bioRxiv Science⌕ Search

Biology subjects

George, J.

Publications and source records attributed to George, J..

At least 19 recordsLinked to original sources

Mapping the immune landscape in small cell lung cancer unveils a distinct tumor-reactive CD8+ T cell molecular signature

Small cell lung cancer (SCLC) is a highly aggressive malignancy with limited therapeutic advances. Unlike many other cancers, its immune landscape, particularly immune competence and T cell recognition, remains poorly characterized. Here, we generate a single-cell transcriptome atlas of the SCLC immune microenvironment with paired T cell receptor (TCR) sequencing. By linking T cell states with clonality and a multilayered functional screening, we identify 6 tumor-reactive TCRs that recognize and eradicate autologous SCLC cell lines. We delineate a novel SCLC-reactive CD8+ T cell signature (SCLC_TR), enabling the identification of 47 further SCLC-reactive TCRs. The SCLC_TR signature performs extremely well in pancreatic ductal adenocarcinoma (PDAC), another immune-cold tumor indication, and, most strikingly, patients with elevated SCLC_TR signature scores exhibited significantly improved survival, underlining its prognostic potential. Comparative cell-cell interaction analyses implicate several immunosuppressive mechanisms, with myeloid cells and CD4 regulatory T cells possibly acting as counterbalances to effector T cell activity in SCLC. In summary, our study challenges the prevailing notion of SCLC as an immune-cold tumor type by providing direct evidence of tumor-reactive T cell responses and introduces the SCLC_TR signature as a tool to identify tumor-specific T cells and their microenvironmental restraints and escape mechanisms, ultimately shaping next-generation immunotherapeutic strategies. O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/735200v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@1c25f2eorg.highwire.dtl.DTLVardef@1f6f519org.highwire.dtl.DTLVardef@553e57org.highwire.dtl.DTLVardef@6fe7e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

Translational Opportunity of Engineered IFNγ-eEVs Through Targeted Inhibition of JAK/STAT1 Signaling, Mimicking IVIg Therapy

Immunoglobulin (Ig) replacement therapies (IgRT) including intravenous (IVIg) and subcutaneous (SCIg), are pooled IgG preparations widely used to restore humoral immunity and to suppress pathological inflammation in autoimmune and inflammatory disorders. Despite broad clinical use, the mechanisms underlying their immunomodulatory effects remain incompletely defined. Here, we identify extracellular vesicle (EV)-associated cytokines as mediators of IVIg activity. Multiplex bead-based flow cytometry revealed that EVs isolated by size exclusion followed by ultracentrifugation from IVIg were CD63 positive but depleted of platelet-derived and HLA markers relative to EVs from unprocessed human plasma. Luminex profiling demonstrated substantial reduction of pro-inflammatory cytokines in IVIg EVs. Notably, although IVIg EVs contained abundant IFN{gamma}, they failed to activate IFNGR/JAK/STAT1 signaling. Instead, prolonged exposure to IVIg EVs suppressed subsequent IFN{gamma}-induced STAT1 activation. Engineered IFN{gamma}-coated EVs (IFN{gamma}-eEVs) recapitulated both activating and inhibitory effects indicating context-dependent signaling bias. Critically, cold ethanol precipitation, a key step in IVIg manufacturing, selectively abrogated the activating function of IFN{gamma}-eEVs while preserving their inhibitory capacity. These findings define a previously unrecognized mechanism where IVIg processing generates EVs that bias IFN{gamma} signaling toward suppression. EV-associated cytokines therefore represent a generalizable pathway through which IVIg exerts anti-inflammatory effects across immune-mediated diseases.

bioengineering↗

Extracellular vesicles from wild-type Epstein-Barr virus-transformed B-cells export host DNA and the viral lncRNA EBER1

