KSHV vIL-6 enhances inflammatory responses by epigenetic reprogramming.

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.

Virally encoded interleukin-6 facilitates KSHV replication in monocytes and induction of dysfunctional macrophages.

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.

KSHV vIL-6 Enhances Inflammatory Responses by Epigenetic Reprogramming.

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 possesses greater risks of having other complications, which include 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-Linked Alkylation for the Metabolic Sequencing and Cleavage Under Target & Release Using Nuclease analysis, 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 RNAPII 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 inhibitor, OTX015, 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.

KSHV uses viral IL6 to expand infected immunosuppressive macrophages.

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.

Multiomic analysis for optimization of combined focal and immunotherapy protocols in murine pancreatic cancer.

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.

A distinct subpopulation of leukemia initiating cells in acute precursor B lymphoblastic leukemia: quiescent phenotype and unique transcriptomic profile.

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.

KSHV Topologically Associating Domains in Latent and Reactivated Viral Chromatin.

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 bp resolution and constructed a 3D KSHV genomic structural model with 2 kb 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 structural maintenance of chromosomes (SMC1) cohesin complex and CCCTC-binding factor (CTCF), and the KSHV transactivator was recruited to those sites during reactivation. Triggering KSHV gene expression decreased prewired 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 noncoding 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 facilitate 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 bp 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 life cycle.

Pharmacogenetic Gene-Drug Associations in Pediatric Burn and Surgery Patients.

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.

KSHV episome tethering sites on host chromosomes and regulation of latency-lytic switch by CHD4.

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.

KSHV transactivator-derived small peptide traps coactivators to attenuate MYC and inhibits leukemia and lymphoma cell growth.

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.

Improving mAb capture productivity on batch and continuous downstream processing using nanofiber PrismA adsorbents.

Improving productivity and decreasing costs for biotherapeutic agents has been a focal driving force in the manufacturing of biologics. Advances in upstream processes have been continuously outpacing the ability for downstream operations to purify biologics, especially monoclonal antibodies. Continuous chromatography has several benefits for biologic purification including automated control, decreased labor, improved productivity, and more consistent product attributes. The goal of this study was to improve productivity and decrease costs associated with batch-mode and continuous purification processes. Productivities using cellulose nanofibers with a protein A ligand offer greater than 30-fold higher productivities than their resin-based equivalents using periodic countercurrent technology with multiple column chromatography. The smaller columns needed for convective mass transfer, faster processing times, and decreased costs allow for a more efficient mAb capture step. Additionally, high throughput purification (grams of mAbs/day) can be achieved from the scale-down model developed using periodic countercurrent technology. These advancements will help drive the evolution of downstream operations to manage the higher workloads due to increased upstream titers in a cost-effective manner.

Longitudinal profiling of the burn patient cutaneous and gastrointestinal microbiota: a pilot study.

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.

Immunofocusing humoral immunity potentiates the functional efficacy of the AnAPN1 malaria transmission-blocking vaccine antigen.

Malaria transmission-blocking vaccines (TBVs) prevent the completion of the developmental lifecycle of malarial parasites within the mosquito vector, effectively blocking subsequent infections. The mosquito midgut protein Anopheline alanyl aminopeptidase N (AnAPN1) is the leading, mosquito-based TBV antigen. Structure-function studies identified two Class II epitopes that can induce potent transmission-blocking (T-B) antibodies, informing the design of the next-generation AnAPN1. Here, we functionally screened new immunogens and down-selected to the UF6b construct that has two glycine-linked copies of the T-B epitopes. We then established a process for manufacturing UF6b and evaluated in outbred female CD1 mice the immunogenicity of the preclinical product with the human-safe adjuvant Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ). UF6b:GLA-LSQ effectively immunofocused the humoral response to one of the key T-B epitopes resulting in potent T-B activity, underscoring UF6b as a prime TBV candidate to aid in malaria elimination and eradication efforts.

Structural Insights into JAK2 Inhibition by Ruxolitinib, Fedratinib, and Derivatives Thereof.

The discovery that aberrant activity of Janus kinase 2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has led to significant efforts to develop small molecule inhibitors for this patient population. Ruxolitinib and fedratinib have been approved for use in MPN patients, while baricitinib, an achiral analogue of ruxolitinib, has been approved for rheumatoid arthritis. However, structural information on the interaction of these therapeutics with JAK2 remains unknown. Here, we describe a new methodology for the large-scale production of JAK2 from mammalian cells, which enabled us to determine the first crystal structures of JAK2 bound to these drugs and derivatives thereof. Along with biochemical and cellular data, the results provide a comprehensive view of the shape complementarity required for chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as therapeutics.

Immune modulation resulting from MR-guided high intensity focused ultrasound in a model of murine breast cancer.

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 Il1ß 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.

Structural mechanism for regulation of DNA binding of BpsR, a Bordetella regulator of biofilm formation, by 6-hydroxynicotinic acid.

