Cytokine and Chemokine Receptor Profiles in Adipose Tissue Vasculature Unravel Endothelial Cell Responses in HIV.

Chronic systemic inflammation contributes to a substantially elevated risk of myocardial infarction in people living with HIV (PLWH). Endothelial cell dysfunction disrupts vascular homeostasis regulation, increasing the risk of vasoconstriction, inflammation, and thrombosis that contribute to cardiovascular disease. Our objective was to study the effects of plasma from PLWH on endothelial cell (EC) function, with the hypothesis that cytokines and chemokines are major drivers of EC activation. We first broadly phenotyped chemokine and cytokine receptor expression on arterial ECs, capillary ECs, venous ECs, and vascular smooth muscle cells (VSMCs) in adipose tissue in the subcutaneous adipose tissue of 59 PLWH using single cell transcriptomic analysis. We used CellChat to predict cell-cell interactions between ECs and other cells in the adipose tissue and Spearman correlation to measure the association between ECs and plasma cytokines. Finally, we cultured human arterial ECs (HAECs) in plasma-conditioned media from PLWH and performed bulk sequencing to study the direct effects ex-vivo. We observed that arterial and capillary ECs expressed higher interferon and tumor necrosis factor (TNF) receptors. Venous ECs had more interleukin (IL)-1R1 and ACKR1 receptors, and VSMCs had high significant IL-6R expression. CellChat predicted ligand-receptor interactions between adipose tissue immune cells as senders and capillary ECs as recipients in TNF-TNFRSF1A/B interactions. Chemokines expressed largely by capillary ECs were predicted to bind ACKR1 receptors on venous ECs. Beyond the adipose tissue, the proportion of venous ECs and VSMCs were positively plasma IL-6. In ex-vivo experiments, HAECs cultured with plasma-conditioned media from PLWH expressed transcripts that enriched for the TNF-α and reactive oxidative phosphorylation pathways. In conclusion, ECs demonstrate heterogeneity in cytokine and chemokine receptor expression. Further research is needed to fully elucidate the role of cytokines and chemokines in EC dysfunction and to develop effective therapeutic strategies.

Proteomic Biomarkers of Quantitative Interstitial Abnormalities in COPDGene and CARDIA Lung Study.

Rationale: Quantitative interstitial abnormalities (QIAs) are early measures of lung injury automatically detected on chest computed tomography scans. QIAs are associated with impaired respiratory health and share features with advanced lung diseases, but their biological underpinnings are not well understood. Objectives: To identify novel protein biomarkers of QIAs using high-throughput plasma proteomic panels within two multicenter cohorts. Methods: We measured the plasma proteomics of 4,383 participants in an older, ever-smoker cohort (COPDGene [Genetic Epidemiology of Chronic Obstructive Pulmonary Disease]) and 2,925 participants in a younger population cohort (CARDIA [Coronary Artery Disease Risk in Young Adults]) using the SomaLogic SomaScan assays. We measured QIAs using a local density histogram method. We assessed the associations between proteomic biomarker concentrations and QIAs using multivariable linear regression models adjusted for age, sex, body mass index, smoking status, and study center (Benjamini-Hochberg false discovery rate-corrected P ⩽ 0.05). Measurements and Main Results: In total, 852 proteins were significantly associated with QIAs in COPDGene and 185 in CARDIA. Of the 144 proteins that overlapped between COPDGene and CARDIA, all but one shared directionalities and magnitudes. These proteins were enriched for 49 Gene Ontology pathways, including biological processes in inflammatory response, cell adhesion, immune response, ERK1/2 regulation, and signaling; cellular components in extracellular regions; and molecular functions including calcium ion and heparin binding. Conclusions: We identified the proteomic biomarkers of QIAs in an older, smoking population with a higher prevalence of pulmonary disease and in a younger, healthier community cohort. These proteomics features may be markers of early precursors of advanced lung diseases.

Atezolizumab in Combination With Carboplatin and Survival Outcomes in Patients With Metastatic Triple-Negative Breast Cancer: The TBCRC 043 Phase 2 Randomized Clinical Trial.

