LAIR1 prevents excess inflammatory tissue damage in S. aureus skin infection and Cutaneous T-cell Lymphoma.

Patients with cutaneous T cell lymphoma (CTCL) experience high morbidity and mortality due to S. aureus skin infections and sepsis, but the causative immune defect is unclear. We previously identified high levels of LAIR2, a decoy protein for the inhibitory receptor LAIR1, in advanced CTCL. Mice do not have a LAIR2 homolog, so we used Lair1 knock-out (KO) mice to model LAIR2 overexpression. In a model of subcutaneous S. aureus skin infection, Lair1 KO mice had significantly larger abscesses and areas of dermonecrosis compared to WT. Lair1 KO exhibited a pattern of increased inflammatory responses in infection and sterile immune stimulation, including increased production of proinflammatory cytokines and myeloid chemokines, neutrophil ROS, and collagen/ECM remodeling pathways. Notably, Lair1 KO infected skin had a similar bacterial burden and neutrophils and monocytes had equivalent S. aureus phagocytosis compared to WT. These findings support a model in which lack of LAIR1 signaling causes an excessive inflammatory response that does not improve infection control. CTCL skin lesions harbored similar patterns of increased expression in cytokine and collagen/ECM remodeling pathways, suggesting that high levels of LAIR2 in CTCL recapitulates Lair1 KO, causing inflammatory tissue damage and compromising host defense against S. aureus infection.

Induced CD8α identifies human NK cells with enhanced proliferative fitness and modulates NK cell activation.

The surface receptor CD8α is present on 20-80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8α expression on donor NK cells was associated with a lack of therapeutic responses for leukemia patients in prior studies, thus we hypothesized that CD8α may impact critical NK cell functions. Here, we discovered that CD8α- NK cells had improved control of leukemia in xenograft models, compared to CD8α+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, CD8α expression was induced on approximately 30% of previously CD8α- NK cells following IL-15 stimulation. These 'induced' CD8α+ ('iCD8α+') NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity, compared to those that sustained existing CD8α expression ('sustained CD8α+) or those that remained CD8α- ('persistent CD8α-'). These iCD8α+ cells originated from an IL-15Rß high NK cell population, with CD8α expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8α status identifies human NK cell capacity for IL-15-induced proliferation and metabolism in a time-dependent fashion and exhibits a suppressive effect on NK cell activating receptors.

LPS and type I and II interferons have opposing effects on epigenetic regulation of LAIR1 expression in mouse and human macrophages.

Inhibitory immune receptors are important for maintaining immune homeostasis. We identified epigenetic alterations in 2 members of this group, LAIR1 and LAIR2, in lymphoma patients with inflammatory tissue damage and susceptibility to infection. We predicted that the expression of LAIR genes is controlled by immune mediators acting on transcriptional regulatory elements. Using flow cytometry, quantitative reverse-transcription polymerase chain reaction, and RNA sequencing, we measured LAIR1 and LAIR2 in human and murine immune cell subsets at baseline and posttreatment with immune mediators, including type I and II interferons, tumor necrosis factor α, and lipopolysaccharide (LPS). We identified candidate regulatory elements using epigenome profiling and measured their regulatory activity using luciferase reporters. LAIR1 expression substantially increases during monocyte differentiation to macrophages in both species. In contrast, murine and human macrophages exhibited opposite changes in LAIR1 in response to immune stimuli: human LAIR1 increased with LPS while mouse LAIR1 increased with interferon γ. LAIR genes had distinct patterns of enhancer activity with variable responses to immune stimuli. To identify relevant transcription factors (TFs), we developed integrative bioinformatic techniques applied to TF chromatin immunoprecipitation sequencing, RNA sequencing, and luciferase activity, revealing distinct sets of TFs for each LAIR gene. Most strikingly, LAIR1 TFs include nuclear factor kappa B factors RELA and RELB, while Lair1 and LAIR2 instead include STAT3 and/or STAT5. Regulation by nuclear factor kappa B factors may therefore explain the LPS-induced increase in LAIR1 expression, in contrast to Lair1 decrease. Our findings reveal new insights into transcriptional mechanisms that control distinct expression patterns of LAIR genes in response to inflammatory stimuli in human and murine myeloid and lymphoid cells.

Genomic landscape of TP53-mutated myeloid malignancies.

