Biology subjects

Liu, M.

Publications and source records attributed to Liu, M..

At least 253 records · Page 14Linked to original sources

bioRxiv2023

KRAS inhibition activates an actionable CD24 do-not-eat-me signal in pancreas cancer

KRASG12C inhibitors (G12Ci) have produced encouraging, albeit modest and transient, clinical benefit in pancreatic ductal adenocarcinoma (PDAC). Identifying and targeting resistance mechanisms to G12Ci treatment is therefore crucial. To better understand the function of KRASG12C and possible G12Ci bypass mechanisms, we developed an autochthonous KRASG12C- driven PDAC model. Compared to the classical KRASG12D PDAC model, the G12C model exhibits slower tumor growth, yet similar histopathological and molecular features. Aligned with clinical experience, G12Ci treatment of KRASG12C tumors produced modest impact despite stimulating a hot tumor immune microenvironment. Immunoprofiling revealed that CD24, a dont eat me signal, is significantly upregulated on cancer cells upon G12Ci treatment. Blocking CD24 enhanced macrophage phagocytosis of cancer cells and significantly sensitized tumors to G12Ci treatment. Similar findings were observed in KRASG12D-driven PDAC. Together, this study reveals common and distinct oncogenic KRAS allele-specific biology and identifies a clinically actionable adaptive mechanism that may improve the efficacy of oncogenic KRAS inhibitor therapy in PDAC. SignificanceGeneration of an autochthonous KRASG12C-driven pancreatic cancer model enabled elucidation of specific effects of KRASG12C during tumor development, revealing CD24 as an actionable adaptive mechanism in cancer cells induced upon KRASG12C inhibition.

cancer biology↗

bioRxiv2023

High-affinity binding to the SARS-CoV-2 spike trimer by a nanostructured, trivalent protein-DNA synthetic antibody

Multivalency enables nanostructures to bind molecular targets with high affinity. Although IgG antibodies can be generated against a wide range of antigens, their shape and size cannot be tuned to match a given target. DNA nanotechnology provides an attractive approach for designing customized multivalent scaffolds due to the addressability and programmability of the nanostructure shape and size. Here, we use computational simulation to guide the design and synthesis of a DNA nanostructure-based synthetic antibody ("nano-synbody"). The nano-synbody is comprised of a three-helix bundle DNA nanostructure with three identical arms terminating in a mini-binder protein that targets the SARS-CoV-2 spike protein. The structure was designed to match the valence and distance between the three receptor binding domains (RBDs) in the spike trimer, in order to enhance binding through avidity effects. Moreover, the design allowed for the display of one, two, or three protein-displaying arms, thereby systematically probing the effect of multivalency on binding affinity. The binding strength of the nano-synbody increased with the increasing number of arms, yielding 11.2 pM affinity ([~]100-fold enhancement over monovalent binding) for the wild-type spike protein for the three-arm structure. Moreover, the multivalency was able to yield a 95 pM affinity for the Omicron variant, a mutant against which the monovalent protein was ineffective. The nano-synbody could also block infection of a spike protein-bearing pseudovirus, and similarly demonstrated effective inhibition of the Omicron variant when trimerized. The structure of the three-arm nano-synbody bound to the Omicron variant spike trimer was solved by negative-stain transmission electron microscopy reconstruction, and shows the protein-DNA nanostructure with all three arms bound to the RBD domains, confirming the intended trivalent attachment. Finally, nano-synbody binding could be reversed by removing one, two, or three arms in a programmable fashion, via toehold-mediated strand displacement. The ability to tune the size and shape of the nano-synbody, as well as its potential ability to attach (and then remove) two or more different binding ligands, will enable the high-affinity binding of a range of proteins, and pave the way towards their manipulation using DNA-based nano-robotic devices.

