HPV16 E7 Protein Research Articles

Design, Synthesis, Characterization, Molecular Docking Studies, In vitro and In vivo Cervical Cancer Activity of Novel N-Substituted Pyrazole Derivatives

The aim of this study is to design, synthesize, characterize few novel N-substituted pyrazole derivatives and evaluated for their anticancer capabilities; these compounds were selected based on their ability to inhibit the HPV E6 protein. The IR, 1H & 13C NMR, mass spectral and elemental analysis were used to determine the structural details of the newly synthesized substances. The molecular docking was performed using Auto Dock Vina, and the protein-ligand interaction was analyzed using Discovery Studio. The sulforhodamine B (SRB) assay was used to determine the in vitro anticancer activity against the HeLa cell line. Body weight analysis, mean survival time and % increase in life span approaches were used to assess in vivo anticancer effectiveness in Swiss albino mice bearing Dalton’s Lymphoma Ascites (DAL) tumor model. Synthesized compounds have excellent docking energies, according to docking experiments. Compounds I, II and III were selected for anticancer activity in vivo DAL-bearing mice based on the findings of their in vitro and SRB assays exhibiting the antiproliferative activity. Pyrazole derivatives, compounds I and III, demonstrated significant potential as anticancer agents.

Spectroscopic, DFT and molecular docking studies of novel diosgenin NSAID conjugates and their in vitro evaluation as potential anti-cancer agents against SiHa cells

In the current study, diosgenin-etodolac and diosgenin-naproxen conjugates have been synthesized using Steglich reaction. The synthesized compound 2 and 3 were purified using column chromatography and characterized with the help of modern spectroscopic techniques like 1H, 13C NMR, FT-IR, UV-Visible spectroscopy and mass spectrometry. The geometries of both the compounds were optimized in the ground state by density function theory at the B3LYP/6-31G(d,p) level. These synthesized compounds were evaluated in vitro for their anti-cancer activity against SiHa cells which demonstrated an increased apoptotic activity in comparison to the parent compound i.e. diosgenin. Molecular docking studies were carried out to investigate the inhibitory action of steroidal derivatives against the HPV16 E7 (PDBID- 2B9D) and HPV18 E2 (PDBID- 1F9F) proteins. The result of molecular docking revealed good interactions between compound 2 and 3 with the selected proteins. The computational analysis data and experimental data were in conformation with each other. Molecular simulation was performed for 50ns to access the conformational stability and fluctuation of protein ligand complexes during the simulation.

Magnetic nanoparticles and quantum dots coupled immuno nano fluorescence assay for visual detection of HPV16-induced cervical cancer cells from cytology/biopsy samples

Biopsy-based histopathology and immunohistochemistry for cervical cancer detection are costly, time-consuming, and require expert personnel for data interpretation. We developed a simple magnetic nanoparticle (MNPs) and quantum dots (QD) coupled immuno nano fluorescence assay (MNPQDCINFA) for visual detection of HPV16-induced cervical cancer cells under UV light from cytology/biopsy samples exploiting host cancer cells expressing viral E7 protein as a biomarker. The E7 domain-specific polyclonal antibodies were generated against the 1–44 amino acid N-terminal (anti-domainN antibody) and 48–98 amino acid C-terminal domain (anti-domainC antibody). These antibodies were bioconjugated with nonfluorescent MNPs (60 % efficiency) and fluorescent QDs (66 % efficiency) to generate capturing (MNPs-anti-domainN antibody) and detecting (QDs-anti-domainC antibody) nano-complex, respectively. Assay conditions, such as concentration of capturing (20 μM) and detecting (50 nM) antibody nano-complexes and incubation duration (30 min), were standardized. The analytical sensitivity using pure HPV16 E7 protein was recorded up to 200 ng with very high specificity to differentiate from other HPV strains E7 proteins. The diagnostic performance characteristics with cytology samples showed 100 % sensitivity and specificity compared to immunofluorescence and biopsy-based histopathology analysis. The present invention can be effectively used for a quick, disposable, rapid cervical cancer cell detection system as an alternate test for immunofluorescence and histopathology.

