression of MicroRNA-145, BCL-2, and BAX in HeLa cancer cells. Results: Bacillus clausii supernatant showed an inhibitory effect on Methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, S. aureus, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii. The Ad5 titers were reduced by about 4.61, 4, 3.9, and 3.1 Log10 TCID50/mL in pre-treatment, pre-incubation, competition, and post-treatment tests (CFS dilution: 1/4), respectively. Similar results of the viral titration were seen when experimental and control E1A expression levels were compared. Also, B. clausii supernatant during 48 h exposure to HeLa cells increased the transcript of the BAX, BCL-2, and miR-145 genes to 9.1, 2.3, and 55 folds, respectively, compared to the untreated condition. Conclusions: Bacillus clausii can be a potent antimicrobial and anticancer agent. Further research is required to learn about the spectrum of anti-bacterial, antiviral, and anti-cancerous activities of B. clausii.

Adenovirus Encoding Tumor Necrosis Factor Alpha and Interleukin 2 Induces a Tertiary Lymphoid Structure Signature in Immune Checkpoint Inhibitor Refractory Head and Neck Cancer.

Immune checkpoint inhibitors (ICI) have provided significant improvement in clinical outcomes for some patients with solid tumors. However, for patients with head and neck cancer, the response rate to ICI monotherapy remains low, leading to the exploration of combinatorial treatment strategies. In this preclinical study, we use an oncolytic adenovirus (Ad5/3) encoding hTNFα and hIL-2 and non-replicate adenoviruses (Ad5) encoding mTNFα and mIL-2 with ICI to achieve superior tumor growth control and improved survival outcomes. The in vitro effect of Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 was characterized through analyses of virus replication, transgene expression and lytic activity using head and neck cancer patient derived cell lines. Mouse models of ICI naïve and refractory oral cavity squamous cell carcinoma were established to evaluate the local and systemic anti-tumor immune response upon ICI treatment with or without the non-replicative adenovirus encoding mTNFα and mIL-2. We delineated the mechanism of action by measuring the metabolic activity and effector function of CD3+ tumor infiltrating lymphocytes (TIL) and transcriptomic profile of the CD45+ tumor immune compartment. Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 demonstrated robust replicative capability in vitro across all head and neck cell lines screened through potent lytic activity, E1a and transgene expression. In vivo, in both ICI naïve and refractory models, we observed improvement to tumor growth control and long-term survival when combining anti-PD-1 or anti-PD-L1 with the non-replicative adenovirus encoding mTNFα and mIL-2 compared to monotherapies. This observation was verified by striking CD3+ TIL derived mGranzyme b and interferon gamma production complemented by increased T cell bioenergetics. Notably, interrogation of the tumor immune transcriptome revealed the upregulation of a gene signature distinctive of tertiary lymphoid structure formation upon treatment of murine anti-PD-L1 refractory tumors with non-replicative adenovirus encoding mTNFα and mIL-2. In addition, we detected an increase in anti-tumor antibody production and expansion of the memory T cell compartment in the secondary lymphoid organs. In summary, a non-replicative adenovirus encoding mTNFα and mIL-2 potentiates ICI therapy, demonstrated by improved tumor growth control and survival in head and neck tumor-bearing mice. Moreover, the data reveals a potential approach for inducing tertiary lymphoid structure formation. Altogether our results support the clinical potential of combining this adenovirotherapy with anti-PD-1 or anti-PD-L1.

