Target Cell Surface Research Articles

Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling.

Many bacterial toxins exert their cytotoxic effects by enzymatically inactivating one or more cytosolic targets in host cells. To reach their intracellular targets, these toxins possess functional domains or subdomains that interact with and exploit various host factors and biological processes. Despite great progress in identifying many of the key host factors involved in the uptake of toxins, significant knowledge gaps remain as to how partially characterized and newly discovered microbial toxins exploit host factors or processes to intoxicate target cells. Proximity-dependent biotinylation (e.g., BioID) is a powerful method to identify nearby host factors in living cells, offering the potential to identify host targets of microbial toxins. Here, we used BioID to interrogate proximal interactors of the multi-domain Clostridioides difficile TcdB toxin. Expressed fusions of TurboID to different fragments of TcdB identified several high-confidence proteins in the cytosol, including members of the Rho GTPase signaling network and the actin cytoskeletal network. Additionally, we developed an extracellular proximity labeling method using recombinant TurboID-toxin chimeras, which uncovered a limited number of cell-surface targets including LRP1, which was previously identified as a cell-surface receptor of TcdB. Our work reveals surface receptors and intracellular components exploited by bacterial toxins, highlighting key vulnerabilities in host cells.IMPORTANCEBacterial toxins are the causative agents of many human diseases. Further characterizing the intoxication mechanisms of these proteins is important for the development of vaccines and treatments for toxin-mediated disease. Proximity-dependent biotinylation approaches offer an orthogonal approach to complement genetic screens. Here, we evaluate the potential of this method to identify host-toxin interactions on the cell surface and in the cytosol, where the toxin modifies essential host targets. Critically, we have highlighted several limitations of this method as applied to protein toxins, which are important for researchers to weigh when considering this technique for exotoxin studies.

Novel Approaches in Targeting Cell Surface and Secreted Proteins for Lysosomal Degradation.

Protein degradation is pivotal for all biochemical aspects of cellular function. In mammalian cells, protein degradation is mediated mainly by the ubiquitin proteasome system (UPS) and the autophagic-lysosomal system (ALS). Over the last two decades, different types of targeted protein degradation approaches have been developed including proteolysis targeting chimeras (PROTACs) and lysosome targeting chimeras (LYTACs), which employ the UPS to degrade intracellular proteins and the ALS to degrade extracellular and membrane proteins respectively. Nevertheless, Current targeted membrane protein degradation approaches face some inherent challenges including limited target protein degradation efficacy and cell type specific applicability. Herein, we highlight some recent developments of novel targeted membrane protein degradation modalities that exhibit wide-applicability and high protein degradation efficiency. These novel membrane protein degraders hold tremendous promise as new pharmacological and biochemical tools in targeting membrane and secretory proteins for lysosomal degradation.

Circulating microRNAs as a Prognostic Tool to Determine Treatment Efficacy in Lung Cancer Patients Undergoing Pembrolizumab PD-1 Blockade Immunotherapy.

Background: Pembrolizumab has recently emerged as a PD-1 blockade immunotherapy treatment for lung cancer. It is critical that such treatment strategies for lung cancer should be chosen not only on the basis of histopathological features and the expression of targetable cell surface proteins (such as PD-1), but should rather be selected based on other determinants of treatment success or risk factors for poor prognosis. One method to forecast cancer trajectory is the identification of biomolecular signatures such as microRNAs (miRNAs), non-protein-coding RNA molecules that play a regulatory role in gene expression by modulating the translation or stability of messenger RNA. Methods: To find out which miRNAs have an important influence on anti-PD-1 treatment outcomes, we evaluated miRNA levels in sera from 38 lung cancer patients undergoing 3 months of pembrolizumab treatment. We selected a panel of miRNAs previously shown to be involved in lung cancer or PD-1 signaling and performed qPCR analysis. Results: Overall, we observed a significant decrease in the levels of miR126-5p (4-fold), let-7a (5-fold), miR133a-3p (4-fold), miR3615 (2-fold), miR4516 (3-fold), miR16 (3-fold), miR34c-5p (2-fold), miR20b-5p (5-fold), miR106b-5p (5-fold), miR146a-5p (3-fold) and miR181b-5p (3-fold) in response to treatment indicating effectiveness of immunotherapy. Within our selected panel of miRNAs, we identified two markers relevant to cancer prognosis: miR-217, which is negatively associated with patient survival, and let-7a, which is positively associated with patient survival. Conclusions: Our findings suggest that circulating miRNAs can be used for future treatment evaluation and lung cancer prognosis, with potential as therapeutic targets.

