Targeting DNA Repair with Combined Inhibition of NHEJ and MMEJ Induces Synthetic Lethality in TP53-Mutant Cancers.

DNA repair pathway inhibitors are a new class of anticancer drugs that are advancing in clinical trials. Peposertib is an inhibitor of DNA-dependent protein kinase (DNA-PK), which is a key driver of nonhomologous end-joining (NHEJ). To identify regulators of response to peposertib, we performed a genome-wide CRISPR knockout screen and found that loss of POLQ (polymerase theta, POLθ) and other genes in the microhomology-mediated end-joining (MMEJ) pathway are key predictors of sensitivity to DNA-PK inhibition. Simultaneous disruption of two DNA repair pathways via combined treatment with peposertib plus a POLθ inhibitor novobiocin exhibited synergistic synthetic lethality resulting from accumulation of toxic levels of DNA double-strand break end resection. TP53-mutant tumor cells were resistant to peposertib but maintained elevated expression of POLQ and increased sensitivity to novobiocin. Consequently, the combination of peposertib plus novobiocin resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models. Thus, the combination of a targeted DNA-PK/NHEJ inhibitor with a targeted POLθ/MMEJ inhibitor may provide a rational treatment strategy for TP53-mutant solid tumors. SIGNIFICANCE: Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.

Histone arginine demethylase JMJD6 is linked to stress granule assembly through demethylation of the stress granule-nucleating protein G3BP1.

Stress granules (SG) are membrane-less organelles that are condensates of stalled translation initiation complexes and mRNAs. SG formation is a cytoprotective response to environmental stress and results from protein interactions involving regions of low amino acid complexity and poorly defined post-translational modifications of SG components. Many RNA-binding proteins are methylated, and we previously demonstrated that the potent SG-nucleating protein G3BP1 is methylated by protein arginine methyltransferase 1 and 5 (PRMT1 and PRMT5). G3BP1 methylation represses SG formation and is reversible. Here we functionally link JMJD6 (Jumonji C domain-containing protein 6) to G3BP1 demethylation. Our findings reveal that JMJD6 is a novel SG component that interacts with G3BP1 complexes, and its expression reduces G3BP1 monomethylation and asymmetric dimethylation at three Arg residues. Knockdown of JMJD6 repressed SG formation and G3BP1 demethylation, but SG formation and G3BP1 demethylation were rescued with catalytically active but not mutant JMJD6. These results suggest that JMJD6 functions directly or indirectly as an arginine demethylase of G3BP1 that promotes SG formation.

Casein Kinase 2 Is Linked to Stress Granule Dynamics through Phosphorylation of the Stress Granule Nucleating Protein G3BP1.

Stress granules (SGs) are large macromolecular aggregates that contain translation initiation complexes and mRNAs. Stress granule formation coincides with translational repression, and stress granules actively signal to mediate cell fate decisions by signaling to the translation apparatus to (i) maintain translational repression, (ii) mount various transcriptional responses, including innate immunity, and (iii) repress apoptosis. Previous work showed that G3BP1 is phosphorylated at serine 149, which regulates G3BP1 oligomerization, stress granule assembly, and RNase activity intrinsic to G3BP1. However, the kinase that phosphorylates G3BP1 was not identified, leaving a key step in stress granule regulation uncharacterized. Here, using chemical inhibition, genetic depletion, and overexpression experiments, we show that casein kinase 2 (CK2) promotes stress granule dynamics. These results link CK2 activity with SG disassembly. We also show that casein kinase 2 phosphorylates G3BP1 at serine 149 in vitro and in cells. These data support a role for casein kinase 2 in regulation of protein synthesis by downregulating stress granule formation through G3BP1.

Arginine Demethylation of G3BP1 Promotes Stress Granule Assembly.

