Autophagy is a cellular catabolic process responsible for the degradation of cytoplasmic constituents, including organelles and long-lived proteins, that helps maintain cellular homeostasis and protect against various cellular stresses. Verteporfin is a benzoporphyrin derivative used clinically in photodynamic therapy to treat macular degeneration. Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light. We report that verteporfin directly targets and modifies p62, a scaffold and adaptor protein that binds both polyubiquitinated proteins destined for degradation and LC3 on autophagosomal membranes. Western blotting experiments revealed that exposure of cells or purified p62 to verteporfin causes the formation of covalently crosslinked p62 oligomers by a mechanism involving low-level singlet oxygen production. Rose bengal, a singlet oxygen producer structurally unrelated to verteporfin, also produced crosslinked p62 oligomers and inhibited autophagosome formation. Co-immunoprecipitation experiments demonstrated that crosslinked p62 oligomers retain their ability to bind to LC3 but show defective binding to polyubiquitinated proteins. Mutations in the p62 PB1 domain that abolish self-oligomerization also abolished crosslinked oligomer formation. Interestingly, small amounts of crosslinked p62 oligomers were detected in untreated cells, and other groups noted the accumulation of p62 forms with reduced SDS-PAGE mobility in cellular and animal models of oxidative stress and aging. These data indicate that p62 is particularly susceptible to oxidative crosslinking and lead us to propose a model whereby oxidized crosslinked p62 oligomers generated rapidly by drugs like verteporfin or over time during the aging process interfere with autophagy.

Nonsense mutations introduce premature termination codons and underlie 11% of genetic disease cases. High concentrations of aminoglycosides can restore gene function by eliciting premature termination codon readthrough but with low efficiency. Using a high-throughput screen, we identified compounds that potentiate readthrough by aminoglycosides at multiple nonsense alleles in yeast. Chemical optimization generated phthalimide derivative CDX5-1 with activity in human cells. Alone, CDX5-1 did not induce readthrough or increase TP53 mRNA levels in HDQ-P1 cancer cells with a homozygous TP53 nonsense mutation. However, in combination with aminoglycoside G418, it enhanced readthrough up to 180-fold over G418 alone. The combination also increased readthrough at all three nonsense codons in cancer cells with other TP53 nonsense mutations, as well as in cells from rare genetic disease patients with nonsense mutations in the CLN2, SMARCAL1 and DMD genes. These findings open up the possibility of treating patients across a spectrum of genetic diseases caused by nonsense mutations.

The Pax6 transcription factor is expressed in cerebellar granule cells and when mutated, as in the Sey/Sey mouse, produces granule cells with disturbed survival and migration and with defects in neurite extension. The impact of Pax6 on other genes in the context of cerebellar development has not been identified. In this study, we performed transcriptome comparisons between wildtype and Pax6-null whole cerebellar tissue at embryonic day (E) 13.5, 15.5 and 18.5 using Affymetrix arrays (U74Av2). Statistical analyses identified 136 differentially regulated transcripts (FDR 0.05, 1.2-fold change cutoff) over time in Pax6-null cerebellar tissue. In parallel we examined the Math1-null granuloprival cerebellum and identified 228 down-regulated transcripts (FDR 0.05, 1.2-fold change cutoff). The intersection of these two microarray datasets produced a total of 21 differentially regulated transcripts. For a subset of the identified transcripts, we used qRT-PCR to validate the microarray data and demonstrated the expression in the rhombic lip lineage and differential expression in Pax6-null cerebellum with in situ hybridisation analysis. The candidate genes identified in this way represent direct or indirect Pax6-downstream genes involved in cerebellar development.

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

Glioblastoma (GBM) is the most common primary malignant brain tumor, and it has a uniformly poor prognosis. Hypoxia is a feature of the GBM microenvironment, and previous work has shown that cancer cells residing in hypoxic regions resist treatment. Hypoxia can trigger the formation of stress granules (SGs), sites of mRNA triage that promote cell survival. A screen of 1120 FDA-approved drugs identified 129 candidates that delayed the dissolution of hypoxia-induced SGs following a return to normoxia. Amongst these candidates, the selective estrogen receptor modulator (SERM) raloxifene delayed SG dissolution in a dose-dependent manner. SG dissolution typically occurs by 15 min post-hypoxia, however pre-treatment of immortalized U251 and U3024 primary GBM cells with raloxifene prevented SG dissolution for up to 2 h. During this raloxifene-induced delay in SG dissolution, translational silencing was sustained, eIF2α remained phosphorylated and mTOR remained inactive. Despite its well-described role as a SERM, raloxifene-mediated delay in SG dissolution was unaffected by co-administration of β-estradiol, nor did β-estradiol alone have any effect on SGs. Importantly, the combination of raloxifene and hypoxia resulted in increased numbers of late apoptotic/necrotic cells. Raloxifene and hypoxia also demonstrated a block in late autophagy similar to the known autophagy inhibitor chloroquine (CQ). Genetic disruption of the SG-nucleating proteins G3BP1 and G3BP2 revealed that G3BP1 is required to sustain the raloxifene-mediated delay in SG dissolution. Together, these findings indicate that modulating the stress response can be used to exploit the hypoxic niche of GBM tumors, causing cell death by disrupting pro-survival stress responses and control of protein synthesis.

