E2 Gene Research Articles

Malignancy Associated with Low-Risk HPV6 and HPV11: A Systematic Review and Implications for Cancer Prevention.

High-risk human papillomavirus (HPV) is etiologically related to cervical cancer, other anogenital cancers and oropharyngeal carcinomas. Low-risk HPV, especially HPV6 and HPV11, cause genital warts and laryngeal papillomas. However, the accumulating data suggests that HPV6 and HPV11 may cause malignant lesions at non-cervical anatomic sites. This review aims to estimate the proportions of single and dual HPV6/11 infections in multiple cancers reported in the last 10 years in the Cochrane, Embasa and PubMed databases. Secondly, the genomes of HPV6/11 were compared with the most common high-risk genotype, HPV16, to determine the similarities and differences. A total of 11 articles were selected, including between one and 334 HPV+ cancer patients. The frequencies of single or dual HPV6/11 infections ranged between 0-5.5% for penile and 0-87.5% for laryngeal cancers and were null for vulvar, vaginal and oral cancers. The genomic similarities between HPV6/11 and HPV16 mainly involved the E7 gene, indicating a limited ability to block cell differentiation. The presence of single or dual HPV6/11 infections in variable proportions of penile and laryngeal cancers support the vaccination strategies that cover these genotypes, not only for preventing genital warts but also for cancer prevention. Other risk factors and co-carcinogens are likely to participate in epithelial carcinogenesis associated with low-risk HPV.

Cervical carcinogenesis risk association of HPV33 E6 and E7 genetic variations in Taizhou, Southeast China

BackgroundHuman papillomavirus (HPV) 33 belongs to the Alphapapillomavirus 9 (α-9 HPV) species group, which also contains types 16, 31, 35, 52, 58 and 67. The purpose of this study was to investigate the genetic variations of HPV33 and to explore its carcinogenicity among women in Taizhou, Southeast China.MethodsExfoliated cervical cells were collected for HPV genotyping. Only single HPV33 infection cases were selected, and their E6 and E7 genes were sequenced using the ABI 3730xl sequencer and then analysed using MEGA X.ResultsFrom 2014 to 2020, a total of 185 single HPV33-positive specimens were successfully amplified. We obtained 15 distinct HPV33 E6/E7 variants, which were published in GenBank under accession numbers OQ672665-OQ672679. Phylogenetic analysis revealed that all HPV33 E6/E7 variants belonged to lineage A, of which 75.7% belonged to lineage A1. Compared with CIN1, the proportion of sublineage A1 in CIN2/3 was higher, but there was no significant difference (76.5% vs. 80.6%, P > 0.05). Altogether, 20 single nucleotide substitutions were identified, of which 6 were novel substitutions, including T196G (C30G), A447T, G458T (R117L), G531A, A704A, and C740T. In addition, no significant trends were observed between the nucleotide substitutions of HPV33 E6/E7 variants and the risk of cervical lesions.ConclusionThis study provides the most comprehensive data on genetic variations, phylogenetics and carcinogenicity of HPV33 E6/E7 variants in Southeast China to date. The data confirmed that cervical lesions among women in Taizhou are attributable to HPV33, which may be due to the high infection rate of sublineage A1 in the population.

Marker-Assisted Selection for Early Maturing E Loci in Soybean Yielded Prospective Breeding Lines for High Latitudes of Northern Kazakhstan.

The photoperiodic sensitivity of soybean (Glycine max L.) is one of the limiting factors affecting plant growth and yield. At higher latitudes, early flowering and maturity with neutral reaction to photoperiods are required for adaptation of soybean plants to long-day conditions. Currently, the production and distribution of new varieties of soybeans adapted to widespread agricultural regions in northern Kazakhstan is in strong demand. Eleven soybean hybrid populations were obtained from crosses between 17 parents with four maturity groups, MG 000, 00, 0, and I. Marker-assisted selection (MAS) was assessed for suitable SSR markers and successfully applied for genes E1, E3, E4, and E7, targeting homozygous genotypes with recessive alleles. The identified and selected genotypes were propagated and tested in the conditions of 53° N latitude in the Kostanay region of northern Kazakhstan. Finally, 20 early maturing F4 breeding lines were identified and developed with genotypes e1 e3 E4 e7, e1 E3 E4 e7, and e1 E3 e4 e7, all completing their growth period within 92-102 days. These breeding lines were developed by MAS and should provide very prospective superior varieties of soybean for northern Kazakhstan through a strategy that may be very helpful to other countries with high latitudes.