Stress granules (SGs) are cytoplasmic condensates of stalled messenger ribonucleoprotein complexes (mRNPs) that form when eukaryotic cells encounter environmental stress. RNA-binding proteins are enriched for arginine methylation and facilitate SG assembly through interactions involving regions of low amino acid complexity. How methylation of specific RNA-binding proteins regulates RNA granule assembly has not been characterized. Here, we examined the potent SG-nucleating protein Ras-GAP SH3-binding protein 1 (G3BP1), and found that G3BP1 is differentially methylated on specific arginine residues by protein arginine methyltransferase (PRMT) 1 and PRMT5 in its RGG domain. Several genetic and biochemical interventions that increased methylation repressed SG assembly, whereas interventions that decreased methylation promoted SG assembly. Arsenite stress quickly and reversibly decreased asymmetric arginine methylation on G3BP1. These data indicate that arginine methylation in the RGG domain prevents large SG assembly and rapid demethylation is a novel signal that regulates SG formation.

Multiple Poliovirus Proteins Repress Cytoplasmic RNA Granules.

We have previously shown that poliovirus (PV) infection induces stress granule (SG) formation early in infection and then inhibits the formation of SG and disperses processing bodies (PBs) by the mid-phase of infection. Loss of SG was linked to cleavage of G3BP1 by viral 3C proteinase (3C(pro)), however dispersal of PBs was not strongly linked to cleavage of specific factors by viral proteinases, suggesting other viral proteins may play roles in inhibition of SG or PB formation. Here we have screened all viral proteins for roles in inducing or inhibiting the formation of RNA granules by creating fusions with mCherry and expressing them individually in cells. Expression of viral proteins separately revealed that the capsid region P1, 2A(pro), 3A, 3C(pro), the protease precursor 3CD and 3D polymerase all affect RNA granules to varying extents, whereas 2BC does not. 2A(pro), which cleaves eIF4GI, induced SGs as expected, and entered novel foci containing the SG nucleating protein G3BP1. Of the two forms of G3BP, only G3BP1 is cleaved by a virus proteinase, 3C(pro), whereas G3BP2 is not cleaved by 3C(pro) or 2A(pro). Surprisingly, 3CD, which contains proteinase activity, differentially repressed PBs but not SGs. Further, both 2A(pro) and 3C(pro) expression dispersed PBs, however molecular targets were different since PB dispersal due to 2A(pro) and heat shock protein (Hsp)90 inhibition but not 3C(pro), could be rescued by application of oxidative stress to cells. The data indicate that PV repression of SGs and PBs is multifactorial, though protease function is dominant.

Cytoplasmic RNA Granules and Viral Infection.

RNA granules are dynamic cellular structures essential for proper gene expression and homeostasis. The two principal types of cytoplasmic RNA granules are stress granules, which contain stalled translation initiation complexes, and processing bodies (P bodies), which concentrate factors involved in mRNA degradation. RNA granules are associated with gene silencing of transcripts; thus, viruses repress RNA granule functions to favor replication. This article discusses the breadth of viral interactions with cytoplasmic RNA granules, focusing on mechanisms that modulate the functions of RNA granules and that typically promote viral replication. Currently, mechanisms for virus manipulation of RNA granules can be loosely grouped into three nonexclusive categories: (a) cleavage of key RNA granule factors, (b) regulation of PKR activation, and (c) co-opting of RNA granule factors for new roles in viral replication. Viral modulation of RNA granules supports productive infection by inhibiting their gene-silencing functions and counteracting their role in linking stress sensing with innate immune activation.

High-performance ZnO thin-film transistors with location-controlled crystal grains fabricated by low-temperature hydrothermal method.

In this paper, high-performance bottom-gate (BG) thin-film transistors (TFTs) with zinc oxide (ZnO) artificially location-controlled lateral grain growth have been prepared via low-temperature hydrothermal method. For the proper design of source/drain structure of ZnO/Ti/Pt thin films, the grains can be laterally grown from the under-cut ZnO beneath the Ti/Pt layer. Consequently, the single one vertical grain boundary perpendicular to the current flow will be produced in the channel region as the grown grains from the source/drain both sides are impinged. As compared with the conventional sputtered ZnO BG-TFTs, the proposed location-controlled hydrothermal ZnO BG-TFTs (W/L = 250 microm/10 microm) demonstrated the higher field-effect mobility of 6.09 cm2/V x s, lower threshold voltage of 3.67 V, higher on/off current ratio above 10(6), and superior current drivability, reflecting the high-quality ZnO thin films with less grain boundary effect in the channel region.

MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis.

MicroRNA-122 (miR-122), which accounts for 70% of the liver's total miRNAs, plays a pivotal role in the liver. However, its intrinsic physiological roles remain largely undetermined. We demonstrated that mice lacking the gene encoding miR-122a (Mir122a) are viable but develop temporally controlled steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). These mice exhibited a striking disparity in HCC incidence based on sex, with a male-to-female ratio of 3.9:1, which recapitulates the disease incidence in humans. Impaired expression of microsomal triglyceride transfer protein (MTTP) contributed to steatosis, which was reversed by in vivo restoration of Mttp expression. We found that hepatic fibrosis onset can be partially attributed to the action of a miR-122a target, the Klf6 transcript. In addition, Mir122a(-/-) livers exhibited disruptions in a range of pathways, many of which closely resemble the disruptions found in human HCC. Importantly, the reexpression of miR-122a reduced disease manifestation and tumor incidence in Mir122a(-/-) mice. This study demonstrates that mice with a targeted deletion of the Mir122a gene possess several key phenotypes of human liver diseases, which provides a rationale for the development of a unique therapy for the treatment of chronic liver disease and HCC.

Effect of oxygen annealing on the ultraviolet photoresponse of P-NiO-nanoflower/n-ZnO-nanowire heterostructures.

A transparent ultraviolet (UV) sensor using nanoheterojunctions (NHJs) composed of p-type NiO nanoflowers (NFs) and n-type ZnO nanowires (NWs) was prepared through a sequential low-temperature hydrothermal-growth process. The devices that were annealed in an oxygen (O2) ambient exhibited better rectification behavior (I forward/I reverse = 427), a lower forward threshold voltage (V(th) = 0.98 V), a lower leakage current (1.68 x 10(-5) A/cm2), and superior sensitivity (I uv/I dark = 57.8; I visible/I dark = 1.25) to UV light (lambda = 325 nm) than the unannealed devices. The remarkably improved device performances and optoelectronic characteristics of the annealed p-NiO-NF/n-ZnO-NW NHJs can be associated with their fewer structural defects, fewer interfacial defects, and better crystallinity. A stable and repeatable operation of dynamic photoresponse was also observed in the annealed devices. The excellent sensitivity and repeatable photoresponse to UV light of the hydrothermally grown p-NiO-NF/n-ZnO-NW NHJs annealed in a suitable O2 ambient indicate that they can be applied to nano-integrated optoelectronic devices.

Field-emission characteristics of Al-doped ZnO nanostructures hydrothermally synthesized at low temperature.

The aluminum-doped ZnO (AZO) nanostructures with different Al concentrations were synthesized on AZO/glass substrate via a simple hydrothermal growth method at a temperature as low as 85 degrees C. The morphologies, crystallinity, optical emission properties, and chemical bonding states of AZO nanostructures show evident dependence on the aluminum dosage. The morphologies of AZO nanostructures were changed from vertically aligned nanowires (NWs), and NWs coexisted with nanosheets (NSs), to complete NSs in respect of the Al-dosages of 0-3 at.%, 5 at.%, and 7 at.%, correspondingly. The undoped ZnO and lightly Al-doped AZO (< or = 3 at.%) NWs are single-crystalline wurtzite structure. In contrast, heavily Al-doped AZO sample is polycrystalline. The AZO nanostructure with 3 at.% Al-dosages reveals the optimal crystallinity and less structural defects, reflecting the longest carrier lifetime and highest conductivity. Consequently, the field-emission characteristics of such an AZO emitter can exhibit the higher current density, larger field-enhancement factor (beta) of 3131, lower turn-on field of 2.17 V/microm, and lower threshold field of 3.43 V/microm.