Targeting Caveolin-1 in Multiple Myeloma Cells Enhances Chemotherapy and Natural Killer Cell-Mediated Immunotherapy.

The cell membrane transport capacity and surface targets of multiple myeloma (MM) cells heavily influence chemotherapy and immunotherapy. Here, it is found that caveolin-1 (CAV1), a primary component of membrane lipid rafts and caveolae, is highly expressed in MM cells and is associated with MM progression and drug resistance. CAV1 knockdown decreases MM cell adhesion to stromal cells and attenuates cell adhesion-mediated drug resistance to bortezomib. CAV1 inhibition in MM cells enhances natural killer cell-mediated cytotoxicity through increasing CXCL10, SLAMF7, and CD112. CAV1 suppression reduces mitochondrial membrane potential, increases reactive oxygen species, and inhibits autophagosome-lysosome fusion, resulting in the disruption of redox homeostasis. Additionally, CAV1 knockdown enhances glutamine addiction by increasing ASCT2 and LAT1 and dysregulates glutathione metabolism. As a result of CAV1 inhibition, MM cells are more sensitive to starvation, glutamine depletion, and glutamine transporter inhibition, and grow more slowly in vivo in a mouse model treated with bortezomib. The observation that CAV1 inhibition modulated by 6-mercaptopurine, daidzin, and statins enhances the efficacy of bortezomib in vitro and in vivo highlights the translational significance of these FDA-approved drugs in improving MM outcomes. These data demonstrate that CAV1 serves as a potent therapeutic target for enhancing chemotherapy and immunotherapy for MM.

Conditionally Activatable Chimeras for Tumor-Specific Membrane Protein Degradation.

The recent advancements on membrane protein degraders (MPDs) have broadened the applicability of proteolysis-targeting chimeras (PROTACs) beyond intracellular proteins to include the previously "undruggable" cell-surface targets. However, the potential toxicity of MPDs caused by undesired off-target degradation poses a significant challenge to clinical deployment, mirroring concerns associated with PROTACs. Here, we introduce a conditionally activatable membrane protein degrader (Pro-MPD), which leverages the specificity and high affinity of biparatopic nanobodies combined with a tumor microenvironment-activated cell-penetrating peptide (Pro-CPP) to achieve on-target activated internalization and degradation of PD-L1 within tumor sites. This modularly designed Pro-MPD demonstrated a high target degradation efficiency and T cell reactivation, as well as sustained inhibition of tumor growth in xenograft models, highlighting its potential as a safer and highly efficient MPD for in vivo applications. Our work provides a general strategy for the development of conditionally activatable MPDs, which offers a new avenue for reducing the undesired systemic toxicity of MPDs due to the off-tumor degradation.

Engineering an αCD206-synNotch Receptor: Insights into the Development of Novel Synthetic Receptors.

Immune cells play a pivotal role in the establishment, growth, and progression of tumors at primary and metastatic sites. Macrophages, in particular, play a critical role in suppressing immune responses and promoting an anti-inflammatory environment through both direct and indirect cell-cell interactions. However, our understanding of the mechanisms underlying such interactions is limited due to a lack of reliable tools for studying transient interactions between cancer cells and macrophages within the tumor microenvironment. Recent advances in mammalian synthetic biology have introduced a wide range of synthetic receptors that have been used in diverse biosensing applications. One such synthetic receptor is the synNotch receptor, which can be tailored to sense specific ligands displayed on the surface of target cells. With this study, we aimed at developing a novel αCD206-synNotch receptor, targeting CD206+ macrophages, a population of macrophages that play a crucial role in promoting metastatic seeding and persistent growth. Engineered in cancer cells and used in mouse metastasis models, such a tool could help monitor─and provide an understanding of─the effects that cell-cell interactions between macrophages and cancer cells have on metastasis establishment. Here, we report the development of cancer landing-pad cells for versatile applications and the engineering of αCD206-synNotch cancer cells in particular. We report the measurement of their activity and specificity, and discuss unexpected caveats regarding their in vivo applications.

Engineered receptors for soluble cellular communication and disease sensing.