Genome-wide identification and transcriptome-based expression profiling of E2 gene family: Implication for potential roles in gonad development of Crassostrea gigas

In this study, we investigated the role of E2 ubiquitin conjugating enzymes (E2) in the Pacific oyster Crassostrea gigas, with a focus on their involvement in gonad development. We identified 34 E2 genes clustered into nine subgroups and 24 subfamilies. The gene structure and intron-exon location were conserved within the same subfamily, but motif variation suggested functional diversity. Tissue transcriptome analyses revealed that most E2 genes were broadly expressed, with UBE2CL showing specific expression in the female gonad. Expression profiling of E2 genes during early embryo-larvae development stages suggested that five E2 genes were highly expressed in early embryo development, indicating their involvement in cell division processes. Furthermore, by profiling the expression of E2 genes in different gonadal developmental stages, we observed a gradual increase in expression for four genes during gametogenesis, with significantly higher expression in the female gonad at the maturation stage. Similarly, five E2 genes displayed elevated expression levels in the male gonad at the maturation stage, indicating their crucial roles in gonadal development and gametogenesis. Our study provides valuable insights into the potential functions of the E2 gene family in C. gigas, shedding light on the molecular mechanisms underlying gonad development in oysters.

Influence of genotype and bacterization on growth, development, and soluble carbohydrate content in soybean E-genes isogenic lines

Photoperiod, which regulates the duration of vegetative and generative development, and the plant-microorganism interaction, which influences the metabolic status of plant organisms, are important factors in the regulating plant growth and development. The aim of the study was to determine the influence of Glycine max (L.) Merr. genotype and seed pre-bacterization with a virulent and active strain of Bradyrhizobium japonicum 634b on the plant growth and development, and on the soluble carbohydrate content in leaves of isogenic by E-genes lines under field conditions. Nearly isogenic lines (NILs) of soybean, in which the E1, E2, and E3 genes are located at different allelic loci, were used. Sterile seeds were pretreated with distilled water (control) and Bradyrhizobium japonicum 634b cell suspension (experiment). Plants were grown under natural long-day conditions (16 hours). The growth and development of the soybean were evaluated by phenological observations, morphometric indicators fixed at the V3 and V5 developmental stages, relative growth rate (RGR), and the content of soluble sugars ‒ mono- and oligosaccharides. The effect of the factors studied (genotype, bacterization, and their interaction) was calculated. The results of the experiment and the calculation of the effect of the factor showed that the isoline genotype has the greatest effect on seed germination, phenological development of the plant and duration of the VE-R1 phase, growth of the root system in the V3 and V5 phases, and the content of monosaccharides involved in forming the plant-microorganism interaction. The effect of bacterization is most evident in the RGR, shoot development, and the oligosaccharide content of the leaves of NILs in the V3 and V5 phases. Among the isolines studied, L 80-5879, which has the E1 gene (flowering repressor) in a dominant state, was characterized by minimal sensitivity to bacterization. It was found that bacterization and genotype interaction didn't influence the VE-R1 duration stage and the shoot and root length. The results obtained therefore prove that the E-series genes, which determine the photoperiodic sensitivity of soya beans, can also be indirectly involved in establishing plant-microorganism interactions.

Genetic Variability in the E6/E7 Region of Human Papillomavirus 16 in Women from Ecuador.

Human Papillomavirus (HPV) infection is associated with intraepithelial neoplasia and cervical cancer (CC). Ecuador has a high prevalence of cervical cancer, with more than 1600 new cases diagnosed annually. This study aimed to analyze oncogenes E6 and E7 of HPV16 in samples collected from women with cancerous and precancerous cervical lesions from the Ecuadorian coast. Twenty-nine women, including six with ASCUS, three with LSIL, thirteen with HSIL, and seven with Cacu, were analyzed. The most common SNPs were E6 350G or L83V (82.6%) and E6 145T/286A/289G/335T/350G or Q14H/F78Y/L83V (17.4%). Both variants are reported to be associated with an increased risk of cervical cancer in worldwide studies. In contrast, all E7 genes have conserved amino-acid positions. Phylogenetic trees showed the circulation of the D (26.1%) and A (73.9) lineages. The frequency of D was higher than that reported in other comparable studies in Ecuador and Latin America, and may be related to the ethnic composition of the studied populations. This study contributes to the characterization of the potential risk factors for cervical carcinogenesis associated with Ecuadorian women infected with HPV16.