Identifying transcriptional start sites of human microRNAs based on high-throughput sequencing data.

MicroRNAs (miRNAs) are critical small non-coding RNAs that regulate gene expression by hybridizing to the 3'-untranslated regions (3'-UTR) of target mRNAs, subsequently controlling diverse biological processes at post-transcriptional level. How miRNA genes are regulated receives considerable attention because it directly affects miRNA-mediated gene regulatory networks. Although numerous prediction models were developed for identifying miRNA promoters or transcriptional start sites (TSSs), most of them lack experimental validation and are inadequate to elucidate relationships between miRNA genes and transcription factors (TFs). Here, we integrate three experimental datasets, including cap analysis of gene expression (CAGE) tags, TSS Seq libraries and H3K4me3 chromatin signature derived from high-throughput sequencing analysis of gene initiation, to provide direct evidence of miRNA TSSs, thus establishing an experimental-based resource of human miRNA TSSs, named miRStart. Moreover, a machine-learning-based Support Vector Machine (SVM) model is developed to systematically identify representative TSSs for each miRNA gene. Finally, to demonstrate the effectiveness of the proposed resource, an important human intergenic miRNA, hsa-miR-122, is selected to experimentally validate putative TSS owing to its high expression in a normal liver. In conclusion, this work successfully identified 847 human miRNA TSSs (292 of them are clustered to 70 TSSs of miRNA clusters) based on the utilization of high-throughput sequencing data from TSS-relevant experiments, and establish a valuable resource for biologists in advanced research in miRNA-mediated regulatory networks.

MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma.

UNLABELLED: MicroRNAs (miRNAs), which are inhibitors of gene expression, participate in diverse biological functions and in carcinogenesis. In this study, we show that liver-specific microRNA-122 (miR-122) is significantly down-regulated in liver cancers with intrahepatic metastasis and negatively regulates tumorigenesis. Restoration of miR-122 in metastatic Mahlavu and SK-HEP-1 cells significantly reduced in vitro migration, invasion, and anchorage-independent growth as well as in vivo tumorigenesis, angiogenesis, and intrahepatic metastasis in an orthotopic liver cancer model. Because an inverse expression pattern is often present between an miRNA and its target genes, we used a computational approach and identified multiple miR-122 candidate target genes from two independent expression microarray datasets. Thirty-two target genes were empirically verified, and this group of genes was enriched with genes regulating cell movement, cell morphology, cell-cell signaling, and transcription. We further showed that one of the miR-122 targets, ADAM17 (a disintegrin and metalloprotease 17) is involved in metastasis. Silencing of ADAM17 resulted in a dramatic reduction of in vitro migration, invasion, in vivo tumorigenesis, angiogenesis, and local invasion in the livers of nude mice, which is similar to that which occurs with the restoration of miR-122. CONCLUSION: Our study suggests that miR-122, a tumor suppressor microRNA affecting hepatocellular carcinoma intrahepatic metastasis by angiogenesis suppression, exerts some of its action via regulation of ADAM17. Restoration of miR-122 has a far-reaching effect on the cell. Using the concomitant down-regulation of its targets, including ADAM17, a rational therapeutic strategy based on miR-122 may prove to be beneficial for patients with hepatocellular carcinoma.

Photoluminescence from n-type porous silicon layer enhanced by a forward-biased np-junction.

A new approach for the fabrication of n-type porous silicon layer is proposed. A hole-rich p-layer is arranged underneath the n-layer, and the np-junction is under forward biased condition in the etching process. Therefore sufficient holes can drift straight-upward and pass across the np-junction from p-region to n-region to participate in electrochemical reaction during the etching process with an unfailing supply. Illumination is an optional hole-supplier in this approach, so the problem of illumination-depth limitation can be overcome. Strong visible photoluminescence emissions are demonstrated on the hole-poor n-type porous layer at about 650 nm.