Long-read transcriptome sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations.

Mutations in splicing factor 3B subunit 1 (SF3B1) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS). These mutations have different effects on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options. We applied long-read transcriptome sequencing (LRTS) to 44 MDS and CLL patients, as well as two pairs of isogenic cell lines with and without SF3B1 mutations, and found >60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3' splice sites. Our data highlighted a constrained window at canonical 3' splice sites in which dynamic splice site switches occurred in SF3B1-mutated patients. Using transcriptome-wide RNA binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3' splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1K700E Our work presents the hitherto most complete LRTS study of the SF3B1 mutation in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosis most likely results from the different cell types expanded during carcinogenesis rather than different mechanisms of action of the mutated SF3B1 These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases.

Oncogenic role and target properties of the lysine-specific demethylase KDM1A in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown in Eµ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and proapoptotic pathways. Genetic KDM1A depletion also affected milieu components (T, stromal, and monocytic cells), resulting in significant reductions in their capacity to support CLL-cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA sequencing) and H3K4me3 marks (chromatin immunoprecipitation sequencing) in Eµ-TCL1A vs iKdm1aKD;Eµ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL which alters histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti-B-cell leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL via tumor-cell intrinsic mechanisms and its impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.

Altered DNA Methylation Profiles in SF3B1 Mutated CLL Patients.

Mutations in splicing factor genes have a severe impact on the survival of cancer patients. Splicing factor 3b subunit 1 (SF3B1) is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL); patients carrying these mutations have a poor prognosis. Since the splicing machinery and the epigenome are closely interconnected, we investigated whether these alterations may affect the epigenomes of CLL patients. While an overall hypomethylation during CLL carcinogenesis has been observed, the interplay between the epigenetic stage of the originating B cells and SF3B1 mutations, and the subsequent effect of the mutations on methylation alterations in CLL, have not been investigated. We profiled the genome-wide DNA methylation patterns of 27 CLL patients with and without SF3B1 mutations and identified local decreases in methylation levels in SF3B1mut CLL patients at 67 genomic regions, mostly in proximity to telomeric regions. These differentially methylated regions (DMRs) were enriched in gene bodies of cancer-related signaling genes, e.g., NOTCH1, HTRA3, and BCL9L. In our study, SF3B1 mutations exclusively emerged in two out of three epigenetic stages of the originating B cells. However, not all the DMRs could be associated with the methylation programming of B cells during development, suggesting that mutations in SF3B1 cause additional epigenetic aberrations during carcinogenesis.

Hedgehog dysregulation contributes to tissue-specific inflammaging of resident macrophages.

Age-associated low-grade sterile inflammation, commonly referred to as inflammaging, is a recognized hallmark of aging, which contributes to many age-related diseases. While tissue-resident macrophages are innate immune cells that secrete many types of inflammatory cytokines in response to various stimuli, it is not clear whether they have a role in driving inflammaging. Here we characterized the transcriptional changes associated with physiological aging in mouse resident macrophage populations across different tissues and sexes. Although the age-related transcriptomic signatures of resident macrophages were strikingly tissue-specific, the differentially expressed genes were collectively enriched for those with important innate immune functions such as antigen presentation, cytokine production, and cell adhesion. The brain-resident microglia had the most wide-ranging age-related alterations, with compromised expression of tissue-specific genes and relatively exaggerated responses to endotoxin stimulation. Despite the tissue-specific patterns of aging transcriptomes, components of the hedgehog (Hh) signaling pathway were decreased in aged macrophages across multiple tissues. In vivo suppression of Hh signaling in young animals increased the expression of pro-inflammatory cytokines, while in vitro activation of Hh signaling in old macrophages, in turn, suppressed the expression of these inflammatory cytokines. This suggests that hedgehog signaling could be a potential intervention axis for mitigating age-associated inflammation and related diseases. Overall, our data represent a resourceful catalog of tissue-specific and sex-specific transcriptomic changes in resident macrophages of peritoneum, liver, and brain, during physiological aging.

Integrative analysis suggests cell type-specific decoding of NF-κB dynamics.

