Estrogen/ERα signaling is critical for breast cancer progression and therapeutic treatments. Thus, identifying new regulators of this pathway will help to develop new therapeutics to overcome chemotherapy resistance of the breast cancer cells. Here, we report Ajuba directly interacts with ERα to potentiate ERα target gene expression, and biologically Ajuba promotes breast cancer cell growth and contributes to tamoxifen resistance of these cells. Ajuba constitutively binds the DBD and AF2 regions of ERα, and these interactions can be markedly enhanced by estrogen treatment. Mechanistically, Ajuba recruits DBC1 and CBP/p300 and forms a ternary complex to co-activate ERα transcriptional activity and concomitantly enhances ERα acetylation. Moreover, components of this complex can be found at endogenous promoters containing functional ERα responsive elements. Taken together, these data demonstrate that Ajuba functions as a novel co-activator of ERα and that Ajuba/DBC1/CBP/p300 ternary complex may be a new target for developing therapeutics to treat breast cancer.

Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.

The identification of the human homologue of the yeast CST in 2009 posed a new challenge in our understanding of the mechanism of telomere capping in higher eukaryotes. The high-resolution structure of the human Stn1-Ten1 (hStn1-Ten1) complex presented here reveals that hStn1 consists of an OB domain and tandem C-terminal wHTH motifs, while hTen1 consists of a single OB fold. Contacts between the OB domains facilitate formation of a complex that is strikingly similar to the replication protein A (RPA) and yeast Stn1-Ten1 (Ten1) complexes. The hStn1-Ten1 complex exhibits non-specific single-stranded DNA activity that is primarily dependent on hStn1. Cells expressing hStn1 mutants defective for dimerization with hTen1 display elongated telomeres and telomere defects associated with telomere uncapping, suggesting that the telomeric function of hCST is hTen1 dependent. Taken together the data presented here show that the structure of the hStn1-Ten1 subcomplex is conserved across species. Cell based assays indicate that hTen1 is critical for the telomeric function of hCST, both in telomere protection and downregulation of telomerase function.

Alphaviruses are a large group of re-emerging arthropod-borne RNA viruses. The compact viral RNA genomes harbor diverse structures that facilitate replication. These structures can be recognized by antiviral cellular RNA-binding proteins, including DExD-box (DDX) helicases, that bind viral RNAs to control infection. The full spectrum of antiviral DDXs and the structures that are recognized remain unclear. Genetic screening identified DDX39A as antiviral against the alphavirus chikungunya virus (CHIKV) and other medically relevant alphaviruses. Upon infection, the predominantly nuclear DDX39A accumulates in the cytoplasm inhibiting alphavirus replication, independent of the canonical interferon pathway. Biochemically, DDX39A binds to CHIKV genomic RNA, interacting with the 5' conserved sequence element (5'CSE), which is essential for the antiviral activity of DDX39A. Altogether, DDX39A relocalization and binding to a conserved structural element in the alphavirus genomic RNA attenuates infection, revealing a previously unknown layer to the cellular control of infection.

Zika virus is an emerging arthropod-borne flavivirus for which there are no vaccines or specific therapeutics. We screened a library of 2,000 bioactive compounds for their ability to block Zika virus infection in three distinct cell types with two different strains of Zika virus. Using a microscopy-based assay, we validated 38 drugs that inhibited Zika virus infection, including FDA-approved nucleoside analogs. Cells expressing high levels of the attachment factor AXL can be protected from infection with receptor tyrosine kinase inhibitors, while placental-derived cells that lack AXL expression are insensitive to this inhibition. Importantly, we identified nanchangmycin as a potent inhibitor of Zika virus entry across all cell types tested, including physiologically relevant primary cells. Nanchangmycin also was active against other medically relevant viruses, including West Nile, dengue, and chikungunya viruses that use a similar route of entry. This study provides a resource of small molecules to study Zika virus pathogenesis.

