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Background Inhibitory immune receptors are important for maintaining immune homeostasis. We recently identified a member of this group, LAIR1, in advanced cutaneous T-cell lymphoma, a cancer of skin-invasive T cells associated with recurrent and often fatal S. aureus skin and soft tissue infections. Based on these data, we predicted that LAIR1 plays a role in immune response to S. aureus infection. Objective Determine the impact of LAIR1 loss on S. aureus skin infection. Methods Wild type (WT) and Lair1 knock-out (KO) mice were subjected to subcutaneous S. aureus skin infection. In skin lesions, we measured size, bacterial clearance, immune cell recruitment (flow cytometry), and cytokine production (ELISA). We compared changes in gene expression (qRT-PCR), cytokine production (ELISA), and complement-mediated phagocytosis (flow cytometry) in WT and Lair1 KO bone marrow-derived macrophages (BMDMs) infected with S. aureus. Finally, we conducted RNAseq from S. aureus skin lesions and bone marrow neutrophils and monocytes 2 and 5 days post-infection (dpi), and from in vitro-infected BMDMs. Results Lair1 KO mice had significantly larger areas of abscess and dermonecrosis by 1 dpi and decreased bacterial clearance at 2 dpi. BMDMs showed defects in proinflammatory cytokine production and complement-mediated phagocytosis. Conclusions Taken together, these data support a previously unrecognized role for LAIR1 in myeloid response to bacterial infection.

Human respiratory syncytial virus (hRSV) causes severe acute respiratory tract infections in infants, elderly and immunocompromised people worldwide and few effective treatments are available. The hRSV nonstructural protein NS1 is known to inhibit host interferon responses in the cytoplasm. We and others have used in vitro interactome studies to identify host proteins that interact with NS1. Among these is the Mediator complex, a transcriptional coactivator localized in the nucleus, suggesting that NS1 may have a more direct role in modulating IFN-stimulated gene expression. We performed chromatin immunoprecipitation of NS1 or Mediator followed by sequencing (ChIP-seq) to map the genomic localization of hRSV NS1 or Mediator in A549 cells infected with hRSV or exogenously expressing hRSV-NS1. We identified 1756 NS1 binding sites, of which 82% (1442) of these are located within 10 KB of at least one gene. 42% (739 / 1756) of NS1 binding sites coincide with Mediator binding sites identified in the same cells, and 73% (1277 / 1756) overlap a known enhancer element (Enhancer Atlas). Moreover, NS1 binding sites often coincide with transcription factor binding sites in A549 cells (ENCODE). Comparison of RNA-seq from hRSV infected cells showed that nearly one-fourth of NS1 binding sites are located near genes significantly up- or down-regulated by hRSV infection. Moreover, these genes are enriched for IFN response genes. Finally, expression of NS1 reduced reporter gene expression when regulated by NS1-bound genetic elements. Together, our findings suggest that NS1 partially suppresses the expression of IFN immune response genes via directly interfering with transcriptional mechanisms, thereby contributing to immune evasion by hRSV.

Cutaneous T-cell lymphomas (CTCL) are cancers of mature skin-homing T lymphocytes, but the molecular drivers remain poorly understood. We performed whole genome, exome, epigenome, bulk and single cell (sc) transcriptome analyses of serial peripheral blood, skin, and lymph node biopsies from 20 CTCL patients. Initial studies defined markers of therapy resistance and disease progression in pathways that mediate cell migration, cytokine signaling, and epigenetic regulation. Several were novel to CTCL, including BIRC5 (anti-apoptotic); RRM2 (cell cycle); CXCR4 and LAIR2 (migration). On average 7413 cells from 16 serial samples (>100,000 cells total) passed quality thresholds for scRNA- + TCR-seq. We used Seurat, dittoSeq, and SingleR for quality control, dimensionality reduction (TSNE/UMAP), clustering, marker detection, cell type inference, and visualizations, and confirmed the therapy-resistant expression patterns we previously identified, including LAIR2. TCR clonotypes confirmed patient-specific malignant cell populations and defined clonal changes in serial samples. Pseudotime analysis of serial samples with Slingshot identified significant changes in expression related to changes in therapy, particularly in chemokine, TNF, and NFKB signaling. We identified mutations in STAT3&5 in 3/10 and in epigenetic modifiers (KMT2C/D and DNMT3A) in 6/10 patients. Loss of DNMT3A perturbs T cell differentiation and causes leukemia/lymphoma in mouse models. DNMT3A deficiency and additional epigenetic alterations may be previously unrecognized “drivers” of CTCL. These studies define mechanisms of therapy resistance, map clonal evolution, and reveal novel mechanisms of pathogenesis and progression in CTCL.

