Distinct molecular profiles drive multifaceted characteristics of colorectal cancer metastatic seeds.

Metastasis of primary tumors remains a challenge for early diagnosis and prevention. The cellular properties and molecular drivers of metastatically competent clones within primary tumors remain unclear. Here, we generated 10-16 single cell-derived lines from each of three colorectal cancer (CRC) tumors to identify and characterize metastatic seeds. We found that intrinsic factors conferred clones with distinct metastatic potential and cellular communication capabilities, determining organ-specific metastasis. Poorly differentiated or highly metastatic clones, rather than drug-resistant clones, exhibited poor clinical prognostic impact. Personalized genetic alterations, instead of mutation burden, determined the occurrence of metastatic potential during clonal evolution. Additionally, we developed a gene signature for capturing metastatic potential of primary CRC tumors and demonstrated a strategy for identifying metastatic drivers using isogenic clones with distinct metastatic potential in primary tumors. This study provides insight into the origin and mechanisms of metastasis and will help develop potential anti-metastatic therapeutic targets for CRC patients.

A Novel Dual-Fc Bispecific Antibody with Enhanced Fc Effector Function.

Bispecific antibodies (BsAbs) are undergoing continued development for applications in oncology and autoimmune diseases. While increasing activity by having more than one targeting arm, most BsAb engineering employs single Fc engagement as monoclonal antibodies. Here, we designed a novel immunoglobulin gamma-1 (IgG1)-derived dual-Fc BsAb containing two Fc regions and two distinct asymmetric antigen binding arms comprising a Fab arm and another VHH domain. In conjunction with the knob-into-hole technology, dual-Fc BsAbs could be produced with a high yield and good stability. We explore how Fc engineering effects on dual-Fc constructs could boost the desired therapeutic efficacy. This new format enabled simultaneous bispecific binding to corresponding antigens. Furthermore, compared to the one-Fc control molecules, dual-Fc BsAbs were shown to increase the avidity-based binding to FcγRs to result in higher ADCC and ADCP activities by potent avidity via binding to two antigens and Fc receptors. Overall, this novel BsAb format with enhanced effector functionalities provides a new option for antibody-based immunotherapy.

Colorectal Cancer Patient-Derived 2D and 3D Models Efficiently Recapitulate Inter- and Intratumoral Heterogeneity.

Pre-existing drug resistance and tumorigenicity of cancer cells are highly correlated with therapeutic failure and tumor growth. However, current cancer models are limited in their application to the study of intratumor functional heterogeneity in personalized oncology. Here, an innovative two-dimensional (2D) and three-dimensional (3D) model for patient-derived cancer cells (PDCCs) and air-liquid interface (ALI) organotypic culture is established from colorectal cancer (CRC). The PDCCs recapitulate the genomic landscape of their parental tumors with high efficiency, high proliferation rate, and long-term stability, while corresponding ALI organotypic cultures retain histological architecture of their original tumors. Interestingly, both 2D and 3D models maintain the transcriptomic profile of the corresponding primary tumors and display the same trend in response to 5-Fluoruracil, regardless of their difference in gene expression profiles. Furthermore, single-cell-derived clones() are efficiently established and pre-existing drug-resistant clones and highly tumorigenic clones within individual CRC tumors are identified. It is found that tumorigenic cancer cells do not necessarily possess the stem cells characteristics in gene expression. This study provides valuable platform and resource for exploring the molecular mechanisms underlying the pre-existing drug resistance and tumorigenicity in cancer cells, as well as for developing therapeutic targets specifically for pre-existing drug-resistant or highly tumorigenic clones.

Structural basis for the tethered peptide activation of adhesion GPCRs.

Adhesion G-protein-coupled receptors (aGPCRs) are important for organogenesis, neurodevelopment, reproduction and other processes1-6. Many aGPCRs are activated by a conserved internal (tethered) agonist sequence known as the Stachel sequence7-12. Here, we report the cryogenic electron microscopy (cryo-EM) structures of two aGPCRs in complex with Gs: GPR133 and GPR114. The structures indicate that the Stachel sequences of both receptors assume an α-helical-bulge-ß-sheet structure and insert into a binding site formed by the transmembrane domain (TMD). A hydrophobic interaction motif (HIM) within the Stachel sequence mediates most of the intramolecular interactions with the TMD. Combined with the cryo-EM structures, biochemical characterization of the HIM motif provides insight into the cross-reactivity and selectivity of the Stachel sequences. Two interconnected mechanisms, the sensing of Stachel sequences by the conserved 'toggle switch' W6.53 and the constitution of a hydrogen-bond network formed by Q7.49/Y7.49 and the P6.47/V6.47φφG6.50 motif (φ indicates a hydrophobic residue), are important in Stachel sequence-mediated receptor activation and Gs coupling. Notably, this network stabilizes kink formation in TM helices 6 and 7 (TM6 and TM7, respectively). A common Gs-binding interface is observed between the two aGPCRs, and GPR114 has an extended TM7 that forms unique interactions with Gs. Our structures reveal the detailed mechanisms of aGPCR activation by Stachel sequences and their Gs coupling.

