E2 Protein Research Articles

Small molecule NMD and MDM2 inhibitors synergistically trigger apoptosis in HeLa cells

The nonsense-mediated mRNA decay (NMD) pathway and the p53 pathway, linked to tumorgenesis and are also promising targets for cancer treatment. NMD plays an important role in RNA quality control, while the p53 pathway is involved in cancer suppression. However, their individual and combined effects on cervical cancer (CC) are poorly understood. In this study, we evaluated the impacts of NMD inhibitor, MDM2 inhibitor, and their combination on cell apoptosis, cell cycle, and p53 target genes in HPV-18-positive HeLa cells. Our findings revealed that XR-2failed to activate p53 or induce apoptosis in HeLa cells, whereas SMG1i repressed cell proliferation at high concentrations. Notably the combination of these two agents significantly inhibited cell proliferation, arrested the cell cycle, and triggered cell apoptosis. Mechanistically, MDM2 inhibitor and NMD inhibitor likely exert a synergistically through the truncated E6 protein. These results underscore the potential of employing a combination of MDM2 inhibitor and NMD inhibitor as a promising candidate for the clinical treatment of HPV-infected tumors.

Structure-Based Discovery of Potential HPV E6 and EBNA1 Inhibitors: Implications for Cervical Cancer Treatment

Cervical cancer is the fourth most diagnosed cancer and the fourth leading cause of cancer death in women globally. Its onset and progression have been attributed to high-risk human papillomavirus (HPV) types, especially 16 and 18, while the Epstein–Barr virus (EBV) is believed to also significantly contribute to cervical cancer growth. The E6 protein associated with high-risk HPV strains, such as HPV16 and HPV18, is known for its role in promoting cervical cancer and other anogenital cancers. E6 proteins contribute to the malignant transformation of infected cells by targeting and degrading tumor suppressor proteins, especially p53. On the other hand, EBV nuclear antigen 1 (EBNA1) plays a crucial role in the maintenance and replication of the EBV genome in infected cells. EBNA1 is believed to increase HPV E6 and E7 levels, as well as c-MYC, and BIRC5 cellular genes in the HeLa cell line, implying that HPV/EBV co-infection accelerates cervical cancer onset and growth. Thus, the E6 and EBNA1 antigens of HPV and EBV, respectively, are attractive targets for cervical cancer immunotherapy. This study, therefore, virtually screened for potential drug candidates with good binding affinity to all three oncoviral proteins, HPV16 E6, HPV18 E6, and EBNA1. The compounds were further subjected to ADMET profiling, biological activity predictions, molecular dynamics (MD) simulations, and molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations. A total of six compounds comprising ZINC000013380012, ZINC000070454124, ZINC000014588133, ZINC000085568136, ZINC000095909247, and ZINC000085597263 demonstrated very strong affinity (≤−60 kJ/mol) to the three oncoviral proteins (EBNA1, HPV16 E6, and HPV18 E6) after being subjected to docking, MD, and MM/PBSA. These compounds demonstrated relatively stronger binding than the controls used, inhibitors of EBNA1 (VK-1727) and HPV E6 (baicalein and gossypetin). Biological activity predictions also corroborated their antineoplastic, p53-enhancing, Pin1 inhibitory, and JAK2 inhibitory activities. Further experimental testing is required to validate the ability of the shortlisted compounds to silence the insidious effects of HPV E6 and EBNA1 proteins in cervical cancers.

Serum IgG antibodies to HPV6 L1, E2, E4, E6, and E7 proteins among children prospectively followed-up for three years.

Current knowledge implicates that human papillomavirus (HPV) infection can be acquired at early age. However, the role of HPV-specific passive immunization from mother to neonate is nearly unexplored, especially against the HPV early proteins. We analyzed IgG antibodies against HPV6 early (E2, E4, E6, E7) and late (L1) proteins in children prospectively followed-up for three years. A total of 272 children and their mothers from the Finnish Family HPV Study were included in these analyses. Serum samples were obtained from pregnant mothers at their third trimester and from newborn/infants at 1-, 2-, 6-, 12-, 24-, and 36-month visits after birth. Antibodies to the early and late proteins were analyzed by multiplex serology based on glutathione S-transferase fusion protein capture to fluorescent beads. Maternal antibodies to all tested HPV6 proteins were transferred to neonates, concordance between maternal and neonates' antibody levels being highly significant (p<0.001). Seropositivity of HPV6 L1 in the neonates declined during the first six months of life, whereas changes in the E-protein antibodies were less obvious. After the maternal antibodies have vanished, seroconversion to HPV6 L1 at 12 months (median) and to the HPV6 E-proteins between 23-35 months was observed. IgG antibodies against HPV6 E- and L-proteins are transferred from mothers to their children. Seroconversion against HPV6 L1, E2, E4, E6, and E7 does occur in early childhood, as a sign of acquired HPV6 infection by vertical or horizontal transmission starting at 12 months of age.

