Pig is an important model for biomedical studies, yet the immune cell heterogeneity, the subtle relationship between immune cells and the inter-breed immune variation in pigs remains poorly characterized. Here, we generate single-cell transcriptomes for the Peripheral Blood Mononuclear Cells (PBMCs) of two pig breeds with distinct immune traits including Duroc and Meishan pigs – with the latter have better immune capacity. Dozens of cell types are annotated, with γδ T lymphocytes making up one-fifth of all PBMCs. A list of putative novel markers is identified in each cell type, such as GATA3, SYTL3 and C9H1orf186 which are enriched in γδ T lymphocytes. The cellular composition and immune gene expression abundance are compared across breeds, showing that Meishan pigs have higher proportion of monocytes and their monocytes have enhanced expression of immune-related genes. Re-clustering analysis further identified the sub-populations of T, B and monocytes, with their inter-breed differences further characterized. We also generate single-cell chromatin maps for PBMCs of Duroc pigs and identify a list of transcription factors associated with each immune lineage. Comparison with human data demonstrates that while most cell populations and the corresponding markers match between pig and human, CD4+CD8+ DPTCs and γδ T cells are absent in human and one monocyte subset are absent in pigs. Overall, the present study examined the transcription levels and constructed the single-cell maps of blood immune cells between Duroc and Meishan pigs, which revealed the heterogeneity of immune cells in pigs and their differential characteristics among Duroc and Meishan pigs. This observation would be valuable for better understanding of the immune heterogeneity and immune traits in pigs.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide, primarily due to its low immunogenicity and immunosuppressive tumor microenvironment. Inducing immunogenic cell death (ICD), a regulated form of cell death with the capacity to enhance tumor immunogenicity and activate antitumor immune responses, has emerged as a pivotal anticancer strategy. Mixed lineage kinase domain-like pseudokinase (MLKL) is a terminal-known obligate effector in the process of necroptosis, a programmed cell death pathway. Although several ATP competitive inhibitors of MLKL were reported, these inhibitors were unable to prevent the function of MLKL, rendering MLKL seemingly "undruggable." Moreover, the majority of research on MLKL focused on its biological role in necroptosis, and the investigation of its non-necroptotic functions has rarely been reported. Here, we report the discovery of C116 as a potent and selective MLKL degrader through leveraging artificial intelligence-assisted ligand discovery combined with targeted protein degradation technology. Notably, C116 effectively induces MLKL degradation and promotes parthanatos in HCC cells. More importantly, C116 was able to induce in vivo MLKL degradation and exerts strong antitumor activities in an orthotopic HCC tumor model, positioning it as a promising starting point for the treatment of HCC and for investigating the non-necroptotic functions of MLKL.

Background:Antiplatelet and anticoagulation are the cornerstones for arterial and venous thrombosis, respectively; however, hemorrhage remains a significant clinical challenge. Platelets are crucial for arterial thrombosis and contribute to venous thrombosis. Integrin β3 mediates outside-in signaling, which is critical for thrombosis, while inside-out signaling maintains hemostasis. Targeting the β3/Src interactions to selectively inhibit outside-in signaling offers a promising antithrombotic strategy without compromising hemostasis. Objectives:To develop more potent small molecules that selectively disrupt the β3/Src interaction, thereby inhibiting arterial and venous thrombogenesis without increasing bleeding risk. Methods:Building on the previously identified compound DCDBS84, we developed the structurally modified small molecules C109 and C116, with enhanced affinity for the Src SH3 domain. Their antithrombotic effects on both arterial and venous thrombosis were systematically evaluated through in vitro and in vivo studies. The impact on hemostatic function was assessed using a tail-bleeding model. Additionally, the drug developability of C109 was assessed via pharmacokinetic (PK) and metabolite analysis. Results:C109 and C116 exhibited superior efficacy in disrupting the β3/Src interaction. In vitro and in vivo studies demonstrated that C109 and C116 effectively suppress thrombosis at levels comparable to high doses of the αIIbβ3 antagonist integrilin, without elevating bleeding risk. In the Stenosis Model, C109 and C116 significantly reduced venous thrombogenesis by suppressing platelet activation and neutrophil extracellular trap formation. Additionally, C109 displayed favorable PK properties and robust metabolic stability. Conclusion:These findings identify promising small molecules that inhibit thrombosis while maintaining hemostasis, providing new avenues for safer and more effective clinical management.

