Skeletal muscle transcriptomics dissects the pathogenesis of Friedreich's ataxia.

OBJECTIVE: In Friedreich's ataxia (FRDA), the most affected tissues are not accessible to sampling and available transcriptomic findings originate from blood-derived cells and animal models. Herein, we aimed at dissecting for the first time the pathophysiology of FRDA by means of RNA-sequencing in an affected tissue sampled in vivo. METHODS: Skeletal muscle biopsies were collected from seven FRDA patients before and after treatment with recombinant human Erythropoietin (rhuEPO) within a clinical trial. Total RNA extraction, 3'-mRNA library preparation and sequencing were performed according to standard procedures. We tested for differential gene expression with DESeq2 and performed gene set enrichment analysis with respect to control subjects. RESULTS: FRDA transcriptomes showed 1873 genes differentially expressed from controls. Two main signatures emerged: (1) a global downregulation of the mitochondrial transcriptome as well as of ribosome/translational machinery and (2) an upregulation of genes related to transcription and chromatin regulation, especially of repressor terms. Downregulation of the mitochondrial transcriptome was more profound than previously shown in other cellular systems. Furthermore, we observed in FRDA patients a marked upregulation of leptin, the master regulator of energy homeostasis. RhuEPO treatment further enhanced leptin expression. INTERPRETATION: Our findings reflect a double hit in the pathophysiology of FRDA: a transcriptional/translational issue and a profound mitochondrial failure downstream. Leptin upregulation in the skeletal muscle in FRDA may represent a compensatory mechanism of mitochondrial dysfunction, which is amenable to pharmacological boosting. Skeletal muscle transcriptomics is a valuable biomarker to monitor therapeutic interventions in FRDA.

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer.

BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.

Smooth Muscle Specific Ablation of CXCL12 in Mice Downregulates CXCR7 Associated with Defective Coronary Arteries and Cardiac Hypertrophy.

The chemokine CXCL12 plays a fundamental role in cardiovascular development, cell trafficking, and myocardial repair. Human genome-wide association studies even have identified novel loci downstream of the CXCL12 gene locus associated with coronary artery disease and myocardial infarction. Nevertheless, cell and tissue specific effects of CXCL12 are barely understood. Since we detected high expression of CXCL12 in smooth muscle (SM) cells, we generated a SM22-alpha-Cre driven mouse model to ablate CXCL12 (SM-CXCL12-/-). SM-CXCL12-/- mice revealed high embryonic lethality (50%) with developmental defects, including aberrant topology of coronary arteries. Postnatally, SM-CXCL12-/- mice developed severe cardiac hypertrophy associated with fibrosis, apoptotic cell death, impaired heart function, and severe coronary vascular defects characterized by thinned and dilated arteries. Transcriptome analyses showed specific upregulation of pathways associated with hypertrophic cardiomyopathy, collagen protein network, heart-related proteoglycans, and downregulation of the M2 macrophage modulators. CXCL12 mutants showed endothelial downregulation of the CXCL12 co-receptor CXCR7. Treatment of SM-CXCL12-/- mice with the CXCR7 agonist TC14012 attenuated cardiac hypertrophy associated with increased pERK signaling. Our data suggest a critical role of smooth muscle-specific CXCL12 in arterial development, vessel maturation, and cardiac hypertrophy. Pharmacological stimulation of CXCR7 might be a promising target to attenuate adverse hypertrophic remodeling.

NR4A1-mediated apoptosis suppresses lymphomagenesis and is associated with a favorable cancer-specific survival in patients with aggressive B-cell lymphomas.

