Targeted inhibition of BET proteins in HPV-16 associated head and neck squamous cell carcinoma reveals heterogeneous transcription response.

Integrated human papillomavirus (HPV-16) associated head and neck squamous cell carcinoma (HNSCC) tumors have worse survival outcomes compared to episomal HPV-16 HNSCC tumors. Therefore, there is a need to differentiate treatment for HPV-16 integrated HNSCC from other viral forms. We analyzed TCGA data and found that HPV+ HNSCC expressed higher transcript levels of the bromodomain and extra terminal domain (BET) family of transcriptional coregulators. However, the mechanism of BET protein-mediated transcription of viral-cellular genes in the integrated viral-HNSCC genomes needs to be better understood. We show that BET inhibition downregulates E6 significantly independent of the viral transcription factor, E2, and there was overall heterogeneity in the downregulation of viral transcription in response to the effects of BET inhibition across HPV-associated cell lines. Chemical BET inhibition was phenocopied with the knockdown of BRD4 and mirrored downregulation of viral E6 and E7 expression. Strikingly, there was heterogeneity in the reactivation of p53 levels despite E6 downregulation, while E7 downregulation did not alter Rb levels significantly. We identified that BET inhibition directly downregulated c-Myc and E2F expression and induced CDKN1A expression. Overall, our studies show that BET inhibition provokes a G1-cell cycle arrest with apoptotic activity and suggests that BET inhibition regulates both viral and cellular gene expression in HPV-associated HNSCC.

HPV16 E6 induces chromosomal instability due to polar chromosomes caused by E6AP-dependent degradation of the mitotic kinesin CENP-E.

Chromosome segregation during mitosis is highly regulated to ensure production of genetically identical progeny. Recurrent mitotic errors cause chromosomal instability (CIN), a hallmark of tumors. The E6 and E7 oncoproteins of high-risk human papillomavirus (HPV), which causes cervical, anal, and head and neck cancers (HNC), cause mitotic defects consistent with CIN in models of anogenital cancers, but this has not been studied in the context of HNC. Here, we show that HPV16 induces a specific type of CIN in patient HNC tumors, patient-derived xenografts, and cell lines, which is due to defects in chromosome congression. These defects are specifically induced by the HPV16 oncogene E6 rather than E7. We show that HPV16 E6 expression causes degradation of the mitotic kinesin CENP-E, whose depletion produces chromosomes that are chronically misaligned near spindle poles (polar chromosomes) and fail to congress. Though the canonical oncogenic role of E6 is the degradation of the tumor suppressor p53, CENP-E degradation and polar chromosomes occur independently of p53. Instead, E6 directs CENP-E degradation in a proteasome-dependent manner via the E6-associated ubiquitin protein ligase E6AP/UBE3A. This study reveals a mechanism by which HPV induces CIN, which may impact HPV-mediated tumor initiation, progression, and therapeutic response.

HIV-1 Protease Inhibitors Slow HPV16-Driven Cell Proliferation through Targeted Depletion of Viral E6 and E7 Oncoproteins.

High-risk human papillomavirus strain 16 (HPV16) causes oral and anogenital cancers through the activities of two viral oncoproteins, E6 and E7, that dysregulate the host p53 and pRb tumor suppressor pathways, respectively. The maintenance of HPV16-positive cancers requires constitutive expression of E6 and E7. Therefore, inactivating these proteins could provide the basis for an anticancer therapy. Herein we demonstrate that a subset of aspartyl protease inhibitor drugs currently used to treat HIV/AIDS cause marked reductions in HPV16 E6 and E7 protein levels using two independent cell culture models: HPV16-transformed CaSki cervical cancer cells and NIKS16 organotypic raft cultures (a 3-D HPV16-positive model of epithelial pre-cancer). Treatment of CaSki cells with some (lopinavir, ritonavir, nelfinavir, and saquinavir) but not other (indinavir and atazanavir) protease inhibitors reduced E6 and E7 protein levels, correlating with increased p53 protein levels and decreased cell viability. Long-term (>7 day) treatment of HPV16-positive NIKS16 raft cultures with saquinavir caused epithelial atrophy with no discernible effects on HPV-negative rafts, demonstrating selectivity. Saquinavir also reduced HPV16's effects on markers of the cellular autophagy pathway in NIKS16 rafts, a hallmark of HPV-driven pre-cancers. Taken together, these data suggest HIV-1 protease inhibitors be studied further in the context of treating or preventing HPV16-positive cancers.