Phylogenetic analysis and antigenic epitope prediction for E6 and E7 of Alpha-papillomavirus 9 in Taizhou, China.

Alpha-papillomavirus 9 (α-9) is a member of the human papillomavirus (HPV) α genus, causing 75% invasive cervical cancers worldwide. The purpose of this study was to provide data for effective treatment of HPV-induced cervical lesions in Taizhou by analysing the genetic variation and antigenic epitopes of α-9 HPV E6 and E7. Cervical exfoliated cells were collected for HPV genotyping. Positive samples of the α-9 HPV single type were selected for E6 and E7 gene sequencing. The obtained nucleotide sequences were translated into amino acid sequences (protein primary structure) using MEGA X, and positive selection sites of the amino acid sequences were evaluated using PAML. The secondary and tertiary structures of the E6 and E7 proteins were predicted using PSIPred, SWISS-MODEL, and PyMol. Potential T/B-cell epitopes were predicted by Industrial Engineering Database (IEDB). From 2012 to 2023, α-9 HPV accounted for 75.0% (7815/10423) of high-risk HPV-positive samples in Taizhou, both alone and in combination with other types. Among these, single-type-positive samples of α-9 HPV were selected, and the entire E6 and E7 genes were sequenced, including 298 HPV16, 149 HPV31, 185 HPV33, 123 HPV35, 325 HPV52, and 199 HPV58 samples. Compared with reference sequences, 34, 12, 10, 2, 17, and 17 nonsynonymous nucleotide mutations were detected in HPV16, 31, 33, 35, 52, and 58, respectively. Among all nonsynonymous nucleotide mutations, 19 positive selection sites were selected, which may have evolutionary significance in rendering α-9 HPV adaptive to its environment. Immunoinformatics predicted 57 potential linear and 59 conformational B-cell epitopes, many of which are also predicted as CTL epitopes. The present study provides almost comprehensive data on the genetic variations, phylogenetics, positive selection sites, and antigenic epitopes of α-9 HPV E6 and E7 in Taizhou, China, which will be helpful for local HPV therapeutic vaccine development.

Evolutionary Profile of Mayaro Virus in the Americas: An Update into Genome Variability

The Mayaro virus (MAYV) is an arbovirus with emerging potential, though with a limited understanding of its epidemiology and evolution due to the lack of studies and surveillance. Here, we investigated 71 MAYV genome sequences from the Americas available at GenBank and characterized the phylogenetic relationship among virus strains. A phylogenetic analysis showed that sequences were grouped according to the genotypes L, D, and N. Genotype D sequences were closely related to sequences collected in adjacent years and from their respective countries, suggesting that isolates may have originated from circulating lineages. The coalescent analysis demonstrated similar results, indicating the continuous circulation of the virus between countries as well. An unidentified sequence from the USA was grouped with genotype D, suggesting the insertion of this genotype in the country. Furthermore, the recombination analysis detected homologous and three heterologous hybrids which presented an insertion into the nsP3 protein. Amino acid substitutions among sequences indicated selective pressure sites, suggesting viral adaptability. This also impacted the binding affinity between the E1–E2 protein complex and the Mxra8 receptor, associated with MAYV entry into human cells. These results provide information for a better understanding of genotypes circulating in the Americas.

Exosomes-mediated transmission of standard bovine viral diarrhea strain OregonC24Va in bovine trophoblast cells

Bovine viral diarrhoea virus (BVDV) can infect cows on days 30–110 of gestation and crossing the placental barrier, resulting in persistently infected (PI) and causing significant economic losses to dairy farming. Bovine placental trophoblast cells (BTCs) are the major cells in the early chorionic tissue of the placenta and play important roles in placental resistance to viral transmission. In this study, we have confirmed that BTCs is among a groups of cell types those could be infected by BVDV in vivo, and BVDV infection stimulates the autophagic responses in BTCs and promotes the release of exosomes. Meanwhile, the exosomes derived from BTCs can be used by BVDV to spread between placental trophoblast cells, and this mode of transmission cannot be blocked by antibodies against the BVDV E2 protein, whereas the replication and spread of BVDV in BTCs can be blocked by inhibiting autophagy and exosomogenesis. Our study provides a theoretical and practical basis for scientific prediction and intervention of reproductive disorders caused by BVDV infection in cows of different gestation periods from a novel perspective.