DEAD-Box Helicase 3 X-Linked (DDX3X) is frequently mutated in the Wingless (WNT) and Sonic hedghog (SHH) subtypes of medulloblastoma-the commonest malignant childhood brain tumor, but whether DDX3X functions as a medulloblastoma oncogene or tumor suppressor gene is not known. Here, we show that Ddx3x regulates hindbrain patterning and development by controlling Hox gene expression and cell stress signaling. In mice predisposed to Wnt- or Shh medulloblastoma, Ddx3x sensed oncogenic stress and suppressed tumor formation. WNT and SHH medulloblastomas normally arise only in the lower and upper rhombic lips, respectively. Deletion of Ddx3x removed this lineage restriction, enabling both medulloblastoma subtypes to arise in either germinal zone. Thus, DDX3X is a medulloblastoma tumor suppressor that regulates hindbrain development and restricts the competence of cell lineages to form medulloblastoma subtypes.

Premature termination codon (PTC) readthrough is considered a potential treatment for genetic diseases caused by nonsense mutations. High concentrations of aminoglycosides induce low levels of PTC readthrough but also elicit severe toxicity. Identifying compounds that enhance PTC readthrough by aminoglycosides or reduce their toxicity is a continuing challenge. In humans, a binary complex of eukaryotic release factors 1 (eRF1) and 3 (eRF3a or eRF3b) mediates translation termination. They also participate in the SURF (SMG1-UPF1-eRF1-eRF3) complex assembly involved in nonsense-mediated mRNA decay (NMD). We show that PTC readthrough by aminoglycoside G418 is considerably enhanced by eRF3a and eRF3b siRNAs and cereblon E3 ligase modulators CC-885 and CC-90009, which induce proteasomal degradation of eRF3a and eRF3b. eRF3 degradation also reduces eRF1 levels and upregulates UPF1 and selectively stabilizes TP53 transcripts bearing a nonsense mutation over WT, indicating NMD suppression. CC-90009 is considerably less toxic than CC-885 and it enhances PTC readthrough in combination with aminoglycosides in mucopolysaccharidosis type I-Hurler, late infantile neuronal ceroid lipofuscinosis, Duchenne muscular dystrophy and junctional epidermolysis bullosa patient-derived cells with nonsense mutations in the IDUA, TPP1, DMD and COL17A1 genes, respectively. Combination of CC-90009 with aminoglycosides such as gentamicin or ELX-02 may have potential for PTC readthrough therapy.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemotherapy. It has been described as requiring elevated autophagy for growth and inhibiting autophagy has been proposed as a treatment strategy. To date, all preclinical reports and clinical trials investigating pharmacological inhibition of autophagy have used chloroquine or hydroxychloroquine, which interfere with lysosomal function and block autophagy at a late stage. Verteporfin is a newly discovered autophagy inhibitor that blocks autophagy at an early stage by inhibiting autophagosome formation. Here we report that PDAC cell lines show variable sensitivity to verteporfin in vitro, suggesting cell-line specific autophagy dependence. Using image-based and molecular analyses, we show that verteporfin inhibits autophagy stimulated by gemcitabine, the current standard treatment for PDAC. Pharmacokinetic and efficacy studies in a BxPC-3 xenograft mouse model demonstrated that verteporfin accumulated in tumors at autophagy-inhibiting levels and inhibited autophagy in vivo, but did not reduce tumor volume or increase survival as a single agent. In combination with gemcitabine verteporfin moderately reduced tumor growth and enhanced survival compared to gemcitabine alone. While our results do not uphold the premise that autophagy inhibition might be widely effective against PDAC as a single-modality treatment, they do support autophagy inhibition as an approach to sensitize PDAC to gemcitabine.

