TIRAP p.R81C is a novel lymphoma risk variant which enhances cell proliferation via NF-κB mediated signaling in B-cells.

Diffuse large B-cell lymphoma is the most common malignant lymphoma in adults. By gene-expression profiling, this lymphoma is divided in three cell-of-origin subtypes with distinct molecular and clinical features. Most lymphomas arise sporadically, yet familial clustering is known, suggesting a genetic contribution to disease risk. Familial lymphoma cases are a valuable tool to investigate risk genes. We studied a Swiss/Japanese family with 2 sisters affected by a primary mediastinal B-cell lymphoma and a non-germinal center diffuse large B-cell lymphoma not otherwise specified, respectively. The somatic landscape of both lymphomas was marked by alterations affecting multiple components of the JAK-STAT pathway. Consequently, this pathway was constitutively activated as evidenced by high pJAK2 as well as increased nuclear pSTAT3 and pSTAT6 in malignant cells. Potential lymphoma risk variants were identified by whole exome sequencing of the germline DNA derived from siblings and unaffected family members. This analysis revealed a pathogenic variant in TIRAP, an upstream regulator of NF-κB, in both affected siblings and their mother. We observed increased B-cell proliferation in family members harboring the TIRAP p.R81C variant. B-cell proliferation correlated with TIRAP and NF-κB target gene expression, suggesting enhanced NF-κB pathway activity in TIRAP p.R81C individuals. TIRAP knockdown reduced B-cell survival and NF-κB target gene expression, particularly in individuals with TIRAP p.R81C. Functional studies revealed significantly increased NF-κB activity and resistance to stress-induced cell-death by TIRAP p.R81C. The identification of an inherited TIRAP variant provides evidence for a novel link between genetic alterations affecting the NF-κB pathway and lymphomagenesis.

AKAP-Lbc mediates protection against doxorubicin-induced cardiomyocyte toxicity.

Doxorubicin (DOX) is a chemotherapic agent that is widely used to treat hematological and solid tumors. Despite its efficacy, DOX displays significant cardiac toxicity associated with cardiomyocytes death and heart failure. Cardiac toxicity is mainly associated with the ability of DOX to alter mitochondrial function. The current lack of treatments to efficiently prevent DOX cardiotoxicity underscores the need of new therapeutic approaches. Our current findings show that stimulation of cardiomyocytes with the α1-adrenergic receptor (AR) agonist phenylephrine (PE) significantly inhibits the apoptotic effect of DOX. Importantly, our results indicate that AKAP-Lbc is critical for transducing protective signals downstream of α1-ARs. In particular, we could show that suppression of AKAP-Lbc expression by infecting primary cultures of ventricular myocytes with lentiviruses encoding AKAP-Lbc specific short hairpin (sh) RNAs strongly impairs the ability of PE to reduce DOX-induced apoptosis. AKAP-Lbc-mediated cardiomyocyte protection requires the activation of anchored protein kinase D1 (PKD1)-dependent prosurvival pathways that promote the expression of the anti-apoptotic protein Bcl2 and inhibit the translocation of the pro-apoptotic protein Bax to mitochondria. In conclusion, AKAP-Lbc emerges as a coordinator of signals that protect cardiomyocytes against the toxic effects of DOX.

Emerging roles of A-kinase anchoring proteins in cardiovascular pathophysiology.

Heart and blood vessels ensure adequate perfusion of peripheral organs with blood and nutrients. Alteration of the homeostatic functions of the cardiovascular system can cause hypertension, atherosclerosis, and coronary artery disease leading to heart injury and failure. A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that are crucially involved in modulating the function of the cardiovascular system both under physiological and pathological conditions. AKAPs assemble multifunctional signaling complexes that ensure correct targeting of the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to precise subcellular compartments. This allows local regulation of specific effector proteins that control the function of vascular and cardiac cells. This review will focus on recent advances illustrating the role of AKAPs in cardiovascular pathophysiology. The accent will be mainly placed on the molecular events linked to the control of vascular integrity and blood pressure as well as on the cardiac remodeling process associated with heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination.

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism.

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion.

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic control.

A-Kinase Anchoring Protein 2 Promotes Protection against Myocardial Infarction.

