Publications

Cutaneous T-cell lymphoma (CTCL) remains a challenging disease due to its significant heterogeneity, therapy resistance, and relentless progression. Multiomics technologies offer the potential to provide uniquely precise views of disease progression and response to therapy. Here, we present a comprehensive multiomics view of CTCL clonal evolution, incorporating exome, whole-genome, epigenome, bulk, single-cell T-cell receptor, and single-cell RNA sequencing of 99 clinically annotated serial skin, peripheral blood, and lymph node samples from 34 patients with CTCL. We leveraged this extensive data set to define the molecular underpinnings of CTCL progression in individual patients at single-cell resolution with the goal of identifying clinically useful biomarkers and therapeutic targets. Our studies identified recurrent progression-associated clonal genomic alterations; we highlight mutation of CCR4, phosphoinositide 3-kinase inhibitor signaling, and programmed cell death protein 1 (PD-1) checkpoint pathways as evasion tactics deployed by malignant T cells. We identified a gain-of-function mutation in STAT3 (D661Y) and demonstrated, using cleavage under targets and release using nuclease (CUT&RUN) and RNA sequencing, that it enhances binding to and transcription of genes in Rho GTPase pathways. With our previous work implicating this pathway in histone deacetylase inhibitor-resistant CTCL, these data provide further support for a previously unrecognized role for Rho GTPase pathway dysregulation in CTCL progression. Recurrent progression-associated mutations were common in the epigenetic modifier EZH2, suggesting that EZH2 inhibition may benefit patients with CTCL. Our findings support an approach in which genomic analysis is widely used for improved disease monitoring, biomarker-informed clinical trial design, and genome-guided therapeutic decision-making. Moreover, these molecular changes present new opportunities for therapeutic targeting in this challenging and incurable cancer.

BACKGROUND: The infiltration of tumor-infiltrating B cells and plasma cells in early-stage breast cancer has been associated with a reduced risk of distant metastasis. However, the influence of B-cell tumor infiltration on overall patient survival remains unclear. MATERIALS AND METHODS: This study explored the relationship between an antitumor immune response, measured by a 14-gene B-cell/immunoglobulin (IGG) signature, and mortality risk in 9638 breast cancer patients across three datasets. Associations with tumor subtype, stage, and age were examined. IGG was characterized using spatial GeoMx profiling and single-cell RNA sequencing, and its relationship with tertiary lymphoid structures (TLSs) was evaluated. The predictive value of each of the 14 IGG genes for B-cell receptor (BCR) and T-cell receptor (TCR) clonality and longevity was also assessed, along with its association with longevity in other cancer types. RESULTS: High IGG signature expression was significantly associated with a 41%-47% reduction in death risk in breast cancer survivors (P < 0.001), regardless of age, tumor stage, or subtype. Similar associations were observed in other cancers, including melanoma. In breast cancer, the IGG signature was significantly linked to overall survival without relapse in patients aged 41-70 years at diagnosis. Additionally, IGG expression correlated with the presence of TLSs and higher B- and T-cell polyclonality. A specific subset of seven IGG genes strongly correlated with BCR and TCR clonality, with predictive power for identifying clonality and improved longevity, especially when combining two of these genes. CONCLUSIONS: This study uncovers a significant link between immune gene expression in tumors and extended longevity in breast cancer survivors, even in the absence of recurrence. The IGG signature, particularly its key gene subset, emerges as a powerful marker of sustained antitumor immunity and overall patient fitness. These findings pave the way for personalized treatment strategies that enhance both survival and long-term health outcomes.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is linked to human papillomavirus (HPV) infection. HPV-positive and HPV-negative HNSCC exhibit distinct molecular and clinical characteristics. Although checkpoint inhibitors have shown efficiency in recurrent/metastatic HNSCC, response variability persists regardless of HPV status. This study aimed to explore the CD8⁺ T-cell landscape in HPV-negative HNSCC. METHODS: We performed simultaneous single-cell RNA and TCR sequencing of CD8⁺ tumor-infiltrating lymphocytes (TILs) from treatment-naïve HPV-negative HNSCC patients. Additionally, cells were stimulated ex vivo, which allowed for the tracking of clonal transcriptomic responses. RESULTS: Our analysis identified a subset of CD8⁺ TILs highly enriched for interferon-stimulated genes (ISG). TCR analysis revealed ISG cells are clonally related to a population of granzyme K (GZMK)-expressing cells. However, unlike GZMK cells, which exhibited rapid effector-like phenotypes following stimulation, ISG cells were transcriptionally inert. Additionally, ISG cells showed specific enrichment within tumor and were found across multiple tumor entities. CONCLUSIONS: ISG-enriched CD8⁺ TILs are a consistent feature of various tumor entities. These cells are poorly understood but possess characteristics that may impact antitumor immunity. Understanding the unique properties and functionality of ISG cells could offer innovative treatment approaches to improve patient outcomes in HPV-negative HNSCC and other cancer types.

Solid-phase immunoassays (SPIs) are the current standard of care for detecting and identifying anti-HLA antibodies in transplantation. However, whether SPIs with different configurations provide comprehensive antigen and eplet coverage has not been documented in the literature. We systematically compared four commercial SPI panels - Mixed, PRA (Panel Reactive Antibody), SAB (Single Antigen Bead), and ExPlex - to assess their coverage of antigens and eplets cataloged in the HLA Eplet Registry. Our results show overlapping and variable antigen and eplet representation across these assays. Out of a total of 252 class I and 260 class II eplets in the HLA Eplet Registry, 251 (99.6 %) and 260 (100 %) were covered by one or more assays; 209 (82.9 %) class I and 189 (72.7 %) of class II eplets were covered by all four assays. All antibody-verified eplets in Class I antigens are represented in three or more assays, whereas two Class II eplets were exclusive to SAB. Although ExPlex did not significantly increase the number of eplets represented beyond the SAB panel, it expanded the diversity of eplets within unique antigenic contexts. We also examined the relationship between eplet dose per bead and antibody reactivity in the PRA assay. Positive correlations were observed between bead-level eplet numbers and mean fluorescence intensity (MFI) in a collection of sera with 82LR (Bw4) or 80 N (Bw6) eplet patterns. Our findings indicate broad coverage of HLA eplets by current SPIs, which offer acceptable antigenic coverage for pre-transplant immune risk screening. We also share the R script to enable ongoing analyses and monitoring of antigen and eplet coverage in future screening reagents.

INTRODUCTION: Single-cell RNA sequencing (scRNA-seq) enables high-resolution profiling of immune heterogeneity. Although previous studies have mapped the single-cell transcriptomic atlases of peripheral leukocytes in healthy dogs, the identification and functional characterization of distinct immune subsets remain incomplete. METHODS: We constructed a single-cell atlas of peripheral leukocytes from six healthy small-breed dogs using the 10x Genomics platform and the updated canFam4 genome. RESULTS AND DISCUSSION: Analysis of 30,040 high-quality transcriptomes revealed 51 distinct immune subsets, including CD14 ⁺ CD33 ⁺ monocytes, XCR1 ⁺ CD1D ⁺ dendritic cells, CEACAM1 ⁺ CD24 ⁺ neutrophils, and IL32 ⁺ BATF ⁺ regulatory T cells, which were underrepresented in canFam3.1-based studies. Interferon-enriched CD14 ⁺ monocytes and CD4 ⁺ T subsets associated with myxomatous mitral valve disease were also identified. Functional enrichment analyses suggested that PDCD1 is associated with attenuated TCR signaling, whereas LAG3 was associated with malate metabolism pathways in CD4 ⁺ T cells and reduced TBX21 expression in CD8 ⁺ T cells linked to antiviral responses. CD274, which encodes PD-L1 was linked to IL-10 production in neutrophils, and CTLA4 represented an initial activation of double-negative T subsets. T cell exhaustion scores and proliferative fractions varied across cohorts, reflecting differences in environmental antigenic exposures. CONCLUSION: To our knowledge, this study represents the first comprehensive, gene-resolved single-cell analysis to reveal immunoregulatory checkpoint mechanisms underlying immune homeostasis in healthy dogs. Our dataset will serve as a valuable resource for future comparative and translational immunology research in dogs.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive and chemo-resistant sarcomas with poor survival rates. Loss of CDKN2A or P53 following NF1 disruption is a key event in MPNST development. Here, we used CRISPR-Cas9 somatic tumorigenesis in mice to identify transcriptomic and metabolomic features distinguishing CDKN2A- versus P53-deleted MPNSTs. Convergent, multiomic analyses revealed that CDKN2A-deleted MPNSTs are especially dependent on the pentose phosphate pathway (PPP) and NADPH metabolism for growth and viability. Disruption of glucose-6-phosphate dehydrogenase (G6PD), the PPP rate-limiting enzyme, slowed CDKN2A-deleted MPNST growth and sensitized MPNSTs to standard-of-care chemotherapy. Knockdown of the redox-regulated transcription factor NRF2 slowed MPNST growth and decreased G6PD transcription. Analysis of patient MPNSTs identified a NRF2 gene signature correlating with tumor transformation. Furthermore, G6PD and NRF2 expression in PanCancer TCGA samples correlates with patient survival. This work identifies NRF2-PPP dependency as a targetable vulnerability in these difficult-to-treat MPNSTs, particularly in the NF1/CDKN2A-deleted majority.

Multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system, is modeled in mice as experimental autoimmune encephalomyelitis (EAE). While CD4⁺ T cells, primarily Th1 and Th17 subsets, drive disease pathogenesis, the exact function of CD8⁺ T cells remains unclear. We previously demonstrated that adoptively transferred myelin-reactive CD8⁺ T cells (PLP-CD8) prevent EAE induction and suppress ongoing disease through the engagement of MHC Class-I in recipient mice. Here, we show that PLP-CD8 induce regulatory changes in both subsets of conventional dendritic cells (cDC1 and CD11b⁺ cDC) in vivo and in vitro. Adoptively transferred PLP-CD8 promoted both cDC subsets to adopt a mature and regulatory phenotype with an anti-inflammatory cytokine profile and a reduced capacity to support CD4⁺ T cell proliferation. In vitro, PLP-CD8 induced similar phenotypic changes in both cDC subsets in an antigen-specific, dose-dependent manner. PLP-CD8 directly interacted with cDC1 and indirectly influenced CD11b⁺ cDC through paracrine signaling. Notably, direct interaction with PLP-CD8 had detrimental effects on CD11b⁺ cDC. Single-cell RNA sequencing revealed upregulation of key immunoregulatory genes, such as Foxo3, in both cDC subsets with enrichment of pathways involved in immune regulation and T cell differentiation. Our study highlights a novel mechanism in which myelin-reactive CD8⁺ T cells directly interact with cDC1 and modulate CD11b⁺ cDC through paracrine mechanisms to induce mature, regulatory dendritic cells, which leads to inhibited CD4⁺ T cell responses and reduced EAE pathogenesis.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain B (TIFAB), an inhibitor of NF-κB signaling, plays critical roles in hematopoiesis, myelodysplastic neoplasms, and leukemia. We previously demonstrated that Tifab enhances KMT2A::MLLT3-driven acute myeloid leukemia (AML) by either upregulating Hoxa9 or through ubiquitin-specific peptidase 15-mediated downregulation of p53 signaling. In this study, we show that Tifab deletion in KMT2A::MLLT3-induced AML impairs leukemia stem/progenitor cell (LSPC) engraftment, glucose uptake, and mitochondrial function. Gene set enrichment analysis reveals that Tifab deletion downregulates MYC, HOXA9/MEIS1, mTORC1 signaling, and genes involved in glycolysis and oxidative phosphorylation. By comparing genes upregulated in TIFAB-overexpressing LSPCs with those downregulated upon Tifab deletion, we identify hepatocyte nuclear factor 4 alpha (Hnf4a) as a key TIFAB target, regulated through the inhibition of NF-κB component RelB, which suppresses Hnf4a in leukemia cells. HNF4A, a nuclear receptor involved in organ development, metabolism, and tumorigenesis, rescues the metabolic defects caused by Tifab deletion and enhances leukemia cell engraftment. Conversely, Hnf4a knockdown attenuates TIFAB-mediated enhancement of LSPC function. These findings highlight the critical role of the TIFAB-HNF4A axis in KMT2A::MLLT3-induced AML and uncover a novel regulator in leukemia biology.

