Loss of synergistic transcriptional feedback loops drives diverse B-cell cancers.

BACKGROUND: The most common B-cell cancers, chronic lymphocytic leukemia/lymphoma (CLL), follicular and diffuse large B-cell (FL, DLBCL) lymphomas, have distinct clinical courses, yet overlapping "cell-of-origin". Dynamic changes to the epigenome are essential regulators of B-cell differentiation. Therefore, we reasoned that these distinct cancers may be driven by shared mechanisms of disruption in transcriptional circuitry. METHODS: We compared purified malignant B-cells from 52 patients with normal B-cell subsets (germinal center centrocytes and centroblasts, naïve and memory B-cells) from 36 donor tonsils using >325 high-resolution molecular profiling assays for histone modifications, open chromatin (ChIP-, FAIRE-seq), transcriptome (RNA-seq), transcription factor (TF) binding, and genome copy number (microarrays). FINDINGS: From the resulting data, we identified gains in active chromatin in enhancers/super-enhancers that likely promote unchecked B-cell receptor signaling, including one we validated near the immunoglobulin superfamily receptors FCMR and PIGR. More striking and pervasive was the profound loss of key B-cell identity TFs, tumor suppressors and their super-enhancers, including EBF1, OCT2(POU2F2), and RUNX3. Using a novel approach to identify transcriptional feedback, we showed that these core transcriptional circuitries are self-regulating. Their selective gain and loss form a complex, iterative, and interactive process that likely curbs B-cell maturation and spurs proliferation. INTERPRETATION: Our study is the first to map the transcriptional circuitry of the most common blood cancers. We demonstrate that a critical subset of B-cell TFs and their cognate enhancers form self-regulatory transcriptional feedback loops whose disruption is a shared mechanism underlying these diverse subtypes of B-cell lymphoma. FUNDING: National Institute of Health, Siteman Cancer Center, Barnes-Jewish Hospital Foundation, Doris Duke Foundation.

BCALM (AC099524.1) Is a Human B Lymphocyte-Specific Long Noncoding RNA That Modulates B Cell Receptor-Mediated Calcium Signaling.

Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell-specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell-specific phospholipase C γ 2 (PLCG2), a B cell-specific enzyme that stimulates intracellular Ca2+ signaling in response to BCR activation. AC099524.1 (B cell-associated lncRNA modulator of BCR-mediated Ca+ signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca+ flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that downmodulates BCR-mediated Ca+ signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca+ signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR-dependent cancers.

PLAIDOH: a novel method for functional prediction of long non-coding RNAs identifies cancer-specific LncRNA activities.

BACKGROUND: Long non-coding RNAs (lncRNAs) exhibit remarkable cell-type specificity and disease association. LncRNA's functional versatility includes epigenetic modification, nuclear domain organization, transcriptional control, regulation of RNA splicing and translation, and modulation of protein activity. However, most lncRNAs remain uncharacterized due to a shortage of predictive tools available to guide functional experiments. RESULTS: To address this gap for lymphoma-associated lncRNAs identified in our studies, we developed a new computational method, Predicting LncRNA Activity through Integrative Data-driven 'Omics and Heuristics (PLAIDOH), which has several unique features not found in other methods. PLAIDOH integrates transcriptome, subcellular localization, enhancer landscape, genome architecture, chromatin interaction, and RNA-binding (eCLIP) data and generates statistically defined output scores. PLAIDOH's approach identifies and ranks functional connections between individual lncRNA, coding gene, and protein pairs using enhancer, transcript cis-regulatory, and RNA-binding protein interactome scores that predict the relative likelihood of these different lncRNA functions. When applied to 'omics datasets that we collected from lymphoma patients, or to publicly available cancer (TCGA) or ENCODE datasets, PLAIDOH identified and prioritized well-known lncRNA-target gene regulatory pairs (e.g., HOTAIR and HOX genes, PVT1 and MYC), validated hits in multiple lncRNA-targeted CRISPR screens, and lncRNA-protein binding partners (e.g., NEAT1 and NONO). Importantly, PLAIDOH also identified novel putative functional interactions, including one lymphoma-associated lncRNA based on analysis of data from our human lymphoma study. We validated PLAIDOH's predictions for this lncRNA using knock-down and knock-out experiments in lymphoma cell models. CONCLUSIONS: Our study demonstrates that we have developed a new method for the prediction and ranking of functional connections between individual lncRNA, coding gene, and protein pairs, which were validated by genetic experiments and comparison to published CRISPR screens. PLAIDOH expedites validation and follow-on mechanistic studies of lncRNAs in any biological system. It is available at https://github.com/sarahpyfrom/PLAIDOH .

cis-Regulatory Circuits Regulating NEK6 Kinase Overexpression in Transformed B Cells Are Super-Enhancer Independent.

