DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene DNAJB1-PRKACA (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long noncoding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and in vivo disease models. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, which indicates mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease.

Activation of human STING by a molecular glue-like compound.

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

An Empirical Antigen Selection Method Identifies Neoantigens That Either Elicit Broad Antitumor T-cell Responses or Drive Tumor Growth.

Neoantigens are critical targets of antitumor T-cell responses. The ATLAS bioassay was developed to identify neoantigens empirically by expressing each unique patient-specific tumor mutation individually in Escherichia coli, pulsing autologous dendritic cells in an ordered array, and testing the patient's T cells for recognition in an overnight assay. Profiling of T cells from patients with lung cancer revealed both stimulatory and inhibitory responses to individual neoantigens. In the murine B16F10 melanoma model, therapeutic immunization with ATLAS-identified stimulatory neoantigens protected animals, whereas immunization with peptides associated with inhibitory ATLAS responses resulted in accelerated tumor growth and abolished efficacy of an otherwise protective vaccine. A planned interim analysis of a clinical study testing a poly-ICLC adjuvanted personalized vaccine containing ATLAS-identified stimulatory neoantigens showed that it is well tolerated. In an adjuvant setting, immunized patients generated both CD4+ and CD8+ T-cell responses, with immune responses to 99% of the vaccinated peptide antigens. SIGNIFICANCE: Predicting neoantigens in silico has progressed, but empirical testing shows that T-cell responses are more nuanced than straightforward MHC antigen recognition. The ATLAS bioassay screens tumor mutations to uncover preexisting, patient-relevant neoantigen T-cell responses and reveals a new class of putatively deleterious responses that could affect cancer immunotherapy design.This article is highlighted in the In This Issue feature, p. 521.

Multiplexed detection of mRNA using porosity-tuned hydrogel microparticles.

Transcriptional profiling, which is directly or indirectly associated with expressed protein levels, has been used in various applications including clinical prognosis and pharmaceutical investigation of drug activities. Although the widely used reverse transcription polymerase chain reaction (RT-PCR) allows for the quantification of absolute amounts of mRNA (mRNA) from inputs as small as a single cell, it is an indirect detection method that requires the amplification of cDNA copies of target mRNAs. Here, we report the quantification of unmodified full-length transcripts, using poly(ethylene) glycol diacrylate (PEGDA) hydrogel microparticles synthesized via stop flow lithography (SFL). We show that PEG600 serves as an effective porogen to allow for the capture of large (∼1000-3700 nt long) mRNAs. Our relatively simple hydrogel-based mRNA detection scheme uses a multibiotinylated universal label probe and provides assay performance (limit of detection of ∼6 amol of an in-vitro-transcribed model target) comparable to an existing commercial bead-based technology that uses branched DNA (bDNA) signal amplification. We also demonstrate a 3-plex mRNA detection, without cross-reactivity, using shape-encoded "intraplex" hydrogel microparticles. Our ability to tune the porosity of encoded hydrogel microparticles expands the utility of this platform to now quantify biomacromolecules ranging in size from large mRNAs to small miRNAs.

DNA sequence analysis of the conserved region around the SOD1 gene locus in recessively inherited ALS.

Familial amyotrophic lateral sclerosis (ALS) accounts for 10% of all ALS cases; 12-23% are associated with mutations in the Cu/Zn superoxide dismutase gene (SOD1). All ALS-linked SOD1 mutations present with a dominant pattern of inheritance apart from the aspartate to alanine mutation in exon 4 (D90A). This mutation has been observed in dominant, recessive and apparently sporadically cases. SOD1(D90A/D90A) ALS cases have a very slow disease progression (>10 years), raising the hypothesis that modifier genes linked to SOD1 ameliorate the phenotype of recessively inherited SOD1(D90A/D90A) mutations. Previous sequence analysis of a conserved haplotype region around the SOD1 gene did not reveal any functional polymorphisms within known coding or putative regulatory regions. In the current study we expanded the previous analyses by sequencing the entire SOD1 conserved haplotypic region. Although many polymorphisms were identified, none of these variants explain the slowly progressive phenotype observed in patients with recessive SOD1(D90A) mutations. This study disproves the hypothesis that there is a tightly linked genetic protective factor specifically located close to the SOD1 gene in SOD1(D90A) mediated ALS.

