Wild-type Hosts Research Articles

The Transcriptional Program of Staphylococcus aureus Phage K Is Affected by a Host rpoC Mutation That Confers Phage K Resistance.

To better understand host-phage interactions and the genetic bases of phage resistance in a model system relevant to potential phage therapy, we isolated several spontaneous mutants of the USA300 S. aureus clinical isolate NRS384 that were resistant to phage K. Six of these had a single missense mutation in the host rpoC gene, which encodes the RNA polymerase β' subunit. To examine the hypothesis that mutations in the host RNA polymerase affect the transcription of phage genes, we performed RNA-seq analysis on total RNA samples collected from NRS384 wild-type (WT) and rpoCG17D mutant cultures infected with phage K, at different timepoints after infection. Infection of the WT host led to a steady increase of phage transcription relative to the host. Our analysis allowed us to define 53 transcriptional units and to categorize genes based on their temporal expression patterns. Predicted promoter sequences defined by conserved -35, -10, and, in some cases, extended -10 elements, were found upstream of early and middle genes. However, in many cases, sequences upstream of late genes did not contain clear, complete, canonical promoter sequences, suggesting that factors in addition to host RNA polymerase are required for their expression. Infection of the rpoCG17D mutant host led to a transcriptional pattern that was similar to that of the WT at early timepoints. However, beginning at 20 min after infection, transcription of late genes (such as phage structural genes and host lysis genes) was severely reduced. Our data indicate that the rpoCG17D mutation prevents the expression of phage late genes, resulting in a failed infection cycle for phage K. In addition to illuminating the global transcriptional landscape of phage K throughout the infection cycle, this study will inform our investigations into the basis of phage K's control of its transcriptional program as well as mechanisms of phage resistance.

Aire mediates tolerance to insulin through thymic trimming of high-affinity T cell clones

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

Abstract 1540: The dendritic cell receptor DNGR-1 shapes immunity to cancer

Abstract Background: Central to the success of CD8+ T cell-mediated anti-cancer response are type 1 conventional dendritic cells (cDC1s), acting as professional antigen presenting cells. In both mouse and human, DNGR-1 (a.k.a. CLEC9A) is a dedicated cDC1 receptor that, upon binding to its physiological ligand filamentous-actin (F-actin) exposed upon cell death, prime cytotoxic CD8+ T cells specifically against dead cell-associated antigens. Here, we interrogate the role of DNGR-1 in shaping the tumor microenvironment during carcinogenesis. Methods: We utilized two models of chemical carcinogenesis: 3-methylcholanthrene (MCA)-induced sarcoma, and azoxymethane (AOM) and dextran sodium sulfate colitis (DSS)-associated carcinoma. Rag1−/- (no B and T cells), Clec9agfp/gfp, Clec9acre/cre and WT (wildtype) mice on C57BL/6 background were used. Primary MCA-sarcoma cell lines were generated and subsequently tested in a series of transplantation experiments in naïve syngeneic WT mice. Whole exome and RNA sequencing were performed to elucidate the mutational landscape and decipher the immunopeptidome of these primary cell lines. Results: Upon challenge with MCA, there is shorter latency for tumorigenesis in DNGR-1-deficient mice (n=46), like in Rag1−/− mice (n=43), in contrast to immunocompetent WT hosts (n=59) (median: 95 vs 113 days, hazard ratio: 1.72, 95% CI 1.11-2.68, p=0.008). DNGR-1-deficient mice also display increased susceptibility to AOM-DSS carcinogenesis compared to co-housed WT mice. Majority (n=16/24, 68%) of MCA-induced primary sarcoma cell lines derived from DNGR-1-deficient hosts are highly immunogenic and were controlled/rejected at a similar rate, upon challenge in secondary naïve WT recipients, like those derived from Rag1−/− mice (n=13/19, 68%). Whole-exome sequencing of cell lines derived from DNGR-1-deficient hosts revealed that they harbor high mutational burden, including mutations in F-actin-binding proteins that have been previously associated with better survival outcomes in patients with cancer (Giampazolias et al. Cell, 2021). Collectively, these data argue that DNGR-1 protects against carcinogenesis and selects for less immunogenic tumor cells. To evaluate the degree of immunoediting which occurred in the respective primary host mice, we applied the evolutionary metric based on the ratio of nonsynonymous to synonymous mutations (dN/dS), and found that they are less immunoedited compared to cell lines derived from WT hosts. Conclusion: Our findings highlight DNGR-1 as a novel immune checkpoint that couples cell death sensing and (neo-)antigen cross-presentation to functional tumor-specific CD8+ T cell responses during cancer immunoediting. Understanding the mechanism by which DNGR-1 sculpts the mutational landscape of tumors may further inform the dynamics of cancer evolution and may have translational therapeutic implications in directing effective immunotherapy strategies. Citation Format: Kok Haw Lim, Evangelos Giampazolias, Bruno Frederico, Neil C. Rogers, Probir Chakravarty, Gavin Kelly, Ana Cardoso, Emma Nye, Alejandro Suárez-Bonnet, Simon Priestnall, Robert Goldstone, Cell Services Science Technology Platform, Sonia Lee, Oliver Schulz, Jessica Strid, Luis Zapata, Caetano Reis e Sousa. The dendritic cell receptor DNGR-1 shapes immunity to cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1540.