bioengineering↗

bioRxiv2023

General Transcription Factor from E. coli with a Distinct Mechanism of Action

Gene expression in E. coli is controlled by well-established mechanisms that activate or repress transcription. Here, we identify CedA as an unconventional transcription factor specifically associated with the RNA polymerase (RNAP) {sigma}70 holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of {beta} and {sigma}70 subunits to stabilize an open-promoter complex. Remarkably, CedA does so without contacting DNA. We further show that cedA is strongly induced in response to amino acid starvation, oxidative stress, and aminoglycosides. CedA provides a basal level of tolerance to these clinically relevant antibiotics, as well as to rifampicin and peroxide. Finally, we show that CedA modulates transcription of hundreds of bacterial genes, which explains its pleotropic effect on cell physiology and pathogenesis. One sentence summaryAn integrated structure-function approach uncovers CedA as a general transcription initiation factor in E. coli and elucidates its multifaceted role and unique mechanism.

molecular biology↗

bioRxiv2023

NGAL contributes to hemostasis and thrombosis by potentiating or suppressing coagulant or anticoagulant factors

Coagulation is related to inflammation, but the key pathways, especially innate immunity inflammatory response-coagulation, hemostasis, and thrombosis regulation is poorly understood and need to be further explored. In the current study, we showed that innate immunity inflammatory mediator neutrophil gelatinase-associated apolipoprotein (NGAL) which was upregulated in plasma of deep vein thrombosis patients interacted with and potentiated thrombin, kallikrein, FXIa, and FVIIa and suppressed antithrombin to induce coagulation, hemostasis, and thrombosis. Furthermore, NGAL can augment thrombin-induced platelet aggregation. In multiple mice hemostasis and thrombosis models, NGAL overexpression or intravenous administration promoted coagulation and hemostasis and aggravated thrombus, whereas NGAL knockout or treatment with anti-NGAL monoclonal antibody significantly prolonged bleeding time and alleviated thrombus formation. Notably, NGAL knockout prolonged both mice tail bleeding time and artery occlusion time to over 40 min, resembling uncontrollable bleeding and clotting disorder seen in Hemophilia mice. Furthermore, anti-NGAL monoclonal antibody treatment markedly reduced the formation of blood clots in a mouse-tail thrombosis model induced by carrageenan, which is linked to inflammation. Collectively, these findings suggest NGAL is a crucial coagulation regulator and mediates the crosstalk between innate immunity inflammation and coagulation, hemostasis, and thrombus, and provide new target and strategy for the development of innovative antithrombotic drugs.

biochemistry↗

bioRxiv2023

Combining Bulk and Single-Cell RNA Sequencing Data to Identify RNA methylation and Autophagy-Related Signatures in Patients with Chronic Obstructive Pulmonary Disease

BackgroundChronic Obstructive Pulmonary Disease (COPD) is a heterogeneous lung condition associated with RNA methylation and autophagy. However, the specific autophagy-related genes and RNA methylation regulators involved in COPD development remain unknown. MethodsWe analyzed COPD and non-COPD patients datasets obtained from the Gene Expression Omnibus database, including Tissue Sequencing Transcriptome (bulk-seq) and single-cell sequencing (scRNA-seq) data. Differentially expressed genes (DEGs) were identified through differential genetic analysis using non-COPD bulk-seq data as the control group and COPD samples were used as the experimental group. Animal experiments were conducted to validate the expression of key genes. COPD model mice were exposed to smoke for four months, and lung function and histopathological changes were assessed. The mRNA and protein expression levels of FTO, IGF2BP2, DDIT3, DNAJB1, and YTHDF3 were measured using RT-qPCR and Western blotting, respectively. ResultsWe identified FTO, IGF2BP2, and YTHDF3 as key methylation genes, along with autophagy hub genes DDIT3 and DNAJB1. Animal experiments showed significantly increased mRNA and protein levels of FTO, YTHDF3 and DNAJB1 and significantly decreased levels of IGF2BP2 in lung tissue of COPD mice compared to the control group. ConclusionOur findings suggest that DDIT3 and DNAJB1 as autophagy hub genes, along with FTO, IGF2BP2 and YTHDF3 as RNA methylation genes, may play crucial roles in the development of COPD. These findings, supported by bulk-seq and scRNA-seq data, contribute novel genetic evidence for understanding the epigenetics of COPD.

cell biology↗

bioRxiv2023

Adult Mouse Leptomeninges Exhibit Regional and Age-related Cellular Heterogeneity and Gene Expression Differences Implicating Mental Disorders