NR4A1 (Nur77) and NR4A3 (Nor-1) function as tumor suppressor genes as demonstrated by the rapid development of acute myeloid leukemia in the NR4A1 and NR4A3 knockout mouse. The aim of our study was to investigate NR4A1 and NR4A3 expression and function in lymphoid malignancies. We found a vastly reduced expression of NR4A1 and NR4A3 in chronic lymphocytic B-cell leukemia (71%), in follicular lymphoma (FL, 70%), and in diffuse large B-cell lymphoma (DLBCL, 74%). In aggressive lymphomas (DLBCL and FL grade 3), low NR4A1 expression was significantly associated with a non-germinal center B-cell subtype and with poor overall survival. To investigate the function of NR4A1 in lymphomas, we overexpressed NR4A1 in several lymphoma cell lines. Overexpression of NR4A1 led to a higher proportion of lymphoma cells undergoing apoptosis. To test the tumor suppressor function of NR4A1 in vivo, the stable lentiviral-transduced SuDHL4 lymphoma cell line harboring an inducible NR4A1 construct was further investigated in xenografts. Induction of NR4A1 abrogated tumor growth in the NSG mice, in contrast to vector controls, which formed massive tumors. Our data suggest that NR4A1 has proapoptotic functions in aggressive lymphoma cells and define NR4A1 as a novel gene with tumor suppressor properties involved in lymphomagenesis.

Co-expressed genes prepositioned in spatial neighborhoods stochastically associate with SC35 speckles and RNA polymerase II factories.

Chromosomally separated, co-expressed genes can be in spatial proximity, but there is still debate about how this nuclear organization is achieved. Proposed mechanisms include global genome organization, preferential positioning of chromosome territories, or gene-gene sharing of various nuclear bodies. To investigate this question, we selected a set of genes that were co-expressed upon differentiation of human multipotent stem cells. We applied a novel multi-dimensional analysis procedure which revealed that prior to gene expression, the relative position of these genes was conserved in nuclei. Upon stem cell differentiation and concomitant gene expression, we found that co-expressed genes were closer together. In addition, we found that genes in the same 1-µm-diameter neighborhood associated with either the same splicing speckle or to a lesser extent with the same transcription factory. Dispersal of speckles by overexpression of the serine-arginine (SR) protein kinase cdc2-like kinase Clk2 led to a significant drop in the number of genes in shared neighborhoods. We demonstrate quantitatively that the frequencies of speckle and factory sharing can be explained by assuming stochastic selection of a nuclear body within a restricted sub-volume defined by the original global gene positioning present prior to gene expression. We conclude that the spatial organization of these genes is a two-step process in which transcription-induced association with nuclear bodies enhances and refines a pre-existing global organization.

Spatial transcriptomics in embryonic mouse diaphragm muscle reveals regional gradients and subdomains of developmental gene expression.

The murine embryonic diaphragm is a primary model for studying myogenesis and neuro-muscular synaptogenesis, both representing processes regulated by spatially organized genetic programs of myonuclei located in distinct myodomains. However, a spatial gene expression pattern of embryonic mouse diaphragm has not been reported. Here, we provide spatially resolved gene expression data for horizontally sectioned embryonic mouse diaphragms at embryonic days E14.5 and E18.5. These data reveal gene signatures for specific muscle regions with distinct maturity and fiber type composition, as well as for a central neuromuscular junction (NMJ) and a peripheral myotendinous junction (MTJ) compartment. Comparing spatial expression patterns of wild-type mice with those of transgenic mice lacking either the skeletal muscle calcium channel CaV1.1 or ß-catenin, reveals curtailed muscle development and dysregulated expression of genes potentially involved in NMJ formation. Altogether, these datasets provide a powerful resource for further studies of muscle development and NMJ formation in the mouse.

Comparative analysis of 10X Chromium vs. BD Rhapsody whole transcriptome single-cell sequencing technologies in complex human tissues.