Abstract A084: AMH paracrine signaling between cancer and mesothelial cells modulates the ovarian tumor immune microenvironment

Abstract A084: AMH paracrine signaling between cancer and mesothelial cells modulates the ovarian tumor immune microenvironment

Abstract B028: Fibroblast PIN1 regulates growth factor and metabolic crosstalk to impact a subset of pancreatic cancer cells

Abstract Pancreatic cancer associated fibroblasts (CAFs) play multiple, often conflicting, roles to support or restrict tumor growth. Recent work suggests that heterogeneous differentiation states of fibroblasts contribute to these diverse functions and that reprogramming of fibroblast states can influence tumor outcomes. How distinct fibroblast subpopulations affect heterogeneous tumor development and growth, however, remains incompletely understood. PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. While PIN1 is known to be overexpressed in cancer and to contribute to cancer cell-intrinsic pro-tumorigenic behaviors, the direct effects of PIN1 inhibition on tumor-associated stromal phenotypes are poorly understood. We have identified a role for PIN1 in regulating fibroblast state and function. Loss or inhibition of PIN1 alters the epigenetic response to TGF-beta, resulting in lower expression of alpha-SMA, a marker of myofibroblast-like CAFs. PIN1 low pancreatic stellate cells (PSCs) or CAFs display decreased proliferation, decreased ECM deposition, and altered paracrine signaling to cancer cells and other stromal cells. In particular, HGF expression was markedly decreased in PIN1 low fibroblasts. Conditioned media from control PSCs was found to activate the MET receptor on cancer cells, driving changes consistent with an epithelial to mesenchymal transition, while conditioned media from shPIN1 PSCs failed to drive these changes. In vivo loss or inhibition of PIN1 significantly decreased tumor growth in multiple mouse models, and consistent with our in vitro data, the smaller lesions in the autochthonous PIN1 knockout KPC mice also displayed decreased expression of alpha-SMA and decreased activation of pMET. To determine the impact of stromal PIN1 on tumor growth, we orthotopically injected KPC pancreatic tumor cell lines into syngeneic PIN1 wildtype or knockout hosts. We found that growth of some cell lines (e.g. KPC8060) were significantly reduced in PIN1 knockout compared to wildtype hosts, while growth of other cells (e.g. KPC7107) were similar in PIN1 wildtype and knockout hosts. In vitro, we found that the KPC8060 cell line increased proliferation and lactate production in response to exogenous HGF, while the KPC7107 cell line did not. This work supports a model in which a subset of pancreatic tumors depends on crosstalk from PIN1 expressing fibroblasts in the tumor microenvironment. We are currently using 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to further understand how PIN1 loss in fibroblasts influences the full tumor microenvironment and to further characterize the dependency of heterogenous pancreatic cancer cells on specific components from a PIN1 sufficient microenvironment. Citation Format: Chloe L. Bowman, Eric J. Carlson, Vidhi Shah, Colin J. Daniel, Xiaoyan Wang, Motoyuki Tsuda, Rosalie C. Sears, Ellen M. Langer. Fibroblast PIN1 regulates growth factor and metabolic crosstalk to impact a subset of pancreatic cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B028.

