James A. Wells

Lung squamous cell carcinoma (LUSC) is an aggressive subtype of non–small cell lung cancer (NSCLC) with limited targeted therapeutic options and incompletely defined immunoregulatory dependencies. In this study, we identified atypical chemokine receptor 3 (ACKR3) as a squamous-enriched regulator that restrains malignant progression in LUSC. Integrative analyses of human LUSC datasets, autochthonous genetically engineered mouse models (GEMM), and ex vivo organoid systems showed that ACKR3 loss accelerated tumor growth and remodeled the immune microenvironment, marked by reduced CD8+ T-cell infiltration and increased T-cell exhaustion. Mechanistically, ACKR3 limited the activation of the macrophage migration inhibitory factor (MIF)–CD74–PD-L1 axis, thereby constraining tumor-cell PD-L1 expression and preserving antitumor immune surveillance. In preclinical LUSC models, treatment with the ACKR3-directed PD-L1 degrader, cytokine receptor–targeting chimera (KineTAC), a bispecific molecule that coengages ACKR3 and PD-L1 to promote lysosomal degradation of PD-L1, enhanced antitumor efficacy, particularly in combination with chemotherapy. Together, these findings define ACKR3 as a squamous-associated immune checkpoint regulator and nominate ACKR3-directed PD-L1 degradation as a tumor-selective therapeutic strategy for LUSC. Significance: ACKR3 is a lineage-imprinted checkpoint restraining MIF–CD74–driven PD-L1 induction in lung squamous cell carcinoma that can be harnessed for PD-L1 degradation to induce tumor-restricted checkpoint dismantling.

The reaction of thioethers in biological macromolecules with oxaziridines enables their functionalization with moieties having utility in both basic and applied science. Exploitation of that reaction has advantages over common bioconjugations due to the relatively low abundance and weak reactivity of thioethers. Herein, we report a novel and improved route to 1-ureal piperidine,3-aryl oxaziridines that enables the preparation of bioconjugation reagents that are either electronically activated or deactivated. We leveraged the differential reactivities of the oxaziridine reagents and engineered methionine residues in distinct environments within a therapeutically relevant antibody fragment (Fab) to effect site-selective bioconjugations. We capitalized on this novel means of kinetic resolution to effect dual functionalization of a HER2-targeted Fab with distinct fluorophores for imaging receptor internalization. This work broadens the scope of what is possible in bioconjugations of thioethers and demonstrates that site-selective bioconjugations of the same residue at more than one site in a protein can be realized via reactivity-based reagent control.

The cell surface is a critical nexus in cellular communication and a rich landscape yielding the majority of current therapeutic targets, yet only a small fraction of cell surface proteins have been targeted therapeutically. The cell surface proteome (surfaceome) comprises only ~1% of the human proteome by mass, motivating enrichment strategies to detect surfaceome alterations in disease for drug target discovery. Developments in photocatalytic technology have revolutionized the study of protein neighborhoods around specific targets of interest, but photocatalysis has been underutilized for global surfaceome analysis. Here, we describe five distinct photocatalytic surfaceomics strategies, all without the need for cellular engineering, that apply the photocatalyst Eosin Y attached to different cell surface structures: direct glycan ligation, glycan attachment via two different lectins, membrane insertion, and free in extracellular space. We systematically compare these photocatalytic surfaceomics methods to each other as well as to three established chemical and enzymatic surfaceomics methods. We demonstrate that the different photocatalytic surfaceomics techniques achieve broadly similar canonical cell surface protein enrichment but show some important differences reflective of cell surface attachment mechanism. These mechanistically orthogonal surfaceomics techniques consistently identified numerous nonglycosylated proteins with high concordance, many of which represent an underappreciated class of noncanonical cell surface proteins lacking traditional cell surface protein features that may represent potential drug targets. This study substantially expands options for surfaceomics methods using photocatalytic proximity labeling.

Tumor reliance on antioxidant defenses creates a vulnerability to ferroptosis, yet strategies to therapeutically disable these systems remain limited. Here, we identify targeted degradation of the selenium uptake receptor lipoprotein receptor-related protein 8 (LRP8) as an effective approach to decrease the abundance of the ferroptosis-protective enzyme glutathione peroxidase 4 (GPX4). Using bispecific cytokine receptor–targeting chimeras (KineTACs) that couple LRP8 to cytokine receptor internalization pathways, we selectively direct LRP8 to the lysosome for degradation. LRP8 degradation reduces the abundance of several selenoproteins, including GPX4, lowering the cellular threshold for lipid peroxidation and sensitizing cancer cells to ferroptosis. These findings establish receptor-mediated selenium uptake as a critical, targetable node in ferroptosis resistance and demonstrate that extracellular protein degradation can be leveraged to reprogram intracellular translational dependencies in cancer cells. More broadly, this work provides a framework for exploiting nutrient acquisition pathways to overcome therapy resistance.

Protein arginine deiminase-4 (PAD4) catalyzes hydrolysis of arginine to citrulline in proteins that promote widespread cellular changes that can induce innate immunity and promote cancer. Hyperactivity of PAD4 leads to a form of cell death called NETosis, releasing PAD4 to the extracellular space to promote various autoimmune diseases through the generation of anticitrulline protein antibodies (ACPAs). Little is known about the specific citrullinated substrates that lead to autoimmunity, but there is growing evidence that PAD4 is localized to the cell surface in response to inflammation. Here, we characterize the cellular consequences of exogenous PAD4, showing that it induces morphological changes that increase cell migration, a hallmark of cancer. We then devised a robust proteomics approach to identify PAD4 substrates. We identified ∼ 3000 citrullinated peptides from 1300 proteins upon exogenous addition of PAD4 both inside and outside of cells. This extracellular set can be further augmented by targeting PAD4 to cancer cells using a HER2 binding protein conjugate. Finally, we studied how citrullinated cells can induce a humoral response in vivo to produce ACPAs. We believe these studies further our understanding of cellular consequences of extracellular PAD4 and identify new PAD4 substrates that are potential neoepitopes for ACPA generation.

Photo-proximity labeling proteomics (PLP) has emerged as a powerful method for rapid and temporal mapping of transient and fragile protein-protein interactions, especially those in membranes. Numerous catalysts can trigger highly reactive diffusive biotinylated probes to label protein neighborhoods at various length scales. Photocatalysts can also trigger protein crosslinking, predominantly between neighboring tyrosines or between histidine and lysine residues. We have exploited this sidechain directed crosslinking for photo-PLP, in a method we call contactMAP. Using biotinylated antibody binders to photocrosslink Her2 or EGFR neighborhoods, contactMAP enriched high resolution maps of these cancer-associated protein neighborhoods in a manner that rivals or outperforms established photo-PLP methods. ContactMAP is an extraordinarily simple and democratic photo-PLP labeling method with broad applications for probing biomolecular interactions in complex mixtures.

Supplementary Figure S5: Combined loss of PVRL2 and PVR loss does not further inhibit tumor growth.

Supplementary Figure S4: PVRL2 functions independently of PVRIG and TIGIT pathways.

Supplementary Figure S1: Proteomic analysis identifies PVRL2 on tumor-derived exosomes.

Supplementary Figure S3: PVRL2 regulates CD8 T cell and NK cell activation.