Despite recent advances in mammalian synthetic biology, there remains a lack of modular synthetic receptors that can robustly respond to soluble ligands and in turn activate bespoke cellular functions. Such receptors would have extensive clinical potential to regulate the activity of engineered therapeutic cells, but to date only receptors against cell surface targets have approached clinical translation1. To address this gap, we developed a receptor architecture called synthetic intramembrane proteolysis receptor (SNIPR), that has the added ability to be activated by soluble ligands, both natural and synthetic, with remarkably low baseline activity and high fold activation, through an endocytic, pH-dependent cleavage mechanism. We demonstrate the therapeutic capabilities of the receptor platform by localizing the activity of CAR T cells to solid tumors where soluble disease-associated factors are expressed, bypassing the major hurdle of on-target off-tumor toxicity in bystander organs. We further applied the SNIPR platform to engineer fully synthetic signaling networks between cells orthogonal to natural signaling pathways, expanding the scope of synthetic biology. Our design framework enables cellular communication and environmental interactions, extending the capabilities of synthetic cellular networking in clinical and research contexts.

Induction of ectosome formation by binding of phospholipases D from Loxosceles venoms to endothelial cell surface: Mechanism of interaction

Members of the phospholipase D (PLD) superfamily found in Loxosceles spider venoms are potent toxins with inflammatory and necrotizing activities. They degrade phospholipids in cell membranes, generating bioactive molecules that activate skin cells. These skin cells, in turn, activate leukocytes involved in dermonecrosis, characterized by aseptic coagulative necrosis. Although the literature has advanced in understanding the structure-function relationship, the cell biology resulting from the interactions of these molecules with cells remains poorly understood. In this study, we show that different cells exposed to recombinant PLDs bind these molecules to their plasma membrane, leading to the subsequent organization of extracellular microvesicles/ectosomes. The binding occurs as quickly as five minutes or less after exposure, increases over time, and eventually, the PLDs are expelled from the cell surface without generating cytotoxicity. PLDs are not endocytosed, nor do they spatially colocalize with acidic organelles in the intracellular environment. At least two regions of PLDs – the domain involved in magnesium ion coordination and the choline binding site – appear to play a role in cell surface binding and ectosome organization. However, the amino acids involved in catalysis do not participate in these events. The binding of these PLDs to the cell membrane, independent of catalytic activity, is sufficient to trigger intracellular signaling and enhance the expression of the pro-inflammatory IL-8 gene. These results are supported by the observation that isoforms of PLDs lacking catalytic activity induce an inflammatory response in vivo when injected into the skin of rabbits, without causing dermonecrosis. Our data indicate that these PLDs bind to the surface of target cells, promoting the organization of extracellular vesicles/ectosomes. Subsequently, these events activate pro-inflammatory genes and induce an inflammatory response in vivo. The binding to cells is not dependent on amino acids involved in catalysis but rather on amino acids involved in magnesium coordination. The binding of PLDs to the cell surface, formation of ectosomes, and activation of cells appear to initiate signals involved in inflammatory responses that can lead to dermonecrosis in accidents. This correlation is supported by experimental observations indicating that the events of toxin binding to cells, formation of microvesicles, and inflammatory responses observed both in vitro and in vivo are interconnected.

Abstract B042: Molecular assessment of cell surface targets using integrated circulating tumor cell (CTC) RNAseq and single CTC phenotyping