A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels and activates the DNA damage response.

Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here, we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. We also demonstrate that the E2-TopBP1 interaction activates the DDR. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

In vitro study of HPV18-positive cervical cancer HeLa cells based on CRISPR/Cas13a system

The E6 protein is a known oncogene in cervical cancer and plays a key role in the development and progression of cervical cancer by reducing the expression level of the tumor suppressor protein P53 and ultimately leading to enhanced cell proliferation and reduced apoptosis. Therefore, antiviral agents that inhibit the expression of E6 oncoprotein are expected to be potential therapies for human cervical cancer. Here we developed CRISPR/Cas13a: crRNA dual plasmid system and demonstrated that CRISPR/Cas13a could effectively and specifically knock down human papillomavirus 18 E6 mRNA, downregulate the expression level of E6 protein, and restore the expression of the tumor suppressor gene P53 protein, thereby inhibiting the growth of cervical cancer cells and increasing their apoptosis, the E6-2, E6-3, and E6-5 groups resulted in apoptosis rates of 25.4%, 22.4%, and 22.2% in HeLa cells. Moreover, CRISPR/Cas13a enhances the proliferation inhibition and apoptosis induction of cisplatin in cervical cancer HeLa cells. The CRISPR/Cas13a system targeting HPV E6 mRNA may be a promising therapeutic approach for the treatment of human papillomavirus-associated cervical cancer.

Development of an effective single-chain variable fragment recognizing a novel epitope in the hepatitis C virus E2 protein that restricts virus entry into hepatocytes.

Previously, we reported a neutralizing monoclonal antibody, A8A11, raised against a novel conserved epitope within the hepatitis C virus (HCV) E2 protein, that could significantly reduce HCV replication. Here, we report the nucleotide sequence of A8A11 and demonstrate the efficacy of a single-chain variable fragment (scFv) protein that mimics the antibody, inhibits the binding of an HCV virus-like particle to hepatocytes, and reduces viral RNA replication in a cell culture system. More importantly, scFv A8A11 was found to effectively restrict the increase of viral RNA levels in the serum of HCV-infected chimeric mice harbouring human hepatocytes. These results suggest a promising approach to neutralizing-antibody-based therapeutic interventions against HCV infection.

Interferon-γ-induced GBP1 is an inhibitor of human papillomavirus 18

BackgroundHuman papillomavirus (HPV) infection is an important factor leading to cervical cell abnormalities. 90% of cervical cancers are closely associated with persistent infection of high-risk HPV, with the highest correlation with HPV16 and 18. Currently available vaccines and antivirals have limited effectiveness and coverage. Guanylate binding protein 1 (GBP1) was induced by interferon gamma and involved in many important cellular processes such as clearance of various microbial pathogens. However, whether GBP1 can inhibit human papillomavirus infection is unclear.ResultsIn this study, we found that GBP1 can effectively degrade HPV18 E6, possibly through its GTPase activity or other pathways, and E6 protein degrades GBP1 through the ubiquitin-proteasome pathway to achieve immune escape.ConclusionTherefore, GBP1 is an effector of IFN-γ anti-HPV activity. Our findings provided new insights into the treatment of HPV 18 infections.

In silico design of a novel multi-epitope vaccine against HCV infection through immunoinformatics approaches

Infection with the hepatitis C virus (HCV) is one of the causes of liver cancer, which is the world's sixth most prevalent and third most lethal cancer. The current treatments do not prevent reinfection; because they are expensive, their usage is limited to developed nations. Therefore, a prophylactic vaccine is essential to control this virus. Hence, in this study, an immunoinformatics method was applied to design a multi-epitope vaccine against HCV. The best B- and T-cell epitopes from conserved regions of the E2 protein of seven HCV genotypes were joined with the appropriate linkers to design a multi-epitope vaccine. In addition, cholera enterotoxin subunit B (CtxB) was included as an adjuvant in the vaccine construct. This study is the first to present this epitopes-adjuvant combination. The vaccine had acceptable physicochemical characteristics. The vaccine's 3D structure was predicted and validated. The vaccine's binding stability with Toll-like receptor 2 (TLR2) and TLR4 was confirmed using molecular docking and molecular dynamics (MD) simulation. The immune simulation revealed the vaccine's efficacy by increasing the population of B and T cells in response to vaccination. In silico expression in Escherichia coli (E. coli) was also successful.