Epstein-Barr virus (EBV) infection is nearly ubiquitous and strongly linked to multiple sclerosis (MS), but how EBV-infected B cells communicate with distal tissues remains unclear. We performed an integrated multiomic characterization of small extracellular vesicles (sEVs) released from spontaneous lymphoblastoid cell lines (SLCLs) derived from healthy donors and patients with MS, transformed ex vivo by endogenous wild-type EBV. Proteomics identified over 6,000 shared proteins enriched in nucleic acid-binding and chromatin-associated factors. EV-associated DNA resolved into two structurally distinct compartments: DNase-sensitive, high-molecular weight DNA associated with the vesicle corona and DNase-resistant, nucleosome-sized ([~]130-150 bp) DNA. Both compartments were overwhelmingly host-derived and broadly genomically distributed, whereas EBV DNA was minimal. In contrast, viral RNA cargo was dominated by the EBV noncoding RNA EBER1, which was strikingly enriched across all lines and confirmed within individual vesicles by ddPCR and super-resolution microscopy. EBER1 has previously been detected in MS brain tissue, yet its route to the CNS has remained unexplained. Our findings identify sEVs as a plausible vehicle for disseminating this immunostimulatory viral ncRNA beyond sites of latency, pointing to EV-mediated export of EBER1 as a candidate mechanism linking peripheral EBV infection to distal tissue signaling in MS and beyond.

immunology↗

MRPL47 as a Novel Mitochondrial Biomarker for Early Detection and Therapeutic Response in Ovarian Cancer

MRPL47 (Mitochondrial Ribosomal Protein Large Subunit 47) gene in chromosome 3q26 encodes a protein that is part of the large subunit of the mitochondrial ribosome. We observed that MRPL47 is frequently amplified and overexpressed in ovarian cancer samples. Importantly, increased expression of MRPL47 mRNA is associated with high levels of MRPL47 protein in ovarian cancer patients. High expression of MRPL47 is also associated with poor overall and recurrence free survival of ovarian cancer patients. Notably, MRPL47 improved metabolic fitness by enhancing cellular respiration, and glycolysis in cancer cells. Gene set enrichment analysis and target specific knockdown assays revealed that MYC transcription factor regulates MRPL47 expression. Furthermore, MRPL47 was identified very high in the plasma samples of ovarian cancer patients compared to those of healthy volunteers. MRPL47 was also associated with cisplatin resistance, whereas its expression predicted sensitivity to cisplatin therapy. Taken together, we demonstrated that MRPL47 can be used as a diagnostic biomarker for ovarian cancer and other cancers with 3q26 chromosomal amplification.

cancer biology↗

Clinical and Cross-Domain Validation of an LLM-Guided, Literature-Based Gene Prioritization Framework