Constitutive signal bias mediated by the human GHRHR splice variant 1.

Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone-releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to ß-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus ß-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward ß-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

Structural mechanism of calcium-mediated hormone recognition and Gß interaction by the human melanocortin-1 receptor.

Melanocortins are peptide hormones critical for the regulation of stress response, energy homeostasis, inflammation, and skin pigmentation. Their functions are mediated by five G protein-coupled receptors (MC1R-MC5R), predominately through the stimulatory G protein (Gs). MC1R, the founding member of melanocortin receptors, is mainly expressed in melanocytes and is involved in melanogenesis. Dysfunction of MC1R is associated with the development of melanoma and skin cancer. Here we present three cryo-electron microscopy structures of the MC1R-Gs complexes bound to endogenous hormone α-MSH, a marketed drug afamelanotide, and a synthetic agonist SHU9119. These structures reveal the orthosteric binding pocket for the conserved HFRW motif among melanocortins and the crucial role of calcium ion in ligand binding. They also demonstrate the basis of differential activities among different ligands. In addition, unexpected interactions between MC1R and the Gß subunit were discovered from these structures. Together, our results elucidate a conserved mechanism of calcium-mediated ligand recognition, a specific mode of G protein coupling, and a universal activation pathway of melanocortin receptors.

A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor.

Glucagon-like peptides (GLP-1 and GLP-2) are two proglucagon-derived intestinal hormones that mediate distinct physiological functions through two related receptors (GLP-1R and GLP-2R) which are important drug targets for metabolic disorders and Crohn's disease, respectively. Despite great progress in GLP-1R structure determination, our understanding on the differences of peptide binding and signal transduction between these two receptors remains elusive. Here we report the electron microscopy structure of the human GLP-2R in complex with GLP-2 and a Gs heterotrimer. To accommodate GLP-2 rather than GLP-1, GLP-2R fine-tunes the conformations of the extracellular parts of transmembrane helices (TMs) 1, 5, 7 and extracellular loop 1 (ECL1). In contrast to GLP-1, the N-terminal histidine of GLP-2 penetrates into the receptor core with a unique orientation. The middle region of GLP-2 engages with TM1 and TM7 more extensively than with ECL2, and the GLP-2 C-terminus closely attaches to ECL1, which is the most protruded among 9 class B G protein-coupled receptors (GPCRs). Functional studies revealed that the above three segments of GLP-2 are essential for GLP-2 recognition and receptor activation, especially the middle region. These results provide new insights into the molecular basis of ligand specificity in class B GPCRs and may facilitate the development of more specific therapeutics.

Structural basis for activation of the growth hormone-releasing hormone receptor.

Growth hormone-releasing hormone (GHRH) regulates the secretion of growth hormone that virtually controls metabolism and growth of every tissue through its binding to the cognate receptor (GHRHR). Malfunction in GHRHR signaling is associated with abnormal growth, making GHRHR an attractive therapeutic target against dwarfism (e.g., isolated growth hormone deficiency, IGHD), gigantism, lipodystrophy and certain cancers. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GHRHR bound to its endogenous ligand and the stimulatory G protein at 2.6 Å. This high-resolution structure reveals a characteristic hormone recognition pattern of GHRH by GHRHR, where the α-helical GHRH forms an extensive and continuous network of interactions involving all the extracellular loops (ECLs), all the transmembrane (TM) helices except TM4, and the extracellular domain (ECD) of GHRHR, especially the N-terminus of GHRH that engages a broad set of specific interactions with the receptor. Mutagenesis and molecular dynamics (MD) simulations uncover detailed mechanisms by which IGHD-causing mutations lead to the impairment of GHRHR function. Our findings provide insights into the molecular basis of peptide recognition and receptor activation, thereby facilitating the development of structure-based drug discovery and precision medicine.

Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir.

The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo-electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.