Canis Familiaris Papillomavirus Type 26: A Novel Papillomavirus of Dogs and the First Canine Papillomavirus within the Omegapapillomavirus Genus.

Domestic dogs are currently recognized as being infected by 25 different canine papillomavirus (CPV) types classified into three genera. A short sequence from a novel CPV type was amplified, along with CPV1, from a papilloma (wart) from the mouth of a dog. The entire 7499 bp genome was amplified, and CPV26 contained putative coding regions that were predicted to produce four early proteins and two late ones. The ORF L1 showed less than 62% similarity for all previously sequenced CPV types but over 69% similarity to multiple Omegapapillomavirus types from a variety of Caniform species including the giant panda, Weddel seal, and polar bear. Phylogenetic analysis confirmed CPV26 clusters within the Omegapapillomavirus genus. Specific primers were used to investigate the presence of CPV26 DNA within a series of 37 canine proliferative lesions. CPV26 DNA was amplified from one lesion, a cutaneous papilloma that also contained CPV6. This is the first time a PV type within the Omegapapillomavirus genus has been detected in a non-domestic species and this provides evidence that the omegapapillomaviruses infected a common ancestor of, and then co-evolved with, the Caniform species. Whether CPV26 causes disease is uncertain, but the absence of an E7 protein may suggest low pathogenicity.

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

Immunoinformatics Design and In Vivo Immunogenicity Evaluation of a Conserved CTL Multi-Epitope Vaccine Targeting HPV16 E5, E6, and E7 Proteins.

Human papillomavirus type 16 (HPV16) infection is responsible for more than 50% of global cervical cancer cases. The development of a vaccine based on cytotoxic T-lymphocyte (CTL) epitopes is a promising strategy for eliminating pre-existing HPV infections and treating patients with cervical cancer. In this study, an immunoinformatics approach was used to predict HLA-I-restricted CTL epitopes in HPV16 E5, E6, and E7 proteins, and a set of conserved CTL epitopes co-restricted by human/murine MHCs was screened and characterized, with the set containing three E5, four E6, and four E7 epitopes. Subsequently, the immunogenicity of the epitope combination was assessed in mice, and the anti-tumor effects of the multi-epitope peptide vaccine E5E6E7pep11 and the recombinant protein vaccine CTB-Epi11E567 were evaluated in the TC-1 mouse tumor model. The results demonstrated that mixed epitope peptides could induce antigen-specific IFN-γ secretion in mice. Prophylactic immunization with E5E6E7pep11 and CTB-Epi11E567 was found to provide 100% protection against tumor growth in mice. Moreover, both types of the multi-epitope vaccine significantly inhibited tumor growth and prolonged mouse survival. In conclusion, in this study, a multi-epitope vaccine targeting HPV16 E5, E6, and E7 proteins was successfully designed and evaluated, demonstrating potential immunogenicity and anti-tumor effects and providing a promising strategy for immunotherapy against HPV-associated tumors.

Antibodies against high-risk human papillomavirus proteins as markers for noncervical HPV-related cancers in a Black South African population, according to HIV status.