Impact of Allelic Variation in Maturity Genes E1–E4 on Soybean Adaptation to Central and West Siberian Regions of Russia

Four maturity genes, namely, E1, E2, E3 and E4, have been found to play major roles in controlling the flowering and maturity time of soybean. Which genotypes of E1–E4 genes provide effective adaptation to the varied conditions of Russia are unknown. To clarify this issue, we have studied the allele variation in soybean E1–E4 genes in terms of both flowering and maturity time under the natural day-length conditions of Central Russia and Western Siberia in a collection of 176 soybean accessions, including 142 Russian and 34 foreign accessions. As a result, a high frequency of previously determined E1–E4 alleles has been identified. The field experiment showed that genotypes with all recessive alleles from e1-nl/e2/e3/e4 and e1-as/e2/e3/e4 provide the effective adaptation of soybean to the mentioned conditions. Cultivars with these genotypes are considered to be most suitable for cultivation in Central Russia and Western Siberia.

Directed natural evolution generates a next-generation oncolytic virus with a high potency and safety profile

Oncolytic viruses (OVs) represent a type of encouraging multi-mechanistic drug for the treatment of cancer. However, attenuation of virulence, which is generally required for the development of OVs based on pathogenic viral backbones, is frequently accompanied by a compromised killing effect on tumor cells. By exploiting the property of viruses to evolve and adapt in cancer cells, we perform directed natural evolution on refractory colorectal cancer cell HCT-116 and generate a next-generation oncolytic virus M1 (NGOVM) with an increase in the oncolytic effect of up to 9690-fold. The NGOVM has a broader antitumor spectrum and a more robust oncolytic effect in a range of solid tumors. Mechanistically, two critical mutations are identified in the E2 and nsP3 genes, which accelerate the entry of M1 virus by increasing its binding to the Mxra8 receptor and antagonize antiviral responses by inhibiting the activation of PKR and STAT1 in tumor cells, respectively. Importantly, the NGOVM is well tolerated in both rodents and nonhuman primates. This study implies that directed natural evolution is a generalizable approach for developing next-generation OVs with an expanded scope of application and high safety.

Genomic landscapes to characterize mismatch-repair deficiency (dMMR)/microsatellite instability-high (MSI-H) gastrointestinal (GI) cancers stratified by tumor mutation burden (TMB).

2618 Background: TMB is a predictive biomarker for immune checkpoint inhibitor treatment in dMMR/MSI-H cancers. We aimed to characterize specific molecular features of a large cohort of dMMR/MSI-H GI tumors with a range of TMB levels. Methods: A total of 2272 GI tumors tested as dMMR/MSI-H were analyzed using Next-generation sequencing (NGS) and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). MMR/MSI status was evaluated by a combination of IHC, Fragment Analysis and NGS. TMB-H were defined using differing TMB cutoffs (10, 20, 50 mutations/Mb). Molecular features were compared in four groups (TMB < 10 vs 10-20 vs 20-50 vs ≥50mutations/Mb) using Fisher-Exact or Chi-square and adjusted for multiple comparison by Benjamini-Hochberg. The impact of TMB on tumor immune environment was analyzed using a TCGA cohort. Significance was determined by adjusted (adj) P < 0.05. Results: Tumors with TMB over 10, 20, 50 mutations/Mb were observed in 95.38%, 86.05% and 14.47% respectively in the dMMR/MSI-H GI cohort. Compared to other groups, dMMR/MSI-H GI tumors with TMB over 50 mutations/Mb exhibited the highest mutations rates in RNF43, MSH6, KMT2D, BRCA2 and ASXL1 (Top 5, all Padj< 0.0001). Among the four MMR genes, only MLH1 mutation had no association with higher TMB. The rates of CNNE1 amplification (i.e. the Cycle E1 gene) and HER2 overexpression were the highest in tumors with TMB below 10 mutations/Mb (all Padj< 0.05). The association between PD-L1 positivity and TMB levels was only observed in gastroesophageal cancers ( P= 0.03). The lymphocyte infiltration signature score, IFNγ response and M1 macrophage infiltration were significantly lower, while fraction of genome alterations was significantly increased in dMMR/MSI-H tumors with TMB below 10 mutations/Mb, compared to other subgroups (all P< 0.05). Neoantigens were significantly enriched in those with TMB over 50 mutations/Mb, compared to other subgroups ( P< 0.0001). Conclusions: This is the largest study to investigate the distinct molecular landscape of dMMR/MSI-H GI cancers with different degrees (magnitude) of TMB. These data may inform our understanding of the efficacy of ICB in dMMR/MSI-H GI tumors.