TP53-mutated myeloid malignancies are associated with complex cytogenetics and extensive structural variants, which complicates detailed genomic analysis by conventional clinical techniques. We performed whole-genome sequencing (WGS) of 42 acute myeloid leukemia (AML)/myelodysplastic syndromes (MDS) cases with paired normal tissue to better characterize the genomic landscape of TP53-mutated AML/MDS. WGS accurately determines TP53 allele status, a key prognostic factor, resulting in the reclassification of 12% of cases from monoallelic to multihit. Although aneuploidy and chromothripsis are shared with most TP53-mutated cancers, the specific chromosome abnormalities are distinct to each cancer type, suggesting a dependence on the tissue of origin. ETV6 expression is reduced in nearly all cases of TP53-mutated AML/MDS, either through gene deletion or presumed epigenetic silencing. Within the AML cohort, mutations of NF1 are highly enriched, with deletions of 1 copy of NF1 present in 45% of cases and biallelic mutations in 17%. Telomere content is increased in TP53-mutated AMLs compared with other AML subtypes, and abnormal telomeric sequences were detected in the interstitial regions of chromosomes. These data highlight the unique features of TP53-mutated myeloid malignancies, including the high frequency of chromothripsis and structural variation, the frequent involvement of unique genes (including NF1 and ETV6) as cooperating events, and evidence for altered telomere maintenance.

Genomic landscape of TP53 -mutated myeloid malignancies.

TP53 -mutated myeloid malignancies are most frequently associated with complex cytogenetics. The presence of complex and extensive structural variants complicates detailed genomic analysis by conventional clinical techniques. We performed whole genome sequencing of 42 AML/MDS cases with paired normal tissue to characterize the genomic landscape of TP53 -mutated myeloid malignancies. The vast majority of cases had multi-hit involvement at the TP53 genetic locus (94%), as well as aneuploidy and chromothripsis. Chromosomal patterns of aneuploidy differed significantly from TP53 -mutated cancers arising in other tissues. Recurrent structural variants affected regions that include ETV6 on chr12p, RUNX1 on chr21, and NF1 on chr17q. Most notably for ETV6 , transcript expression was low in cases of TP53 -mutated myeloid malignancies both with and without structural rearrangements involving chromosome 12p. Telomeric content is increased in TP53 -mutated AML/MDS compared other AML subtypes, and telomeric content was detected adjacent to interstitial regions of chromosomes. The genomic landscape of TP53 -mutated myeloid malignancies reveals recurrent structural variants affecting key hematopoietic transcription factors and telomeric repeats that are generally not detected by panel sequencing or conventional cytogenetic analyses. Key Points: WGS comprehensively determines TP53 mutation status, resulting in the reclassification of 12% of cases from mono-allelic to multi-hit Chromothripsis is more frequent than previously appreciated, with a preference for specific chromosomes ETV6 is deleted in 45% of cases, with evidence for epigenetic suppression in non-deleted cases NF1 is mutated in 48% of cases, with multi-hit mutations in 17% of these cases TP53 -mutated AML/MDS is associated with altered telomere content compared with other AMLs.

Proteomic and phosphoproteomic landscapes of acute myeloid leukemia.

We have developed a deep-scale proteome and phosphoproteome database from 44 representative acute myeloid leukemia (AML) patients from the LAML TCGA dataset and 6 healthy bone marrow-derived controls. After confirming data quality, we orthogonally validated several previously undescribed features of AML revealed by the proteomic data. We identified examples of posttranscriptionally regulated proteins both globally (ie, in all AML samples) and also in patients with recurrent AML driver mutations. For example, samples with IDH1/2 mutations displayed elevated levels of the 2-oxoglutarate-dependent histone demethylases KDM4A/B/C, despite no changes in messenger RNA levels for these genes; we confirmed this finding in vitro. In samples with NPMc mutations, we identified several nuclear importins with posttranscriptionally increased protein abundance and showed that they interact with NPMc but not wild-type NPM1. We identified 2 cell surface proteins (CD180 and MRC1/CD206) expressed on AML blasts of many patients (but not healthy CD34+ stem/progenitor cells) that could represent novel targets for immunologic therapies and confirmed these targets via flow cytometry. Finally, we detected nearly 30 000 phosphosites in these samples; globally, AML samples were associated with the abnormal phosphorylation of specific residues in PTPN11, STAT3, AKT1, and PRKCD. FLT3-TKD samples were associated with increased phosphorylation of activating tyrosines on the cytoplasmic Src-family tyrosine kinases FGR and HCK and related signaling proteins. PML-RARA-initiated AML samples displayed a unique phosphorylation signature, and TP53-mutant samples showed abundant phosphorylation of serine-183 on TP53 itself. This publicly available database will serve as a foundation for further investigations of protein dysregulation in AML pathogenesis.