Bordetella bacteria are respiratory pathogens of humans, birds, and livestock. Bordetella pertussis the causative agent of whopping cough remains a significant health issue. The transcriptional regulator, BpsR, represses a number of Bordetella genes relating to virulence, cell adhesion, cell motility, and nicotinic acid metabolism. DNA binding of BpsR is allosterically regulated by interaction with 6-hydroxynicotinic acid (6HNA), the first product in the nicotinic acid degradation pathway. To understand the mechanism of this regulation, we have determined the crystal structures of BpsR and BpsR in complex with 6HNA. The structures reveal that BpsR binding of 6HNA induces a conformational change in the protein to prevent DNA binding. We have also identified homologs of BpsR in other Gram negative bacteria in which the amino acids involved in recognition of 6HNA are conserved, suggesting a similar mechanism for regulating nicotinic acid degradation.

A Syngeneic ErbB2 Mammary Cancer Model for Preclinical Immunotherapy Trials.

In order to develop a practical model of breast cancer, with in vitro and syngeneic, immune-intact, in vivo growth capacity, we established a primary cell line derived from a mammary carcinoma in the transgenic FVB/N-Tg(MMTV-ErbB2*)NDL2-5Mul mouse, referred to as "NDLUCD". The cell line is adapted to standard cell culture and can be transplanted into syngeneic FVB/N mice. The line maintains a stable phenotype over multiple in vitro passages and rounds of in vivo transplantation. NDLUCD tumors in FVB/N mice exhibit high expression of ErbB2 and ErbB3 and signaling molecules downstream of ErbB2. The syngeneic transplant tumors elicit an immune reaction in the adjacent stroma, detected and characterized using histology, immunophenotyping, and gene expression. NDLUCD cells also express PD-L1 in vivo and in vitro, and in vivo transplants are reactive to anti-immune checkpoint therapy with responses conducive to immunotherapy studies. This new NDLUCD cell line model is a practical alternative to the more commonly used 4T1 cells, and our previously described FVB/N-Tg(MMTV-PyVT)634Mul derived Met-1fvb2 and FVB/NTg(MMTV-PyVTY315F/Y322F) derived DB-7fvb2 cell lines. The NDLUCD cells have, so far, remained genetically and phenotypically stable over many generations, with consistent and reproducible results in immune intact preclinical cohorts.

Distinct immune signatures in directly treated and distant tumors result from TLR adjuvants and focal ablation.

Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments. Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components. Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45+ cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45+ cells) compared to the ablation-only (~0.1% of CD45+ cells) and immunotherapy-only cohorts (~10% of CD45+ cells). Macrophages and DCs process and present this antigen to CD8+ T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169+ macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation. Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response.

KIBRA (WWC1) Is a Metastasis Suppressor Gene Affected by Chromosome 5q Loss in Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBCs) display a complex spectrum of mutations and chromosomal aberrations. Chromosome 5q (5q) loss is detected in up to 70% of TNBCs, but little is known regarding the genetic drivers associated with this event. Here, we show somatic deletion of a region syntenic with human 5q33.2-35.3 in a mouse model of TNBC. Mechanistically, we identify KIBRA as a major factor contributing to the effects of 5q loss on tumor growth and metastatic progression. Re-expression of KIBRA impairs metastasis in vivo and inhibits tumorsphere formation by TNBC cells in vitro. KIBRA functions co-operatively with the protein tyrosine phosphatase PTPN14 to trigger mechanotransduction-regulated signals that inhibit the nuclear localization of oncogenic transcriptional co-activators YAP/TAZ. Our results argue that the selective advantage produced by 5q loss involves reduced dosage of KIBRA, promoting oncogenic functioning of YAP/TAZ in TNBC.

High frequency of the PNPLA3 rs738409 [G] single-nucleotide polymorphism in Hmong individuals as a potential basis for a predisposition to chronic liver disease.

BACKGROUND: An exploratory study was performed to determine the prevalence of the patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs78409 [G] allele among the Hmong as a risk factor for nonalcoholic fatty liver disease (NAFLD). NAFLD/nonalcoholic steatohepatitis is the world's most common chronic liver disease and is expected to replace viral hepatitis as the leading cause of cirrhosis and potential precursor to hepatocellular carcinoma (HCC). Of all populations in California, the Hmong experience the highest risk of death from HCC and the highest prevalence of metabolic syndrome risk factors among Asians that predispose them to NAFLD. Here a genetic explanation was sought for the high rates of chronic liver disease among the Hmong. The literature pointed to the PNPLA3 rs738409 [G] allele as a potential genetic culprit. METHODS: Cell-free DNA was isolated from 26 serum samples previously collected in community settings. Quantitative polymerase chain reaction-based single-nucleotide polymorphism (SNP) genotyping was performed with a validated TaqMan SNP genotyping assay, and results were analyzed with TaqMan Genotyper software. RESULTS: The PNPLA3 rs738409 [C>G] variant occurred at a frequency of 0.46 (12 of 26; 95% confidence interval, 0.27-0.67). This carrier rate would rank the Hmong as the third highest population in the 1000 Genomes Project. CONCLUSIONS: Although this small sample size limits the generalizability, the high frequency rates of this allele along with the presence of metabolic syndrome risk factors warrant further studies into the etiology of NAFLD among the Hmong. Cancer 2018;124:1583-9. © 2018 American Cancer Society.