Abstract Background: Androgen-targeted therapies are a mainstay of treatment for metastatic prostate cancer and have significantly improved patient outcomes. However, development of treatment resistance remains universal, occurring through androgen receptor (AR) alterations driving constitutive AR signaling, or lineage state transitions that bypass AR entirely and culminate in a small cell/neuroendocrine phenotype (NEPC). Cell surface targeted therapies, including antibody drug conjugates and targeted radioligand therapies, represent a new therapeutic class that has shown promise for treatment of androgen-resistant metastatic prostate cancer (mCRPC). Circulating tumor cells (CTCs) present an accessible source of tumor material to identify expression of known and novel targets for therapeutic development, as well as the unique potential for longitudinal profiling to understand the evolution of target expression and association with clinical resistance to cell surface targeted therapies. We have developed a CTC platform allowing for gene expression and protein profiling of cell surface targets on CTCs. Methods: Live CTCs were isolated with our automated microfluidic technology integrating negative and positive selection for CTC enrichment with a cohort of 273 samples. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA-seq, or protein staining on chip for single cell fluorescent quantification of prostate adenocarcinoma and NEPC cell surface targets including PSMA, TROP2, B7H3 and DLL3. Results: CTC RNA sequencing can be used to understand the expression of cell surface targets across different CTC phenotypes. In CTCs from patient with mCRPC, canonical prostate adenocarcinoma cell surface targets STEAP1, KLK2 and FOLH1 and epithelial cell surface target TACSTD2 had the highest expression, while prostate adenocarcinoma target STEAP2, pan-tumor target ERBB2, and tumor immune checkpoint cell surface protein CD276 (B7H3) had intermediate expression. Expression of DLL3 and SSTR2, targets associated with neuroendocrine differentiation, was lowest, though still detected in a subset of CRPC CTCs. Comparison of expression levels between CRPC and NEPC CTCs demonstrated lower expression of most adenocarcinoma cell surface targets in NEPC CTCs as expected, and a trend towards higher expression of DLL3 and SSTR2. Cell surface expression is being assessed for proteins including Trop-2, PSMA and DLL3. Evaluation of DLL3 at the protein level in a subset of patients including both CRPC (n=13) and NEPC (n=12) confirmed that DLL3 was detected in both adenocarcinoma and neuroendocrine CTCs, with a median of 45.8% DLL3-positive CTCs per sample. Conclusions: Biomarkers are needed to better predict response and resistance to targeted therapies. CTC RNAseq and single CTC phenotyping hold potential for predictive and on-treatment biomarkers of response to cell surface targeted therapies. Further study of mechanisms driving response and resistance can be evaluated with these biomarkers. Citation Format: Jamie M. Sperger, Marina N. Sharifi, Katherine R. Kaufmann, Matthew L. Bootsma, Shannon R. Reese, Viridiana Carreno, Alex H. Chang, Luke A. Nunamaker A. Nunamaker, Charlotte Linebarger, Amy K. Taylor, Katherine E Tippins, Kyle T. Helzer, Shuang G. Zhao, Joshua M Lang. Molecular assessment of cell surface targets using integrated circulating tumor cell (CTC) RNAseq and single CTC phenotyping [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B042.

2D Nano-Photosensitizer Facilitates Proximity Labeling for Living Cells Surfaceome Deciphering.

Photocatalytic proximity labeling has shown great promise for mapping the spatiotemporal dynamics of surfaceome. Although cell-surface targeting photosensitizers relying on antibodies, lipid molecules, and metabolic labeling have gained effects, the development of simpler and stable methods that avoid complex chemical synthesis and biosynthesis steps is still a huge challenge. Here, the study has introduced 2D nanomaterials with the ability of cell surface engineering to perform the in situ anchoring of photosensitizer on living cell surface. Photosensitizer can be stabilized on nanomaterials by coordination after one-step mixing, resulting in the nano-photosensitizer combining cell surface targeting ability and photosensitivity that allowing surface-specific proximity labeling. Nano-photosensitizer can be dispersed stably in aqueous solution, avoiding the defects of poor water solubility and aggregation of traditional organic photosensitizers. Singlet oxygen is generated locally under light irradiation, enabling spatiotemporally-resolved activating and labeling of cell surface proteome. Further application in the brain metastatic lung cancer has been found effective with numerous quantified differential cell surfaces proteins highly correlated with cancer metastasis and three potential players have been validated via immunoblotting and immunofluorescence, providing important insights for metastasis supported molecular mechanism.

Enhancing Fc-mediated effector functions of monoclonal antibodies: The example of HexaBodies.

Since the approval of the CD20-targeting monoclonal antibody (mAb) rituximab for the treatment of lymphoma in 1997, mAb therapy has significantly transformed cancer treatment. With over 90 FDA-approved mAbs for the treatment of various hematological and solid cancers, modern cancer treatment relies heavily on these therapies. The overwhelming success of mAbs as cancer therapeutics is attributed to their broad applicability, high safety profile, and precise targeting of cancer-associated surface antigens. Furthermore, mAbs can induce various anti-tumor cytotoxic effector mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), all of which are mediated via their fragment crystallizable (Fc) domain. Over the past decades, these effector mechanisms have been substantially improved through Fc domain engineering. In this review, we will outline the different approaches to enhance Fc effector functions via Fc engineering of mAbs, with a specific emphasis on the so-called "HexaBody" technology, which is designed to enhance the hexamerization of mAbs on the target cell surface, thereby inducing greater complement activation, CDC, and receptor clustering. The review will summarize the development, preclinical, and clinical testing of several HexaBodies designed for the treatment of B-cell malignancies, as well as the potential use of the HexaBody technology beyond Fc-mediated effector functions.