Importance: Agents targeting programmed death ligand 1 (PD-L1) have demonstrated efficacy in triple-negative breast cancer (TNBC) when combined with chemotherapy and are now the standard of care in patients with PD-L1-positive metastatic disease. In contrast to microtubule-targeting agents, the effect of combining platinum compounds with programmed cell death 1 (PD-1)/PD-L1 immunotherapy has not been extensively determined. Objective: To evaluate the efficacy of atezolizumab with carboplatin in patients with metastatic TNBC. Design, Setting, and Participants: This phase 2 randomized clinical trial was conducted in 6 centers from August 2017 to June 2021. Interventions: Patients with metastatic TNBC were randomized to receive carboplatin area under the curve (AUC) 6 alone or with atezolizumab, 1200 mg, every 3 weeks until disease progression or unacceptable toxic effects with a 3-year duration of follow-up. Main Outcome and Measures: The primary end point was investigator-assessed progression-free survival (PFS). Secondary end points included overall response rate (ORR), clinical benefit rate (CBR), and overall survival (OS). Other objectives included correlation of response with tumor PD-L1 levels, tumor-infiltrating lymphocytes (TILs), tumor DNA- and RNA-sequenced biomarkers, TNBC subtyping, and multiplex analyses of immune markers. Results: All 106 patients with metastatic TNBC who were enrolled were female with a mean (range) age of 55 (27-79) years, of which 12 (19%) identified as African American/Black, 1 (1%) as Asian, 73 (69%) as White, and 11 (10%) as unknown. Patients were randomized and received either carboplatin (n = 50) or carboplatin and atezolizumab (n = 56). The combination improved PFS (hazard ratio [HR], 0.66; 95% CI, 0.44-1.01; P = .05) from a median of 2.2 to 4.1 months, increased ORR from 8.0% (95% CI, 3.2%-18.8%) to 30.4% (95% CI, 19.9%-43.3%), increased CBR at 6 months from 18.0% (95% CI, 9.8%-30.1%) to 37.5% (95% CI, 26.0%-50.6%), and improved OS (HR, 0.60; 95% CI, 0.37-0.96; P = .03) from a median of 8.6 to 12.6 months. Subgroup analysis showed PD-L1-positive tumors did not benefit more from adding atezolizumab (HR, 0.62; 95% CI, 0.23-1.65; P = .35). Patients with high TILs (HR, 0.12; 95% CI, 0.30-0.50), high mutation burden (HR, 0.50; 95% CI, 0.23-1.06), and prior chemotherapy (HR, 0.59; 95% CI, 0.36-0.95) received greater benefit on the combination. Patients with obesity and patients with more than 125 mg/dL on-treatment blood glucose levels were associated with better PFS (HR, 0.35; 95% CI, 0.10-1.80) on the combination. TNBC subtypes benefited from adding atezolizumab, except the luminal androgen receptor subtype. Conclusions and Relevance: In this randomized clinical trial, the addition of atezolizumab to carboplatin significantly improved survival of patients with metastatic TNBC regardless of PD-L1 status. Further, lower risk of disease progression was associated with increased TILs, higher mutation burden, obesity, and uncontrolled blood glucose levels. Trial Registration: ClinicalTrials.gov Identifier: NCT03206203.

Anti-Inflammatory Effect of High-Density Lipoprotein Blunted by Delivery of Altered MicroRNA Cargo in Patients With Rheumatoid Arthritis.