Human papillomavirus (HPV) proteins may elicit antibody responses in the process toward HPV-related malignancy. However, HPV seroepidemiology in noncervical HPV-related cancers remains poorly understood, particularly in populations with a high prevalence of human immunodeficiency virus (HIV). Using a glutathione S-transferase-based multiplex serology assay, antibodies against E6, E7 and L1 proteins of HPV16 and HPV18 were measured in sera of 535 cases of noncervical HPV-related cancers (anal (n = 104), vulval (n = 211), vaginal (n = 49), penile (n = 37) and oropharyngeal (n = 134)) and 6651 non-infection-related cancer controls, from the Johannesburg Cancer Study that recruited Black South African with newly diagnosed cancer between 1995 and 2016. Logistic and Poisson regression models were used to calculate adjusted odds ratios (aOR) and prevalence ratios (aPR) and 95% confidence intervals (CI) in cases versus controls. HPV16 E6 was more strongly associated with noncervical HPV-related cancers than HPV16 L1 or E7, or HPV18 proteins: anal (females (HPV16 E6 aOR = 11.50;95%CI:6.0-22.2), males (aOR = 10.12;95%CI:4.9-20.8), vulval (aOR = 11.69;95%CI:7.9-17.2), vaginal (aOR = 10.26;95%CI:5.0-21), penile (aOR = 18.95;95%CI:8.9-40), and oropharyngeal (females (aOR = 8.95;95%CI:2.9-27.5), males (aOR = 3.49;95%CI:1.8-7.0)) cancers. HPV16-E6 seropositivity ranged from 24.0% to 35.1% in anal, vulval, vaginal and penile cancer but was significantly lower (11.2%) in oropharyngeal cancer. After adjustment for HIV, prevalence of which increased from 22.2% in 1995-2005 to 54.1% in 2010-2016, HPV16 E6 seropositivity increased by period of diagnosis (aPR for 2010-2016 vs. 1995-2006 = 1.84;95%CI:1.1-3.0). Assuming HPV16 E6 seroprevalence reflects HPV attributable fraction, the proportion of certain noncervical-HPV-related cancers caused by HPV is increasing over time in South Africa. This is expected to be driven by the increasing influence of HIV.

Designing a Novel Multiepitope Vaccine from the Human Papilloma Virus E1 and E2 Proteins for Indonesia with Immunoinformatics and Molecular Dynamics Approaches.

One of the deadliest malignant cancer in women globally is cervical cancer. Specifically, cervical cancer is the second most common type of cancer in Indonesia. The main infectious agent of cervical cancer is the human papilloma virus (HPV). Although licensed prophylactic vaccines are available, cervical cancer cases are on the rise. Therapy using multiepitope-based vaccines is a very promising therapy for cervical cancer. This study aimed to develop a multiepitope vaccine based on the E1 and E2 proteins of HPV 16, 18, 45, and 52 using in silico. In this study, we develop a novel multiepitope vaccine candidate using an immunoinformatic approach. We predicted the epitopes of the cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) and evaluated their immunogenic properties. Population coverage analysis of qualified epitopes was conducted to determine the successful use of the vaccine worldwide. The epitopes were constructed into a multiepitope vaccine by using AAY linkers between the CTL epitopes and GPGPG linkers between the HTL epitopes. The tertiary structure of the multiepitope vaccine was modeled with AlphaFold and was evaluated by Prosa-web. The results of vaccine construction were analyzed for B-cell epitope prediction, molecular docking with Toll like receptor-4 (TLR4), and molecular dynamics simulation. The results of epitope prediction obtained 4 CTL epitopes and 7 HTL epitopes that are eligible for construction of multiepitope vaccines. Prediction of the physicochemical properties of multiepitope vaccines obtained good results for recombinant protein production. The interaction showed that the interaction of the multiepitope vaccine-TLR4 complex is stable based on the binding free energy value -106.5 kcal/mol. The results of the immune response simulation show that multiepitope vaccine candidates could activate the adaptive and humoral immune systems and generate long-term B-cell memory. According to these results, the development of a multiepitope vaccine with a reverse vaccinology approach is a breakthrough to develop potential cervical cancer therapeutic vaccines.

Therapeutic Effect Analysis of Cervical Cancer Vaccine

Cervical cancer is a very widespread carcinogenic condition that poses a significant threat to women's health. The primary cause of this condition is mostly linked to persistent infection with high-risk strains of human papillomavirus (HPV), which are recognized for their ability to promote the development of cancer. Due to HPV infection, both therapeutic and preventative HPV vaccinations have been developed. Therapeutic vaccines can eradicate viruses and virus-infected cells by stimulating cellular immunity. Notwithstanding the availability of preventive HPV vaccinations, HPV screening, surgery, radiotherapy, and chemotherapy, the therapeutic effect on cervical cancer patients is limited. Therefore, therapeutic vaccines have emerged as a new treatment option. The cancer-causing proteins E6 and E7, which are consistently detected in cervical cancer and precancerous lesions, are crucial in the progression and sustenance of cervical cancer. Consequently, these cancer-causing proteins present an encouraging opportunity for the research and creation of therapeutic cervical cancer vaccines. The multiple therapeutic vaccines against cervical cancer will be briefly discussed in this article, including their mechanisms, development methods, and clinical effects.

