Zhijie Li

Uveal melanoma is the most common intraocular cancer in adults, driven by activating mutations in GNAQ or GNA11 , which encode the G proteins Gαq and Gα11. Currently, therapeutic options for uveal melanoma, particularly metastatic uveal melanoma, remain limited. Through an isogenic CRISPR screening approach, we identify EP300 , which encodes the protein lysine acetyltransferase p300, as a top synthetic lethal vulnerability in the context of GNAQ mutation. Notably, CREBBP , which encodes the closely related paralogue CBP, also emerges among the top-ranked candidates. Mechanistically, we find that p300 and CBP support YAP-dependent transcription downstream of oncogenic Gαq in uveal melanoma through interactions with YAP at chromatin. These interactions enhance H3K27 acetylation at the promoters and putative enhancers of YAP target genes and promote the recruitment of RNA polymerase II and CDK9. Using multiple in vivo models representing clinically relevant stages of Gαq-mutant uveal melanoma, we find that p300 inhibition, by both genetic and pharmacological approaches, profoundly impairs tumor growth and metastasis. Taken together, this study establishes p300 as a potential therapeutic target for Gαq-driven uveal melanoma, which is dependent on unrestrained YAP signaling.

Potent and sustained inhibition of epidermal growth factor receptor (EGFR) signaling is critical for suppressing colorectal cancer growth, yet current EGFR therapies are often limited by incomplete EGFR blockade and on-target/off-tumor toxicities, particularly skin rash. In this study, we cotargeted EGFR and cadherin-17 (CDH17) with a bispecific antibody (bsAb), overcoming these limitations by reducing skin-related toxicities and achieving superior tumor growth inhibition compared with EGFR- or c-Met/EGFR-targeting antibodies. Mechanistically, the high-affinity CDH17-binding arm anchored the EGFR/CDH17 bsAb to the tumor-cell surface, facilitating rapid engagement of unbound or newly synthesized EGFR. This “anchor and capture” mechanism allowed the EGFR/CDH17 bsAb to achieve superior and more sustained suppression of the EGFR pathway than cetuximab and amivantamab. Further incorporation of an anti-CD16A nanobody transformed the EGFR/CDH17 bsAb into a trispecific natural killer (NK) cell engager. Extensive format screening revealed that NK-cell activation is heavily influenced by the spatial distance between the tumor antigen–binding Fab and the anti-CD16A nanobody, with longer distances impairing the bulky CD45 phosphatase exclusion from the immunologic synapse and leading to significantly reduced cytotoxicity. Consequently, the widely adopted Morrison-type antibody consistently underperformed compared with architectures with shorter CD16A–tumor-associated antigen (TAA)–Fab spacing. The final optimized molecule was IBI3019, a CDH17/EGFR/CD16A trispecific antibody that integrates potent CDH17-enhanced EGFR blockade with optimal architecture for efficient NK-cell engagement. It demonstrated superior in vivo efficacy and a good safety profile in cynomolgus monkeys, with no observable skin toxicity. These promising preclinical findings warrant the clinical development of IBI3019. Significance: IBI3019 integrates two key underappreciated mechanisms for potent anti-EGFR therapy: the “anchor and capture” strategy for enhanced EGFR antagonism and the necessity of a narrow immunological synapse for optimal ADCC.

Mapping the subcellular partitioning of therapeutics is important for understanding their trafficking, mechanisms of action, and off-target effects. NanoSIMS provides chemical images of labeled therapeutics, but assigning these signals to cellular structures requires correlation with electron microscopy (EM). This correlation is commonly performed by manual landmark selection and is therefore time-consuming and operator-dependent. Here, we present an automated computational pipeline for registering chemical and ultrastructural images across multiple spatial scales. The method combines bidirectional RAFT optical-flow estimation, confidence-guided affine fitting, and template matching to locate a NanoSIMS field of view within a larger EM map. A morphology-rich ion channel (for example, 32S) is used to estimate the transformation, which is then applied to molecule-specific channels (for example, 79Br or 15N). Agreement with expert-guided manual registration was evaluated using PSNR and SSIM, and the workflow was applied to several cell and tissue specimens. Applications to labeled oligonucleotide and antibody-based therapeutics demonstrate how the registered images can support organelle-level interpretation of molecular distributions in vitro and in vivo.