Most normal cells have two centrosomes that form bipolar spindles during mitosis, while cancer cells often contain more than two, or "supernumerary" centrosomes. Such cancer cells achieve bipolar division by clustering their centrosomes into two functional poles, and inhibiting this process then leads to cancer-specific cell death. A major problem with clinically used anti-mitotic drugs, such as paclitaxel, is their toxicity in normal cells. To discover new compounds with greater specificity for cancer cells, we established a high-content screen for agents that block centrosome clustering in BT-549 cells, a breast cancer cell line that harbors supernumerary centrosomes. Using this screen, we identified 14 compounds that inhibit centrosome clustering and induce mitotic arrest. Some of these compounds were structurally similar, suggesting a common structural motif important for preventing centrosome clustering. We next compared the effects of these compounds on the growth of several breast and other cancer cell lines, an immortalized normal human mammary epithelial cell line, and progenitor-enriched primary normal human mammary epithelial cells. From these comparisons, we found some compounds that kill breast cancer cells, but not their normal epithelial counterparts, suggesting their potential for targeted therapy. One of these compounds, N2-(3-pyridylmethyl)-5-nitro-2-furamide (Centrosome Clustering Chemical Inhibitor-01, CCCI-01), that showed the greatest differential response in this screen was confirmed to have selective effects on cancer as compared to normal breast progenitors using more precise apoptosis induction and clonogenic growth endpoints. The concentration of CCCI-01 that killed cancer cells in the clonogenic assay spared normal human bone marrow hematopoietic progenitors in the colony-forming cell assay, indicating a potential therapeutic window for CCCI-01, whose selectivity might be further improved by optimizing the compound. Immunofluorescence analysis showed that treatment with CCCI-01 lead to multipolar spindles in BT-549, while maintaining bipolar spindles in the normal primary human mammary epithelial cells. Since centrosome clustering is a complex process involving multiple pathways, the 14 compounds identified in this study provide a potentially novel means to developing non-cross-resistant anti-cancer drugs that block centrosome clustering.

Math1 is the defining molecule of the cerebellar rhombic lip and Pax6 is downstream in the Math1 pathway. In the present study, we discover that Wntless (Wls) is a novel molecular marker of the cells in the interior face of the rhombic lip throughout normal mouse cerebellar development. Wls expression is found complementary to the expression of Math1 and Pax6, which are localized to the exterior face of the rhombic lip. To determine the interaction between these molecules, we examine the loss-of-Math1 or loss-of-Pax6 in the cerebellum, i.e., the Math1-null and Pax6-null (Sey) mutant cerebella. The presence of Wls-positive cells in the Math1-null rhombic lip indicates that Wls expression is independent of Math1. In the Sey mutant cerebellum, there is an expansion of Wls-expressing cells into regions that are normally colonized by Pax6-expressing cells. The ectopic expression of Wls in the Pax6-null cerebellum suggests a negative interaction between Wls-expressing cells and Pax6-positive cells. These findings suggest that the rhombic lip is dynamically patterned by the expression of Wls, Math1, and Pax6. We also examine five rhombic lip cell markers (Wls, Math1, Pax6, Lmx1a, and Tbr2) to identify four molecularly distinct compartments in the rhombic lip during cerebellar development. The existence of spatial compartmentation in the rhombic lip and the interplay between Wls, Math1, and Pax6 in the rhombic lip provides novel views of early cerebellar development.

Choroid plexus carcinomas (CPCs) are poorly understood and frequently lethal brain tumors with few treatment options. Using a mouse model of the disease and a large cohort of human CPCs, we performed a cross-species, genome-wide search for oncogenes within syntenic regions of chromosome gain. TAF12, NFYC, and RAD54L co-located on human chromosome 1p32-35.3 and mouse chromosome 4qD1-D3 were identified as oncogenes that are gained in tumors in both species and required for disease initiation and progression. TAF12 and NFYC are transcription factors that regulate the epigenome, whereas RAD54L plays a central role in DNA repair. Our data identify a group of concurrently gained oncogenes that cooperate in the formation of CPC and reveal potential avenues for therapy.

Wntless (Wls) is implicated in the Wnt signaling pathway by regulating the secretion of Wnt molecules. During brain development, Wls is expressed in the isthmic organizer (ISO) and rhombic lip (RL). Wls regulates Wnt1 secretion at the ISO which is required to induce midbrain-hindbrain structures. However, Wls function in the RL is not known. Here, we employed Nestin-cre to delete Wls specifically in the RL during mid-gestation. The loss-of-Wls leads to an abnormal RL during development and cerebellar vermis hypoplasia at birth. The Wls conditional knockout (cKO) has rudimentary foliation with an absence of Bergmann glia fibers in the external germinal layer (EGL). The Wls-cKO cerebellum also exhibits ectopia of several cell types and aberrations in granule cell organization. Finally, there is a loss of 85% of unipolar brush cells. From these findings, Wls-expressing cells in the rhombic lip are implicated in the orchestration of cerebellar development.