Getting around an Early Lethal Phenotype in Mice with Chimeras.

The same gene can have many different functions in different places in the body and/or at different times in development and adult life. Often only one organ or one developmental stage is of particular interest to an investigator. If, however, lethality or severe detrimental effects of a mutation prevent the study of the organ or stage of interest, there are a number of ways to circumvent an early effect. In this overview, we discuss one way of getting around an early lethal phenotype by using chimeras, a method that is also useful for studying the mutant cells in the context of a wild-type host as part of the phenotypic analysis. The composition of chimeras with respect to embryonic cell lineages can be controlled to some extent to produce lineage-restricted chimeras with, for example, mutant cells restricted to certain lineages. Depending on the site of action of the mutant gene, this could result in chimeric "rescue." Details of how to distinguish mutant cells from wild type, an essential part of any chimera experiment, are discussed as well as methods to genotype the chimeras with respect to both component cell types.

Uncovering Phenotypes in Mutant Mice by Determining Embryo, Organ, Tissue, and Cell Developmental Potential.

The death of an embryo during gestation does not necessarily preclude the study of the mutant embryo or the developmental potential of its individual cells, tissues, or organs. Whole-embryo in vitro culture prior to the time of death will allow real-time observation of living embryos and direct comparisons with controls. Organ anlage can be removed from embryos and cultured in vitro beyond the time of death of the whole embryo. In both whole embryos and organ anlage culture, fluorescent protein reporters may be used productively to follow cell types or specific gene expression changes. Some cells, such as hematopoietic cells, and organ anlage, may be suitable for transplantation to wild-type hosts for further analysis of their potential. Additionally, cell lines, including embryonic stem (ES) cells, trophoblast stem (TS) cells, extraembryonic endoderm (XEN) stem cells, and epiblast-derived stem cells (EpiSC), can be derived from mutant embryos to reveal the potential of the mutant cells outside the context of the whole organism. Mutant stem cells or even whole mutant embryos can be used to test potential in chimeras or in teratomas.

Role of tenascin C in lesion formation in early peritoneal endometriosis

To identify cytokines or extracellular matrix components that contribute to adhesion to, and invasion of, the peritoneum, proximal to lesions in the early phase of endometriosis. Laboratory-based study. University Hospital and Laboratory of Animal Science. Five women with ovarian endometrioma, 138 wild-type (WT) C57BL/6N mice, and 48 Tenascin C (Tnc) knockout (TncKO) mice. To establish a murine endometriosis model, 20 pieces of minced uterine tissue fragments from each horn were administered intraperitoneally to syngeneic mice. Three days later, endometriotic lesions and peritoneal tissues were collected. Separately, we transfected human peritoneal mesothelial cells (HMrSV5) or human endometrial stromal cells (hESCs) with Tnc small interfering ribonucleic acid. We employed a polymerase chain reaction array to profile gene expression in the murine peritoneum, in both peritoneum distal to lesions and peritoneum surrounding lesions (PSL). The expression of upregulated genes in the PSL was verified in the peritoneal samples by real-time reverse transcription-polymerase chain reaction. TncKO mice were used to investigate the role of Tnc in the development of endometriosis. We evaluated the proliferative activity or inflammatory state of lesions by Ki67 or CD3 immunostaining. Intraperitoneal distribution of macrophages was assessed by fluorescence-activated cell sorting. Using Tnc small interfering ribonucleic acid, we examined the invasive capacity of hESCs in a coculture system with HMrSV5. Tnc gene expression was significantly higher in PSL than in peritoneum distal to lesions. The weight and number of TncKO lesions in TncKO hosts were lower than those of WT lesions in WT hosts. In contrast, the weight and number of nonattached TncKO lesions in TncKO hosts were higher than those of nonattached WT lesions in WT hosts. We observed decreased Ki67-positive cells or H-scores for CD3, a lower proportion of M1 macrophages, and a higher proportion of M2 macrophages in TncKO lesions in TncKO recipients. Silencing of Tnc expression in hESCs and HMrSV5 diminished the invasivity of hESCs. Tnc may be a crucial factor in the development of early peritoneal endometriosis.