Upregulation of endogenous utrophin offers great promise for treating DMD, as it can functionally compensate for the lack of dystrophin caused by DMD gene mutations, without the immunogenic concerns associated with delivering dystrophin. However, post-transcriptional repression mechanisms targeting the 5' and 3' untranslated regions (UTRs) of utrophin mRNA significantly limit the magnitude of utrophin upregulation achievable by promoter activation. Using a utrophin 5'3'UTR reporter assay, we performed a high-throughput screen (HTS) for small molecules capable of relieving utrophin post-transcriptional repression. We identified 27 hits that were ranked using a using an algorithm that we designed for hit prioritization that we call Hit to Lead Prioritization Score (H2LPS). The top 10 hits were validated using an orthogonal assay for endogenous utrophin expression. Evaluation of the top scoring hit, Trichostatin A (TSA), demonstrated utrophin upregulation and functional improvement in the mdx mouse model of DMD. TSA and the other small molecules identified here represent potential starting points for DMD drug discovery efforts.

Most genes associated with acute myeloid leukemia (AML) are mutated in less than 10% of patients, suggesting that alternative mechanisms of gene disruption contribute to this disease. Here, we find a set of splicing events that alter the expression of a subset of AML-associated genes independent of known somatic mutations. In particular, aberrant splicing triples the number of patients with reduced functional EZH2 compared with that predicted by somatic mutation alone. In addition, we unexpectedly find that the nonsense-mediated decay factor DHX34 exhibits widespread alternative splicing in sporadic AML, resulting in a premature stop codon that phenocopies the loss-of-function germline mutations observed in familial AML. Together, these results demonstrate that classical mutation analysis underestimates the burden of functional gene disruption in AML and highlight the importance of assessing the contribution of alternative splicing to gene dysregulation in human disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths, posing a substantial threat to global public health. Viruses evolve different strategies to antagonize or evade host immune responses. While ectopic expression of SARS-CoV-2 accessory protein ORF6 blocks interferon (IFN) production and downstream IFN signaling, the role of ORF6 in IFN signaling during bona fide viral infection of respiratory cells is unclear. By comparing wild-type (WT) and ORF6-deleted (ΔORF6) SARS-CoV-2 infection and IFN signaling in respiratory cells, we found that ΔORF6 SARS-CoV-2 replicates more efficiently than WT virus and, thus, stimulates more robust immune signaling. Loss of ORF6 does not alter innate signaling in infected cells: both WT and ΔORF6 virus induce delayed IFN responses only in bystander cells. Moreover, expression of ORF6 in the context of SARS-CoV-2 infection has no effect on Sendai virus-stimulated IFN induction: robust translocation of IRF3 is observed in both SARS-CoV-2 infected and bystander cells. Furthermore, IFN pretreatment potently blocks WT and ΔORF6 virus replication similarly, and both viruses fail to suppress the induction of interferon-stimulated genes (ISGs) upon IFN-β treatment. However, upon treatment with IFN-β, only bystander cells induce STAT1 translocation during infection with WT virus, whereas ΔORF6 virus-infected cells now show translocation. This suggests that under conditions of high IFN activation, ORF6 can attenuate STAT1 activation. These data provide evidence that ORF6 is not sufficient to antagonize IFN production or IFN signaling in SARS-CoV-2-infected respiratory cells but may impact the efficacy of therapeutics that stimulate innate immune pathways. IMPORTANCE Previous studies identified several SARS-CoV-2 proteins, including ORF6, that antagonize host innate immune responses in the context of overexpression of viral proteins in non-respiratory cells. We set out to determine the role of ORF6 in IFN responses during SARS-CoV-2 infection of respiratory cells. Using a deletion strain, we observed no reduction of infection and no difference in evasion of IFN signaling, with responses limited to bystander cells. Moreover, stimulation of Sendai virus-induced IFN production or IFN-β-stimulated ISG expression was comparable between SARS-CoV-2 virus and SARS-CoV-2 lacking ORF6 virus, suggesting that ORF6 is not sufficient to counteract IFN induction or IFN signaling during viral infection.

