Reversal of malignant ADAR1 splice isoform switching with Rebecsinib.

Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses. However, inflammatory-microenvironment-induced ADAR1p110 to p150 splice isoform switching drives cancer stem cell (CSC) generation and therapeutic resistance in 20 malignancies. Previously, predicting and preventing ADAR1p150-mediated malignant RNA editing represented a significant challenge. Thus, we developed lentiviral ADAR1 and splicing reporters for non-invasive detection of splicing-mediated ADAR1 adenosine-to-inosine (A-to-I) RNA editing activation; a quantitative ADAR1p150 intracellular flow cytometric assay; a selective small-molecule inhibitor of splicing-mediated ADAR1 activation, Rebecsinib, which inhibits leukemia stem cell (LSC) self-renewal and prolongs humanized LSC mouse model survival at doses that spare normal hematopoietic stem and progenitor cells (HSPCs); and pre-IND studies showing favorable Rebecsinib toxicokinetic and pharmacodynamic (TK/PD) properties. Together, these results lay the foundation for developing Rebecsinib as a clinical ADAR1p150 antagonist aimed at obviating malignant microenvironment-driven LSC generation.

Inflammation-driven deaminase deregulation fuels human pre-leukemia stem cell evolution.

Inflammation-dependent base deaminases promote therapeutic resistance in many malignancies. However, their roles in human pre-leukemia stem cell (pre-LSC) evolution to acute myeloid leukemia stem cells (LSCs) had not been elucidated. Comparative whole-genome and whole-transcriptome sequencing analyses of FACS-purified pre-LSCs from myeloproliferative neoplasm (MPN) patients reveal APOBEC3C upregulation, an increased C-to-T mutational burden, and hematopoietic stem and progenitor cell (HSPC) proliferation during progression, which can be recapitulated by lentiviral APOBEC3C overexpression. In pre-LSCs, inflammatory splice isoform overexpression coincides with APOBEC3C upregulation and ADAR1p150-induced A-to-I RNA hyper-editing. Pre-LSC evolution to LSCs is marked by STAT3 editing, STAT3ß isoform switching, elevated phospho-STAT3, and increased ADAR1p150 expression, which can be prevented by JAK2/STAT3 inhibition with ruxolitinib or fedratinib or lentiviral ADAR1 shRNA knockdown. Conversely, lentiviral ADAR1p150 expression enhances pre-LSC replating and STAT3 splice isoform switching. Thus, pre-LSC evolution to LSCs is fueled by primate-specific APOBEC3C-induced pre-LSC proliferation and ADAR1-mediated splicing deregulation.

Trends and Perspectives in Immunosensors for Determination of Currently-Used Pesticides: The Case of Glyphosate, Organophosphates, and Neonicotinoids.

Pesticides, due to their intensive use and their peculiar chemical features, can persist in the environment and enter the trophic chain, thus representing an environmental risk for the ecosystems and human health. Although there are several robust and reliable standard analytical techniques for their monitoring, the high frequency of contamination caused by pesticides requires methods for massive monitoring campaigns that are capable of rapidly detecting these compounds in many samples of different origin. Immunosensors represent a potential tool for simple, rapid, and sensitive monitoring of pesticides. Antibodies coupled to electrochemical or optical transducers have resulted in effective detection devices. In this review, the new trends in immunosensor development and the application of immunosensors for the detection of pesticides of environmental concern-such as glyphosate, organophosphates, and neonicotinoids-are described.

Hyper-Editing of Cell-Cycle Regulatory and Tumor Suppressor RNA Promotes Malignant Progenitor Propagation.

Adenosine deaminase associated with RNA1 (ADAR1) deregulation contributes to therapeutic resistance in many malignancies. Here we show that ADAR1-induced hyper-editing in normal human hematopoietic progenitors impairs miR-26a maturation, which represses CDKN1A expression indirectly via EZH2, thereby accelerating cell-cycle transit. However, in blast crisis chronic myeloid leukemia progenitors, loss of EZH2 expression and increased CDKN1A oppose cell-cycle transit. Moreover, A-to-I editing of both the MDM2 regulatory microRNA and its binding site within the 3' UTR region stabilizes MDM2 transcripts, thereby enhancing blast crisis progenitor propagation. These data reveal a dual mechanism governing malignant transformation of progenitors that is predicated on hyper-editing of cell-cycle-regulatory miRNAs and the 3' UTR binding site of tumor suppressor miRNAs.

Thioredoxin-1 actively maintains the pseudokinase MLKL in a reduced state to suppress disulfide bond-dependent MLKL polymer formation and necroptosis.