Reactivation of Epstein-Barr Virus by HIF-1α Requires p53.

We previously reported that the cellular transcription factor hypoxia-inducible factor 1α (HIF-1α) binds a hypoxia response element (HRE) located within the promoter of Epstein-Barr virus's (EBV's) latent-lytic switch BZLF1 gene, Zp, inducing viral reactivation. In this study, EBV-infected cell lines derived from gastric cancers and Burkitt lymphomas were incubated with HIF-1α-stabilizing drugs: the iron chelator deferoxamine (Desferal [DFO]), a neddylation inhibitor (pevonedistat [MLN-4924]), and a prolyl hydroxylase inhibitor (roxadustat [FG-4592]). DFO and MLN-4924, but not FG-4592, induced accumulation of both lytic EBV proteins and phosphorylated p53 in cell lines that contain a wild-type p53 gene. FG-4592 also failed to activate transcription from Zp in a reporter assay despite inducing accumulation of HIF-1α and transcription from another HRE-containing promoter. Unexpectedly, DFO failed to induce EBV reactivation in cell lines that express mutant or no p53 or when p53 expression was knocked down with short hairpin RNAs (shRNAs). Likewise, HIF-1α failed to activate transcription from Zp when p53 was knocked out by CRISPR-Cas9. Importantly, DFO induced binding of p53 as well as HIF-1α to Zp in chromatin immunoprecipitation (ChIP) assays, but only when the HRE was present. Nutlin-3, a drug known to induce accumulation of phosphorylated p53, synergized with DFO and MLN-4924 in inducing EBV reactivation. Conversely, KU-55933, a drug that inhibits ataxia telangiectasia mutated, thereby preventing p53 phosphorylation, inhibited DFO-induced EBV reactivation. Lastly, activation of Zp transcription by DFO and MLN-4924 mapped to its HRE. Thus, we conclude that induction of BZLF1 gene expression by HIF-1α requires phosphorylated, wild-type p53 as a coactivator, with HIF-1α binding recruiting p53 to Zp.IMPORTANCE EBV, a human herpesvirus, is latently present in most nasopharyngeal carcinomas, Burkitt lymphomas, and some gastric cancers. To develop a lytic-induction therapy for treating patients with EBV-associated cancers, we need a way to efficiently reactivate EBV into lytic replication. EBV's BZLF1 gene product, Zta, usually controls this reactivation switch. We previously showed that HIF-1α binds the BZLF1 gene promoter, inducing Zta synthesis, and HIF-1α-stabilizing drugs can induce EBV reactivation. In this study, we determined which EBV-positive cell lines are reactivated by classes of HIF-1α-stabilizing drugs. We found, unexpectedly, that HIF-1α-stabilizing drugs only induce reactivation when they also induce accumulation of phosphorylated, wild-type p53. Fortunately, p53 phosphorylation can also be provided by drugs such as nutlin-3, leading to synergistic reactivation of EBV. These findings indicate that some HIF-1α-stabilizing drugs may be helpful as part of a lytic-induction therapy for treating patients with EBV-positive malignancies that contain wild-type p53.

Hypoxia-inducible factor-1α plays roles in Epstein-Barr virus's natural life cycle and tumorigenesis by inducing lytic infection through direct binding to the immediate-early BZLF1 gene promoter.

When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs). We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV). Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE) located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK) and gingival epithelial (hGET) cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV's natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for development of a lytic-induction therapy for treating some EBV-associated malignancies.