Convergent Clonal Evolution of Signaling Gene Mutations Is a Hallmark of Myelodysplastic Syndrome Progression.

Progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML) is associated with the acquisition and expansion of subclones. Our understanding of subclone evolution during progression, including the frequency and preferred order of gene mutation acquisition, remains incomplete. Sequencing of 43 paired MDS and secondary AML samples identified at least one signaling gene mutation in 44% of MDS and 60% of secondary AML samples, often below the level of standard sequencing detection. In addition, 19% of MDS and 47% of secondary AML patients harbored more than one signaling gene mutation, almost always in separate, coexisting subclones. Signaling gene mutations demonstrated diverse patterns of clonal evolution during disease progression, including acquisition, expansion, persistence, and loss of mutations, with multiple patterns often coexisting in the same patient. Multivariate analysis revealed that MDS patients who had a signaling gene mutation had a higher risk of AML progression, potentially providing a biomarker for progression. SIGNIFICANCE: Subclone expansion is a hallmark of progression from MDS to secondary AML. Subclonal signaling gene mutations are common at MDS (often at low levels), show complex and convergent patterns of clonal evolution, and are associated with future progression to secondary AML. See related article by Guess et al., p. 316 (33). See related commentary by Romine and van Galen, p. 270. This article is highlighted in the In This Issue feature, p. 265.

Genetic and Transcriptional Contributions to Relapse in Normal Karyotype Acute Myeloid Leukemia.

To better understand clonal and transcriptional adaptations after relapse in patients with acute myeloid leukemia (AML), we collected presentation and relapse samples from six normal karyotype AML cases. We performed enhanced whole-genome sequencing to characterize clonal evolution, and deep-coverage single-cell RNA sequencing on the same samples, which yielded 142,642 high-quality cells for analysis. Identifying expressed mutations in individual cells enabled us to discriminate between normal and AML cells, to identify coordinated changes in the genome and transcriptome, and to identify subclone-specific cell states. We quantified the coevolution of genetic and transcriptional heterogeneity during AML progression, and found that transcriptional changes were significantly correlated with genetic changes. However, transcriptional adaptation sometimes occurred independently, suggesting that clonal evolution does not represent all relevant biological changes. In three cases, we identified cells at diagnosis that likely seeded the relapse. Finally, these data revealed a conserved relapse-enriched leukemic cell state bearing markers of stemness, quiescence, and adhesion. SIGNIFICANCE: These data enabled us to identify a relapse-enriched leukemic cell state with distinct transcriptional properties. Detailed case-by-case analyses elucidated the complex ways in which the AML genome, transcriptome, and immune microenvironment interact to evade chemotherapy. These analyses provide a blueprint for evaluating these factors in larger cohorts.This article is highlighted in the In This Issue feature, p. 1.

Focal disruption of DNA methylation dynamics at enhancers in IDH-mutant AML cells.

Recurrent mutations in IDH1 or IDH2 in acute myeloid leukemia (AML) are associated with increased DNA methylation, but the genome-wide patterns of this hypermethylation phenotype have not been comprehensively studied in AML samples. We analyzed whole-genome bisulfite sequencing data from 15 primary AML samples with IDH1 or IDH2 mutations, which identified ~4000 focal regions that were uniquely hypermethylated in IDHmut samples vs. normal CD34+ cells and other AMLs. These regions had modest hypermethylation in AMLs with biallelic TET2 mutations, and levels of 5-hydroxymethylation that were diminished in IDH and TET-mutant samples, indicating that this hypermethylation results from inhibition of TET-mediated demethylation. Focal hypermethylation in IDHmut AMLs occurred at regions with low methylation in CD34+ cells, implying that DNA methylation and demethylation are active at these loci. AML samples containing IDH and DNMT3AR882 mutations were significantly less hypermethylated, suggesting that IDHmut-associated hypermethylation is mediated by DNMT3A. IDHmut-specific hypermethylation was highly enriched for enhancers that form direct interactions with genes involved in normal hematopoiesis and AML, including MYC and ETV6. These results suggest that focal hypermethylation in IDH-mutant AML occurs by altering the balance between DNA methylation and demethylation, and that disruption of these pathways at enhancers may contribute to AML pathogenesis.

Immunosuppression and outcomes in adult patients with de novo acute myeloid leukemia with normal karyotypes.