No genetic causal association between human papillomavirus and lung cancer risk: a bidirectional two-sample Mendelian randomization analysis

IntroductionSeveral observational or retrospective studies have previously been conducted to explore the possible association between lung cancer and human papillomavirus (HPV) infection. However, there may be inconsistencies in the data and conclusions due to differences in study design and HPV testing methods. There are currently no studies that provide conclusive evidence to support the involvement of HPV in the occurrence and development of lung cancer. Therefore, the relationship between HPV and lung cancer remains controversial and uncertain. This study aimed to explore whether HPV infection is causally related to lung cancer risk by systematically performing a two-way Two-Sample Mendelian Randomization (TSMR) analysis.MethodsIn the International Lung Cancer Consortium (ILCCO) genome-wide association study dataset, we included 11,348 lung cancer (LUCA) cases, including 3275 squamous cell carcinoma (LUSC) cases, 3442 adenocarcinoma (LUAD) cases, and 15,861 cases of control. Using genetic variants associated with the HPV E7 protein as instrumental variables, we summarized statistics associated with HPV infection in the MRC IEU OpenGWAS database, which included the HPV-16 E7 protein and the HPV-18 E7 protein. Two-sample Mendelian randomization (MR) results are expressed as odds ratios (OR) and 95% confidence intervals (CI).ResultsBased on a comprehensive analysis of genome-wide association study (GWAS) data from public databases, we mainly used inverse-variance weighted (IVW) to estimate causal relationships, while using MR-Egger, weighted median, simple mode, and weighted mode, and other four methods as supplements. Two-sample MR Analysis revealed no causal relationship between exposure factors (HPV-16 E7 protein and HPV-18 E7 protein) and outcome factors (lung cancer (LUCA) and its subtypes squamous cell carcinoma (LUSC) and adenocarcinoma (LUAD)) in forward MR Analysis using the IVW approach.HPV-16 E7 protein and LUCA and its subtypes LUSC and LUAD by IVW method results: [OR] = 1.002; 95% [CI]: 0.961 − 1.045; p = 0.920; [OR] = 1.023; 95% [CI]: 0.966 − 1.084; p = 0.438; [OR] = 0.994; 95% [CI]: 0.927 − 1.066; p = 0.872); HPV-18 E7 protein and LUCA and its subtypes LUSC and LUAD by IVW method results: [OR] = 0.965; 95% [CI]: 0.914 − 1.019; p = 0.197; [OR] = 0.933; 95% [CI]: 0.834 − 1.043; p = 0.222; [OR] = 1.028; 95% [CI]: 0.945 − 1.118; p = 0.524. It was observed through reverse MR that LUCA and its subtypes LUSC and LUAD were used as exposure factors, and HPV infection (HPV-16 E7 protein and HPV-18 E7 protein) was used as the outcome factors, the results of the IVW method are also invalid.LUCA and HPV-16 E7 protein and HPV-18 E7 protein by IVW method results: [OR] = 1.036; 95% [CI]: 0.761 − 1.411; p = 0.82; [OR] = 1.318; 95% [CI]: 0.949 − 1.830; p = 0.099; LUSC and HPV-16 E7 protein and HPV-18 E7 protein by IVW method results: [OR] = 1.123; 95% [CI]0.847 − 1.489; p = 0.421; [OR] = 0.931; 95% [CI]: 0.660 − 1.313; p = 0.682; LUAD and HPV-16 E7 protein and HPV-18 E7 protein by IVW method results: [OR] = 1.182; 95% [CI] 0.983 − 1.421; p = 0.075; [OR] = 1.017; 95% [CI]: 0.817 − 1.267; p = 0.877.Our results indicate that there is no causal relationship between genetically predicted HPV infection and LUCA and its subtypes LUSC and LUAD. In addition, in the reverse MR analysis, we did not observe a significant causal relationship between LUCA and its subtypes LUSC and LUAD on HPV infection.ConclusionsOur findings do not support a genetic association between HPV infection and lung cancer.

Cationic Liposomes Carrying HPV16 E6-siRNA Inhibit the Proliferation, Migration, and Invasion of Cervical Cancer Cells.

The E6 and E7 oncoproteins of high-risk types of human papillomavirus (HR-HPV) are crucial for the development of cervical cancer (CC). Small interfering RNAs (siRNAs) are explored as novel therapies that silence these oncogenes, but their clinical use is hampered by inefficient delivery systems. Modification (pegylation) with polyethylene glycol (PEG) of liposomal siRNA complexes (siRNA lipoplexes) may improve systemic stability. We studied the effect of siRNA targeting HPV16 E6, delivered via cationic liposomes (lipoplexes), on cellular processes in a cervical carcinoma cell line (CaSki) and its potential therapeutic use. Lipoplexes-PEG-HPV16 E6, composed of DOTAP, Chol, DOPE, and DSPE-PEG2000 were prepared. The results showed that pegylation (5% DSPE-PEG2000) provided stable siRNA protection, with a particle size of 86.42 ± 3.19 nm and a complexation efficiency of over 80%; the siRNA remained stable for 30 days. These lipoplexes significantly reduced HPV16 E6 protein levels and restored p53 protein expression, inhibiting carcinogenic processes such as proliferation by 25.74%, migration (95.7%), and cell invasion (97.8%) at concentrations of 20 nM, 200 nM, and 80 nM, respectively. In conclusion, cationic lipoplexes-PEG-HPV16 E6 show promise as siRNA carriers for silencing HPV16 E6 in CC.