Traditional strategies for developing small-molecule inhibitors of protein-protein interactions (PPIs) are time-consuming and often yield low success rates due to the flat and dynamic interfaces of PPIs. To enable the rapid design of highly potent PPI inhibitors, we proposed a novel strategy named "Fulcrum Occupancy-Leverage Perturbation (FOLP)". In this strategy, high-affinity fragments serve as the "Fulcrum" by binding to the orthosteric pocket, while suitable moieties extend into allosteric sites near the PPI interface as "Leverage" to modulate the protein-protein interaction. As a proof of concept, the potent CDK2-Cyclin A2 PPI inhibitor LC-K2CAin-3, which fits the "FOLP" paradigm, was discovered with an IC50 of 32.1 nM for inhibiting the interaction. Molecular dynamics simulations and cryptic pocket identification were employed, revealing the activation loop (A-loop) of CDK2 was flexible and targetable. X-ray crystallography and hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis showed that LC-K2CAin-3 indeed bound to and stabilized the A-loop. LC-K2CAin-3 effectively inhibited the CDK2-Cyclin A2 interaction in CDK2 highly expressed melanoma cells, leading to cell cycle arrest and apoptosis and inhibition of CDK2 mediated signaling. In conclusion, the "FOLP" strategy offers a novel approach for PPI inhibitor discovery and could accelerate the development of PPI inhibitors.

Bromodomain protein BRD4 binds to acetylated histones to regulate transcription. BRD4 also drives cancer cell proliferation. However, the role of BRD4 in normal cell growth has remained unclear. Here we investigated this question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells: they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. In summary, BRD4 epigenetically marks above genes and serves as a master regulator of normal cell growth.

The development of enantioselective C-H macrocyclizations to efficiently access structurally diversified macrocycles is highly desirable, but remain a big challenge. Herein, we reported the first rhodium(III)-catalyzed asymmetric intramolecular C-H macrocyclization, enabling the efficient synthesis of structurally diverse enantioenriched macrocycles. This robust enantioselective C-H macrocyclization has a broad functional group tolerance, excellent enantioselectivities (up to 98.5:1.5 e.r.) and a mild reaction condition, releasing CO2 as the single by-product. More significantly, the resulting unique enantioenriched 19-membered macrocycle 2f was found to demonstrate a potent in vitro anti-Zika virus (ZIKV) activity without obvious cytotoxicity. Further investigation revealed that the anti-ZIKV activity is presumably attributed to an autophagy inhibition in the early stage of viral infection by down-regulating the expression of autophagy related gene Atg12.

Itaconate which is discovered as a mammalian metabolite possessing antimicrobial and immunoregulatory activity has attracted much attention in the field of immunometabolism. Itaconate is synthesized by myeloid cells under conditions of pathogen infection and sterile inflammation. In addition to regulating immune response of myeloid cells, itaconate secreted from myeloid cells can also be taken up by non-myeloid cells to exert immunoregulatory effects in a cell non-autonomous manner. In this review, we recap the discovery of itaconate as a distinct immunologic regulator and effector, describe the development of itaconate biosensor, and detail the recent findings that decipher the mechanism underlying intercellular transport of itaconate. Based on these knowledges, we propose itaconate is a messenger transmitting immunologic signals from myeloid cells to other types of cells during host inflammation and immune defense.

