Elimination of cancer stem cells (CSCs) and reinvigoration of antitumor immunity remain unmet challenges for cancer therapy. Tumor-associated macrophages (TAMs) constitute the prominant population of immune cells in tumor tissues, contributing to the formation of CSC niches and a suppressive immune microenvironment. Here, we report that high expression of inhibitor of differentiation 1 (ID1) in TAMs correlates with poor outcome in patients with colorectal cancer (CRC). ID1 expressing macrophages maintain cancer stemness and impede CD8+ T cell infiltration. Mechanistically, ID1 interacts with STAT1 to induce its cytoplasmic distribution and inhibits STAT1-mediated SerpinB2 and CCL4 transcription, two secretory factors responsible for cancer stemness inhibition and CD8+ T cell recruitment. Reducing ID1 expression ameliorates CRC progression and enhances tumor sensitivity to immunotherapy and chemotherapy. Collectively, our study highlights the pivotal role of ID1 in controlling the protumor phenotype of TAMs and paves the way for therapeutic targeting of ID1 in CRC.

Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dual agonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their multiplexed pharmacological actions over monoagonists such as semaglutide, we determine cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR. The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the near-atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility of tirzepatide and peptide 20.

During mesenchymal development, the sources of mechanical forces transduced by cells transition over time from predominantly cell-cell interactions to predominantly cell-extracellular matrix (ECM) interactions. Transduction of the associated mechanical signals is critical for development, but how these signals converge to regulate human mesenchymal stem cells (hMSCs) mechanosensing is not fully understood, in part because time-evolving mechanical signals cannot readily be presented in vitro. Here, we established a DNA-driven cell culture platform that could be programmed to present the RGD peptide from fibronectin, mimicking cell-ECM interactions, and the HAVDI peptide from N-cadherin, mimicking cell-cell interactions, through DNA hybridization and toehold-mediated strand displacement reactions. The platform could be programmed to mimic the evolving cell-ECM and cell-cell interactions during mesenchymal development. We applied this platform to reveal that RGD/integrin ligation promoted cofilin phosphorylation, while HAVDI/N-cadherin ligation inhibited cofilin phosphorylation. Cofilin phosphorylation upregulated perinuclear apical actin fibers, which deformed the nucleus and thereby induced YAP nuclear localization in hMSCs, resulting in subsequent osteogenic differentiation. Our programmable culture platform is broadly applicable to the study of dynamic, integrated mechanobiological signals in development, healing, and tissue engineering.

Angiogenesis is a critical pathophysiological process involved in organ growth and various diseases. Transcription factors Sp1/Sp3 are necessary for fetal development and tumor growth. Sp1/Sp3 proteins were downregulated in the capillaries of the gastrocnemius in patients with critical limb ischemia samples. Endothelial-specific Sp1/Sp3 knockout reduces angiogenesis in retinal, pathological, and tumor models and induced activation of the Notch1 pathway. Further, the inactivation of VEGFR2 signaling by Notch1 contributes to the delayed angiogenesis phenotype. Mechanistically, endothelial Sp1 binds to the promoter of Notch1 and inhibits its transcription, which is enhanced by Sp3. The proangiogenic effect of ACEI is abolished in Sp1/Sp3-deletion male mice. We identify USP7 as an ACEI-activated deubiquitinating enzyme that translocated into the nucleus binding to Sp1/Sp3, which are deacetylated by HDAC1. Our findings demonstrate a central role for endothelial USP7-Sp1/Sp3-Notch1 signaling in pathophysiological angiogenesis in response to ACEI treatment.