Abstract 4719: Modulation of HR-HPV viral protein E7 by SHetA2 in cervical cancer

Abstract Introduction: The purpose of this study is to determine the effect of the chemotherapeutic sulfur heteroarotinoid A2 (SHetA2) on high-risk human papillomavirus (HR-HPV) proteins and the modulation of cell survival and proliferation in cervical cancer. Cervical cancer is caused by persistent HR-HPV infection. HR-HPV tumorigenesis is driven by its early 6 and 7 (E6, E7) oncoproteins, which increase cell proliferation and survival. Cellular pathways regulated by E6 and E7 are counter-regulated by the investigational new drug SHetA2, which is currently in phase 1 clinical trial for advanced and recurrent cancers. SHetA2 disrupts complexes of 70-kDa heat shock protein (HSP70) family members, hsc70, GRP78, and mortalin, with their client proteins. We hypothesized that SHetA2 reduces E6 and E7 levels and their binding to HSP70 chaperones in association with the reduction of cervical cancer cells and tumor growth. Methods: SHetA2 effects on specific genes and proteins in HR-HPV-positive cervical cancer cell lines and xenograft tumors were assessed by western blot, quantitative polymerase chain reaction, immunofluorescence ligation assays, and coimmunoprecipitation assays. Results: SHetA2 significantly reduced E6 and E7 mRNA, and protein levels of E7 and the E7-regulated p16, but not E6 in cervical cancer cells and xenograft tumors. MG132 inhibition of proteasome function attenuated the E7 reduction. HSP70/hsc70 proteins bound E7 and SHetA2 disrupted these complexes. Conclusions/Implications: SHetA2 reduces E7 proteins in cervical cancer cells through a mechanism that involves proteasomal degradation in association with disruption of HSP70/E7 complexes, p16 reduction, and decreased cell and tumor growth. This is the first demonstration of HSP70/E7 complexes. SHetA2-induced release of E7 from HSP70s could be causing increased susceptibility of E7 to proteasomal degradation. This data supports the development of SHetA2 and HSP70 inhibitors for cervical cancer. Citation Format: Justin Garland, Showket Hussain, Doris M. Benbrook. Modulation of HR-HPV viral protein E7 by SHetA2 in cervical cancer [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 4719.

Abstract 2471: State-of-the-art Specific T cell Activating Modulator on ePENDY platform (STAM.eP) activates specific CD8+ T cell by stimulating TCR signal

Abstract After decades of research, therapeutic cancer vaccine approach now sheds light on developing efficient cancer therapy treating patients who have resistance to other cancer immunotherapy. However, cancer vaccine approach using conventional modalities such as protein, peptide, and mRNA is considered a powerful cancer treatment only when the patients have healthy immune systems. In addition, dendritic cell (DC) cancer vaccine can be a great alternative therapy, but manufacture of DC vaccines in vitro is a labor-intensive, time- and money-consuming procedure. Here, we developed Specific T cell Activating Modulator (STAM) on our engineered IgM backbone, ePENDY (engineered PENtamer boDY), with high avidity based on multivalent binding sites to overcome the disadvantages of other therapeutic cancer vaccines. STAM on ePENDY (STAM.eP) consists of ten peptide-loaded-HLAs linked to ePENDY to simulate priming signal of CD8+ T cell by TCR cluster and a costimulatory signal molecule is linked to J chain of ePENDY to prevent CD8+ T cell from becoming anergic status. With these features, STAM.eP mimics cognately licensed DC that can present tumor peptide-loaded MHC class I molecule to CD8+ T cell and promote its priming. Thus, one of the greatest advantages of STAM.eP is to be a potential treatment for immunosuppressive cancer patients with DCs that do not respond appropriately. Another advantage is that any tumor specific antigen (TSA), including from intracellular proteins, can be applied to STAM.eP concept to activate specific CD8+ T cells for targeting cancer cells. In this study, we used Human Papillomavirus (HPV) E7-derived peptide which is an oncoprotein associated with cervical and head/neck cancers. We observed that HPV E7-derived peptide loaded STAM.eP, named IMB-401, increases antigen-specific CD8+ T cell population and shows cytotoxicity activity against cancer cells expressing HPV E7 protein in vitro test model. In vivo test model, it was also observed that population of specific CD8+ T cells and memory T cells were increased in the blood and spleens of transgenic mice treated with IMB-401. Furthermore, we confirmed that IMB-401 shows anti-tumor efficacy due to an increase of antigen-specific CD8+ T cell in vivo humanized mouse model. These results suggest that IMB-401 can be a new therapeutic cancer vaccine technology that replaces DC function and is expected to have robust anti-tumor activity in combination with immune checkpoint inhibitors. Citation Format: Kyung-gi Hyun, Sungmuk Kang, Yongjun Kang, Jihye Koo, Suho Park, Sunghyun Byun, Sunghyun Yoon, Jaebeom Lee, Hong Jai Lee, Hong Kyu Lee, Eunyoung Cho, Chungmin Lee, Kyung-Chul Choi, Gyongsik Ha. State-of-the-art Specific T cell Activating Modulator on ePENDY platform (STAM.eP) activates specific CD8+ T cell by stimulating TCR signal [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 2471.

