Background/Objectives: Though PARP inhibition has improved the management of advanced prostate cancer, patient outcomes may be modest and disease progression on treatment is common. We sought to improve understanding of tumor cell response to PARP inhibition to support development of novel strategies to enhance and/or prolong PARP inhibitor (PARPi) efficacy. Methods: Cell viability assays and microscopy were used for initial characterization of PARPi response in models of advanced prostate cancer. RNA sequencing was performed to investigate time-dependent transcriptomic changes induced by PARP inhibition. Western blots, flow cytometry, and both additional viability assays and microscopy were used to validate RNA sequencing results and test potential therapeutic strategies. Results: Characterization of responses to PARP inhibition reveals time-dependent changes which may be targeted to improve treatment efficacy. In line with the expected PARPi mechanism of action, short-term treatment is largely associated with activation of ATM and the DNA damage response and cell cycle checkpoint signaling. Targeting ATM with clinical stage inhibitors significantly enhances reduction of tumor cell viability by PARP inhibition. Tumor cells exposed to longer-term treatment exhibit SLUG-dependent epithelial-mesenchymal transition (EMT) and evidence for altered fatty acid metabolism, both of which may be targeted to enhance PARPi anti-tumor cell effects. Conclusions: This study provides insight into both short and longer-term cellular response to PARPi treatment and provides a foundation for additional efforts to explore effective strategies to maximize the utility of PARP inhibition for managing prostate cancer.

Kaposi sarcoma associated herpesvirus (KSHV) is the causative agent of Kaposi sarcoma (KS) and two human B cell lymphoproliferative diseases. KSHV-encoded latency-associated nuclear antigen (LANA) is expressed in KSHV-infected cancer cells. Thus, LANA is an attractive target for therapeutic intervention for KSHV-associated diseases. Here, we devised a cancer gene therapy vector using the adeno-associated virus (AAV), which capitalizes on the LANA's function to maintain terminal repeat (TR)-containing circular genomes and the TR's enhancer function for KSHV-inducible gene promoters. By including two TR copies with a lytic inducible gene promoter (TR2-OriP), we prepared an AAV vector, which expresses an engineered thymidine kinase (TK) selectively in KSHV-infected cells. Ganciclovir (GCV), an anti-herpesvirus drug, effectively eradicated multiple KSHV-infected cells that include induced pluripotent stem cell-derived epithelial colony-forming cells but not non-KSHV-infected counterparts in the presence of AAV8-TR2-OriP-TK. In addition, AAV8-TR2-OriP-TK prevents KSHV from producing virions from reactivated cells. Anti-cancer drugs, known to reactivate KSHV, stimulated TK expression from the vector and, therefore, synergized with AAV8-TR2-OriP-TK/GCV. Finally, the AAV8-TR2-OriP-TK/GCV effectively suppressed the growth of KSHV-infected cancer cells in the xenograft tumor model, whereas systemic intravenous AAV8-TR2-OriP-TK injection/GCV showed no detectable side effects. Our proof-of-concept studies highlight a promising strategy for targeting cancers driven by herpesviruses.

Canine oral squamous cell carcinoma (COSCC) is the second most common oral tumor in dogs and the most relevant for comparative human trials as a spontaneous large animal model of disease. Historical genomic work has focused primarily on bulk sequencing. The present study describes the complete transcriptomic landscape of COSCC with spatial distinction between the surface tumor, deep invasive tumor, peritumoral dysplastic epithelium, and tumor microenvironment compared to matched normal oral samples. Each region demonstrated distinct molecular signatures. Genes related to epithelial growth factor (EGFR) and epithelial-mesenchymal transformation (EMT) were upregulated in both peritumoral dysplasia and surface cancer. Additionally, the KRAS gene set, KRT17, and SSP1 were enriched in cancer. We identified five genes that represent dysplastic lesion with high potential for malignant transformation (FZD4, GAS1, HACD2, NOG, and SLC39A6). Also, three genes, SFRP4, FZD1, and IL34 represented a specific signature of the invasive portion of the COSCC that should be explored for prognostic value as a biomarker of malignancy. Lastly, we verified the immunomodulatory tumor microenvironment detecting an increase in macrophages and an abundance of IL-10 secretion. The other predominant leukocytes were T-cells, with CD4+ T-cells being the most prevalent. CD4+ T cells expressed transcripts for both stimulatory (Inducible T-cell Co-Stimulator (ICOS) and inhibitory molecules (CTLA4). The observed high CTLA4 suggests that this inhibitory signal may be preventing a robust antitumor immune response. Taken together, this study identified multiple targets to be explored for biomarkers of malignancy, prediction of tumor behavior, and potential targets for development of novel therapies.

Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection, and viral genes are poised to be transcribed in the latent chromatin. In the poised chromatins, KSHV latency-associated nuclear antigen (LANA) interacts with cellular chromodomain-helicase-DNA-binding protein 4 (CHD4) and inhibits viral promoter activation. CHD4 is known to regulate cell differentiation by preventing enhancers from activating promoters. Here, we identified a putative CHD4 inhibitor peptide (VGN73) from the LANA sequence corresponding to the LANA-CHD4 interaction surface. The VGN73 interacts with CHD4 at its PHD domain with a dissociation constant (KD) of 14 nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.

S-Figure 1. KSHV reactivation by PEP005; S-Figure 2. Chromatin immunoprecipitations; S-Figure 3. Intersection analysis of genes differentially-expressed in response to each treatment in different PEL cell lines.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic double-stranded DNA virus and the etiologic agent of Kaposi's sarcoma and hyperinflammatory lymphoproliferative disorders. Understanding the mechanism by which KSHV increases the infected cell population is crucial for curing KSHV-associated diseases. Using scRNA-seq, we demonstrate that KSHV preferentially infects CD14+ monocytes, sustains viral lytic replication through the viral interleukin-6 (vIL-6), which activates STAT1 and 3, and induces an inflammatory gene expression program. To study the role of vIL-6 in monocytes upon KSHV infection, we generated recombinant KSHV with premature stop codon (vIL-6(-)) and its revertant viruses (vIL-6(+)). Infection of the recombinant viruses shows that both vIL-6(+) and vIL-6(-) KSHV infection induced indistinguishable host anti-viral response with STAT1 and 3 activations in monocytes; however, vIL-6(+), but not vIL-6(-), KSHV infection promoted the proliferation and differentiation of KSHV-infected monocytes into macrophages. The macrophages derived from vIL-6(+) KSHV infection showed a distinct transcriptional profile of elevated IFN-pathway activation with immune suppression and were compromised in T-cell stimulation function compared to those from vIL-6(-) KSHV infection or uninfected control. Notably, a viral nuclear long noncoding RNA (PAN RNA), which is required for sustaining KSHV gene expression, was substantially reduced in infected primary monocytes upon vIL-6(-) KSHV infection. These results highlight the critical role of vIL-6 in sustaining KSHV transcription in primary monocytes. Our findings also imply a clever strategy in which KSHV utilizes vIL-6 to secure its viral pool by expanding infected monocytes via differentiating into longer-lived dysfunctional macrophages. This mechanism may facilitate KSHV to escape from host immune surveillance and to support a lifelong infection.