Single-cell adaptive immune receptor repertoire sequencing (scAIRR-seq) and single-cell RNA sequencing (scRNA-seq) provide a transformative approach to profiling immune responses at unprecedented resolution across diverse pathophysiologic contexts. This work presents scRepertoire 2, a substantial update to our R package for analyzing and visualizing single-cell immune receptor data. This new version introduces an array of features designed to enhance both the depth and breadth of immune receptor analysis, including improved workflows for clonotype tracking, repertoire diversity metrics, and novel visualization modules that facilitate longitudinal and comparative studies. Additionally, scRepertoire 2 offers seamless integration with contemporary single-cell analysis frameworks like Seurat and SingleCellExperiment, allowing users to conduct end-to-end single-cell immune profiling with transcriptomic data. Performance optimizations in scRepertoire 2 resulted in a 85.1% increase in speed and a 91.9% reduction in memory usage from the first version over the range repertoire size tested in benchmarking, addressing the demands of the ever-increasing size and scale of single-cell studies. This release marks an advancement in single cell immunogenomics, equipping researchers with a robust toolset to uncover immune dynamics in health and disease.

Integration of single-cell RNA-sequencing (scRNA-seq) and adaptive immune receptor (AIR) sequencing (scVDJ-seq) is extremely powerful in studying lymphocyte development. A python-based package, Dandelion, introduced the VDJ-feature space method, which addresses the challenge of integrating single-cell AIR data with gene expression data and enhances trajectory analysis results. However, no R-based equivalent or similar methods currently exist. To fill this gap, we present dandelionR, an R implementation of Dandelion's trajectory analysis workflow, bringing the VDJ feature space construction and trajectory analysis using diffusion maps and absorbing Markov chains to R, offering a new option for scRNA-seq and scVDJ-seq analysis to R users.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and mRNA vaccination induce robust CD4⁺ T cell responses. Using single-cell transcriptomics, here, we evaluated CD4⁺ T cells specific for the SARS-CoV-2 spike protein in the blood and draining lymph nodes (dLNs) of individuals 3 months and 6 months after vaccination with the BNT162b2 mRNA vaccine. We analyzed 1,277 spike-specific CD4⁺ T cells, including 238 defined using Trex, a deep learning-based reverse epitope mapping method to predict antigen specificity. Human dLN spike-specific CD4⁺ follicular helper T (T_FH) cells exhibited heterogeneous phenotypes, including germinal center CD4⁺ T_FH cells and CD4⁺IL-10⁺ T_FH cells. Analysis of an independent cohort of SARS-CoV-2-infected individuals 3 months and 6 months after infection found spike-specific CD4⁺ T cell profiles in blood that were distinct from those detected in blood 3 months and 6 months after BNT162b2 vaccination. Our findings provide an atlas of human spike-specific CD4⁺ T cell transcriptional phenotypes in the dLNs and blood following SARS-CoV-2 vaccination or infection.

Single-cell T cell and B cell antigen receptor-sequencing data analysis can potentially perform in-depth assessments of adaptive immune cells that inform on understanding immune cell development to tracking clonal expansion in disease and therapy. However, it has been extremely challenging to analyze and interpret T cells and B cells and their adaptive immune receptor repertoires at the single-cell level due to not only the complexity of the data but also the underlying biology. In this Review, we delve into the computational breakthroughs that have transformed the analysis of single-cell T cell and B cell antigen receptor-sequencing data.

INTRODUCTION: Single-cell RNA sequencing (scRNA-seq) has become an essential tool for uncovering the complexities of various physiological and immunopathological conditions in veterinary medicine. However, there is currently limited information on immune-suppressive cancer subsets in canine breast cancers. In this study, we aimed to identify and characterize immune-suppressive subsets of triple-negative canine breast cancer (TNBC) by utilizing integrated scRNA-seq data from published datasets. METHODS: Published scRNA-seq datasets, including data from six groups of 30 dogs, were subjected to integrated bioinformatic analysis. RESULTS: Immune modulatory TNBC subsets were identified through functional enrichment analysis using immune-suppressive gene sets, including those associated with anti-inflammatory and M2-like macrophages. Key immune-suppressive signaling, such as viral infection, angiogenesis, and leukocyte chemotaxis, was found to play a role in enabling TNBC to evade immune surveillance. In addition, interactome analysis revealed significant interactions between distinct subsets of cancer cells and effector T cells, suggesting potential T-cell suppression. DISCUSSION: The present study demonstrates a versatile and scalable approach to integrating and analyzing scRNA-seq data, which successfully identified immune-modulatory subsets of canine TNBC. It also revealed potential mechanisms through which TNBC promotes immune evasion in dogs. These findings are crucial for advancing the understanding of the immune pathogenesis of canine TNBC and may aid in the development of new immune-based therapeutic strategies.

Childhood Sjögren's disease represents critically unmet medical needs due to a complete lack of immunological and molecular characterizations. This study presents key immune cell subsets and their interactions in the periphery in childhood Sjögren's disease. Here we show that single-cell RNA sequencing identifies the subsets of IFN gene-enriched monocytes, CD4⁺ T effector memory, and XCL1⁺ NK cells as potential key players in childhood Sjögren's disease, and especially in those with recurrent parotitis, which is the chief symptom prompting clinical visits from young children. A unique cluster of monocytes with type I and II IFN-related genes is identified in childhood Sjögren's disease, compared to the age-matched control. In vitro regulatory T cell functional assay demonstrates intact functionality in childhood Sjögren's disease in contrast to reduced suppression in adult Sjögren's disease. Mapping this transcriptomic landscape and interplay of immune cell subsets will expedite the understanding of childhood Sjögren's disease pathogenesis and set the foundation for precision medicine.

The interplay between T-cell states of differentiation, dysfunction, and treatment response in acute myeloid leukemia (AML) remains unclear. Here, we leveraged a multimodal approach encompassing high-dimensional flow cytometry and single-cell transcriptomics and found that early memory CD8+ T cells are associated with therapy response and exhibit a bifurcation into 2 distinct terminal end states. One state is enriched for markers of activation, whereas the other expresses natural killer (NK)-like and senescence markers. The skewed clonal differentiation trajectory toward CD8+ senescence was also a hallmark indicative of therapy resistance. We validated these findings by generating an AML CD8+ single-cell atlas integrating our data and other independent data sets. Finally, our analysis revealed that an imbalance between CD8+ early memory and senescent-like cells is linked to AML treatment refractoriness and poor survival. Our study provides crucial insights into the dynamics of CD8+ T-cell differentiation and advances our understanding of CD8+ T-cell dysfunction in AML.

Mitochondria transfer is a recently described phenomenon in which donor cells deliver mitochondria to acceptor cells^1-3. One possible consequence of mitochondria transfer is energetic support of neighbouring cells; for example, exogenous healthy mitochondria can rescue cell-intrinsic defects in mitochondrial metabolism in cultured ρ⁰ cells or Ndufs4^-/- peritoneal macrophages^4-7. Exposing haematopoietic stem cells to purified mitochondria before autologous haematopoietic stem cell transplantation allowed for treatment of anaemia in patients with large-scale mitochondrial DNA mutations^8,9, and mitochondria transplantation was shown to minimize ischaemic damage to the heart^10-12, brain^13-15 and limbs¹⁶. However, the therapeutic potential of using mitochondria transfer-based therapies to treat inherited mitochondrial diseases is unclear. Here we demonstrate improved morbidity and mortality of the Ndufs4^-/- mouse model of Leigh syndrome (LS) in multiple treatment paradigms associated with mitochondria transfer. Transplantation of bone marrow from wild-type mice, which is associated with release of haematopoietic cell-derived extracellular mitochondria into circulation and transfer of mitochondria to host cells in multiple organs, ameliorates LS in mice. Furthermore, administering isolated mitochondria from wild-type mice extends lifespan, improves neurological function and increases energy expenditure of Ndufs4^-/- mice, whereas mitochondria from Ndufs4^-/- mice did not improve neurological function. Finally, we demonstrate that cross-species administration of human mitochondria to Ndufs4^-/- mice also improves LS. These data suggest that mitochondria transfer-related approaches can be harnessed to treat mitochondrial diseases, such as LS.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is refractory to immune checkpoint inhibitor therapy. However, intratumoral T-cell infiltration correlates with improved overall survival (OS). Herein, we characterized the diversity and antigen specificity of the PDAC T-cell receptor (TCR) repertoire to identify novel immune-relevant biomarkers. Demographic, clinical, and TCR-beta sequencing data were collated from 353 patients across three cohorts that underwent surgical resection for PDAC. TCR diversity was calculated using Shannon Wiener index, Inverse Simpson index, and "True entropy." Patients were clustered by shared repertoire specificity. TCRs predictive of OS were identified and their associated transcriptional states were characterized by single-cell RNAseq. In multivariate Cox regression models controlling for relevant covariates, high intratumoral TCR diversity predicted OS across multiple cohorts. Conversely, in peripheral blood, high abundance of T-cells, but not high diversity, predicted OS. Clustering patients based on TCR specificity revealed a subset of TCRs that predicts OS. Interestingly, these TCR sequences were more likely to encode CD8⁺ effector memory and CD4⁺ T-regulatory (Tregs) T-cells, all with the capacity to recognize beta islet-derived autoantigens. As opposed to T-cell abundance, intratumoral TCR diversity was predictive of OS in multiple PDAC cohorts, and a subset of TCRs enriched in high-diversity patients independently correlated with OS. These findings emphasize the importance of evaluating peripheral and intratumoral TCR repertoires as distinct and relevant biomarkers in PDAC.

T cells are endowed with T-cell antigen receptors (TCR) that give them the capacity to recognize specific antigens and mount antigen-specific adaptive immune responses. Because TCR sequences are distinct in each naïve T cell, they serve as molecular barcodes to track T cells with clonal relatedness and shared antigen specificity through proliferation, differentiation, and migration. Single-cell RNA sequencing provides coupled information of TCR sequence and transcriptional state in individual cells, enabling T-cell clonotype-specific analyses. In this protocol, we outline a computational workflow to perform T-cell states and clonal analysis from scRNA-seq data based on the R packages Seurat, ProjecTILs, and scRepertoire. Given a scRNA-seq T-cell dataset with TCR sequence information, cell states are automatically annotated by reference projection using the ProjecTILs method. TCR information is used to track individual clonotypes, assess their clonal expansion, proliferation rates, bias towards specific differentiation states, and the clonal overlap between T-cell subtypes. We provide fully reproducible R code to conduct these analyses and generate useful visualizations that can be adapted for the needs of the protocol user. Key features Computational analysis of paired scRNA-seq and scTCR-seq data Characterizing T-cell functional state by reference-based analysis using ProjecTILs Exploring T-cell clonal structure using scRepertoire Linking T-cell clonality to transcriptomic state to study relationships between clonal expansion and functional phenotype Graphical overview.

Tissue-resident macrophages (TRMs) are long-lived cells that maintain locally and can be phenotypically distinct from monocyte-derived macrophages. Whether TRMs and monocyte-derived macrophages have district roles under differing pathologies is not understood. Here, we showed that a substantial portion of the macrophages that accumulated during pancreatitis and pancreatic cancer in mice had expanded from TRMs. Pancreas TRMs had an extracellular matrix remodeling phenotype that was important for maintaining tissue homeostasis during inflammation. Loss of TRMs led to exacerbation of severe pancreatitis and death, due to impaired acinar cell survival and recovery. During pancreatitis, TRMs elicited protective effects by triggering the accumulation and activation of fibroblasts, which was necessary for initiating fibrosis as a wound healing response. The same TRM-driven fibrosis, however, drove pancreas cancer pathogenesis and progression. Together, these findings indicate that TRMs play divergent roles in the pathogenesis of pancreatitis and cancer through regulation of stromagenesis.