Anti-Tumor Effect of a Novel Oncolytic Virus Based on Chimpanzee Adenovirus Type 6

To construct, with chimpanzee adenovirus serotype 6 (AdC6) as the vector, a novel oncolytic adenovirus, enabling it to selectively replicate intratumorally, to test its tumor suppressive effect in vitro and in vivo, and to study its oncolytic mechanism. Based on the AdC6 vector, the human telomerase reverse transcriptase (hTERT) promoter was used to drive the expression of E1A, the adenovirus replication-related gene, and the recombinant oncolytic virus AdC6-htertΔE1A-ΔE3 was thus obtained. The oncolytic virus AdC6-htertE1A-ΔE3 (CSF 2) expressing granulocyte macrophage colony-stimulating factor (GM-CSF/CSF 2) and replication-deficient adenovirus AdC6-ΔE1-ΔE3 were constructed by homologous recombination, respectively. The recombinant adenovirus was packaged in HEK293 cells, purified and then identified with restriction enzyme digestion. Different types of tumor cells, including RD, SW-620, HeLa, Huh7, RM-1 and MC-38 were infected with the three adenoviruses. Twenty-four hours after infection, Western blot was used to determine the expression of CSF 2 24 hours after infection. CCK8 assay was used to determine the survival rate of tumor cells 72 hours after infection. HeLa cells were infected with the three adenoviruses, and the expression levels of apoptosis signaling pathway proteins were examined with Western blot at 12 h, 24 h, and 48 h. C57BL/6 mice were subcutaneously injected with cell suspension containing 1×10 6 MC38 murine colon cancer cells and RM-1 murine prostate cancer cells to construct two tumor-bearing mice models. The tumor-bearing mice were divided into 4 groups, receiving intratumoral injection of 50 μL of PBS, AdC6-ΔE1-ΔE3 (1×10 8 PFU), AdC6-htertE1A-ΔE3 (1×10 8PFU), and AdC6-htertE1A-ΔE3 (CSF 2) (1×10 8 PFU), respectively. When the tumor size of PBS group reached 2 500 mm 3, all the mice were sacrificed and the tumor tissue was collected for TUNEL staining. Then, apoptosis-positive cells were observed and counted under a microscope. Restriction digestion revealed that the oncolytic viruses AdC6-htertE1A-ΔE3, AdC6-htertE1A-ΔE3 (CSF 2) and AdC6-ΔE1-ΔE3 were successfully constructed. Western blot confirmed that AdC6-htertE1A-ΔE3 (CSF 2) could infect different tumor cells and stably express CSF 2, the exogenous gene. CCK8 results showed that AdC6-htertE1A-ΔE3 and AdC6-htertE1A-ΔE3 (CSF 2) had obvious killing effects on RD, SW-620, HeLa, Huh7, RM-1and MC-38. Compared with the replication-deficient adenovirus AdC6-ΔE1-ΔE3, AdC6-htertE1A-ΔE3 and AdC6-htertE1A-ΔE3 (CSF 2) at a multiplicity of infection of 100 MOI had extremely obvious killing effects on tumor cells ( P<0.05). Western blot showed that AdC6-htertE1A-ΔE3 and AdC6-htertE1A-ΔE3 (CSF 2) induced tumor cell apoptosis by activating the P53-dependent pathway. Injection of oncolytic virus in tumor-bearing mouse models of prostate cancer and colorectal cancer could significantly inhibit the tumor growth and even clear the tumor. Oncolytic virus based on AdC6 could eliminate tumor in vivoand in vitro through mechanisms that induced apoptosis, showing great potential for the treatment of tumors.

Differential expression of pyruvate dehydrogenase E1A and its inactive phosphorylated form among breast cancer subtypes

