Deregulation of the Host Cell Cycle Induced by SARS‐CoV‐2 Nucleocapsid Protein

Introduction: The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARSCoV2) is the causative agent responsible for the COVID-19 pandemic and has resulted in devastating impacts on global public health. The nucleocapsid (N) protein of other coronaviruses, such as SARS-CoV-1, have been previously implicated in the deregulation of the host cell cycle through interactions with cell cycle checkpoint proteins, Cyclin-Dependent Kinases (CDKs) or cyclins. In this study, we highlight the role of SARS-CoV-2 N-protein in modulating CDK expression, thereby, deregulating the host cell cycle. Methods: A549 cells were transfected with pCMV plasmids, harbouring the SARS-CoV-2 N-protein. Protein extracts of control and Nprotein transfected cells were electrophoresed on SDS-PAGE, transferred onto a nitrocellulose membrane and incubated with CDK2 and CDK4 antibodies. The blots were visualized and protein quantification was performed using ImageJ analysis. Results: Transfection of SARS-CoV-2 N resulted in differential expression of CDK2 and CDK4, which are key regulators that drive cell cycle progression through G0 or G1 phase into S phase. Notably, preliminary findings also demonstrate that N protein results in decreased CDK2 and CDK4 expression. Conclusion: The differential expression of CDKs caused by SARS-CoV-2 N-protein suggests its role in inducing cell cycle arrest at the S phase to facilitate SARS-CoV-2 replication. The results from this research may aid in future characterisation of the mechanisms for SARS-CoV-2-mediated cell cycle arrest, and contribute towards the development of novel antiviral strategies and therapies.

Oral Covid-19 Antiviral Candidate LAU-7b Demonstrates 100[percnt] Reduction of the Risk of Progression to Mechanical Ventilation and Death in Hospitalized Moderate-to-Severe Covid-19 Patients

Rationale: LAU-7b is developed as a broadly effective oral COVID-19 therapeutic targeting membrane lipids to exert dual antiviral and inflammation-controlling activity. SARS-CoV-2 reprograms host cellular lipid metabolism to favor entry and replication, a mechanism shared by all lipid-enveloped viruses. LAU-7b decreases host cell membrane lipids fluidity, inhibits de-novo cell lipogenesis, and modulates phospholipid signaling promoting resolution of inflammation. Due to its host-directed mutation-agnostic mechanism, LAU-7b utility could span across future variants, as demonstrated in-vitro against multiple SARS-CoV-2 strains and MERS-CoV. RESOLUTION, a large Phase 2/3 study evaluating LAU-7b in hospitalized COVID-19 patients, is ongoing in the US and Canada, and preliminary Phase 2 results are presented. Methods: RESOLUTION is a placebocontrolled study of oral LAU-7b, once-a-day for 14 days on top of standard of care, in hospitalized COVID-19 patients at risk of developing pulmonary complications. The Phase 2 portion of the study randomized 148 patients with moderate-to-severe COVID-19 and 84 patients in critical condition, but not on invasive ventilation. Key endpoints included proportion of patients alive and free of respiratory failure at Day 29, rates of progression to mechanical ventilation and all-causes death by Day 60, time to recovery and length of hospitalization. Results: Both study arms were highly comparable in terms of mean age, number of comorbidities and concomitant medications. LAU-7b demonstrated a 100% reduction in the risk of progressing to mechanical ventilation or death by Day 60 in moderate-to-severe COVID-19 patients. None of the 76 patients on LAU-7b required mechanical ventilation and none died, while 5 out of 72 patients on placebo progressed to mechanical ventilation (6.9% difference, p=0.025), and 4 patients died (5.6% difference, p=0.053). LAU-7b group also showed an increase of 6.9% (p=0.055) in the proportion of patients alive and free of respiratory failure at Day 29, versus placebo. Patients on LAU-7b tended to recover more rapidly and leave hospital faster. LAU-7b was well-tolerated, with safety comparable to placebo. Critically ill patients treated with LAU-7b did not show improvement over placebo, suggesting that COVID-19 patients in respiratory failure at baseline are too severely affected to benefit. Conclusion: LAU-7b showed positive results in the trial's Phase 2 portion on both survival and avoidance of mechanical ventilation in moderate-to-severe COVID-19. The confirmatory Phase 3 portion was triggered and received approval from the FDA and Health Canada, focusing on moderate-to-severe COVID-19 and using the “Proportion of patients requiring mechanical ventilation and/or death by Day 60” as primary efficacy endpoint.

