A small-molecule allele-selective transcriptional inhibitor of the MIF immune susceptibility locus.

Functional variants of the gene for the cytokine macrophage migration inhibitory factor (MIF) are defined by a 4-nucleotide promoter microsatellite (-794 CATT5-8, rs5844572) and confer risk for autoimmune, infectious, and oncologic diseases. We describe herein the discovery of a prototypic, small molecule inhibitor of MIF transcription with selectivity for high microsatellite repeat number and correspondingly high gene expression. Utilizing a high-throughput luminescent proximity screen, we identify 1-carbomethoxy-5-formyl-4,6,8-trihydroxyphenazine (CMFT) to inhibit the functional interaction between the transcription factor ICBP90 (namely, UHRF1) and the MIF -794 CATT5-8 promoter microsatellite. CMFT inhibits MIF mRNA expression in a -794 CATT5-8 length-dependent manner with an IC50 of 470 nM, and preferentially reduces ICBP90-dependent MIF mRNA and protein expression in high-genotypic versus low-genotypic MIF-expressing macrophages. RNA expression analysis also showed CMFT to downregulate MIF-dependent, inflammatory gene expression with little evidence of off-target metabolic toxicity. These findings provide proof-of-concept for advancing the pharmacogenomic development of precision-based MIF inhibitors for diverse autoimmune and inflammatory conditions.

Discovery of novel microRNA mimic repressors of ribosome biogenesis.

While microRNAs and other non-coding RNAs are the next frontier of novel regulators of mammalian ribosome biogenesis (RB), a systematic exploration of microRNA-mediated RB regulation has not yet been undertaken. We carried out a high-content screen in MCF10A cells for changes in nucleolar number using a library of 2603 mature human microRNA mimics. Following a secondary screen for nucleolar rRNA biogenesis inhibition, we identified 72 novel microRNA negative regulators of RB after stringent hit calling. Hits included 27 well-conserved microRNAs present in MirGeneDB, and were enriched for mRNA targets encoding proteins with nucleolar localization or functions in cell cycle regulation. Rigorous selection and validation of a subset of 15 microRNA hits unexpectedly revealed that most of them caused dysregulated pre-rRNA processing, elucidating a novel role for microRNAs in RB regulation. Almost all hits impaired global protein synthesis and upregulated CDKN1A (p21) levels, while causing diverse effects on RNA Polymerase 1 (RNAP1) transcription and TP53 protein levels. We provide evidence that the MIR-28 siblings, hsa-miR-28-5p and hsa-miR-708-5p, potently target the ribosomal protein mRNA RPS28 via tandem primate-specific 3' UTR binding sites, causing a severe pre-18S pre-rRNA processing defect. Our work illuminates novel microRNA attenuators of RB, forging a promising new path for microRNA mimic chemotherapeutics.

Human nucleolar protein 7 (NOL7) is required for early pre-rRNA accumulation and pre-18S rRNA processing.

The main components of the essential cellular process of eukaryotic ribosome biogenesis are highly conserved from yeast to humans. Among these, the U3 Associated Proteins (UTPs) are a small subunit processome subcomplex that coordinate the first two steps of ribosome biogenesis in transcription and pre-18S processing. While we have identified the human counterparts of most of the yeast Utps, the homologs of yeast Utp9 and Bud21 (Utp16) have remained elusive. In this study, we find that NOL7 is the likely ortholog of Bud21. Previously described as a tumour suppressor through regulation of antiangiogenic transcripts, we now show that NOL7 is required for early pre-rRNA accumulation and pre-18S rRNA processing in human cells. These roles lead to decreased protein synthesis and induction of the nucleolar stress response upon NOL7 depletion. Beyond Bud21's nonessential role in yeast, we establish human NOL7 as an essential UTP that is necessary to maintain both early pre-rRNA levels and processing.

Engineered cardiac tissue model of restrictive cardiomyopathy for drug discovery.

