E1A Protein Research Articles

Bioselection Reveals miR-99b and miR-485 as Enhancers of Adenoviral Oncolysis in Pancreatic Cancer

Oncolytic viruses are designed for cancer treatment. Cell-virus interactions are key determinants for successful viral replication. Therefore, the extensive reprogramming of gene expression that occurs in tumor cells might create a hurdle for viralpropagation. We used a replication-based approach of a microRNA (miRNA) adenoviral library encoding up to 243 human miRNAs as a bioselection strategy to identify miRNAs that facilitate adenoviral oncolytic activity in pancreatic ductal adenocarcinoma. We identify two miRNAs, miR-99b and miR-485, that function as enhancers of adenoviral oncolysis by improving the intra- and extracellular yield of mature virions. An increased adenoviral activity is the consequence of enhanced E1A and late viral protein expression, which is probably mediated by the downregulation of the transcriptional repressors ELF4, MDM2, and KLF8, which we identify as miR-99b or miR-485 target genes. Arming the oncolytic adenovirus ICOVIR15 with miR-99b or miR-485 enhances its fitness and its antitumoral activity. Our results demonstrate the potential of this strategy to improve oncolytic adenovirus potency, and they highlight miR-99b and miR-485 as sensitizers of adenoviral replication.

Interplay between sequence, structure and linear motifs in the adenovirus E1A hub protein

E1A is the main transforming protein in mastadenoviruses. This work uses bioinformatics to extrapolate experimental knowledge from Human adenovirus serotype 5 and 12 E1A proteins to all known serotypes. A conserved domain architecture with a high degree of intrinsic disorder acts as a scaffold for multiple linear motifs with variable occurrence mediating the interaction with over fifty host proteins. While linear motifs contribute strongly to sequence conservation within intrinsically disordered E1A regions, motif repertoires can deviate significantly from those found in prototypical serotypes. Close to one hundred predicted residue-residue contacts suggest the presence of stable structure in the CR3 domain and of specific conformational ensembles involving both short- and long-range intramolecular interactions. Our computational results suggest that E1A sequence conservation and co-evolution reflect the evolutionary pressure to maintain a mainly disordered, yet non-random conformation harboring a high number of binding motifs that mediate viral hijacking of the cell machinery.

Do the two transcription factors form a positive feedback amplifier circuit in their common fight against pathogens?

Do the two transcription factors form a positive feedback amplifier circuit in their common fight against pathogens?

Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters.

How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters.

Inhibition of androgen receptor transactivation function by adenovirus type 12 E1A undermines prostate cancer cell survival.

Mutations or truncation of the ligand-binding domain (LBD) of androgen receptor (AR) underlie treatment resistance for prostate cancer (PCa). Thus, targeting the AR N-terminal domain (NTD) could overcome such resistance. Luciferase reporter assays after transient transfection of various DNA constructs were used to assess effects of E1A proteins on AR-mediated transcription. Immunofluorescence microscopy and subcellular fractionation were applied to assess intracellular protein localization. Immunoprecipitation and mammalian two-hybrid assays were used to detect protein-protein interactions. qRT-PCR was employed to determine RNA levels. Western blotting was used to detect protein expression in cells. Effects of adenoviruses on prostate cancer cell survival were evaluated with CellTiter-Glo assays. Adenovirus 12 E1A (E1A12) binds specifically to the AR. Interestingly, the full-length E1A12 (266 aa) preferentially binds to full-length AR, while the small E1A12 variant (235 aa) interacts more strongly with AR-V7. E1A12 promotes AR nuclear translocation, likely through mediating intramolecular AR NTD-LBD interactions. In the nucleus, AR and E1A12 co-expression in AR-null PCa cells results in E1A12 redistribution from nuclear foci containing CBX4 (also known as Pc2), suggesting a preferential AR-E1A12 interaction over other E1A12 interactors. E1A12 represses AR-mediated transcription in reporter gene assays and endogenous AR target genes such as ATAD2 and MYC in AR-expressing PCa cells. AR-expressing PCa cells are more sensitive to death induced by a recombinant adenovirus expressing E1A12 (Ad-E1A12) than AR-deficient PCa cells, which could be attributed to the increased viral replication promoted by androgen stimulation. Targeting the AR by E1A12 promotes apoptosis in PCa cells that express the full-length AR or C-terminally truncated AR variants. Importantly, inhibition of mTOR signaling that blocks the expression of anti-apoptotic proteins markedly augments Ad-E1A12-induced apoptosis of AR-expressing cells. Mechanistically, Ad-E1A12 infection triggers apoptotic response while activating the PI3K-AKT-mTOR signaling axis; thus, mTOR inhibition enhances apoptosis in AR-expressing PCa cells infected by Ad-E1A12. Ad12 E1A inhibits AR-mediated transcription and suppresses PCa cell survival, suggesting that targeting the AR by E1A12 might have therapeutic potential for treating advanced PCa with heightened AR signaling.