Proteolysis-targeting chimera (PROTAC) is a powerful technology that can effectively trigger the degradation of target proteins. The intricate interplay among various factors leads to a heterogeneous drug response, bringing about significant challenges in comprehending drug mechanisms. Our study applied data-independent acquisition-based mass spectrometry to multidimensional proteome profiling of PROTAC (DIA-MPP) to uncover the efficacy and sensitivity of the PROTAC compound. We profiled the signal transducer and activator of transcription 3 (STAT3) PROTAC degrader in six leukemia and lymphoma cell lines under multiple conditions, demonstrating the pharmacodynamic properties and downstream biological responses. Through comparison between sensitive and insensitive cell lines, we revealed that STAT1 can be regarded as a biomarker for STAT3 PROTAC degrader, which was validated in cells, patient-derived organoids, and mouse models. These results set an example for a comprehensive description of the multidimensional PROTAC pharmacodynamic response and PROTAC drug sensitivity biomarker exploration.

Gene expression profiles play an important role in biology and translational medicine. Due to the vast combinations of drugs and cell lines, the exhaustive measurements of transcriptional perturbation responses become challenging. There are some computational methods to predict drug responses, but the mapping between the drug responses of different cell lines is largely overlooked. We propose CDDTR, a cross-domain autoencoders based method, that uses the drug induced response of one cell line to predict the response of another cell line. For the perturbation profiles of 1570 drugs on seven cell lines in Phase II LINCS-L1000 data, the Pearson correlation coefficient (PCC) value of CDDTR achieves significant performance improvements compared to other state-of-the-art methods, including DeepCellState, VAE and MAGAN. The latent features of transcriptional perturbations of A375 and PC3 cells extracted by 10-fold cross-validation have a 0.663 PCC, revealing the competence of CDDTR to predict the cross-cell type responses. By integrating perturbations from multiple cell lines and incorporating pre-training, the predictive performance of CDDTR can be further improved. Source code is available at https://github.com/hanjunbaba/CDDTR.

Acute liver injury (ALI) is characteristic of abrupt hepatic dysfunction and inflammatory response, and currently the main treatment for ALI is merely supportive rather than curative. Therefore, the development of novel and effective therapeutic strategies for ALI therapy is highly desirable. The emerging biological understanding of the role of BET Bromodomains has opened up an exciting opportunity to develop potent BET Bromodomain inhibitors as an effective therapeutic strategy for the treatment of acute liver injury. Herein, we synthesized a series of potent BET Bromodomain inhibitors with a tetracyclic scaffold, exemplified by compound 28 which showed good in vitro anti-inflammatory activity and good therapeutic effects in the LPS-induced acute liver injury model without obvious cytotoxicity, suggesting that compound 28 is a highly promising candidate worthy for further development.

BRD4 binds to acetylated histones to regulate transcription and drive cancer cell proliferation. However, the role of BRD4 in normal cell growth remains to be elucidated. Here we investigated the question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells: they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. We suggest that BRD4 epigenetically marks those genes and serves as a master regulator of normal cell growth.

Mitochondria use different substrates for energy production and intermediatory metabolism according to the availability of nutrients and oxygen levels. The role of mitochondrial metabolic flexibility for CD8+ T cell immune response is poorly understood. Here, we report that the deletion or pharmacological inhibition of protein tyrosine phosphatase, mitochondrial 1 (PTPMT1) significantly decreased CD8+ effector T cell development and clonal expansion. In addition, PTPMT1 deletion impaired stem-like CD8+ T cell maintenance and accelerated CD8+ T cell exhaustion/dysfunction, leading to aggravated tumor growth. Mechanistically, the loss of PTPMT1 critically altered mitochondrial fuel selection-the utilization of pyruvate, a major mitochondrial substrate derived from glucose-was inhibited, whereas fatty acid utilization was enhanced. Persistent mitochondrial substrate shift and metabolic inflexibility induced oxidative stress, DNA damage, and apoptosis in PTPMT1 knockout cells. Collectively, this study reveals an important role of PTPMT1 in facilitating mitochondrial utilization of carbohydrates and that mitochondrial flexibility in energy source selection is critical for CD8+ T cell antitumor immunity.