The development of single-cell omics tools has enabled scientists to study the tumor microenvironment (TME) in unprecedented detail. However, each of the different techniques may have its unique strengths and limitations. Here we directly compared two commercially available high-throughput single-cell RNA sequencing (scRNA-seq) technologies - droplet-based 10X Chromium vs. microwell-based BD Rhapsody - using paired samples from patients with localized prostate cancer (PCa) undergoing a radical prostatectomy. Although high technical consistency was observed in unraveling the whole transcriptome, the relative abundance of cell populations differed. Cells with low mRNA content such as T cells were underrepresented in the droplet-based system, at least partly due to lower RNA capture rates. In contrast, microwell-based scRNA-seq recovered less cells of epithelial origin. Moreover, we discovered platform-dependent variabilities in mRNA quantification and cell-type marker annotation. Overall, our study provides important information for selection of the appropriate scRNA-seq platform and for the interpretation of published results.

Prediction of Clinically Significant Prostate Cancer by a Specific Collagen-related Transcriptome, Proteome, and Urinome Signature.

BACKGROUND AND OBJECTIVE: While collagen density has been associated with poor outcomes in various cancers, its role in prostate cancer (PCa) remains elusive. Our aim was to analyze collagen-related transcriptomic, proteomic, and urinome alterations in the context of detection of clinically significant PCa (csPCa, International Society of Urological Pathology [ISUP] grade group ≥2). METHODS: Comprehensive analyses for PCa transcriptome (n = 1393), proteome (n = 104), and urinome (n = 923) data sets focused on 55 collagen-related genes. Investigation of the cellular source of collagen-related transcripts via single-cell RNA sequencing was conducted. Statistical evaluations, clustering, and machine learning models were used for data analysis to identify csPCa signatures. KEY FINDINGS AND LIMITATIONS: Differential expression of 30 of 55 collagen-related genes and 34 proteins was confirmed in csPCa in comparison to benign prostate tissue or ISUP 1 cancer. A collagen-high cancer cluster exhibited distinct cellular and molecular characteristics, including fibroblast and endothelial cell infiltration, intense extracellular matrix turnover, and enhanced growth factor and inflammatory signaling. Robust collagen-based machine learning models were established to identify csPCa. The models outcompeted prostate-specific antigen (PSA) and age, showing comparable performance to multiparametric magnetic resonance imaging (mpMRI) in predicting csPCa. Of note, the urinome-based collagen model identified four of five csPCa cases among patients with Prostate Imaging-Reporting and Data System (PI-IRADS) 3 lesions, for which the presence of csPCa is considered equivocal. The retrospective character of the study is a limitation. CONCLUSIONS AND CLINICAL IMPLICATIONS: Collagen-related transcriptome, proteome, and urinome signatures exhibited superior accuracy in detecting csPCa in comparison to PSA and age. The collagen signatures, especially in cases of ambiguous lesions on mpMRI, successfully identified csPCa and could potentially reduce unnecessary biopsies. The urinome-based collagen signature represents a promising liquid biopsy tool that requires prospective evaluation to improve the potential of this collagen-based approach to enhance diagnostic precision in PCa for risk stratification and guiding personalized interventions. PATIENT SUMMARY: In our study, collagen-related alterations in tissue, and urine were able to predict the presence of clinically significant prostate cancer at primary diagnosis.

Arxes: retrotransposed genes required for adipogenesis.

Retrotransposed sequences arise from messenger RNAs (mRNAs) that have been reinserted into genomic DNA by reverse transcription. Usually, these sequences are embedded in dormant regions, collect missense mutations over time and constitute processed, nonfunctional pseudogenes. There are thousands of processed pseudogenes in the mouse and human genome. Here, we report evidence for two paralog genes (termed Arxes1 and Arxes2), which arose by retrotransposition of the signal peptidase Spcs3 followed by a segmental duplication event. They gained a functional promoter that we show to be transactivated by adipogenic transcription factors. We further show that the Arxes mRNAs are highly expressed in adipose tissue and strongly upregulated during adipogenesis in different cell models. Additionally, their expression is elevated by an anti-diabetic agent in vitro and in vivo. Importantly, we provide evidence that the Arxes genes are translated and that the proteins are located in the endoplasmic reticulum. Although the sequence similarity and subcellular location are reminiscent of their parental gene, our data suggest that the Arxes have developed a different function, since their expression is required for adipogenesis, whereas Spcs3 is dispensable. In summary, we report retrotransposed-duplicated genes that evolved from a parental gene to function in a tissue and adipogenesis-specific context.