Mimicry of Cellular A Kinase-Anchoring Proteins Is a Conserved and Critical Function of E1A across Various Human Adenovirus Species.

The E1A proteins of the various human adenovirus (HAdV) species perform the critical task of converting an infected cell into a setting primed for virus replication. While E1A proteins differ in both sequence and mechanism, the evolutionary pressure on viruses with limited coding capacity ensures that these proteins often have significant overlap in critical functions. HAdV-5 E1A is known to use mimicry to rewire cyclic AMP (cAMP) signaling by decoupling protein kinase A (PKA) from cellular A kinase-anchoring proteins (AKAPs) and utilizing PKA to its own advantage. We show here that E1As from other species of HAdV also possess this viral AKAP (vAKAP) function and examine how they manipulate PKA. E1A from most species of HAdV examined contain a small AKAP-like motif in their N terminus which targets the docking-dimerization domain of PKA as the binding interface for a conserved protein-protein interaction. This motif is also responsible for an E1A-mediated relocalization of PKA regulatory subunits from the cytoplasm into the nucleus, with species-specific E1A proteins having preference for one particular isoform of PKA subunit over another. Importantly, we showed that these newly characterized vAKAPs can integrate into cAMP-responsive transcription as well as contribute to viral genome replication and infectious progeny production for several distinct HAdV species.IMPORTANCE These data enhance the mechanistic knowledge on how HAdV E1A manipulates cellular PKA to benefit infection. The work establishes that mimicry of AKAPs and subversion of PKA-mediated cAMP signaling are conserved features for numerous human adenoviruses. This study also highlights the molecular determinants conferring selective protein-protein interactions between distinct PKA regulatory subunits and the different E1A proteins of these viruses. Additionally, it further emphasizes the utility of using viral proteins like E1A as tools for studying the molecular biology of cellular regulatory pathways.

A First-in-Class TWIST1 Inhibitor with Activity in Oncogene-Driven Lung Cancer.

TWIST1, an epithelial-mesenchymal transition (EMT) transcription factor, is critical for oncogene-driven non-small cell lung cancer (NSCLC) tumorigenesis. Given the potential of TWIST1 as a therapeutic target, a chemical-bioinformatic approach using connectivity mapping (CMAP) analysis was used to identify TWIST1 inhibitors. Characterization of the top ranked candidates from the unbiased screen revealed that harmine, a harmala alkaloid, inhibited multiple TWIST1 functions, including single-cell dissemination, suppression of normal branching in 3D epithelial culture, and proliferation of oncogene driver-defined NSCLC cells. Harmine treatment phenocopied genetic loss of TWIST1 by inducing oncogene-induced senescence or apoptosis. Mechanistic investigation revealed that harmine targeted the TWIST1 pathway through its promotion of TWIST1 protein degradation. As dimerization is critical for TWIST1 function and stability, the effect of harmine on specific TWIST1 dimers was examined. TWIST1 and its dimer partners, the E2A proteins, which were found to be required for TWIST1-mediated functions, regulated the stability of the other heterodimeric partner posttranslationally. Harmine preferentially promoted degradation of the TWIST1-E2A heterodimer compared with the TWIST-TWIST1 homodimer, and targeting the TWIST1-E2A heterodimer was required for harmine cytotoxicity. Finally, harmine had activity in both transgenic and patient-derived xenograft mouse models of KRAS-mutant NSCLC. These studies identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC.Implications: TWIST1 is required for oncogene-driven NSCLC tumorigenesis and EMT; thus, harmine and its analogues/derivatives represent a novel therapeutic strategy to treat oncogene-driven NSCLC as well as other solid tumor malignancies. Mol Cancer Res; 15(12); 1764-76. ©2017 AACR.