Alterations in distal regulatory elements that control gene expression underlie many diseases, including cancer. Epigenomic analyses of normal and diseased cells have produced correlative predictions for connections between dysregulated enhancers and target genes involved in pathogenesis. However, with few exceptions, these predicted cis-regulatory circuits remain untested. Here, we dissect cis-regulatory circuits that lead to overexpression of NEK6, a mitosis-associated kinase, in human B cell lymphoma. We find that only a minor subset of predicted enhancers is required for NEK6 expression. Indeed, an annotated super-enhancer is dispensable for NEK6 overexpression and for maintaining the architecture of a B cell-specific regulatory hub. A CTCF cluster serves as a chromatin and architectural boundary to block communication of the NEK6 regulatory hub with neighboring genes. Our findings emphasize that validation of predicted cis-regulatory circuits and super-enhancers is needed to prioritize transcriptional control elements as therapeutic targets.

Distinct Gene Regulatory Pathways for Human Innate versus Adaptive Lymphoid Cells.

Innate lymphoid cells (ILCs) serve as sentinels in mucosal tissues, sensing release of soluble inflammatory mediators, rapidly communicating danger via cytokine secretion, and functioning as guardians of tissue homeostasis. Although ILCs have been extensively studied in model organisms, little is known about these "first responders" in humans, especially their lineage and functional kinships to cytokine-secreting T helper (Th) cell counterparts. Here, we report gene regulatory circuitries for four human ILC-Th counterparts derived from mucosal environments, revealing that each ILC subset diverges as a distinct lineage from Th and circulating natural killer cells but shares circuitry devoted to functional polarization with their Th counterparts. Super-enhancers demarcate cohorts of cell-identity genes in each lineage, uncovering new modes of regulation for signature cytokines, new molecules that likely impart important functions to ILCs, and potential mechanisms for autoimmune disease SNP associations within ILC-Th subsets.

Enhancer sequence variants and transcription-factor deregulation synergize to construct pathogenic regulatory circuits in B-cell lymphoma.

Most B-cell lymphomas arise in the germinal center (GC), where humoral immune responses evolve from potentially oncogenic cycles of mutation, proliferation, and clonal selection. Although lymphoma gene expression diverges significantly from GC B cells, underlying mechanisms that alter the activities of corresponding regulatory elements (REs) remain elusive. Here we define the complete pathogenic circuitry of human follicular lymphoma (FL), which activates or decommissions REs from normal GC B cells and commandeers enhancers from other lineages. Moreover, independent sets of transcription factors, whose expression was deregulated in FL, targeted commandeered versus decommissioned REs. Our approach revealed two distinct subtypes of low-grade FL, whose pathogenic circuitries resembled GC B or activated B cells. FL-altered enhancers also were enriched for sequence variants, including somatic mutations, which disrupt transcription-factor binding and expression of circuit-linked genes. Thus, the pathogenic regulatory circuitry of FL reveals distinct genetic and epigenetic etiologies for GC B-cell transformation.

The genetic architecture of degenerin/epithelial sodium channels in Drosophila.

Degenerin/epithelial sodium channels (DEG/ENaC) represent a large family of animal-specific membrane proteins. Although the physiological functions of most family members are not known, some have been shown to act as nonvoltage gated, amiloride-sensitive sodium channels. The DEG/ENaC family is exceptionally large in genomes of Drosophila species relative to vertebrates and other insects. To elucidate the evolutionary history of the DEG/ENaC family in Drosophila, we took advantage of the genomic and genetic information available for 12 Drosophila species that represent all the major species groups in the Drosophila clade. We have identified 31 family members (termed pickpocket genes) in Drosophila melanogaster, which can be divided into six subfamilies, which are represented in all 12 species. Structure prediction analyses suggested that some subunits evolved unique structural features in the large extracellular domain, possibly supporting mechanosensory functions. This finding is further supported by experimental data that show that both ppk1 and ppk26 are expressed in multidendritic neurons, which can sense mechanical nociceptive stimuli in larvae. We also identified representative genes from five of the six DEG/ENaC subfamilies in a mosquito genome, suggesting that the core DEG/ENaC subfamilies were already present early in the dipteran radiation. Spatial and temporal analyses of expression patterns of the various pickpocket genes indicated that paralogous genes often show very different expression patterns, possibly indicating that gene duplication events have led to new physiological or cellular functions rather than redundancy. In summary, our analyses support a rapid early diversification of the DEG/ENaC family in Diptera followed by physiological and/or cellular specialization. Some members of the family may have diversified to support the physiological functions of a yet unknown class of ligands.