Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS) results from mutations in spermine synthase (SMS), which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonia, and seizures. Symptom management is the only treatment. Reduced SMS activity causes spermidine accumulation while spermine levels are reduced. The resulting exaggerated spermidine:spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity. Our studies aim to pharmacologically manipulate polyamine metabolism to correct this imbalance as a therapeutic strategy for SRS. Here we report the repurposing of 2-difluoromethylornithine (DFMO), an FDA-approved inhibitor of polyamine biosynthesis, in rebalancing spermidine:spermine ratios in SRS patient cells. Mechanistic in vitro studies demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of spermidine into spermine in hypomorphic SMS cells and induces uptake of exogenous spermine, altogether reducing the aberrant ratios. In a Drosophila SRS model characterized by reduced lifespan, DFMO improves longevity. As nearly all SRS patient mutations are hypomorphic, these studies form a strong foundation for translational studies with significant therapeutic potential.

Phenylbutyrate modulates polyamine acetylase and ameliorates Snyder-Robinson syndrome in a Drosophila model and patient cells.

Polyamine dysregulation plays key roles in a broad range of human diseases from cancer to neurodegeneration. Snyder-Robinson syndrome (SRS) is the first known genetic disorder of the polyamine pathway, caused by X-linked recessive loss-of-function mutations in spermine synthase. In the Drosophila SRS model, altered spermidine/spermine balance has been associated with increased generation of ROS and aldehydes, consistent with elevated spermidine catabolism. These toxic byproducts cause mitochondrial and lysosomal dysfunction, which are also observed in cells from SRS patients. No efficient therapy is available. We explored the biochemical mechanism and discovered acetyl-CoA reduction and altered protein acetylation as potentially novel pathomechanisms of SRS. We repurposed the FDA-approved drug phenylbutyrate (PBA) to treat SRS using an in vivo Drosophila model and patient fibroblast cell models. PBA treatment significantly restored the function of mitochondria and autolysosomes and extended life span in vivo in the Drosophila SRS model. Treating fibroblasts of patients with SRS with PBA ameliorated autolysosome dysfunction. We further explored the mechanism of drug action and found that PBA downregulates the first and rate-limiting spermidine catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), reduces the production of toxic metabolites, and inhibits the reduction of the substrate acetyl-CoA. Taken together, we revealed PBA as a potential modulator of SAT1 and acetyl-CoA levels and propose PBA as a therapy for SRS and potentially other polyamine dysregulation-related diseases.

NMNAT promotes glioma growth through regulating post-translational modifications of P53 to inhibit apoptosis.

Gliomas are highly malignant brain tumors with poor prognosis and short survival. NAD+ has been shown to impact multiple processes that are dysregulated in cancer; however, anti-cancer therapies targeting NAD+ synthesis have had limited success due to insufficient mechanistic understanding. Here, we adapted a Drosophila glial neoplasia model and discovered the genetic requirement for NAD+ synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) in glioma progression in vivo and in human glioma cells. Overexpressing enzymatically active NMNAT significantly promotes glial neoplasia growth and reduces animal viability. Mechanistic analysis suggests that NMNAT interferes with DNA damage-p53-caspase-3 apoptosis signaling pathway by enhancing NAD+-dependent posttranslational modifications (PTMs) poly(ADP-ribosyl)ation (PARylation) and deacetylation of p53. Since PARylation and deacetylation reduce p53 pro-apoptotic activity, modulating p53 PTMs could be a key mechanism by which NMNAT promotes glioma growth. Our findings reveal a novel tumorigenic mechanism involving protein complex formation of p53 with NAD+ synthetic enzyme NMNAT and NAD+-dependent PTM enzymes that regulates glioma growth.

One of the most common types of brain cancer, glioma, emerges when harmful mutations take place in the 'glial' cells tasked with supporting neurons. When these genetically damaged cells are not fixed or eliminated, they can go on to multiply uncontrollability. A protein known as p53 can help to repress emerging tumors by stopping mutated cells in their tracks. Glioma is a highly deadly cancer, and treatments are often ineffective. Some of these approaches have focused on a protein involved in the creation of the coenzyme NAD+, which is essential to the life processes of all cells. However, these drugs have had poor outcomes. Instead, Liu et al. focused on NMNAT, the enzyme that participates in the final stage of the creation of NAD+. NMNAT is known to protect neurons, but it is unclear how it involved in cancer. Experiments in fruit flies which were then validated in human glioma cells showed that increased NMNAT activity allowed glial cells with harmful mutations to survive and multiply. Detailed molecular analysis showed that NMNAT orchestrates chemical modifications that inactivate p53. It does so by working with other molecular actors to direct NAD+ to add and remove chemical groups that control the activity of p53. Taken together, these results show how NMNAT can participate in the emergence of brain cancers. They also highlight the need for further research on whether drugs that inhibit this enzyme could help to suppress tumors before they become deadly.

Stress-Induced Low Complexity RNA Activates Physiological Amyloidogenesis.

Amyloid bodies (A-bodies) are inducible membrane-less nuclear compartments composed of heterogeneous proteins that adopt an amyloid-like state. A-bodies are seeded by noncoding RNA derived from stimuli-specific loci of the rDNA intergenic spacer (rIGSRNA). This raises the question of how rIGSRNA recruits a large population of diverse proteins to confer A-body identity. Here, we show that long low-complexity dinucleotide repeats operate as the architectural determinants of rIGSRNA. On stimulus, clusters of rIGSRNA with simple cytosine/uracil (CU) or adenosine/guanine (AG) repeats spanning hundreds of nucleotides accumulate in the nucleolar area. The low-complexity sequences facilitate charge-based interactions with short cationic peptides to produce multiple nucleolar liquid-like foci. Local concentration of proteins with fibrillation propensity in these nucleolar foci induces the formation of an amyloidogenic liquid phase that seeds A-bodies. These results demonstrate the physiological importance of low-complexity RNA and repetitive regions of the genome often dismissed as "junk" DNA.

Tristetraprolin Recruits Eukaryotic Initiation Factor 4E2 To Repress Translation of AU-Rich Element-Containing mRNAs.

Tristetraprolin (TTP) regulates the expression of AU-rich element-containing mRNAs through promoting the degradation and repressing the translation of target mRNA. While the mechanism for promoting target mRNA degradation has been extensively studied, the mechanism underlying translational repression is not well established. Here, we show that TTP recruits eukaryotic initiation factor 4E2 (eIF4E2) to repress target mRNA translation. TTP interacted with eIF4E2 but not with eIF4E. Overexpression of eIF4E2 enhanced TTP-mediated translational repression, and downregulation of endogenous eIF4E2 or overexpression of a truncation mutant of eIF4E2 impaired TTP-mediated translational repression. Overexpression of an eIF4E2 mutant that lost the cap-binding activity also impaired TTP's activity, suggesting that the cap-binding activity of eIF4E2 is important in TTP-mediated translational repression. We further show that TTP promoted eIF4E2 binding to target mRNA. These results imply that TTP recruits eIF4E2 to compete with eIF4E to repress the translation of target mRNA. This notion is supported by the finding that downregulation of endogenous eIF4E2 increased the production of tumor necrosis factor alpha (TNF-α) protein without affecting the mRNA levels in THP-1 cells. Collectively, these results uncover a novel mechanism by which TTP represses target mRNA translation.