AimsPyruvate dehydrogenase E1A (PDH-E1A) is one of the key regulators of metabolic pathways that determines pyruvate entry into the citric acid cycle or glycolysis. When PDH-E1A is phosphorylated (P-PDH-E1A), it loses its activity, shifting the metabolism towards glycolysis. Breast cancer (BC) is a highly heterogeneous disease by which different breast cancer subtypes acquire distinct metabolic profiles. Assessing PDH-E1A and P-PDH-E1A expressions among BC subtypes might reveal their association with the distinct molecular profiles of BCs. MethodsThe expressions of PDH-E1A and P-PDH-E1A were investigated in BC cell lines and 115 BC tissues using Western blot and immunohistochemistry, respectively. Besides, PDHE1A mRNA expression was assessed in 1084 BCE patients' transcriptomics data retrieved from Cancer Genome Atlas database. Statistical analyses were performed to assess the correlation of PDH-E1A and P-PDH-E1A expressions with patients' clinicopathological characteristics. Kaplan-Meier method was used to evaluate their prognostic value. Key findingsMultivariate analysis revealed a significant association between PDH-E1A/P-PDH-E1A expressions and the molecular subtype, histological type, and tumor size of breast cancer tissues. The hormonal receptors (ER and PR), HER-2, and Ki67 protein expressions were significantly associated with PDH-E1A and P-PDH-E1A protein expressions. Similar findings were observed when PDHA1 mRNA expression was assessed. The increased protein expression of PDH-E1A could be an independent prognostic factor for unfavorable overall survival (OS). In contrast, high PDHA1 mRNA expression had better OS. SignificanceThis study revealed the differential expression of PDH-E1A and P-PDH-E1A among breast cancer subtypes and suggested PDH-E1A expression as a prognostic factor for BC patients' OS.

Immediate-early transcriptional response to insulin receptor stimulation

Insulin binding to the insulin receptor triggers intracellular signaling cascades involving the activation of protein and lipid kinases. As a result, multiple biological functions of the cells are changed. Here, we analyzed the regulation and signaling cascades leading to insulin-induced activation of the stimulus-responsive transcription factors. For the analyses, we used chromatin-embedded reporter genes having a cellular nucleosomal organisation, and fibroblasts expressing human insulin receptors (HIRcB cells). The results show that stimulation of the insulin receptor induced the expression of the transcription factor Egr-1. Attenuation of Egr-1 promoter activation was observed following expression of a dominant-negative mutant of the ternary complex factor Elk-1. These data were corroborated by experiments showing that insulin receptor stimulation increased the transcriptional activation potential of Elk-1. In addition, the transcriptional activity of AP-1 was significantly elevated in insulin-stimulated HIRcB cells. Expression of the dominant-negative mutant of Elk-1 reduced insulin-induced activation of AP-1, indicating that Elk-1 controls both serum response element and AP-1-regulated transcription. Moreover, we show that stimulation of the insulin receptor activates cyclic AMP response element (CRE)-controlled transcription, involving the transcription factor CREB. Insulin-induced transcription of Elk-1 and CREB-controlled reporter genes was attenuated by overexpression of MAP kinase phosphatase-1 or a constitutively active mutant of calcineurin A, indicating that both phosphatases are part of a negative feedback loop for reducing insulin-mediated gene transcription. Finally, we show that expression of the adenoviral protein E1A selectively reduced CRE-mediated transcription following stimulation of the insulin receptor. These data indicate that insulin-regulated transcription of CRE-containing genes is under epigenetic control.

Abstract 1153: Targeting CA125 transcription for ovarian cancer treatment

Abstract CA125 coded by MUC16 gene is highly expressed in most ovarian cancer cells and serves as a tumor marker to monitor disease progression or response to treatment of ovarian cancer patients. However, targeting CA125 for ovarian cancer treatment has never been successful. We performed multiple steps of high-fidelity PCR and obtained a 4.2kb DNA fragment upstream of the human MUC16 gene, which included a region upstream from the transcription start site (TSS) and a region downstream from the TSS that contains both the 5' untranslated region and the first 136bp of the open reading frame. Reporter assays showed that this DNA fragment possesses strong transactivation activity in CA125-high cancer cells, but not in CA125-low cells, indicating that the DNA fragment we cloned contains the transactivation region that controls specific expression of MUC16 gene in ovarian cancer cells. We further refined the promoter and found a 1040bp fragment with similar transcriptional activity and specificity. We used this refined MUC16 promoter to replace the E1A promoter in the adenovirus type 5 genome DNA to control expression of E1A, an essential gene for adenovirus replication, and successfully generated a conditionally replication-competent adenovirus that can replicate in and lyse CA125-high-expressing cancer cells, including CAOV3, Kuramochi, MADH2774, OVCAR3, OVCAR4, and TOV112D cells, but not in CA125-low or -negative cancer cell lines, such as A2780, OVCAR5, PEO4, or SKOV3 cells. In vivo studies showed that intraperitoneal virus injection prolonged survival of NSG mice inoculated intraperitoneally with MADH2774 cells. Preliminary experiment showed that a mouse ovarian cancer cell line, ID8, infected with the virus might elicit a protective immune response to rechallenge of parental cancer cells. Our data indicate that targeting MUC16 transactivation for ovarian cancer treatment by conditionally replicative adenovirus development is feasible and practical. Citation Format: Er Yue, Guangchao Yang, Yuanfei Yao, Guangyu Wang, Yanqiao Zhang, Edward W. Wang. Targeting CA125 transcription for ovarian cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1153.