Comparison of Liver Detargeting Strategies for Systemic Therapy with Oncolytic Adenovirus Serotype 5.

Oncolytic viruses would ideally be of use for systemic therapy to treat disseminated cancer. To do this safely, this may require multiple layers of cancer specificity. The pharmacology and specificity of oncolytic adenoviruses can be modified by (1) physical retargeting, (2) physical detargeting, (3) chemical shielding, or (4) by modifying the ability of viral early gene products to selectively activate in cancer versus normal cells. We explored the utility of these approaches with oncolytic adenovirus serotype 5 (Ad5) in immunocompetent Syrian hamsters bearing subcutaneous HaK tumors. After a single intravenous injection to reach the distant tumors, the physically hepatocyte-detargeted virus Ad5-hexon-BAP was more effective than conditionally replicating Ad5-dl1101/07 with mutations in its E1A protein. When these control or Ad5 treated animals were treated a second time by intratumoral injection, prior exposure to Ad5 did not affect tumor growth, suggesting that anti-Ad immunity neither prevented treatment nor amplified anti-tumor immune responses. Ad5-dl1101/07 was next chemically shielded with polyethylene glycol (PEG). While 5 kDa of PEG blunted pro-inflammatory IL-6 production induced by Ad5-dl1101/07, this shielding reduced Ad oncolytic activity.

Cell Cycle-Dependent Kinase Cdk9 Is a Postexposure Drug Target against Human Adenoviruses.

Human adenoviruses (HAdVs) infect respiratory, gastrointestinal, and urinary tracts and give rise to eye infections and epidemic keratoconjunctivitis (EKC). They persist in lymphoid tissue and cause morbidity and mortality in immunocompromised people. Treatments with significant postexposure efficacy are not available. Here, we report that inhibition of the cell cycle-dependent kinase 9 (Cdk9) by RNA interference, or the compound flavopiridol, blocked infections with HAdV-C2/5, EKC-causing HAdV-D8/37, and progeny formation in human corneal epithelial and cancer cells. Flavopiridol abrogated the production of the immediate early viral transactivating protein E1A without affecting nuclear import of viral DNA. In morphometric plaque assays, the compound exhibited antiviral efficacy in both pre- and postexposure regimens with therapeutic indexes exceeding 10. The study identifies Cdk9 as a postexposure drug target against adenovirus infections in vitro and suggests that the clinically tested anticancer drug flavopiridol is a candidate for treating adenoviral EKC or adenovirus emergence upon immune suppression.

Suppression of Adenovirus Replication by Cardiotonic Steroids.

Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.

Inhibitory effect of oncolytic adenovirus ZD55-special AT rich sequence binding protein 1 on proliferation and invasion of andragon-independent prostate cancer DU145 cells

