NOS2 and COX-2 Co-Expression Promotes Cancer Progression: A Potential Target for Developing Agents to Prevent or Treat Highly Aggressive Breast Cancer.

Nitric oxide (NO) and reactive nitrogen species (RNS) exert profound biological impacts dictated by their chemistry. Understanding their spatial distribution is essential for deciphering their roles in diverse biological processes. This review establishes a framework for the chemical biology of NO and RNS, exploring their dynamic reactions within the context of cancer. Concentration-dependent signaling reveals distinctive processes in cancer, with three levels of NO influencing oncogenic properties. In this context, NO plays a crucial role in cancer cell proliferation, metastasis, chemotherapy resistance, and immune suppression. Increased NOS2 expression correlates with poor survival across different tumors, including breast cancer. Additionally, NOS2 can crosstalk with the proinflammatory enzyme cyclooxygenase-2 (COX-2) to promote cancer progression. NOS2 and COX-2 co-expression establishes a positive feed-forward loop, driving immunosuppression and metastasis in estrogen receptor-negative (ER-) breast cancer. Spatial evaluation of NOS2 and COX-2 reveals orthogonal expression, suggesting the unique roles of these niches in the tumor microenvironment (TME). NOS2 and COX2 niche formation requires IFN-γ and cytokine-releasing cells. These niches contribute to poor clinical outcomes, emphasizing their role in cancer progression. Strategies to target these markers include direct inhibition, involving pan-inhibitors and selective inhibitors, as well as indirect approaches targeting their induction or downstream effectors. Compounds from cruciferous vegetables are potential candidates for NOS2 and COX-2 inhibition offering therapeutic applications. Thus, understanding the chemical biology of NO and RNS, their spatial distribution, and their implications in cancer progression provides valuable insights for developing targeted therapies and preventive strategies.

Adjuvant COX inhibition augments STING signaling and cytolytic T cell infiltration in irradiated 4T1 tumors.

Immune therapy is the new frontier of cancer treatment. Therapeutic radiation is a known inducer of immune response and can be limited by immunosuppressive mediators including cyclooxygenase-2 (COX2) that is highly expressed in aggressive triple negative breast cancer (TNBC). A clinical cohort of TNBC tumors revealed poor radiation therapeutic efficacy in tumors expressing high COX2. Herein, we show that radiation combined with adjuvant NSAID (indomethacin) treatment provides a powerful combination to reduce both primary tumor growth and lung metastasis in aggressive 4T1 TNBC tumors, which occurs in part through increased antitumor immune response. Spatial immunological changes including augmented lymphoid infiltration into the tumor epithelium and locally increased cGAS/STING1 and type I IFN gene expression were observed in radiation-indomethacin-treated 4T1 tumors. Thus, radiation and adjuvant NSAID treatment shifts "immune desert phenotypes" toward antitumor M1/TH1 immune mediators in these immunologically challenging tumors. Importantly, radiation-indomethacin combination treatment improved local control of the primary lesion, reduced metastatic burden, and increased median survival when compared with radiation treatment alone. These results show that clinically available NSAIDs can improve radiation therapeutic efficacy through increased antitumor immune response and augmented local generation of cGAS/STING1 and type I IFNs.

The Chemical Biology of NO that Regulates Oncogenic Signaling and Metabolism: NOS2 and Its Role in Inflammatory Disease.

Nitric oxide (NO) and the enzyme that synthesizes it, nitric oxide synthase 2 (NOS2), have emerged as key players in inflammation and cancer. Expression of NOS2 in tumors has been correlated both with positive outcomes and with poor prognoses. The chemistry of NO is the major determinate to the biological outcome and the concentration of NO, which can range over five orders of magnitude, is critical in determining which pathways are activated. It is the activation of specific oncogenic and immunological mechanisms that shape the outcome. The kinetics of specific reactions determine the mechanisms of action. In this review, the relevant reactions of NO and related species are discussed with respect to these oncogenic and immunological signals.

Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction.

The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations.