13C tracer analysis reveals the landscape of metabolic checkpoints in human CD8+ T cell differentiation and exhaustion.

Introduction: Naïve T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start to proliferate and generate effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells. Methods: Here we used 13C metabolomics and transcriptomics to study the metabolism of CD8+ T cells in their complete course of differentiation from naïve over stem-like memory to effector cells and in exhaustion-inducing conditions. Results: The quiescence of naïve T cells was evident in a profound suppression of glucose oxidation and a decreased expression of ENO1, downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naïve T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of metabolic enzymes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which was, however, not linked to changes in mitochondrial abundance. In exhaustion, decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of quiescence-associated genes, TXNIP and KLF2, and the T cell suppressive metabolites succinate and itaconate. Discussion: Overall, these results identify multiple metabolic features that regulate quiescence, proliferation and effector function, but also exhaustion of CD8+ T cells during differentiation. Thus, targeting these metabolic checkpoints may be a promising therapeutic strategy for both prevention of exhaustion and promotion of stemness of anti-tumor T cells.

SARS-CoV-2 activates the TLR4/MyD88 pathway in human macrophages: A possible correlation with strong pro-inflammatory responses in severe COVID-19.

Background: Toll-like receptors (TLRs) play a pivotal role in the immunologic response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Exaggerated inflammatory response of innate immune cells, however, may drive morbidity and death in Coronavirus disease 19 (COVID-19). Objective: We investigated the engagement of SARS-CoV-2 with TLR4 in order to better understand how to tackle hyperinflammation in COVID-19. Methods: We combined RNA-sequencing data of human lung tissue and of bronchoalveolar lavage fluid cells derived from COVID-19 patients with functional studies in human macrophages using SARS-CoV-2 spike proteins and viable SARS-CoV-2. Pharmacological inhibitors as well as gene editing with CRISPR/Cas9 were used to delineate the signalling pathways involved. Results: We found TLR4 to be the most abundantly upregulated TLR in human lung tissue irrespective of the underlying pathology. Accordingly, bronchoalveolar lavage fluid cells from patients with severe COVID-19 showed an NF-κB-pathway dominated immune response, whereas they were mostly defined by type I interferon signalling in moderate COVID-19. Mechanistically, we found the Spike ectodomain, but not receptor binding domain monomer to induce TLR4-dependent inflammation in human macrophages. By using pharmacological inhibitors as well as CRISPR/Cas9 deleted macrophages, we identify SARS-CoV-2 to engage canonical TLR4-MyD88 signalling. Importantly, we demonstrate that TLR4 blockage prevents exaggerated inflammatory responses in human macrophages infected with different SARS-CoV-2 variants, including immune escape variants B.1.1.7.-E484K and B.1.1.529 (omicron). Conclusion: Our study critically extends the current knowledge on TLR-mediated hyperinflammatory responses to SARS-CoV-2 in human macrophages, paving the way for novel approaches to tackle severe COVID-19. Take-home message: Our study combining human lung transcriptomics with functional studies in human macrophages clearly supports the design and development of TLR4 - directed therapeutics to mitigate hyperinflammation in severe COVID-19.

Functional and spatial proteomics profiling reveals intra- and intercellular signaling crosstalk in colorectal cancer.