Mitochondria are believed to have originated through an ancient endosymbiotic process in which proteobacteria were captured and co-opted for energy production and cellular metabolism. Mitochondria segregate during cell division and differentiation, with vertical inheritance of mitochondria and the mitochondrial DNA genome from parent to daughter cells. However, an emerging body of literature indicates that some cell types export their mitochondria for delivery to developmentally unrelated cell types, a process called intercellular mitochondria transfer. In this Review, we describe the mechanisms by which mitochondria are transferred between cells and discuss how intercellular mitochondria transfer regulates the physiology and function of various organ systems in health and disease. In particular, we discuss the role of mitochondria transfer in regulating cellular metabolism, cancer, the immune system, maintenance of tissue homeostasis, mitochondrial quality control, wound healing and adipose tissue function. We also highlight the potential of targeting intercellular mitochondria transfer as a therapeutic strategy to treat human diseases and augment cellular therapies.

Cutaneous T-cell lymphomas (CTCLs) are a spectrum of diseases with varied clinical courses caused by malignant clonal proliferation of skin-tropic T cells. Most patients have an indolent disease course managed with skin-directed therapies. In contrast, others, especially in advanced stages of disease or with specific forms, have aggressive progression and poor median survival. Sézary syndrome (SS), a leukemic variant of CTCL, lacks highly consistent phenotypic and genetic markers that may be leveraged to prevent the delay in diagnosis experienced by most patients with CTCL and could be useful for optimal treatment selection. Using single-cell mRNA and T-cell receptor sequencing of peripheral blood immune cells in SS, we extensively mapped the transcriptomic variations of nearly 50 000 T cells of both malignant and nonmalignant origins. We identified potential diverging SS cell populations, including quiescent and proliferative populations shared across multiple patients. In particular, the expression of AIRE was the most highly upregulated gene in our analysis, and AIRE protein expression could be observed over a variety of CTCLs. Furthermore, within a single patient, we were able to characterize differences in cell populations by comparing malignant T cells over the course of treatment with histone deacetylase inhibition and photopheresis. New cellular clusters after progression of the therapy notably exhibited increased expression of the transcriptional factor FOXP3, a master regulator of regulatory T-cell function, raising the potential implication of an evolving mechanism of immune evasion.

CD4+ T-helper 17 (Th17) T cells are a key population in protective immunity during infection and in self-tolerance/autoimmunity. Through the secretion of IL-17, Th17 cells act in promotion of inflammation and are thus a major potential therapeutic target in autoimmune disorders. Recent reports have brought to light that the IL-17 family cytokines, IL-17A, IL-17F and IL-17AF, can directly act on CD4+ T-cells, both in murine and human systems, inducing functional changes in these cells. Here we show that this action is preferentially targeted toward naïve, but not memory, CD4+ T-cells. Naïve cells showed transcriptome changes as early as 48 hours post-IL-17 exposure, whereas memory cells remained unaffected as late as 7 days. These functional differences occurred despite similar IL-17 receptor expression on these subsets and were maintained in co-culture/transwell systems, with each subset maintaining its functional response to IL-17. Importantly, there were differences in downstream transcriptional signaling by the three IL-17 cytokines, with the IL-17AF heterodimer conferring both the greatest transcriptional change and most altered functional consequences. Detailed transcriptome analysis provides important insights into the genes and pathways that are modulated as a result of IL-17-mediated signaling and may serve as targets of future therapies.

The induction of proinflammatory T cells by dendritic cell (DC) subtypes is critical for antitumor responses and effective immune checkpoint blockade (ICB) therapy. Here, we show that human CD1c⁺CD5⁺ DCs are reduced in melanoma-affected lymph nodes, with CD5 expression on DCs correlating with patient survival. Activating CD5 on DCs enhanced T cell priming and improved survival after ICB therapy. CD5⁺ DC numbers increased during ICB therapy, and low interleukin-6 (IL-6) concentrations promoted their de novo differentiation. Mechanistically, CD5 expression by DCs was required to generate optimally protective CD5^hi T helper and CD8⁺ T cells; further, deletion of CD5 from T cells dampened tumor elimination in response to ICB therapy in vivo. Thus, CD5⁺ DCs are an essential component of optimal ICB therapy.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. Immune checkpoint blockade has improved survival for many patients with NSCLC, but most fail to obtain long-term benefit. Understanding the factors leading to reduced immune surveillance in NSCLC is critical in improving patient outcomes. Here, we show that human NSCLC harbors large amounts of fibrosis that correlates with reduced T cell infiltration. In murine NSCLC models, the induction of fibrosis led to increased lung cancer progression, impaired T cell immune surveillance, and failure of immune checkpoint blockade efficacy. Associated with these changes, we observed that fibrosis leads to numerically and functionally impaired dendritic cells and altered macrophage phenotypes that likely contribute to immunosuppression. Within cancer-associated fibroblasts, distinct changes within the Col13a1-expressing population suggest that these cells produce chemokines to recruit macrophages and regulatory T cells while limiting recruitment of dendritic cells and T cells. Targeting fibrosis through transforming growth factor-β receptor signaling overcame the effects of fibrosis to enhance T cell responses and improved the efficacy of immune checkpoint blockade but only in the context of chemotherapy. Together, these data suggest that fibrosis in NSCLC leads to reduced immune surveillance and poor responsiveness to checkpoint blockade and highlight antifibrotic therapies as a candidate strategy to overcome immunotherapeutic resistance.

Cutaneous 5T cell lymphoma (CTCL), characterized by malignant T cells infiltrating the skin with potential for dissemination, remains a challenging disease to diagnose and treat due to disease heterogeneity, treatment resistance, and lack of effective and standardized diagnostic and prognostic clinical tools. Currently, diagnosis of CTCL practically relies on clinical presentation, histopathology, and immunohistochemistry. These methods are collectively fraught with limitations in sensitivity and specificity. Fortunately, recent advances in flow cytometry, polymerase chain reaction, high throughput sequencing, and other molecular techniques have shown promise in improving diagnosis and treatment of CTCL. Examples of these advances include T cell receptor clonotyping via sequencing to detect CTCL earlier in the disease course and single-cell RNA sequencing to identify gene expression patterns that commonly drive CTCL pathogenesis. Experience with these techniques has afforded novel insights which may translate into enhanced diagnostic and therapeutic approaches for CTCL.

Chronic activation of inflammatory pathways and suppressed interferon are hallmarks of immunosuppressive tumors. Previous studies have shown that CD11b integrin agonists could enhance anti-tumor immunity through myeloid reprograming, but the underlying mechanisms remain unclear. Herein we find that CD11b agonists alter tumor-associated macrophage (TAM) phenotypes by repressing NF-κB signaling and activating interferon gene expression simultaneously. Repression of NF-κB signaling involves degradation of p65 protein and is context independent. In contrast, CD11b agonism induces STING/STAT1 pathway-mediated interferon gene expression through FAK-mediated mitochondrial dysfunction, with the magnitude of induction dependent on the tumor microenvironment and amplified by cytotoxic therapies. Using tissues from phase I clinical studies, we demonstrate that GB1275 treatment activates STING and STAT1 signaling in TAMs in human tumors. These findings suggest potential mechanism-based therapeutic strategies for CD11b agonists and identify patient populations more likely to benefit.

SARS-CoV-2 infection and mRNA vaccination induce robust CD4⁺ T cell responses that are critical for the development of protective immunity. Here, we evaluated spike-specific CD4⁺ T cells in the blood and draining lymph node (dLN) of human subjects following BNT162b2 mRNA vaccination using single-cell transcriptomics. We analyze multiple spike-specific CD4⁺ T cell clonotypes, including novel clonotypes we define here using Trex, a new deep learning-based reverse epitope mapping method integrating single-cell T cell receptor (TCR) sequencing and transcriptomics to predict antigen-specificity. Human dLN spike-specific T follicular helper cells (T_FH) exhibited distinct phenotypes, including germinal center (GC)-T_FH and IL-10⁺ T_FH, that varied over time during the GC response. Paired TCR clonotype analysis revealed tissue-specific segregation of circulating and dLN clonotypes, despite numerous spike-specific clonotypes in each compartment. Analysis of a separate SARS-CoV-2 infection cohort revealed circulating spike-specific CD4⁺ T cell profiles distinct from those found following BNT162b2 vaccination. Our findings provide an atlas of human antigen-specific CD4⁺ T cell transcriptional phenotypes in the dLN and blood following vaccination or infection.

Cytokine production by memory T cells is a key mechanism of T cell mediated protection. However, we have limited understanding of the persistence of cytokine producing T cells during memory cell maintenance and secondary responses. We interrogated antigen-specific CD4 T cells using a mouse influenza A virus infection model. Although CD4 T cells detected using MHCII tetramers declined in lymphoid and non-lymphoid organs, we found similar numbers of cytokine⁺ CD4 T cells at days 9 and 30 in the lymphoid organs. CD4 T cells with the capacity to produce cytokines expressed higher levels of pro-survival molecules, CD127 and Bcl2, than non-cytokine⁺ cells. Transcriptomic analysis revealed a heterogeneous population of memory CD4 T cells with three clusters of cytokine⁺ cells. These clusters match flow cytometry data and reveal an enhanced survival signature in cells capable of producing multiple cytokines. Following re-infection, multifunctional T cells expressed low levels of the proliferation marker, Ki67, whereas cells that only produce the anti-viral cytokine, interferon-γ, were more likely to be Ki67⁺ . Despite this, multifunctional memory T cells formed a substantial fraction of the secondary memory pool. Together these data indicate that survival rather than proliferation may dictate which populations persist within the memory pool.

PURPOSE: To investigate IL-17 related mechanisms for developing dry eye disease in the Pinkie mouse strain with a loss of function RXRα mutation. METHODS: Measures of dry eye disease were assessed in the cornea and conjunctiva. Expression profiling was performed by single-cell RNA sequencing (scRNA-seq) to compare gene expression in conjunctival immune cells. Conjunctival immune cells were immunophenotyped by flow cytometry and confocal microscopy. The activity of RXRα ligand 9-cis retinoic acid (RA) was evaluated in cultured monocytes and γδ T cells. RESULTS: Compared to wild type (WT) C57BL/6, Pinkie has increased signs of dry eye disease, including decreased tear volume, corneal barrier disruption, corneal/conjunctival cornification and goblet cell loss, and corneal vascularization, opacification, and ulceration with aging. ScRNA-seq of conjunctival immune cells identified γδ T cells as the predominant IL-17 expressing population in both strains and there is a 4-fold increased percentage of γδ T cells in Pinkie. Compared to WT, IL-17a, and IL-17f significantly increased in Pinkie with conventional T cells and γδ T cells as the major producers. Flow cytometry revealed an increased number of IL-17⁺ γδ T cells in Pinkie. Tear concentration of the IL-17 inducer IL-23 is significantly higher in Pinkie. 9-cis RA treatment suppresses stimulated IL-17 production by γδ T and stimulatory activity of monocyte supernatant on γδ T cell IL-17 production. Compared to WT bone marrow chimeras, Pinkie chimeras have increased IL-17⁺ γδ T cells in the conjunctiva after desiccating stress and anti-IL-17 treatment suppresses dry eye induced corneal MMP-9 production/activity and conjunctival goblet cell loss. CONCLUSION: These findings indicate that RXRα suppresses generation of dry eye disease-inducing IL-17 producing lymphocytes s in the conjunctiva and identifies RXRα as a potential therapeutic target in dry eye.