Genetic variations underlying Gilbert syndrome and HBV infection outcomes: a cross-sectional study.

Background: Constant cellular damage causes a poor prognosis of hepatitis B virus (HBV) infection. Accumulating evidence indicates the cytoprotective properties of bilirubin. Here, we investigated the association of UDP glucuronosyltransferase family 1 member A1 (UGT1A1), the genetic cause of Gilbert syndrome (GS), a common condition of mild unconjugated bilirubinemia, with HBV infection outcomes. Methods: Patients (n = 2,792) with unconjugated hyperbilirubinemia were screened for HBV infection and host UGT1A1 variations in Ruijin Hospital from January 2015 to May 2023, and those with confirmed HBV exposure were included. The promoter/exons/adjacent intronic regions of UGT1A1 were sequenced. HBV infection outcomes were compared between hosts with wild-type and variant-type UGT1A1. The effect magnitudes of UGT1A1 variations were evaluated using three classification approaches. Results: In total, 175 patients with confirmed HBV exposure were recruited for final analysis. Age, gender, level of HBV serological markers, and antiviral treatment were comparable between UGT1A1 wild-type and disease-causing variation groups. Five known disease-causing mutations (UGT1A1*28, UGT1A1*6, UGT1A1*27, UGT1A1*63, and UGT1A1*7) were detected. The incidence of cirrhosis or hepatocellular carcinoma (LC/HCC) was significantly lower in UGT1A1 variant hosts than in UGT1A1 wild-type hosts (13.14% vs. 78.95%, p < 0.0001). The rarer the UGT1A1 variation a patient possessed, the higher the age at which LC/HCC was diagnosed (R = 0.34, p < 0.05). In contrast, patients without cirrhosis achieving HBsAg clearance were identified only in the UGT1A1 variant group (12.32% vs. 0%). Conclusion: The findings of this study provide insights into the association between preexisting genetically mild bilirubin elevation and viral infection outcome. We showed that the accumulation of UGT1A1 variants or the rarity of the variation is associated with a better prognosis, and the effect magnitude correlates with UGT1A1 deficiency. This study demonstrates the therapeutic potential of host UGT1A1 variations underlying GS against HBV infection outcomes.

Mutant fixation in the presence of a natural enemy

The literature about mutant invasion and fixation typically assumes populations to exist in isolation from their ecosystem. Yet, populations are part of ecological communities, and enemy-victim (e.g. predator-prey or pathogen-host) interactions are particularly common. We use spatially explicit, computational pathogen-host models (with wild-type and mutant hosts) to re-visit the established theory about mutant fixation, where the pathogen equally attacks both wild-type and mutant individuals. Mutant fitness is assumed to be unrelated to infection. We find that pathogen presence substantially weakens selection, increasing the fixation probability of disadvantageous mutants and decreasing it for advantageous mutants. The magnitude of the effect rises with the infection rate. This occurs because infection induces spatial structures, where mutant and wild-type individuals are mostly spatially separated. Thus, instead of mutant and wild-type individuals competing with each other, it is mutant and wild-type “patches” that compete, resulting in smaller fitness differences and weakened selection. This implies that the deleterious mutant burden in natural populations might be higher than expected from traditional theory.

Crosstalk between tumor and stroma modifies CLIC4 cargo in extracellular vesicles.

Mouse models of breast cancer have revealed that tumor-bearing hosts must express the oxidoreductase CLIC4 to develop lung metastases. In the absence of host CLIC4, primary tumors grow but the lung premetastatic niche is defective for metastatic seeding. Primary breast cancer cells release EVs that incorporate CLIC4 as cargo and circulate in plasma of wildtype tumor-bearing hosts. CLIC4-deficient breast cancer cells also form tumors in wildtype hosts and release EVs in plasma, but these EVs lack CLIC4, suggesting that the tumor is the source of the plasma-derived EVs that carry CLIC4 as cargo. Paradoxically, circulating EVs are also devoid of CLIC4 when CLIC4-expressing primary tumors are grown in CLIC4 knockout hosts. Thus, the incorporation of CLIC4 (and perhaps other factors) as EV cargo released from tumors involves specific signals from the surrounding stroma determined by its genetic composition. Since CLIC4 is also detected in circulating EVs from human breast cancer patients, future studies will address its association with disease.