Kaposi sarcoma-associated herpesvirus (KSHV) inflammatory cytokine syndrome (KICS) is a newly described chronic inflammatory disease condition caused by KSHV infection and is characterized by high KSHV viral load and sustained elevations of serum KSHV-encoded IL-6 (vIL-6) and human IL-6 (hIL-6). KICS has significant immortality and greater risks of other complications, including malignancies. Although prolonged inflammatory vIL-6 exposure by persistent KSHV infection is expected to have key roles in subsequent disease development, the biological effects of prolonged vIL-6 exposure remain elusive. Using thiol(SH)-linked alkylation for the metabolic (SLAM) sequencing and Cleavage Under Target & Release Using Nuclease analysis (CUT&RUN), we studied the effect of prolonged vIL-6 exposure in chromatin landscape and resulting cytokine production. The studies showed that prolonged vIL-6 exposure increased Bromodomain containing 4 (BRD4) and histone H3 lysine 27 acetylation co-occupancies on chromatin, and the recruitment sites were frequently co-localized with poised RNA polymerase II with associated enzymes. Increased BRD4 recruitment on promoters was associated with increased and prolonged NF-κB p65 binding after the lipopolysaccharide stimulation. The p65 binding resulted in quicker and sustained transcription bursts from the promoters; this mechanism increased total amounts of hIL-6 and IL-10 in tissue culture. Pretreatment with the BRD4 inhibitors, OTX015 and MZ1, eliminated the enhanced inflammatory cytokine production. These findings suggest that persistent vIL-6 exposure may establish a chromatin landscape favorable for the reactivation of inflammatory responses in monocytes. This epigenetic memory may explain the greater risk of chronic inflammatory disease development in KSHV-infected individuals.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic double-stranded DNA virus and the etiologic agent of Kaposi's sarcoma and hyperinflammatory lymphoproliferative disorders. Understanding the mechanism by which KSHV increases the infected cell population is crucial for curing KSHV-associated diseases. Here we demonstrate that KSHV preferentially infects CD14 + monocytes and sustains viral replication through the viral interleukin-6 (vIL6)-mediated activation of STAT1 and 3. Using vIL6-sufficient and vIL6-deficient recombinant KSHV, we demonstrated that vIL6 plays a critical role in promoting the proliferation and differentiation of KSHV-infected monocytes into macrophages. The macrophages derived from vIL6-sufficient KSHV infection showed a distinct transcriptional profile of elevated IFN-pathway activation with immune suppression and were compromised in T-cell stimulation function compared to those from vIL6-deficient KSHV infection or uninfected control. These results highlight a clever strategy, in which KSHV utilizes vIL6 to secure its viral pool by expanding infected dysfunctional macrophages. This mechanism also facilitates KSHV to escape from host immune surveillance and to establish a lifelong infection. 160. Summary:KSHV causes multiple inflammatory diseases, however, the underlying mechanism is not clear. Shimoda et al. demonstrate that KSHV preferentially infects monocytes and utilizes virally encoded interleukin-6 to expand and deregulate infected monocytes. This helps the virus escape from host immune surveillance.

Prolonged Kaposi's Sarcoma-associated Herpesvirus vIL-6 Exposure 1 Enhances Inflammatory Responses by Epigenetic Reprogramming 2 3 Tomoki Inagaki, Kang-Hsin Wang, Ashish Kumar, Chie Izumiya, Hiroki Miura, 4 Somayeh Komaki, Ryan R. Davis, Clifford G. Tepper, Harutaka Katano, 5 Michiko Shimoda, & Yoshihiro Izumiya 3, * 6 7 1 Department of Dermatology, School of Medicine, the University of California Davis (UC Davis), 8

In leukemia, a distinct subpopulation of cancer-initiating cells called leukemia stem cells (LSCs) is believed to drive population expansion and tumor growth. Failing to eliminate LSCs may result in disease relapse regardless of the amount of non-LSCs destroyed. The first step in targeting and eliminating LSCs is to identify and characterize them. Acute precursor B lymphoblastic leukemia (B-ALL) cells derived from patients were incubated with fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose (NBDG) and sorted based on NBDG uptake. Cell subpopulations defined by glucose uptake were then serially transplanted into mice and evaluated for leukemia initiating capacity. Gene expression profiles of these cells were characterized using RNA-Sequencing (RNA-Seq). A distinct population of NBDG-low cells was identified in patient B-ALL samples. These cells are a small population (1.92% of the entire leukemia population), have lower HLA expression, and are smaller in size (4.0 to 7.0 μm) than the rest of the leukemia population. All mice transplanted with NBDG-low cells developed leukemia between 5 and 14 weeks, while those transplanted with NBDG-high cells did not develop leukemia (p ≤ 0.0001-0.002). Serial transplantation of the NBDG-low mouse model resulted in successful leukemia development. NBDG-medium (NBDG-med) populations also developed leukemia. Interestingly, comprehensive molecular characterization of NBDG-low and NBDG-med cells from patient-derived xenograft (PDX) models using RNA-Seq revealed a distinct profile of 2,162 differentially-expressed transcripts (DETs) (p<0.05) with 70.6% down-regulated in NBDG-low cells. Hierarchical clustering of DETs showed distinct segregation of NBDG-low from NBDG-med and NBDG-high groups with marked transcription expression alterations in the NBDG-low group consistent with cancer survival. In conclusion, A unique subpopulation of cells with low glucose uptake (NBDG-low) in B-ALL was discovered. These cells, despite their quiescence characteristics, once transplanted in mice, showed potent leukemia initiating capacity. Although NBDG-med cells also initiated leukemia, gene expression profiling revealed a distinct signature that clearly distinguishes NBDG-low cells from NBDG-med and the rest of the leukemia populations. These results suggest that NBDG-low cells may represent quiescent LSCs. These cells can be activated in the appropriate environment in vivo, showing leukemia initiating capacity. Our study provides insight into the biologic mechanisms of B-ALL initiation and survival.