OBJECTIVE: High-density lipoprotein (HDL) has well-characterized anti-atherogenic cholesterol efflux and antioxidant functions. Another function of HDL uncharacterized in rheumatoid arthritis (RA) is its ability to transport microRNAs (miRNAs) between cells and thus alter cellular function. The study's purpose was to determine if HDL-miRNA cargo is altered and affects inflammation in RA. METHODS: HDL-microRNAs were characterized in 30 RA and 30 control participants by next generation sequencing and quantitative polymerase chain reaction. The most abundant differentially expressed miRNA was evaluated further. The function of miR-1246 was assessed by miRNA mimics, antagomiRs, small interfering RNA knockdown, and luciferase assays. Monocyte-derived macrophages were treated with miR-1246-loaded HDL and unmodified HDL from RA and control participants to measure delivery of miR-1246 and its effect on interleukin-6 (IL-6). RESULTS: The most abundant miRNA on HDL was miR-1246; it was significantly enriched two-fold on HDL from RA versus control participants. HDL-mediated miR-1246 delivery to macrophages significantly increased IL6 expression 43-fold. miR-1246 delivery significantly decreased DUSP3 1.5-fold and DUSP3 small interfering RNA knockdown increased macrophage IL6 expression. Luciferase assay indicated DUSP3 is a direct target of miR-1246. Unmodified HDL from RA delivered 1.6-fold more miR-1246 versus control participant HDL. Unmodified HDL from both RA and control participants attenuated activated macrophage IL6 expression, but this effect was significantly blunted in RA so that IL6 expression was 3.4-fold higher after RA versus control HDL treatment. CONCLUSION: HDL-miR-1246 was increased in RA versus control participants and delivery of miR-1246 to macrophages increased IL-6 expression by targeting DUSP3. The altered HDL-miRNA cargo in RA blunted HDL's anti-inflammatory effect.

Molecular signature incorporating the immune microenvironment enhances thyroid cancer outcome prediction.

Genomic and transcriptomic analysis has furthered our understanding of many tumors. Yet, thyroid cancer management is largely guided by staging and histology, with few molecular prognostic and treatment biomarkers. Here, we utilize a large cohort of 251 patients with 312 samples from two tertiary medical centers and perform DNA/RNA sequencing, spatial transcriptomics, and multiplex immunofluorescence to identify biomarkers of aggressive thyroid malignancy. We identify high-risk mutations and discover a unique molecular signature of aggressive disease, the Molecular Aggression and Prediction (MAP) score, which provides improved prognostication over high-risk mutations alone. The MAP score is enriched for genes involved in epithelial de-differentiation, cellular division, and the tumor microenvironment. The MAP score also identifies aggressive tumors with lymphocyte-rich stroma that may benefit from immunotherapy. Future clinical profiling of the stromal microenvironment of thyroid cancer could improve prognostication, inform immunotherapy, and support development of novel therapeutics for thyroid cancer and other stroma-rich tumors.

Systematic Assessment of Small RNA Profiling in Human Extracellular Vesicles.

MOTIVATION: Extracellular vesicles (EVs) are produced and released by most cells and are now recognized to play a role in intercellular communication through the delivery of molecular cargo, including proteins, lipids, and RNA. Small RNA sequencing (small RNA-seq) has been widely used to characterize the small RNA content in EVs. However, there is a lack of a systematic assessment of the quality, technical biases, RNA composition, and RNA biotypes enrichment for small RNA profiling of EVs across cell types, biofluids, and conditions. METHODS: We collected and reanalyzed small RNA-seq datasets for 2756 samples from 83 studies involving 55 with EVs only and 28 with both EVs and matched donor cells. We assessed their quality by the total number of reads after adapter trimming, the overall alignment rate to the host and non-host genomes, and the proportional abundance of total small RNA and specific biotypes, such as miRNA, tRNA, rRNA, and Y RNA. RESULTS: We found that EV extraction methods varied in their reproducibility in isolating small RNAs, with effects on small RNA composition. Comparing proportional abundances of RNA biotypes between EVs and matched donor cells, we discovered that rRNA and tRNA fragments were relatively enriched, but miRNAs and snoRNA were depleted in EVs. Except for the export of eight miRNAs being context-independent, the selective release of most miRNAs into EVs was study-specific. CONCLUSION: This work guides quality control and the selection of EV isolation methods and enhances the interpretation of small RNA contents and preferential loading in EVs.

Inflammatory characteristics of central compartment atopic disease.