Roles of PDGF/PDGFR signaling in various organs.

Platelet-derived growth factors (PDGFs) ligands and their corresponding receptors, PDGF receptor (PDGFR)α and PDGFRβ, play a crucial role in controlling diverse biological functions, including cell growth, viability and migration. These growth factors bind to PDGFRs, which are receptor tyrosine kinases present on the surface of target cells. The interaction between PDGFs and PDGFRs induces receptor dimerization and subsequent activation through auto-phosphorylation, which in turn triggers a cascade of intracellular signaling pathways. PDGF/PDGFR signaling is essential for maintaining normal physiological functions, including tissue regeneration and growth. However, dysregulation of this signaling pathway leads to pathological conditions, including fibrosis, atherosclerosis, and cancer development in various organs. The pathological impact of PDGF/PDGFR signaling primarily stems from its capacity to promote excessive cell proliferation, enhanced migration, and increased extracellular matrix deposition, resulting in tissue overgrowth, scarring, and abnormal vessel formation. These processes are integral to the pathogenesis of fibrotic, neoplastic, and vascular disorders. Therefore, understanding these pathways is crucial for developing targeted treatments designed to inhibit PDGF/PDGFR signaling in these diseases. This review delves into the dual role of PDGF/PDGFR signaling in both physiological and pathophysiological contexts across different organs and provides insights into current pharmacological therapies designed to target the PDGF signaling pathway. INTRODUCTION Platelet-derived growth factors (PDGFs) are key signaling molecules that interact with specific cell to modulate various cellular responses. Upon binding to their receptors (PDGFRs), PDGFs initiate dimerization and tyrosine phosphorylation, which activates downstream signaling pathways. The PDGF signaling network comprises four ligands-PDGF-A, PDGF-B, PDGFC, and PDGF-D, that interact with two receptors, PDGFRα and PDGFRβ [1-6]. PDGFRα exhibits broader ligand specificity, binding to PDGF-A, PDGF-B, PDGF-C homodimers, and PDGFAB heterodimers, whereas PDGFRβ specifically binds to PDGFB and PDGF-D homodimers. Under both physiological and pathol.

Targeting DLK1 in neuroblastoma

An ideal cell surface target has ubiquitously high cancer expression, absence from healthy tissues, and an essential role cancer initiation and/or maintenance. In this issue of Cancer Cell, Hamilton etal. combine proteomics, transcriptomics, epigenomics, and dependency databases to identify DLK1, a novel immunotherapeutic target for neuroblastoma.

Alpha-1 antitrypsin inhibits pertussis toxin

Pertussis (whooping cough) is a vaccine-preventable but re-emerging, highly infectious respiratory disease caused by Bordetella pertussis. There are currently no effective treatments for pertussis, complicating care for non-vaccinated individuals, especially newborns. Disease manifestations are predominantly caused by pertussis toxin (PT), a pivotal virulence factor classified as an ADP-ribosylating AB-type protein toxin. In this work, an unbiased approach using peptide libraries, bioassay-guided fractionation and mass spectrometry revealed α1-antitrypsin (α1AT) as a potent PT inhibitor. Biochemistry-, cell culture- and molecular modeling-based in vitro experimentation demonstrated that the α1AT mode of action is based on blocking PT-binding to the host target cell surface. In the infant mouse model of severe pertussis, α1AT expression was reduced upon infection. Further, systemic administration of α1AT significantly reduced B. pertussis-induced leukocytosis, which is a hallmark of infant infection and major risk factor for fatal pertussis. Taken together our data demonstrates that α1AT is a novel PT inhibitor and that further evaluation and development of α1AT as a therapeutic agent for pertussis is warranted. Importantly, purified α1AT is already in use clinically as an intravenous augmentation therapy for those with genetic α1AT deficiency and could be repurposed to clinical management of pertussis.

Probing the Depths of Molecular Complexity: STAT3 as a Key Architect in Colorectal Cancer Pathogenesis.