Single genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug.

Frontotemporal lobar degeneration (FTLD) is a devastating and progressive disorder, and a common cause of early onset dementia. Progranulin (PGRN) haploinsufficiency due to autosomal dominant mutations in the progranulin gene (GRN) is an important cause of FTLD (FTLD-GRN), and nearly a quarter of these genetic cases are due to a nonsense mutation. Premature termination codons (PTC) can be therapeutically targeted by compounds allowing readthrough, and aminoglycoside antibiotics are known to be potent PTC readthrough drugs. Restoring endogenous PGRN through PTC readthrough has not previously been explored as a therapeutic intervention in FTLD.

The work of the FANTOM5 Consortium has brought forth a new level of understanding of the regulation of gene transcription and the cellular processes involved in creating diversity of cell types. In this study, we extended the analysis of the FANTOM5 Cap Analysis of Gene Expression (CAGE) transcriptome data to focus on understanding the genetic regulators involved in mouse cerebellar development.

Schimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder caused by biallelic mutations in the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) gene. Changes in gene expression underlie the arteriosclerosis and T-cell immunodeficiency of SIOD; therefore, we hypothesized that SMARCAL1 deficiency causes the focal segmental glomerulosclerosis (FSGS) of SIOD by altering renal gene expression. We tested this hypothesis by gene expression analysis of an SIOD patient kidney and verified these findings through immunofluorescent analysis in additional SIOD patients and a genetic interaction analysis in Drosophila.

ETV6 is an E26 transformation specific family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein-protein interaction. Herein, we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, along with amide hydrogen exchange measurements, we demonstrate that monomeric PNT domain variants adopt very stable helical bundle folds that do not change in conformation upon self-association into heterodimer models of the ETV6 polymer. Surface plasmon resonance-monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.

Nonsense mutations, which occur in ∼11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as G418 permit PTC readthrough and so may be used to address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we tested whether TRPC nonselective cation channels contribute to the variable PTC readthrough effect of aminoglycosides by controlling their cellular uptake. Indeed, a recently reported selective TRPC5 inhibitor, AC1903, consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cancer cell line and junctional epidermolysis bullosa (JEB) patient-derived keratinocytes. Interestingly, the effect of AC1903 in DMS-114 cells was mimicked by nonselective TRPC inhibitors, but not by well-characterized inhibitors of TRPC1/4/5 (Pico145, GFB-8438) or TRPC3/6/7 (SAR7334), suggesting that AC1903 may work through additional or undefined targets. Indeed, in our experiments, AC1903 inhibited multiple TRPC channels including TRPC3, TRPC4, TRPC5, TRPC6, TRPC4-C1, and TRPC5-C1, as well as endogenous TRPC1:C4 channels in A498 renal cancer cells, all with low micromolar IC50 values (1.8-18 μM). We also show that AC1903 inhibited TRPV4 channels, but had weak or no effects on TRPV1 and no effect on the nonselective cation channel PIEZO1. Our study reveals that AC1903 has previously unrecognized targets, which need to be considered when interpreting results from experiments with this compound. In addition, our data strengthen the hypothesis that nonselective calcium channels are involved in aminoglycoside uptake.

The development of the brain requires precise coordination of molecular processes across many cell-types. Underpinning these events are gene expression programs which require intricate regulation by non-coding regulatory sequences known as enhancers. In the context of the developing brain, transcribed enhancers (TEs) regulate temporally-specific expression of genes critical for cell identity and differentiation. Transcription of non-coding RNAs at active enhancer sequences, known as enhancer RNAs (eRNAs), is tightly associated with enhancer activity and has been correlated with target gene expression. TEs have been characterized in a multitude of developing tissues, however their regulatory role has yet to be described in the context of embryonic and early postnatal brain development. In this study, eRNA transcription was analyzed to identify TEs active during cerebellar development, as a proxy for the developing brain. Cap Analysis of Gene Expression followed by sequencing (CAGE-seq) was conducted at 12 stages throughout embryonic and early postnatal cerebellar development.

The lack of new anthelmintic agents is of growing concern because it affects human health and our food supply, as both livestock and plants are affected. Two principal factors contribute to this problem. First, nematode resistance to anthelmintic drugs is increasing worldwide and second, many effective nematicides pose environmental hazards. In this paper we address this problem by deploying a high throughput screening platform for anthelmintic drug discovery using the nematode Caenorhabditis elegans as a surrogate for infectious nematodes. This method offers the possibility of identifying new anthelmintics in a cost-effective and timely manner.