In silico Analysis of Natural Inhibitors against HPV E6 Protein.

Drug re-purposing is one of the cost-effective methods to establish novel therapeutics against many diseases. Established natural products are collected from databases and used to potentially screen them against HPV E6 protein, a critical viral protein. This study aims to design potential small molecule inhibitors against HPV E6 protein using structure-based approaches. Ten natural anti-cancerous compounds (Apigenin, Baicalein, Baicalin, Ponicidin, Oridonin, Lovastatin, Triterpenoid, Narirutin, Rosmarinic Acid, and Xanthone) were selected by review of the literature. These compounds were screened using Lipinski Rule of Five. Out of ten compounds, seven were found to satisfy Rule of five. Docking of these seven compounds was carried out using AutoDock software and corresponding Molecular Dynamics Simulations were performed by GROMACS. Among the seven compounds docked with the E6 target protein, six compounds showed lesser binding energy than the reference compound, Luteolin. The three-dimensional structures of E6 protein and the corresponding ligand complexes were visualised and analysed using PyMOL whereas the two-dimensional images of protein-ligand interactions were obtained by LigPlot⁺ software to study the specific interactions. ADME analysis using SwissADME software revealed that all the compounds except Rosmarinic acid have good gastrointestinal absorption and solubility characteristics while Xanthone and Lovastatin showed blood brain barrier penetration properties. Considering the binding energy and ADME analysis, Apigenin and Ponicidin are found to be most suitable for de novo</i> designing of potential inhibitors against the HPV16 E6 protein. Further, synthesis and characterization of these potential HPV16 E6 inhibitors will be carried out and their functional evaluation using cell culture-based assays will be undertaken.

A phase 1 dose-escalation and expansion study of CUE-101, given as monotherapy and in combination with pembrolizumab, in patients with recurrent/metastatic HPV16+ head and neck squamous cell cancer (R/M HNSCC).

6004 Background: Immuno-STATs are modular T cell engagers engineered to selectively activate tumor-antigen specific CD8+ T cells via targeted delivery of cytokines. CUE-101, the first Immuno-STAT in clinical trials, is composed of a human leukocyte antigen (HLA) complex, HLA-A*0201, a peptide epitope derived from the HPV16 E7 protein and 4 molecules of attenuated human interleukin-2 (IL-2), to bind, expand, and activate HPV16-specific CD8+ T cells for the treatment of HPV16+ HNSCC. Methods: CUE-101-01 is an ongoing first-in-human study in HLA-A*0201 patients with HPV16+ R/M HNSCC. Escalating doses of CUE-101 monotherapy (0.06 mg/kg to 8 mg/kg) were evaluated in R/M HNSCC refractory to ≥ 1 platinum or checkpoint inhibitor (CPI) based therapy, alone or combined with pembrolizumab (1 mg/kg to 4 mg/kg + 200 mg pembrolizumab) in the first line treatment of PD-L1+ R/M HNSCCs. Enrollment at the recommended phase 2 dose (RP2D) was expanded. Therapy was administered every 3 weeks (Q3W) until disease progression or intolerable toxicity. Safety, PK/PD, and antitumor activity were assessed. Results: Enrollment in both monotherapy and combination cohorts is now completed (N=80 patients, 49 in monotherapy and 31 CUE-101 plus pembrolizumab). Following dose escalation, 4 mg/kg Q3W of CUE-101 was selected for RP2D for both monotherapy and pembrolizumab combination cohorts. At data cut-off, adverse events (AEs) have been manageable and 92% grade ≤2. The most frequent grade 3 AEs reported include lymphocyte count decreased (7.6%), anemia (6.3%), decreased appetite (5.1%) and infusion-related reactions (5.1%). In combination with pembrolizumab no unanticipated significant safety concerns have emerged. Exposure-dependent PD effects of CUE-101 are consistent with IL-2 pharmacology and indicate preferential expansion of CUE-101 on E7-specific T cells. Among the 19 evaluable patients treated with the RP2D of CUE-101 plus pembrolizumab, an ORR of 47% (1 CR, 8 PRs), a Disease Control Rate (ORR + durable SDs) of 74%, and mPFS of 5.8 months [95% CI 2.56; NA] were observed. A median OS has not been reached. Of the 9 patients with confirmed objective responses, all achieved &gt;99% reduction in HPV16 cfDNA in plasma during their treatment course. Among the 19 evaluable monotherapy RP2D patients, 1 PR and 6 durable SD (SD ≥ 12 weeks) and mOS of 20.8 months [95% CI 11.0; NA] were observed. Conclusions: An ORR of 47% and a mPFS of 5.8 months were observed in R/M HNSCC patients treated with CUE-101 4 mg/kg + pembrolizumab as 1L therapy. A median OS of 20.8 was observed in patients treated with CUE-101 monotherapy as post-platinum/CPI therapy. CUE-101 continues to demonstrate safety, tolerability and meaningful clinical benefit in patients with HPV16+ R/M HNSCC. Clinical trial information: NCT03978689 .

