The Flowering Repressor SVP recruits the TOPLESS co-repressor to control flowering in chrysanthemum and Arabidopsis

Plant flowering time is a consequence of the perception of environmental and endogenous signals. The MCM1-AGAMOUSDEFICIENS-SRF-box (MADS-box) gene SHORT VEGETATIVE PHASE (SVP) is a pivotal repressor that negatively regulates the floral transition during the vegetative phase. The transcriptional corepressor TOPLESS (TPL) plays critical roles in many aspects of plant life. An interaction first identified between the second LXLXLX motif (LRLGLP) of CmSVP with CmTPL1-2, which can repress the expression of a key flowering factor CmFTL3 by binding its promotor CArG element in chrysanthemum. Genetic analysis suggested that the CmSVP-CmTPL1-2 transcriptional complex is a prerequisite for SVP to act as a floral repressor, which reduces CmFTL3 transcriptional activity. CmSVP rescued the phenotype of the svp-31 mutant in Arabidopsis, and overexpression of AtSVP or CmSVP in the Arabidopsis dominant negative mutation tpl-1 led to a loss-of-function in late flowering, which confirmed the highly conserved function of SVP in the two completely different species. Thus, we have validated a conserved machinery wherein SVP relies on TPL to inhibit flowering through the direct regulation of FT, which is more meaningful for the evolution of species and could be translated to high-quality cultivation and breeding of crops.

EXTH-30. HARNESSING CELLULAR STRESS FOR IMMUNE TARGETING OF DIPGS

Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor. An almost ubiquitous dominant negative mutation at lysine (K)-27 in genes encoding histone genes HIST1H3B and H3F3A found in patient tumors is a driver of DIPG development. ONC201, a small molecule DRD2 antagonist and ClpP agonist developed by Chimerix Inc, targets the unfolded protein response (UPR) and integrated stress response (ISR) signaling. It is under clinical investigation in patients with recurrent H3K27M DMGs. In adults, single agent studies have shown durable objective responses when administered orally. A multi-arm, non-randomized multi-institutional Phase I clinical trial (NCT03416530) for pediatric patients with H3K27M DMGs is open and accruing. Preliminary results suggest that the drug has a favorable safety profile and holds promise for patients with DIPGs and other midline gliomas. However, the mechanism of action of ONC201 against DIPGs warrants further study. Here, we show that ONC201 is cytotoxic to DIPGs in vitro and in vivo. RNA Seq analyses revealed cell context specific deployment of PERK-activated UPR and calcium signaling-associated RON tyrosine kinase-macrophage stimulating protein (MSP) signaling in DIPGs. Single cell proteomic assays revealed substantial heterogeneity in the sensitivity of DIPG cells to ONC201, and identified stem-like sub-populations of H3K27M DIPGs with intrinsic insensitivity to the drug. ONC201 treatment, which induces cellular stress, also sensitized DIPG cells to cytolytic activity by ex-vivo expanded and activated innate immune cells, in vitro. Ongoing in vivo experiments are expected to support a novel investigational study in patients with midline gliomas.

Porcine deltacoronavirus enters porcine IPI-2I intestinal epithelial cells via macropinocytosis and clathrin-mediated endocytosis dependent on pH and dynamin

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhoea in suckling piglets and has the potential for cross-species transmission. Although extensive studies have been reported on the biology and pathogenesis of PDCoV, the mechanisms by which PDCoV enters cells are not well characterized. In this study, we investigated how PDCoV enters IPI-2I cells, a line of porcine intestinal epithelial cells derived from pig ileum. Immunofluorescence assays, siRNA interference, specific pharmacological inhibitors and dominant-negative mutation results revealed that PDCoV entry into IPI-2I cells depended on clathrin, dynamin and a low-pH environment, but was independent of caveolae. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) and the Na + /H + exchanger (NHE) revealed that PDCoV entry involves macropinocytosis and depends on NHE rather than on PI3K. Additionally, Rab5 and Rab7, but not Rab11, regulated PDCoV endocytosis. This is the first study to demonstrate that PDCoV uses clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter porcine intestinal epithelial cells. We also discussed the entry pathways of PDCoV into other porcine cell lines. Our findings reveal the entry mechanisms of PDCoV and provide new insight into the PDCoV life cycle. IMPORTANCE An emerging enteropathogenic coronavirus, PDCoV has the potential for cross-species transmission, attracting extensive attenuation. Characterizing the detailed process of PDCoV entry into cells will deepen our understanding of the viral infection and pathogenesis, and provide the clues for therapeutic intervention against PDCoV. With the objective, we used complementary approaches to dissect the process in PDCoV-infected IPI-2I cells, a line of more physiologically relevant intestinal epithelial cells to PDCoV infection in vivo. Here, we demonstrate that PDCoV enters IPI-2I cells via macropinocytosis that does not require a specific receptor and clathrin-mediated endocytosis that requires a low-pH environment and dynamin, while a caveola-mediated endocytic pathway is used by PDCoV to enter swine testicular (ST) cells and porcine kidney (LLC-PK1) cells. These findings provide a molecular detail of the cellular entry pathways of PDCoV and may direct us toward novel antiviral drug development.