LFA-1/ICAM-1 Adhesion Pathway Mediates the Homeostatic Migration of Lymphocytes from Peripheral Tissues into Lymph Nodes through Lymphatic Vessels.

Lymphocyte function-associated antigen-1 (LFA-1) and its endothelial ligand intercellular adhesion molecule-1 (ICAM-1) are important for the migration of lymphocytes from blood vessels into lymph nodes. However, it is largely unknown whether these molecules mediate the homeostatic migration of lymphocytes from peripheral tissues into lymph nodes through lymphatic vessels. In this study, we find that, in naive mice, ICAM-1 is expressed on the sinus endothelia of lymph nodes, but not on the lymphatic vessels of peripheral tissues. In in vivo lymphocyte migration assays, memory CD4+ T cells migrated to lymph nodes from peripheral tissues much more efficiently than from blood vessels, as compared to naive CD4+ T cells. Moreover, ICAM-1 deficiency in host mice significantly inhibited the migration of adoptively transferred wild-type donor lymphocytes from peripheral tissues, but not from blood vessels, into lymph nodes. The migration of LFA-1-deficient donor lymphocytes from peripheral tissues into the lymph nodes of wild-type host mice was also significantly reduced as compared to wild-type donor lymphocytes. Furthermore, the number of memory T cells in lymph nodes was significantly reduced in the absence of ICAM-1 or LFA-1. Thus, our study extends the functions of the LFA-1/ICAM-1 adhesion pathway, indicating its novel role in controlling the homeostatic migration of lymphocytes from peripheral tissues into lymph nodes and maintaining memory T cellularity in lymph nodes.

Cancer-associated mesothelial cells are regulated by the anti-Müllerian hormone axis

Cancer-associated mesothelial cells (CAMCs) in the tumor microenvironment are thought to promote growth and immune evasion. We find that, in mouse and human ovarian tumors, cancer cells express anti-Müllerian hormone (AMH) while CAMCs express its receptor AMHR2, suggesting a paracrine axis. Factors secreted by cancer cells induce AMHR2 expression during their reprogramming into CAMCs in mouse and human invitro models. Overexpression of AMHR2 in the Met5a mesothelial cell line is sufficient to induce expression of immunosuppressive cytokines and growth factors that stimulate ovarian cancer cell growth in an AMH-dependent way. Finally, syngeneic cancer cells implanted in transgenic mice with Amhr2-/- CAMCs grow significantly slower than in wild-type hosts. The cytokine profile of Amhr2-/- tumor-bearing mice is altered and their tumors express less immune checkpoint markers programmed-cell-death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Taken together, these data suggest that the AMH/AMHR2 axis plays a critical role in regulating the pro-tumoral function of CAMCs in ovarian cancer.

Home-site advantage for host species-specific gut microbiota.

Mammalian species harbor compositionally distinct gut microbial communities, but the mechanisms that maintain specificity of symbionts to host species remain unclear. Here, we show that natural selection within house mice (Mus musculus domesticus) drives deterministic assembly of the house-mouse gut microbiota from mixtures of native and non-native microbiotas. Competing microbiotas from wild-derived lines of house mice and other mouse species (Mus and Peromyscus spp.) within germ-free wild-type (WT) and Rag1-knockout (Rag1-/-) house mice revealed widespread fitness advantages for native gut bacteria. Native bacterial lineages significantly outcompeted non-native lineages in both WT and Rag1-/- mice, indicating home-site advantage for native microbiota independent of host adaptive immunity. However, a minority of native Bacteriodetes and Firmicutes favored by selection in WT hosts were not favored or disfavored in Rag1-/- hosts, indicating that Rag1 mediates fitness advantages of these strains. This study demonstrates home-site advantage for native gut bacteria, consistent with local adaptation of gut microbiota to their mammalian species.

Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection.