Characterization of novel compounds that directly bind to and inactivate the HPV E6 protein for treatment of HPV-associated cervical and oropharyngeal cancers.

e15122 Background: The human papillomavirus (HPV) E6 protein is necessary for viral genome replication and is always expressed in HPV-induced malignancies. We performed in silico screening of chemical libraries for molecules that bind to a pocket on the cancer associated HPV-16 E6 protein. We focused on the protein-protein interaction of E6 with one of its cellular binding partners, the ubiquitin ligase E6AP. In a trimolecular complex with E6, E6AP ubiquitinates p53, leading to its destruction by the proteasome. We designed, synthesized, and characterized &gt;300 compounds that 1) bind to the E6AP interaction surface of the HPV-16 E6 protein; 2) are armed with a ‘warhead’ to make an irreversible bond with a specific cysteine in this HPV-16 E6 binding pocket; 3) and restore levels of p53 and selectively decrease viability of HPV-16 expressing tumor derived cells. Methods: E6 inhibitor optimization was guided by in silico docking strategies, co-crystallization, and medicinal chemistry, followed by biochemical and cellular assays to iteratively design and synthesize molecules with increased E6 inhibitory activities. Results: We used whole protein mass spectrometry to interrogate covalent bonding to E6, showing irreversible binding to the cysteine in the targeted E6 binding pocket without the other 13 cysteines in E6, with Ki in the range of seconds. Biolayer interferometry and fluorescence polarimetry measured blockade of E6 with E6AP, with IC50 in the range of 400-600 nM. To establish in cell inactivation of E6, human cervical cancer derived SiHa cells that express HPV-16 E6 were incubated with compounds, showing dose dependent increase of p53 levels up to 8-fold after 24 hrs, an effect that was not observed in the HPV-16- cell line RPE-1 expressing wild-type p53. Viability of two cervical (SiHa, CaSki) and two oropharyngeal HPV-16+ cancer cell lines (UM-SC-47, UM-SCC-104) was reduced with an IC50 of ~2-3 µM, with an IC50&gt;30µM in RPE-1 cells and telomerase immortalized human keratinocytes. These in vitro and cell-based experiments revealed dependence on the cysteine residue in the E6AP binding pocket of E6. For in vivo proof-of-concept studies, we developed mouse xenograft models, performed by engrafting SiHa, UM-SC-47 and UM-SCC-104 cancer derived cells subcutaneously into the flank of immunodeficient mice. Lead compounds were administered by intraperitoneal injection and demonstrated nearly complete inhibition (&gt; 70% tumor reduction) in HPV-16 tumor growth compared to vehicle. Anti-tumor activity was not observed with the HPV-negative tumor cell line C33A. Conclusions: HPV-16 is identified in about 50% of all cervical cancers and the majority of head and neck malignancies. Inactivation of the HPV E6 protein represents the opportunity for a novel molecular targeted approach for medical treatment of HPV-16 expressing cancers of the cervix and oropharynx.

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.

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.

Adenosine Increases the Immunosuppressive Capacity of Cervical Cancer Cells by Increasing PD-L1 Expression and TGF-β Production through Its Interaction with A2AR/A2BR.