Abstract 1748: Armed oncolytic adenovirus expressing the positive immune checkpoint OX40L for gastric cancer therapy

Abstract Gastric cancer is the third leading cause of cancer-related mortality worldwide, with a 5-year survival rate of 30% in the US despite standard treatment. Black, Asian, and non-white Hispanic populations have a 40-50% higher risk of gastric cancer than white people. The poor prognosis and limited therapies associated with gastric cancer has led us to investigate the use of these oncolytic adenoviruses as an alternative treatment. Viroimmunotherapy is showing promising results in preclinical and clinical studies. Our group developed an oncolytic adenovirus, Delta-24-RGD, also named DNX-2401, which has been tested in clinical trials for recurrent glioblastoma, with complete responses and durable survival in 20% of patients, which was associated with an anti-tumor immune response. To further enhance the immune effect of Delta-24-RGD, we developed the next generation virus armed with T cell co-stimulatory agonist, OX40L, termed Delta-24-RGDOX, clinically known as DNX-2440. To explore the sensitivity of gastric cancer to oncolytic adenoviruses, we performed viral infection and replication experiments in a panel of human gastric cancer cells (AGS, MKN-45, SNU-1) and a murine gastric cancer cell line, M12. Cell lines were infected in a dose-dependent experiment using an Ad-GFP-RGD vector, showing that &amp;gt;80% of the culture was infected using a multiplicity of infection of 25 for AGS, 50 for MKN-45 and SNU-1, and 100 for M12. Viral replication in gastric cancer was assessed by hexon staining of 293HEK cells incubated with cell lysates obtained from infected gastric cancer cells, illustrating that infection with either virus resulted in the production of new virions. Consistent with these data, we detected in Delta-24-RGD- and Delta-24-RGDOX-infected gastric cancer cells expression of E1A and hexon, confirming viral infection and replication, respectively. Of interest, 74% to 81% Delta-24-RGDOX-infected gastric cancer cells expressed in their surface OX40L 48 hours post-infection as assessed by flow cytometry. Efficacy of the proposed virotherapy in gastric cancer was visualized by using cell titer blue viability assay, which showed anti-cancer efficacy of the proposed therapy. Results on the efficacy and immune effects of armed-oncolytic adenovirus expressing OX40L in a M12-C57BL/6 gastric cancer immunocompetent mouse model are in progress. Current results suggest that armed oncolytic adenoviruses with positive immunomodulators can effectively infect, replicate, and lyse gastric cancer cells. Ultimately, we aim to translate these oncolytic adenoviruses to the clinic to improve the survival of patients with gastric cancer. Citation Format: Ashley Ossimetha, Teresa Nguyen, Natalie Melendez, Xuejun Fan, Marta Alonso, Juan Fueyo, Filipa Godoy-Vitorino, Candelaria Gomez-Manzano. Armed oncolytic adenovirus expressing the positive immune checkpoint OX40L for gastric cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1748.

Cell-to-cell and genome-to-genome variability of adenovirus transcription tuned by the cell cycle.