Kaposi sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with CHD4 and ADNP proteins, components of the cellular ChAHP complex. The CHD4 and ADNP proteins occupy the 5'-region of the highly inducible lncRNAs and terminal repeats of the KSHV genome together with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding, and KSHV reactivation sequesters CHD4 from the KSHV genome, which is also accompanied by detachment of KSHV episomes from host chromosome docking sites. We propose a model in which robust KSHV lncRNA expression determines the latency-lytic decision by regulating LANA/CHD4 binding to KSHV episomes.

The tumor microenvironment (TME) subtype is a critical factor when combining immunotherapy with tumor debulking strategies such as ultrasound ablation. To understand the effects of the TME subtype, we studied two multi-site murine cancer models, an immune-suppressed KPC (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) pancreatic adenocarcinoma (MT4) model and a neu deletion HER2+ (NDL) mammary adenocarcinoma model with a larger naïve lymphocyte population. After profiling the two models with histology and single-cell sequencing, we designed immunotherapy combinations with Magnetic Resonance-guided Focused Ultrasound (MRgFUS) thermal ablation that were specific to each TME subtype. To understand the impact of the molecular and cellular differences upon each model, we characterized the treatment effect with transcriptomic analysis and digital cytometry. We found that combining MRgFUS ablation with immunotherapy reduced tumor growth and extended survival by at least 2-fold in the MT4 pancreatic cancer model. Gene set enrichment analysis (GSEA) of bulk RNA sequencing data indicated that the combination of ablation with immunotherapy preferentially enriched functional annotations for leukocyte migration in the MT4 model and wound healing and inflammatory cytokines in the NDL model (e.g. for IL6: 1.6 fold increase in MT4 vs 10.3 fold in NDL tumors). We then particularly focused on whether dendritic cell (DC) activation and CD4 T cell populations could be enhanced when ablating pancreatic tumors. By CIBERSORTx digital cytometry, aCD40 + aPD-1 combinations increased the presence of activated DCs in MT4 tumors by 2.4-fold. Activated DCs were also increased by CP4 (aCD40 + aPD-1 + CTLA-4) alone 1.7 fold or in combination with ablation in both treated (2.4 fold) and distant (2.2 fold) lesions. Ablation combined with aPD-1 alone did not significantly impact DCs in the MT4 model. In addition, CD4 lymphocytes increased 5 and 8 fold in treated and distant tumors by ablation combined with CP4 in the MT4 model. Taken together, inflammatory cytokines were preferentially elevated by ablation in a dense epithelial breast cancer as compared with a pancreatic tumor model. Further, the results suggest that aCD40 immunotherapy retains efficacy when combined with thermal ablation in pancreatic tumors. This work can inform clinical translation of ablative and immunotherapy combination protocols. Citation Format: James Wang, Brett Z. Fite, Aris Kare, Bo Wu, Marina N. Raie, Spencer Tumbale, Ryan R. Davis, Clifford G. Tepper, Katherine Ferrara. TME subtype impacts response to combined thermal ablation and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6314.

Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 base pair resolution and constructed a 3D KSHV genomic structural model with 2kb binning. The latent KSHV genome formed very similar genomic architectures in three different naturally infected PEL cell lines and in an experimentally infected epithelial cell line. The majority of the TAD boundaries were occupied by CTCF and SMC1, and the KSHV transactivator was recruited to these sites during reactivation. Triggering KSHV gene expression decreased pre-wired genomic loops within the regulatory unit, while contacts extending outside of regulatory borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D genomic structural model proposes that the immediate-early promoter region is localized on the periphery of the 3D viral genome during latency, while highly inducible non-coding RNA regions moved toward the inner space of the structure, resembling the configuration of a “bird cage” during reactivation. The compartment-like properties of viral episomal chromatin structure and its reorganization during the transition from latency may help coordinate viral gene transcription. Importance The 3D architecture of chromatin allows for efficient arrangement, expression, and replication of genetic material. The genomes of all organisms studied to date have been found to be organized through some form of tiered domain structures. However, the architectural framework of the genomes of large double-stranded DNA viruses such as the herpesvirus family has not been reported. Prior studies with Kaposi’s sarcoma-associated herpesvirus (KSHV) have indicated that the viral chromatin shares many biological properties exhibited by the host cell genome, essentially behaving as a mini human chromosome. Thus, we hypothesized that the KSHV genome may be organized in a similar manner. In this report, we describe the domain structure of the latent and lytic KSHV genome at 500 base pair resolution and present a 3D genomic structural model for KSHV under each condition. These results add new insights into the complex regulation of the viral lifecycle.

Background: Although combination immunotherapies incorporating local and systemic components have shown promising results in treating solid tumors, varied tumor microenvironments (TMEs) can impact immunotherapeutic efficacy.Method: We designed and evaluated treatment strategies for breast and pancreatic cancer combining magnetic resonance-guided focused ultrasound (MRgFUS) ablation and antibody therapies. With a combination of single-cell sequencing, spectral flow cytometry, and histological analyses, we profiled an immune-suppressed KPC (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) pancreatic adenocarcinoma (MT4) model and a dense epithelial neu deletion (NDL) HER2+ mammary adenocarcinoma model with a greater fraction of lymphocytes, natural killer cells and activated dendritic cells. We then performed gene ontology analysis, spectral and digital cytometry to assess the immune response to combination immunotherapies and correlation with survival studies.Result: Based on gene ontology analysis, adding ablation to immunotherapy enriched immune cell migration pathways in the pancreatic cancer model and extensively enriched wound healing pathways in the breast cancer model. With CIBERSORTx digital cytometry, aCD40 + aPD-1 immunotherapy combinations enhanced dendritic cell activation in both models. In the MT4 TME, adding the combination of aCD40 antibody and checkpoint inhibitors (aPD-1 and aCTLA-4) with ablation was synergistic, increasing activated natural killer cells and T cells in distant tumors. Furthermore, ablation with immunotherapy upregulated critical Ly6c myeloid remodeling phenotypes that enhance T-cell effector function and increased granzyme and protease encoding genes by as much as 100-fold. Ablation combined with immunotherapy then extended survival in the MT4 model to a greater extent than immunotherapy alone.Conclusion: In summary, TME profiling informed a successful multicomponent treatment protocol incorporating ablation and facilitated differentiation of TMEs in which ablation is most effective.

Management of critically ill patients requires simultaneous administration of many medications. Treatment for patient comorbidities may lead to drug-drug interactions which decrease drug efficacy or increase adverse reactions. Current practices rely on a one-size-fits-all dosing approach. Pharmacogenetic testing is generally reserved for addressing problems rather than used proactively to optimize care. We hypothesized that burn and surgery patients will have one or more genetic variants in drug metabolizing pathways used by one or more medications administered during the patient's hospitalization. The aim of this study was to determine the frequency of variants with abnormal function in the primary drug pathways and identify which medications may be impacted. Genetic (19 whole exome and 11 whole genome) and medication data from 30 pediatric burn and surgery patients were analyzed to identify pharmacogene-drug associations. Nineteen patients were identified with predicted altered function in one or more of the following genes: CYP2C9, CYP2C19, CYP2D6, and CYP3A4. The majority had decreased function, except for several patients with CYP2C19 rapid or ultrarapid variants. Some drugs administered during hospitalization that rely on these pathways include hydrocodone, oxycodone, methadone, ibuprofen, ketorolac, celecoxib, diazepam, famotidine, diphenhydramine, and glycopyrrolate. Approximately one-third of the patients tested had functionally impactful genotypes in each of the primary drug metabolizing pathways. This study suggests that genetic variants may in part explain the vast variability in drug efficacy and suggests that future pharmacogenetics research may optimize dosing regimens.