Colorectal cancer (CRC) has become a significant threat in recent decades, and its incidence is predicted to continue rising. Despite notable advancements in therapeutic strategies, managing CRC poses complex challenges, primarily due to the lack of clinically feasible therapeutic targets. Among the myriad molecules implicated in CRC, the signal transducer and activator of transcription 3 (STAT3) stands out as a promising target tightly regulated by various genes. This intracellular transcription factor, spanning 750-795 amino acids and weighing approximately 92 kDa, is crucial in key cellular activities such as growth, migration, invasion, inflammation, and angiogenesis. Aberrant activation of STAT3 signaling has been linked to various cancers, including CRC. Therefore, targeting this signaling pathway holds significance for potential CRC treatment strategies.STAT3, as a central intracellular transcription factor, is implicated in colorectal cancer development by activating aberrant signaling pathways. Numerous studies have demonstrated that the abnormal hyperactivation of STAT3 in CRC tissues enhances cell proliferation, suppresses apoptosis, promotes angiogenesis, and facilitates tumor invasion and metastasis. As a focal point in colorectal cancer research, STAT3 emerges as a promising candidate for detecting and treating CRC. This review aims to present recent data on STAT3, emphasizing the activation and functions of STAT3 inhibitors in CRC. Indeed, STAT3 inhibitors have been identified to have therapeutic potential in CRC, especially inhibitors targeting the DNA-binding domain (DBD). Indeed, STAT3 inhibitors have been identified to have a therapeutic potential in CRC, especially the inhibitors targeting the DNA binding domain (DBD). For example, imatinib acts by targeting cell surface receptors, and these inhibitors have shown potential for the control and treatment of tumor growth, angiogenesis, and metastasis. Imatinib, for example acts by targeting cell surface receptors, and these inhibitors have shown the future direction toward the control and treatment of tumor growth, angiogenesis, and metastasis.

Selective Recruitment of Antibodies to Cancer Cells and Immune Cell-mediated Killing via In Situ Click Chemistry.

Many current cancer immunotherapies function by redirecting immune system components to recognize cancer biomarkers and initiate a cytotoxic attack. The lack of a universal tumor biomarker limits the therapeutic potential of these approaches. However, one feature characteristic of nearly all solid tumors is extracellular acidity. This inherent acidity provides the basis for targeted drug delivery via the pH-low insertion peptide (pHLIP), which selectively accumulates in tumors in vivo due to a pH-dependent membrane insertion propensity. Previously, we established that we could selectively decorate cancer cells with antigen-pHLIP conjugates to facilitate antibody recruitment and subsequent killing by engineered effector cells via antibody-dependent cellular cytotoxicity (ADCC). Here, we present a novel strategy for opsonizing antibodies on target cell surfaces using click chemistry. We utilize pHLIP to facilitate selective tetrazine - trans-cyclooctene ligation of human IgGs to the cancer cell surface and induce ADCC. We demonstrate that our approach activates the primary ADCC signaling pathway via CD16a (FcγRIIIa) receptors on effector cells and induces the killing of cancer cell targets by engineered NK cells.

Towards a switchable nanoparticle behavior using inverse electron-demand Diels-Alder chemistry and ectoenzyme-based ligand activation

Nanoparticles (NPs) as drug delivery platforms encounter numerous obstacles on their journey from administration to the target site. Often, diametrically opposing particle properties are desirable to overcome biological and physical barriers. Therefore, stimuli-responsive NPs have been developed to allow for specific particle adaptation. In this work, it was demonstrated that NPs can be rendered switchable with respect to their interaction with a receptor through an external chemical stimulus. A combination of the inverse electron-demand Diels-Alder (iEDDA) reaction for subsequent NP functionalization and ectoenzyme-based ligand activation allowed for specific particle tailoring. Building on this, a two-step process for target cell recognition was developed. First, NPs were functionalized with Angiotensin-I (Ang-I) as inactive ligand using iEDDA chemistry. At the target site, the ligand was enzymatically processed to Angiotensin-ll (Ang-II) by cellular ectoenzymes. Ang-ll binds as active ligand to the angiotensin ll type 1 (AT1) receptor on the target cell surface. This enzymatic activation aims to minimize the biological effect of the ligand prior to particle binding, while the NP target cell specificity is increased by a two-step recognition with enzymatic processing and receptor binding.

