Targeting the NF-κB pathway enhances responsiveness of mammary tumors to JAK inhibitors.

Interactions between tumor cells and the tumor microenvironment are critical for tumor growth, progression, and response to therapy. Effective targeting of oncogenic signaling pathways in tumors requires an understanding of how these therapies impact both tumor cells and cells within the tumor microenvironment. One such pathway is the janus kinase (JAK)/signal transducer and activator or transcription (STAT) pathway, which is activated in both breast cancer cells and in tumor associated macrophages. This study demonstrates that exposure of macrophages to JAK inhibitors leads to activation of NF-κB signaling, which results in increased expression of genes known to be associated with therapeutic resistance. Furthermore, inhibition of the NF-κB pathway improves the ability of ruxolitinib to reduce mammary tumor growth in vivo. Thus, the impact of the tumor microenvironment is an important consideration in studying breast cancer and understanding such mechanisms of resistance is critical to development of effective targeted therapies.

Type III interferon drives thymic B cell activation and regulatory T cell generation.

The activation of thymic B cells is critical for their licensing as antigen presenting cells and resulting ability to mediate T cell central tolerance. The processes leading to licensing are still not fully understood. By comparing thymic B cells to activated Peyer's patch B cells at steady state, we found that thymic B cell activation starts during the neonatal period and is characterized by TCR/CD40-dependent activation, followed by immunoglobulin class switch recombination (CSR) without forming germinal centers. Transcriptional analysis also demonstrated a strong interferon signature, which was not apparent in the periphery. Thymic B cell activation and CSR were primarily dependent on type III IFN signaling, and loss of type III IFN receptor in thymic B cells resulted in reduced thymocyte regulatory T cell (Treg) development. Finally, from TCR deep sequencing, we estimate that licensed B cells induce development of a substantial fraction of the Treg cell repertoire. Together, these findings reveal the importance of steady-state type III IFN in generating licensed thymic B cells that induce T cell tolerance to activated B cells.

Type 2 cytokines in the thymus activate Sirpα+ dendritic cells to promote clonal deletion.

The thymus contains a diversity of dendritic cells (DCs) that exist in defined locations and have different antigen-processing and -presenting features. This suggests that they play nonredundant roles in mediating thymocyte selection. In an effort to eliminate SIRPα+ classic DC2 subsets, we discovered that a substantial proportion expresses the surface lectin, CD301b, in the thymus. These cells resemble the CD301b+ type 2 immune response promoting DCs that are present in the skin-draining lymph nodes. Transcriptional and phenotypic comparison to other DC subsets in the thymus revealed that thymic CD301b+ cDCs represent an activated state that exhibits enhanced antigen processing and presentation. Furthermore, a CD301b+ cDC2 subset demonstrated a type 2 cytokine signature and required steady-state interleukin-4 receptor signaling. Selective ablation of CD301b+ cDC2 subsets impaired clonal deletion without affecting regulatory T cells (Treg cells). The T cell receptor α repertoire sequencing confirmed that a cDC2 subset promotes deletion of conventional T cells with minimal effect on Treg cell selection. Together, these findings suggest that cytokine-induced activation of DCs in the thymus substantially enforces central tolerance.

Chondroitin sulfate proteoglycan 4, a targetable oncoantigen that promotes ovarian cancer growth, invasion, cisplatin resistance and spheroid formation.

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease encompassing several distinct molecular subtypes and clinical entities. Despite the initial success of surgical debulking and adjuvant chemotherapy, recurrence with chemotherapy resistant tumors is common in patients with EOC and leads to poor overall survival. The extensive genetic and phenotypic heterogeneity associated with ovarian cancers has hindered the identification of effective prognostic and predictive biomarkers in EOC patients. In the current studies, we identify a tumor cell surface oncoantigen, chondroitin sulfate proteoglycan 4 (CSPG4), as an independent risk factor for decreased survival of patients with EOC. Our results show that CSPG4 promotes EOC cell invasion, cisplatin resistance and spheroid formation in vitro and tumor expansion in vivo. Mechanistically, spheroid formation and tumor cell invasion are due to CSPG4-stimulated expression of the mesenchymal transcription factor ZEB1. Furthermore, we have developed a novel monoclonal anti-CSGP4 antibody against the juxtamembrane domain of the core protein that limits CSPG4-stimulated ZEB1 expression, tumor cell invasion and promotes EOC apoptosis within spheroid cultures. We therefore propose that CSPG4 expression drives phenotypic heterogeneity and malignant progression in EOC tumors. These studies further demonstrate that CSPG4 expression levels are a potential diagnostic biomarker in EOC and indicate that targeting cells which express this oncoantigen could limit recurrence and improve outcomes in patients with EOC.

STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment.