In ovarian cancer (OC), IL-17-producing T cells (Th17s) predict improved survival, whereas regulatory T cells predict poorer survival. We previously developed a vaccine whereby patient-derived dendritic cells (DCs) are programmed to induce Th17 responses to the OC antigen folate receptor alpha (FRα). Here we report the results of a single-arm open-label phase I clinical trial designed to determine vaccine safety and tolerability (primary outcomes) and recurrence-free survival (secondary outcome). Immunogenicity is also evaluated. Recruitment is complete with a total of 19 Stage IIIC-IV OC patients in first remission after conventional therapy. DCs are generated using our Th17-inducing protocol and are pulsed with HLA class II epitopes from FRα. Mature antigen-loaded DCs are injected intradermally. All patients have completed study-related interventions. No grade 3 or higher adverse events are seen. Vaccination results in the development of Th1, Th17, and antibody responses to FRα in the majority of patients. Th1 and antibody responses are associated with prolonged recurrence-free survival. Antibody-dependent cell-mediated cytotoxic activity against FRα is also associated with prolonged RFS. Of 18 patients evaluable for efficacy, 39% (7/18) remain recurrence-free at the time of data censoring, with a median follow-up of 49.2 months. Thus, vaccination with Th17-inducing FRα-loaded DCs is safe, induces antigen-specific immunity, and is associated with prolonged remission.

Ghrelin, also called "the hunger hormone", is a gastric peptide hormone that regulates food intake, body weight, as well as taste sensation, reward, cognition, learning and memory. One unique feature of ghrelin is its acylation, primarily with an octanoic acid, which is essential for its binding and activation of the ghrelin receptor, a G protein-coupled receptor. The multifaceted roles of ghrelin make ghrelin receptor a highly attractive drug target for growth retardation, obesity, and metabolic disorders. Here we present two cryo-electron microscopy structures of Gq-coupled ghrelin receptor bound to ghrelin and a synthetic agonist, GHRP-6. Analysis of these two structures reveals a unique binding pocket for the octanoyl group, which guides the correct positioning of the peptide to initiate the receptor activation. Together with mutational and functional data, our structures define the rules for recognition of the acylated peptide hormone and activation of ghrelin receptor, and provide structural templates to facilitate drug design targeting ghrelin receptor.

Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs). Here, we report three cryo-electron microscopy structures of RXFP4-Gi protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053. The B chain of INSL5 adopts a single α-helix that penetrates into the orthosteric pocket, while the A chain sits above the orthosteric pocket, revealing a peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. Our findings not only provide insights into ligand recognition and subtype selectivity among class A G protein-coupled receptors, but also expand the knowledge of signaling mechanisms in the insulin superfamily.

Super-enhancers regulate genes with important functions in processes that are cell type-specific or define cell identity. Mouse embryonic fibroblasts establish 40 senescence-associated super-enhancers regardless of how they become senescent, with 50 activated genes located in the vicinity of these enhancers. Here we show, through gene knockdown and analysis of three core biological properties of senescent cells that a relatively large number of senescence-associated super-enhancer-regulated genes promote survival of senescent mouse embryonic fibroblasts. Of these, Mdm2, Rnase4, and Ang act by suppressing p53-mediated apoptosis through various mechanisms that are also engaged in response to DNA damage. MDM2 and RNASE4 transcription is also elevated in human senescent fibroblasts to restrain p53 and promote survival. These insights identify key survival mechanisms of senescent cells and provide molecular entry points for the development of targeted therapeutics that eliminate senescent cells at sites of pathology.

MRGPRX1, a Mas-related GPCR (MRGPR), is a key receptor for itch perception and targeting MRGPRX1 may have potential to treat both chronic itch and pain. Here we report cryo-EM structures of the MRGPRX1-Gi1 and MRGPRX1-Gq trimers in complex with two peptide ligands, BAM8-22 and CNF-Tx2. These structures reveal a shallow orthosteric pocket and its conformational plasticity for sensing multiple different peptidic itch allergens. Distinct from MRGPRX2, MRGPRX1 contains a unique pocket feature at the extracellular ends of TM3 and TM4 to accommodate the peptide C-terminal "RF/RY" motif, which could serve as key mechanisms for peptidic allergen recognition. Below the ligand binding pocket, the G6.48XP6.50F6.51G6.52X(2)F/W6.55 motif is essential for the inward tilting of the upper end of TM6 to induce receptor activation. Moreover, structural features inside the ligand pocket and on the cytoplasmic side of MRGPRX1 are identified as key elements for both Gi and Gq signaling. Collectively, our studies provide structural insights into understanding itch sensation, MRGPRX1 activation, and downstream G protein signaling.