AbstractDevelopment of normal blood cells is often suppressed in juvenile myelomonocytic leukemia (JMML), a myeloproliferative neoplasm (MPN) of childhood, causing complications and impacting therapeutic outcomes. However, the mechanism underlying this phenomenon remains uncharacterized. To address this question, we induced the most common mutation identified in JMML (Ptpn11E76K) specifically in the myeloid lineage with hematopoietic stem cells (HSCs) spared. These mice uniformly developed a JMML-like MPN. Importantly, HSCs in the same bone marrow (BM) microenvironment were aberrantly activated and differentiated at the expense of self-renewal. As a result, HSCs lost quiescence and became exhausted. A similar result was observed in wild-type (WT) donor HSCs when co-transplanted with Ptpn11E76K/+ BM cells into WT mice. Co-culture testing demonstrated that JMML/MPN cells robustly accelerated differentiation in mouse and human normal hematopoietic stem/progenitor cells. Cytokine profiling revealed that Ptpn11E76K/+ MPN cells produced excessive IL-1β, but not IL-6, T NF-α, IFN-γ, IL-1α, or other inflammatory cytokines. Depletion of the IL-1β receptor effectively restored HSC quiescence, normalized their pool size, and rescued them from exhaustion in Ptpn11E76K/+/IL-1R−/− double mutant mice. These findings suggest IL-1β signaling as a potential therapeutic target for preserving normal hematopoietic development in JMML.

Abstract Pig is a widely-used for biomedical studies and xenotransplantation, yet its immune cell heterogeneity remains poorly characterized. Here, we generate single-cell transcriptomes and chromatin maps for the Peripheral Blood Mononuclear Cells (PBMCs) of two porcine breeds with distinct immune traits. Dozens of cell types are annotated, with γδ T lymphocytes making up one-fifth of all PBMCs. Both the cellular composition and immune gene expression abundance underlie the immune trait variations across breeds. Transcription factors associated with each immune lineage are also identified, including many known to be critical for T, B and myeloid development. Furthermore, most cell populations and the corresponding markers match between pigs and human. Our study would be valuable not only for better understanding of the immune heterogeneity and immune traits in pigs, but also for identifying novel immune cell subsets, markers and regulators to facilitate future biomedical studies about human immunology and organ xenotransplantation.

Lysosomes are the main organelles in macrophages for killing invading bacteria. However, the precise mechanism underlying lysosomal biogenesis upon bacterial infection remains enigmatic. We demonstrate here that LPS stimulation increases IRG1-dependent itaconate production, which promotes lysosomal biogenesis by activating the transcription factor, TFEB. Mechanistically, itaconate directly alkylates human TFEB at cysteine 212 (Cys270 in mice) to induce its nuclear localization by antagonizing mTOR-mediated phosphorylation and cytosolic retention. Functionally, abrogation of itaconate synthesis by IRG1/Irg1 knockout or expression of an alkylation-deficient TFEB mutant impairs the antibacterial ability of macrophages in vitro. Furthermore, knockin mice harboring an alkylation-deficient TFEB mutant display elevated susceptibility to Salmonella typhimurium infection, whereas in vivo treatment of OI, a cell-permeable itaconate derivative, limits inflammation. Our study identifies itaconate as an endogenous metabolite that functions as a lysosomal inducer in macrophages in response to bacterial infection, implying the potential therapeutic utility of itaconate in treating human bacterial infection.

Amino acid-mediated metabolism is one of the key catabolic and anabolic processes involved in diverse cellular functions. However, the role of the semi-essential amino acid arginine in normal and malignant hematopoietic cell development is poorly understood. Here we report that a continuous supply of exogenous arginine is required for the maintenance/function of normal hematopoietic stem cells (HSCs). Surprisingly, knockout of Slc7a3 (CAT3), a major L-arginine transporter, does not affect HSCs in steady-state or under stress. Although Slc7a3 is highly expressed in naïve and activated CD8 T cells, neither T cell development nor activation/proliferation is impacted by Slc7a3 depletion. Furthermore, the Slc7a3 deletion does not attenuate leukemia development driven by Pten loss or the oncogenic Ptpn11 E76K mutation. Arginine uptake assays reveal that L-arginine uptake is not disrupted in Slc7a3 knockout cells. These data suggest that extracellular arginine is critically important for HSCs, but CAT3 is dispensable for normal hematopoiesis and leukemogenesis.