Reduced expression of the Kinesin-Associated Protein 3 (KIFAP3) gene increases survival in sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a degenerative disorder of motor neurons that typically develops in the 6th decade and is uniformly fatal, usually within 5 years. To identify genetic variants associated with susceptibility and phenotypes in sporadic ALS, we performed a genome-wide SNP analysis in sporadic ALS cases and controls. A total of 288,357 SNPs were screened in a set of 1,821 sporadic ALS cases and 2,258 controls from the U.S. and Europe. Survival analysis was performed using 1,014 deceased sporadic cases. Top results for susceptibility were further screened in an independent sample set of 538 ALS cases and 556 controls. SNP rs1541160 within the KIFAP3 gene (encoding a kinesin-associated protein) yielded a genome-wide significant result (P = 1.84 x 10(-8)) that withstood Bonferroni correction for association with survival. Homozygosity for the favorable allele (CC) conferred a 14.0 months survival advantage. Sequence, genotypic and functional analyses revealed that there is linkage disequilibrium between rs1541160 and SNP rs522444 within the KIFAP3 promoter and that the favorable alleles of rs1541160 and rs522444 correlate with reduced KIFAP3 expression. No SNPs were associated with risk of sporadic ALS, site of onset, or age of onset. We have identified a variant within the KIFAP3 gene that is associated with decreased KIFAP3 expression and increased survival in sporadic ALS. These findings support the view that genetic factors modify phenotypes in this disease and that cellular motor proteins are determinants of motor neuron viability.

Variants of the elongator protein 3 (ELP3) gene are associated with motor neuron degeneration.

Amyotrophic lateral sclerosis (ALS) is a spontaneous, relentlessly progressive motor neuron disease, usually resulting in death from respiratory failure within 3 years. Variation in the genes SOD1 and TARDBP accounts for a small percentage of cases, and other genes have shown association in both candidate gene and genome-wide studies, but the genetic causes remain largely unknown. We have performed two independent parallel studies, both implicating the RNA polymerase II component, ELP3, in axonal biology and neuronal degeneration. In the first, an association study of 1884 microsatellite markers, allelic variants of ELP3 were associated with ALS in three human populations comprising 1483 people (P=1.96 x 10(-9)). In the second, an independent mutagenesis screen in Drosophila for genes important in neuronal communication and survival identified two different loss of function mutations, both in ELP3 (R475K and R456K). Furthermore, knock down of ELP3 protein levels using antisense morpholinos in zebrafish embryos resulted in dose-dependent motor axonal abnormalities [Pearson correlation: -0.49, P=1.83 x 10(-12) (start codon morpholino) and -0.46, P=4.05 x 10(-9) (splice-site morpholino), and in humans, risk-associated ELP3 genotypes correlated with reduced brain ELP3 expression (P=0.01). These findings add to the growing body of evidence implicating the RNA processing pathway in neurodegeneration and suggest a critical role for ELP3 in neuron biology and of ELP3 variants in ALS.

50bp deletion in the promoter for superoxide dismutase 1 (SOD1) reduces SOD1 expression in vitro and may correlate with increased age of onset of sporadic amyotrophic lateral sclerosis.

The objective was to test the hypothesis that a described association between homozygosity for a 50bp deletion in the SOD1 promoter 1684bp upstream of the SOD1 ATG and an increased age of onset in SALS can be replicated in additional SALS and control sample sets from other populations. Our second objective was to examine whether this deletion attenuates expression of the SOD1 gene. Genomic DNA from more than 1200 SALS cases from Ireland, Scotland, Quebec and the USA was genotyped for the 50bp SOD1 promoter deletion. Reporter gene expression analysis, electrophoretic mobility shift assays and chromatin immunoprecipitation studies were utilized to examine the functional effects of the deletion. The genetic association for homozygosity for the promoter deletion with an increased age of symptom onset was confirmed overall in this further study (p=0.032), although it was only statistically significant in the Irish subset, and remained highly significant in the combined set of all cohorts (p=0.001). Functional studies demonstrated that this polymorphism reduces the activity of the SOD1 promoter by approximately 50%. In addition we revealed that the transcription factor SP1 binds within the 50bp deletion region in vitro and in vivo. Our findings suggest the hypothesis that this deletion reduces expression of the SOD1 gene and that levels of the SOD1 protein may modify the phenotype of SALS within selected populations.

Analysis of a genetic defect in the TATA box of the SOD1 gene in a patient with familial amyotrophic lateral sclerosis.

We report a patient with autosomal-dominant amyotrophic lateral sclerosis (ALS) and a sequence variation in the SOD1 promoter region, located in the conserved TATA box motif (TATAAA-->TGTAAA). Functional promoter studies of this variant in an in vitro system showed moderate reduction in transcriptional activity of SOD1. This variant was present in only two of 301 individuals with sporadic amyotrophic lateral sclerosis, was not detected in 396 matched controls, and was recently reported in dbSNP (rs7277748). Our data suggest that this TATA box defect is not a disease-causing mutation or susceptibility factor for ALS but rather a rare polymorphism with a potential effect on SOD1 gene expression.

Two approaches to drug discovery in SOD1-mediated ALS.