The present study provides evidence showing that adenosine (Ado) increases the expression of programmed death ligand 1 (PD-L1) in cervical cancer (CeCa) cells by interacting with A2AR/A2BR and that TGF-β1 acts in an autocrine manner to induce PD-L1 expression, enhancing the immunosuppressive effects of CeCa cells on activated T lymphocytes (ATLs) and CD8+ cytotoxic T lymphocytes (CTLs) specific for antigenic peptides derived from E6 and E7 proteins of HPV-16. Interestingly, the addition of the antagonists ZM241385 and MRS1754, which are specific for A2AR and A2BR, respectively, or SB-505124, which is a selective TGF-β1 receptor inhibitor, to CeCa cell cultures significantly inhibited PD-L1 expression. In addition, supernatants from CeCa cells that were treated with Ado (CeCa-Ado Sup) increased the expression of PD-1, TGF-β1, and IL-10 and decreased the expression of IFN-γ in ATLs. Interestingly, the addition of an anti-TGF-β neutralizing antibody strongly reversed the effect of CeCa-Ado Sup on PD-1 expression in ATLs. These results strongly suggest the presence of a feedback mechanism that involves the adenosinergic pathway, the production of TGF-β1, and the upregulation of PD-L1 expression in CeCa cells that suppresses the antitumor response of CTLs. The findings of this study suggest that this pathway may be clinically important and may be a therapeutic target.

Structural analysis of PTPN21 reveals a dominant-negative effect of the FERM domain on its phosphatase activity.

PTPN21 belongs to the four-point-one, ezrin, radixin, moesin (FERM) domain-containing protein tyrosine phosphatases (PTP) and plays important roles in cytoskeleton-associated cellular processes like cell adhesion, motility, and cargo transport. Because of the presence of a WPE loop instead of a WPD loop in the phosphatase domain, it is often considered to lack phosphatase activity. However, many of PTPN21's biological functions require its catalytic activity. To reconcile these findings, we have determined the structures of individual PTPN21 FERM, PTP domains, and a complex between FERM-PTP. Combined with biochemical analysis, we have found that PTPN21 PTP is weakly active and is autoinhibited by association with its FERM domain. Disruption of FERM-PTP interaction results in enhanced ERK activation. The oncogenic HPV18 E7 protein binds to PTP at the same location as PTPN21 FERM, indicating that it may act by displacing the FERM domain from PTP. Our results provide mechanistic insight into PTPN21 and benefit functional studies of PTPN21-mediated processes.

HPV16 E7 protein antagonizes TNF-α-induced apoptosis of cervical cancer cells via Daxx/JNK pathway

Human papillomavirus (HPV) E7 protein as an important viral factor was involved in the progression of cervical cancer by mediating the cellular signaling pathways. Daxx (Death domain-associated protein) can interact with a variety of proteins to affect the viral infection process. However, the interaction and its related function between HPV16 E7 and Daxx have not been adequately investigated. Here, it was found that HPV16 E7 can interact with Daxx in HeLa or C33A cells by co-immunoprecipitation. HPV16 E7 protein treatment can up-regulate Daxx protein expression, while the interference in Daxx expression and the agonists for JNK can both reduce the antagonistic effects of HPV16 E7 on TNF-α-induced apoptosis, suggesting that Daxx/JNK pathway may be involved in the anti-apoptotic activity of HPV16 E7.

Investigating the Potential Role of IKZF3 in HPV-Associated Carcinogenesis

HPVs cause approximately 5% of all human cancers. They encode two viral transforming proteins, E6 and E7, that are consistently expressed in these cancers. The HPV E6 and E7 proteins contribute to carcinogenesis by binding and functionally compromising multiple cellular regulatory proteins, most prominently the p53 and retinoblastoma (pRB) tumor suppressors, respectively. To discover additional cellular regulatory circuits that are dysregulated by E6 and E7 expression, we performed RNA sequencing analyses of HPV16 E6 and E7 expressing primary human genital epithelial cells. These experiments revealed that the Ikaros Zinc Finger 3 (IKZF3) transcription factor is highly upregulated in HPV16 E6/E7 expressing keratinocytes as compared to control keratinocytes. IKZF3 plays a key role in lymphocyte development, and when over-expressed or mutated, contributes to hematopoietic malignancies. IKZF3 is expressed at a very low level in normal epithelial cells, but the expression is upregulated in solid tumors, including invasive breast, cervical, and lung cancers. IKZF3 expression in these tumors causes upregulation of lymphocyte-specific genes, conferring lymphocyte-like behavior referred to as "lymphocyte mimicry," which presumably enables cancer cell survival in the bloodstream, thus potentially contributing to cancer metastasis. There is an FDA-approved drug, lenalidomide, that targets IKZF3 for degradation. Hence, we set out to determine whether IKZF3 might contribute to cervical carcinogenesis. We treated HPV16-positive SiHa and CaSki cervical cancer cells with 10 uM of lenalidomide for 12 hours. Cells were treated with equal amounts of DMSO as a negative control. Then, IKZF3 and E7 mRNA expression were determined using qRT-PCR after 12 hours of treatment. Gene expression was analyzed relative to GAPDH, and the results were analyzed using 2-way ANOVAs with Dunnett's correction for multiple comparisons. Western Blot experiments were performed to examine IKZF3, pRB, E7, and p53 (to indirectly assess E6) protein expression levels after 12 hours of lenalidomide treatment. Actin protein was used as a loading control. Each of these experiments were performed 3 times, qRT-PCR experiments were each performed in triplicate, and the results were graphed using a scientific 2-D graphing and statistics software. Although Western Blot showed that IKZF3 protein levels were significantly decreased by lenalidomide treatment, there was no change in pRB, E7, and p53 protein expression upon IKZF3 depletion. Similarly, there was no decrease in E7 mRNA expression. We showed by western blotting that targeting IKZF3 for proteasome-mediated degradation with lenalidomide effectively decreased the steady-state levels of IKZF3. However, we did not consistently observe an associated decrease in HPV gene expression. We are currently analyzing the transcriptome changes that IKZF3 depletion causes in cervical carcinoma cells to determine whether this may affect anoikis resistance.