BACKGROUND: Central compartment atopic disease (CCAD) is an emerging phenotype of chronic rhinosinusitis with nasal polyposis (CRSwNP) characterized by prominent central nasal inflammatory changes. This study compares the inflammatory characteristics of CCAD relative to other phenotypes of CRSwNP. METHODS: A cross-sectional analysis of data from a prospective clinical study was performed on patients with CRSwNP who were undergoing endoscopic sinus surgery (ESS). Patients with CCAD, aspirin-exacerbated respiratory disease (AERD), allergic fungal rhinosinusitis (AFRS), and non-typed CRSwNP (CRSwNP NOS) were included and mucus cytokine levels and demographic data were analyzed for each group. Chi-squared/Mann-Whitney U tests and partial least squares discriminant analysis (PLS-DA) were performed for comparison and classification. RESULTS: A total of 253 patients were analyzed (CRSwNP, n = 137; AFRS, n = 50; AERD, n = 42; CCAD, n = 24). Patients with CCAD were the least likely to have comorbid asthma (p = 0.0004). The incidence of allergic rhinitis in CCAD patients did not vary significantly compared to patients with AFRS and AERD, but was higher compared to patients with CRSwNP NOS (p = 0.04). On univariate analysis, CCAD was characterized by less inflammatory burden, with reduced levels of interleukin 6 (IL-6), IL-8, interferon gamma (IFN-γ), and eotaxin relative to other groups and significantly lower type 2 cytokines (IL-5, IL-13) relative to both AERD and AFRS. These findings were supported by multivariate PLS-DA, which clustered CCAD patients into a relatively homogenous low-inflammatory cytokine profile. CONCLUSIONS: CCAD has unique endotypic features compared to other patients with CRSwNP. The lower inflammatory burden may be reflective of a less severe variant of CRSwNP.

Localizing Hormone Receptor Expression to Cellular Compartments in Idiopathic Subglottic Stenosis.

OBJECTIVES: Idiopathic subglottic stenosis (iSGS) is an unexplained progressive fibrosis of the upper airway. iSGS almost exclusively affects women; as a result, female hormones (estrogen and progesterone) have been proposed to participate in the pathogenesis of iSGS. Our aim was to localize cell-specific gene expression of estrogen receptors (ESR1 and ESR2) and progesterone receptor (PGR) using an established iSGS single-cell RNA sequencing (scRNAseq) cell atlas. STUDY DESIGN: Ex vivo molecular study of airway scar and healthy mucosa from iSGS patients. METHODS: An established scRNAseq atlas consisting of 25,974 individually sequenced cells from subglottic scar (n = 7) or matched unaffected mucosa (n = 3) in iSGS patients was interrogated for RNA expression of ESR1, ESR2, and PGR. Results were quantified and compared across cell subsets, then visualized using Uniform Manifold Approximation and Projection (UMAP). Confirmatory protein assessment of endocrine receptors in fibroblasts from iSGS patients (n = 5) was performed via flow cytometry. RESULTS: The proximal airway mucosa in iSGS patients demonstrates differential expression of endocrine receptors (ESR1, ESR2, PGR). Within airway scar, endocrine receptors are primarily expressed by fibroblasts, immune cells, and endothelial cells. Fibroblasts show strong ESR1 and PGR expression, while immune cells possess RNA for both ESR1 and ESR2. Endothelial cells predominantly express ESR2. Epithelial cells in unaffected mucosa express all three receptors, which are all reduced in airway scar. CONCLUSIONS: scRNAseq data localized endocrine receptor expression to specific cell subsets. These results provide the foundation for future work interrogating how hormone-dependent mechanisms promote, sustain, or participate in iSGS disease pathogenesis. LEVEL OF EVIDENCE: NA; Basic science Laryngoscope, 133:3506-3511, 2023.

Epithelial innate immune response to Pseudomonas aeruginosa-derived flagellin in chronic rhinosinusitis.