BackgroundWe previously published a literature-based pipeline for sepsis gene prioritization (PS3 and candidate genes) using an LLM-enabled retrieval and judging framework. Here, we extend that work to ask whether these prioritized genes show independent clinical validity and whether the same strategy generalizes to a drug-obesity- infection setting. MethodsUsing the original LLM-guided workflow, we evaluated PS3 and the Candidate set in two new settings. First, we tested 28-day mortality prediction in the independent VANISH sepsis trial, benchmarking PS3 and Candidate against two established immune signatures--the Severe-or-Mild (SoM) signature and Immune Health Metric (IHM)--under a uniform logistic-regression framework with clinical covariates. Second, we applied the same genome-wide screening and tiered judging pipeline to GLP-1/obesity/infection biology centered on semaglutide, comparing Tier-1 and Tier-2 gene sets to STEP trial serum proteomics at gene and Hallmark pathway levels. In parallel, we fine-tuned an open-weight GPT-OSS-20B model on curated sepsis justifications to obtain a domain-aware "LLM-as-judge," and compared its scoring behavior with the base model on semaglutide Tier-2 genes. ResultsIn the full VANISH cohort, PS3 and the Candidate set showed moderate discrimination, whereas SoM remained the strongest single predictor of 28-day mortality. In the Critical/High APACHE II subgroup, PS3 achieved ROC and precision-recall performance comparable to, or slightly better than, SoM despite its smaller, knowledge-derived composition, indicating that literature-prioritized genes capture mortality-relevant immune dysregulation under severe illness. In the semaglutide case study, gene-level overlap between LLM-prioritized genes and differentially abundant serum proteins was modest, but Tier-1 genes recapitulated the main semaglutide-responsive metabolic programs from STEP and highlighted additional immune-metabolic pathways relevant to infection, with discordances largely explained by serum proteome coverage. The fine-tuned judge remained moderately concordant with the base GPT-OSS across mechanistic themes, preserving overall ranking while inducing systematic, biologically interpretable shifts in immune and infection-related scores. ConclusionsAn LLM-guided, literature-based gene prioritization framework yields compact gene sets that show independent sepsis mortality signal and pathway-level concordance in a semaglutide/obesity/infection setting, while a sepsis-aware LLM-as-judge provides domain-specific refinements without overturning core rankings. Together, these findings support knowledge-grounded, LLM-derived gene sets and judges as interpretable components for probing immune dysregulation across diseases and therapies. Short abstractWe previously published a literature-based pipeline for sepsis gene prioritization (Priority Set 3, PS3, and a Candidate set) using an LLM-enabled retrieval and judging framework; here, we test whether these prioritized genes show independent clinical validity and whether the same strategy generalizes to a drug-obesity-infection setting. Using the original workflow, we first evaluated 28-day mortality prediction in the independent VANISH sepsis trial, benchmarking PS3 and the Candidate set against two established immune signatures--the Severe-or-Mild (SoM) signature and Immune Health Metric (IHM)--within a uniform logistic-regression framework adjusted for clinical covariates. In the full cohort, PS3 and Candidate showed moderate discrimination, while SoM remained the strongest single predictor; however, in the Critical/High APACHE II subgroup, PS3 achieved ROC and precision-recall performance comparable to, or slightly better than, SoM despite its smaller, knowledge-driven composition, indicating that literature-prioritized genes capture mortality-relevant immune dysregulation under severe illness. We then applied the same genome-wide screening and tiered judging pipeline to GLP-1/obesity/infection biology centered on semaglutide, comparing Tier-1 and Tier-2 genes with STEP trial serum proteomics; although gene-level overlap with differentially abundant proteins was modest, Tier-1 genes recapitulated key semaglutide-responsive metabolic programs and highlighted additional immune-metabolic pathways relevant to infection, with discordances largely attributable to serum proteome coverage. Finally, supervised fine-tuning of an open-weight GPT-OSS model on curated sepsis justifications yielded a domain-aware "LLM-as-judge" that remained broadly concordant with the base model while inducing systematic, interpretable shifts in immune and infection-related scores. Together, these results support LLM-guided, literature-based gene sets and judges as compact, mechanistically interpretable components for probing immune dysregulation across diseases and therapies.

bioinformatics↗

YAP1 defines an emergent, plastic population of relapsed small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy characterized by rapid onset of chemoresistance and poor clinical outcomes. Transcriptional heterogeneity among treatment-naive SCLC tumors underlies four transcriptional subtypes, each with distinct clinical vulnerabilities. Though previously hypothesized to delineate a distinct subtype, expression of YAP1 is largely absent from treatment-naive, pure SCLC. To characterize relapsed SCLC, circulating tumor DNA, circulating tumor cells, and core needle biopsies from SCLC patients and preclinical models following resistance to standard-of-care therapies were analyzed. In contrast to treatment-naive SCLC, these analyses reveal an emergent YAP1-positive cell population that coincides with treatment resistance. These YAP1-positive cells exhibit characteristics of drug tolerant persister cells, including senescence, stemness, and plasticity, as YAP1 positive cells largely abandon features characteristic of SCLC to adopt those of large-cell neuroendocrine carcinoma (LCNEC). As a result of this SCLC-like to LCNEC-like evolution, YAP1-positive cells lack several clinically relevant SCLC surface targets (i.e., DLL3, SEZ6), but are enriched for others (i.e., B7-H3, TROP2). We propose a model where YAP1 expressing cells emerge with SCLC treatment resistance and characterize a tenacious subpopulation capable of diverging from the treatment naive lineage and adopting features to evade therapeutic response.