Objective To investigate the suppressive effect of oncolytic adenovirus ZD55-special AT rich sequence binding protein 1 (SATB1) expressing small hairpin RNA targeting SATB1 gene on growth and invasion of prostate cancer DU145 cells. Methods The oncolytic adenovirus ZD55-SATB1 was constructed and identifies. The inhibitory effect of ZD55-SATB1 on SATB1 expression was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting analysis at 72 h after infection of DU145 cells with ZD55-SATB1 at a MOI of 10. The cytotoxicity of ZD55-SATB1 was detected by cell counting kit-8 (CCK-8) assay and crystal violet staining assay. Cell invasion was detected by Matrigel invasion assay. The expression of E1A protein in DU145 cell lines infected with viruses respectively was detected by Western blotting. Results The oncolytic adenoviruses ZD55-SATB1 were successfully constructed and identified. RT-PCR and Western blotting assay showed that ZD55-SATB1 could significantly decrease the expression of SATB1 in DU145 cells (P<0.05). The relative protein levels of E1A in DU145 cells treated with ZD55-SATB1 were significantly higher than those in human normal prostate epithelial cell line PZ-HPV-7 cells (P<0.01), which indicated cancer specificity of ZD55-SATB1 for prostate cance cells. Crystal violet staining assay showed that ZD55-SATB1 exhibited obvious cytotoxicity in DU145 cells. The cytopathic effect of ZD55-SATB1 was much stronger than that of ZD55-enhanced green fluorescent protein (EGFP) and Ad-SATB1. CCK-8 assay showed that cell viability of ZD55-SATB1 group [(31.60±3.31)%] was significantly lower than that in ZD55-EGFP group [(61.70±2.42)%, P=0.025] and Ad-SATB1 group [(82.40±3.54)%, P=0.017] for DU145 cells after 4 days. Transwell cell invasive experiment showed that the number of cells that invaded the membrane in ZD55-SATB1 group was (61.30±17.25) cells, significantly less than that in the Ad-SATB1 group [(170.10±25.44) cells, P=0.043], the ZD55-EGFP group [(295.40±23.36) cells, P=0.013] and the PBS group [(490.50±33.36) cells, P=0.009]. Conclusion Oncolytic adenovirus ZD55-SATB1 could effectively knockdown the SATB1 expression in DU145 cells, and dramatically inhibit proliferation and invasion of DU145 cells. Key words: Oncolytic adenovirus; Special AT rich sequence binding protein 1; Proliferation; Invasion

The interaction of adenovirus E1A with the mammalian protein Ku70/XRCC6

Human adenovirus infects terminally differentiated cells and to replicate it must induce S-phase. The chief architects that drive adenovirus-infected cells into S-phase are the E1A proteins, with 5 different isoforms expressed during infection. E1A remodels the infected cell by associating with cellular factors and modulating their activity. The C-terminus of E1A is known to bind to only a handful of proteins. We have identified a novel E1A C-terminus binding protein, Ku70 (XRCC6), which was found to bind directly within the CR4 of E1A from human adenovirus type 5. Depletion of Ku70 reduced virus growth, possibly by activating the DNA damage response pathway. Ku70 was found to localize to viral replication centers and associate with the viral genome. Ku70 was also recruited to cellular cell cycle regulated promoters following viral infection. Our study has identified, for the first time, Ku70 as a novel E1A-binding protein which affects virus life cycle.

Effect of combined treatment of radiation and tissue-specific recombinant oncolytic adenovirus on bladder cancer cells

Purpose: Gene therapy combined with radiation has shown promising potential for the treatment of tumors. This paper aimed to clarify the synergistic effect of radiotherapy combined with the bladder cancer tissue-specific oncolytic adenovirus (Ad-PSCAE-UPII-E1A) on bladder cancer cells and to study the underlying synergy mechanisms of the combined treatment.Materials and methods: The Adenovirus carrying E1A under control of UPII promoter and prostate stem cell antigen enhancer (PSCAE) were successfully constructed. The viability of bladder cancer cells BIU-87 and EJ was determined by MTT assay. The apoptotic assay was demonstrated by flow cytometry and TEM. Virus titer was determined by TCID50 assay, and proteins Mre11, Chk2-Thr68, and E1A were analyzed by Western blot method.Results: Oncolytic adenovirus combined with radiotherapy improved antitumor efficacy compared with the single treatment at a time and was X-ray dosage-dependent. When the adenovirus infection was scheduled at 24 h after irradiation, cancer cells had the lowest viability. Adenovirus and irradiation induced cell death through the caspase-3 related apoptotic pathway, and bladder cancer cells were arrested at the G1 (BIU-87) or S phase (EJ). Autophagic vacuoles were observed in bladder cancer cells treated with radiation and adenovirus. After irradiation, more virus particles were observed in the BIU-87 and EJ cells. However, by a TCID50 assay, there was no difference in virus titter between irradiated bladder cancer cells and unirradiated cells. The proteins Mre11, Chk2-Thr68 which involved in the DNA break repair pathway were decreased while γ-H2AX-Ser139 increased; at the same time, the E1A gene and the hexon proteins of oncolytic adenovirus were increased after irradiation.Conclusions: Our results proved synergistic antitumor effect of adenovirus Ad-PSCAE-UPII-E1A and radiation, which might be a potential therapeutic strategy for bladder cancer.