Acute myeloid leukemia (AML) patients rarely have long first remissions (LFRs; >5 y) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, standard remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA-sequencing, and functional immunologic studies, we characterized 28 normal karyotype (NK)-AML patients with >5 y first remissions after chemotherapy (LFRs) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 y (standard first remissions [SFRs]). Our combined analyses indicated that genetic-risk profiling at presentation (as defined by European LeukemiaNet [ELN] 2017 criteria) was not sufficient to explain the outcomes of many SFR cases. Single-cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T cells expressed an exhaustion signature and were resistant to activation by T cell receptor stimulation in the presence of autologous AML cells. T cell activation could be restored by removing the AML cells or blocking the inhibitory major histocompatibility complex class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ T cell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

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.

Determining subpopulation methylation profiles from bisulfite sequencing data of heterogeneous samples using DXM.

Epigenetic changes, such as aberrant DNA methylation, contribute to cancer clonal expansion and disease progression. However, identifying subpopulation-level changes in a heterogeneous sample remains challenging. Thus, we have developed a computational approach, DXM, to deconvolve the methylation profiles of major allelic subpopulations from the bisulfite sequencing data of a heterogeneous sample. DXM does not require prior knowledge of the number of subpopulations or types of cells to expect. We benchmark DXM's performance and demonstrate improvement over existing methods. We further experimentally validate DXM predicted allelic subpopulation-methylation profiles in four Diffuse Large B-Cell Lymphomas (DLBCLs). Lastly, as proof-of-concept, we apply DXM to a cohort of 31 DLBCLs and relate allelic subpopulation methylation profiles to relapse. We thus demonstrate that DXM can robustly find allelic subpopulation methylation profiles that may contribute to disease progression using bisulfite sequencing data of any heterogeneous sample.

Epigenomic regulation of human T-cell leukemia virus by chromatin-insulator CTCF.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes an aggressive T-cell malignancy and a variety of inflammatory conditions. The integrated provirus includes a single binding site for the epigenomic insulator, CCCTC-binding protein (CTCF), but its function remains unclear. In the current study, a mutant virus was examined that eliminates the CTCF-binding site. The mutation did not disrupt the kinetics and levels of virus gene expression, or establishment of or reactivation from latency. However, the mutation disrupted the epigenetic barrier function, resulting in enhanced DNA CpG methylation downstream of the CTCF binding site on both strands of the integrated provirus and H3K4Me3, H3K36Me3, and H3K27Me3 chromatin modifications both up- and downstream of the site. A majority of clonal cell lines infected with wild type HTLV-1 exhibited increased plus strand gene expression with CTCF knockdown, while expression in mutant HTLV-1 clonal lines was unaffected. These findings indicate that CTCF binding regulates HTLV-1 gene expression, DNA and histone methylation in an integration site dependent fashion.

Genome Sequencing as an Alternative to Cytogenetic Analysis in Myeloid Cancers.

BACKGROUND: Genomic analysis is essential for risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Whole-genome sequencing is a potential replacement for conventional cytogenetic and sequencing approaches, but its accuracy, feasibility, and clinical utility have not been demonstrated. METHODS: We used a streamlined whole-genome sequencing approach to obtain genomic profiles for 263 patients with myeloid cancers, including 235 patients who had undergone successful cytogenetic analysis. We adapted sample preparation, sequencing, and analysis to detect mutations for risk stratification using existing European Leukemia Network (ELN) guidelines and to minimize turnaround time. We analyzed the performance of whole-genome sequencing by comparing our results with findings from cytogenetic analysis and targeted sequencing. RESULTS: Whole-genome sequencing detected all 40 recurrent translocations and 91 copy-number alterations that had been identified by cytogenetic analysis. In addition, we identified new clinically reportable genomic events in 40 of 235 patients (17.0%). Prospective sequencing of samples obtained from 117 consecutive patients was performed in a median of 5 days and provided new genetic information in 29 patients (24.8%), which changed the risk category for 19 patients (16.2%). Standard AML risk groups, as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes. Whole-genome sequencing was also used to stratify patients who had inconclusive results by cytogenetic analysis into risk groups in which clinical outcomes were measurably different. CONCLUSIONS: In our study, we found that whole-genome sequencing provided rapid and accurate genomic profiling in patients with AML or MDS. Such sequencing also provided a greater diagnostic yield than conventional cytogenetic analysis and more efficient risk stratification on the basis of standard risk categories. (Funded by the Siteman Cancer Research Fund and others.).

Validation and interpretation of IGH and TCR clonality testing by Ion Torrent S5 NGS for diagnosis and disease monitoring in B and T cell cancers.