Introduction of E-Score: A gene expression score to characterize immunogenic versus oncogenic HPV expression in HNSCC.

6057 Background: Human Papillomavirus (HPV) integration in the human genome occurs in a majority of HPV(+) head and neck squamous cell carcinomas (HNSCC), with direct and indirect effects on host gene expression and tumorigenesis. Although previous studies showed that HPV integration in HNSCC is overrepresented in genes often somatically mutated in head and neck, lung, and urogenital cancers, reports are mixed for whether patients with HPV integration have worse clinical outcomes. These disparate results may be due to differences in how integration is defined. Integration can often lead to partial loss of the viral genome (e.g. HPV genes E1, E2, or E5) with retention of the oncogenes E6 and E7, which led to a low E2/E7 expression ratio being used as a biomarker for integration. Conversely, others have used the direct detection of DNA or RNA HPV-human breakpoints to identify tumors harboring integrated HPV. HPV integration and the retention or deletion of HPV genes can dramatically change the expression of HPV genes, and we hypothesize it also affects the host immune response. Hypothesis: Loss of non-oncogenic HPV genes relative to E6 and E7 levels reduces immunogenicity, leading to weakened immune cell infiltration and worse clinical outcomes. Methods: Bulk RNA sequencing data from HPV(+) HNSCC tumors (n = 136) was used to characterize the differences in retention or loss of HPV genes and calculate the E-Score as the sum of the logCPM of HPV genes E1, E2, E4, and E5 relative to E7. Cell type deconvolution was performed using ComBat to correct for batch effects, and CIBERSORTx to quantify cell type proportions within the tumor samples. Kaplan-Meier estimates of recurrence and overall survival (OS) were calculated, and Cox proportional hazards tests with covariates were used to test for association with recurrence and OS. To test the correlation between immune cell type inferred proportions and E-Score, T-Tests were performed between samples with high and low E-scores. Results: Low E-Score was significantly associated with recurrence (p = 0.012) but not with OS (p = 0.39) in the multivariate model accounting for age, tumor stage (AJCC8), smoking status, drinking status, BMI, marital status, and molecular tumor subtype (defined as IMU or KRT). For integration positive samples (n = 66), higher E-Score, suggesting lower rates of HPV gene loss, was correlated with higher percentages of dendritic cells (p = 0.044), macrophages (p = 0.021), and CD8+ T Cells (p = 0.024), but not CD4+ T cells (p = 0.14). This significance did not hold when looking at integration negative samples. Conclusions: These results suggest that HPV+ HNSCC patients with a loss of E1, E2, E4 and E5 tend to have less immune cell infiltration and are more likely to have recurrence. We conclude that E-Score is a potential prognostic biomarker of recurrence in HPV+ HNSCC and potential predictive biomarker for immunotherapy.

Targeting Cell Cycle Facilitates E1A-Independent Adenoviral Replication.

DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.