PF-06409577 Activates AMPK Signaling and Inhibits Osteosarcoma Cell Growth

Osteosarcoma (OS) is a common primary bone malignancy. We here investigated the potential activity of PF-06409577, a novel, potent, and direct activator of AMP-activated protein kinase (AMPK), against human OS cells. In established (U2OS, MG-63, and SaOs-2 lines) and primary human OS cells, PF-06409577 inhibited cell viability and proliferation, while inducing cell apoptosis and cell cycle arrest. PF-06409577 induced AMPK activation, mTORC1 inhibition, autophagy induction, and downregulation of multiple receptor tyrosine kinase inOS cells. AMPK inactivation by AMPKα1 shRNA, CRISPR/Cas9 knockout, or dominant negative mutation (T172A) was able to abolish PF-06409577-induced activity in OS cells. In vivo, PF-06409577 oral administration at well-tolerated doses potently inhibited growth of U2OS cells and primary human OS cells in severe combined immunodeficient mice. AMPK activation, mTORC1 inhibition, autophagy induction, as well as RTK degradation and apoptosis activation were detected in PF-06409577-treated xenografts. In conclusion, activation of AMPK by PF-06409577 inhibits OS cell growth.

Geminivirus betasatellite-encoded βC1 protein exhibits novel ATP hydrolysis activity that influences its DNA-binding activity and viral pathogenesis

Plant virus satellites are maintained by their associated helper viruses and satellites influence viral pathogenesis. Diseases caused by geminivirus-betasatellite complexes can become epidemics and therefore have become a threat to economically important crops across the world. Here, we identified a novel molecular function of the betasatellite-encoded pathogenicity determinant βC1. The tomato leaf curl Patna betasatellite (ToLCPaB)-encoded βC1 protein was found to exhibit novel ATPase activity in the presence of the divalent metal ion cofactor MgCl 2 . Moreover, ATPase activity was confirmed to be ubiquitously displayed by βC1 proteins encoded by diverse betasatellites. Mutational and sequence analysis revealed conserved lysine/arginine residues at positions 49/50 and 91 of βC1 proteins to be essential for their ATPase activity. Biochemical studies revealed the DNA-binding activity of the βC1 protein was interfered by the binding of ATP to the protein. Mutating arginine 91 of βC1 to alanine reduced its DNA-binding activity. The results of docking studies provided evidence for an overlap of the ATP-binding and DNA-binding regions of βC1 and for the importance of arginine 91 for both ATP-binding and DNA-binding activities. A mutant betasatellite with a specifically βC1-ATPase dominant negative mutation was found to induce symptoms on Nicotiana benthamiana plants similar to those induced by wild-type betasatellite infection. The ATPase function of βC1 was found to be negatively associated with geminivirus-betasatellite DNA accumulation, despite the positive influence of this ATPase function on the accumulation of replication-associated protein (Rep) and βC1 transcripts. Importance Most satellites influence the pathogenesis of their helper viruses. Here we characterized the novel molecular function of βC1, a non-structural, pathogenicity determinant protein encoded by a betasatellite. Here, we demonstrated the display of ATPase activity by this βC1 protein. Additionally, we confirmed the ubiquitous display of ATPase activity by βC1 proteins encoded by diverse betasatellites. The lysine/arginine residues conserved at positions 49 and 91 of βC1 were found to be crucial for its ATPase function. DNA-binding activity of βC1 was found to be reduced in the presence of ATP. Inhibition of ATPase activity of βC1 in the presence of an excess concentration of cold ATP, GTP, CTP or UTP suggested that the purified βC1 can also hydrolyze other cellular NTPs besides ATP in vitro. These results established the importance of the ATPase and DNA-binding activities of the βC1 protein in regulating the geminivirus-betasatellite DNA accumulation in the infected plant cell.

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.

Spreading and epigenetic inheritance of heterochromatin require a critical density of histone H3 lysine 9 tri-methylation

Heterochromatin assembly requires methylation of histone H3 lysine 9 (H3K9me) and serves as a paradigm for understanding the importance of histone modifications in epigenetic genome control. Heterochromatin is nucleated at specific genomic sites and spreads across extended chromosomal domains to promote gene silencing. Moreover, heterochromatic structures can be epigenetically inherited in a self-templating manner, which is critical for stable gene repression. The spreading and inheritance of heterochromatin are believed to be dependent on preexisting H3K9 tri-methylation (H3K9me3), which is recognized by the histone methyltransferase Clr4/Suv39h via its chromodomain, to promote further deposition of H3K9me. However, the process involving the coupling of the “read” and “write” capabilities of histone methyltransferases is poorly understood. From an unbiased genetic screen, we characterize a dominant-negative mutation in histone H3 (H3G13D) that impairs the propagation of endogenous and ectopic heterochromatin domains in the fission yeast genome. H3G13D blocks methylation of H3K9 by the Clr4/Suv39h methyltransferase and acts in a dosage-dependent manner to interfere with the spreading and maintenance of heterochromatin. Our analyses show that the incorporation of unmethylatable histone H3G13D into chromatin decreases H3K9me3 density and thereby compromises the read-write capability of Clr4/Suv39h. Consistently, enhancing the affinity of Clr4/Suv39h for methylated H3K9 is sufficient to overcome the defects in heterochromatin assembly caused by H3G13D. Our work directly implicates methylated histones in the transmission of epigenetic memory and shows that a critical density threshold of H3K9me3 is required to promote epigenetic inheritance of heterochromatin through the read-write mechanism.