Non-Hodgkin Lymphoma (NHL) is the most common blood cancer in the U.S. Our analysis of gene regulation in >100 NHL and normal B cell samples revealed a distinct profile of long non-coding RNA (lncRNA) expression. Few lncRNAs have been characterized, and there are no guidelines for functional prediction or categorization. Therefore, we developed a novel computational approach: Correlative Recurrent Expression of Predicted Elements (CREPE), which integrates -omics data to create a correlative regression model to predict lncRNA function. CREPE analysis revealed several NHL-associated lncRNAs that may regulate the expression of signaling proteins downstream of B-cell receptor activation. We highlight a multi-exon lncRNA that is highly expressed in NHL and has a top-ranked CREPE transcriptional regulatory score: AC074289.1. Consistent with a role in regulating transcription, qRT-PCR of subcellular fractions demonstrate nuclear localization and chromatin association (3.5–5X : cytoplasm). Expression of AC074289.1 is cell-type specific with 5–10X higher levels in B lymphoid compared to T or epithelial cell lines. AC074289.1 expression positively correlates with a neighboring gene, PELI1, which encodes Pellino 1, an E3 ubiquitin ligase that modulates NF-κb signaling in immune cells. NF-κb signaling also plays a significant role in NHL pathogenesis. Notably, high PELI1 expression is associated with more aggressive NHL. In summary, our global -omics study of normal and NHL samples identified new gene regulatory connections between lncRNAs and potential NHL oncogenes, including AC074289.1 and PELI1. This novel approach will accelerate our understanding of lncRNA function and may uncover new therapeutic targets in NHL.

Long non-coding RNAs (lncRNAs) act as transcriptional regulators, scaffolds, and signaling modulators in development, immune response, and oncogenesis. Our -omics study of >100 human Non-Hodgkin Lymphoma (NHL) and normal B cell samples revealed altered expression of lncRNAs in NHL. LncRNAs have not been characterized in NHL or B cells, and there are few guidelines for functional prediction. To address this gap, we developed a novel computational approach: Correlative Recurrent Expression of Predicted Elements (CREPE). This method calculates and tracks the following for each lncRNA: expression, cell-type specificity, subcellular localization, differential expression (eg, tumor/normal), correlation with neighboring gene transcripts (RNAseq); local enhancer and 3D genome interaction landscapes (ChIPseq, Hi-C, ChIA-PET). From this, CREPE calculates a rankable score to predict the likelihood of potential functions, including 1) transcriptional regulation via lncRNA transcript, 2) enhancer-associated regulation, and 3) scaffold/modulator cytoplasmic function. The likelihood score formula incorporates an if-then logic, correlative regression analysis, and statistical significance. Finally, CREPE enables heuristic ranking of lncRNAs by pathway analysis. CREPE identified lncRNAs that may regulate NHL oncogenes and modulate B-cell receptor signaling. CREPE analysis of data from The Cancer Genome Atlas and a CRISPRi lncRNA growth screen validate its ability to identify lncRNAs with oncogenic or growth-promoting potential in diverse studies. In summary, we developed a novel approach that fills an unmet need for predictive modeling of lncRNA function and identified lncRNAs likely to promote lymphomagenesis.

Epigenetic alterations deregulate gene expression and are a significant contributor to cancer pathogenesis. Unlike gene mutations, epigenetic changes are reversible, and this distinction has been exploited therapeutically for Cutaneous T-cell Lymphomas (CTCL). Histone deacetylase inhibitors (HDACi) are FDA-approved; however, two-thirds of patients do not respond to HDACi. Therefore, we are addressing this gap by 1) establishing new techniques to identify HDACi response elements from all stages of CTCL and 2) developing targeted epigenetic modifiers (TEM) as a novel precision therapy. Our key innovations in this study are the generation of targeted epigenetic modifiers (TEM) as potential therapeutics and the development of a technique called Ultra-Low Input Chromatin Immunoprecipitation (ULI-ChIP) that enables epigenome-wide profiling of histone modifications in 10,000–30,000 cells. We are utilizing this method to identify recurrently altered regulatory elements in HDACi-resistant CTCL patients, which, in conjunction with RNA-seq data, allow us to determine epigenomic and transcriptomic states that predispose patients for resistance to these treatments. We have identified several regulatory elements upstream of CXCR4, a chemokine receptor with known roles in cell survival and metastasis in B cell lymphomas and T cell leukemias, that are dysregulated in HDACi resistant patients. Similar loci were also found near key transcription factor (FOXP1) and anti-apoptotic genes (BCL2). The most promising HDACi response elements will be targeted in CTCL cells using locus-specific epigenetic modifiers to validate and evaluate the potential of each locus to be further developed for TEM protein therapeutics.