The leptomeninges envelop the central nervous system (CNS) and contribute to cerebrospinal fluid (CSF) production and homeostasis. We analyzed the meninges overlying the anterior or posterior forebrain in the adult mouse by single nuclear RNA-sequencing (snucRNA-seq). This revealed regional differences in fibroblast and endothelial cell composition and gene expression. Surprisingly, these non-neuronal cells co-expressed genes implicated in neural functions. The regional differences changed with aging, from 3 to 18 months. Cytokine analysis revealed specific soluble factor production from anterior vs posterior meninges that also altered with age. Secreted factors from the leptomeninges from different regions and ages differentially impacted the survival of anterior or posterior cortical neuronal subsets, neuron morphology, and glia proliferation. These findings suggest that meningeal dysfunction in different brain regions could contribute to specific neural pathologies. The disease-associations of meningeal cell genes differentially expressed with region and age were significantly enriched for mental and substance abuse disorders.

neuroscience↗

bioRxiv2023

Analysis of a mouse germ cell tumor model establishes pluripotency-associated miRNAs as conserved serum biomarkers for germ cell cancer detection

Malignant testicular germ cells tumors (TGCTs) are the most common solid cancers in young men. Current TGCT diagnostics include conventional serum protein markers, but these lack the sensitivity and specificity to serve as accurate markers across all TGCT subtypes. MicroRNAs (miRNAs) are small non-coding regulatory RNAs and informative biomarkers for several diseases. In humans, miRNAs of the miR-371-373 cluster are detectable in the serum of patients with malignant TGCTs and outperform existing serum protein markers for both initial diagnosis and subsequent disease monitoring. We previously developed a genetically engineered mouse model featuring malignant mixed TGCTs consisting of pluripotent embryonal carcinoma (EC) and differentiated teratoma that, like the corresponding human malignancies, originate in utero and are highly chemosensitive. Here, we report that miRNAs in the mouse miR-290-295 cluster, homologs of the human miR-371-373 cluster, were detectable in serum from mice with malignant TGCTs but not from tumor-free control mice or mice with benign teratomas. miR-291-293 were expressed and secreted specifically by pluripotent EC cells, and expression was lost following differentiation induced by the drug thioridazine. Notably, miR-291-293 levels were significantly higher in the serum of pregnant dams carrying tumor-bearing fetuses compared to that of control dams. These findings reveal that expression of the miR-290-295 and miR-371-373 clusters in mice and humans, respectively, is a conserved feature of malignant TGCTs, further validating the mouse model as representative of the human disease. These data also highlight the potential of serum miR-371-373 assays to improve patient outcomes through early TGCT detection, possibly even prenatally.

cancer biology↗

bioRxiv2023

Autoantibodies against eukaryotic translation elongation factor 1 delta (EEF1D) in two patients with autoimmune cerebellar ataxia

To report a novel autoantibody against eukaryotic translation elongation factor 1 delta (EEF1D) in two patients with autoimmune cerebellar ataxia (ACA). The patients on one center with cerebellar ataxia of unknown cause, who were tested positive with tissue-based indirect immunofluorescence assay (TBA) on rat cerebellum sections and negative for comprehensive anti-neural autoantibodies panel, were investigated for novel autoantibody identification. Tissue-immunoprecipitation (TIP) combined with mass spectrometric (MS) analysis was used to identify the target antigen. The EEF1D protein was identified from complex precipitated by serum and cerebrospinal fluid (CSF) of one patient, while the specific binding of autoantibodies and EEF1D were confirmed by subsequent neutralization experiment, recombinant cell-based indirect immunofluorescence assay (CBA) and western blot. In consequently test using CBA, another anti-EEF1D positive ACA patient was identified and confirmed by western blot. All enrolled 30 health doners and 15 connective tissue diseases patients without neurological disorders were anti-EEF1D negative. The two anti-EEF1D positive patients presented similar clinical manifestations, their outcomes supported the effectiveness of immunotherapy, but the cerebellar atrophy occurred before treatment may be irreversible. Symptoms of one patient worsened again after the weaning of pulse corticosteroid therapy. The identification of anti-EEF1D autoantibody provides a new potential biomarker for early diagnosis and recurrence prevention of ACA.

