GB and gH/gL fusion machinery: a promising target for vaccines to prevent Epstein-Barr virus infection.

Epstein‒Barr virus (EBV) is a double-stranded DNA virus belonging to the family Orthoherpesviridae that is associated with the development of various tumors, such as lymphoma, nasopharyngeal carcinoma, and gastric cancer. There are no uniformly effective treatments for human EBV infection, and vaccines and immunotherapies are currently the main research directions. The glycoproteins gB and gH/gL are surface glycoproteins that are common to all herpesviruses, with subtle differences in structure and function between different viruses. The core membrane fusion machinery constituted by EBV gB and gH/gL is an important target of neutralizing antibodies in epithelial EBV infection due to its essential role in the fusion of viral and target cell membranes. In this article, we review the main modes of EBV infection, the structure and function of the core fusion machinery gB and gH/gL, and the development of neutralizing antibodies and prophylactic vaccines based on this target.

Early change of plasma Epstein-Barr virus DNA load and the viral lytic genome level could positively predict clinical outcome in recurrent or metastatic nasopharyngeal carcinoma receiving anti-programmed cell death 1 monotherapy.

PURPOSE: Patients with recurrent or metastatic nasopharyngeal carcinoma (RM-NPC) have proven benefit from anti-programmed cell death 1 (anti-PD-1) monotherapy. Here, we retrospectively analyze the association of plasma Epstein-Barr virus (EBV) DNA load and tumor viral lytic genome with clinical outcome from 2 registered phase I trials. METHODS: Patients with RM-NPC from Checkmate 077 (nivolumab phase I trial in China) and Camrelizumab phase I trial between March 2016 and January 2018 were enrolled. Baseline EBV DNA titers were tested in 68 patients and EBV assessment was performed in 60 patients who had at least 3 post-baseline timepoints of EBV data and at least 1 post-baseline timepoint of radiographic assessment. We defined "EBV response" as 3 consecutive timepoints of load below 50% of baseline, and "EBV progression" as 3 consecutive timepoints of load above 150% of baseline. Whole-exome sequencing was performed in 60 patients with available tumor samples. RESULTS: We found that the baseline EBV DNA load was positively correlated with tumor size (spearman p < 0.001). Both partial response (PR) and stable disease (SD) patients had significantly lower EBV load than progression disease (PD) patients. EBV assessment was highly consistent with radiographic evaluation. Patients with EBV response had significantly improved overall survival (OS) than patients with EBV progression (log-rank p = 0.004, HR = 0.351 [95% CI: 0.171-0.720], median 22.5 vs. 11.9 months). The median time to initial EBV response and progression were 25 and 36 days prior to initial radiographic response and progression, respectively. Patients with high levels of EBV lytic genomes at baseline, including BKRF2, BKRF3 and BKRF4, had better progression-free survival (PFS) and OS. CONCLUSION: In summary, early clearance of plasma EBV DNA load and high levels of lytic EBV genes were associated with better clinical outcome in patients with RM-NPC receiving anti-PD-1 monotherapy.

CD150­dependent activation of EBV­transformed B cells induces the differentiation of peripheral blood monocytes via the secretion of multiple cytokines.

CD150, also termed signaling lymphocyte activation molecule family member 1, is a cell surface receptor expressed on T cells, B cells, dendritic cells (DCs) and some tumors. Stimulation of CD150 on immune cells induces cell proliferation and cytokine production. However, the function of CD150 in Epstein­Barr virus (EBV)­infected B cells is still not fully understood. In the present study, CD150 expression on B cells increased rapidly following EBV infection, and various CD150 antibodies, measles viral proteins and recombinant CD150 proteins induced the secretion of multiple cytokines in both CD150+ EBV­transformed B cells and EBV+ lymphoma cells. Notably, the IL­1α protein level showed the greatest increase among all cytokines measured. The culture supernatant containing these cytokines induced the rapid differentiation of monocytes to DCs after only 2 days in vitro, which was faster than the established DC maturation time. Furthermore, knockdown of CD150 expression led to a reduction in the secretion of multiple cytokines, and monocyte differentiation was partially inhibited by anti­IL­1α and anti­granulocyte­macrophage colony­stimulating factor neutralizing antibodies. Collectively, the results of the present study suggest that CD150 activation triggers cytokine production in EBV­transformed B cells, and that measles virus coinfection might affect immune responses through the production of various cytokines in EBV+ lymphoma cells.