cancer biology↗

γδ T cells modulate anti-tumor immunity in small cell lung cancer

Small cell lung cancer (SCLC) is a highly aggressive neoplasm with limited sensitivity to anti-PD-(L)1 blockade, likely due to the epigenetic silencing of MHC-I. Elucidating MHC-I-independent immune recognition mechanisms is therefore crucial for enhancing treatment responses and improving clinical outcomes in a greater number of patients. Leveraging single cell approaches, we discovered {gamma}{delta} T cell infiltration in biospecimens from patients with SCLC. Despite PD-1 expression, {gamma}{delta} T cells maintained a cytotoxic transcriptional profile, suggestive of an anti-tumor role. Indeed, high {gamma}{delta} T cell infiltration predicted improved response to anti-PD-L1 immunotherapy in patients with SCLC. Moreover, using pre-clinical models, we demonstrated that {gamma}{delta} T cells are effective at tarlatamab (DLL3-CD3 BiTE) redirected SCLC killing and that zoledronate, an FDA-approved compound, can sensitize SCLC cells to {gamma}{delta} T cell-mediated killing. Thus, our findings suggest that engaged {gamma}{delta} T cells are potentially valuable targets for SCLC therapy.

cancer biology↗

Temperature and Excipient Mediated Modulation of Monoclonal Antibody Interactions Revealed by kD, Rheology, and Raman Spectroscopy

High-concentration monoclonal antibody (mAb) formulations are frequently constrained by elevated viscosity and colloidal instability, stemming from enhanced intermolecular interactions under crowded conditions. This study delineates the thermodynamic and rheological consequences of modulating protein-protein interactions through excipient-mediated and temperature-dependent mechanisms. Using an orthogonal analytical framework comprising diffusion interaction parameter (kD) measurements, high-shear rheometry, and Raman spectroscopic profiling, we interrogated mAb solutions at [~]80 and 160 mg/mL across a physiologically and industrially relevant thermal window (5-45 {degrees}C). In the absence of ionic additives, high kD values ([~]60 mL/g) indicated dominant long-range electrostatic repulsions, resulting in suppressed self-association and lower viscosity. Incorporation of NaCl (0.05% w/v) markedly decreased kD ([~]16-20 mL/g), consistent with Debye screening of surface charges and a shift toward short-range hydrophobic and van der Waals attractions, particularly impactful at elevated protein concentrations and low temperatures. Polysorbate 20 (0.05% v/v) mitigated these interactions via preferential surface adsorption, while sucrose exhibited a dualistic, concentration-dependent influence on viscosity via preferential exclusion and entropic crowding. The combination of NaCl and PS20 yielded the most pronounced rheological suppression, reflecting synergistic attenuation of both long-range repulsion and short-range association. Raman spectral analysis of Amide I/III regions confirmed structural invariance under thermal and shear stress, attributing viscosity modulation to colloidal rather than conformational perturbations. Collectively, these data elucidate the multivariate control of interparticle potentials in mAb solutions and provide a predictive basis for engineering subcutaneous formulations that optimize manufacturability, physical stability, and injectability through strategic manipulation of colloidal interaction landscapes.

biophysics↗

Eukaryotic Translation Initiation Factor Loaded Extracellular Vesicles Promotes Macrophage Cholesterol Metabolism in ovarian cancer

Tumor-driven immune suppression poses a significant impediment to the success of immunotherapy in ovarian cancer. Among the various mechanisms contributing to immune suppression, intracellular communication facilitated by tumor-derived extracellular vesicles (EVs) within the tumor microenvironment (TME) emerges as a pivotal factor influencing tumor growth. We discovered that EVs from both ovarian tumor cell lines and the plasma of ovarian cancer patients are encapsulated with eukaryotic translation initiation factor 4E (eIF4E). Our study revealed a new mechanism showing how these EVs are loaded with eIF4E and its impact on ovarian cancer progression. We also demonstrated that eIF4E-containing EVs (eIF4E-EVs) alter protein translation in macrophages, contributing to anti-tumor immune response. Treatment of macrophages with eIF4E-EVs induces an immunosuppressive phenotype marked by the release of cytokines such as IL-6 and an elevated expression of Programmed death-ligand 1 (PD-L1). Notably, eIF4E-packaged EVs enhance the expression of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) a pivotal enzyme in cholesterol biosynthesis, resulting in increased cholesterol levels within macrophages. Inhibition of HMGCR or reduction of cholesterol in macrophages effectively restores their antitumor activity by decreasing PD-L1 on macrophages. Analysis of tumor tissue from ovarian cancer patients revealed a positive correlation between HMGCR and TAM in ovarian cancer. In summary, we have characterized the mechanism of how eIF4E loaded EVs induced cholesterol synthesis, creating an immunosuppressive environment by upregulating PD-L1 expression in macrophages.