Immune cells in the exposed conjunctiva mucosa defend against environmental and microbial stresses. Expression profiling by single-cell RNA sequencing was performed to identify conjunctival immune cell populations expressing homeostatic and regulatory genes. Fourteen distinct clusters were identified, including myeloid cells (neutrophils, monocytes, macrophages), dendritic cells (DC), and lymphoid cells (B, T, γδT, ILC2, and NK) lineages. Novel neutrophil [lipocalin (Lcn2) high and low), and MHCII^lo macrophage (MP) clusters were identified. More than half of the cells map to myeloid and dendritic cell populations with differential expression profiles that include genes with homeostatic and regulatory functions: Serpinb2 (MHCII^lo macrophage), Apoe (monocyte), Cd209a (macrophage), Cst3 (cDC1), and IL4i1 in migratory DC (mDC). ILC2 expresses the goblet cell trophic factor IL-13. Suppressed inflammatory and activated anti-inflammatory/regulatory pathways were observed in certain myeloid and DC populations. Confocal immunolocalization of identity markers showed mDC (CCR7, FASCIN1) located on or within the conjunctival epithelium. Monocyte, macrophage, cDC1 and IL-13/IL-5⁺ ILC2 were located below the conjunctival epithelium and goblet cells. This study found distinct immune cell populations in the conjunctiva and identified cells expressing genes with known homeostatic and immunoregulatory functions.

Adipocytes transfer mitochondria to macrophages in white and brown adipose tissues to maintain metabolic homeostasis. In obesity, adipocyte-to-macrophage mitochondria transfer is impaired, and instead, adipocytes release mitochondria into the blood to induce a protective antioxidant response in the heart. We found that adipocyte-to-macrophage mitochondria transfer in white adipose tissue is inhibited in murine obesity elicited by a lard-based high-fat diet, but not a hydrogenated-coconut-oil-based high-fat diet, aging, or a corn-starch diet. The long-chain fatty acids enriched in lard suppress mitochondria capture by macrophages, diverting adipocyte-derived mitochondria into the blood for delivery to other organs, such as the heart. The depletion of macrophages rapidly increased the number of adipocyte-derived mitochondria in the blood. These findings suggest that dietary lipids regulate mitochondria uptake by macrophages locally in white adipose tissue to determine whether adipocyte-derived mitochondria are released into systemic circulation to support the metabolic adaptation of distant organs in response to nutrient stress.

Acute myeloid leukemia (AML) is maintained by self-renewing leukemic stem cells (LSCs). A fundamental problem in treating AML is that conventional therapy fails to eliminate LSCs, which can reinitiate leukemia. Heat shock transcription factor 1 (HSF1), a central regulator of the stress response, has emerged as an important target in cancer therapy. Using genetic Hsf1 deletion and a direct HSF1 small molecule inhibitor, we show that HSF1 is specifically required for the maintenance of AML, while sparing steady-state and stressed hematopoiesis. Mechanistically, deletion of Hsf1 dysregulates multifaceted genes involved in LSC stemness and suppresses mitochondrial oxidative phosphorylation through downregulation of succinate dehydrogenase C (SDHC), a direct HSF1 target. Forced expression of SDHC largely restores the Hsf1 ablation-induced AML developmental defect. Importantly, the growth and engraftment of human AML cells are suppressed by HSF1 inhibition. Our data provide a rationale for developing efficacious small molecules to specifically target HSF1 in AML.

The effects of radiotherapy (RT) on tumor immunity in pancreatic ductal adenocarcinoma (PDAC) are not well understood. To better understand if RT can prime antigen-specific T-cell responses, we analyzed human PDAC tissues and mouse models. In both settings, there was little evidence of RT-induced T-cell priming. Using in vitro systems, we found that tumor-stromal components, including fibroblasts and collagen, cooperate to blunt RT efficacy and impair RT-induced interferon signaling. Focal adhesion kinase (FAK) inhibition rescued RT efficacy in vitro and in vivo, leading to tumor regression, T-cell priming, and enhanced long-term survival in PDAC mouse models. Based on these data, we initiated a clinical trial of defactinib in combination with stereotactic body RT in patients with PDAC (NCT04331041). Analysis of PDAC tissues from these patients showed stromal reprogramming mirroring our findings in genetically engineered mouse models. Finally, the addition of checkpoint immunotherapy to RT and FAK inhibition in animal models led to complete tumor regression and long-term survival. SIGNIFICANCE: Checkpoint immunotherapeutics have not been effective in PDAC, even when combined with RT. One possible explanation is that RT fails to prime T-cell responses in PDAC. Here, we show that FAK inhibition allows RT to prime tumor immunity and unlock responsiveness to checkpoint immunotherapy. This article is highlighted in the In This Issue feature, p. 2711.

Coxsackievirus B3 (CVB3)-induced myocarditis is commonly employed to study viral pathogenesis in mice. Chronically affected mice may develop dilated cardiomyopathy, which may involve the mediation of immune and nonimmune cells. To dissect this complexity, we performed single-cell RNA sequencing on heart cells from healthy and myocarditic mice, leading us to note significant proportions of myeloid cells, T cells, and fibroblasts. Although the transcriptomes of myeloid cells were mainly of M2 phenotype, the Th17 cells, CTLs, and Treg cells had signatures critical for cytotoxic functions. Fibroblasts were heterogeneous expressing genes important in fibrosis and regulation of inflammation and immune responses. The intercellular communication networks revealed unique interactions and signaling pathways in the cardiac cellulome, whereas myeloid cells and T cells had upregulated unique transcription factors modulating cardiac remodeling functions. Together, our data suggest that M2 cells, T cells, and fibroblasts may cooperatively or independently participate in the pathogenesis of viral myocarditis.

OBJECTIVE: Metabolomics as an approach to solve biological problems is exponentially increasing in use. Thus, this a pivotal time for the adoption of best practices. It is well known that disrupted tissue oxygen supply rapidly alters cellular energy charge. However, the speed and extent to which delayed mouse tissue freezing after dissection alters the broad metabolome is not well described. Furthermore, how tissue genotype may modulate such metabolomic drift and the degree to which traced ¹³C-isotopologue distributions may change have not been addressed. METHODS: By combined liquid chromatography (LC)- and gas chromatography (GC)-mass spectrometry (MS), we measured how levels of 255 mouse liver metabolites changed following 30-second, 1-minute, 3-minute, and 10-minute freezing delays. We then performed test-of-concept delay-to-freeze experiments evaluating broad metabolomic drift in mouse heart and skeletal muscle, differential metabolomic change between wildtype (WT) and mitochondrial pyruvate carrier (MPC) knockout mouse livers, and shifts in ¹³C-isotopologue abundances and enrichments traced from ¹³C-labled glucose into mouse liver. RESULTS: Our data demonstrate that delayed mouse tissue freezing after dissection leads to rapid hypoxia-driven remodeling of the broad metabolome, induction of both false-negative and false-positive between-genotype differences, and restructuring of ¹³C-isotopologue distributions. Notably, we show that increased purine nucleotide degradation products are an especially high dynamic range marker of delayed liver and heart freezing. CONCLUSIONS: Our findings provide a previously absent, systematic illustration of the extensive, multi-domain metabolomic changes occurring within the early minutes of delayed tissue freezing. They also provide a novel, detailed resource of mouse liver ex vivo, hypoxic metabolomic remodeling.

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT.

INTRODUCTION: Phytoestrogens found in soy, fruits, peanuts, and other legumes, have been identified as metabolites capable of providing beneficial effects in multiple pathological conditions due to their ability to mimic endogenous estrogen. Interestingly, the health-promoting effects of some phytoestrogens, such as isoflavones, are dependent on the presence of specific gut bacteria. Specifically, gut bacteria can metabolize isoflavones into equol, which has a higher affinity for endogenous estrogen receptors compared to dietary isoflavones. We have previously shown that patients with multiple sclerosis (MS), a neuroinflammatory disease, lack gut bacteria that are able to metabolize phytoestrogen. Further, we have validated the importance of both isoflavones and phytoestrogen-metabolizing gut bacteria in disease protection utilizing an animal model of MS. Specifically, we have shown that an isoflavone-rich diet can protect from neuroinflammatory diseases, and that protection was dependent on the ability of gut bacteria to metabolize isoflavones into equol. Additionally, mice on a diet with isoflavones showed an anti-inflammatory response compared to the mice on a diet lacking isoflavones. However, it is unknown how isoflavones and/or equol mediates their protective effects, especially their effects on host metabolite levels. OBJECTIVES: In this study, we utilized untargeted metabolomics to identify metabolites found in plasma that were modulated by the presence of dietary isoflavones. RESULTS: We found that the consumption of isoflavones increased anti-inflammatory monounsaturated fatty acids and beneficial polyunsaturated fatty acids while reducing pro-inflammatory glycerophospholipids, sphingolipids, phenylalanine metabolism, and arachidonic acid derivatives. CONCLUSION: Isoflavone consumption alters the systemic metabolic landscape through concurrent increases in monounsaturated fatty acids and beneficial polyunsaturated fatty acids plus reduction in pro-inflammatory metabolites and pathways. This highlights a potential mechanism by which an isoflavone diet may modulate immune-mediated disease.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.

Clear cell renal cell carcinoma (ccRCC) is one of the most immunologically distinct tumor types due to high response rate to immunotherapies, despite low tumor mutational burden. To characterize the tumor immune microenvironment of ccRCC, we applied single-cell-RNA sequencing (SCRS) along with T-cell-receptor (TCR) sequencing to map the transcriptomic heterogeneity of 25,688 individual CD45⁺ lymphoid and myeloid cells in matched tumor and blood from three patients with ccRCC. We also included 11,367 immune cells from four other individuals derived from the kidney and peripheral blood to facilitate the identification and assessment of ccRCC-specific differences. There is an overall increase in CD8⁺ T-cell and macrophage populations in tumor-infiltrated immune cells compared to normal renal tissue. We further demonstrate the divergent cell transcriptional states for tumor-infiltrating CD8⁺ T cells and identify a MKI67 + proliferative subpopulation being a potential culprit for the progression of ccRCC. Using the SCRS gene expression, we found preferential prediction of clinical outcomes and pathological diseases by subcluster assignment. With further characterization and functional validation, our findings may reveal certain subpopulations of immune cells amenable to therapeutic intervention.

The Toll-like receptor 8 (TLR8) agonist VTX-2337 (motolimod) is an anti-cancer immunotherapeutic agent that is believed to augment natural killer (NK) and dendritic cell (DC) activity. The goal of this work is to examine the role of TLR8 expression/activity in head and neck squamous cell carcinoma (HNSCC) to facilitate the prediction of responders to VTX-2337-based therapy. The prognostic role of TLR8 expression in HNSCC patients was assessed by TCGA and tissue microarray analyses. The anti-tumor effect of VTX-2337 was determined in SCCVII/C3H, mEERL/C57Bl/6 and TUBO-human EGFR/BALB/c syngeneic mouse models. The effect of combined VTX-2337 and cetuximab treatment on tumor growth, survival and immune cell recruitment was assessed. TLR8 expression was associated with CD8+ T cell infiltration and favorable survival outcomes. VTX-2337 delayed tumor growth in all 3 syngeneic mouse models and significantly increased the survival of cetuximab-treated mice. The anti-tumor effects of VTX-2337+ cetuximab were accompanied by increased splenic lymphoid DCs and IFNγ+ CD4+ and tumor-specific CD8+ T cells. Depletion of CD4+ T cells, CD8+ T cells and NK cells were all able to abolish the anti-tumor effect of VTX-2337+ cetuximab. Altogether, VTX-2337 remains promising as an adjuvant for cetuximab-based therapy however patients with high TLR8 expression may be more likely to derive benefit from this drug combination compared to patients with low TLR8 expression.

The recently identified G-protein-coupled receptor GPR171 and its ligand BigLEN are thought to regulate food uptake and anxiety. Though GPR171 is commonly used as a T cell signature gene in transcriptomic studies, its potential role in T cell immunity has not been explored. Here we show that GPR171 is transcribed in T cells and its protein expression is induced upon antigen stimulation. The neuropeptide ligand BigLEN interacts with GPR171 to suppress T cell receptor-mediated signalling pathways and to inhibit T cell proliferation. Loss of GPR171 in T cells leads to hyperactivity to antigen stimulation and GPR171 knockout mice exhibit enhanced antitumor immunity. Blockade of GPR171 signalling by an antagonist promotes antitumor T cell immunity and improves immune checkpoint blockade therapies. Together, our study identifies the GPR171/BigLEN axis as a T cell checkpoint pathway that can be modulated for cancer immunotherapy.