Long non-coding RNAs (lncRNA) act as transcriptional regulators, scaffolds, and signaling modulators, but their role in normal or malignant lymphocytes is unknown. We identified lncRNAs with altered expression in >100 human Non-Hodgkin Lymphoma (NHL) and normal B cell samples. One of these lncRNA genes is upstream of PLCG2 and parallels its expression, but the transcript is localized in the cytoplasm, making transcriptional regulation of PLCG2 unlikely. As expected, knock-out (KO), knock-down (KD), or over-expression of lncRNA-PLCG2 did not affect PLCG2 levels. PLCG2 is a B-cell specific phospholipase C enzyme that stimulates Ca2+ signaling after BCR activation. We assessed Ca2+ signaling via IgM stimulation of the BCR in multiple lncRNA-PLCG2 KO/KD NHL cell lines and observed defects in Ca2+ efflux from the ER and influx through Orai channels. To identify lncRNA-PLCG2 interacting proteins, we performed RNA pull-down experiments using sense (S) and anti-sense (AS, control) lncRNA-PLCG2 as bait incubated with B cell lysates. Mass spectrometry of eluted proteins showed several proteins significantly enriched in S versus AS, including PLD1, DICER1 and DHX9; we confirmed these by Western blot. We next performed RNA-binding protein Immunoprecipitation (RIP) using antibodies for PLD1, DHX9 and DICER1, which showed 2–10-fold enrichment of lncRNA-PLCG2 compared to IgG isotype control. Ongoing studies are mapping lncRNA-PLCG2-protein interactions and evaluating the impact of lncRNA-PLCG2 on PLD1 activity. Taken together, these results suggest that lncRNA-PLCG2 modulates BCR-mediated Ca2+ signaling via interaction with BCR downstream signaling proteins, a previously unrecognized mechanism that may promote lymphomagenesis.

BACKGROUND—Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear. METHODS—We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis. RESULTS—AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcriptionfactor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumorsuppressor genes (16%), DNA-methylation–related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories. CONCLUSIONS—We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.) The molecular pathogenesis of acute myeloid leukemia (AML) has been studied with the use of cytogenetic analysis for more than three decades. Recurrent chromosomal structural variations are well established as diagnostic and prognostic markers, suggesting that acquired genetic abnormalities (i.e., somatic mutations) have an essential role in pathogenesis. 1,2 However, nearly 50% of AML samples have a normal karyotype, and many of these genomes lack structural abnormalities, even when assessed with high-density comparative genomic hybridization or single-nucleotide polymorphism (SNP) arrays 3-5 (see Glossary). Targeted sequencing has identified recurrent mutations in FLT3, NPM1, KIT, CEBPA, and TET2. 6-8 Massively parallel sequencing enabled the discovery of recurrent mutations in DNMT3A 9,10 and IDH1. 11 Recent studies have shown that many patients with

Reprogramming gene expression via epigenetic perturbation of transcriptional control elements has emerged as a major mechanism of oncogenesis. Non-Hodgkin Lymphoma (NHL) is the most frequently diagnosed hematopoietic tumor. While distinct NHL gene expression patterns were identified a decade ago, underlying changes to the control elements driving these alterations remain poorly understood. Comparative integration of epigenome and transcriptome data from NHL cells and their normal counterpart B cells reveal the oncogenic regulatory circuitry that links differentially regulated control elements to target genes. Although many of these circuits are engaged in GC B cells or during B cell activation, a portion is commandeered from other cell lineages to promote transformation. Independent sets of transcription factors, whose expression are also deregulated in NHL, target augmented or attenuated circuit components. Importantly, these components are enriched for sequence variants that disrupt transcription factor binding and expression of circuit-linked genes. The pathogenic regulatory circuitry of NHL reveals genetic and epigenetic etiologies for B-cell transformation, providing unprecedented opportunity for therapeutic intervention.

Transgenic mice we generated with ubiquitous expression of the NKG2D ligand RAE1ε, and therefore low surface NKG2D expression, developed natural, spontaneous B cell lymphoma at a younger age than wild-type mice. Transplantation of lymphomas from RAE1ε transgenic mice into lymphodeficient mice resulted in expression of NKG2D on immune cells transferred along with the tumor and rejection of the lymphoma cells, suggesting NKG2D is important in controlling lymphoma outgrowth in wild-type mice. In the absence of overt tumor masses, older mice developed lymphoid aggregates in non-lymphoid organs and these aggregates occurred at a younger age in RAE1ε transgenic and NKG2D-deficient mice. The increased incidence of both these lymphoid aggregates and tumor masses in RAE1ε transgenic mice suggests these lymphoid aggregates are slow growing indolent lymphomas. Although NKG2D ligands were undetectable on wild-type tumors, ligands were present on splenic B cells in a similar proportion of 10 month-old wild-type and NKG2D-deficient mice as those with lymphoid aggregates present. These results suggest that 1) induction of NKG2D ligands is an early marker of B cell transformation, 2) NKG2D-NKG2D ligand interaction suppresses B cell lymphoma growth, and 3) loss of NKG2D receptor-ligand interactions leads to the transformation of indolent B cell lymphomas into aggressive tumors in this model. We are now exploring whether these results are relevant to human follicular lymphoma progression.