Precision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology-a strategy that is based on perturbing primary tumor cells from cancer patients-could provide a road forward to personalize treatment. We extend this paradigm to measuring proteome activity landscapes by acquiring quantitative phosphoproteomic data from patient-derived organoids (PDOs). We show that kinase inhibitors induce inhibitor- and patient-specific off-target effects and pathway crosstalk. Reconstruction of the kinase networks revealed that the signaling rewiring is modestly affected by mutations. We show non-genetic heterogeneity of the PDOs and upregulation of stemness and differentiation genes by kinase inhibitors. Using imaging mass-cytometry-based profiling of the primary tumors, we characterize the tumor microenvironment (TME) and determine spatial heterocellular crosstalk and tumor-immune cell interactions. Collectively, we provide a framework for inferring tumor cell intrinsic signaling and external signaling from the TME to inform precision (immuno-) oncology in CRC.

Exosomal mitochondrial tRNAs and miRNAs as potential predictors of inflammation in renal proximal tubular epithelial cells.

Exosomes have emerged as a valuable repository of novel biomarkers for human diseases such as chronic kidney disease (CKD). From a healthy control group, we performed microRNA (miRNA) profiling of urinary exosomes and compared it with a cell culture model of renal proximal tubular epithelial cells (RPTECs). Thereby, a large fraction of abundant urinary exosomal miRNAs could also be detected in exosomes derived from RPTECs, indicating them as a suitable model system for investigation of CKD. We subsequently analyzed exosomes from RPTECs in pro-inflammatory and pro-fibrotic states, mimicking some aspects of CKD. Following cytokine treatment, we observed a significant increase in exosome release and identified 30 dysregulated exosomal miRNAs, predominantly associated with the regulation of pro-inflammatory and pro-fibrotic-related pathways. In addition to miRNAs, we also identified 16 dysregulated exosomal mitochondrial RNAs, highlighting a pivotal role of mitochondria in sensing renal inflammation. Inhibitors of exosome biogenesis and release significantly altered the abundance of selected candidate miRNAs and mitochondrial RNAs, thus suggesting distinct sorting mechanisms of different non-coding RNA (ncRNA) species into exosomes. Hence, these two exosomal ncRNA species might be employed as potential indicators for predicting the pathogenesis of CKD and also might enable effective monitoring of the efficacy of CKD treatment.

Reconstruction of gene association network reveals a transmembrane protein required for adipogenesis and targeted by PPARγ.

We have developed a method for reconstructing gene association networks and have applied this method to gene profiles from 3T3-L1 cells. Priorization of the candidate genes pinpointed a transcript annotated as APMAP (adipocyte plasma membrane-associated protein). Functional studies showed that APMAP is upregulated in murine and human adipogenic cell models as well as in a genetic mouse model of obesity. Silencing APMAP in 3T3-L1 cells strongly impaired the differentiation into adipocytes. Moreover, APMAP expression was strongly induced by the PPARγ ligand rosiglitazone in adipocytes in vitro and in vivo in adipose tissue. Using ChIP-qPCR and luciferase reporter assays, we show a functional PPARγ binding site. In addition, we provide evidence that the extracellular C-terminal domain of APMAP is required for the function of APMAP in adipocyte differentiation. Finally, we demonstrate that APMAP translocates from the endoplasmatic reticulum to the plasma membrane during adipocyte differentiation.

Correction to: Molecular and pharmacological modulators of the tumor immune contexture revealed by deconvolution of RNA-seq data.

It was highlighted that the original article [1] contained a typesetting mistake in the name of Noel Filipe da Cunha Carvalho de Miranda. This was incorrectly captured as Noel Filipe da Cunha Carvahlo de Miranda. It was also highlighted that in Fig. 3C the left panels Y-axis were cropped and in Fig. 5C, CD8 bar was cropped. This Correction article shows the correct Figs. 3 and 5. The original article has been updated.

Molecular and pharmacological modulators of the tumor immune contexture revealed by deconvolution of RNA-seq data.