Mammary gland ductal morphogenesis depends on the differentiation of mammary stem cells (MaSCs) into basal and luminal lineages. The AP-2γ transcription factor, encoded by Tfap2c, has a central role in mammary gland development but its effect in mammary lineages and specifically MaSCs is largely unknown. Here, we utilized an inducible, conditional knockout of Tfap2c to elucidate the role of AP-2γ in maintenance and differentiation of MaSCs. Loss of AP-2γ in the basal epithelium profoundly altered the transcriptomes and decreased the number of cells within several clusters of mammary epithelial cells, including adult MaSCs and luminal progenitors. AP-2γ regulated the expression of genes known to be required for mammary development, including Cebpb, Nfkbia, and Rspo1. As a result, AP-2γ-deficient mice exhibited repressed mammary gland ductal outgrowth and inhibition of regenerative capacity. The findings demonstrate that AP-2γ can regulate development of mammary gland structures potentially regulating maintenance and differentiation of multipotent MaSCs.

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177⁺ TI Treg population that may serve as a target for TI Treg-specific immunotherapy.

Several clinicopathological features of clear cell renal cell carcinomas (ccRCC) contribute to make an "atypical" cancer, including resistance to chemotherapy, sensitivity to anti-angiogenesis therapy and ICIs despite a low mutational burden, and CD8⁺ T cell infiltration being the predictor for poor prognosis-normally CD8⁺ T cell infiltration is a good prognostic factor in cancer patients. These "atypical" features have brought researchers to investigate the molecular and immunological mechanisms that lead to the increased T cell infiltrates despite relatively low molecular burdens, as well as to decipher the immune landscape that leads to better response to ICIs. In the present study, we summarize the past and ongoing pivotal clinical trials of immunotherapies for ccRCC, emphasizing the potential molecular and cellular mechanisms that lead to the success or failure of ICI therapy. Single-cell analysis of ccRCC has provided a more thorough and detailed understanding of the tumor immune microenvironment and has facilitated the discovery of molecular biomarkers from the tumor-infiltrating immune cells. We herein will focus on the discussion of some major immune cells, including T cells and tumor-associated macrophages (TAM) in ccRCC. We will further provide some perspectives of using molecular and cellular biomarkers derived from these immune cell types to potentially improve the response rate to ICIs in ccRCC patients.

Regulatory T cells (Tregs) are critical in maintaining immune homeostasis under various pathophysiological conditions. A growing body of evidence demonstrates that Tregs play an important role in cancer progression and that they do so by suppressing cancer-directed immune responses. Tregs have been targeted for destruction by exploiting antibodies against and small-molecule inhibitors of several molecules that are highly expressed in Tregs-including immune checkpoint molecules, chemokine receptors, and metabolites. To date, these strategies have had only limited antitumor efficacy, yet they have also created significant risk of autoimmunity because most of them do not differentiate Tregs in tumors from those in normal tissues. Currently, immune checkpoint inhibitor (ICI)-based cancer immunotherapies have revolutionized cancer treatment, but the resistance to ICI is common and the elevation of Tregs is one of the most important mechanisms. Therapeutic strategies that can selectively eliminate Tregs in the tumor (i.e. therapies that do not run the risk of causing autoimmunity by affecting normal tissue), are urgently needed for the development of cancer immunotherapies. This chapter discusses specific properties of human Tregs under the context of cancer and the various ways to target Treg for cancer immunotherapy.

Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis and, within tumors, their upregulation is common and promotes an immunosuppressive microenvironment. Therapeutic strategies that can eliminate Tregs in the tumor (i.e., therapies that do not run the risk of affecting normal tissues), are urgently needed for the development of cancer immunotherapies. Here we report our discovery of B-cell lymphoma extra-large (BCL-X_L) as a potential molecular target of tumor-infiltrating (TI) Tregs. We show that pharmacological degradation of BCL-X_L using a newly developed platelet-sparing BCL-X_L Proteolysis-targeting chimera (PROTAC) induces the apoptosis of TI-Tregs and the activation of TI-CD8⁺ T cells. Moreover, these activities result in an effective suppression of syngeneic tumor growth in immunocompetent, but not in immunodeficient or CD8⁺ T cell-depleted mice. Notably, treatment with BCL-X_L PROTAC does not cause detectable damage within several normal tissues or thrombocytopenia. These findings identify BCL-X_L as a target in the elimination of TI-Tregs as a component of cancer immunotherapies, and that the BCL-X_L-specific PROTAC has the potential to be developed as a therapeutic for cancer immunotherapy.

The oncogenic role of estrogen receptor (ER) signaling in breast cancer has long been established. Interaction of estrogen with estrogen receptor (ER) in the nucleus activates genomic pathways of estrogen signaling. In contrast, estrogen interaction with the cell membrane-bound G-protein-coupled estrogen receptor (GPER) activates the rapid receptor-mediated signaling transduction cascades. Aberrant estrogen signaling enhances mammary epithelial cell proliferation, survival, and angiogenesis, hence is an important step towards breast cancer initiation and progression. Meanwhile, a growing number of studies also provide evidence for estrogen's pro- or anti-inflammatory roles. As other articles in this issue cover classic ER and GPER signaling mediated by estrogen, this review will discuss the crucial mechanisms by which estrogen signaling influences chronic inflammation and how that is involved in breast cancer. Xenoestrogens acquired from plant diet or exposure to industrial products constantly interact with and alter innate estrogen signaling at various levels. As such, they can modulate chronic inflammation and breast cancer development. Natural xenoestrogens generally have anti-inflammatory properties, which is consistent with their chemoprotective role in breast cancer. In contrast, synthetic xenoestrogens are proinflammatory and carcinogenic compounds that can increase the risk of breast cancer. This article also highlights important xenoestrogens with a particular focus on their role in inflammation and breast cancer. Improved understanding of the complex relationship between estrogens, inflammation, and breast cancer will guide clinical research on agents that could advance breast cancer prevention and therapy.

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity.

Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma that metastasizes early in its clinical course and lacks an effective medical therapy. The TAZ-CAMTA1 and YAP-TFE3 fusion proteins are chimeric transcription factors and initiating oncogenic drivers of EHE. A combined proteomic/genetic screen in human cell lines identified YEATS2 and ZZZ3, components of the Ada2a-containing histone acetyltransferase (ATAC) complex, as key interactors of both fusion proteins despite the dissimilarity of the C terminal fusion partners CAMTA1 and TFE3. Integrative next-generation sequencing approaches in human and murine cell lines showed that the fusion proteins drive a unique transcriptome by simultaneously hyperactivating a TEAD-based transcriptional program and modulating the chromatin environment via interaction with the ATAC complex. Interaction of the ATAC complex with both fusion proteins indicates that it is a key oncogenic driver and unifying enzymatic therapeutic target for this sarcoma. This study presents an approach to mechanistically dissect how chimeric transcription factors drive the formation of human cancers.

Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or seasonal influenza may lead to respiratory failure requiring intubation and mechanical ventilation. The pathophysiology of this respiratory failure is attributed to local immune dysregulation, but how the immune response to viral infection in the lower airways of the human lung differs between individuals with respiratory failure and those without is not well understood. We used quantitative multiparameter flow cytometry and multiplex cytokine assays to evaluate matched blood and bronchoalveolar lavage (BAL) samples from control human subjects, subjects with symptomatic seasonal influenza who did not have respiratory failure, and subjects with severe seasonal influenza or SARS-CoV-2 infection with respiratory failure. We find that severe cases are associated with an influx of nonclassical monocytes, activated T cells, and plasmablast B cells into the lower airways. Cytokine concentrations were not elevated in the lower airways of moderate influenza patients compared with controls; however, 28 of 35 measured cytokines were significantly elevated in severe influenza, severe SARS-CoV-2 infection, or both. We noted the largest elevations in IL-6, IP-10, MCP-1, and IL-8. IL-1 family cytokines and RANTES were higher in severe influenza infection than severe SARS-CoV-2 infection. Interestingly, only the concentration of IP-10-correlated between blood and BAL during severe infection. Our results demonstrate inflammatory immune dysregulation in the lower airways during severe viral pneumonia that is distinct from lower airway responses seen in human patients with symptomatic, but not severe, illness and suggest that measurement of blood IP-10 concentration may predict this unique dysregulation.

Type 1 diabetes and insulinoma can co-occur in pediatric patients and may present with episodes of hypo- and hyperglycemia, significant glycemic variability, and weight gain. Surgical resection leads to development of fulminant diabetes.

We previously showed stabilization of NIK-induced activation of NF-κB non-canonical signaling suppresses MLL-AF9-induced AML. In the current study, we demonstrate that deletion of NF-κB non-canonical RelB prevents the inhibitory effect of NIK stabilization in MLL-AF9 AML. Mechanistically, RelB suppresses its direct target, TIFAB, which is upregulated in human AML and correlates negatively with the survival of AML patients. Forced expression of TIFAB reverses NIK-induced impaired AML development through downregulation of RelB and upregulation of HOXA9. Consistent with upregulation of HOXA9, gene set enrichment analysis shows that forced expression of TIFAB blocks myeloid cell development, upregulates leukemia stem cell signature and induces similar gene expression patterns to those of HOXA9-MEIS1 and HOXA9-NUP98, and upregulates oxidative phosphorylation. Accordingly, forced expression of HOXA9 also largely releases the inhibitory impact of NIK stabilization via downregulation of RelB and upregulation of RelA. Our data suggest that NIK/RelB suppresses MLL-AF9-induced AML mainly through downregulation of TIFAB/HOXA9.

Single-cell sequencing is an emerging technology in the field of immunology and oncology that allows researchers to couple RNA quantification and other modalities, like immune cell receptor profiling at the level of an individual cell. A number of workflows and software packages have been created to process and analyze single-cell transcriptomic data. These packages allow users to take the vast dimensionality of the data generated in single-cell-based experiments and distill the data into novel insights. Unlike the transcriptomic field, there is a lack of options for software that allow for single-cell immune receptor profiling. Enabling users to easily combine mRNA and immune profiling, scRepertoire was built to process data derived from 10x Genomics Chromium Immune Profiling for both T-cell receptor (TCR) and immunoglobulin (Ig) enrichment workflows and subsequently interacts with a number of popular R packages for single-cell expression, such as Seurat. The scRepertoire R package and processed data are open source and available on GitHub and provides in-depth tutorials on the capability of the package.

Alopecia areata (AA) is a common autoimmune condition, presenting initially with loss of hair without other overt skin changes. The unremarkable appearance of the skin surface contrasts with the complex immune activity occurring at the hair follicle. AA pathogenesis is due to the loss of immune privilege of the hair follicle, leading to autoimmune attack. Although the literature has focused on CD8+ T cells, vital roles for CD4+ T cells and antigen-presenting cells have been suggested. Here, we use single-cell sequencing to reveal distinct expression profiles of immune cells in murine AA. We found clonal expansions of both CD4+ and CD8+ T cells, with shared clonotypes across varied transcriptional states. The murine AA data were used to generate highly predictive models of human AA disease. Finally, single-cell sequencing of T cells in human AA recapitulated the clonotypic findings and the gene expression of the predictive models.