Restrictive cardiomyopathy (RCM) is defined as increased myocardial stiffness and impaired diastolic relaxation leading to elevated ventricular filling pressures. Human variants in filamin C (FLNC) are linked to a variety of cardiomyopathies, and in this study, we investigate an in-frame deletion (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp) in a patient with RCM. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with this variant display impaired relaxation and reduced calcium kinetics in 2D culture when compared with a CRISPR-Cas9-corrected isogenic control line. Similarly, mutant engineered cardiac tissues (ECTs) demonstrate increased passive tension and impaired relaxation velocity compared with isogenic controls. High-throughput small-molecule screening identifies phosphodiesterase 3 (PDE3) inhibition by trequinsin as a potential therapy to improve cardiomyocyte relaxation in this genotype. Together, these data demonstrate an engineered cardiac tissue model of RCM and establish the translational potential of this precision medicine approach to identify therapeutics targeting myocardial relaxation.

Discovery of novel microRNA mimic repressors of ribosome biogenesis.

While microRNAs and other non-coding RNAs are the next frontier of novel regulators of mammalian ribosome biogenesis (RB), a systematic exploration of microRNA-mediated RB regulation has not yet been undertaken. We carried out a high-content screen in MCF10A cells for changes in nucleolar number using a library of 2,603 mature human microRNA mimics. Following a secondary screen for nucleolar rRNA biogenesis inhibition, we identified 72 novel microRNA negative regulators of RB after stringent hit calling. Hits included 27 well-conserved microRNAs present in MirGeneDB, and were enriched for mRNA targets encoding proteins with nucleolar localization or functions in cell cycle regulation. Rigorous selection and validation of a subset of 15 microRNA hits unexpectedly revealed that most of them caused dysregulated pre-rRNA processing, elucidating a novel role for microRNAs in RB regulation. Almost all hits impaired global protein synthesis and upregulated CDKN1A ( p21 ) levels, while causing diverse effects on RNA Polymerase 1 (RNAP1) transcription and TP53 protein levels. We discovered that the MIR-28 siblings, hsa-miR-28-5p and hsa-miR-708-5p, directly and potently target the ribosomal protein mRNA RPS28 via tandem primate-specific 3' UTR binding sites, causing a severe pre-18S pre-rRNA processing defect. Our work illuminates novel microRNA attenuators of RB, forging a promising new path for microRNA mimic chemotherapeutics.

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing.

In eukaryotes, the nucleolus is the site of ribosome biosynthesis, an essential process in all cells. While human ribosome assembly is largely evolutionarily conserved, many of the regulatory details underlying its control and function have not yet been well-defined. The nucleolar protein RSL24D1 was originally identified as a factor important for 60S ribosomal subunit biogenesis. In addition, the PeBoW (BOP1-PES1-WDR12) complex has been well-defined as required for pre-28S rRNA processing and cell proliferation. In this study, we show that RSL24D1 depletion impairs both pre-ribosomal RNA (pre-rRNA) transcription and mature 28S rRNA production, leading to decreased protein synthesis and p53 stabilization in human cells. Surprisingly, each of the PeBoW complex members is also required for pre-rRNA transcription. We demonstrate that RSL24D1 and WDR12 co-immunoprecipitate with the RNA polymerase I subunit, RPA194, and regulate its steady state levels. These results uncover the dual role of RSL24D1 and the PeBoW complex in multiple steps of ribosome biogenesis, and provide evidence implicating large ribosomal subunit biogenesis factors in pre-rRNA transcription control.

RASGRF1 Fusions Activate Oncogenic RAS Signaling and Confer Sensitivity to MEK Inhibition.