A cross-sectional study of human papillomavirus genotype distribution and integration status in penile cancer among Chinese population

Human papillomavirus (HPV) has been recognized as an important risk factor in penile cancer. This study aimed to investigate the HPV subtypes and integration status in Chinese patients. Samples were collected from 103 penile cancer patients aged 24–90 years between 2013 and 2019. We found that HPV infection rate was 72.8%, with 28.0% integration. The aging patients were more susceptible to HPV (p = 0.009). HPV16 was the most frequent subtype observed (52/75) and exhibited the highest frequency of integration events, with 11 out of 30 single infection cases showing integration positive. The HPV integrations sites in the viral genome were not randomly distributed, the breakpoints were enriched in the E1 gene (p = 0.006) but relatively scarce in L1, E6 and E7. Our research might provide some clues how HPV leads to the progression of penile cancer.

Trap and ambush therapy using sequential primary and tumor escape-selective oncolytic viruses

In multiple models of oncolytic virotherapy, it is common to see an early anti-tumor response followed by recurrence. We have previously shown that frontline treatment with oncolytic VSV-IFN-β induces APOBEC proteins, promoting the selection of specific mutations that allow tumor escape. Of these mutations in B16 melanoma escape (ESC) cells, a C-T point mutation in the cold shock domain-containing E1 (CSDE1) gene was present at the highest frequency, which could be used to ambush ESC cells by vaccination with the mutant CSDE1 expressed within the virus. Here, we show that the evolution of viral ESC tumor cells harboring the escape-promoting CSDE1C-T mutation can also be exploited by a virological ambush. By sequential delivery of two oncolytic VSVs invivo, tumors which would otherwise escape VSV-IFN-β oncolytic virotherapy could be cured. This also facilitated the priming of anti-tumor Tcell responses, which could be further exploited using immune checkpoint blockade with the CD200 activation receptor ligand (CD200AR-L) peptide. Our findings here are significant in that they offer the possibility to develop oncolytic viruses as highly specific, escape-targeting viro-immunotherapeutic agents to be used in conjunction with recurrence of tumors following multiple different types of frontline cancer therapies.

Multi-omics data reveals novel impacts of human papillomavirus integration on the epigenomic and transcriptomic signatures of cervical tumorigenesis.

Integration of human papilloma virus (HPV) DNA into the human genome may progressively contribute to cervical carcinogenesis. To explore how HPV integration affects gene expression by altering DNA methylation during carcinogenesis, we analyzed a multiomics dataset for cervical cancer. We obtained multiomics data by HPV-capture sequencing, RNA sequencing, and Whole Genome Bisulfite Sequencing from 50 patients with cervical cancer. We detected 985 and 485 HPV-integration sites in matched tumor and adjacent paratumor tissues. Of these, LINC00486 (n = 19), LINC02425 (n = 11), LLPH (n = 11), PROS1 (n = 5), KLF5 (n = 4), LINC00392 (n = 3), MIR205HG (n = 3) and NRG1 (n = 3) were identified as high-frequency HPV-integrated genes, including five novel recurrent genes. Patients at clinical stage II had the highest number of HPV integrations. E6 and E7 genes of HPV16 but not HPV18 showed significantly fewer breakpoints than random distribution. HPV integrations occurring in exons were associated with altered gene expression in tumor tissues but not in paratumor tissues. A list of HPV-integrated genes regulated at transcriptomic or epigenetic level was reported. We also carefully checked the candidate genes with regulation pattern correlated in both levels. HPV fragments integrated at MIR205HG mainly came from the L1 gene of HPV16. RNA expression of PROS1 was downregulated when HPV integrated in its upstream region. RNA expression of MIR205HG was elevated when HPV integrated into its enhancer. The promoter methylation levels of PROS1 and MIR205HG were all negatively correlated with their gene expressions. Further experimental validations proved that upregulation of MIR205HG could promote the proliferative and migrative abilities of cervical cancer cells. Our data provides a new atlas for epigenetic and transcriptomic regulations regarding HPV integrations in cervical cancer genome. We demonstrate that HPV integration may affect gene expression by altering methylation levels of MIR205HG and PROS1. Our study provides novel biological and clinical insights into HPV-induced cervical cancer.

The possible impact of novel mutations in human papillomavirus 52 on the infection characteristics.