Interferon-gamma is quintessential for NOS2 and COX2 expression in ER- breast tumors that lead to poor outcome.

A strong correlation between NOS2 and COX2 tumor expression and poor clinical outcomes in ER breast cancer has been established. However, the mechanisms of tumor induction of these enzymes are unclear. Analysis of The Cancer Genome Atlas (TCGA) revealed correlations between NOS2 and COX2 expression and Th1 cytokines. Herein, single-cell RNAseq analysis of TNBC cells shows potent NOS2 and COX2 induction by IFNγ combined with IL1ß or TNFα. Given that IFNγ is secreted by cytolytic lymphocytes, which improve clinical outcomes, this role of IFNγ presents a dichotomy. To explore this conundrum, tumor NOS2, COX2, and CD8+ T cells were spatially analyzed in aggressive ER-, TNBC, and HER2 + breast tumors. High expression and clustering of NOS2-expressing tumor cells occurred at the tumor/stroma interface in the presence of stroma-restricted CD8+ T cells. High expression and clustering of COX2-expressing tumor cells extended into immune desert regions in the tumor core where CD8+ T cell penetration was limited or absent. Moreover, high NOS2-expressing tumor cells were proximal to areas with increased satellitosis, suggestive of cell clusters with a higher metastatic potential. Further in vitro experiments revealed that IFNγ + IL1ß/TNFα increased the elongation and migration of treated tumor cells. This spatial analysis of the tumor microenvironment provides important insight into distinct neighborhoods where stroma-restricted CD8+ T cells exist proximal to NOS2-expressing tumor niches that could have increased metastatic potential.

Interferon-gamma is Quintessential for NOS2 and COX2 Expression in ER - Breast Tumors that Lead to Poor Outcome.

A strong correlation between NOS2 and COX2 tumor expression and poor clinical outcomes in ER-breast cancer has been established. However, mechanisms of tumor induction of these enzymes are unclear. Analysis of The Cancer Genome Atlas (TCGA) revealed correlations between NOS2 and COX2 expression and Th1 cytokines. Herein, single cell RNAseq analysis of TNBC cells shows potent NOS2 and COX2 induction by IFNγ combined with IL1ß or TNFα. Given that IFNγ is secreted by cytolytic lymphocytes, which improve clinical outcomes, this role of IFNγpresents a dichotomy. To explore this conundrum, tumor NOS2, COX2, and CD8 + T cells were spatially analyzed in aggressive ER-, TNBC, and HER2+ breast tumors. High expression and clustering of NOS2-expressing tumor cells occurred at the tumor/stroma interface in the presence of stroma-restricted CD8 + T cells. High expression and clustering of COX2-expressing tumor cells extended into immune desert regions in the tumor core where CD8 + T cell penetration was limited or absent. Moreover, high NOS2-expressing tumor cells were proximal to areas with increased satellitosis suggestive of cell clusters with a higher metastatic potential. Further in vitro experiments revealed that IFNγ+IL1ß/TNFα increased elongation and migration of treated tumor cells. This spatial analysis of the tumor microenvironment provides important insight of distinct neighborhoods where stroma-restricted CD8 + T cells exist proximal to NOS2-expressing tumor niches that could have increased metastatic potential.

Chronic Exposure to Nitric Oxide Induces P53 Mutations and Malignant-like Features in Human Breast Epithelial Cells.

The small endogenous signaling molecule nitric oxide (NO) has been linked with chronic inflammation and cancer. The effects of NO are both concentration and temporally dependent; under some conditions, NO protects against damage caused by reactive oxygen species and activates P53 signaling. During chronic inflammation, NO causes DNA damage and inhibits repair proteins. To extend our understanding of the roles of NO during carcinogenesis, we investigated the possible effects of chronic NO exposure on MCF10A breast epithelial cells, as defined by changes in cellular morphology, chromosome/genomic stability, RNA, and protein expression, and altered cell phenotypes. Human MCF10A cells were maintained in varying doses of the NO donor DETANO for three weeks. Distinct patterns of genomic modifications in TP53 and KRAS target genes were detected in NO-treated cells when compared to background mutations. In addition, quantitative real-time PCR demonstrated an increase in the expression of cancer stem cell (CSC) marker CD44 after prolonged exposure to 300 µM DETANO. While similar changes in cell morphology were found in cells exposed to 300-500 µM DETANO, cells cultured in 100 µM DETANO exhibited enhanced motility. In addition, 100 µM NO-treated cells proliferated in serum-free media and selected clonal populations and pooled cells formed colonies in soft agar that were clustered and disorganized. These findings show that chronic exposure to NO generates altered breast epithelial cell phenotypes with malignant characteristics.