BACKGROUND: Pseudomonas aeruginosa is a common colonizing pathogen in the upper respiratory tract and is associated with recalcitrant chronic rhinosinusitis (CRS). Herein we sought to characterize the effect of P. aeruginosa-derived flagellin on human sinonasal epithelial cell (HSNEC) immune responses and determine whether these pathways are disrupted in CRS. METHODS: Air-liquid interface cultures were established from CRS and healthy control donors. Cells were incubated with P. aeruginosa-derived flagellin for 24 hours and transcriptional changes were assessed using whole transcriptome RNA sequencing. Apical and basolateral secretion of the pro-inflammatory cytokines in interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6 were measured after stimulation by lipopolysaccharide or flagellin and responses were compared between CRS and healthy control patients. RESULTS: HSNECs were weakly responsive to lipopolysaccharide, whereas flagellin stimulated a profound innate immune response dominated by TNF-α, IL-1ß, and IL-17 signaling and activation of the IL-17C/IL-23 axis. CRS-derived HNSECs showed an altered innate immune response to flagellin, characterized by a profound increase in TNF-α secretion coupled with reduced IL-6 secretion. CONCLUSIONS: Flagellin activates a potent innate immune response in HSNECs characterized by pro-inflammatory mediators and cytokines/chemokines associated with neutrophilic inflammation. HSNECs from CRS patients have a dysregulated innate immune response to flagellin characterized by an imbalance between IL-6 and TNF-α secretion.

Plasma extracellular vesicle transcriptome as a dynamic liquid biopsy in acute heart failure.

Background: Acute decompensation is associated with increased mortality in heart failure (HF) patients, though the underlying etiology remains unclear. Extracellular vesicles (EVs) and their cargo may mark specific cardiovascular physiologic states. We hypothesized that EV transcriptomic cargo, including long non-coding RNAs (lncRNAs) and mRNAs, is dynamic from the decompensated to recompensated HF state, reflecting molecular pathways relevant to adverse remodeling. Methods: We examined differential RNA expression from circulating plasma extracellular RNA in acute HF patients at hospital admission and discharge alongside healthy controls. We leveraged different exRNA carrier isolation methods, publicly available tissue banks, and single nuclear deconvolution of human cardiac tissue to identify cell and compartment specificity of the topmost significantly differentially expressed targets. EV-derived transcript fragments were prioritized by fold change (-1.5 to + 1.5) and significance (<5% false discovery rate), and their expression in EVs was subsequently validated in 182 additional patients (24 control; 86 HFpEF; 72 HFrEF) by qRT-PCR. We finally examined the regulation of EV-derived lncRNA transcripts in human cardiac cellular stress models. Results: We identified 138 lncRNAs and 147 mRNAs (present mostly as fragments in EVs) differentially expressed between HF and control. Differentially expressed transcripts between HFrEF vs. control were primarily cardiomyocyte derived, while those between HFpEF vs. control originated from multiple organs and different (non-cardiomyocyte) cell types within the myocardium. We validated 5 lncRNAs and 6 mRNAs to differentiate between HF and control. Of those, 4 lncRNAs (AC092656.1, lnc-CALML5-7, LINC00989, RMRP) were altered by decongestion, with their levels independent of weight changes during hospitalization. Further, these 4 lncRNAs dynamically responded to stress in cardiomyocytes and pericytes in vitro , with a directionality mirroring the acute congested state. Conclusion: Circulating EV transcriptome is significantly altered during acute HF, with distinct cell and organ specificity in HFpEF vs. HFrEF consistent with a multi-organ vs. cardiac origin, respectively. Plasma EV-derived lncRNA fragments were more dynamically regulated with acute HF therapy independent of weight change (relative to mRNAs). This dynamicity was further demonstrated with cellular stress in vitro . Prioritizing transcriptional changes in plasma circulating EVs with HF therapy may be a fruitful approach to HF subtype-specific mechanistic discovery. CLINICAL PERSPECTIVE: What is new?: We performed extracellular transcriptomic analysis on the plasma of patients with acute decompensated heart failure (HFrEF and HFpEF) before and after decongestive efforts.Long non-coding RNAs (lncRNAs) within extracellular vesicles (EVs) changed dynamically upon decongestion in concordance with changes within human iPSC-derived cardiomyocytes under stress.In acute decompensated HFrEF, EV RNAs are mainly derived from cardiomyocytes, whereas in HFpEF, EV RNAs appear to have broader, non-cardiomyocyte origins.What are the clinical implications?: Given their concordance between human expression profiles and dynamic in vitro responses, lncRNAs within EVs during acute HF may provide insight into potential therapeutic targets and mechanistically relevant pathways. These findings provide a "liquid biopsy" support for the burgeoning concept of HFpEF as a systemic disorder extending beyond the heart, as opposed to a more cardiac-focused physiology in HFrEF.