Human papillomavirus 52 (HPV52) infection is prevalent in the Chinese population, and variations in HPV52 show correlations with oncogenicity. However, no specific variation in HPV52 was reported to show relevancy to infection characteristics. In this study, we retrieved 222 isolates of E6 and L1 full-length genes from 197 Chinese women with HPV52 infection. After sequence alignment and phylogenetic tree construction, we found that 98.39 % of the collected variants belonged to the sublineage B2 and two variants displayed incongruence between the phylogenetic tree of E6 and L1. The analysis of the infection pattern showed that the presence of C6480A/T mutation in the L1 gene was associated with single infection (P=0.01) and persistent infection (P=0.047) of HPV52, while the A6516G nucleotide change was relevant to transient infection (P=0.018). Our data also indicated that variations T309C in the E6 gene and C6480T, C6600A in L1 were more commonly presented in patients with high-grade cytology (P<0.05). One HPV52 breakthrough infection after vaccination was identified, which hinted at the immune escape post-vaccination. Young coitarche age and non-condom usage were correlated to multiple infections. This study provided insight into the polymorphism of HPV52 and revealed the impact of variations in HPV52 on its infection characteristics.

Abstract LB153: Inhibiting BRD4 as a proposed alternative therapeutic strategy for HPV16-associated head and neck squamous cell carcinoma

Abstract Integration of Human Papilloma Virus (HPV) 16 into human genomes is an integral event for cancer progression in Head and Neck Squamous Cell Carcinoma (HNSCC). There is a need to differentiate treatment for HPV-16 integrated HNSCC from other subtypes. We hypothesize that by inhibiting Bromodomain Extra Terminal (BET) protein BRD4, a transcriptional co-regulator of host and viral genes can advance treatment in patients with the integrated subtype of HPV16 associated HNSCC. Through small molecule inhibition and knockdown of BRD4 in seven model HPV-16 integrated cell lines, we demonstrated that BRD4 down-regulates the expression of viral genes E6, E7, independent of viral E2 type of integration. Given that E6 expression is mediated by BRD4, E6-p53 interactions was abrogated as revealed through increased expression of p53. Furthermore, BRD4 inhibition caused downregulation of c-Myc. This result also suggests the function of BRD4 in the regulation of proteins in cell cycle regulation, including E2F1 and p21. BRD4 inhibition was also shown to delay DNA damage response (DDR) by reducing the expression of RAD51 in 6 out of 7 cell lines. RNA-sequencing results give further insight regarding the downregulation of genes involved in DDR – RAD51, BRCA1 and BRCA2 in all cell lines. RNA-sequencing data also gives us an understanding of the implication of BRD4-inhibition in other genes involved in cell cycle regulation. Knockdown of BRD4 done through small hairpin RNA (shRNA) constructs phenocopied the effects shown through inhibition of BRD4. Additionally, treating the above-mentioned cell lines with BRD4 inhibitor (JQ1) followed by radiation showed further downregulation of viral oncogenes E6, and E7. This suggests that combining radiation and JQ1 treatment can be a viable combination treatment. De-escalating radiation treatment with BRD4 inhibition has potential to be an effective combination treatment for HPV16 integrated HNSCC. Citation Format: Dhruthi Suresh, Chitrasen Mohanty, Albert R. Wang, Christina Kendziorski, Gopal Iyer. Inhibiting BRD4 as a proposed alternative therapeutic strategy for HPV16-associated head and neck squamous cell carcinoma [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 LB153.

Bacterial Species from Vaginal Microbiota Differently Affect the Production of the E6 and E7 Oncoproteins and of p53 and p-Rb Oncosuppressors in HPV16-Infected Cells.