The complex signaling dynamics of transcription factors can encode both qualitative and quantitative information about the extracellular environment, which increases the information transfer capacity and potentially supports accurate cellular decision-making. An important question is how these signaling dynamics patterns are translated into functionally appropriate gene regulation programs. To address this question for transcription factors of the nuclear factor κB (NF-κB) family, we profiled the single-cell dynamics of two major NF-κB subunits, RelA and c-Rel, induced by a panel of pathogen-derived stimuli in immune and nonimmune cellular contexts. Diverse NF-κB-activating ligands produced different patterns of RelA and c-Rel signaling dynamic features, such as variations in duration or time-integrated activity. Analysis of nascent transcripts delineated putative direct targets of NF-κB as compared to genes controlled by other transcriptional and posttranscriptional mechanisms and showed that the transcription of more than half of the induced genes was tightly linked to specific dynamic features of NF-κB signaling. Fibroblast and macrophage cell lines shared a cluster of such "NF-κB dynamics-decoding" genes, as well as cell type-specific decoding genes. Dissecting the subunit specificity of dynamics-decoding genes suggested that target genes were most often linked to both RelA and c-Rel or to RelA alone. Thus, our analysis reveals the cell type-specific interpretation of pathogenic information through the signaling dynamics of NF-κB.

lncRNA expression predicts mRNA abundance.

Aim: Long noncoding RNAs (lncRNAs) have been reported to influence multiple gene regulatory processes. Technological advances in RNA-seq platforms allow detection of low-abundance RNA species such as lncRNAs. This study examined the relationship between expression of lncRNAs and their putative partner mRNAs. Methods: We analyzed total RNA-seq data from mouse macrophages under various inflammatory and intervention conditions. Results: The macrophage expression of lncRNAs is strongly regulated by an inflammatory stimulus. Moreover, the expression of a majority of lncRNAs was correlated or anti-correlated with the partner mRNA(s), across the different treatment conditions. This relationship was maintained even in cells from a distinct genotype. Conclusion: These results suggest a previously unappreciated tight coupling of lncRNA and mRNA expression during macrophage responses to various microenvironmental perturbations.

Differences in miRNA differential expression in whole blood between horses with sarcoid regression and progression.

BACKGROUND: Currently no methods are available to predict the clinical outcome of individual horses with equine sarcoid (ES) disease. OBJECTIVE: To investigate if whole blood microRNA (miRNA) profiles can predict the long-term development of ES tumors. ANIMALS: Five horses with regression and 5 with progression of ES lesions monitored over 5-7 years and 5 control horses free of ES for at least 5 years. METHODS: For this cohort study, RNA extracted from whole blood samples from the regression, progression, and control groups was used for high throughput sequencing. Known and novel miRNAs were identified using miRDeep2 and differential expression analysis was carried out by the DESeq2 algorithm. Target gene and pathway prediction as well as enrichment and network analyses were conducted using TarBase, mirPath, and metaCore from GeneGo. RESULTS: Fourteen miRNAs were differentially expressed between regression and progression groups after accounting for the control condition: 4 miRNAs (28.6%) were upregulated and 10 miRNAs (71.4%) were downregulated with >2-fold change. Seven of the 10 downregulated miRNAs are encoded in an miRNA cluster on equine chromosome 24, homologous to the well-known 14q32 cluster in humans. Their target genes show enrichment for pathways involved in viral carcinogenesis. CONCLUSIONS AND CLINICAL IMPORTANCE: Whole blood miRNA expression profiles are associated with long-term ES growth in horses and warrant further validation as prognostic biomarkers in a larger study cohort. Deregulation of miRNAs on equine chromosome 24 might represent a trigger for ES development.

MicroRNA fingerprints in serum and whole blood of sarcoid-affected horses as potential non-invasive diagnostic biomarkers.

Serum and whole blood microRNA (miRNA) fingerprints have been proposed as a new class of non-invasive human cancer biomarkers. In this study, we compared equine sarcoid (ES) disease-specific serum and whole blood miRNA fingerprints and correlated them to miRNA expression in sarcoid tissue. After high throughput sequencing, miRNA differential expression analysis between six ES-affected and five control horses was carried out in serum and whole blood using a DESeq algorithm, accounting for the influence of hemolysis and the white blood cell count. Target gene, pathway prediction and enrichment analyses were conducted using TarBase, mirPath and GeneCodis. After exclusion of 4 hemolyzed out of a total of 11 serum samples, 9 miRNAs were found to be differentially expressed in serum of ES vs control horses. In whole blood, all 11 samples showed normal white blood cell counts and 19 miRNAs were found to be differentially expressed. A total of 2/9 serum and 7/19 whole blood differentially expressed miRNAs were also highly expressed at the tissue level and their predicted target genes were associated with cancer pathways. Serum and whole blood miRNA expression allowed discrimination between ES and control horses and merits further validation in a larger study cohort. The use of whole blood might be superior because it has higher miRNA content and is less influenced by pre-analytical variables compared to serum. Concurrent dysregulation of single miRNAs in tissue and blood suggests a possible biological function of circulating miRNAs.