cancer biology↗

Federated deep learning enables cancer subtyping by proteomics

Artificial intelligence applications in biomedicine face major challenges from data privacy requirements. To address this issue for clinically annotated tissue proteomic data, we developed a Federated Deep Learning (FDL) approach (ProCanFDL), training local models on simulated sites containing data from a pan-cancer cohort (n=1,260) and 29 cohorts held behind private firewalls (n=6,265), representing 19,930 replicate data-independent acquisition mass spectrometry (DIA-MS) runs. Local parameter updates were aggregated to build the global model, achieving a 43% performance gain on the hold-out test set (n=625) in 14 cancer subtyping tasks compared to local models, and matching centralized model performance. The approachs generalizability was demonstrated by retraining the global model with data from two external DIA-MS cohorts (n=55) and eight acquired by tandem mass tag (TMT) proteomics (n=832). ProCanFDL presents a solution for internationally collaborative machine learning initiatives using proteomic data, e.g., for discovering predictive biomarkers or treatment targets, while maintaining data privacy. Statement of SignificanceA federated deep learning approach applied to human proteomic data, acquired using two distinct proteomic technologies from 40 tumor cohorts from eight countries, enabled accurate cancer histopathological subtyping while preserving data privacy. This approach will enable privacy-compliant development of large-scale proteomic AI models, including foundation models, across institutions globally.

cancer biology↗

Developing, characterizing and modelingCRISPR-based point-of-use pathogen diagnostics

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

synthetic biology↗

Cortical inhibitory parvalbumin interneurons exhibit metabolic specializations coordinated by PGC-1α that are lost in rodents and humans after traumatic brain injury

Parvalbumin-positive interneurons (PV-INs) regulate neuronal and circuit activity, and their dysfunction is observed across neurological conditions, including traumatic brain injury (TBI), epilepsy, Alzheimers disease, and schizophrenia. PV-INs are particularly vulnerable to cell loss, potentially due to their increased metabolic demands arising from their uniquely high level of electrical activity, which render them susceptible to metabolic pressure. Here, we use single-nucleus RNA-sequencing (snRNAseq) data from a rodent model of TBI, as well as human TBI data, and demonstrate PV-INs have unique metabolic specializations that are lost after injury and can be rescued by in vivo treatment with the glycolytic inhibitor, 2-deoxyglucose. We generated a novel PV-IN transcriptional identity module comprised primarily of genes encoding specialized ion channels, metabolic enzymes, and synaptic machinery, that identifies heterogenous subsets of injury-associated PV-INs with loss of PV-IN transcriptional identity. We show that changes in metabolic specialization are coupled to changes in transcriptional identity in PV-INs and implicate the PV-IN-enriched transcriptional co-activator, Ppargc1a, as a key driver of PV-IN transcriptional metabolic dysfunction. We also identify a family of long non-coding RNAs enriched in this subset of transcriptionally dysfunctional PV-INs that negatively correlates with PV-IN metabolic specialization. Lastly, we utilize these tools to interrogate a published human TBI snRNAseq data set and find nearly identical changes, underscoring the importance of PV-IN metabolic dysfunction in the pathology of TBI.

neuroscience↗

Causal network perturbation analysis identifies known and novel type-2 diabetes driver genes

The molecular pathogenesis of type 2 diabetes mellitus (T2DM) involves environmental and genetic factors that remain poorly understood. Increased insulin resistance leads to pancreatic {beta}-islet cell exhaustion and failure, leading to more severe T2DM. As such, targeting {beta}-cell dysfunction is an attractive pathway for T2DM treatment. Single-cell RNA sequencing can help identify differentially expressed genes (DEGs) that may contribute to disease-associated pathophysiology, but cannot capture the full spectrum of disease pathophysiology, as some genes that are not differentially expressed may still play important roles in disease progression or mechanisms. As such, we investigated single-cell gene expression changes in {beta}-cells from healthy (C57BL/6J) and diabetic (NZO/HlLtJ) mice fed with normal or high-fat, high-sugar diet (HFHS) across the spectrum of T2DM development (prediabetic, mild or severely diabetic) using an innovative integration of the causal network perturbation assessment (ssNPA) framework with meta-cell transcriptome analysis to identify driver genes for T2DM. By generating a reference causal network and conducting in silico perturbations, we identified three classes of genes implicated in T2DM pathophysiology: (1) DEGs that were not perturbed (e.g, Igf2bp2 strongly linked to {beta} cell dysfunction and glucose regulation), (2) perturbed non-DEGs (e.g., Glp1r a therapeutic target of modern diabetes drugs) that were perturbed, and (3) DEGs that were perturbed (e.g., Pdx1 a master {beta} cell transcription factor), and validated some of the targets using the KOMP database. Together, these results show that each analytic layer captures complementary mechanisms underlying T2DM severity, and their synthesis provides a more complete view of the disease pathophysiology.