NOS2 and COX2 Provide Key Spatial Targets that Determine Outcome in ER- Breast Cancer.

Estrogen receptor-negative (ER-) breast cancer is an aggressive breast cancer subtype with limited therapeutic options. Upregulated expression of both inducible nitric oxide synthase (NOS2) and cyclo-oxygenase (COX2) in breast tumors predicts poor clinical outcomes. Signaling molecules released by these enzymes activate oncogenic pathways, driving cancer stemness, metastasis, and immune suppression. The influence of tumor NOS2/COX2 expression on the landscape of immune markers using multiplex fluorescence imaging of 21 ER- breast tumors were stratified for survival. A powerful relationship between tumor NOS2/COX2 expression and distinct CD8+ T cell phenotypes was observed at 5 years post-diagnosis. These results were confirmed in a validation cohort using gene expression data showing that ratios of NOS2 to CD8 and COX2 to CD8 are strongly associated with poor outcomes in high NOS2/COX2-expressing tumors. Importantly, multiplex imaging identified distinct CD8+ T cell phenotypes relative to tumor NOS2/COX2 expression in Deceased vs Alive patient tumors at 5-year survival. CD8+NOS2-COX2- phenotypes defined fully inflamed tumors with significantly elevated CD8+ T cell infiltration in Alive tumors expressing low NOS2/COX2. In contrast, two distinct phenotypes including inflamed CD8+NOS2+COX2+ regions with stroma-restricted CD8+ T cells and CD8-NOS2-COX2+ immune desert regions with abated CD8+ T cell penetration, were significantly elevated in Deceased tumors with high NOS2/COX2 expression. These results were supported by applying an unsupervised nonlinear dimensionality-reduction technique, UMAP, correlating specific spatial CD8/NOS2/COX2 expression patterns with patient survival. Moreover, spatial analysis of the CD44v6 and EpCAM cancer stem cell (CSC) markers within the CD8/NOS2/COX2 expression landscape revealed positive correlations between EpCAM and inflamed stroma-restricted CD8+NOS2+COX2+ phenotypes at the tumor/stroma interface in deceased patients. Also, positive correlations between CD44v6 and COX2 were identified in immune desert regions in deceased patients. Furthermore, migrating tumor cells were shown to occur only in the CD8-NOS2+COX2+ regions, identifying a metastatic hot spot. Taken together, this study shows the strength of spatial localization analyses of the CD8/NOS2/COX2 landscape, how it shapes the tumor immune microenvironment and the selection of aggressive tumor phenotypes in distinct regions that lead to poor clinical outcomes. This technique could be beneficial for describing tumor niches with increased aggressiveness that may respond to clinically available NOS2/COX2 inhibitors or immune-modulatory agents.

Spatial analysis of NOS2 and COX2 interaction with T-effector cells reveals immunosuppressive landscapes associated with poor outcome in ER- breast cancer patients.