immunology↗

bioRxiv2023

Alpha Oscillations Encode Bayesian Belief Updating Underlying Attentional Allocation in Dynamic Environments

In a dynamic environment, expectations of the future constantly change based on updated evidence and affect the dynamic allocation of attentional resources.To further investigate the neural mechanisms underlying efficient allocation of attention, we employed a modified Central Cue Posner paradigm in which the probability of cues being valid (that is, accurately indicated the upcoming target location) was manipulated. The proportion of attentional resources allocated to the cued location (), which was sensitive to precision of predictions on previous trials, was estimated using a hierarchical Bayesian model and was included as a regressor in the analyses of electrophysiological (EEG) data. Our results revealed that before the target appeared, the attentional modulation of alpha oscillations (8[~]13Hz) for high-certainty cues (88% valid) were significantly predicted by the attentional resources measure (). This relationship was not observed under low-certainty conditions (69% and 50% valid cues). After the target appeared, the attentional resources measure () correlated with alpha band oscillations only in the valid cue condition and not in the invalid condition. These results provide new insights on how trial-by-trial Bayesian belief updating relates to alpha band encoding environmentally-sensitive allocation of visual spatial attention.

neuroscience↗

bioRxiv2023

Structural basis for coupling of the WASH subunit FAM21 with the endosomal SNX27-Retromer complex

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the WASH complex which nucleates actin filaments establishing the endosomal reycling domain. Crystal structures, modelling, biochemical and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling, however it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains. Significance StatementCell surface transmembrane proteins are regulated by a constant cycle of internalization and recycling from intracellular endosomal compartments. The Retromer protein complex and the sorting nexin adaptor protein play a critical role in the retrieval of hundreds of proteins responsible for ion transport, glucose metabolism, neurotransmission, and other cell functions. We have defined the mechanism by which both Retromer and SNX27 engage the actin-nucleating complex called WASH through multiple repeated sequences in the subunit FAM21. Dysfunction in WASH, Retromer and SNX27 are implicated in various disorders including Parkinsons disease, Alzheimers disease, hereditary spastic paraplegia, and this work provides important insights into the assembly of these essential endosomal sorting machineries.

cell biology↗

bioRxiv2023

Parvalbumin and somatostatin: biomarkers for two parallel tectothalamic pathways in the auditory midbrain

The inferior colliculus (IC) represents a crucial relay station in the auditory pathway, located in the midbrains tectum and primarily projecting to the thalamus. Despite the identification of distinct cell types based on various biomarkers in the IC, their specific contributions to the organization of auditory tectothalamic pathways have remained poorly understood. In this study, we demonstrate that IC neurons expressing parvalbumin (ICPV+) or somatostatin (ICSOM+) represent major, non-overlapping cell types throughout the three IC subdivisions. Strikingly, regardless of their location within the IC, these neurons predominantly project to the primary and secondary auditory thalamic nuclei, respectively. Retrograde tracing data specific to cell types indicate that ICPV+ neurons primarily receive auditory inputs, whereas ICSOM+ neurons integrate polymodal inputs that hold behavioral significance. Furthermore, ICPV+ neurons exhibit significant heterogeneity in both intrinsic electrophysiological properties and presynaptic terminal size compared to ICSOM+ neurons. Notably, approximately one quarter of ICPV+ neurons are inhibitory neurons, whereas all ICSOM+ neurons are excitatory neurons. Collectively, our findings suggest that parvalbumin and somatostatin expression in the IC can serve as biomarkers for two functionally distinct, parallel tectothalamic pathways. This discovery challenges the conventional IC subdivision-based definition of tectothalamic pathways and calls for a reassessment of their functional roles.