Hyphal growth is essential for host colonization during Aspergillus infection. The transcription factor ZfpA regulates A. fumigatus hyphal development including branching, septation, and cell wall composition. However, how ZfpA affects fungal growth and susceptibility to host immunity during infection has not been investigated. Here, we use the larval zebrafish-Aspergillus infection model and primary human neutrophils to probe how ZfpA affects A. fumigatus pathogenesis and response to antifungal drugs in vivo. ZfpA deletion promotes fungal clearance and attenuates virulence in wild-type hosts and this virulence defect is abrogated in neutrophil-deficient zebrafish. ZfpA deletion also increases susceptibility to human neutrophils ex vivo while overexpression impairs fungal killing. Overexpression of ZfpA confers protection against the antifungal caspofungin by increasing chitin synthesis during hyphal development, while ZfpA deletion reduces cell wall chitin and increases caspofungin susceptibility in neutrophil-deficient zebrafish. These findings suggest a protective role for ZfpA activity in resistance to the innate immune response and antifungal treatment during A. fumigatus infection.

PARP1 Regulates Circular RNA Biogenesis though Control of Transcriptional Dynamics.

Circular RNAs (circRNAs) are a recently discovered class of RNAs derived from protein-coding genes that have important biological and pathological roles. They are formed through backsplicing during co-transcriptional alternative splicing; however, the unified mechanism that accounts for backsplicing decisions remains unclear. Factors that regulate the transcriptional timing and spatial organization of pre-mRNA, including RNAPII kinetics, the availability of splicing factors, and features of gene architecture, have been shown to influence backsplicing decisions. Poly (ADP-ribose) polymerase I (PARP1) regulates alternative splicing through both its presence on chromatin as well as its PARylation activity. However, no studies have investigated PARP1's possible role in regulating circRNA biogenesis. Here, we hypothesized that PARP1's role in splicing extends to circRNA biogenesis. Our results identify many unique circRNAs in PARP1 depletion and PARylation-inhibited conditions compared to the wild type. We found that while all genes producing circRNAs share gene architecture features common to circRNA host genes, genes producing circRNAs in PARP1 knockdown conditions had longer upstream introns than downstream introns, whereas flanking introns in wild type host genes were symmetrical. Interestingly, we found that the behavior of PARP1 in regulating RNAPII pausing is distinct between these two classes of host genes. We conclude that the PARP1 pausing of RNAPII works within the context of gene architecture to regulate transcriptional kinetics, and therefore circRNA biogenesis. Furthermore, this regulation of PARP1 within host genes acts to fine tune their transcriptional output with implications in gene function.

Abstract 3276: Tumor cell-intrinsic PGE2-EP4 signaling is a major immunosuppressive pathway in pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the US partly because of its resistance to conventional chemotherapies. Immunotherapies that improve survival in many cancers, almost universally fail in PDA. The resistance to immune checkpoint blockade is at least partially explained by the lack of T cells in the tumor microenvironment (TME) in PDA. We have previously demonstrated that tumor cell-derived prostaglandin E2 (PGE2) is one of the tumor intrinsic factors that creates/supports immunosuppressive TME in PDA presumably through its paracrine effect on the stromal cells. In the current study, we investigate the role of autocrine PGE2 signaling through its cognate EP4 receptor in shaping the T cell low, myeloid cell rich PDA TME. The knockdown (EP4 KD) or knockout (EP4 KO) of the receptors on KrasG12D/p53R172H/YFP/PdxCre (KPCY) tumor cells was sufficient to suppress or abolish the growth of implanted tumors, respectively (control vs EP4 KD, p=0.01, and control vs EP4 KO, p&amp;lt;0.0001). The rejection of the EP4 KO PDA cell lines was T cell dependent as it did not happen in hosts depleted of CD4+ and CD8+ T cells. Upon rechallenge, 80-90% of mice that rejected the primary EP4 KO tumors were able to reject the control, EP4-sufficient tumors in a T cell-dependent manner. While EP4 KO tumors were universally rejected by wild-type (WT) or EP4 global KO (KOgl) hosts, control cell lines formed tumors and grew in both hosts albeit with significantly slower growth in EP4 KOgl hosts (p&amp;lt;0.0001). The TME analysis revealed changes in the T cell presence, subtypes, and functional state in EP4 KO tumors demonstrating more T cell inflamed TME with increased proportions of central memory CD4+ and CD8+ cells (p=0.02 both) along with downregulation of exhaustion markers PD-1 (p=0.02) and CD39 (p=0.009) on CD4+ and CD8+ cells, respectively. Deletion of EP4 on tumor cells changed the systemic anti-tumor immunity by inducing T cell proliferation in lymph nodes (EP4 KO vs control, Ki67+CD8+ p&amp;lt;0.0001 and Ki67+CD4+ p=0.003) and depleting gMDSC population in the spleens of EP4 KO tumor bearing mice (p&amp;lt;0.0001). RNAseq analysis of tumor cells (cultured and in vivo) as well as bulk tumors identified TNFa, IFNa, and IFNg pathways as top upregulated ones in EP4 KO tumors while TGFb and Myc pathways were among the significantly downregulated pathways. The study demonstrates a primary role of tumor cell PGE2-EP4 signaling in creating immunosuppressive TME in PDA suggesting EP4 blockade as a novel approach in treatment of pancreatic cancer. Citation Format: Nune Markosyan, Charu Arora, Liz Quinones, Il-Kyu Kim, Nikhil Joshi, Noah Cheng, John Tobias, Ben Stanger, Robert Vonderheide. Tumor cell-intrinsic PGE2-EP4 signaling is a major immunosuppressive pathway in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3276.