Vaginal dysbiosis is characterized by a decrease in the relative abundance of Lactobacillus species in favor of other species. This condition facilitates infections by sexually transmitted pathogens including high risk (HR)-human papilloma viruses (HPVs) involved in the development of cervical cancer. Some vaginal dysbiosis bacteria contribute to the neoplastic progression by inducing chronic inflammation and directly activating molecular pathways involved in carcinogenesis. In this study, SiHa cells, an HPV-16-transformed epithelial cell line, were exposed to different representative vaginal microbial communities. The expression of the HPV oncogenes E6 and E7 and the production of relative oncoproteins was evaluated. The results showed that Lactobacillus crispatus and Lactobacillus gasseri modulated the basal expression of the E6 and E7 genes of SiHa cells and the production of the E6 and E7 oncoproteins. Vaginal dysbiosis bacteria had contrasting effects on E6/E7 gene expression and protein production. The expression of the E6 and E7 genes and the production of the relative oncoproteins was increased by strains of Gardnerella vaginalis and, to a lesser extent, by Megasphaera micronuciformis. In contrast, Prevotella bivia decreased the expression of oncogenes and the production of the E7 protein. A decreased amount of p53 and pRb was found in the cultures of SiHa cells with M. micronuciformis, and accordingly, in the same cultures, a higher percentage of cells progressed to the S-phase of the cell cycle compared to the untreated or Lactobacillus-stimulated cultures. These data confirm that L. crispatus represents the most protective component of the vaginal microbiota against neoplastic progression of HR-HPV infected cells, while M. micronuciformis and, to a lesser extent, G. vaginalis may directly interfere in the oncogenic process, inducing or maintaining the production of viral oncoproteins.

The First Genomic Characterization of the Chikungunya Virus in Saudi Arabia

BackgroundChikungunya is an arboviral infection caused by the Chikungunya virus (CHIKV) transmitted to humans by mosquitoes of Aedes spp. CHIKV has been confined to African countries and South-East Asia up to 2004, but since then, the pathogen has become more global, and its high morbidity rate has become more visible. Saudi Arabia is not an endemic region of CHIKV, and the virus’s origin is not yet fully understood. This study aimed to characterize the genome of CHIKV from samples detected in Jeddah in 2018.MethodTwenty-two sets of primers were designed to amplify near-full length genome of CHIKV. RT-PCR was conducted from clinical samples. Two samples were used for studying near complete genome sequence while the remaining samples were used to study the E1 gene. Different bioinformatics tools were utilized.ResultsPhylogenetic analysis showed that the CHIKV strains clustered with strains isolated from Kenya during 2017–2018 and belonged to ECSA genotype. E1: L136F, K211E and I317V mutations were identified in our strains. Also, E2: M74I, A76T, and V264A mutations were documented. Additionally, the capsid N79S substitution was also detected.ConclusionThe genome of CHIKV was analyzed for the first time in Saudi Arabia to better understand the origin of the CHIKV and its genetic diversity, which showed high similarity with IE—a subclade of CHIKV strains detected in Mombasa (Kenya) indicating its possible origin.

Abstract 5469: Characterization of CCNE1 amplifications and associated genomic features in ovarian and uterine cancers

Abstract Amplification of the Cyclin E1 gene (CCNE1) is a recurrent genetic alteration in ovarian and uterine cancers. Therapeutic approaches to target cancers harboring CCNE1 amplification are being developed (e.g. PKMYT1, WEE1, and CDK2 inhibitors). In this study, we report the genomic landscape and architecture of CCNE1 amplifications using a combination of whole-exome (WES) and whole-genome sequencing (WGS) approaches. WES data from 361 ovarian and 580 uterine cancers was retrieved from TCGA. WGS data from 113 ovarian and 51 uterine cancers was retrieved from PCAWG. For both WES and WGS, amplification was defined as CN minus ploidy (CN-ploidy) &amp;gt;= 4. CCNE1 amplification was detected in up to 21.2% of ovarian and 13.7% of uterine cancers from both methods. A review of the co-alteration landscape using WES revealed that TP53 alterations frequently co-occurred with CCNE1 amplifications in both ovarian and uterine cancers (ovarian: 95.8% vs 87.6%, OR = 3.24, p=0.1; uterine: 89.6% vs 36.0%, OR = 15.2, p =2.14e-13) and BRCA1 alterations were mutually exclusive with CCNE1 amplifications in ovarian cancer (2.1% vs 17.9%, OR=0.2, p = 3.1e-3). In addition, alterations in ARID1A (0% vs 40.5%, OR = 0, p = 1.3e-10) and PTEN (4.2% vs 60.1%, OR = 0.03, p =2.7e-15) were mutually exclusive with CCNE1 amplifications in uterine cancer. The number of CCNE1 copies ranged widely, with median CN-ploidy values of 9.3 (range: 4.0 -35.0) in ovarian and 7.7 (range: 4.0 -40.3) in uterine cancers from the WES cohort, and 7.4 (range: 4.1 -34.3) in ovarian and 11.0 (range: 4.6 - 81.9) in uterine cancers from WGS. We also observed a broad range of CCNE1 amplicon sizes, from highly focal to chromosome arm-level amplifications. WGS data showed frequent loss of heterozygosity (LOH) of chromosome 19 arms and an enrichment of fold-back inversions proximal to the CCNE1 amplicon implicating breakage-fusion-bridge in formation of CCNE1 amplicons. Whole genome duplication (WGD) was detected significantly more frequently in ovarian and uterine cancers with CCNE1 amplification than the wild-type group (ovarian: 90.9% vs 48.3%, p = 6.4e-10; uterine: 78.7% vs 21.9%, p = 7.1e-15). As compared to CCNE1 wild-type cancers, CCNE1-amplified cancers were found to display an enrichment for certain structural variant signatures: RS1, dominated by 100Kb-1Mb duplications (median = 30, p=0.08); RS2, dominated by translocations (median = 16, p=0.001); and RS7, dominated by 100Kb-1Mb deletions (median=23, p=0.0002). In conclusion, CCNE1-amplified ovarian and uterine cancers are enriched for TP53 alterations and display a high degree of genome instability manifested as WGD and characteristic structural variants. Fold-back inversions and LOH were frequently observed at the CCNE1 locus in tumors with CCNE1 amplification, suggesting a potential breakage-fusion-bridge mechanism in the genesis of this amplicon. Citation Format: Sunantha Sethuraman, Dominik Glodzik, Pier Selenica, Adrienne Johnson, Jorge S. Reis-Filho, Artur Veloso, Ian M. Silverman. Characterization of CCNE1 amplifications and associated genomic features in ovarian and uterine cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5469.