bioinformatics↗

p300/CBP degradation is required to disable the active AR enhanceosome in prostate cancer

Prostate cancer is an exemplar of an enhancer-binding transcription factor-driven disease. The androgen receptor (AR) enhanceosome complex comprised of chromatin and epigenetic coregulators assembles at enhancer elements to drive disease progression. The paralog lysine acetyltransferases p300 and CBP deposit histone marks that are associated with enhancer activation. Here, we demonstrate that p300/CBP are determinant cofactors of the active AR enhanceosome in prostate cancer. Histone H2B N-terminus multisite lysine acetylation (H2BNTac), which is exclusively reliant on p300/CBP catalytic function, marked active enhancers and was notably elevated in prostate cancer lesions relative to the adjacent benign epithelia. Degradation of p300/CBP rapidly depleted acetylation marks associated with the active AR enhanceosome, which was only partially phenocopied by inhibition of their reader bromodomains. Notably, H2BNTac was effectively abrogated only upon p300/CBP degradation, which led to a stronger suppression of p300/CBP-dependent oncogenic gene programs relative to bromodomain inhibition or the inhibition of its catalytic domain. In vivo experiments using an orally active p300/CBP proteolysis targeting chimera (PROTAC) degrader (CBPD-409) showed that p300/CBP degradation potently inhibited tumor growth in preclinical models of castration-resistant prostate cancer and synergized with AR antagonists. While mouse p300/CBP orthologs were effectively degraded in host tissues, prolonged treatment with the PROTAC degrader was well tolerated with no significant signs of toxicity. Taken together, our study highlights the pivotal role of p300/CBP in maintaining the active AR enhanceosome and demonstrates how target degradation may have functionally distinct effects relative to target inhibition, thus supporting the development of p300/CBP degraders for the treatment of advanced prostate cancer.

cancer biology↗

NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis

The androgen receptor (AR) is a ligand-responsive transcription factor that binds at enhancers to drive terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to drive hyper-proliferative, metastatic, or therapy-resistant phenotypes, the molecular mechanisms of which remain poorly understood. Here, we show that the tumor-specific enhancer circuitry of AR is critically reliant on the activity of Nuclear Receptor Binding SET Domain Protein 2 (NSD2), a histone 3 lysine 36 di-methyltransferase. NSD2 expression is abnormally gained in prostate cancer cells and its functional inhibition impairs AR trans-activation potential through partial off-loading from over 40,000 genomic sites, which is greater than 65% of the AR tumor cistrome. The NSD2-dependent AR sites distinctly harbor a chimeric AR-half motif juxtaposed to a FOXA1 element. Similar chimeric motifs of AR are absent at the NSD2-independent AR enhancers and instead contain the canonical palindromic motifs. Meta-analyses of AR cistromes from patient tumors uncovered chimeric AR motifs to exclusively participate in tumor-specific enhancer circuitries, with a minimal role in the physiological activity of AR. Accordingly, NSD2 inactivation attenuated hallmark cancer phenotypes that were fully reinstated upon exogenous NSD2 re-expression. Inactivation of NSD2 also engendered increased dependency on its paralog NSD1, which independently maintained AR and MYC hyper-transcriptional programs in cancer cells. Concordantly, a dual NSD1/2 PROTAC degrader, called LLC0150, was preferentially cytotoxic in AR-dependent prostate cancer as well as NSD2-altered hematologic malignancies. Altogether, we identify NSD2 as a novel subunit of the AR neo-enhanceosome that wires prostate cancer gene expression programs, positioning NSD1/2 as viable paralog co-targets in advanced prostate cancer.

cancer biology↗

Single-cell and bulk transcriptional profiling of mouse ovaries reveals novel genes and pathways associated with DNA damage response in oocytes.