The novel coronavirus disease 2019 (COVID-19) pandemic has caused catastrophic damage to human life across the globe along with social and financial hardships. According to the Johns Hopkins University Coronavirus Resource Center, more than 41.3 million people worldwide have been infected, and more than 1,133,000 people have died as of October 22, 2020. At present, there is no available vaccine and a scarcity of efficacious therapies. However, there is tremendous ongoing effort towards identifying effective drugs and developing novel vaccines. Early data from Adaptive COVID-19 Treatment Trials (ACTT) sponsored by the National Institute of Allergy and Infectious Diseases (NIAID) and compassionate use study have shown promise for remdesivir, leading to emergency authorization by the Food and Drug Administration (FDA) for treatment of hospitalized COVID-19 patients. However, several randomized studies have now shown no benefit or increased adverse events associated with remdesivir treatment. Drug development is a time-intensive process and requires extensive safety and efficacy evaluations. In contrast, drug repurposing is a time-saving and cost-effective drug discovery strategy geared towards using existing drugs instead of de novo drug discovery. Treatments for cancer and COVID-19 often have similar goals of controlling inflammation, inhibiting cell division, and modulating the host microenvironment to control the disease. In this review, we focus on anti-cancer drugs that can potentially be repurposed for COVID-19 and are currently being tested in clinical trials.

Untoward effector CD4+ T cell responses are kept in check by immune regulatory mechanisms mediated by CD4+ and CD8+ T cells. CD4+ T helper 17 (Th17) cells, characterized by IL-17 production, play important roles in the pathogenesis of autoimmune diseases (such as arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, among others) and in the host response to infection and cancer. Here, we demonstrate that human CD4+ T cells cells exposed to a Th17-differentiating milieu are significantly more resistant to immune suppression by CD8+ T cells compared to control Th0 cells. This resistance is mediated, in part, through the action of IL-17A, IL-17F, and IL-17AF heterodimer through their receptors (IL-17RA and IL-17RC) on CD4+ T cells themselves, but not through their action on CD8+ T cells or APC. We further show that IL-17 can directly act on non-Th17 effector CD4+ T cells to induce suppressive resistance, and this resistance can be reversed by blockade of IL-1β, IL-6, or STAT3. These studies reveal a role for IL-17 cytokines in mediating CD4-intrinsic immune resistance. The pathways induced in this process may serve as a critical target for future investigation and immunotherapeutic intervention.

Aiming to identify immune molecules with a novel function in cancer pathogenesis, we found the cluster of differentiation 177 (CD177), a known neutrophil antigen, to be positively correlated with relapse-free, metastasis-free, or overall survival in breast cancer. In addition, CD177 expression is correlated with good prognosis in several other solid cancers including prostate, cervical, and lung. Focusing on breast cancer, we found that CD177 is expressed in normal breast epithelial cells and is significantly reduced in invasive cancers. Loss of CD177 leads to hyperproliferative mammary epithelium and contributes to breast cancer pathogenesis. Mechanistically, we found that CD177-deficiency is associated with an increase in β-catenin signaling. Here we identified CD177 as a novel regulator of mammary epithelial proliferation and breast cancer pathogenesis likely via the modulation of Wnt/β-catenin signaling pathway, a key signaling pathway involved in multiple cancer types.

BACKGROUND: HER2 plays a critical role in tumourigenesis and is associated with poor prognosis of patients with HER2-positive breast cancers. Although anti-HER2 drugs are beneficial for treating breast cancer, de novo, or acquired resistance often develops. Epigenetic factors are increasingly targeted for therapy; however, such mechanisms that interact with HER2 signalling are poorly understood. METHODS: RNA sequencing was performed to identify PHF8 targets downstream of HER2 signalling. CHIP-qPCR were used to investigate how PHF8 regulates HER2 transcription. ELISA determined cytokine secretion. Cell-based assay revealed a feed forward loop in HER2 signalling and then evaluated in vivo. FINDINGS: We report the synergistic interplay between histone demethylase PHF8 and HER2 signalling. Specifically, PHF8 levels were elevated in HER2-positive breast cancers and upregulated by HER2. PHF8 functioned as a coactivator that regulated the expression of HER2, markers of the HER2-driven epithelial-to-mesenchymal transition and cytokines. The HER2-PHF8-IL-6 regulatory axis was active in cell lines and in newly established MMTV-Her2/MMTV-Cre/Phf8^fl°^x/fl°^x mouse models, which revealed the oncogenic function of Phf8 in breast cancer for the first time. Further, the PHF8-IL-6 axis contributed to the resistance to trastuzumab in vitro and may play a critical role in the infiltration of T cells in HER2-driven breast cancers. INTERPRETATION: These findings provided informative mechanistic insight into the potential application of PHF8 inhibitors to overcome resistance to anti-HER2 therapies. FUNDING: This work was supported by Carver Trust Young Investigator Award (01-224 to H.H.Q); and a Breast Cancer Research Award (to H.H.Q.).

Autoimmune diseases are characterized by regulatory deficit in both the CD4+ and CD8+ T-cell compartments. We have shown that CD8+ T-cells associated with acute relapse of multiple sclerosis are significantly deficient in their immune suppressive ability. We hypothesized that distinct CD8+ cytotoxic T-cell (Tc) lineages, determined by cytokine milieu during naïve T-cell differentiation, may harbor differential ability to suppress effector CD4+ T-cells. We differentiated purified human naïve CD8+ T-cells in vitro toward Tc0 (media control), Tc1 and Tc17 lineages. Using in vitro flow cytometric suppression assays, we observed that Tc0 and Tc17 cells had similar suppressive ability. In contrast, Tc1 cells showed significant loss of suppressive ability against ex vivo CD4+ T-cells and in vitro-differentiated Th0, Th1 and Th17 cells. Of note, Tc1 cells were also suboptimal in suppressing CD4-induced acute xenogeneic graft versus host disease (xGVHD) in vivo. Tc subtypes derived under various cytokine combinations revealed that IL-12-containing conditions resulted in less suppressive cells exhibiting dysregulated cytotoxic degranulation. RNA sequencing transcriptome analyses indicated differential regulation of inflammatory genes and enrichment in GM-CSF-associated pathways. These studies provide insights into the role of T-cell differentiation in CD8 suppressive biology and may reveal therapeutically targetable pathways to reverse suppressive deficit during immune-mediated disease.

Recently antibiotic exposure has been associated with worse outcomes in patients undergoing treatment with antibodies directed against programmed cell death protein-1 (PD-1). We reviewed data of 1264 patients enrolled at Melanoma Skin and Ocular Tissue Repositories at University of Iowa Hospitals and Clinic. Reviewed data included patient demographics, prior medical history, baseline hematologic and disease parameters and outcomes including progression-free survival (PFS) and overall survival (OS). Cox regression models were used to determine predictive markers. Overall, 169 patients with advanced cutaneous melanoma received anti-PD-1 based therapies. Median follow up was 18.46 (range 0.89 to 62.52) months. On multivariable analysis brain metastasis, higher absolute neutrophil count (ANC) and lower absolute lymphocyte count were associated with poorer PFS while brain and liver metastasis and lower albumin were associated with poorer OS. Prior antibiotics, radiation as well as age, gender, basal metabolic index (BMI), smoking status, BRAF mutation, line of therapy (first or latter), prior treatments (ipilimumab or BRAF inhibitors), hemoglobin, neutrophil-to-lymphocyte ratio, white blood cell, platelet and eosinophil counts were not associated with PFS or OS in multivariable analysis. Contrary to some prior studies BMI, radiation, and antibiotics were not associated with PFS or OS.

Immunotherapy has revolutionized the treatment of both hematological malignancies and solid tumors. The use of immunotherapy has improved outcome for patients with cancer across multiple tumor types, including lung, melanoma, ovarian, genitourinary, and more recently breast cancer with durable responses seen even in patients with widespread metastatic disease. Despite the promising results, immunotherapy still helps only a subset of patients due to overall low response rates. Moreover, the response to immunotherapy is highly cancer specific and results have not been as promising in cancers that are considered less immunogenic. The strategies to improve immunotherapy responses have focused on biomarker selection, like PD-L1 status, and usage of combinatorial agents, such as chemotherapy, targeted therapy, and radiotherapy. Of particular interest, DNA-damaging agents have the potential to enhance the response to immunotherapy by promoting neoantigen release, increasing tumor mutational burden, and enhancing PD-L1 expression. Poly-ADP-ribose polymerase (PARP) inhibitors are one such class of drugs that has shown synergy with immunotherapy in preclinical and early clinical studies. PARP-based therapies work through the inhibition of single-strand DNA repair leading to DNA damage, increased tumor mutational burden, making the tumor a more attractive target for immunotherapy. Of the solid tumors reviewed, breast, ovarian, and prostate cancers have demonstrated efficacy in the combination of PARP inhibition and immunotherapy, predominately in BRCA-mutated tumors. However, initial investigations into wildtype BRCA and gastrointestinal tumors have shown moderate overall response or disease control rates, dependent on the tumor type. In contrast, although a number of clinical trials underway, there is a paucity of published results for the use of the combination in lung or urothelial cancers. Overall this article focuses on the promise of combinatorial PARP inhibition and immunotherapy to improve patient outcomes in solid tumors, summarizing both early results and looking toward ongoing trials.

The AP-2γ transcription factor, encoded by the TFAP2C gene, regulates the expression of estrogen receptor-alpha (ERα) and other genes associated with hormone response in luminal breast cancer. Little is known about the role of AP-2γ in other breast cancer subtypes. A subset of HER2⁺ breast cancers with amplification of the TFAP2C gene locus becomes addicted to AP-2γ. Herein, we sought to define AP-2γ gene targets in HER2⁺ breast cancer and identify genes accounting for physiologic effects of growth and invasiveness regulated by AP-2γ. Comparing HER2⁺ cell lines that demonstrated differential response to growth and invasiveness with knockdown of TFAP2C, we identified a set of 68 differentially expressed target genes. CDH5 and CDKN1A were among the genes differentially regulated by AP-2γ and that contributed to growth and invasiveness. Pathway analysis implicated the MAPK13/p38δ and retinoic acid regulatory nodes, which were confirmed to display divergent responses in different HER2⁺ cancer lines. To confirm the clinical relevance of the genes identified, the AP-2γ gene signature was found to be highly predictive of outcome in patients with HER2⁺ breast cancer. We conclude that AP-2γ regulates a set of genes in HER2⁺ breast cancer that drive cancer growth and invasiveness. The AP-2γ gene signature predicts outcome of patients with HER2⁺ breast cancer and pathway analysis predicts that subsets of patients will respond to drugs that target the MAPK or retinoic acid pathways. IMPLICATIONS: A set of genes regulated by AP-2γ in HER2⁺ breast cancer that drive proliferation and invasion were identified and provided a gene signature that is predictive of outcome in HER2⁺ breast cancer.

NF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.

Ferroptosis is an iron-dependent, non-apoptotic form of regulated cell death driven by lipid hydroperoxides within biological membranes. Although therapy-resistant mesenchymal-high cancers are particularly vulnerable to ferroptosis inducers, especially phospholipid glutathione peroxidase 4 (GPx4) inhibitors, the underlying mechanism is yet to be deciphered. As such, the full application of GPx4 inhibitors in cancer therapy remains challenging. Here we demonstrate that metadherin (MTDH) confers a therapy-resistant mesenchymal-high cell state and enhanced sensitivity to inducers of ferroptosis. Mechanistically, MTDH inhibited GPx4, as well as the solute carrier family 3 member 2 (SLC3A2, a system X_c^- heterodimerization partner), at both the messenger RNA and protein levels. Our metabolomic studies demonstrated that MTDH reduced intracellular cysteine, but increased glutamate levels, ultimately decreasing levels of glutathione and setting the stage for increased vulnerability to ferroptosis. Finally, we observed an enhanced antitumor effect when we combined various ferroptosis inducers both in vitro and in vivo; the level of MTDH correlated with the ferroptotic effect. We have demonstrated for the first time that MTDH enhances the vulnerability of cancer cells to ferroptosis and may serve as a therapeutic biomarker for future ferroptosis-centered cancer therapy.