Focal Adhesion Kinase Binds to the HPV E2 Protein to Regulate Initial Replication after Infection.

Human papillomaviruses are small DNA tumor viruses that infect cutaneous and mucosal epithelia. The viral lifecycle is linked to the differentiation status of the epithelium. During initial viral infection, the genomes replicate at a low copy number but the mechanism(s) the virus uses to control the copy number during this stage is not known. In this study, we demonstrate that the tyrosine kinase focal adhesion kinase (FAK) binds to and phosphorylates the high-risk viral E2 protein, the key regulator of HPV replication. The depletion of FAK with a specific PROTAC had no effect on viral DNA content in keratinocytes that already maintain HPV-16 and HPV-31 episomes. In contrast, the depletion of FAK significantly increased HPV-16 DNA content in keratinocytes infected with HPV-16 quasiviruses. These data imply that FAK prevents the over-replication of the HPV genome after infection through the interaction and phosphorylation of the E2 protein.

HPV16 E7 oncoprotein test as a triage strategy for HPV16-positive women in cervical cancer screening: long-term follow-up outcome.

Colposcopy is recommended once human papillomavirus (HPV)16/18 infection is detected. However, not all HPV16/18-positive women will necessarily develop cervical lesions. Therefore, this study aimed to investigate the application of quantitative HPV16 E7 oncoprotein detection as a cervical cancer screening method for more efficient screening while minimizing unnecessary colposcopy. E7 oncoprotein (HPV16) was quantitatively detected in cervical exfoliated cells of HPV16-positive women. The levels of HPV16 E7 oncoprotein in different degrees of cervical lesions were compared, and the optimal cut-off value for identifying HSIL+ was determined by receiver operating characteristic (ROC) curve analysis. With a pathological diagnosis as the gold standard, the sensitivity (SEN), specificity (SPE), positive predictive value (PPV), negative predictive value (NPV), and Kappa value were calculated to verify the diagnostic value of the method. Women diagnosed with low-grade squamous intraepithelial lesions (LSIL) and normal women were followed up for 5 years to evaluate the predictive value of HPV16 E7 protein for disease progression/persistent infection. The expression level of HPV16 E7 oncoprotein was positively correlated with the degree of the cervical lesion (r = 0.589, P < 0.01). The area under the ROC curve (AUC) was 0.817 (confidence interval: 0.729-0.904). The cut-off value of E7 oncoprotein for identifying HSIL+ was 8.68 ng/ml. The SEN, SPE, PPV, NPV, and Kappa values of HPV16 E7 oncoprotein for the identification of HSIL+ were 87.1%,70.0%, 87.1%, 70.0%, and 0.571, respectively, which were higher than those of ThinPrep cytology test (TCT). The SEN, SPE, PPV, and NPV of HPV16 E7 oncoprotein in predicting disease progression/persistent infection were 93.75%, 91.30%, 88.24%, and 95.45%, respectively. The quantitative detection of HPV 16 E7 oncoprotein can not only accurately screen cervical lesions but also achieve efficient colposcopy referral. Additionally, HPV16 E7 oncoprotein can accurately predict the progression of cervical lesions and persistent HPV infection.

The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis.

