Proteolytic activity of the mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) paracaspase is required for survival of the activated B cell subtype of diffuse large B cell lymphoma (ABC-DLBCL). We have identified distinct derivatives of medicinal active phenothiazines, namely mepazine, thioridazine, and promazine, as small molecule inhibitors of the MALT1 protease. These phenothiazines selectively inhibit cleavage activity of recombinant and cellular MALT1 by a noncompetitive mechanism. Consequently, the compounds inhibit anti-apoptotic NF-κB signaling and elicit toxic effects selectively on MALT1-dependent ABC-DLBCL cells in vitro and in vivo. Our data provide a conceptual proof for a clinical application of distinct phenothiazines in the treatment of ABC-DLBCL.

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) represents a very aggressive human lymphoma entity. Constitutive NF-κB activation caused by chronic active B-cell receptor (BCR) signaling is common feature of many ABC DLBCL cells; however, the pathways linking BCR signaling to the NF-κB prosurvival network are largely unknown. Here we report that constitutive activity of PI3K and the downstream kinase PDK1 are essential for the viability of two ABC DLBCL cell lines that carry mutations in the BCR proximal signaling adaptor CD79B. In these cells, PI3K inhibition reduces NF-κB activity and decreases the expression of NF-κB target genes. Furthermore, PI3K and PDK1 are required for maintaining MALT1 protease activity, which promotes survival of the affected ABC DLBCL cells. These results demonstrate a critical function of PI3K-PDK1 signaling upstream of MALT1 protease and NF-κB in distinct ABC DLBCL cells and provide a rationale for the pharmacologic use of PI3K inhibitors in DLBCL therapy.

Current challenges in capturing naive human pluripotent stem cells (hPSCs) suggest that the factors regulating human naive versus primed pluripotency remain incompletely defined. Here we demonstrate that the widely used Essential 8 minimal medium (E8) captures hPSCs at a naive-to-primed intermediate state of pluripotency expressing several naive-like developmental, bioenergetic, and epigenomic features despite providing primed-statesustaining growth factor conditions. Transcriptionally, E8 hPSCs are marked by activated lipid biosynthesis and suppressed MAPK/TGF-b gene expression, resulting in endogenous ERK inhibition. These features are dependent on lipidfree culture conditions and are lost upon lipid exposure, whereas short-term pharmacological ERK inhibition restores naive-to-primed intermediate traits even in the presence of lipids. Finally, we identify de novo lipogenesis as a common transcriptional signature of E8 hPSCs and the pre-implantation human epiblast in vivo. These findings implicate exogenous lipid availability in regulating human pluripotency and define E8 hPSCs as a stable, naive-to-primed intermediate (NPI) pluripotent state.

The discovery of constitutive nuclear factor-κB (NF-κB) activation in Hodgkin's lymphoma tumor cells almost two decades ago was one of the first reports that directly connected deregulated NF-κB signaling to human cancer. Subsequent studies demonstrated that enhanced NF-κB signaling is a common hallmark of many lymphoid malignancies, including Hodgkin lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large B-cell lymphoma and multiple myeloma. By inducing an anti-apoptotic and pro-proliferative gene program, NF-κB is involved in lymphoma survival and growth. Identification of somatic mutations that led to activation of oncogenes and inactivation of tumor suppressor genes in the pathway revealed that specific pathogenic mechanisms are responsible for constitutive NF-κB activation in different lymphoma entities. Thus, the identification of distinct oncogenic events is reflecting the diverse cellular origins of the different lymphomas. Further, elucidation of the mechanisms that drive NF-κB in lymphoma is of high clinical relevance as it will allow the design of target-directed precision therapy. Indeed, a number of drugs that impair constitutive NF-κB activation in lymphoid malignancies are currently in preclinical or clinical development.