BACKGROUND: In breast cancer, complex interactions between tumor cells and cells within the surrounding stroma, such as macrophages, are critical for tumor growth, progression, and therapeutic response. Recent studies have highlighted the complex nature and heterogeneous populations of macrophages associated with both tumor-promoting and tumor-inhibiting phenotypes. Defining the pathways that drive macrophage function is important for understanding their complex phenotypes within the tumor microenvironment. Signal transducer and activator of transcription (STAT) transcription factors, such as STAT5, are key regulators of immune cell function. The studies described here investigate the functional contributions of STAT5 to tumor-associated macrophage function in breast cancer. METHODS: Initial studies were performed using a panel of human breast cancer and mouse mammary tumor cell lines to determine the ability of tumor cell-derived factors to induce STAT5 activation in macrophages. Further studies used these models to identify soluble factors that activate STAT5 in macrophages. To delineate STAT5-specific contributions to macrophage function, a conditional model of myeloid STAT5 deletion was used for in vitro, RNA-sequencing, and in vivo studies. The effects of STAT5 deletion in macrophages on tumor cell migration and metastasis were evaluated using in vitro co-culture migration assays and an in vivo tumor cell-macrophage co-injection model. RESULTS: We demonstrate here that STAT5 is robustly activated in macrophages by tumor cell-derived factors and that GM-CSF is a key cytokine stimulating this pathway. The analysis of RNA-seq studies reveals that STAT5 promotes expression of immune stimulatory genes in macrophages and that loss of STAT5 in macrophages results in increased expression of tissue remodeling factors. Finally, we demonstrate that loss of STAT5 in macrophages promotes tumor cell migration in vitro and mammary tumor metastasis in vivo. CONCLUSIONS: Breast cancer cells produce soluble factors, such as GM-CSF, that activate the STAT5 pathway in macrophages and drive expression of inflammatory factors. STAT5 deletion in myeloid cells enhances metastasis, suggesting that STAT5 activation in tumor-associated macrophages protects against tumor progression. Understanding mechanisms that drive macrophage function in the tumor microenvironment will ultimately lead to new approaches that suppress tumor-promoting functions while enhancing their anti-tumor functions.

Whole-genome variation of transposable element insertions in a maize diversity panel.

Intact transposable elements (TEs) account for 65% of the maize genome and can impact gene function and regulation. Although TEs comprise the majority of the maize genome and affect important phenotypes, genome-wide patterns of TE polymorphisms in maize have only been studied in a handful of maize genotypes, due to the challenging nature of assessing highly repetitive sequences. We implemented a method to use short-read sequencing data from 509 diverse inbred lines to classify the presence/absence of 445,418 nonredundant TEs that were previously annotated in four genome assemblies including B73, Mo17, PH207, and W22. Different orders of TEs (i.e., LTRs, Helitrons, and TIRs) had different frequency distributions within the population. LTRs with lower LTR similarity were generally more frequent in the population than LTRs with higher LTR similarity, though high-frequency insertions with very high LTR similarity were observed. LTR similarity and frequency estimates of nested elements and the outer elements in which they insert revealed that most nesting events occurred very near the timing of the outer element insertion. TEs within genes were at higher frequency than those that were outside of genes and this is particularly true for those not inserted into introns. Many TE insertional polymorphisms observed in this population were tagged by SNP markers. However, there were also 19.9% of the TE polymorphisms that were not well tagged by SNPs (R2 < 0.5) that potentially represent information that has not been well captured in previous SNP-based marker-trait association studies. This study provides a population scale genome-wide assessment of TE variation in maize and provides valuable insight on variation in TEs in maize and factors that contribute to this variation.

De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes.

We report de novo genome assemblies, transcriptomes, annotations, and methylomes for the 26 inbreds that serve as the founders for the maize nested association mapping population. The number of pan-genes in these diverse genomes exceeds 103,000, with approximately a third found across all genotypes. The results demonstrate that the ancient tetraploid character of maize continues to degrade by fractionation to the present day. Excellent contiguity over repeat arrays and complete annotation of centromeres revealed additional variation in major cytological landmarks. We show that combining structural variation with single-nucleotide polymorphisms can improve the power of quantitative mapping studies. We also document variation at the level of DNA methylation and demonstrate that unmethylated regions are enriched for cis-regulatory elements that contribute to phenotypic variation.

Genetic control of kernel compositional variation in a maize diversity panel.