In clonal cultures, not all cells are equally susceptible to virus infection, and the mechanisms underlying this are poorly understood. Here, we developed image-based single-cell measurements to scrutinize the heterogeneity of adenovirus (AdV) infection. AdV delivers, transcribes and replicates a linear double-stranded DNA genome in the nucleus. We measured the abundance of viral transcripts using single-molecule RNA fluorescence in situ hybridization (FISH) and the incoming 5-ethynyl-2'-deoxycytidine (EdC)-tagged viral genomes using a copper(I)-catalyzed azide-alkyne cycloaddition (click) reaction. Surprisingly, expression of the immediate early gene E1A only moderately correlated with the number of viral genomes in the cell nucleus. Intranuclear genome-to-genome heterogeneity was found at the level of viral transcription and, in accordance, individual genomes exhibited heterogeneous replication activity. By analyzing the cell cycle state, we found that G1 cells exhibited the highest E1A gene expression and displayed increased correlation between E1A gene expression and viral genome copy numbers. The combined image-based single-molecule procedures described here are ideally suited to explore the cell-to-cell variability in viral gene expression in a range of different settings, including the innate immune response.

Efficacy of a novel double-controlled oncolytic adenovirus driven by the Ki67 core promoter and armed with IL-15 against glioblastoma cells

BackgroundGlioblastoma (GBM) is an immunosuppressive, highly vascular and devastating malignant brain tumor. Even with progressive combination treatment that includes surgery, radiotherapy, and chemotherapy, the prognosis for GBM patients is still extremely poor. Oncolytic adenovirus (OAd) can specifically replicate in GBM cells, permitting the rapid copy of the therapeutic genes it carries. Moreover, E1A is an essential gene in adenoviral replication and is the first gene expressed upon viral infection. E1A expression can be regulated by the Ki67 promoter, while the CMV promoter drives therapeutic gene expression. However, the efficacy of a double-controlled OAd driven by the Ki67 core promoter and armed with IL-15 against GBM cells has not been investigated.MethodsFluorescence microscopy was performed to evaluate infection ability in the viruses. Cell viability was detected by CCK-8 assay. Levels of cytokines in different supernatants were determined by ELISA, and IL-15 gene expression was measured by RT-PCR. Angiogenic capacity was analyzed by tube formation assay.ResultsWe successfully constructed a double-controlled oncolytic adenovirus driven by the Ki67 core promoter and armed with IL-15 that selectively infected and killed GBM cells while sparing normal cells. The adenoviruses prime IL-15 gene expression to significantly enhance anti-GBM efficacy through effective activation of microglial cells. Moreover, OAd not only directly inhibits angiogenesis but exhibits potent antiangiogenic capacity mediated by the reduction of VEGF secretion.ConclusionsThese results provide new insight into the effects of a novel double-controlled OAd driven by the Ki67 core promoter and armed with IL-15 in glioblastoma treatment, which may help in the development of novel therapies in solid tumors.

Advantages of Using Paclitaxel in Combination with Oncolytic Adenovirus Utilizing RNA Destabilization Mechanism.

Oncolytic virotherapy is a novel approach to cancer therapy. Ad-fosARE is a conditionally replicative adenovirus engineered by inserting AU-rich elements (ARE) in the 3’-untranslated region of the E1A gene. In this study, we examined the oncolytic activity of Ad-fosARE and used it in a synergistic combination with the chemotherapeutic agent paclitaxel (PTX) for treating cancer cells. The expression of E1A was high in cancer cells due to stabilized E1A-ARE mRNA. As a result, the efficiency of its replication and cytolytic activity in cancer cells was higher than in normal cells. PTX treatment increased the cytoplasmic HuR relocalization in cancer cells, enhanced viral replication through elevated E1A expression, and upregulated CAR (Coxsackie-adenovirus receptor) required for viral uptake. Furthermore, PTX altered the instability of microtubules by acetylation and detyrosination, which is essential for viral internalization and trafficking to the nucleus. These results indicate that PTX can provide multiple advantages to the efficacy of Ad-fosARE both in vitro and in vivo, and provides a basis for designing novel clinical trials. Thus, this virus has a lot of benefits that are not found in other oncolytic viruses. The virus also has the potential for treating PXT-resistant cancers.