Second site: In the crystal structure of human MALT1casp‐Ig3 (mucosa‐associated lymphoid tissue lymphoma translocation protein 1) in complex with the tricyclic phenothiazine derivative thioridazine (violet in the picture), the inhibitor is bound in a hydrophobic pocket far from the active site. This explains the action of phenothiazine derivatives as noncompetitive, reversible inhibitors.

Sam68 (Src-associated during mitosis, 68 kDa) is a prototypical member of the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. STAR proteins bind mRNA targets and modulate cellular processes such as cell cycle regulation and tissue development in response to extracellular signals. Sam68 has been shown to modulate alternative splicing of the pre-mRNAs of CD44 and Bcl-xL, which are linked to tumor progression and apoptosis. Sam68 and other STAR proteins recognize bipartite RNA sequences and are thought to function as homodimers. However, the structural and functional roles of the self-association are not known. Here, we present the solution structure of the Sam68 Qua1 homodimerization domain. Each monomer consists of two antiparallel ␣-helices connected by a short loop. The two subunits are arranged perpendicular to each other in an unusual four-helix topology. Mutational analysis of Sam68 in vitro and in a cell-based assay revealed that the Qua1 domain and residues within the dimerization interface are essential for alternative splicing of a CD44 minigene. Together, our results indicate that the Qua1 homodimerization domain is required for regulation of alternative splicing by Sam68. Sam682 (Src-associated during mitosis, 68 kDa) (1) belongs to the STAR (signal transducer and activator of RNA) family of RNAbinding proteins, which also includes Qk1 (quaking 1), SF1 (splicing factor 1), and Gld-1 (germline development defective-1) (2). STAR family proteins link signaling pathways and many aspects of RNA metabolism (splicing, localization, and translation). They are regulated by post-translational modifications such as phosphorylation, acetylation, and arginine methylation (2).Sam68 acts in post-transcriptional regulation of pre-mRNA splicing in response to extracellular signals (3). It is involved in a variety of pathways, including insulin and T-cell receptor signaling (4), and plays a key role in cell cycle regulation (5). Sam68 exhibits binding specificity for homopolymeric poly(U) RNA and has been shown to recognize UAAA or UUUA sequences with high affinity as determined by Systematic Evolution of Ligands by EXponential Enrichment (SELEX) and in vivo crosslinking (6, 7). Post-translational modifications can regulate Sam68 function by critically affecting the accessibility to RNA (8, 9). Tyrosine phosphorylation by Src kinase during mitosis enhances the interaction of Sam68 with Ras-GAP (10) but prevents its association with RNA. On the other hand, acetylation of lysine residues by histone acetyltransferases enhances RNA binding (11). Finally, overexpression of Sam68 has been linked to prostate cancer, cell proliferation, and survival (12).Sam68 has been identified as a key determinant in the alternative splicing of various important RNA targets, like CD44 (13) and Bcl-x L (14), which are linked to apoptosis and cancer. In particular, alternative splicing of CD44 impacts embryonic development and immune response (15-18). Up to 10 variant exon sequences can be included in the mature C...