Epstein-Barr virus deubiquitinating enzyme BPLF1 is involved in EBV carcinogenesis by affecting cellular genomic stability.

Increased mutational burden and EBV load have been revealed from normal tissues to Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs). BPLF1, encoded by EBV, is a lytic cycle protein with deubiquitinating activity has been found to participate in disrupting repair of DNA damage. We first confirmed that BPLF1 gene in gastric cancer (GC) significantly increased the DNA double strand breaks (DSBs). Ubiquitination mass spectrometry identified histones as BPLF1 interactors and potential substrates, and co-immunoprecipitation and in vitro experiments verified that BPLF1 regulates H2Bub by targeting Rad6. Over-expressing Rad6 restored H2Bub but partially reduced γ-H2AX, suggesting that other downstream DNA repair processes were affected. mRNA expression of BRCA2 were significantly down-regulated by next-generation sequencing after over-expression of BPLF1, and over-expression of p65 facilitated the repair of DSBs. We demonstrated BPLF1 may lead to the accumulation of DSBs by two pathways, reducing H2B ubiquitination (H2Bub) and blocking homologous recombination which may provide new ideas for the treatment of gastric cancer.

Epstein-Barr Virus Lytic Transcripts Correlate with the Degree of Myocardial Inflammation in Heart Failure Patients.

The Epstein-Barr virus (EBV) is frequently found in endomyocardial biopsies (EMBs) from patients with heart failure, but the detection of EBV-specific DNA has not been associated with progressive hemodynamic deterioration. In this paper, we investigate the use of targeted next-generation sequencing (NGS) to detect EBV transcripts and their correlation with myocardial inflammation in EBV-positive patients with heart failure with reduced ejection fraction (HFrEF). Forty-four HFrEF patients with positive EBV DNA detection and varying degrees of myocardial inflammation were selected. EBV-specific transcripts from EMBs were enriched using a custom hybridization capture-based workflow and, subsequently, sequenced by NGS. The short-read sequencing revealed the presence of EBV-specific transcripts in 17 patients, of which 11 had only latent EBV genes and 6 presented with lytic transcription. The immunohistochemical staining for CD3+ T lymphocytes showed a significant increase in the degree of myocardial inflammation in the presence of EBV lytic transcripts, suggesting a possible influence on the clinical course. These results imply the important role of EBV lytic transcripts in the pathogenesis of inflammatory heart disease and emphasize the applicability of targeted NGS in EMB diagnostics as a basis for specific treatment.

Association between Epstein-Barr virus LMP-1 and Hodgkin lymphoma LMP-1 mechanisms in Hodgkin lymphoma development.

Hodgkin lymphoma is histologically characterised by the presence of Hodgkin (H) and Reed-Sternberg (RS) cells originating from germinal centre B-cells rearranged in the IgV gene. The formation of multinucleated RS cells is a product of telomere organisation in a process initiated by telomere aggregate accumulation in mononuclear H cells and may be mediated by latent membrane protein 1 (LMP-1) expression. LMP-1 is the main oncoprotein of EBV and supports several tumourigenic processes. LMP-1 may rescue proapoptotic B-cells through downregulation of B-cell receptor (BCR) components, mimicking and inducing multiple distinct B-cell signalling pathways to promote proliferation and survival, such as Janus kinase-signal transducer and activator of transcription (JAK-STAT), nuclear factor-kappa b (NF-кB), and cellular MYC (c-MYC), and inducing telomere instability mainly through Telomere repeat binding factor 2 (TRF2) downregulation to promote the formation of multinucleated RS cells. This review presents recent discoveries regarding the influence of LMP-1 on the surviving cellular signalling, genomic instability and mecanical formation of HRS cells.

Heightened Epstein-Barr virus immunity and potential cross-reactivities in multiple sclerosis.