Excessive neutrophil activation and neutrophil extracellular trap (NET) release drive systemic inflammation and organ injury in sepsis, yet the upstream regulatory pathways remain incompletely defined. Here, we identify epidermal growth factor receptor (EGFR) as a critical neutrophil-intrinsic regulator of NETosis. EGFR expression was markedly elevated in neutrophils from patients with sepsis and correlated with disease severity. Neutrophil-specific EGFR deletion in mice improved survival after polymicrobial sepsis by reducing cytokine storm, tissue injury, and NET formation. Mechanistically, EGFR associated with CCAAT/enhancer-binding protein beta (CEBPβ) and recruited Mitogen-activated protein kinase 14 (MAPK14) to phosphorylate CEBPβ, promoting its nuclear localization and transcriptional activation of peptidoglycan recognition protein 1 (PGLYRP1). Elevated PGLYRP1, in turn, amplified NETs release via autocrine engagement of triggering receptor expressed on myeloid cell-1 (TREM-1), establishing a feed-forward inflammatory loop. Administration of recombinant PGLYRP1 or forced CEBPβ overexpression reversed the protection conferred by EGFR deficiency, confirming the centrality of this axis. These findings define an unrecognized EGFR-MAPK14-CEBPβ-PGLYRP1-TREM1 circuit that links receptor signaling to pathological NETosis and highlight a promising therapeutic target to attenuate neutrophil-driven immunopathology in sepsis.

Yes-associated protein (YAP), a key effector of the Hippo pathway, regulates gene expression and promotes tumorigenesis. YAP is conventionally considered “undruggable”, however, targeted protein degradation offers a promising approach to address the challenges associated with targeting this oncogenic protein. In this study, through naïve nanobody phage library screening, we identify multiple nanobodies against human YAP with high affinity and specificity. The YAP nanobody is then fused to the RING domain of RNF4, creating a bio-Proteolysis-Targeting Chimera (bioPROTAC) molecule capable of selectively targeting endogenous YAP for ubiquitin-mediated degradation. Notably, the constructed YAP bioPROTAC demonstrates significant YAP degradation and anticancer efficacy in various YAP-dependent cancers both in vitro and in vivo. Nanoparticles and adeno-associated virus (AAV) can effectively deliver the encoding gene of YAP bioPROTAC, achieving YAP degradation in tumors. Collectively, our study provides a proof-of-concept that the YAP nanobody-bioPROTAC approach can effectively degrade endogenous YAP via the ubiquitin-proteasome system, highlighting a feasible strategy for “undruggable” YAP-dependent cancers.

Aggressive variant prostate cancer (AVPC) originates from metastatic prostate cancer (mPCa) following androgen receptor-targeted therapies, leading to diverse pathological subtypes, notably castration-resistant prostate cancer (CRPC). Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), is consistently expressed across AVPC phenotypes, including neuroendocrine prostate carcinoma (NEPC) and double-negative prostate carcinoma (DNPC), which are significant subtypes of CRPC, making it a promising therapeutic target. In this study, A high-affinity nanobody, B12, specific to CEACAM5, was discovered through phage library screening. B12 exhibited robust binding capabilities, enhanced tumor accumulation, and effective tissue penetration, facilitating rapid in vivo imaging of AVPC. The conjugation of B12 with PE38 to create the immunotoxin B12-PE38 showed significant anti-tumor activity in AVPC xenograft models, including one that mimics bone metastasis. When B12-PE38 was combined with docetaxel, it elicited enhanced tumor inhibitory effects, effectively inhibiting tumor progression. This study underscores CEACAM5 as a target for precise imaging and targeted therapy in AVPC, introducing novel diagnostic and therapeutic strategies for a disease that currently faces a dearth of effective treatment options due to the scarcity of well-defined targets.