Abstract 1649: Large scale multi-omics concordance study of cyclin E1 identifies high expressing tumors lacking CCNE1 gene alterations across multiple tumor indications

Abstract Cyclin E1 is a key regulator of the G1/S transition and is thought to cause excessive entry into the cell cycle. CCNE1 is amplified in a range of tumor indications, and its amplification is associated with poorer prognosis and resistance to chemotherapy. Cyclin E1 is also being investigated as a potential biomarker for inhibitors targeting the replication stress response. Understanding the best way to quantify cyclin E1 overexpression is fundamental to support patient selection approaches for these inhibitors. In many cases, gene amplification correlates with increased cyclin E1 protein expression; however, there are a proportion of high-grade serous ovarian cancer and basal-like breast cancer cases where cyclin E1 protein overexpression does not correlate with gene amplification. In this study, we investigate if this phenomenon is observed in other cancers and some of the potential mechanisms causing cyclin E1 overexpression. We used a multi-omics approach to assess cyclin E1 expression in 1417 formalin-fixed paraffin-embedded (FFPE) patient tumor resections across 6 indications (SCCHN, Bladder, NSCLC, TNBC, Ovarian &amp; SCLC). We used copy number variation (CNV) determined by next generation sequencing (NGS), NanoString nCounter RNA analysis and protein expression by immunohistochemistry (IHC). 830 were evaluable for all 3 methods and demonstrated CCNE1 gene amplification by NGS (CNV normalized log2 ratio ≥1.5) in 3.3% cases. Using a threshold of mRNA z-score &amp;gt;2, 17% cases had high CCNE1 mRNA and 29% had high cyclin E1 protein (H-score ≥100). 15.7% (130/830) cases had high protein expression without gene amplification or gain, crucially this was found across all 6 indications (n=20/102 SCCHN, n=29/166 Bladder, n=34/232 NSCLC, n=15/76 SCLC, n=15/143 TNBC &amp; n=17/118 Ovarian). Impaired protein degradation has been proposed as a mechanism that may result in high Cyclin E1 expression. From a panel of 1600 genes, we assessed the relationship between cyclin E1 and mRNA expression of 57 genes involved in the ubiquitin-proteasome pathway in 1239 samples across the 6 indications. We assessed mutations which could impair ubiquitination in 915 samples across the 6 indications. We also assessed the methylation status of 893 genes associated with ubiquitin ligases or deubiquitinases in a cohort of 170 TNBC samples. Further investigation into other mechanisms causing cyclin E1 overexpression is required. IHC provides an ideal platform to build our understanding of cyclin E1 expression spatially. Our results indicate that assessing Cyclin E1 protein overexpression instead of CCNE1 gene amplification could greatly broaden the population of patients who could benefit from replication stress response kinase inhibitors, as cyclin E1 overexpression correlates with genomic instability and replication stress. Citation Format: Sophie E. Willis, Gemma N. Jones, Shaan Gill, Emma V. Jones, Amelia Raymond, Barrett Nuttall, Sophie Kirschner, Sakshi Gulati, Paola Marco-Casanova, Benedetta Lombardi, David Jenkins, Felicia S. Ng, James Hadfield, Paul Waring, Helen K. Angell, Heike Laman. Large scale multi-omics concordance study of cyclin E1 identifies high expressing tumors lacking CCNE1 gene alterations across multiple tumor indications [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 1649.