BACKGROUND: Epstein-Barr virus (EBV) is a likely prerequisite for multiple sclerosis (MS) but the underlying mechanisms are unknown. We investigated antibody and T cell responses to EBV in persons with MS (pwMS), healthy EBV-seropositive controls (HC) and post-infectious mononucleosis (POST-IM) individuals up to 6 months after disease resolution. The ability of EBV-specific T cell responses to target antigens from the central nervous system (CNS) was also investigated. METHODS: Untreated persons with relapsing-remitting MS, POST-IM individuals and HC were, as far as possible, matched for gender, age and HLA-DRB1*15:01. EBV load was determined by qPCR, and IgG responses to key EBV antigens were determined by ELISA, immunofluorescence and Western blot, and tetanus toxoid antibody responses by multiplex bead array. EBV-specific T cell responses were determined ex vivo by intracellular cytokine staining (ICS) and cross-reactivity of in vitro-expanded responses probed against 9 novel Modified Vaccinia Ankara (MVA) viruses expressing candidate CNS autoantigens. RESULTS: EBV load in peripheral blood mononuclear cells (PBMC) was unchanged in pwMS compared to HC. Serologically, while tetanus toxoid responses were unchanged between groups, IgG responses to EBNA1 and virus capsid antigen (VCA) were significantly elevated (EBNA1 p = 0.0079, VCA p = 0.0298) but, importantly, IgG responses to EBNA2 and the EBNA3 family antigens were also more frequently detected in pwMS (EBNA2 p = 0.042 and EBNA3 p = 0.005). In ex vivo assays, T cell responses to autologous EBV-transformed B cells and to EBNA1 were largely unchanged numerically, but significantly increased IL-2 production was observed in response to certain stimuli in pwMS. EBV-specific polyclonal T cell lines from both MS and HC showed high levels of autoantigen recognition by ICS, and several neuronal proteins emerged as common targets including MOG, MBP, PLP and MOBP. DISCUSSION: Elevated serum EBV-specific antibody responses in the MS group were found to extend beyond EBNA1, suggesting a larger dysregulation of EBV-specific antibody responses than previously recognised. Differences in T cell responses to EBV were more difficult to discern, however stimulating EBV-expanded polyclonal T cell lines with 9 candidate CNS autoantigens revealed a high level of autoreactivity and indicate a far-reaching ability of the virus-induced T cell compartment to damage the CNS.

A cocktail nanovaccine targeting key entry glycoproteins elicits high neutralizing antibody levels against EBV infection.

Epstein-Barr virus (EBV) infects more than 95% of adults worldwide and is closely associated with various malignancies. Considering the complex life cycle of EBV, developing vaccines targeting key entry glycoproteins to elicit robust and durable adaptive immune responses may provide better protection. EBV gHgL-, gB- and gp42-specific antibodies in healthy EBV carriers contributed to sera neutralizing abilities in vitro, indicating that they are potential antigen candidates. To enhance the immunogenicity of these antigens, we formulate three nanovaccines by co-delivering molecular adjuvants (CpG and MPLA) and antigens (gHgL, gB or gp42). These nanovaccines induce robust humoral and cellular responses through efficient activation of dendritic cells and germinal center response. Importantly, these nanovaccines generate high levels of neutralizing antibodies recognizing vulnerable sites of all three antigens. IgGs induced by a cocktail vaccine containing three nanovaccines confer superior protection from lethal EBV challenge in female humanized mice compared to IgG elicited by individual NP-gHgL, NP-gB and NP-gp42. Importantly, serum antibodies elicited by cocktail nanovaccine immunization confer durable protection against EBV-associated lymphoma. Overall, the cocktail nanovaccine shows robust immunogenicity and is a promising candidate for further clinical trials.

Flow Cytometry as the Tool to Define Peripheral Blood Leukocyte Signatures in Acute EBV Infection.

Primary Epstein-Barr virus (EBV) infection which can manifest as infectious mononucleosis (IM) is commonly acquired during childhood. EBV primarily invades B cells leading to a lytic reaction; the control of the infection is handled by natural killer and T cells in immunocompetent individuals. The infection has a wide spectrum of clinical findings and can lead to serious complications in patients with certain underlying immunological dysfunctions. We retrospectively investigated peripheral white blood cell populations' surface marker characteristics in IM using a comprehensive flow cytometry marker panel. Twenty-one cases of IM and seventeen EBV-seropositive cases without IM serving as controls were included. We observed novel alterations in lymphocyte, neutrophil, and monocyte populations. In addition to increased activated cytotoxic T cells and low B cells, we demonstrated high T-large granular lymphocyte (T-LGL) populations in IM cases. Furthermore, despite T cells' increased HLA-DR expression, another activation marker, CD11b, was lower in T-LGL populations. Monocytes showed increased CD16 expression; CD64 was higher in neutrophils. Our findings point to monocyte and neutrophil activation which may account for acute clinical features and may contribute to the understanding of IM immunobiology. Furthermore, they may serve as a useful tool in investigating inherited and post-transplant conditions characterized by deficiencies in controlling EBV infection.