LDL delivery of microbial small RNAs drives atherosclerosis through macrophage TLR8.

Macrophages present a spectrum of phenotypes that mediate both the pathogenesis and resolution of atherosclerotic lesions. Inflammatory macrophage phenotypes are pro-atherogenic, but the stimulatory factors that promote these phenotypes remain incompletely defined. Here we demonstrate that microbial small RNAs (msRNA) are enriched on low-density lipoprotein (LDL) and drive pro-inflammatory macrophage polarization and cytokine secretion via activation of the RNA sensor toll-like receptor 8 (TLR8). Removal of msRNA cargo during LDL re-constitution yields particles that readily promote sterol loading but fail to stimulate inflammatory activation. Competitive antagonism of TLR8 with non-targeting locked nucleic acids was found to prevent native LDL-induced macrophage polarization in vitro, and re-organize lesion macrophage phenotypes in vivo, as determined by single-cell RNA sequencing. Critically, this was associated with reduced disease burden in distinct mouse models of atherosclerosis. These results identify LDL-msRNA as instigators of atherosclerosis-associated inflammation and support alternative functions of LDL beyond cholesterol transport.

Single cell analysis of cribriform prostate cancer reveals cell intrinsic and tumor microenvironmental pathways of aggressive disease.

Cribriform prostate cancer, found in both invasive cribriform carcinoma (ICC) and intraductal carcinoma (IDC), is an aggressive histological subtype that is associated with progression to lethal disease. To delineate the molecular and cellular underpinnings of ICC/IDC aggressiveness, this study examines paired ICC/IDC and benign prostate surgical samples by single-cell RNA-sequencing, TCR sequencing, and histology. ICC/IDC cancer cells express genes associated with metastasis and targets with potential for therapeutic intervention. Pathway analyses and ligand/receptor status model cellular interactions among ICC/IDC and the tumor microenvironment (TME) including JAG1/NOTCH. The ICC/IDC TME is hallmarked by increased angiogenesis and immunosuppressive fibroblasts (CTHRC1+ASPN+FAP+ENG+) along with fewer T cells, elevated T cell dysfunction, and increased C1QB+TREM2+APOE+-M2 macrophages. These findings support that cancer cell intrinsic pathways and a complex immunosuppressive TME contribute to the aggressive phenotype of ICC/IDC. These data highlight potential therapeutic opportunities to restore immune signaling in patients with ICC/IDC that may afford better outcomes.

Combined Dusp4 and p53 loss with Dbf4 amplification drives tumorigenesis via cell cycle restriction and replication stress escape in breast cancer.

AIM: Deregulated signaling pathways are a hallmark feature of oncogenesis and driver of tumor progression. Dual specificity protein phosphatase 4 (DUSP4) is a critical negative regulator of the mitogen-activated protein kinase (MAPK) pathway and is often deleted or epigenetically silenced in tumors. DUSP4 alterations lead to hyperactivation of MAPK signaling in many cancers, including breast cancer, which often harbor mutations in cell cycle checkpoint genes, particularly in TP53. METHODS: Using a genetically engineered mouse model, we generated mammary-specific Dusp4-deleted primary epithelial cells to investigate the necessary conditions in which DUSP4 loss may drive breast cancer oncogenesis. RESULTS: We found that Dusp4 loss alone is insufficient in mediating tumorigenesis, but alternatively converges with loss in Trp53 and MYC amplification to induce tumorigenesis primarily through chromosome 5 amplification, which specifically upregulates Dbf4, a cell cycle gene that promotes cellular replication by mediating cell cycle checkpoint escape. CONCLUSIONS: This study identifies a novel mechanism for breast tumorigenesis implicating Dusp4 loss and p53 mutations in cellular acquisition of Dbf4 upregulation as a driver of cellular replication and cell cycle checkpoint escape.

scMRMA: single cell multiresolution marker-based annotation.