Myocardial infarction (MI) is a leading cause of maladaptive cardiac remodeling and heart failure. In the damaged heart, loss of function is mainly due to cardiomyocyte death and remodeling of the cardiac tissue. The current study shows that A-kinase anchoring protein 2 (AKAP2) orchestrates cellular processes favoring cardioprotection in infarcted hearts. Induction of AKAP2 knockout (KO) in cardiomyocytes of adult mice increases infarct size and exacerbates cardiac dysfunction after MI, as visualized by increased left ventricular dilation and reduced fractional shortening and ejection fraction. In cardiomyocytes, AKAP2 forms a signaling complex with PKA and the steroid receptor co-activator 3 (Src3). Upon activation of cAMP signaling, the AKAP2/PKA/Src3 complex favors PKA-mediated phosphorylation and activation of estrogen receptor α (ERα). This results in the upregulation of ER-dependent genes involved in protection against apoptosis and angiogenesis, including Bcl2 and the vascular endothelial growth factor a (VEGFa). In line with these findings, cardiomyocyte-specific AKAP2 KO reduces Bcl2 and VEGFa expression, increases myocardial apoptosis and impairs the formation of new blood vessels in infarcted hearts. Collectively, our findings suggest that AKAP2 organizes a transcriptional complex that mediates pro-angiogenic and anti-apoptotic responses that protect infarcted hearts.

Metoclopramide treatment blocks CD93-signaling-mediated self-renewal of chronic myeloid leukemia stem cells.

Self-renewal is a key characteristic of leukemia stem cells (LSCs) responsible for the development and maintenance of leukemia. In this study, we identify CD93 as an important regulator of self-renewal and proliferation of murine and human LSCs, but not hematopoietic stem cells (HSCs). The intracellular domain of CD93 promotes gene transcription via the transcriptional regulator SCY1-like pseudokinase 1 independently of ligation of the extracellular domain. In a drug library screen, we identify the anti-emetic agent metoclopramide as an efficient blocker of CD93 signaling. Metoclopramide treatment reduces murine and human LSCs in vitro and prolongs survival of chronic myeloid leukemia (CML) mice through downregulation of pathways related to stemness and proliferation in LSCs. Overall, these results identify CD93 signaling as an LSC-specific regulator of self-renewal and proliferation and a targetable pathway to eliminate LSCs in CML.

Molecular modulation of autophagy: New venture to target resistant cancer stem cells.

Autophagy is a highly regulated catabolic process in which superfluous, damaged organelles and other cytoplasmic constituents are delivered to the lysosome for clearance and the generation of macromolecule substrates during basal or stressed conditions. Autophagy is a bimodal process with a context dependent role in the initiation and the development of cancers. For instance, autophagy provides an adaptive response to cancer stem cells to survive metabolic stresses, by influencing disease propagation via modulation of essential signaling pathways or by promoting resistance to chemotherapeutics. Autophagy has been implicated in a cross talk with apoptosis. Understanding the complex interactions provides an opportunity to improve cancer therapy and the clinical outcome for the cancer patients. In this review, we provide a comprehensive view on the current knowledge on autophagy and its role in cancer cells with a particular focus on cancer stem cell homeostasis.

Functional diversification of Dicer-like proteins and small RNAs required for genome sculpting.

In eukaryotes, small RNAs (sRNAs) have key roles in development, gene expression regulation, and genome integrity maintenance. In ciliates, such as Paramecium, sRNAs form the heart of an epigenetic system that has evolved from core eukaryotic gene silencing components to selectively target DNA for deletion. In Paramecium, somatic genome development from the germline genome accurately eliminates the bulk of typically gene-interrupting, noncoding DNA. We have discovered an sRNA class (internal eliminated sequence [IES] sRNAs [iesRNAs]), arising later during Paramecium development, which originates from and precisely delineates germline DNA (IESs) and complements the initial sRNAs ("scan" RNAs [scnRNAs]) in targeting DNA for elimination. We show that whole-genome duplications have facilitated successive differentiations of Paramecium Dicer-like proteins, leading to cooperation between Dcl2 and Dcl3 to produce scnRNAs and to the production of iesRNAs by Dcl5. These innovations highlight the ability of sRNA systems to acquire capabilities, including those in genome development and integrity.

Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium.

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.