neuroscience↗

bioRxiv2023

An adult clock component links circadian rhythms to pancreatic β-cell maturation

Circadian clocks attune metabolism to daily energy cycles, but how they regulate functional maturation of metabolic tissues is poorly understood. Here we show that DEC1, a clock transcription factor induced in adult islet {beta} cells, coordinates their glucose responsiveness by synchronizing energetic and secretory rhythms. DEC1 binds and regulates maturity-linked genes to integrate insulin exocytosis with {beta}-cell energy metabolism, and {beta}-cell-specific Dec1 ablation disrupts their transcription synchrony. Dec1-disrupted mice develop lifelong glucose intolerance and insulin deficiency, despite normal islet formation. Metabolic dysfunction upon {beta}-cell Dec1 loss stems from poor coupling of islet insulin secretion to glucose metabolism, reminiscent of fetal/neonatal islet immaturity. We link stunted maturation to a deficit in circadian bioenergetics, prompted by compromised glucose utilization, mitochondrial dynamics, and respiratory metabolism, which is rescued by increased metabolic flux. Thus, DEC1 links circadian clockwork to {beta}-cell metabolic maturation, revealing a hierarchy for how the clock programs metabolic tissue specialization.

cell biology↗

bioRxiv2023

A novel architecture of PT neuron-based corticothalamic connectivity in the auditory system

Largely topographical projections from different modules of the thalamus, such as the primary, secondary and association sensory thalamus, to hierarchically defined cortical areas have been recognized across sensory systems. However, how corticothalamic projections, which are believed to be crucial for the remarkable flexibility and precision exhibited by our sensory systems, are organized remained poorly understood compared with the thalamocortical counterpart. Here we report that, first, the primary auditory thalamus received direct inputs from cortical L5 neurons. Second, in contrast to the robust thalamocortical topography, L5 neurons in each of the primary, secondary and association auditory cortical regions project to each individual module of the auditory thalamus at the macroscale. Third, the association cortex provided the most L5 inputs to all thalamic modules followed by the secondary and primary auditory cortices. Lastly, L5 axon terminals were mainly varicosity-type and evenly distributed across thalamic modules, but those in the polymodal association module were the largest. Our data suggest that all the modules of the auditory thalamus may be under the modulation of common L5 inputs. This fully-connected-like corticothalamic architecture urges a revision of the traditional hierarchical model in the sensory systems.

neuroscience↗

bioRxiv2023

A genome-wide single-cell 3D genome atlas of lung cancer progression

Although three-dimensional (3D) genome structures are altered in cancer cells, little is known about how these changes evolve and diversify during cancer progression. Leveraging genome-wide chromatin tracing to visualize 3D genome folding directly in tissues, we generated 3D genome cancer atlases of murine lung and pancreatic adenocarcinoma. Our data reveal stereotypical, non-monotonic, and stage-specific alterations in 3D genome folding heterogeneity, compaction, and compartmentalization as cancers progress from normal to preinvasive and ultimately to invasive tumors, discovering a potential structural bottleneck in early tumor progression. Remarkably, 3D genome architectures distinguish histologic cancer states in single cells, despite considerable cell-to-cell heterogeneity. Gene-level analyses of evolutionary changes in 3D genome compartmentalization not only showed compartment-associated genes are more homogeneously regulated, but also elucidated prognostic and dependency genes in lung adenocarcinoma and a previously unappreciated role for polycomb-group protein Rnf2 in 3D genome regulation. Our results demonstrate the utility of mapping the single-cell cancer 3D genome in tissues and illuminate its potential to identify new diagnostic, prognostic, and therapeutic biomarkers in cancer.

cell biology↗

bioRxiv2023

Genome editing induces the transfer of mitochondrial DNA into the nuclear genome

Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause broad transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, retinal cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that the newly developed mitochondrial base editor DdCBE can also cause widespread mtDNA integrations. However, we provide a practical solution to suppress the transfer of mtDNA by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings also shed light on the origins of mitochondrial-nuclear DNA segments.