Accumulation of senescent cells during aging contributes to chronic inflammation and age-related diseases. While senescence is associated with profound alterations of the epigenome, a systematic view of epigenetic factors in regulating senescence is lacking. Here, we curated a library of short hairpin RNAs for targeted silencing of all known epigenetic proteins and performed a high-throughput screen to identify key candidates whose downregulation can delay replicative senescence of primary human cells. This screen identified multiple new players including the histone acetyltransferase p300 that was found to be a primary driver of the senescent phenotype. p300, but not the paralogous CBP, induces a dynamic hyper-acetylated chromatin state and promotes the formation of active enhancer elements in the non-coding genome, leading to a senescence-specific gene expression program. Our work illustrates a causal role of histone acetyltransferases and acetylation in senescence and suggests p300 as a potential therapeutic target for senescence and age-related diseases.

Basic helix-loop-helix (bHLH) transcription factors recognize the canonical E-box (CANNTG) to regulate gene transcription; however, given the prevalence of E-boxes in a genome, it has been puzzling how individual bHLH proteins selectively recognize E-box sequences on their targets. TWIST is a bHLH transcription factor that promotes epithelial-mesenchymal transition (EMT) during development and tumor metastasis. High-resolution mapping of TWIST occupancy in human and Drosophila genomes reveals that TWIST, but not other bHLH proteins, recognizes a unique double E-box motif with two E-boxes spaced preferentially by 5 nucleotides. Using molecular modeling and binding kinetic analyses, we found that the strict spatial configuration in the double E-box motif aligns two TWIST-E47 dimers on the same face of DNA, thus providing a high-affinity site for a highly stable intramolecular tetramer. Biochemical analyses showed that the WR domain of TWIST dimerizes to mediate tetramer formation, which is functionally required for TWIST-induced EMT. These results uncover a novel mechanism for a bHLH transcription factor to recognize a unique spatial configuration of E-boxes to achieve target specificity. The WR-WR domain interaction uncovered here sets an example of target gene specificity of a bHLH protein being controlled allosterically by a domain outside of the bHLH region.

Sulindac is an FDA-approved non-steroidal anti-inflammatory drug with documented anticancer activities. Our recent studies showed that sulindac selectively enhanced the killing of cancer cells exposed to oxidizing agents via production of reactive oxygen species (ROS) resulting in mitochondrial dysfunction. This effect of sulindac and oxidative stress on cancer cells could be related to the defect in respiration in cancer cells, first described by Warburg 50 years ago, known as the Warburg effect. We postulated that sulindac might enhance the selective killing of cancer cells when combined with any compound that alters mitochondrial respiration. To test this hypothesis we have used dichloroacetate (DCA), which is known to shift pyruvate metabolism away from lactic acid formation to respiration. One might expect that DCA, since it stimulates aerobic metabolism, could stress mitochondrial respiration in cancer cells, which would result in enhanced killing in the presence of sulindac. In this study, we have shown that the combination of sulindac and DCA enhances the selective killing of A549 and SCC25 cancer cells under the conditions used. As predicted, the mechanism of killing involves ROS production, mitochondrial dysfunction, JNK signaling and death by apoptosis. Our results suggest that the sulindac-DCA drug combination may provide an effective cancer therapy.

Cervical cancer is the sixth most common cancer in women worldwide and the leading cause of women's death in developing countries. Nearly all cervical cancers are associated with infection of the human papillomavirus (HPV). This sexually transmitted pathogen disrupts the cell cycle via two oncoproteins: E6 and E7. Cells respond to E7-mediated degradation of pRB by upregulating the p53 tumor suppressor pathway. However, E6 thwarts this response by binding to the cellular E6-Associating Protein (E6AP) and targeting p53 for degradation. These two virus-facilitated processes pave the way for cellular transformation. Prophylactic HPV vaccines are available, but individuals already infected with HPV lack drug-based therapeutic options. To fill this void, we sought to identify small molecule inhibitors of the E6-E6AP interaction. We designed an ELISA-based high throughput assay to rapidly screen compound libraries, and hits were confirmed in several orthogonal biochemical and cell-based assays. Over 88,000 compounds were screened; 30 had in vitro potencies in the mid-nanomolar to mid-micromolar range and were classified as validated hits. Seven of these hits inhibited p53 degradation in cell lines with HPV-integrated genomes. Two compounds of similar scaffold successfully blocked p53 degradation and inhibited cell proliferation in cells stably transfected with E6. Together, these studies suggest that small molecules can successfully block E6-dependent p53 degradation and restore p53 activity. The compounds identified here constitute attractive starting points for further medicinal chemistry efforts and development into beneficial therapeutics.