Post-transplant lymphoproliferative disorder associated Epstein-Barr virus DNAemia after liver transplantation in children: Experience from single center.

The most prevalent malignancy that complicates both adult and pediatric solid organ transplantation is post-transplant lymphoproliferative disorder (PTLD). This study aimed to analyze the clinical and pathological characteristics, treatments, and outcomes of Epstein-Barr virus (EBV) DNAemia and PTLD in pediatric liver transplant recipients. A retrospective chart review was performed on 112 patients less than 18 years of age who underwent isolated orthotopic liver transplantation (OLT) between 2010 and 2022 at Ege University Children's Hospital. Data gathered for 1-year post-OLT included age at OLT, EBV, immunoglobulin (Ig)M/IgG status of the donor and recipient, indication for OLT, induction regimen, all immunosuppression levels, date and result of EBV polymerase chain reaction testing, rejection episodes documented by liver biopsy, and the development of PTLD. Forty-nine patients (43.75%) developed EBV DNAemia (median interval from surgery: 2 months, min-max: 2-36), of which 43 (87.8%) grafts came from living donors, and 6 (12.2%) came from deceased donors. Nine (18.4%) patients died during follow-up, and eight (16.3%) developed PTLD. Of these 8 patients; five patients developed EBV-related disease, one child developed hemophagocytic lymphohistiocytosis, one developed aplastic anemia, and one child developed B cell lymphoma. When PTLD patients and without-PTLD patients were compared, pediatric intensive care unit hospitalization, abnormal bone marrow biopsy findings, lymphadenopathy, age at diagnosis of EBV DNAemia, EBV viral load, tacrolimus (FK 506) pre-infection, were higher and tacrolimus 1-month levels were lower in patients with PTLD (p < 0.05). In logistic regression analysis, we showed that the age at diagnosis of EBV DNAemia was significantly higher in children with PTLD (p = 0.045; OR: 1.389; 95% CI: 1.007-1.914). PTLD is a rare but severe complication associated with EBV after OLT. This study demonstrated that PTLD is associated with older age, higher tacrolimus blood levels before EBV DNAemia, and higher peak EBV viral load at 1 month of EBV DNAemia.

Chronic active Epstein-Barr virus infection with reinfection of SARS-CoV-2: a case report.

We describe the case of a 57-year-old male with jaundice, abdominal distension and fatigue. He was diagnosed as chronic active Epstein-Barr virus infection (CAEBV) due to intermittent elevated liver enzymes, hepatosplenomegaly and pancytopenia, with persistent positive of EBV biomarkers in blood and also positive in liver tissue. The patient was reinfected by SARS-CoV-2 within 2 months companied with CAEBV. The patient's second infection with SARS-CoV-2 led to the aggravated liver dysfunction with pneumonia and re-admission. After receiving symptomatic treatment, the patient showed significantly improvement of symptoms with partially restoration of liver function. After discharge, the patient's health status continued to deteriorate and eventually died. The instances of SARS-CoV-2 co-infection with the original chronic virus are not uncommon, but the exact mechanism of EBV and SARS-CoV-2 coinfection and the relationship between them are still unclear. Since co-infection of SARS-CoV-2 with original chronic virus might affect each other and lead disease aggravated and complicated, it is necessary to differentiate in the diagnosis of disease and it is important to be aware of the re-infection signs of SARS-CoV-2 in people with chronic virus infection diseases, as well as the risk of co-infection of SARS-CoV-2 with other viruses.

KSHV infection of B cells primes protective T cell responses in humanized mice.