Abstract 4442: Dual benefit of MALT1 blockade in glioblastoma

Abstract Background: Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor. Despite advances in treatment modalities, GBM remains largely incurable and new treatment approaches are desperately needed. An innovative strategy for cancer therapy is to combine the inhibition of cancer cell-intrinsic oncogenic signaling with cancer cell-extrinsic immunological activation of the tumor microenvironment (TME). MALT1 represents a candidate target whose inhibition may provide this dual benefit. Originally identified as a proto-oncoprotein in lymphoma, MALT1 has recently also been implicated in multiple solid tumors, including GBM, and significant effort has been made to develop MALT1 protease inhibitors as anti-cancer agents. Interestingly, blockade of MALT1 protease has also been shown to selectively reprogram tumor infiltrating Treg cells into antitumor effector cells. Based on these observations, we hypothesized that in GBM, MALT1 acts both within tumor cells and within cells of the TME to promote tumorigenesis and MALT1 inhibitor treatment could thus have dual benefit. Methods/Results: We assembled a panel of mouse and human GBM cell lines and performed western-blot analysis to show that these cells demonstrate constitutive MALT1 expression and proteolytic activity. MALT1 blockade in GBM cells, using siRNA or MALT1 protease inhibitors, reduces viability and clonogenic potential, suggesting that inhibition of MALT1 could impair tumor cell survival. Single-cell RNAseq data from public datasets demonstrated that myeloid cells have the highest MALT1 expression in the GBM TME. Our data indicate that co-culture of macrophages with GBM cells induces MALT1-NF-κB activation within the macrophages and this is associated with polarization towards an M2-like immunosuppressive phenotype. Additionally, treating primary human or mouse macrophages with MALT1-protease inhibitor prevents GBM-induced M2-polarization, increases M1-polarization and enhances GBM cell killing. In a syngeneic orthotopic mouse model, GBM tumor growth is impaired, and survival is extended when tumor cells are implanted into MALT1-protease dead (PD) host mice (mice which harbor a point mutation in the MALT1-protease catalytic cysteine) as compared to when implanted into wild-type host mice. MALT1-PD mice demonstrate a less immunosuppressive GBM TME with increased M1-macrophages and decreased M2-macrophages, MDSCs and Tregs. Taken together, our findings suggest that inhibiting MALT1 protease in TME cells promotes anti-tumor immunity against brain tumors. Conclusions: Our studies suggest MALT1 protease as a new therapeutic target in GBM whose inhibition could have dual benefit via both direct effect on tumor cells and by reversing the immunosuppressive TME. Citation Format: Juliana Hofstatter Azambuja, Saigopalakrishna S. Yerneni, Gabriela N. Debom, Lisa M. Maurer, Hannah E. Butterfield, Linda R. Klei, Gary Kohanbash, Peter C. Lucas, Linda M. McAllister-Lucas. Dual benefit of MALT1 blockade in glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4442.