We introduce quanTIseq, a method to quantify the fractions of ten immune cell types from bulk RNA-sequencing data. quanTIseq was extensively validated in blood and tumor samples using simulated, flow cytometry, and immunohistochemistry data.quanTIseq analysis of 8000 tumor samples revealed that cytotoxic T cell infiltration is more strongly associated with the activation of the CXCR3/CXCL9 axis than with mutational load and that deconvolution-based cell scores have prognostic value in several solid cancers. Finally, we used quanTIseq to show how kinase inhibitors modulate the immune contexture and to reveal immune-cell types that underlie differential patients' responses to checkpoint blockers.Availability: quanTIseq is available at http://icbi.at/quantiseq .

Targeting immune checkpoints potentiates immunoediting and changes the dynamics of tumor evolution.

The cancer immunoediting hypothesis postulates a dual role of the immune system: protecting the host by eliminating tumor cells, and shaping the tumor by editing its genome. Here, we elucidate the impact of evolutionary and immune-related forces on editing the tumor in a mouse model for hypermutated and microsatellite-instable colorectal cancer. Analyses of wild-type and immunodeficient RAG1 knockout mice transplanted with MC38 cells reveal that upregulation of checkpoint molecules and infiltration by Tregs are the major tumor escape mechanisms. Our results show that the effects of immunoediting are weak and that neutral accumulation of mutations dominates. Targeting the PD-1/PD-L1 pathway using immune checkpoint blocker effectively potentiates immunoediting. The immunoediting effects are less pronounced in the CT26 cell line, a non-hypermutated/microsatellite-instable model. Our study demonstrates that neutral evolution is another force that contributes to sculpting the tumor and that checkpoint blockade effectively enforces T-cell-dependent immunoselective pressure.

Nuclear receptor NR2F6 inhibition potentiates responses to PD-L1/PD-1 cancer immune checkpoint blockade.

Analyzing mouse tumor models in vivo, human T cells ex vivo, and human lung cancer samples, we provide direct evidence that NR2F6 acts as an immune checkpoint. Genetic ablation of Nr2f6, particularly in combination with established cancer immune checkpoint blockade, efficiently delays tumor progression and improves survival in experimental mouse models. The target genes deregulated in intratumoral T lymphocytes upon genetic ablation of Nr2f6 alone or together with PD-L1 blockade reveal multiple advantageous transcriptional alterations. Acute Nr2f6 silencing in both mouse and human T cells induces hyper-responsiveness that establishes a non-redundant T-cell-inhibitory function of NR2F6. NR2F6 protein expression in T-cell-infiltrating human NSCLC is upregulated in 54% of the cases (n = 303) and significantly correlates with PD-1 and CTLA-4 expression. Our data define NR2F6 as an intracellular immune checkpoint that suppresses adaptive anti-cancer immune responses and set the stage for clinical validation of targeting NR2F6 for next-generation immuno-oncological regimens.

NR4A3 Suppresses Lymphomagenesis through Induction of Proapoptotic Genes.

Nuclear orphan receptor NR4A1 exerts an essential tumor suppressor function in aggressive lymphomas. In this study, we investigated the hypothesized contribution of the related NR4A family member NR4A3 to lymphomagenesis. In aggressive lymphoma patients, low expression of NR4A3 was associated with poor survival. Ectopic expression or pharmacological activation of NR4A3 in lymphoma cell lines led to a significantly higher proportion of apoptotic cells. In a mouse NSG xenograft model of lymphoma (stably transduced SuDHL4 cells), NR4A3 expression abrogated tumor growth, compared with vector control and uninduced cells that formed massive tumors. Transcript analysis of four different aggressive lymphoma cell lines overexpressing either NR4A3 or NR4A1 revealed that apoptosis was driven similarly by induction of BAK, Puma, BIK, BIM, BID, and Trail. Overall, our results showed that NR4A3 possesses robust tumor suppressor functions of similar impact to NR4A1 in aggressive lymphomas. Cancer Res; 77(9); 2375-86. ©2017 AACR.