Immature oocytes enclosed in primordial follicles stored in female ovaries are under constant threat of DNA damage induced by endogenous and exogenous factors. Checkpoint kinase 2 (CHEK2) is a key mediator of the DNA damage response in all cells. Genetic studies have shown that CHEK2 and its downstream targets, p53 and TAp63, regulate primordial follicle elimination in response to DNA damage, however the mechanism leading to their demise is still poorly characterized. Single-cell and bulk RNA sequencing were used to determine the DNA damage response in wildtype and Chek2-deficient ovaries. A low but oocyte-lethal dose of ionizing radiation induces a DNA damage response in ovarian cells that is solely dependent on CHEK2. DNA damage activates multiple ovarian response pathways related to apoptosis, p53, interferon signaling, inflammation, cell adhesion, and intercellular communication. These pathways are differentially employed by different ovarian cell types, with oocytes disproportionately affected by radiation. Novel genes and pathways are induced by radiation specifically in oocytes, shedding light on their sensitivity to DNA damage, and implicating a coordinated response between oocytes and pre-granulosa cells within the follicle. These findings provide a foundation for future studies on the specific mechanisms regulating oocyte survival in the context of aging, as well as therapeutic and environmental genotoxic exposures.

cell biology↗

Adaptation to ER Stress by Slt2, Counterpart of Human MAP Kinase ERK1/2, via Enhancing Splicing and Translation of HAC1 mRNA in Saccharomyces cerevisiae

Unfolded protein response (UPR) is a cellular strategy to increase the protein folding capacity of cells in response to stress within the endoplasmic reticulum (ER). In metazoan cells, three major UPR sensors Ire1, PERK and ATF6 work in concert by simultaneously activating intracellular signaling pathways and modulating a series of physiological processes such as attenuation of the general protein synthesis and expression of protein chaperones. In yeast Saccharomyces cerevisiae, Ire1 is known to be the only UPR sensor, which mediates splicing of HAC1 mRNA in the cytoplasm and derepresses its translation. Hac1 is a transcription factor that increases the expression of protein folding enzymes and chaperones, thus enhancing the protein folding capacity of cells. In this study, we provide compelling evidence that kinase Slt2 plays a significant role in facilitating both the splicing and translation of HAC1 mRNA, while also serving as a key mediator in the activation of UPR genes through an alternative route. We also provide evidence that human extracellular signal-regulated kinase 1 (ERK1) or ERK2 served as a functional substitute for yeast Slt2 in the context of UPR. Furthermore, ERK1 exhibits an enhanced activation in human primary cells when grown in the presence of ER stressor. These findings collectively suggest that Slt2 responds to ER stress by activating the Ire1 pathway as well as initiating a parallel signaling pathway.

molecular biology↗

Immunosuppressive tumor microenvironment of osteosarcoma

Osteosarcoma is the most common malignant bone tumor in children, characterized by a high degree of genomic instability, resulting in copy-number alterations and genomic rearrangements without disease-defining recurrent mutations. Clinical trials based on molecular characterization have failed to find new effective therapies or improve outcomes over the last 40 years. To better understand the immune microenvironment of osteosarcoma, we performed single-cell RNA sequencing on six tumor biopsy samples, combined with a previously-published cohort of six samples. Additional osteosarcoma samples were profiled using spatial transcriptomics for validation of discovered subtypes and to add spatial context. Analysis revealed immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs), regulatory and exhausted T-cells, and LAMP3+ dendritic cells. Using cell-cell communication modeling, we identified robust interactions between MDSCs and other cells, leading to NF-{kappa}B upregulation and an immunosuppressive microenvironment, as well as interactions involving regulatory T-cells and osteosarcoma cells that promoted tumor progression and a proangiogenic niche. Statement of SignificanceOsteosarcoma patient survival has remained stagnant for several decades due to lack of successful therapy for high-risk patients, including those with metastatic disease. Identifying novel therapeutics including immunotherapies is of great clinical importance. Our study highlights several important immunosuppressive mechanisms within osteosarcoma that should be considered when developing future immunotherapies.

cancer biology↗