Cancers of B and T lymphocytes are the most common hematologic malignancies in the US. Molecular assays for assessing clonal rearrangements of the immunoglobulin receptor (IGH) and T-cell receptor (TCR), commonly referred to as B- and T-cell clonality, as well as determination of IGH somatic mutation status, enables improved diagnostic accuracy and disease monitoring. Here we describe validation of NGS LymphoTrack (IGH, TCRG, Invivoscribe, Inc) with Ion Torrent S5 sequencing, which employs a different sequencing chemistry and has not been previously reported for NGS clonality to our knowledge. We also demonstrate the concordance of clonality by LymphoTrack with S5 sequencing with other molecular methodologies and with clinical measurements of disease. We show that LymphoTrack with S5 sequencing identifies previously detected IGH and TCRG clonal sequences across matched biopsy specimens and clinical timepoints, enabling more precise and sensitive disease monitoring for B- and T-cell cancers compared to PCR fragment or capillary sequencing. In sum, our study demonstrates that the LymphoTrack assays with Ion Torrent S5 sequencing 1) can be used successfully for IGH and TCR clonality with reproducible results; 2) generates and quantifies clonal sequences to enable highly precise comparison of samples; 3) are substantially more sensitive than PCR fragment and return clonality results in specimens that failed PCR fragment assays; and 4) the TCRG assays are highly concordant with clinical and histopathologic diagnoses. Taken together, the LymphoTrack with Ion S5 NGS clonality assays offer a sensitive and precise method for diagnostic testing and disease monitoring in B- and T-cell cancers.

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.

RAG-Mediated DNA Breaks Attenuate PU.1 Activity in Early B Cells through Activation of a SPIC-BCLAF1 Complex.

Early B cell development is regulated by stage-specific transcription factors. PU.1, an ETS-family transcription factor, is essential for coordination of early B cell maturation and immunoglobulin gene (Ig) rearrangement. Here we show that RAG DNA double-strand breaks (DSBs) generated during Ig light chain gene (Igl) rearrangement in pre-B cells induce global changes in PU.1 chromatin binding. RAG DSBs activate a SPIC/BCLAF1 transcription factor complex that displaces PU.1 throughout the genome and regulates broad transcriptional changes. SPIC recruits BCLAF1 to gene-regulatory elements that control expression of key B cell developmental genes. The SPIC/BCLAF1 complex suppresses expression of the SYK tyrosine kinase and enforces the transition from large to small pre-B cells. These studies reveal that RAG DSBs direct genome-wide changes in ETS transcription factor activity to promote early B cell development.

Novel cell adhesion/migration pathways are predictive markers of HDAC inhibitor resistance in cutaneous T cell lymphoma.

BACKGROUND: Treatment for Cutaneous T Cell Lymphoma (CTCL) is generally not curative. Therefore, selecting therapy that is effective and tolerable is critical to clinical decision-making. Histone deacetylase inhibitors (HDACi), epigenetic modifier drugs, are commonly used but effective in only ~30% of patients. There are no predictive markers of HDACi response and the CTCL histone acetylation landscape remains unmapped. We sought to identify pre-treatment molecular markers of resistance in CTCL that progressed on HDACi therapy. METHODS: Purified T cells from 39 pre/post-treatment peripheral blood samples and skin biopsies from 20 patients were subjected to RNA-seq and ChIP-seq for histone acetylation marks (H3K14/9 ac, H3K27ac). We correlated significant differences in histone acetylation with gene expression in HDACi-resistant/sensitive CTCL. We extended these findings in additional CTCL patient cohorts (RNA-seq, microarray) and using ELISA in matched CTCL patient plasma. FINDINGS: Resistant CTCL exhibited high levels of histone acetylation, which correlated with increased expression of 338 genes (FDR < 0·05), including some novel to CTCL: BIRC5 (anti-apoptotic); RRM2 (cell cycle); TXNDC5, GSTM1 (redox); and CXCR4, LAIR2 (cell adhesion/migration). Several of these, including LAIR2, were elevated pre-treatment in HDACi-resistant CTCL. In CTCL patient plasma (n = 6), LAIR2 protein was also elevated (p < 0·01) compared to controls. INTERPRETATION: This study is the first to connect genome-wide differences in chromatin acetylation and gene expression to HDACi-resistance in primary CTCL. Our results identify novel markers with high pre-treatment expression, such as LAIR2, as potential prognostic and/or predictors of HDACi-resistance in CTCL. FUNDING: NIH:CA156690, CA188286; NCATS: WU-ICTS UL1 TR000448; Siteman Cancer Center: CA091842.

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 .