Necroptosis is an immunogenic cell death program that is associated with a host of human diseases, including inflammation, infections, and cancer. Receptor-interacting protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like protein (MLKL) are required for necroptosis activation. Specifically, RIPK3-dependent MLKL phosphorylation promotes the assembly of disulfide bond-dependent MLKL polymers that drive the execution of necroptosis. However, how MLKL disulfide bond formation is regulated is not clear. In this study we discovered that the MLKL-modifying compound necrosulfonamide cross-links cysteine 86 of human MLKL to cysteine 32 of the thiol oxidoreductase thioredoxin-1 (Trx1). Recombinant Trx1 preferentially binds to monomeric MLKL and blocks MLKL disulfide bond formation and polymerization in vitro Inhibition of MLKL polymer formation requires the reducing activity of Trx1. Importantly, shRNA-mediated knockdown of Trx1 promotes MLKL polymerization and sensitizes cells to necroptosis. Furthermore, pharmacological inhibition of Trx1 with compound PX-12 induces necroptosis in multiple cancer cell lines. Altogether, these findings demonstrate that Trx1 is a critical regulator of necroptosis that suppresses cell death by maintaining MLKL in a reduced inactive state. Our results further suggest new directions for targeted cancer therapy in which thioredoxin inhibitors like PX-12 could potentially be used to specifically target cancers expressing high levels of MLKL or MLKL short isoforms.

MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α-induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis.

Proteomics studies of the interactome of RNA polymerase II C-terminal repeated domain.

BACKGROUND: Eukaryotic RNA polymerase II contains a C-terminal repeated domain (CTD) consisting of 52 consensus heptad repeats of Y1S2P3T4S5P6S7 that mediate interactions with many cellular proteins to regulate transcription elongation, RNA processing and chromatin structure. A number of CTD-binding proteins have been identified and the crystal structures of several protein-CTD complexes have demonstrated considerable conformational flexibility of the heptad repeats in those interactions. Furthermore, phosphorylation of the CTD at tyrosine, serine and threonine residues can regulate the CTD-protein interactions. Although the interactions of CTD with specific proteins have been elucidated at the atomic level, the capacity and specificity of the CTD-interactome in mammalian cells is not yet determined. RESULTS: A proteomic study was conducted to examine the mammalian CTD-interactome. We utilized six synthetic peptides each consisting of four consensus CTD-repeats with different combinations of serine and tyrosine phosphorylation as affinity-probes to pull-down nuclear proteins from HeLa cells. The pull-down fractions were then analyzed by MUDPIT mass spectrometry, which identified 100 proteins with the majority from the phospho-CTD pull-downs. Proteins pulled-down by serine-phosphorylated CTD-peptides included those containing the previously defined CTD-interacting domain (CID). Using SILAC mass spectrometry, we showed that the in vivo interaction of RNA polymerase II with the mammalian CID-containing RPRD1B is disrupted by CID mutation. We also showed that the CID from four mammalian proteins interacted with pS2-phosphorylated but not pY1pS2-doubly phosphorylated CTD-peptides. However, we also found proteins that were preferentially pulled-down by pY1pS2- or pY1pS5-doubly phosphorylated CTD-peptides. We prepared an antibody against tyrosine phosphorylated CTD and showed that ionizing radiation (IR) induced a transient increase in CTD tyrosine phosphorylation by immunoblotting. Combining SILAC and IMAC purification of phospho-peptides, we found that IR regulated the phosphorylation at four CTD tyrosine sites in different ways. CONCLUSION: Upon phosphorylation, the 52 repeats of the CTD have the capacity to generate a large number of binding sites for cellular proteins. This study confirms previous findings that serine phosphorylation stimulates whereas tyrosine phosphorylation inhibits the protein-binding activity of the CTD. However, tyrosine phosphorylation of the CTD can also stimulate other CTD-protein interactions. The CTD-peptide affinity pull-down method described here can be adopted to survey the mammalian CTD-interactome in various cell types and under different biological conditions.

Primary B cell lymphoma of the rectum in a patient coinfected with HIV-1 and HTLV-I

Este artículo describe el caso clínico de un enfermo con SIDA coinfectado por HTLV-1 que desarrolló un linfoma B del recto, variedad sarcoma inmunoblástico con diferenciación plasmacitoide. Las células malignas mostraron arreglo clonal de los genes de las CP (Jh) y CLk. La infección por el VEB fue demostrada serológicamente y por hibridación de un monitor específico con el ADN genómico de las células cancerosas. No se detectaron secuencias de HTLV-1 en el seno del tumor. Una remisión clínica completa, pero temporal, se obtuvo con siete ciclos de VACO-B. El enfermo abandonó el tratamiento y la sobrevida se desconoce.