PURPOSE: Cutaneous T-cell lymphomas (CTCL), encompassing a spectrum of T-cell lymphoproliferative disorders involving the skin, have collectively increased in incidence over the last 40 years. Sézary syndrome is an aggressive form of CTCL characterized by significant presence of malignant cells in both the blood and skin. The guarded prognosis for Sézary syndrome reflects a lack of reliably effective therapy, due, in part, to an incomplete understanding of disease pathogenesis. EXPERIMENTAL DESIGN: Using single-cell sequencing of RNA and the machine-learning reverse graph embedding approach in the Monocle package, we defined a model featuring distinct transcriptomic states within Sézary syndrome. Gene expression used to differentiate the unique transcriptional states were further used to develop a boosted tree classification for early versus late CTCL disease. RESULTS: Our analysis showed the involvement of FOXP3 ⁺ malignant T cells during clonal evolution, transitioning from FOXP3 ⁺ T cells to GATA3 ⁺ or IKZF2 ⁺ (HELIOS) tumor cells. Transcriptomic diversities in a clonal tumor can be used to predict disease stage, and we were able to characterize a gene signature that predicts disease stage with close to 80% accuracy. FOXP3 was found to be the most important factor to predict early disease in CTCL, along with another 19 genes used to predict CTCL stage. CONCLUSIONS: This work offers insight into the heterogeneity of Sézary syndrome, providing better understanding of the transcriptomic diversities within a clonal tumor. This transcriptional heterogeneity can predict tumor stage and thereby offer guidance for therapy.

Obesity is a risk factor for breast cancer and also predicts poor clinical outcomes regardless of menopausal status. Contributing to the poor clinical outcomes is the suboptimal efficacy of standard therapies due to dose limiting toxicities and obesity-related complications, highlighting the need to develop novel therapeutic approaches for treating obese patients. We recently found that obesity leads to an increase in tumor-infiltrating macrophages with activated NLRC4 inflammasome and increased interleukin (IL)-1β production. IL-1β, in turn, leads to increased angiogenesis and cancer progression. Using Next Generation RNA sequencing, we identified an NLRC4/IL-1β-dependent upregulation of angiopoietin-like 4 (ANGPTL4), a known angiogenic factor in cancer, in tumors from obese mice. ANGPTL4-deficiency by genetic knockout or treatment with a neutralizing antibody led to a significant reduction in obesity-induced angiogenesis and tumor growth. At a mechanistic level, ANGPTL4 expression is induced by IL-1β from primary adipocytes in a manner dependent on NF-κB- and MAP kinase-activation, which is further enhanced by hypoxia. This report shows that adipocyte-derived ANGPTL4 drives disease progression under obese conditions and is a potential therapeutic target for treating obese breast cancer patients.

Transforming growth factor-β (TGF-β) signaling plays fundamental roles in the development and homeostasis of somatic cells. Dysregulated TGF-β signaling contributes to cancer progression and relapse to therapies by inducing epithelial-to-mesenchymal transition (EMT), enriching cancer stem cells, and promoting immunosuppression. Although many TGF-β-regulated genes have been identified, only a few datasets were obtained by next-generation sequencing. In this study, we performed RNA-sequencing analysis of MCF10A cells and identified 1166 genes that were upregulated and 861 genes that were downregulated by TGF-β. Gene set enrichment analysis revealed that focal adhesion and metabolic pathways were the top enriched pathways of the up- and downregulated genes, respectively. Genes in these pathways also possess significant predictive value for renal cancers. Moreover, we confirmed that TGF-β induced expression of MICAL1 and 2, and the histone demethylase, KDM7A, and revealed their regulatory roles on TGF-β-induced cell migration. We also show a critical effect of KDM7A in regulating the acetylation of H3K27 on TGF-β-induced genes. In sum, this study identified novel effectors that mediate the pro-migratory role of TGF-β signaling, paving the way for future studies that investigate the function of MICAL family members in cancer and the novel epigenetic mechanisms downstream TGF-β signaling.

Among all breast cancer types, basal-like breast cancer (BLBC) represents an aggressive subtype that lacks targeted therapy. We and others have found that receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in BLBC and other types of cancer and that ROR1 is significantly correlated with patient prognosis. In addition, using primary patient-derived xenografts (PDXs) and ROR1-knockout BLBC cells, we found that ROR1⁺ cells form tumors in immunodeficient mice. We developed an anti-ROR1 immunotoxin and found that targeting ROR1 significantly kills ROR1⁺ cancer cells and slows down tumor growth in ROR1⁺ xenografts. Our bioinformatics analysis revealed that ROR1 expression is commonly associated with the activation of FGFR-mediated signaling pathway. Further biochemical analysis confirmed that ROR1 stabilized FGFR expression at the posttranslational level by preventing its degradation. CRISPR/Cas9-mediated ROR1 knockout significantly reduced cancer cell invasion at cellular levels by lowering FGFR protein and consequent inactivation of AKT. Our results identified a novel signaling regulation from ROR1 to FGFR and further confirm that ROR1 is a potential therapeutic target for ROR1⁺ BLBC cells.

Reverse-phase protein arrays (RPPAs) are a highthroughput approach to protein quantification utilizing antibody-based micro-to-nano scale dot blot. Within the Cancer Genome Atlas (TCGA), RPPAs were used to quantify over 200 proteins in 8,167 tumor and metastatic samples. Protein-level data has particular advantages in assessing putative prognostic or therapeutic targets in tumors. However, many of the available pipelines do not allow for the partitioning of clinical and RPPA information to make meaningful conclusions. We developed a cloud-based application, TRGAted to enable researchers to better examine patient survival based on single or multiple proteins across 31 cancer types in the TCGA. TRGAted contains up-to-date overall survival, disease-specific survival, disease-free interval and progression-free interval information. Furthermore, survival information for primary tumor samples can be stratified based on gender, age, tumor stage, histological type, and subtype, allowing for highly adaptive and intuitive user experience. The code and processed data are open sourced and available on github and contains a tutorial built into the application for assisting users.

E-cadherin is conventionally considered to be a good prognostic marker in cancer. The loss of E-cadherin is one of the key hallmarks of epithelial-to-mesenchymal transition, a biological process that promotes cancer cell invasiveness and metastasis. Recent evidence has cast doubt on the importance of epithelial-to-mesenchymal transition in metastasis. The availability of protein-level data in the Cancer Genome Atlas allows for the quantitative analysis of protein and prognosis. The prognostic values of E-cadherin and β-catenin were revisited across 19 cancer types, and high E-cadherin was found to correlate with good prognosis in most cancers. Conversely, higher E-cadherin and β-catenin correlated with shorter survival in invasive breast carcinoma. Stratifying breast cancers by histologic subtype revealed that the poor prognosis of E-cadherin and β-catenin proteins was characteristic of infiltrating ductal, but not lobular, carcinomas. To further corroborate the protein findings and examine cellular localization, immunohistochemistry was used for E-cadherin and β-catenin in 163 breast patient samples from the Iowa cohort. Most previous studies showing that reduced or absent E-cadherin and β-catenin was inversely associated with tumor stages in ductal carcinomas were confirmed. Taken together, these results lead us to question the prognostic values of E-cadherin and β-catenin in ductal carcinomas and indicate a complicated role of E-cadherin and β-catenin in breast cancer progression.

Immune checkpoints are a diverse set of inhibitory signals to the immune system that play a functional role in adaptive immune response and self-tolerance. Dysregulation of these pathways is a vital mechanism in the avoidance of immune destruction by tumor cells. Immune checkpoint blockade (ICB) refers to targeted strategies to disrupt the tumor co-opted immune suppression to enhance anti-tumor immunity. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) are two immune checkpoints that have the widest range of antibody-based therapies. These therapies have gone from promising approaches to Food and Drug Administration-approved first- and second-line agents for a number of immunogenic cancers. The burgeoning investigations of ICB efficacy in blood and solid cancers have underscored the importance of identifying the predictors of response and resistance to ICB. Identification of response correlates is made complicated by the observations of mixed reactions, or different responses in multiple lesions from the same patient, and delayed responses that can occur over a year after the induction therapy. Factors that can influence response and resistance in ICB can illuminate underlying molecular mechanisms of immune activation and suppression. These same response predictors can guide the identification of patients who would benefit from ICB, reduce off-target immune-relate adverse events, and facilitate the use of combinatorial therapies to increase efficacy. Here we review the underlying principles of immune checkpoint therapy and results of single-agent ICB clinical trials, and summarize the predictors of response and resistance.

To improve student preparedness for anatomy laboratory dissection, the dental gross anatomy laboratory was transformed using flipped classroom pedagogy. Instead of spending class time explaining the procedures and anatomical structures for each laboratory, students were provided online materials to prepare for laboratory on their own. Eliminating in-class preparation provided the opportunity to end each period with integrative group activities that connected laboratory and lecture material and explored clinical correlations. Materials provided for prelaboratory preparation included: custom-made, three-dimensional (3D) anatomy videos, abbreviated dissection instructions, key atlas figures, and dissection videos. Data from three years of the course (n = 241 students) allowed for analysis of students' preferences for these materials and detailed tracking of usage of 3D anatomy videos. Students reported spending an average of 27:22 (±17:56) minutes preparing for laboratory, similar to the 30 minutes previously allocated for in-class dissection preparation. The 3D anatomy videos and key atlas figures were rated the most helpful resources. Scores on laboratory examinations were compared for the three years before the curriculum change (2011-2013; n = 242) and three years after (2014-2016; n = 241). There was no change in average grades on the first and second laboratory examinations. However, on the final semi-cumulative laboratory examination, scores were significantly higher in the post-flip classes (P = 0.04). These results demonstrate an effective model for applying flipped classroom pedagogy to the gross anatomy laboratory and illustrate a meaningful role for 3D anatomy visualizations in a dissection-based course. Anat Sci Educ 11: 385-396. © 2017 American Association of Anatomists.

The transcriptional regulation of the human epidermal growth factor receptor-2 (HER2) contributes to an enhanced HER2 expression in HER2-positive breast cancers with HER2 gene amplification and HER2-low or HER2-negative breast cancers following radiotherapy or endocrine therapy, and this drives tumorigenesis and the resistance to therapy. Epigenetic mechanisms are critical for transcription regulation, however, such mechanisms in the transcription regulation of HER2 are limited to the involvement of tri-methylated histone 3 lysine 4 (H3K4me3) and acetylated histone 3 lysine 9 (H3K9ac) at the HER2 promoter region. Here, we report the identification of a novel enhancer in the HER2 3' gene body, which we have termed HER2 gene body enhancer (HGE). The HGE starts from the 3' end of intron 19 and extends into intron 22, possesses enhancer histone modification marks in specific cells and enhances the transcriptional activity of the HER2 promoters. We also found that TFAP2C, a known regulator of HER2, binds to HGE and is required for its enhancer function and that DNA methylation in the HGE region inhibits the histone modifications characterizing enhancer and is inversely correlated with HER2 expression in breast cancer samples. The identification of this novel enhancer sheds a light on the roles of epigenetic mechanisms in HER2 transcription, in both HER2-positive breast cancer samples and individuals with HER2-low or HER2-negative breast cancers undergoing radiotherapy or endocrine therapy.

Multiple GWAS studies have shown that the SNP rs2281808 TT variant, present within the SIRPG gene, is associated with autoimmune diseases, such as type 1 diabetes. However, the role of SIRPγ in human T-cells is not known, neither is the functional significance of TT variant. Here we investigated SIRPG genotypes and their effects on the fate and function of human T-cells. We found that the presence of T variant resulted in reduction of SIRPγ expression on T-cells. Functionally, SIRPγ^low CD8 T-cells in CT and TT individuals existed in a heightened effector state with lower activation threshold and had greater expression of genes and molecules associated with migratory and cytotoxic potential. Further, SIRPγ^low CD8 T-cells were deficient in transcription factors associated with long-term functional memory formation. Our study reveals biological consequences of the SNP rs2281808 and provides novel insights into the potential mechanisms by which SIRPγ might regulate human immune responses.