Single-cell RNA sequencing has become a powerful tool for identifying and characterizing cellular heterogeneity. One essential step to understanding cellular heterogeneity is determining cell identities. The widely used strategy predicts identities by projecting cells or cell clusters unidirectionally against a reference to find the best match. Here, we develop a bidirectional method, scMRMA, where a hierarchical reference guides iterative clustering and deep annotation with enhanced resolutions. Taking full advantage of the reference, scMRMA greatly improves the annotation accuracy. scMRMA achieved better performance than existing methods in four benchmark datasets and successfully revealed the expansion of CD8 T cell populations in squamous cell carcinoma after anti-PD-1 treatment.

SETD2 regulates the methylation of translation elongation factor eEF1A1 in clear cell renal cell carcinoma.

BACKGROUND: SET domain-containing protein 2 (SETD2) is commonly mutated in renal cell carcinoma. SETD2 methylates histone H3 as well as a growing list of non-histone proteins. OBJECTIVE: Initially, we sought to explore SETD2-dependent changes in lysine methylation of proteins in proximal renal tubule cells. Subsequently, we focused on changes in lysine methylation of the translation elongation factor eEF1A1. METHODS: To accomplish these objectives, we initially performed a systems-wide analysis of protein lysine-methylation and expression in wild type (WT) and SETD2-knock out (KO) kidney cells and later focused our studies on eEF1A1 as well as the expression of lysine methyltransferases that regulate its lysine methylation. RESULTS: We observed decreased lysine methylation of the translation elongation factor eEF1A1. EEF1AKMT2 and EEF1AKMT3 are known to methylate eEF1A1, and we show here that their expression is dependent on SET-domain function of SETD2. Globally, we observe differential expression of hundreds of proteins in WT versus SETD2-KO cells, including increased expression of many involved in protein translation. Finally, we observe decreased progression free survival and loss of EEF1AKMT2 gene expression in SETD2-mutated tumors predicted to have loss of function of the SET domain. CONCLUSION: Overall, these data suggest that SETD2-mutated ccRCC, via loss of enzymatic function of the SET domain, displays dysregulation of protein translation as a potentially important component of the transformed phenotype.

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

A pan-cancer immunogenomic atlas for immune checkpoint blockade immunotherapy.

The ability to identify robust genomic signatures that predict response to immune checkpoint blockade is restricted by limited sample sizes and ungeneralizable performance across cohorts. To address these challenges, we established Cancer-Immu (http://bioinfo.vanderbilt.edu/database/Cancer-Immu/) a comprehensive platform that integrates large-scale multidimensional omics data, including genetic, bulk, and single-cell transcriptomic, proteomic, and dynamic genomic profiles, with clinical phenotypes to explore consistent and rare immunogenomic connections. Currently Cancer-Immu has incorporated data for 3,652 samples for 16 cancer types. It provides easy access to immunogenomic data and empowers researchers to translate omics datasets into biological insights and clinical applications.

Acquisition of aneuploidy drives mutant p53-associated gain-of-function phenotypes.

p53 is mutated in over half of human cancers. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. To study mutant p53 GOF mechanisms and phenotypes, we genetically engineered non-transformed and tumor-derived WT p53 cell line models to express endogenous missense mutant p53 (R175H and R273H) or to be deficient for p53 protein (null). Characterization of the models, which initially differed only by TP53 genotype, revealed that aneuploidy frequently occurred in mutant p53-expressing cells. GOF phenotypes occurred clonally in vitro and in vivo, were independent of p53 alteration and correlated with increased aneuploidy. Further, analysis of outcome data revealed that individuals with aneuploid-high tumors displayed unfavorable prognoses, regardless of the TP53 genotype. Our results indicate that genetic variation resulting from aneuploidy accounts for the diversity of previously reported mutant p53 GOF phenotypes.