Kaposi sarcoma associated herpesvirus (KSHV) is associated with around 1% of all human tumors, including the B cell malignancy primary effusion lymphoma (PEL), in which co-infection with the Epstein Barr virus (EBV) can almost always be found in malignant cells. Here, we demonstrate that KSHV/EBV co-infection of mice with reconstituted human immune systems (humanized mice) leads to IgM responses against both latent and lytic KSHV antigens, and expansion of central and effector memory CD4+ and CD8+ T cells. Among these, KSHV/EBV dual-infection allows for the priming of CD8+ T cells that are specific for the lytic KSHV antigen K6 and able to kill KSHV/EBV infected B cells. This suggests that K6 may represent a vaccine antigen for the control of KSHV and its associated pathologies in high seroprevalence regions, such as Sub-Saharan Africa.

MicroRNA-focused CRISPR/Cas9 screen identifies miR-142 as a key regulator of Epstein-Barr virus reactivation.

Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection. To gain insight into the host miRNAs regulating transitions from EBV latency into the lytic stage, we conducted a CRISPR/Cas9-based screen in EBV+ Burkitt lymphoma (BL) cells using anti-Ig antibodies to crosslink the B cell receptor (BCR) and induce reactivation. Using a gRNA library against >1500 annotated human miRNAs, we identified miR-142 as a key regulator of EBV reactivation. Genetic ablation of miR-142 enhanced levels of immediate early and early lytic gene products in infected BL cells. Ago2-PAR-CLIP experiments with reactivated cells revealed miR-142 targets related to Erk/MAPK signaling, including components directly downstream of the B cell receptor (BCR). Consistent with these findings, disruption of miR-142 enhanced SOS1 levels and Mek phosphorylation in response to surface Ig cross-linking. Effects could be rescued by inhibitors of Mek (cobimetinib) or Raf (dabrafenib). Taken together, these results show that miR-142 functionally regulates SOS1/Ras/Raf/Mek/Erk signaling initiated through the BCR and consequently, restricts EBV entry into the lytic cycle.

EBV promotes TCR-T-cell therapy resistance by inducing CD163+M2 macrophage polarization and MMP9 secretion.

BACKGROUND: Epstein-Barr virus (EBV) is a double-stranded DNA oncogenic virus. Several types of solid tumors, such as nasopharyngeal carcinoma, EBV-associated gastric carcinoma, and lymphoepithelioma-like carcinoma of the lung, have been linked to EBV infection. Currently, several TCR-T-cell therapies for EBV-associated tumors are in clinical trials, but due to the suppressive immune microenvironment of solid tumors, the clinical application of TCR-T-cell therapy for EBV-associated solid tumors is limited. Figuring out the mechanism by which EBV participates in the formation of the tumor immunosuppressive microenvironment will help T cells or TCR-T cells break through the limitation and exert stronger antitumor potential. METHODS: Flow cytometry was used for analyzing macrophage differentiation phenotypes induced by EBV-infected and EBV-uninfected tumors, as well as the function of T cells co-cultured with these macrophages. Xenograft model in mice was used to explore the effects of M2 macrophages, TCR-T cells, and matrix metalloprotein 9 (MMP9) inhibitors on the growth of EBV-infected tumors. RESULTS: EBV-positive tumors exhibited an exhaustion profile of T cells, despite the presence of a large T-cell infiltration. EBV-infected tumors recruited a large number of mononuclear macrophages with CCL5 and induced CD163+M2 macrophages polarization through the secretion of CSF1 and the promotion of autocrine IL10 production by mononuclear macrophages. Massive secretion of MMP9 by this group of CD163+M2 macrophages induced by EBV infection was an important factor contributing to T-cell exhaustion and TCR-T-cell therapy resistance in EBV-positive tumors, and the use of MMP9 inhibitors improved the function of T cells cocultured with M2 macrophages. Finally, the combination of an MMP9 inhibitor with TCR-T cells targeting EBV-positive tumors significantly inhibited the growth of xenografts in mice. CONCLUSIONS: MMP9 inhibitors improve TCR-T cell function suppressed by EBV-induced M2 macrophages. TCR-T-cell therapy combined with MMP9 inhibitors was an effective therapeutic strategy for EBV-positive solid tumors.

Targeted eradication of EBV-positive cancer cells by CRISPR/dCas9-mediated EBV reactivation in combination with ganciclovir.