EXTH-22. TARGETING A NEWLY DISCOVERED IMMUNE CHECKPOINT, SIGLEC-15, INCREASES ONCOLYTIC EFFICACY OF ZIKA VIRUS (ZIKV) IN GLIOBLASTOMA (GBM)

Abstract We are developing ZIKV as a therapy for GBM. We previously demonstrated ZIKV specifically kills GBM stem cells. Using GL261 and CT2A mouse models, ZIKV induces a CD8+ T-cell mediated anti-tumor response and leads to 65% and 40% long term survivors, respectively (0%-untreated controls). ZIKV treatment significantly increases the number of myeloid cells in the tumor microenvironment (with about 10,000 infiltrating macrophages per ZIKV-treated brain, 1,000 cells per untreated brain). We hypothesized that by targeting a myeloid immune checkpoint, we would further enhance efficacy. Siglec-15 is a newly described myeloid checkpoint, and anti-siglec-15 antibody is currently in clinical trial for patients with advanced or metastatic solid tumors (NCT03665285). While there was no effect of anti-siglec-15 treatment alone, we observed increased efficacy when combined with ZIKV (cure rate: GL261-70%, CT2A-60% with ZIKV+anti-siglec-15 antibody; GL261-20%, CT2A-0% with anti-siglec-15 antibody alone; GL261-30%, CT2A-25% ZIKV alone; GL261-0%, CT2A-0% without treatment). Since recurrence is a major problem, we performed re-challenge experiments in cured mice at 6 months. Mice previously treated with anti-siglec-15 antibody and ZIKV had 70% long-term survival, compared to 30% of age matched untreated controls. This supports our earlier findings that an immunological response after ZIKV engenders long-term, tumor-specific, immune surveillance. Lastly, we used Siglec-15 knockout mice to confirm our observations. After treating Siglec-15 knockout mice bearing CT2A tumors with ZIKV, 70% animals were cured. There was no significant difference in survival between CT2A-bearing Siglec-15 knockout mice without ZIKV treatment compared to CT2A-bearing wild type hosts. Taken together, our work suggests targeting putative myeloid suppressor cells, combined with oncolytic ZIKV and its ensuing anti-cancer stem cell and activated CD8+ T-cell effects, may be an effective tool in neuro-oncology. Targeting Siglec-15 may also enhance other oncolytic or cytotoxic therapies.

The two-component regulatory system CenK-CenR regulates expression of a previously uncharacterized protein required for salinity and oxidative stress tolerance in Sinorhizobium meliloti.

Genes of unknown function constitute a considerable fraction of most bacterial genomes. In a Tn5-based search for stress response genes in the nitrogen-fixing facultative endosymbiont Sinorhizobium (Ensifer) meliloti, we identified a previously uncharacterized gene required for growth on solid media with increased NaCl concentrations. The encoded protein carries a predicted thioredoxin fold and deletion of the gene also results in increased sensitivity to hydrogen peroxide and cumene hydroperoxide. We have designated the gene srlA (stress resistance locus A) based on these phenotypes. A deletion mutant yields phenotypic revertants on high salt medium and genome sequencing revealed that all revertants carry a mutation in genes homologous to either cenK or cenR. srlA promoter activity is abolished in these revertant host backgrounds and in a strain carrying a deletion in cenK. We also observed that the srlA promoter is autoregulated, displaying low activity in a wildtype (wt) host background and high activity in the srl deletion mutant background. The srlA promoter includes a conserved inverted repeat directly upstream of the predicted −35 subsequence. A mutational analysis demonstrated that the site is required for the high promoter activity in the srlA deletion background. Electromobility shift assays using purified wildtype CenR response regulator and a D55E phosphomimetic derivative suggest this protein acts as a likely Class II activator by binding promoter DNA. These results document the first identified CenK–CenR regulon member in S. meliloti and demonstrate this two-component regulatory system and gene srlA influences cellular growth and persistence under certain stress-inducing conditions.