Familial amyotrophic lateral sclerosis (ALS) accounts for 10% of all ALS cases; approximately 25% of these cases are due to mutations in the Cu/Zn superoxide dismutase gene (SOD1). To date, 105 different mutations spanning all 5 exons have been identified in the SOD1 gene. Mutant SOD1-associated ALS is caused by a toxic gain of function of the mutated protein. Therefore, regardless of the specific mechanism whereby mutant SOD1 initiates motor neuron death, the authors hypothesize that measures that decrease levels of mutant SOD1 protein should ameliorate the phenotype in transgenic mice and potentially in patients with SOD1-mediated disease. They have designed 2 cell-based screening assays to identify small, brain-permeant molecules that inactivate expression of the SOD1 gene or increase the degradation of the SOD1 protein. Here they describe the development and optimization of these assays and the results of high-throughput screening using a variety of compound libraries, including a total of more than 116,000 compounds. The majority of the hit compounds identified that down-regulated SOD1 were shown to be toxic in a cell-based viability assay or were nonselective transcription inhibitors, but work is continuing on a number of nonspecific inhibitors of SOD1 expression. Ultimately, the authors believe that these 2 cell-based assays will provide powerful strategies to identify novel therapies for the treatment of inherited SOD1-associated forms of ALS.

Variants in candidate ALS modifier genes linked to Cu/Zn superoxide dismutase do not explain divergent survival phenotypes.

Familial amyotrophic lateral sclerosis (ALS) accounts for 10% of all ALS cases; approximately 25% are due to mutations in the Cu/Zn superoxide dismutase gene (SOD1). In North America, SOD1(A4V) is the most common SOD1 mutation. A4V ALS cases typically have a very short survival (1-1.5 years versus 3-5 years for other dominant SOD1 mutations). A recent study of A4V carriers identified a common haplotype around the SOD1 locus, suggesting the hypothesis that genetic variations within the haplotypic region might accelerate the course of A4V cases. By contrast, SOD1(D90A/D90A) ALS cases have a very slow progression (>10 years), raising the reciprocal hypothesis that modifier genes linked to SOD1 ameliorate the phenotype of recessively inherited SOD1(D90A/D90A) mutations. In the present study, DNA sequencing of four genes within the haplotypic region shared in A4V and D90A ALS patients revealed 15 novel variants, but none result in changes in amino acid sequences specifically associated with SOD1(D90A/D90A) or SOD1(A4V) ALS. We conclude that mutations within coding regions of genes around the SOD1 locus are not responsible for the more aggressive and more benign natures of the SOD1(A4V) and SOD1(D90A/D90A) mutations, respectively.

Inhibition of SOD1 expression by mitomycin C is a non-specific consequence of cellular toxicity.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative condition that results in the death of the large motor neurons of the brain and spinal cord. Familial ALS accounts for 10% of all ALS cases. Approximately 25% of these cases are due to mutations in the SOD1 gene. Several lines of evidence argue that mutant SOD1 causes ALS by a toxic gain of function. We therefore anticipate that measures that reduce the levels of mutant SOD1 expression should be beneficial in mutant SOD1-associated ALS patients. Mitomycin C (MC) is an antitumor antibiotic previously demonstrated to reduce SOD1 expression in a reporter gene system. We investigated whether MC reduces endogenous SOD1 expression levels both in vitro and in vivo. MC reduced human and rat SOD1 protein levels in vitro, with a concomitant decrease in actin and increase in p53 protein levels, as detected by Western blotting. However, this decrease in SOD1 protein levels was paralleled by a similar decrease in cell viability. In contrast, intracerebroventricular administration of MC to rats and mice failed to produce any effect on brain or spinal cord SOD1 protein levels. Our data indicate the apparent inhibition of SOD1 expression by MC is a non-specific consequence of MC-induced cellular toxicity.

D90A-SOD1 mediated amyotrophic lateral sclerosis: a single founder for all cases with evidence for a Cis-acting disease modifier in the recessive haplotype.

More than 100 different heterozygous mutations in copper/zinc superoxide dismutase (SOD1) have been found in patients with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Uniquely, D90A-SOD1 has been identified in recessive, dominant and apparently sporadic pedigrees. The phenotype of homozygotes is stereotyped with an extended survival, whereas that of affected heterozygotes varies. The frequency of D90A-SOD1 is 50 times higher in Scandinavia (2.5%) than elsewhere, though ALS prevalence is not raised there. Our earlier study indicated separate founders for recessive and dominant/sporadic ALS and we proposed a disease-modifying factor linked to the recessive mutation. Here we have doubled our sample set and employed novel markers to characterise the mutation's origin and localise any modifying factor. Linkage disequilibrium analysis indicates that D90A homozygotes and heterozygotes share a rare haplotype and are all descended from a single ancient founder (alpha 0.974) c.895 generations ago. Homozygotes arose subsequently only c.63 generations ago (alpha 0.878). Recombination has reduced the region shared by recessive kindreds to 97-265 kb around SOD1, excluding all neighbouring genes. We propose that a cis-acting regulatory polymorphism has arisen close to D90A-SOD1 in the recessive founder, which decreases ALS susceptibility in heterozygotes and slows disease progression.