Abstract 2020: Structure-function analysis of twist1-e2a transcriptional activity in kras-driven non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer death in the United States and in the world. Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. We previously demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1, in KRAS mutant, EGFR mutant, and MET amplified/mutant NSCLC can induce either oncogene induced senescence or apoptosis. However, the key functions of TWIST1 that are required for its transcriptional activity in cancer are unknown. In the current study, we engineered domain-specific mutations in TWIST1 to determine the impact of altered DNA binding, dimerization and post-translational modifications on its transcriptional activity by luciferase reporter assays. Testing the promoter activity at three TWIST1-regulated loci involved in tumorigenesis, YBX1, SNAI2, and FLIP, we found that DNA binding and nuclear localization were uniformly required for TWIST1 transcriptional function. However, phosphorylation, TWIST1 box function, and ability to form homo- versus hetero- dimers impacted TWIST1 activity in a locus-specific manner. Previous studies have demonstrated that TWIST1 dimerizes with the E2A proteins, E12 and E47. We have shown that silencing of E2A phenocopies loss of TWIST1 in NSCLC and that formation of the TWIST1-E2A heterodimer results in a reciprocal stabilization of the binding partner. Therefore, we sought to determine how differential dimerization by TWIST1 might modulate tumorigenic gene expression. We utilized tethered TWIST1 dimers with either TWIST1 or the E2A proteins to form TWIST1-TWIST1 (homodimer), TWIST1-E12 or -E47 (heterodimers), and tested their transcriptional activities at several TWIST1-regulated promoters. We found that TWIST1-E2A heterodimers could enhance TWIST1 transcriptional activity compared to that of the TWIST-TWIST homodimer. In addition, we determined that the TWIST-E2A heterodimer is degraded by harmine, a harmala alkaloid that we have shown degrades TWIST1, whereas the TWIST-TWIST homodimer is resistant to harmine degradation. This suggests that the TWIST1-E2A heterodimer is the key target of harmine. Furthermore, we have found that overexpression of the TWIST1, E2A proteins, or the TWIST1-E2A heterodimer is able to partially rescue harmine induced cytotoxicity in KRAS mutant NSCLC. Taken together, these data suggest that E2A is essential for TWIST1 mediated tumorigenesis and that targeting of the TWIST1-E2A axis may be an effective therapeutic strategy against oncogene-driven NSCLC. Citation Format: Susheel K. Khetarpal. Structure-function analysis of twist1-e2a transcriptional activity in kras-driven non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2020.

Abstract 3573: TWIST1/E2A signaling axis suppresses apoptosis in oncogene driven non-small cell lung cancer