Human papillomavirus (HPV) encoded E7 oncoprotein plays an important role in supporting the viral productive cycle and inducing cancer phenotypes. The ability of E7 to exercise these functions, partly, depends upon its steady-state level. HPV manipulates the host de-ubiquitination pathway to maintain the stability of its viral proteins. In this study, we uncovered that HPV interacts with the host ubiquitin specific protease 7 (USP7), a universal de-ubiquitinating enzyme, leading to the stabilization of E7 oncoprotein. We observed that HPV16E7 complexes with USP7 via the E7-CR3 domain, and this E7-USP7 complex exists predominantly in the nucleus. Our results showed that USP7 stabilizes and prolongs the half-life of HPV16E7 by antagonizing ubiquitination and proteasomal degradation. Consistently, when we inhibited USP7 activity using HBX 19818, HPV16E7 protein level was reduced and its turnover was increased. We also provide evidence that HBX 19818-induced USP7 inhibition can halt HPV-mediated carcinogenesis, including cell proliferation, invasion, migration and transformation. These findings indicate that USP7 plays an essential role in stabilizing E7. The specific and potent inhibitory effects of HBX 19818 on HPV-induced carcinogenesis provide a molecular insight, suggesting the potential of targeting USP7 as a new therapeutic approach for the treatment of HPV-associated cancers.

A phase 1 dose-escalation and expansion study of CUE-101, a novel HPV16 E7-pHLA-IL2-Fc fusion protein, given as monotherapy and in combination with pembrolizumab in patients with recurrent/metastatic HPV16+ head and neck cancer.

6013 Background: Immuno-STATs are modular T cell engagers engineered to selectively activate tumor-antigen specific CD8+ T cells via targeted delivery of cytokines. CUE-101, the first Immuno-STAT in clinical trials, is composed of a human leukocyte antigen (HLA) complex, HLA-A*0201, a peptide epitope derived from the HPV16 E7 protein, and 4 molecules of attenuated human interleukin-2 (IL-2) designed to bind, expand, and activate HPV16-specific CD8+ T cells for the treatment of HPV16+ cancers. Methods: CUE-101-01 is an ongoing first-in-human study in HLA-A*0201 patients with HPV16+ recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). Escalating doses of CUE-101 monotherapy (0.06 mg/kg to 8 mg/kg) and in combination with pembrolizumab (1 mg/kg to 4 mg/kg + 200 mg pembrolizumab) were evaluated, followed by expanded enrollment at the recommended phase 2 dose (RP2D). Patients with R/M HNSCC refractory to ≥ 1 platinum-based or checkpoint inhibitor therapy received CUE-101 monotherapy. Patients with previously untreated PD-L1+ R/M HNSCC received CUE-101 + pembrolizumab as first-line treatment. Therapy was administered every 3 weeks until disease progression or intolerable toxicity. Safety, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity were assessed. Results: As of January 12, 2023, 67 patients were enrolled. A MTD was not established in monotherapy or combination-treated patients; 4 mg/kg of CUE-101, alone or in combination with pembrolizumab, was chosen as the RP2D dose in both cohorts. Monotherapy RP2D enrollment is complete and combination RP2D enrollment is ongoing. Frequent adverse events include fatigue (45%), anemia (34%), chills (27%), infusion related reactions (25%), constipation (22%), lymphopenia (22%) and nausea (22%). CUE-101 PK data demonstrate dose-dependent increases in drug exposure sustained with repeat dosing. PD data demonstrate selective expansion of HPV-16 E711-20-specific CD8+ T cells, sustained increase in NK cells and transient increase in Treg cells. Among 19 evaluable monotherapy RP2D patients, 1 PR and 6 durable SD (SD ≥ 12 weeks) were observed, with mOS of 24.4 months (9.1, NA). Among 12 evaluable RP2D combination patients to date, 5 PRs, and 2 durable SDs have been observed, with 71% (5/7) of patients expressing PD-L1 CPS ≤ 20. Conclusions: CUE-101 facilitates the targeted delivery of high concentrations of IL-2 to relevant tumor-specific CD8+ T cells. We demonstrate safety and tolerability with encouraging PD signals and antitumor activity with monotherapy and in combination with pembrolizumab in HPV+ R/M HNSCC patients. Enrollment continues in the CUE-101 and pembrolizumab combination cohort. Clinical trial information: Clinical trial information: NCT03978689 .

The Role of the p16 and p53 Tumor Suppressor Proteins and Viral HPV16 E6 and E7 Oncoproteins in the Assessment of Survival in Patients with Head and Neck Cancers Associated with Human Papillomavirus Infections.