Abstract 1212: Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma

Abstract Human papillomavirus (HPV) infections are currently associated with more than 70% of oropharyngeal squamous cell carcinomas (OPSCCs). HPV-positive OPSCCs display a distinct biologic behavior and treatment response and thus are now considered a distinct entity than HPV-negative OPSCCs. However, the underlying process of HPV-related OPSCC carcinogenesis is not entirely clear. HPV as a driver of cervical carcinogenesis is well-characterized with identification of a canonical mechanism dependent on HPV DNA integration, loss of HPV E2 gene expression, and increased expression of HPV E6 and E7 oncogenes with no active infection present in malignant lesions. However, whole genome sequencing found no HPV DNA integration in ~25% of HPV-associated OPSCC, suggesting an alternative mechanism of HPV carcinogenesis. Expression of all HPV genes, including self-assembling capsid proteins, in a substantial portion of HPV+ OPSCC, led us to hypothesize that tumors with episomal HPV are capable of transferring HPV genes to surrounding cells and that HPV gene transfer contributes to carcinogenesis in this subset of OPSCC. Our data support this hypothesis, showing that growth media from HPV+ OPSCC cell lines, as well as isolates from HPV+ tumors or nodal metastases, effectively transferred functional HPV genes to cells lacking HPV. The HPV DNA Transferring Agent (HDTA) derived from HPV+ OPSCC contained HPV L1 capsid protein and intact HPV genome protected from DNAase degradation. Initial characterization revealed that anti-L1 antibody blocked gene transfer and that exposure of cells to the HDTA initiated a robust innate immune response in recipient cells. Negative staining of HDTA isolate from metastatic lymph nodes revealed many pleomorphic, electron-dense particles with diameter ranging between 20 and 80nM, with approximately 40% of particles being between 50 and 60nM, consistent with the size of HPV. We propose that development and/or maintenance of OPSCCs depend on continuous production of HDTA capable of transferring HPV genes into neighboring cells. This paradigm of tumorigenesis/maintenance in OPSCC is most likely to apply to cancers lacking HPV integration, but may also apply to tumors with integration, since recent data suggested that integrated HPV may excise from the genome to recreate a transient episomal like structure, even containing cellular DNA. Our findings offer novel insights into oropharyngeal carcinogenesis and suggest targeting HDTA as a new therapeutic strategy for HPV-positive OPSCCs. Citation Format: Jason Tasoulas, Wendell Gray Yarbrough, Natalia Issaeva. Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1212.