BackgroundThe human genome contains multiple LTR elements including human endogenous retroviruses (HERVs) that together account for approximately 8–9% of the genomic DNA. At least 40 different HERV groups have been assigned to three major HERV classes on the basis of their homologies to exogenous retroviruses. Although most HERVs are silenced by a variety of genetic and epigenetic mechanisms, they may be reactivated by environmental stimuli such as exogenous viruses and thus may contribute to pathogenic conditions. The objective of this study was to perform an in-depth analysis of the influence of HIV-1 infection on HERV activity in different cell types.ResultsA retrovirus-specific microarray that covers major HERV groups from all three classes was used to analyze HERV transcription patterns in three persistently HIV-1 infected cell lines of different cellular origins and in their uninfected counterparts. All three persistently infected cell lines showed increased transcription of multiple class I and II HERV groups. Up-regulated transcription of five HERV taxa (HERV-E, HERV-T, HERV-K (HML-10) and two ERV9 subgroups) was confirmed by quantitative reverse transcriptase PCR analysis and could be reversed by knock-down of HIV-1 expression with HIV-1-specific siRNAs. Cells infected de novo by HIV-1 showed stronger transcriptional up-regulation of the HERV-K (HML-2) group than persistently infected cells of the same origin. Analysis of transcripts from individual members of this group revealed up-regulation of predominantly two proviral loci (ERVK-7 and ERVK-15) on chromosomes 1q22 and 7q34 in persistently infected KE37.1 cells, as well as in de novo HIV-1 infected LC5 cells, while only one single HML-2 locus (ERV-K6) on chromosome 7p22.1 was activated in persistently infected LC5 cells.ConclusionsOur results demonstrate that HIV-1 can alter HERV transcription patterns of infected cells and indicate a correlation between activation of HERV elements and the level of HIV-1 production. Moreover, our results suggest that the effects of HIV-1 on HERV activity may be far more extensive and complex than anticipated from initial studies with clinical material.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0156-6) contains supplementary material, which is available to authorized users.

The Rev protein is a key regulator of human immunodeficiency virus type 1 (HIV-1) gene expression. Rev is primarily known as an adaptor protein for nuclear export of HIV RNAs. However, Rev also contributes to numerous other processes by less well known mechanisms. Understanding the functional nature of Rev requires extensive knowledge of its cellular interaction partners. Here we demonstrate that Rev interacts with members of a large family of multifunctional host cell factors called hnRNPs. Rev employs amino acids 9 -14 for specific binding to the heterogeneous nuclear ribonucleoproteins (hnRNP) A1, Q, K, R, and U. In addition, Rev interacts with hnRNP E1 and E2 by a different mechanism. The set of hnRNPs recognized by the N terminus of Rev feature RGG boxes. Exemplary testing of hnRNP A1 revealed a critical role of arginine residues within the RGG box for interaction with Rev. Finally, we demonstrate that expression levels of hnRNP A1, Q, K, R, and U influence HIV-1 production by persistently infected astrocytes, linking these hnRNPs to HIV replication. The novel interaction of HIV-1 Rev with functionally diverse hnRNPs lends further support to the idea that Rev is a multifunctional protein and may be involved in coupling HIV replication to diverse cellular processes and promoting virus-host cell interactions. During human immunodeficiency virus (HIV)3 replication, the transcripts that encode viral structural proteins and the viral RNA genome contain introns and would normally be eliminated by the host cell. The production of these RNAs and their utilization is achieved by strictly controlled alternative splicing mechanisms and the regulatory activities of the HIV trans-activator protein Rev (reviewed in Refs. 1-3).Rev is an RNA-binding protein that specifically binds a recognition element (RRE) within intron-containing HIV RNAs. One of the best studied functions of Rev is the recruitment of cellular factors that mediate nuclear export of Rev-bound RNAs (2, 3). Rev has also been shown to influence splicing (4), stability (3,5,6), and translation (7-9) of the viral RNAs as well as their packaging (10, 11). These regulatory activities of the Rev protein render it a key player in the HIV replication cycle. Detailed and sophisticated studies of the Rev protein identified at least three functional domains in Rev (2): (i) an arginine richmotif that functions both as nuclear localization signal and RNA-binding sequence (AA 35-50), (ii) a bipartite multimerization domain (AA 12-29 and AA 52-60), and (iii) a nuclear export signal (AA 75-83). Host factors shown to bind to these functional domains include B23 and Importin ␤ for the nuclear localization signal and CRM1/Exportin-1 and eIF-5A for the nuclear export signal (2, 12). In addition, Rev has been shown to interact with several RNA helicases (13-15).However, the overall number of cellular interactor proteins identified for Rev is still surprisingly small, compared with, for example, Tat (16). The many activities of Rev (17) and evidence for host-cell regulation of ...