Structural basis of synthetic agonist activation of the nuclear receptor REV-ERB.

The nuclear receptor REV-ERB plays an important role in a range of physiological processes. REV-ERB behaves as a ligand-dependent transcriptional repressor and heme has been identified as a physiological agonist. Our current understanding of how ligands bind to and regulate transcriptional repression by REV-ERB is based on the structure of heme bound to REV-ERB. However, porphyrin (heme) analogues have been avoided as a source of synthetic agonists due to the wide range of heme binding proteins and potential pleotropic effects. How non-porphyrin synthetic agonists bind to and regulate REV-ERB has not yet been defined. Here, we characterize a high affinity synthetic REV-ERB agonist, STL1267, and describe its mechanism of binding to REV-ERB as well as the method by which it recruits transcriptional corepressor both of which are unique and distinct from that of heme-bound REV-ERB.

LXR-inverse agonism stimulates immune-mediated tumor destruction by enhancing CD8 T-cell activity in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype that is untreatable with hormonal or HER2-targeted therapies and is also typically unresponsive to checkpoint-blockade immunotherapy. Within the tumor microenvironment dysregulated immune cell metabolism has emerged as a key mechanism of tumor immune-evasion. We have discovered that the Liver-X-Receptors (LXRα and LXRß), nuclear receptors known to regulate lipid metabolism and tumor-immune interaction, are highly activated in TNBC tumor associated myeloid cells. We therefore theorized that inhibiting LXR would induce immune-mediated TNBC-tumor clearance. Here we show that pharmacological inhibition of LXR activity induces tumor destruction primarily through stimulation of CD8+ T-cell cytotoxic activity and mitochondrial metabolism. Our results imply that LXR inverse agonists may be a promising new class of TNBC immunotherapies.

Evolution of mitotic spindle behavior during the first asymmetric embryonic division of nematodes.

Asymmetric cell division is essential to generate cellular diversity. In many animal cells, the cleavage plane lies perpendicular to the mitotic spindle, and it is the spindle positioning that dictates the size of the daughter cells. Although some properties of spindle positioning are conserved between distantly related model species and different cell types, little is known of the evolutionary robustness of the mechanisms underlying this event. We recorded the first embryonic division of 42 species of nematodes closely related to Caenorhabditis elegans, which is an excellent model system to study the biophysical properties of asymmetric spindle positioning. Our recordings, corresponding to 128 strains from 27 Caenorhabditis and 15 non-Caenorhabditis species (accessible at http://www.ens-lyon.fr/LBMC/NematodeCell/videos/), constitute a powerful collection of subcellular phenotypes to study the evolution of various cellular processes across species. In the present work, we analyzed our collection to the study of asymmetric spindle positioning. Although all the strains underwent an asymmetric first cell division, they exhibited large intra- and inter-species variations in the degree of cell asymmetry and in several parameters controlling spindle movement, including spindle oscillation, elongation, and displacement. Notably, these parameters changed frequently during evolution with no apparent directionality in the species phylogeny, with the exception of spindle transverse oscillations, which were an evolutionary innovation at the base of the Caenorhabditis genus. These changes were also unrelated to evolutionary variations in embryo size. Importantly, spindle elongation, displacement, and oscillation each evolved independently. This finding contrasts starkly with expectations based on C. elegans studies and reveals previously unrecognized evolutionary changes in spindle mechanics. Collectively, these data demonstrate that, while the essential process of asymmetric cell division has been conserved over the course of nematode evolution, the underlying spindle movement parameters can combine in various ways. Like other developmental processes, asymmetric cell division is subject to system drift.

Pharmacological inhibition of REV-ERB stimulates differentiation, inhibits turnover and reduces fibrosis in dystrophic muscle.

Duchenne muscular dystrophy (DMD) is a debilitating X-linked disorder that is fatal. DMD patients lack the expression of the structural protein dystrophin caused by mutations within the DMD gene. The absence of functional dystrophin protein results in excessive damage from normal muscle use due to the compromised structural integrity of the dystrophin associated glycoprotein complex. As a result, DMD patients exhibit ongoing cycles of muscle destruction and regeneration that promote inflammation, fibrosis, mitochondrial dysfunction, satellite cell (SC) exhaustion and loss of skeletal and cardiac muscle function. The nuclear receptor REV-ERB suppresses myoblast differentiation and recently we have demonstrated that the REV-ERB antagonist, SR8278, stimulates muscle regeneration after acute injury. Therefore, we decided to explore whether the REV-ERB antagonist SR8278 could slow the progression of muscular dystrophy. In mdx mice SR8278 increased lean mass and muscle function, and decreased muscle fibrosis and muscle protein degradation. Interestingly, we also found that SR8278 increased the SC pool through stimulation of Notch and Wnt signaling. These results suggest that REV-ERB is a potent target for the treatment of DMD.

Biallelic mutation of UNC50, encoding a protein involved in AChR trafficking, is responsible for arthrogryposis.

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Homozygosity mapping of disease loci combined with whole exome sequencing in a consanguineous family presenting with lethal AMC allowed the identification of a homozygous frameshift deletion in UNC50 gene (c.750_751del:p.Cys251Phefs*4) in the index case. To assess the effect of the mutation, an equivalent mutation in the Caenorhabditis elegans orthologous gene was created using CRISPR/Cas9. We demonstrated that unc-50(kr331) modification caused the loss of acetylcholine receptor (AChR) expression in C. elegans muscle. unc-50(kr331) animals were as resistant to the cholinergic agonist levamisole as unc-50 null mutants suggesting that AChRs were no longer expressed in this animal model. This was confirmed by using a knock-in strain in which a red fluorescent protein was inserted into the AChR locus: no signal was detected in unc-50(kr331) background, suggesting that UNC-50, a protein known to be involved in AChR trafficking, was no longer functional. These data indicate that biallelic mutation in the UNC50 gene underlies AMC through a probable loss of AChR expression at the neuromuscular junction which is essential for the cholinergic transmission during human muscle development.

Scaling, selection, and evolutionary dynamics of the mitotic spindle.

BACKGROUND: Cellular structures such as the nucleus, Golgi, centrioles, and spindle show remarkable diversity between species, but the mechanisms that produce these variations in cell biology are not known. RESULTS: Here we investigate the mechanisms that contribute to variations in morphology and dynamics of the mitotic spindle, which orchestrates chromosome segregation in all Eukaryotes and positions the division plane in many organisms. We use high-throughput imaging of the first division in nematodes to demonstrate that the measured effects of spontaneous mutations, combined with stabilizing selection on cell size, are sufficient to quantitatively explain both the levels of within-species variation in the spindle and its diversity over ∼100 million years of evolution. Furthermore, our finding of extensive within-species variation for the spindle demonstrates that there is not just one "wild-type" form, rather that cellular structures can exhibit a surprisingly broad diversity of naturally occurring behaviors. CONCLUSIONS: Our results argue that natural selection acts predominantly on cell size and indirectly influences the spindle through the scaling of the spindle with cell size. Previous studies have shown that the spindle also scales with cell size during early development. Thus, the scaling of the spindle with cell size controls its variation over both ontogeny and phylogeny.