The role of HPV in the survival prognosis of patients with head and neck squamous cell carcinoma, especially patients with laryngeal squamous cell carcinoma (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC), is still somewhat ambiguous. The present study aimed to explore the significance of tumor suppressor proteins and HPV16 E6 and E7 oncoproteins in the assessment of survival in patients with oropharyngeal squamous cell carcinoma (OPSCC), LSCC, and HPSCC associated with high-risk (HR-) and low-risk (LR-) HPV infections. By utilizing molecular and immunohistochemical investigations of HNSCC samples and patient data, univariate and multivariate survival analyses were conducted. The presence of HPV DNA (LR- and HR-HPV) was associated with a better 5-year OS and DSS for OPSCC and LSCC. The IHC overexpression of HPV16 E6 protein and p16 protein was associated with better survival in the univariate (for OPSCC) and multivariate (OPSCC and HPSCC) survival analyses. The overexpression of p53 was associated with better survival in OPSCC. HPV infection plays a significant role in the tumorigenesis of HNSCC, and the immunohistochemical assessment of HPV16 E6 protein expression should be interpreted as a useful prognostic marker for OPSCC and HPSCC.

Abstract CT045: A randomized phase II trial of TG4001 plus avelumab versus avelumab alone in recurrent/metastatic (R/M) human papilloma virus (HPV)-16 positive anogenital cancers

Abstract Background: Immune checkpoint inhibitors targeting the PD1/PD-L1 axis have made a breakthrough in advanced solid tumors, leading to improved overall survival (OS) in several cancer types. However, only a minority of patients experience durable responses, requiring further development of anti-tumoral immunotherapies to overcome tumor immune escape. We hypothesized that priming the immune system with TG4001, a vaccine using the highly attenuated Modified Vaccinia virus Ankara as vector expressing the HPV16 E6 and E7 proteins (rendered non-oncogenic) and interleukin-2, would increase the clinical benefit associated with PD-L1 blockade in HPV16+ cancer patients. In the single-arm part of the trial evaluating the combination of TG4001 and avelumab in terms of response rate, the overall response rate (ORR) was 22%. Presence of liver metastases had a profound impact on outcome in terms of ORR and Progression Free Survival (PFS). In patients (pts) without liver metastases an ORR of 32% was observed versus 0% in pts with liver metastases, warranting further development of this combination in this patient population (SITC 2020, Abstract ID 793). Methods: We have designed a randomized open-label two-arms trial evaluating the efficacy of the combination of TG4001 and avelumab versus avelumab alone (NCT03260023). Eligible pts have R/M HPV-16 positive anogenital cancer including cervical, vulvar, vaginal, penile, and anal cancers with no more than one prior line of chemotherapy for R/M disease and no previous exposure to cancer immunotherapy. HPV-16 positivity is determined in a central laboratory. Patients are randomly assigned 1:1 to receive either TG4001 plus avelumab or avelumab alone. Randomization is stratified by primary tumor type (cervical, anal, genital). TG4001 is administered subcutaneously (SC) at 5 × 107 plaque forming units (pfu) Q1w for 5 weeks, Q2w until month 6 and Q12w until progressive disease and avelumab intravenously (IV) at 800 mg Q2w until progressive disease. A total of up to 140 pts are currently being recruited into two cohorts (A and B) in France, Spain, and USA. For Cohort A, consisting of pts without liver metastases at Baseline (BL), a 2-stage sample size adjustment design is used with PFS by RECIST 1.1 as primary endpoint. Plans are to accrue 120 patients following Interim Analysis and Independent Data Monitoring Committee (IDMC) review. Cohort B recruits pts with liver metastases at BL. To be eligible, hepatic disease is restricted in terms of extent and number of lesions. A total of 20 pts will be recruited and analyzed for the percentage of early progressors. Secondary endpoints include overall survival, objective response rate according to RECIST1.1, duration of response, disease control rate. Exploratory endpoints comprise analyses of blood-based parameters and tumor-based proteins and RNA expression and their impact on clinical outcomes. Citation Format: Christophe Le Tourneau, Frédéric Rolland, Amaury Daste, Philippe Cassier, Sébastien Salas, Luis Manso Sánchez, Gerardo Colon-Otero, Lauriane Eberst, Olivier Lantz, Ana Lalanne, Annette Tavernaro, Hakim Makhloufi, Kaïdre Bendjama, Maud Brandely, Jean-Pierre Delord. A randomized phase II trial of TG4001 plus avelumab versus avelumab alone in recurrent/metastatic (R/M) human papilloma virus (HPV)-16 positive anogenital cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT045.