PURPOSE: The identification of actionable oncogenic alterations has enabled targeted therapeutic strategies for subsets of patients with advanced malignancies, including lung adenocarcinoma (LUAD). We sought to assess the frequency of known drivers and identify new candidate drivers in a cohort of LUAD from patients with minimal smoking history. EXPERIMENTAL DESIGN: We performed genomic characterization of 103 LUADs from patients with ≤10 pack-year smoking history. Tumors were subjected to targeted molecular profiling and/or whole-exome sequencing and RNA sequencing in search of established and previously uncharacterized candidate drivers. RESULTS: We identified an established oncogenic driver in 98 of 103 tumors (95%). From one tumor lacking a known driver, we identified a novel gene rearrangement between OCLN and RASGRF1. The encoded OCLN-RASGRF1 chimera fuses the membrane-spanning portion of the tight junction protein occludin with the catalytic RAS-GEF domain of the RAS activator RASGRF1. We identified a similar SLC4A4-RASGRF1 fusion in a pancreatic ductal adenocarcinoma cell line lacking an activating KRAS mutation and an IQGAP1-RASGRF1 fusion from a sarcoma in The Cancer Genome Atlas. We demonstrate these fusions increase cellular levels of active GTP-RAS, induce cellular transformation, and promote in vivo tumorigenesis. Cells driven by RASGRF1 fusions are sensitive to targeting of the RAF-MEK-ERK pathway in vitro and in vivo. CONCLUSIONS: Our findings credential RASGRF1 fusions as a therapeutic target in multiple malignancies and implicate RAF-MEK-ERK inhibition as a potential treatment strategy for advanced tumors harboring these alterations. See related commentary by Moorthi and Berger, p. 2983.

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis.

Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis (RB), and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting RB. However, there is a need for a more direct high-throughput assay for a nucleolar function to further evaluate hits. Previous reports have monitored nucleolar rRNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay that enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay uses two siRNA controls: a negative non-targeting siRNA control and a positive siRNA control targeting RNA Polymerase 1 (RNAP1; POLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small-molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dispersion. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of RB in health and disease.

Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function.

Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery.

Restriction of SARS-CoV-2 replication by targeting programmed -1 ribosomal frameshifting.

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires a programmed -1 ribosomal frameshift (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor for SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other betacoronaviruses. Consistent with the essential role of -1 PRF in viral gene expression, merafloxacin impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing proof-of-principle for targeting -1 PRF as a plausible and effective antiviral strategy for SARS-CoV-2 and other coronaviruses.

Increased numbers of nucleoli in a genome-wide RNAi screen reveal proteins that link the cell cycle to RNA polymerase I transcription.

Nucleoli are dynamic nuclear condensates in eukaryotic cells that originate through ribosome biogenesis at loci that harbor the ribosomal DNA. These loci are known as nucleolar organizer regions (NORs), and there are 10 in a human diploid genome. While there are 10 NORs, however, the number of nucleoli observed in cells is variable. Furthermore, changes in number are associated with disease, with increased numbers and size common in aggressive cancers. In the near-diploid human breast epithelial cell line, MCF10A, the most frequently observed number of nucleoli is two to three per cell. Here, to identify novel regulators of ribosome biogenesis we used high-throughput quantitative imaging of MCF10A cells to identify proteins that, when depleted, increase the percentage of nuclei with ≥5 nucleoli. Unexpectedly, this unique screening approach led to identification of proteins associated with the cell cycle. Functional analysis on a subset of hits further revealed not only proteins required for progression through the S and G2/M phase, but also proteins required explicitly for the regulation of RNA polymerase I transcription and protein synthesis. Thus, results from this screen for increased nucleolar number highlight the significance of the nucleolus in human cell cycle regulation, linking RNA polymerase I transcription to cell cycle progression.

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.

Restriction of SARS-CoV-2 Replication by Targeting Programmed -1 Ribosomal Frameshifting In Vitro.

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires programmed -1 ribosomal frameshifting (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor of SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other beta coronaviruses. Importantly, frameshift inhibition by merafloxacin substantially impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing the proof of principle of targeting -1 PRF as an effective antiviral strategy for SARS-CoV-2.

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-ß signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.

An allosteric site on MKP5 reveals a strategy for small-molecule inhibition.