Epstein-Barr Virus (EBV) latent infection is associated with several human malignancies and is a causal agent of lymphoproliferative diseases during immunosuppression. While inhibitors of herpesvirus DNA polymerases, like gancyclovir, reduce EBV lytic cycle infection, these treatments have limited efficacy for treating latent infection. EBNA1 is an EBV-encoded DNA-binding protein required for viral genome maintenance during latent infection.

MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The MeiobD383A/D383A mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB-SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB-SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein-protein interactions or promote degradation of meiosis-specific proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide outbreak, known as coronavirus disease 2019 (COVID-19). Alongside vaccines, antiviral therapeutics is an important part of the healthcare response to COVID-19. We previously reported that TEMPOL, a small molecule stable nitroxide, inactivated the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 by causing the oxidative degradation of its iron-sulfur cofactors. Here, we demonstrate that TEMPOL is effective in vivo in inhibiting viral replication in the Syrian hamster model. The inhibitory effect of TEMPOL on SARS-CoV-2 replication was observed in animals when the drug was administered 2 h before infection in a high-risk exposure model. These data support the potential application of TEMPOL as a highly efficacious antiviral against SARS-CoV-2 infection in humans.

Arthropod-borne viruses, including the alphavirus chikungunya virus (CHIKV), cause acute disease in millions of people and utilize potent mechanisms to antagonize and circumvent innate immune pathways including the type I interferon (IFN) pathway. In response, hosts have evolved antiviral counterdefense strategies that remain incompletely understood. Recent studies have found that long noncoding RNAs (lncRNAs) regulate classical innate immune pathways; how lncRNAs contribute to additional antiviral counterdefenses remains unclear. Using high-throughput genetic screening, we identified a cytoplasmic antiviral lncRNA that we named antiviral lncRNA prohibiting human alphaviruses (ALPHA), which is transcriptionally induced by alphaviruses and functions independently of IFN to inhibit the replication of CHIKV and its closest relative, O'nyong'nyong virus (ONNV), but not other viruses. Furthermore, we showed that ALPHA interacts with CHIKV genomic RNA and restrains viral RNA replication. Together, our findings reveal that ALPHA and potentially other lncRNAs can mediate non-canonical antiviral immune responses against specific viruses.

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP in vivo and for formation of SDS-resistant complexes in vitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.

Hepatocytes store triglycerides (TGs) in the form of lipid droplets (LDs), which are increased in hepatosteatosis. The regulation of hepatic LDs is poorly understood and new therapies to reduce hepatosteatosis are needed. We performed a siRNA kinase and phosphatase screen in HuH-7 cells using high-content automated imaging of LDs. Changes in accumulated lipids were quantified with developed pipeline that measures intensity, area, and number of LDs. Selected "hits," which reduced lipid accumulation, were further validated with other lipid and expression assays. Among several siRNAs that resulted in significantly reduced LDs, one was targeted to the nuclear adapter protein, transformation/transcription domain-associated protein (TRRAP). Knockdown of TRRAP reduced triglyceride accumulation in HuH-7 hepatocytes, in part by reducing C/EBPα-mediated de novo synthesis of TGs. These findings implicate TRRAP as a novel regulator of hepatic TG metabolism and nominate it as a potential drug target for hepatosteatosis.

The gene encoding ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently have no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K-AKT signaling. Importantly, EZH2 inhibition caused regression of ARID1A-mutated ovarian tumors in vivo. To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition. Our data indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for cancers involving ARID1A mutations.

The molecular determinants of breast cancer resistance to first-line anthracycline-containing chemotherapy are unknown.