Abstract Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. In addition, acquired resistance to currently available targeted therapies is inevitable. We previously demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1 in KRAS mutant, EGFR mutant, and MET amplified/mutant NSCLC can induce apoptosis, which suggests that a subset of oncogene dependent NSCLC are potentially “addicted” to TWIST1. Importantly, we have identified the harmala alkaloid, harmine, as a novel TWIST1 inhibitor which could inhibit growth in several oncogene driver defined NSCLC cell lines and decrease levels of TWIST1 and its dimerization partners, the E2A proteins, via degradation. We examined the target genes and pathways required for suppression of apoptosis by TWIST1 and E2A. Genetic or pharmacological (harmine) inhibition of TWIST1 or E2A resulted in apoptosis in several oncogenic driver dependent cell lines. Additionally, treatment with a pan-caspase inhibitor resulted in rescue of growth inhibition following TWIST1 or E2A silencing or harmine treatment. This suggests that apoptosis is the mechanism of growth inhibition following TWIST1 inhibition. TWIST1 or E2A inhibition resulted in increased levels of Bid, Bim, and DR5, as well as, reduced c-FLIP and Bcl-2 levels. Conversely, we demonstrated that TWIST1 overexpression leads to increased levels of c-FLIP and anti-apoptotic Bcl-2 family members as well as decreased levels of Bim and Bid. c-FLIP appears to be a direct transcriptional target of TWIST1 as TWIST1 overexpression leads to transactivation of the c-FLIP promoter and is dependent on the ability of TWIST1 to bind DNA. Interestingly, the TWIST1-E2A heterodimer results in greater promoter transactivation when compared to the TWIST1 homodimer. Furthermore, knockdown of Bim, overexpression of Bcl-2, or overexpression of c-FLIPs resulted in partial rescue of growth inhibition and apoptosis following TWIST1 silencing. However, only knockdown of Bim or Bcl-2 overexpression was able to rescue apoptosis following harmine treatment. This suggests that apoptosis following harmine treatment only requires the intrinsic machinery, while specifically silencing TWIST1 also engages the extrinsic pathway. In summary, we found that the apoptosis observed after TWIST1/E2A inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through its novel target genes, c-FLIP and Bim. Our studies will establish the target genes of TWIST1 that are required for suppression of apoptosis with the ultimate goal of identifying biomarkers of response to TWIST1 inhibitors. We also aim to determine if TWIST1, through its apoptotic target genes, modulates response to targeted therapies or standard chemotherapies. Citation Format: Zachary A. Yochum, Susheel Khetarpal, Timothy F. Burns. TWIST1/E2A signaling axis suppresses apoptosis in oncogene driven non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3573.

The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2.

DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation.

Mouse Adenovirus Type 1 Early Region 1A Effects on the Blood-Brain Barrier.

Mouse adenovirus type 1 (MAV-1) infects endothelial cells and disrupts the blood-brain barrier (BBB), causing encephalitis in inbred and outbred mice. Using a virus mutant that does not produce the early region 1A protein E1A, we investigated whether the activity of this known viral transcriptional regulator is needed for BBB disruption and other phenotypes associated with encephalitis. The wild-type (wt) virus and E1A mutant virus caused similar levels of permeability of sodium fluorescein in brains of infected mice. In an in vitro assay of BBB integrity, wt and mutant virus caused similar decreases in transendothelial electrical resistance in primary mouse brain endothelial cell monolayers. These results indicate that E1A protein does not contribute to disruption of BBB integrity in animals or cultured cells. Both wt and E1A mutant virus infection of mice led to similar increases in the activity of two matrix metalloproteinases known to correlate with BBB disruption, MMP2 and MMP9, while causing no increase in the steady-state expression of MMP2 or MMP9 mRNA. In contrast, the amount of MMP3 transcripts increased upon infection by both viruses and to a higher level in infections by the mutant virus lacking E1A protein production. There was no difference in the levels of steady-state expression of mRNA for tight junction proteins among mock virus, wt virus, and mutant virus infections. Thus, the MAV-1 E1A protein does not measurably affect BBB integrity in the parameters assayed, although it reduces the amount of MMP3 mRNA steady-state expression induced in brains upon infection. IMPORTANCE Encephalitis can be caused by viruses, and it is potentially life-threatening because of the vital nature of the brain and the lack of treatment options. MAV-1 produces viral encephalitis in its natural host, providing a model for investigating factors involved in development of encephalitis. MAV-1 infection disrupts the BBB and increases activity of matrix metalloproteinases in brains of infected mice. We investigated whether the major transcriptional regulator of adenoviruses, E1A protein, is responsible for any of the specific phenotypes that result from MAV-1 infection. For some of the functions assayed, an E1A mutant virus behaved like wild-type virus. However, expression of mRNA for one matrix metalloproteinase was higher in the virus lacking E1A protein production. This highlights the complex nature of encephalitis and suggests that E1A may have transcriptional effects on host genes important for the development of encephalitis.