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) have been considered "undruggable," but their position as regulators of the MAPKs makes them promising therapeutic targets. MKP5 has been suggested as a potential target for the treatment of dystrophic muscle disease. Here, we identified an inhibitor of MKP5 using a p38α MAPK-derived, phosphopeptide-based small-molecule screen. We solved the structure of MKP5 in complex with this inhibitor, which revealed a previously undescribed allosteric binding pocket. Binding of the inhibitor to this pocket collapsed the MKP5 active site and was predicted to limit MAPK binding. Treatment with the inhibitor recapitulated the phenotype of MKP5 deficiency, resulting in activation of p38 MAPK and JNK. We demonstrated that MKP5 was required for TGF-ß1 signaling in muscle and that the inhibitor blocked TGF-ß1-mediated Smad2 phosphorylation. TGF-ß1 pathway antagonism has been proposed for the treatment of dystrophic muscle disease. Thus, allosteric inhibition of MKP5 represents a therapeutic strategy against dystrophic muscle disease.

Development and optimization of a high-throughput screening method utilizing Ancylostoma ceylanicum egg hatching to identify novel anthelmintics.

Hookworms remain a major health burden in the developing world, with hundreds of millions currently afflicted by these blood-feeding parasites. There exists a vital need for the discovery of novel drugs and identification of parasite drug targets for the development of chemotherapies. New drug development requires the identification of compounds that target molecules essential to parasite survival and preclinical testing in robust, standardized animal models of human disease. This process can prove costly and time consuming using conventional, low-throughput methods. We have developed a novel high-throughput screen (HTS) to identify anthelmintics for the treatment of soil-transmitted helminths. Our high-throughput, plate reader-based assay was used to rapidly assess compound toxicity to Ancylostoma ceylanicum L1 larva. Using this method, we screened 39,568 compounds from several small molecule screening libraries at 10 µM and identified 830 bioactive compounds that inhibit egg hatching of the human hookworm A. ceylanicum by >50%. Of these, 132 compounds inhibited hookworm egg hatching by >90% compared to controls. The nematicidal activities of 268 compounds were verified by retesting in the egg hatching assay and were also tested for toxicity against the human HeLa cell line at 10 µM. Fifty-nine compounds were verified to inhibit A. ceylanicum egg hatching by >80% and were <20% toxic to HeLa cells. Half-maximal inhibitory concentration (IC50) values were determined for the 59 hit compounds and ranged from 0.05-8.94 µM. This stringent advancement of compounds was designed to 1) systematically assess the nematicidal activity of novel compounds against the egg stage of A. ceylanicum hookworms in culture and 2) define their chemotherapeutic potential in vivo by evaluating their toxicity to human cells. Information gained from these experiments may directly contribute to the development of new drugs for the treatment of human hookworm disease.

High-throughput screening identifies compounds that protect RPE cells from physiological stressors present in AMD.