genomics↗

bioRxiv2023

SALL4B, not targeted by IMiD, is important for SALL4-mediated tumorigenesis

Immunomodulatory (IMiD) drugs have shown a prominent therapeutic activity in hematologic malignancies; however, their usage in solid tumors is limited. The oncofetal protein SALL4 is essential for cancer cell survival. While IMiDs can induce SALL4 degradation, they fail to induce cell death in SALL4-expressing cancer cell lines. Here, we observed that this inefficacy arose from their selective degradation of the long SALL4 isoform, while sparing the short SALL4B isoform. Selective silencing of SALL4B phenocopied total SALL4 depletion by inducing cancer apoptosis, underscoring the critical role of SALL4B in cancer maintenance. Recognizing that IMiDs cant degrade SALL4B, we performed a high-throughput screen to identify compound(s) that could achieve this. We identified a small molecule compound that degrades both SALL4 isoforms with enhanced potency towards SALL4B in a cereblon- and proteasome-dependent manner. This compound suppressed cancer cell proliferation and attenuated tumor development in both cell line and patient-derived xenograft models. Transcriptomic analyses further revealed convergent effects of genetic and pharmacologic SALL4B depletion on DNA damage response and replication pathways. Together, these findings identify SALL4B as the therapeutically relevant isoform in SALL4-dependent cancers and establish isoform-aware targeted degradation as a strategy to overcome the limitation of IMiDs in solid tumors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/548071v2_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@99670dorg.highwire.dtl.DTLVardef@13ca58forg.highwire.dtl.DTLVardef@998e7eorg.highwire.dtl.DTLVardef@15a1875_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract | Identification of QE: a non-IMiDs degrader capable of degrading both SALL4A and SALL4B, triggers anti-cancer effects beyond IMiDs, and Impacts of QE on Key Validated SALL4B Targets and Pathways C_FIG

cancer biology↗

bioRxiv2023

Unconventional Activation of IRE1 Enhances TH17 Responses and Promotes Neutrophilic Airway Inflammation

Treatment-refractory severe asthma manifests a neutrophilic phenotype associated with TH17 responses. Heightened unfolded protein responses (UPRs) are associated with the risk of asthma, including severe asthma. However, how UPRs participate in the deregulation of TH17 cells leading to this type of asthma remains elusive. In this study, we investigated the role of the UPR sensor IRE1 in TH17 cell function and neutrophilic airway inflammation. We found that IRE1 is induced in fungal asthma and is highly expressed in TH17 cells relative to naive CD4+ T cells. Cytokine (e.g. IL-23) signals induce the IRE1-XBP1s axis in a JAK2-dependent manner. This noncanonical activation of the IRE1-XBP1s pathway promotes UPRs and cytokine secretion by TH17 cells. Ern1 (encoding IRE1)-deficiency decreases the expression of ER stress factors and impairs the differentiation and cytokine secretion of TH17 cells. Genetic ablation of Ern1 leads to alleviated TH17 responses and airway neutrophilia in a Candida albicans asthma model. Consistently, IL-23 activates the JAK2-IRE1-XBP1s pathway in vivo and enhances TH17 responses and neutrophilic infiltration into the airway. Taken together, our data indicate that IRE1, noncanonically activated by cytokine signals, promotes neutrophilic airway inflammation through the UPR- mediated secretory function of TH17 cells. The findings provide a novel insight into the fundamental understanding of IRE1 in TH17-biased TH2-low asthma.

immunology↗

bioRxiv2023

Structural insights into dual-antagonize mechanism of AB928 on adenosine A2 receptors

The adenosine subfamily G protein-coupled receptors A2AR and A2BR were identified as promising candidates for cancer immunotherapy within recent years. One of the A2AR/A2BR dual antagonist, AB928, has progressed to phase II clinic trial for the treatment of rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of A2AR in complex with AB928 and a selective A2AR antagonist, 2-118. The structures reveal a common binding mode on A2AR, wherein the ligands establish extensive interactions with residues from both the orthosteric pocket and the secondary pocket. Conversely, the cAMP assay together with molecular dynamics simulations conducted on both A2AR and A2BR indicate that the ligands adopt distinct binding modes on A2BR. Detailed analysis of their chemical structures suggests that AB928 can readily adapt to the A2BR pocket, while 2-118 cannot due to its intrinsic differences. This disparity potentially accounts for their divergent inhibitory efficacies between A2BR and A2AR. The findings from this study can serve as valuable structural templates for future development of selective or dual inhibitors targeting A2AR/A2BR in the context of cancer therapy.

cell biology↗