Abstract A182: Zbtb7b is involved in the association of EBF1 enhancer to the nuclear lamina during early B cell commitment

Abstract It is well established that E2A proteins activate the expression of tumor suppressors, EBF1, PAX5 and FOXO1, to promote the B cell fate. E2A and EBF1 are frequently mutated either by chromosomal translocations, point mutations or gene deletions in B-cell precursor acute lymphoblastic leukemia. However, it remains an enigma why EBF1 is not transcribed in multipotent progenitors, in which E2A is expressed at high levels. It has been shown that, by HiC and 3D-fluorescence in situ hybridization (3D-FISH), in multipotent progenitors the EBF1 locus was located at the nuclear lamina, in pro-B cells the EBF1 locus was located away from the lamina. In this study, using gain-of-function approaches, we show that enforced Zbtb7b expression inhibited B cell differentiation and caused rapid loss of EBF1 and Pax5 mRNA. Notably, Using 3D-FISH, we found enforced expression of Zbtb7b sequestered EBF1 locus to the nuclear lamina, and using live cell imaging, we show that, Zbtb7b primarily localized at nuclear lamina. We are now investigating whether and how the nuclear envelope and its interaction with the genome change during early B cell commitment using Damseq; and anti-Zbtb7b ChIP-seq provide fine mapping of potential interaction regions with nuclear lamina. These data indicate a novel role of Zbtb7b in B cell lineage commitment and potentially link EBF1 and Zbtb7b into a common pathway. Experiments are ongoing to further examine how sequestration of genes to the nuclear lamina relates to the development of lymphoid cells. Citation Format: Yina Zhu, Cornelis Murre. Zbtb7b is involved in the association of EBF1 enhancer to the nuclear lamina during early B cell commitment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A182.

The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity.

Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication by enhancing the action of E1A products.

A CCAAT/enhancer-binding protein site within antioxidant/electrophile response element along with CREB-binding protein participate in the negative regulation of rat GST-Ya gene in vascular smooth muscle cells.

Studies were conducted to evaluate the negative regulatory function of rat (r)GST-Ya antioxidant/electrophile response element (ARE/EpRE) in vascular smooth muscle cells (vSMCs). We report that CCAAT/enhancer-binding protein (C/EBP)-beta interacts with ARE/EpRE in the rGST-Ya promoter and that aryl hydrocarbon receptor (AhR) is present within the protein complex binding to the C/EBP site. Overexpression of C/EBP-beta or C/EBP-alpha repressed, whereas AhR enhanced, 1.6CAT reporter activity in cells treated with benzo(a)pyrene (BaP). Overexpression of CREB-binding protein (CBP) nullified repression of rGST-Ya transcription. Human adenovirus E1A protein abrogated cotransactivation by CBP but an E1A mutant did not. Overexpression of C/EBPs abrogated stimulation of 1.6CAT by CBP or AhR alone, or in combination, regardless of BaP treatment. Similar profiles were observed using an AhRECAT construct. The C/EBP site within the ARE/EpRE inhibited chemical inducibility of the AhRE. The pattern of mouse GST-Ya regulation by BaP was similar to that of rGST-Ya. We conclude that multiple mechanisms mediate negative regulation of GST-Ya gene in vSMCs, most significant of which are that C/EBP-beta inhibits AhRE or ARE/EpRE inducibility of GST-Ya, limiting CBP levels compromise gene induction, functional interference exists between AhRE and ARE/EpRE, and AhR alone, or in combination with C/EBP-beta, functions as a repressor of the ARE/EpRE.