Genomic profiling of KRAS wide-type pancreatic ductal adenocarcinomas identifies targetable genetic alterations.

4121 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and the 5-year survival of PDAC patients is below 10%. The oncogenic KRASmutations account for about 90% of PDAC cases. Unfortunately, there is no FDA-approved targeted therapy for KRAS mutations. Therefore, the genomic profiling of KRAS wide-type PDACs can provide invaluable sights to the etiology of these patients and offer them the opportunity of precision therapy trials. Methods: To characterize actionable targets in 521 Chinese PDAC patients, deep sequencing of a 831-gene panel (OncoPanscan, Genetronhealth) was applied to assess somatic mutations of their tumor tissues including SNV, insertions/deletions, CNV and re-arrangements, as well as possible pathogenic germline variants of paired genomic DNA sample. Results: There were 89% (463/521) of patients in our PDAC cohort harbored KRAS mutations. Among the remaining 58 patients in KRAS wild-type subgroup, 33% (19/58) carried activating mutations in the RTK/Ras/MAPK pathway. Targetable BRAF mutations were seen in five (9%) patients: V600E (1/5), G464V (1/5), N486_P490del (2/5) and BRAF fusion (1/5). The frequency of BRAF mutations was 9% (5/58) in KRAS wild-type PDACs but only 0.4% (2/463) in KRAS-mutated PDACs (P < 0.001). We found one classic EGFR activing mutation (L747_A750delinsP) and one MAP2K1 activating mutation (F53_Q58delinsL), which can be targeted by EGFR-TKIs and MEK inhibitor trametinib, respectively. An oncogenic ERBB3 mutation (V104L) was seen in one patient, who was eligible for HER2-targeted therapy clinical trials. We also found STK11/TSC2 inactivating mutations and a dominant-negative mutation of PTEN (R130Q) which could be targeted by mTOR inhibitor everolimus and AKT inhibitor capivasertib, respectively. Additionally, we observed a patient with high level amplification of MET and another patient with the NCOA4-RET fusion gene which can be targeted by MET inhibitor carbozantinib. Interestingly, we also identified two patients with inactivating mutations in ELF3 (one frameshift and one in splicing-site), which is a driver gene of ampullary carcinoma. Lastly, two patients carried deleterious germline mutations in BRCA1 and PALB2, which may be targeted with PARP inhibitors. Overall, at least 29% (17/58) KRAS wide-type patients harbored potentially actionable genomic alterations to currently used anticancer drugs. Conclusions: The mutational landscape of our PDAC cohort provided compelling evidence that targetable driver mutations accounted for a significant portion of KRAS wide-type tumors. Our findings demonstrated that genomic profiling of PDAC patients can enable physicians to optimize their clinical management and enroll them into genomically matched clinical trials.

The Role of Alcohol, LPS Toxicity, and ALDH2 in Dental Bony Defects

It is estimated that 560 million people carry an East Asian-specific ALDH2*2 dominant-negative mutation which leads to enzyme inactivation. This common ALDH2 polymorphism has a significant association with osteoporosis. We hypothesized that the ALDH2*2 mutation in conjunction with periodontal Porphyromonas gingivalis bacterial infection and alcohol drinking had an inhibitory effect on osteoblasts and bone regeneration. We examined the prospective association of ALDH2 activity with the proliferation and mineralization potential of human osteoblasts in vitro. The ALDH2 knockdown experiments showed that the ALDH2 knockdown osteoblasts lost their proliferation and mineralization capability. To mimic dental bacterial infection, we compared the dental bony defects in wild-type mice and ALDH2*2 knockin mice after injection with purified lipopolysaccharides (LPS), derived from P. gingivalis which is a bacterial species known to cause periodontitis. Micro-computed tomography (micro-CT) scan results indicated that bone regeneration was significantly affected in the ALDH2*2 knockin mice with about 20% more dental bony defects after LPS injection than the wild-type mice. Moreover, the ALDH2*2 knockin mutant mice had decreased osteoblast growth and more dental bone loss in the upper left jaw region after LPS injection. In conclusion, these results indicated that the ALDH2*2 mutation with alcohol drinking and chronic exposure to dental bacterial-derived toxin increased the risk of dental bone loss.