Triple-negative breast cancer (TNBC) is a heterogeneous disease with poorer outcomes compared to other breast cancer subtypes. Contributing to the worse prognosis in TNBC is the higher rates of relapse and rapid progression after relapse. Advances in targeted therapeutics and conventional chemotherapy for TNBC have been stymied due to the lack of specific targets. Moreover, the responses to chemotherapy in TNBC lack durability, partially accounting for the higher rates of relapse. Immunotherapy, notably immune-checkpoint blockade, has shown to improve survival and maintain robust antitumor responses in both hematologic and solid malignancies. Unlike lung cancer, melanoma, and bladder cancer, most breast cancers are not inherently immunogenic and typically have low T cell infiltration. However, among breast cancer subtypes, TNBC is characterized by greater tumor immune infiltrate and higher degree of stromal and intratumoral tumor-infiltrating lymphocytes (TILs), a predictive marker for responses to immunotherapy. Moreover, in TNBC, the high number of stromal TILs is predictive of more favorable survival outcomes and response to chemotherapy. Immunotherapy is being extensively explored in TNBC and clinical trials are showing some promising results. This article focuses on the rationale for immunotherapy in TNBC, to explore and discuss preclinical data, results from early clinical trials, and to summarize some ongoing trials. We will also discuss the potential application of immunotherapy in TNBC from a clinician's perspective.

Canonical NF-κB signaling is constitutively activated in acute myeloid leukemia (AML) stem cells and is required for maintenance of the self-renewal of leukemia stem cells (LSCs). However, any potential role for NF-κB non-canonical signaling in AML has been largely overlooked. Here, we report that stabilization of NF-κB-inducing kinase (NIK) suppresses AML. Mechanistically, stabilization of NIK activates NF-κB non-canonical signaling and represses NF-κB canonical signaling. In addition, stabilization of NIK-induced activation of NF-κB non-canonical signaling upregulates Dnmt3a and downregulates Mef2c, which suppresses and promotes AML development, respectively. Importantly, by querying the connectivity MAP using up- and downregulated genes that are present exclusively in NIK-stabilized LSCs, we discovered that verteporfin has anti-AML effects, suggesting that repurposing verteporfin to target myeloid leukemia is worth testing clinically. Our data provide a scientific rationale for developing small molecules to stabilize NIK specifically in myeloid leukemias as an attractive therapeutic option.

Obesity is associated with an increased risk of developing breast cancer and is also associated with worse clinical prognosis. The mechanistic link between obesity and breast cancer progression remains unclear, and there has been no development of specific treatments to improve the outcome of obese cancer patients. Here we show that obesity-associated NLRC4 inflammasome activation/ interleukin (IL)-1 signalling promotes breast cancer progression. The tumour microenvironment in the context of obesity induces an increase in tumour-infiltrating myeloid cells with an activated NLRC4 inflammasome that in turn activates IL-1β, which drives disease progression through adipocyte-mediated vascular endothelial growth factor A (VEGFA) expression and angiogenesis. Further studies show that treatment of mice with metformin inhibits obesity-associated tumour progression associated with a marked decrease in angiogenesis. This report provides a causal mechanism by which obesity promotes breast cancer progression and lays out a foundation to block NLRC4 inflammasome activation or IL-1β signalling transduction that may be useful for the treatment of obese cancer patients.

Paracrine signaling between mammary epithelial cells has long been appreciated. Recently, we found that Wnt5a, a novel noncanonical Wnt ligand of luminal origin, counteracts canonical Wnt signaling in basal mammary epithelial cells through a paracrine pathway, inhibits the expansion of Erbb2-induced tumor-initiating cells, and suppresses tumor incidence and metastasis.

PURPOSE: We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. METHODS: CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. RESULTS: Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. CONCLUSIONS: CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.

Breast cancer is the leading cause of cancer-related mortality for females worldwide.¹ Improving early screening strategies and understanding the events that lead to tumor initiation have led to demonstrable improvements in clinical outcome. Our previous work revealed a variance in the tumorigenic capacity between different mammary epithelial cell populations in an MMTV-ErbB2 mouse model. In order to greater understand how different mammary epithelial cells influence the tumorigenic capacity in ErbB2-induced breast cancer, we transplanted different cell populations from pre-neoplastic MMTV-ErbB2 female mice into recipient mice for tumorigenic study. We found that different mammary epithelial cells bear different tumorigenic potential even when induced by the same ErbB2 proto-oncogene. To understand the difference in tumors formed from different epithelial cells, we performed gene expression profiling using these tumors (GSE64487). Several genes were further validated using real-time reverse transcription polymerase chain reaction (RT-PCR). Here we provide further details on the experimental methods and microarray analysis. This data provides a resource to further understanding how different mammary cell populations can initiate ErbB2-driven tumors and the role of these cell populations as putative tumor-initiating cells (TICs).

It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared with the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of the TGFβ-SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal cross-talk in normal mammary tissue.

BACKGROUND: Bone marrow stromal antigen 2 (BST-2) is a known anti-viral gene that has been recently identified to be overexpressed in many cancers, including breast cancer. BST-2 is critical for the invasiveness of breast cancer cells and the formation of metastasis in vivo. Although the regulation of BST-2 in immune cells is unraveling, it is unknown how BST-2 expression is regulated in breast cancer. We hypothesized that meta-analyses of BST-2 gene expression and BST-2 DNA methylation profiles would illuminate mechanisms regulating elevated BST-2 expression in breast tumor tissues and cells. MATERIALS AND METHODS: We performed comprehensive meta-analyses of BST-2 gene expression and BST-2 DNA methylation in The Cancer Genome Atlas (TCGA) and various Gene Expression Omnibus (GEO) datasets. BST-2 expression levels and BST-2 DNA methylation status at specific CpG sites on the BST-2 gene were compared for various breast tumor molecular subtypes and breast cancer cell lines. RESULTS: We show that BST-2 gene expression is inversely associated with the methylation status at specific CpG sites in primary breast cancer specimens and breast cancer cell lines. BST-2 demethylation is significantly more prevalent in primary tumors and cancer cells than in normal breast tissues or normal mammary epithelial cells. Demethylation of the BST-2 gene significantly correlates with its mRNA expression. These studies provide the initial evidence that significant differences exist in BST-2 DNA methylation patterns between breast tumors and normal breast tissues, and that BST-2 expression patterns in tumors and cancer cells correlate with hypomethylated BST-2 DNA. CONCLUSION: Our study suggests that the DNA methylation pattern and expression of BST-2 may play a role in disease pathogenesis and could serve as a biomarker for the diagnosis of breast cancer.

Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the ROR family consisting of ROR1 and ROR2. RORs contain two distinct extracellular cysteine-rich domains and one transmembrane domain. Within the intracellular portion, ROR1 possesses a tyrosine kinase domain, two serine/threonine-rich domains and a proline-rich domain. RORs have been studied in the context of embryonic patterning and neurogenesis through a variety of homologs. These physiologic functions are dichotomous based on the requirement of the kinase domain. A growing literature has established ROR1 as a marker for cancer, such as in CLL and other blood malignancies. In addition, ROR1 is critically involved in progression of a number of blood and solid malignancies. ROR1 has been shown to inhibit apoptosis, potentiate EGFR signaling, and induce epithelial-mesenchymal transition (EMT). Importantly, ROR1 is only detectable in embryonic tissue and generally absent in adult tissue, making the protein an ideal drug target for cancer therapy.

Nuclear Interactor of ARF and Mdm2 (NIAM, gene designation Tbrg1) is a largely unstudied inhibitor of cell proliferation that helps maintain chromosomal stability. It is a novel activator of the ARF-Mdm2-Tip60-p53 tumor suppressor pathway as well as other undefined pathways important for genome maintenance. To examine its predicted role as a tumor suppressor, we generated NIAM mutant (NIAM(m/m)) mice homozygous for a β-galactosidase expressing gene-trap cassette in the endogenous gene. The mutant mice expressed significantly lower levels of NIAM protein in tissues compared to wild-type animals. Fifty percent of aged NIAM deficient mice (14 to 21 months) developed proliferative lesions, including a uterine hemangioma, pulmonary papillary adenoma, and a Harderian gland adenoma. No age-matched wild-type or NIAM(+/m) heterozygous animals developed lesions. In the spleen, NIAM(m/m) mice had prominent white pulp expansion which correlated with enhanced increased reactive lymphoid hyperplasia and evidence of systemic inflammation. Notably, 17% of NIAM mutant mice had splenic white pulp features indicating early B-cell lymphoma. This correlated with selective expansion of marginal zone B cells in the spleens of younger, tumor-free NIAM-deficient mice. Unexpectedly, basal p53 expression and activity was largely unaffected by NIAM loss in isolated splenic B cells. In sum, NIAM down-regulation in vivo results in a significant predisposition to developing benign tumors or early stage cancers. These mice represent an outstanding platform for dissecting NIAM's role in tumorigenesis and various anti-cancer pathways, including p53 signaling.

Metastatic spread is the leading cause of cancer mortality. Breast cancer (BCa) metastatic recurrence can happen years after removal of the primary tumor. Here we show that Ubc13, an E2 enzyme that catalyzes K63-linked protein polyubiquitination, is largely dispensable for primary mammary tumor growth but is required for metastatic spread and lung colonization by BCa cells. Loss of Ubc13 inhibited BCa growth and survival only at metastatic sites. Ubc13 was dispensable for transforming growth factor β (TGFβ)-induced SMAD activation but was required for activation of non-SMAD signaling via TGFβ-activating kinase 1 (TAK1) and p38, whose activity controls expression of numerous metastasis promoting genes. p38 activation restored metastatic activity to Ubc13-deficient cells, and its pharmacological inhibition attenuated BCa metastasis in mice, suggesting it is a therapeutic option for metastatic BCa.

Diabetes is a rapidly growing epidemic affecting millions of Americans and has been implicated in a number of devastating secondary complications. We previously demonstrated that type 2 diabetic rats exhibit vitamin D deficiency due to aberrant megalin-mediated endocytosis and excessive urinary excretion of 25-hydroxycholecalciferol (25D3) and vitamin D-binding protein (DBP). Here, we examined whether a model of type 1 diabetes [T1D; streptozotocin (STZ)-treated Sprague-Dawley rats] would similarly excrete abnormally high concentrations of 25D3 and DBP due to renal damage and compromised expression of megalin and its endocytic partner, disabled-2 (Dab2). Moreover, we tested whether feeding diabetic rats starch that is resistant to digestion could alleviate these abnormalities. Control (n = 12) rats were fed a standard, semipurified diet (AIN-93G) containing 55% total dietary starch and STZ-treated rats were fed the AIN-93G diet (n = 12) or a diet containing 55% high-amylose maize that is partially resistant to digestion [20% total dietary resistant starch (RS); n = 12] for 2 and 5 wk. The RS diet attenuated weight loss and polyuria in STZ-treated rats. Histology and immunohistochemistry revealed that dietary RS also attenuated the loss of Dab2 expression in renal proximal tubules. Moreover, urinary concentrations of both 25D3 and DBP were elevated ∼10-fold in STZ-treated rats (5 wk post STZ injection), which was virtually prevented by the RS. We also observed a ∼1.5-fold increase in megalin mRNA expression in STZ-treated rats, which was attenuated by feeding rats the RS diet for 2 wk. Taken together, these studies indicate that consumption of low-glycemic carbohydrates can attenuate disruption of vitamin D homeostasis in T1D through the rescue of megalin-mediated endocytosis in the kidney.

IκB kinase α (IKKα) activity is required for ErbB2-induced mammary tumorigenesis. Here, we show that IKKα and its activator, NF-κB-inducing kinase (NIK), support the expansion of tumor-initiating cells (TICs) that copurify with a CD24(med)CD49f(hi) population from premalignant ErbB2-expressing mammary glands. Upon activation, IKKα enters the nucleus, phosphorylates the cyclin-dependent kinase (CDK) inhibitor p27/Kip1, and stimulates its nuclear export or exclusion. Reduced p27 expression rescues mammary tumorigenesis in mice deficient in IKKα kinase activity and restores TIC self-renewal. IKKα is also likely to be involved in human breast cancer, where its expression shows an inverse correlation with metastasis-free survival, and its presence in the nucleus of invasive ductal carcinomas (IDCs) is associated with decreased nuclear p27 abundance.