Multiple immunosuppressive mechanisms exist in the tumor microenvironment that drive poor outcomes and decrease treatment efficacy. The co-expression of NOS2 and COX2 is a strong predictor of poor prognosis in ER- breast cancer and other malignancies. Together, they generate pro-oncogenic signals that drive metastasis, drug resistance, cancer stemness, and immune suppression. Using an ER- breast cancer patient cohort, we found that the spatial expression patterns of NOS2 and COX2 with CD3+CD8+PD1- T effector (Teff) cells formed a tumor immune landscape that correlated with poor outcome. NOS2 was primarily associated with the tumor-immune interface, whereas COX2 was associated with immune desert regions of the tumor lacking Teff cells. A higher ratio of NOS2 or COX2 to Teff was highly correlated with poor outcomes. Spatial analysis revealed that regional clustering of NOS2 and COX2 was associated with stromal-restricted Teff, while only COX2 was predominant in immune deserts. Examination of other immunosuppressive elements, such as PDL1/PD1, Treg, B7H4, and IDO1, revealed that PDL1/PD1, Treg, and IDO1 were primarily associated with restricted Teff, whereas B7H4 and COX2 were found in tumor immune deserts. Regardless of the survival outcome, other leukocytes, such as CD4 T cells and macrophages, were primarily in stromal lymphoid aggregates. Finally, in a 4T1 model, COX2 inhibition led to a massive cell infiltration, thus validating the hypothesis that COX2 is an essential component of the Teff exclusion process and, thus, tumor evasion. Our study indicates that NOS2/COX2 expression plays a central role in tumor immunosuppression. Our findings indicate that new strategies combining clinically available NOS2/COX2 inhibitors with various forms of immune therapy may open a new avenue for the treatment of aggressive ER-breast cancers.

Systemic Nos2 Depletion and Cox inhibition limits TNBC disease progression and alters lymphoid cell spatial orientation and density.

Antitumor immune polarization is a key predictor of clinical outcomes to cancer therapy. An emerging concept influencing clinical outcome involves the spatial location of CD8+ T cells, within the tumor. Our earlier work demonstrated immunosuppressive effects of NOS2 and COX2 tumor expression. Here, we show that NOS2/COX2 levels influence both the polarization and spatial location of lymphoid cells including CD8+ T cells. Importantly, elevated tumor NOS2/COX2 correlated with exclusion of CD8+ T cells from the tumor epithelium. In contrast, tumors expressing low NOS2/COX2 had increased CD8+ T cell penetration into the tumor epithelium. Consistent with a causative relationship between these observations, pharmacological inhibition of COX2 with indomethacin dramatically reduced tumor growth of the 4T1 model of TNBC in both WT and Nos2- mice. This regimen led to complete tumor regression in ∼20-25% of tumor-bearing Nos2- mice, and these animals were resistant to tumor rechallenge. Th1 cytokines were elevated in the blood of treated mice and intratumoral CD4+ and CD8+ T cells were higher in mice that received indomethacin when compared to control untreated mice. Multiplex immunofluorescence imaging confirmed our phenotyping results and demonstrated that targeted Nos2/Cox2 blockade improved CD8+ T cell penetration into the 4T1 tumor core. These findings are consistent with our observations in low NOS2/COX2 expressing breast tumors proving that COX2 activity is responsible for limiting the spatial distribution of effector T cells in TNBC. Together these results suggest that clinically available NSAID's may provide a cost-effective, novel immunotherapeutic approach for treatment of aggressive tumors including triple negative breast cancer.

Selective internal radiation therapy for hepatocellular carcinoma: A 15-year multicenter Australian cohort study.

BACKGROUND AND AIM: The exact place for selective internal radiation therapy (SIRT) in the therapeutic algorithm for hepatocellular carcinoma (HCC) is debated. There are limited data on its indications, efficacy, and safety in Australia. METHODS: We performed a multicenter retrospective cohort study of patients undergoing SIRT for HCC in all Sydney hospitals between 2005 and 2019. The primary outcome was overall survival. Secondary outcomes were progression-free survival and adverse events. RESULTS: During the study period, 156 patients underwent SIRT across 10 institutions (mean age 67 years, 81% male). SIRT use progressively increased from 2005 (n = 2), peaking in 2017 (n = 42) before declining (2019: n = 21). Barcelona Clinic Liver Cancer stages at treatment were A (13%), B (33%), C (52%), and D (2%). Forty-four (28%) patients had tumor thrombus. After a median follow-up of 13.9 months, there were 117 deaths. Median overall survival was 15 months (95% confidence interval 11-19). Independent predictors of mortality on multivariable analysis were extent of liver involvement, Barcelona Clinic Liver Cancer stage, baseline ascites, alpha fetoprotein, and model for end-stage liver disease score. Median progression-free survival was 6.0 months (95% confidence interval 5.1-6.9 months). Following SIRT, 11% of patients were downstaged to curative therapy. SIRT-related complications occurred in 17%: radioembolization-induced liver disease (11%), pneumonitis (3%), gastrointestinal ulceration, and cholecystitis (1% each). Baseline ascites predicted for radioembolization-induced liver disease. CONCLUSION: We present the largest Australian SIRT cohort for HCC. We have identified several factors associated with a poor outcome following SIRT. Patients with early-stage disease had the best survival with some being downstaged to curative therapy.

Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation.

Lipopolysaccharides are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex. Detailed insights into the architecture of LptDE transporter complexes have been derived from X-ray crystallography. However, no structure of a laterally open LptD transporter, a transient state that occurs during LPS release, is available to date. Here, we report a cryo-EM structure of a partially opened LptDE transporter in complex with rigid chaperones derived from nanobodies, at 3.4 Å resolution. In addition, a subset of particles allows to model a structure of a laterally fully opened LptDE complex. Our work offers insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a highly rigid chaperone scaffold to enable structural biology of challenging protein targets.

Acoustic levitation and rotation of thin films and their application for room temperature protein crystallography.

Acoustic levitation has attracted attention in terms of chemical and biochemical analysis in combination with various analytical methods because of its unique container-less environment for samples that is not reliant on specific material characteristics. However, loading samples with very high viscosity is difficult. To expand the scope, we propose the use of polymer thin films as sample holders, whereby the sample is dispensed on a film that is subsequently loaded onto an acoustic levitator. When applied for protein crystallography experiments, rotation controllability and positional stability are important prerequisites. We therefore study the acoustic levitation and rotation of thin films with an aspect ratio (the diameter-to-thickness ratio) of 80-240, which is an order of magnitude larger than those reported previously. For films with empirically optimized shapes, we find that it is possible to control the rotation speed in the range of 1-4 rotations per second while maintaining a positional stability of 12 ± 5 µm. The acoustic radiation force acting on the films is found to be a factor of 26-30 higher than that for same-volume water droplets. We propose use cases of the developed films for protein crystallography experiments and demonstrate data collections for large single crystal samples at room temperature.

Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 channel reveal a novel binding site for positive modulators.

Kv3 ion-channels constitute a class of functionally distinct voltage-gated ion channels characterized by their ability to fire at a high frequency. Several disease relevant mutants, together with biological data, suggest the importance of this class of ion channels as drug targets for CNS disorders, and several drug discovery efforts have been reported. Despite the increasing interest for this class of ion channels, no structure of a Kv3 channel has been reported yet. We have determined the cryo-EM structure of Kv3.1 at 2.6 Å resolution using full-length wild type protein. When compared to known structures for potassium channels from other classes, a novel domain organization is observed with the cytoplasmic T1 domain, containing a well-resolved Zinc site and displaying a rotation by 35°. This suggests a distinct cytoplasmic regulation mechanism for the Kv3.1 channel. A high resolution structure was obtained for Kv3.1 in complex with a novel positive modulator Lu AG00563. The structure reveals a novel ligand binding site for the Kv class of ion channels located between the voltage sensory domain and the channel pore, a region which constitutes a hotspot for disease causing mutations. The discovery of a novel binding site for a positive modulator of a voltage-gated potassium channel could shed light on the mechanism of action for these small molecule potentiators. This finding could enable structure-based drug design on these targets with high therapeutic potential for the treatment of multiple CNS disorders.

Nitric Oxide Modulates Metabolic Processes in the Tumor Immune Microenvironment.