The bromodomain and extra-terminal (BET) family proteins have recently emerged as promising drug targets for cancer therapy. In this study, identification of an 8-methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one fragment (47) as a new binder to the BET bromodomains and the subsequent incorporation of fragment 47 to the scaffold of ABBV-075, which recently entered Phase I clinical trials, enabled the generation of a series of highly potent BET bromodomain inhibitors. Further druggability optimization led to the discovery of compound 38 as a potential preclinical candidate. Significantly, compared with ABBV-075, which exhibits a 63-fold selectivity for BRD4(1) over EP300, compound 38 demonstrates an excellent selectivity for the BET bromodomain family over other bromodomains, with an ∼1500-fold selectivity for BRD4(1) over EP300. Orally administered 38 achieves a complete inhibition of tumor growth with a tumor growth inhibition (TGI) of 99.7% accompanied by good tolerability.

HIRA is a histone chaperone that deposits the histone variant H3.3 in transcriptionally active genes. In DiGeorge syndromes, a DNA stretch encompassing HIRA is deleted. The syndromes manifest varied abnormalities, including immunodeficiency and thrombocytopenia. HIRA is essential in mice, as total knockout (KO) results in early embryonic death. However, the role of HIRA in hematopoiesis is poorly understood. We investigate hematopoietic cell-specific Hira deletion in mice and show that it dramatically reduces bone marrow hematopoietic stem cells (HSCs), resulting in anemia, thrombocytopenia, and lymphocytopenia. In contrast, fetal hematopoiesis is normal in Hira-KO mice, although fetal HSCs lack the reconstitution capacity. Transcriptome analysis reveals that HIRA is required for expression of many transcription factors and signaling molecules critical for HSCs. ATAC-seq analysis demonstrates that HIRA establishes HSC-specific DNA accessibility, including the SPIB/PU.1 sites. Together, HIRA provides a chromatin environment essential for HSCs, thereby steering their development and survival.

A vaccine is still urgently needed to overcome the hepatitis C virus (HCV) epidemic. It is estimated that 1.75 million new HCV infections occur each year, many of which will go undiagnosed and untreated. Untreated HCV can lead to continued spread of the disease, progressive liver fibrosis, cirrhosis, and eventually, end-stage liver disease and/or hepatocellular carcinoma (HCC). Previously, our 1a E1/E2 glycoprotein vaccine was shown to elicit broadly cross-neutralizing antibodies; however, there remains variation in the effectiveness of these antibodies against different HCV genotypes. In this study, we investigated determinants of differential neutralization sensitivity between two highly related genotype 2a isolates, J6 and JFH-1. Our data indicate that the HVR1 region determines neutralization sensitivity to vaccine antisera through modulation of sensitivity to antibodies and interactions with SR-B1. Our results provide additional insight into optimizing a broadly neutralizing HCV vaccine.

The variant histone H3.3 is incorporated into the genome in a transcription-dependent manner. This histone is thus thought to play a role in epigenetic regulation. However, our understanding of how H3.3 controls gene expression and epigenome landscape has remained incomplete. This is partly because precise localization of H3.3 in the genome has been difficult to decipher particularly for cells in vivo To circumvent this difficulty, we generated knockin mice, by homologous recombination, to replace both of the two H3.3 loci (H3f3a and H3f3b) with the hemagglutinin-tagged H3.3 cDNA cassette, which also contained a GFP gene. We show here that the hemagglutinin-tagged H3.3 and GFP are expressed in the majority of cells in all adult tissues tested. ChIP-seq data, combined with RNA-seq, revealed a striking correlation between the level of transcripts and that of H3.3 accumulation in expressed genes. Finally, we demonstrate that H3.3 deposition is markedly enhanced upon stimulation by interferon on interferon-stimulated genes, highlighting transcription-coupled H3.3 dynamics. Together, these H3.3 knockin mice serve as a useful experimental model to study epigenome regulation in development and in various adult cells in vivo.