Dysfunction and eventual loss of retinal pigment epithelial (RPE) cells is a hallmark of atrophic age-related macular degeneration (AMD), and linked to oxidative and nitrosative damage. Herein, we use a high-throughput screen (HTS) to identify compounds that protect human RPE cells from oxidative stress-induced cell death and elucidate the possible mechanism of action. HTS was used to identify compounds that protect RPE cells from oxidative damage. We tested the identified compound(s) in models of RPE stress, including tert-butyl hydroperoxide (TBHP) exposure, ultraviolet-B (UV-B)-mediated light damage and nitrosative stress to the basement membrane using ARPE-19 cells, primary human RPE cells and induced-pluripotent stem cell (iPSC)-derived RPE cells from patients with AMD. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect gene expression of oxidative stress- and apoptosis-related genes and mitochondrial function was measured using a Seahorse XF96 analyzer to elucidate possible mechanisms of action. Five thousand and sixty-five compounds were screened, and of these, 12 compounds were active based on their ability to improve cell viability after exposure to TBHP. After chemical structure review, we identified ciclopirox olamine as a potent inhibitor of oxidative damage to RPE cells. Ciclopirox olamine increased cell viability in ARPE-19 cells treated with TBHP, UV-B light or on nitrite-modified extracellular matrix (ECM) by 1.68-fold, 1.54-fold and 4.3-fold, respectively (p < 0.01). Treatment with TBHP altered expression of genes related to oxidative stress and apoptosis, which was reversed by pretreatment with ciclopirox olamine. Ciclopirox olamine improved mitochondrial function in TBHP-exposed ARPE-19 cells and iPSC-derived RPE cells. Ciclopirox olamine protected primary human RPE cells and iPSC-derived RPE cells from the oxidative stress or damaged basement membrane. HTS of bioactive Food and Drug Administration (FDA)-approved libraries and follow-up studies can be used to identify small molecules (including ciclopirox olamine) that protect RPE cells exposed to various stressors associated with disease progression of AMD. This strategy can be used to identify potential compounds for treatment and prevention of AMD.

Macrolides selectively inhibit mutant KCNJ5 potassium channels that cause aldosterone-producing adenoma.

Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT.

DNA Polymerase Beta Germline Variant Confers Cellular Response to Cisplatin Therapy.

Resistance to cancer chemotherapies leads to deadly consequences, yet current research focuses only on the roles of somatically acquired mutations in this resistance. The mutational status of the germline is also likely to play a role in the way cells respond to chemotherapy. The carrier status for the POLB rs3136797 germline mutation encoding P242R DNA polymerase beta (Pol ß) is associated with poor prognosis for lung cancer, specifically in response to treatment with cisplatin. Here, it is revealed that the P242R mutation is sufficient to promote resistance to cisplatin in human cells and in mouse xenografts. Mechanistically, P242R Pol ß acts as a translesion polymerase and prefers to insert the correct nucleotide opposite cisplatin intrastrand cross-links, leading to the activation of the nucleotide excision repair (NER) pathway, removal of crosslinks, and resistance to cisplatin. In contrast, wild-type (WT) Pol ß preferentially inserts the incorrect nucleotide initiating mismatch repair and cell death. Importantly, in a mouse xenograft model, tumors derived from lung cancer cells expressing WT Pol ß displayed a slower rate of growth when treated with cisplatin, whereas tumors expressing P242R Pol ß had no response to cisplatin. Pol ß is critical for mediating crosstalk in response to cisplatin. The current data strongly suggest that the status of Pol ß influences cellular responses to crosslinking agents and that Pol ß is a promising biomarker to predict responses to specific chemotherapies. Finally, these results highlight that the genetic status of the germline is a critical factor in the response to cancer treatment.Implications: Pol ß has prognostic biomarker potential in the treatment of cancer with cisplatin and perhaps other intrastrand crosslinking agents. Mol Cancer Res; 15(3); 269-80. ©2017 AACR.

A novel small molecule that disrupts a key event during the oocyte-to-embryo transition in C. elegans.

The complex cellular events that occur in response to fertilization are essential for mediating the oocyte-to-embryo transition. Here, we describe a comprehensive small-molecule screen focused on identifying compounds that affect early embryonic events in Caenorhabditis elegans We identify a single novel compound that disrupts early embryogenesis with remarkable stage and species specificity. The compound, named C22, primarily impairs eggshell integrity, leading to osmotic sensitivity and embryonic lethality. The C22-induced phenotype is dependent upon the upregulation of the LET-607/CREBH transcription factor and its candidate target genes, which primarily encode factors involved in diverse aspects of protein trafficking. Together, our data suggest that in the presence of C22, one or more key components of the eggshell are inappropriately processed, leading to permeable, inviable embryos. The remarkable specificity and reversibility of this compound will facilitate further investigation into the role and regulation of protein trafficking in the early embryo, as well as serve as a tool for manipulating the life cycle for other studies such as those involving aging.