The metabolic requirements and functions of cancer and normal tissues are vastly different. Due to the rapid growth of cancer cells in the tumor microenvironment, distorted vasculature is commonly observed, which creates harsh environments that require rigorous and constantly evolving cellular adaption. A common hallmark of aggressive and therapeutically resistant tumors is hypoxia and hypoxia-induced stress markers. However, recent studies have identified alterations in a wide spectrum of metabolic pathways that dictate tumor behavior and response to therapy. Accordingly, it is becoming clear that metabolic processes are not uniform throughout the tumor microenvironment. Metabolic processes differ and are cell type specific where various factors promote metabolic heterogeneity within the tumor microenvironment. Furthermore, within the tumor, these metabolically distinct cell types can organize to form cellular neighborhoods that serve to establish a pro-tumor milieu in which distant and spatially distinct cellular neighborhoods can communicate via signaling metabolites from stroma, immune and tumor cells. In this review, we will discuss how biochemical interactions of various metabolic pathways influence cancer and immune microenvironments, as well as associated mechanisms that lead to good or poor clinical outcomes.

Multigenerational Epigenetic Regulation of Allergic Diseases: Utilizing an Experimental Dust Mite-Induced Asthma Model.

Environmental exposures have been linked to increased asthma risk, particularly during pregnancy and in early life. Here we use a mouse model of allergic lung disease to examine the effects of pre- and perinatal house dust mite (HDM) allergen exposure on offspring phenotypic and transcriptional outcomes in three generations. We show that maternal HDM exposure (F0) acts synergistically with adult HDM exposure, leading to enhanced airway hyperresponsiveness (AHR) and lung inflammation when compared to mice exposed solely in adulthood. Additionally, a subset of F1 males were not challenged in adulthood, and used to generate F2 progeny, which was then used to generate F3 progeny. Upon adult challenge to HDM, F2, and F3 males generated from the maternal HDM (F0) exposure lineage displayed increased airway reactivity and inflammation when compared to mice exposed solely in adulthood. These findings indicate that maternal allergen exposure is capable of enhancing either susceptibly to or severity of allergic airway disease. To examine the role of epigenetic inheritance of asthma susceptibility induced by maternal HDM exposure, we utilized a genome-wide MeDIP-seq and hMeDIP-seq analysis to identify genes differentially methylated (DMG) and hydroxymethylated (DHG), and their association with the enhanced AHR. In addition, we validated the relationship between DNA methylation and mRNA expression of the DMGs and DHGs in the male sub-generations (F1-F3). We found the expression of Kchn1, Nron, and Spag17 to be differentially hydroxymethylated and upregulated in the F1 exposed to HDM both in early life and in adulthood when compared to F1 mice exposed solely in adulthood. Kcnh1 remained upregulated in the F2 and F3 from the maternal HDM (F0) exposure lineage, when compared to F1 mice exposed solely in adulthood. In summary, we demonstrated that maternal HDM exposure in early life can alter the gene expression and phenotype of offspring upon adult HDM exposure, resulting in more severe disease. These effects persist at least two generations past the initial insult, transmitted along the paternal line.

Crystal Structure and Subsequent Ligand Design of a Nonriboside Partial Agonist Bound to the Adenosine A2A Receptor.

In this study, we determined the crystal structure of an engineered human adenosine A2A receptor bound to a partial agonist and compared it to structures cocrystallized with either a full agonist or an antagonist/inverse agonist. The interaction between the partial agonist, belonging to a class of dicyanopyridines, and amino acids in the ligand binding pocket inspired us to develop a small library of derivatives and assess their affinity in radioligand binding studies and potency and intrinsic activity in a functional, label-free, intact cell assay. It appeared that some of the derivatives retained the partial agonist profile, whereas other ligands turned into inverse agonists. We rationalized this remarkable behavior with additional computational docking studies.

Advances in long-wavelength native phasing at X-ray free-electron lasers.

Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.

Small molecule AZD4635 inhibitor of A2AR signaling rescues immune cell function including CD103+ dendritic cells enhancing anti-tumor immunity.

Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated. METHODS: We report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition. RESULTS: We provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies. CONCLUSION: We provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.