High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65 °C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3 , Jun, Mefv, Il6 and Il1β and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA . The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2 , Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.

In previous studies, we have shown that expression of a viral lncRNA, polyadenylated nuclear RNA (PAN RNA) is essential for inducible viral genomic looping and distal gene activation during Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation. Here we identify viral lncRNA binding proteins, and show that an underlying molecular mechanism regulating the KSHV latency-lytic replication switch is via a viral lncRNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. Proximity biotin labeling, single cell transcriptomics, and siRNA screening along with complementation studies identified that CHD4's enzymatic activity silences viral gene expression by preventing transcription factory formation. Furthermore, Capture Hi-C, Cleavage Under Targets and Release Using Nuclease (CUT&RUN), and proteomics approaches together identify KSHV episome docking sites on host chromosomes and colocalization with a CHD4 protein complex, ChAHP, at epigenetically active genomic regions. PAN RNA binds and competes with CHD4 DNA binding in vitro, and KSHV episomes detached from these host genomic loci sites when reactivation is triggered. Our studies suggest that CHD4 exhibits strong repressor function by preventing inducible genomic looping, and is therefore important for the ability of KSHV to establish and maintain latency in a "poised" state at selected host genomic loci.

Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with a CHD4 complex, ChAHP, at epigenetically active genomic regions and tethers frequently near centromeric regions of host chromosomes. The ChAHP complex also occupies the 5’-region of a highly-inducible lncRNAs and terminal repeats of KSHV genome with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding , and KSHV reactivation is accompanied by the detachment of KSHV episomes from host chromosome docking sites We propose a model in which elevated lncRNA expression determines the KSHV latency-lytic decision by regulating LANA/ChAHP DNA binding at inducible viral enhancers.

In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here, we show a small peptide derived from the Kaposi’s sarcoma-associated herpesvirus transactivator (K-Rta) sequence, which attenuates cellular MYC expression, reduces cell proliferation, and selectively kills cancer cell lines in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide functions as a decoy to block the recruitment of coactivator complexes consisting of Nuclear receptor coactivator 2 (NCOA2), p300, and SWI/SNF proteins to the MYC promoter in primary effusion lymphoma cells. Thiol(SH)-linked alkylation for the metabolic sequencing of RNA ( SLAM seq ) with target-transcriptional analyses further confirm that the viral peptide directly attenuates MYC and MYC-target gene expression. This study thus provides a unique tool to control MYC activation, which may be used as a therapeutic payload to treat MYC-dependent diseases such as cancers and autoimmune diseases.

Sepsis is a leading cause of morbidity and mortality in patients that have sustained a severe burn injury. Early detection and treatment of infections improves outcomes and understanding changes in the host microbiome following injury and during treatment may aid in burn care. The loss of functional barriers, systemic inflammation, and commensal community perturbations all contribute to a burn patient's increased risk of infection. We sampled 10 burn patients to evaluate cutaneous microbial populations on the burn wound and corresponding spared skin on days 0, 3, 7, 14, 21, and 28 post-intensive care unit admission. In addition, skin samples were paired with perianal and rectal locations to evaluate changes in the burn patient gut microbiome following injury and treatment. We found significant (P = 0.011) reduction in alpha diversity on the burn wound compared to spared skin throughout the sampling period as well as reduction in common skin commensal bacteria such as Propionibacterium acnes and Staphylococcus epidermitis. Compared to healthy volunteers (n = 18), the burn patient spared skin also exhibited a significant reduction in alpha diversity (P = 0.001). Treatments such as systemic or topical antibiotic administration, skin grafting, and nutritional formulations also impact diversity and community composition at the sampling locations. When evaluating each subject individually, an increase in relative abundance of taxa isolated clinically by bacterial culture could be seen in 5/9 infections detected among the burn patient cohort.