Maize (Zea mays L.) is a multi-purpose row crop grown worldwide, which, over time, has often been bred for increased yield at the detriment of lower composition grain quality. Some knowledge of the genetic factors that affect quality traits has been discovered through the study of classical maize mutants; however, much of the underlying genetic control of these traits and the interaction between these traits remains unknown. To better understand variation that exists for grain compositional traits in maize, we evaluated 501 diverse temperate maize inbred lines in five unique environments and predicted 16 compositional traits (e.g., carbohydrates, protein, and starch) based on the output of near-infrared (NIR) spectroscopy. Phenotypic analysis found substantial variation for compositional traits and the majority of variation was explained by genetic and environmental factors. Correlations and trade-offs among traits in different maize types (e.g., dent, sweetcorn, and popcorn) were explored, and significant differences and meaningful correlations were detected. In total, 22.9-71.0% of the phenotypic variation across these traits could be explained using 2,386,666 single nucleotide polymorphism (SNP) markers generated from whole-genome resequencing data. A genome-wide association study (GWAS) was conducted using these same markers and found 72 statistically significant SNPs for 11 compositional traits. This study provides valuable insights in the phenotypic variation and genetic control underlying compositional traits that can be used in breeding programs for improving maize grain quality.

Assessing the regulatory potential of transposable elements using chromatin accessibility profiles of maize transposons.

Transposable elements (TEs) have the potential to create regulatory variation both through the disruption of existing DNA regulatory elements and through the creation of novel DNA regulatory elements. In a species with a large genome, such as maize, many TEs interspersed with genes create opportunities for significant allelic variation due to TE presence/absence polymorphisms among individuals. We used information on putative regulatory elements in combination with knowledge about TE polymorphisms in maize to identify TE insertions that interrupt existing accessible chromatin regions (ACRs) in B73 as well as examples of polymorphic TEs that contain ACRs among four inbred lines of maize including B73, Mo17, W22, and PH207. The TE insertions in three other assembled maize genomes (Mo17, W22, or PH207) that interrupt ACRs that are present in the B73 genome can trigger changes to the chromatin, suggesting the potential for both genetic and epigenetic influences of these insertions. Nearly 20% of the ACRs located over 2 kb from the nearest gene are located within an annotated TE. These are regions of unmethylated DNA that show evidence for functional importance similar to ACRs that are not present within TEs. Using a large panel of maize genotypes, we tested if there is an association between the presence of TE insertions that interrupt, or carry, an ACR and the expression of nearby genes. While most TE polymorphisms are not associated with expression for nearby genes, the TEs that carry ACRs exhibit enrichment for being associated with higher expression of nearby genes, suggesting that these TEs may contribute novel regulatory elements. These analyses highlight the potential for a subset of TEs to rewire transcriptional responses in eukaryotic genomes.

Complex pleiotropic genetic architecture of evolved heat stress and oxidative stress resistance in the nematode Caenorhabditis remanei.

The adaptation of complex organisms to changing environments has been a central question in evolutionary quantitative genetics since its inception. The structure of the genotype-phenotype maps is critical because pleiotropic effects can generate widespread correlated responses to selection and potentially restrict the extent of evolutionary change. In this study, we use experimental evolution to dissect the genetic architecture of natural variation for acute heat stress and oxidative stress response in the nematode Caenorhabiditis remanei. Previous work in the classic model nematode Caenorhabiditis elegans has found that abiotic stress response is controlled by a handful of genes of major effect and that mutations in any one of these genes can have widespread pleiotropic effects on multiple stress response traits. Here, we find that acute heat stress response and acute oxidative response in C. remanei are polygenic, complex traits, with hundreds of genomic regions responding to selection. In contrast to expectation from mutation studies, we find that evolved acute heat stress and acute oxidative stress response for the most part display independent genetic bases. This lack of correlation is reflected at the levels of phenotype, gene expression, and in the genomic response to selection. Thus, while these findings support the general view that rapid adaptation can be generated by changes at hundreds to thousands of sites in the genome, the architecture of segregating variation is likely to be determined by the pleiotropic structure of the underlying genetic networks.

Transposable elements contribute to dynamic genome content in maize.

Transposable elements (TEs) are ubiquitous components of eukaryotic genomes and can create variation in genome organization and content. Most maize genomes are composed of TEs. We developed an approach to define shared and variable TE insertions across genome assemblies and applied this method to four maize genomes (B73, W22, Mo17 and PH207) with uniform structural annotations of TEs. Among these genomes we identified approximately 400 000 TEs that are polymorphic, encompassing 1.6 Gb of variable TE sequence. These polymorphic TEs include a combination of recent transposition events as well as deletions of older TEs. There are examples of polymorphic TEs within each of the superfamilies of TEs and they are found distributed across the genome, including in regions of recent shared ancestry among individuals. There are many examples of polymorphic TEs within or near maize genes. In addition, there are 2380 gene annotations in the B73 genome that are located within variable TEs, providing evidence for the role of TEs in contributing to the substantial differences in annotated gene content among these genotypes. TEs are highly variable in our survey of four temperate maize genomes, highlighting the major contribution of TEs in driving variation in genome organization and gene content. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://github.com/SNAnderson/maizeTE_variation; https://mcstitzer.github.io/maize_TEs.