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma

Cancer immunotherapies produce remarkable results in B cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor and normal tissues to identify biologically relevant cell surface immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer. Proteogenomic analyses reveal sixty high-confidence candidate immunotherapeutic targets, and we prioritize delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlates with a super-enhancer. Immunofluorescence, flow cytometry, and immunohistochemistry show robust cell surface expression of DLK1. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells results in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Since high DLK1 expression is found in several adult and pediatric cancers, our study demonstrates the utility of a proteogenomic approach and credentials DLK1 as an immunotherapeutic target.

Androgen production, uptake, and conversion (APUC) genes define prostate cancer patients with distinct clinical outcomes.

BACKGROUNDProstate cancer (PC) is driven by aberrant signaling of the androgen receptor (AR) or its ligands, and androgen deprivation therapies (ADTs) are a cornerstone of treatment. ADT responsiveness may be associated with germline changes in genes that regulate androgen production, uptake, and conversion (APUC).METHODSWe analyzed whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) data from prostate tissues (SU2C/PCF, TCGA, GETx). We also interrogated the Caris Precision Oncology Alliance (POA) DNA (592-gene/whole exome) and RNA (whole transcriptome) next-generation sequencing databases. Algorithm for Linking Activity Networks (ALAN) was used to quantify all pairwise gene-to-gene associations. Real-world overall survival was determined from insurance claims data using Kaplan-Meier estimates.RESULTSSix APUC genes (HSD3B1, HSD3B2, CYP3A43, CYP11A1, CYP11B1, CYP17A1) exhibited coalescent gene behavior in a cohort of metastatic tumors (n = 208). In the Caris POA dataset, the 6 APUC genes (APUC-6) exhibited robust clustering in primary prostate (n = 4,490) and metastatic (n = 2,593) biopsies. Surprisingly, tumors with elevated APUC-6 expression had statically lower expression of AR, AR-V7, and AR signaling scores, suggesting ligand-driven disease biology. APUC-6 genes instead associated with the expression of alternative steroid hormone receptors, ESR1/2 and PGR. We used RNA expression of AR or APUC-6 genes to define 2 subgroups of tumors with differential association with hallmark pathways and cell surface targets.CONCLUSIONSThe APUC-6-high/AR-low tumors represented a subgroup of patients with good clinical outcomes, in contrast with the AR-high or neuroendocrine PCs. Altogether, measuring the aggregate expression of APUC-6 genes in current genomic tests identifies PCs that are ligand (rather than AR) driven and require distinct therapeutic strategies.FUNDINGNCI/NIH 1R37CA288972-01, NCI Cancer Center Support P30 CA077598, DOD W81XWH-22-2-0025, R01 CA249279.

ROR1 MRNA EXPRESSION IN GLIOMA - A NOVEL PROGNOSTIC BIOMARKER

Abstract AIMS Glioblastoma is the most common and aggressive primary brain tumour in adults. Despite maximal surgical resection followed by concomitant chemo-/radiotherapy, the three-year survival remains less than 5%. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) overexpression is associated with poor prognosis in several cancers. ROR1 therapeutics are in Phase I/II clinical trials making it an exciting novel target with translational potential to explore for glioblastoma treatment. The aim of this study was to examine the association between ROR1 mRNA expression and overall survival (OS), by applying the WHO 2021 classification of transcriptomic glioma datasets. METHOD Clinical, histological, and molecular data were extracted from four independent glioma datasets (TCGA, CGGA, Rembrandt, and Gravendeel) via the GlioVis portal and analysed using the WHO 2021 classification. Only confirmed cases of astrocytoma, oligodendroglioma, or glioblastoma, and ROR1 mRNA expression data were included (2,216 cases). Kaplan-Meier survival curves were produced for OS with low/high grade glioma versus ROR1 mRNA expression. Log-rank (Mantel-Cox) multivariate regression was applied to analyse the effect of selected covariates on OS and ROR1 mRNA expression. RESULTS We observed a significant decrease in median OS in patients with high-ROR1 mRNA expression when compared to the low-ROR1 mRNA expression cohort (20 and 70 months, respectively). High-grade gliomas represented the highest ROR1 mRNA expression across the consortium, resulting in a poor median OS of 16 months. CONCLUSION This study represents the first report of an association between OS and the oncogenic role of ROR1 in glioma, a targetable cell surface marker for novel putative treatment in glioblastoma.