Epstein-Barr virus (EBV) is a ubiquitous human tumor virus that establishes lifelong, persistent infections in B cells. The presence of EBV in cancer cells presents an opportunity to target these cells by reactivating the virus from latency. In this study, we developed a novel approach for EBV reactivation termed clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated EBV reactivation (CMER) strategy. Using modified CRISPR-associated protein 9 (dCas9) fused with VP64, we designed 10 single guide RNAs (sgRNAs) to target and activate the EBV immediate-early gene promoter. In Akata Burkitt lymphoma cells, 9 out of 10 CMER sgRNAs effectively reactivated EBV. Among these, CMER sgRNA-5 triggered robust reactivation across various cell types, including lymphoma, gastric cancer, and nasopharyngeal carcinoma cells. Importantly, the combination of CMER and ganciclovir selectively eliminated EBV-positive cells, regardless of their cell origin. These findings indicate that targeted virus reactivation by CMER, combined with nucleoside analog therapy, holds promise for EBV-associated cancer treatment. IMPORTANCE: This study explores a novel strategy called clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated Epstein-Barr virus (EBV) reactivation (CMER) to reactivate the Epstein-Barr virus in cancer cells. EBV is associated with various cancers, and reactivating EBV from latency offers a potential therapeutic strategy. We utilized an enzymatically inactive CRISPR-associated protein 9 (dCas9) fused with VP64 and designed 10 single guide RNAs to target the EBV immediate-early gene promoter. Nine of these sgRNAs effectively reactivated EBV in Burkitt lymphoma cells, with CMER sgRNA-5 demonstrating strong reactivation across different cancer cell types. Combining CMER with ganciclovir selectively eliminated EBV-positive cells, showing promise for EBV-associated cancer treatment.

Polymerase theta is a synthetic lethal target for killing Epstein-Barr virus lymphomas.

Treatment options for Epstein-Barr virus (EBV)-cancers are limited, underscoring the need for new therapeutic approaches. We have previously shown that EBV-transformed cells and cancers lack homologous recombination (HR) repair, a prominent error-free pathway that repairs double-stranded DNA breaks; instead, EBV-transformed cells demonstrate genome-wide scars of the error-prone microhomology-mediated end joining (MMEJ) repair pathway. This suggests that EBV-cancers are vulnerable to synthetic lethal therapeutic approaches that target MMEJ repair. Indeed, we have previously found that targeting PARP, an enzyme that contributes to MMEJ, results in the death of EBV-lymphoma cells. With the emergence of clinical resistance to PARP inhibitors and the recent discovery of inhibitors of Polymerase theta (POLθ), the polymerase essential for MMEJ, we investigated the role of POLθ in EBV-lymphoma cells. We report that EBV-transformed cell lines, EBV-lymphoma cell lines, and EBV-lymphomas in AIDS patients demonstrate greater abundance of POLθ, driven by the EBV protein EBNA1, compared to EBV-uninfected primary lymphocytes and EBV-negative lymphomas from AIDS patients (a group that also abundantly expresses POLθ). We also find POLθ enriched at cellular DNA replication forks and exposure to the POLθ inhibitor Novobiocin impedes replication fork progress, impairs MMEJ-mediated repair of DNA double-stranded breaks, and kills EBV-lymphoma cells. Notably, cell killing is not due to Novobiocin-induced activation of the lytic/replicative phase of EBV. These findings support a role for POLθ not just in DNA repair but also DNA replication and as a therapeutic target in EBV-lymphomas and potentially other EBV-cancers as EBNA1 is expressed in all EBV-cancers.IMPORTANCEEpstein-Barr virus (EBV) contributes to ~2% of the global cancer burden. With a recent estimate of >200,000 deaths a year, identifying molecular vulnerabilities will be key to the management of these frequently aggressive and treatment-resistant cancers. Building on our earlier work demonstrating reliance of EBV-cancers on microhomology-mediated end-joining repair, we now report that EBV lymphomas and transformed B cell lines abundantly express the MMEJ enzyme POLθ that likely protects cellular replication forks and repairs replication-related cellular DNA breaks. Importantly also, we show that a newly identified POLθ inhibitor kills EBV-cancer cells, revealing a novel strategy to block DNA replication and repair of these aggressive cancers.