Pharmacologic inhibition of SUMOactivating enzyme potentiates interferon response and T cell-mediated anti-tumor immunity in chronic lymphocytic leukemia (CLL) and lymphoma models

Introduction: CLL is characterized by deficient immunity which clinically manifests as an increased predisposition toward malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with a predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Unlike chemotherapy, novel targeted agents may have beneficial immunomodulatory effects, which may be particularly relevant in the COVID-19 era. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription, and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFjB signaling, IFN response, and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in the context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. Activation, proliferation, and apoptosis of CD3+ T cells were studied following T-cell receptor engagement (TCR;aCD3/CD28) with/without 0-1 lM TAK-981. Cytokines were measured after in vitro stimulation. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP;Clariom S), RNA was harvested after 3 and 24 h of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 mg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days before spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1×106 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement. Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels, yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFjB, and NFAT activation detected by immunoblotting, immunocytochemistry, and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4+ T cells following 72 and 96 h of TCR stimulation vs. control. Meanwhile, the expression of CD25, HLA-DR, and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced 2-fold in TAK-981-treated cells after 24 h and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 lM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL- 2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNc and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift toward Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4+ naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNc, and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in express on of FoxP3 and an increased production of IFNc. In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNc secretion in CD8+ TILs. Conclusion: Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies.

Does Investing in Renewable Energy Sources Contribute to Growth? A Preliminary Study on Greece’s National Energy and Climate Plan

As more economies are transitioning away from fossil fuels for their electricity production and towards greener alternatives, many socioeconomic implications of this shift remain actively debated. The present paper attempts to assess the economic impact of investments in renewable energy sources (RESs) for Greece and whether the broader effects of this transition can offset the negative impact that will occur due to the targeted phase-out of lignite plants by 2028, which constitute the predominant power source for Greece. Our methodological approach builds on input–output analysis and the creation of composite RES industries for the estimation of the net effects of a series of monetary shocks that correspond to Greece’s phase-out investment plan, utilizing the most recent national input–output tables and satellite structural business statistics. We focus on the structural effects of these shocks on a series of socioeconomic indicators, including GDP, employment, wages, government income (through taxes), and capital formation. The results indicate that even though lignite power production still provides a significant contribution to the Greek economy, investing in renewables presents a significant opportunity for value added and job creation.

Low-Dose Weekly Decitabine and Venetoclax in TP53-Mutated Myeloid Malignancies

Decitabine (Dec) and Azacitidine (Aza) that target DNA methyltransferase 1 (DNMT1) are hypomethylating agents (HMAs) approved to treat acute myeloid leukemia (AML) in combination with Venetoclax (Ven). The combination is also used to treat high-risk myelodysplastic syndromes, especially TP53-mutated (TP53mut) cases in which responses to HMA alone are short-lived. In most patients (pts), however, myelosuppression from treatment leads to frequent Ven duration and/or dose-reductions, and/or cycle delays. An approach to decrease HMA-mediated myelosuppression but maintain S-phase dependent DNMT1-targeting, evaluated in a previous clinical trial (https://doi.org/10.1111/bjh.16281), is to administer noncytotoxic doses/concentrations of Dec (0.2 mg/kg;~5 mg/m 2) by a frequent-distributed schedule of 1X/week. An approach to decrease Ven mediated myelosuppression but maintain cooperation with HMA, shown in pre-clinical studies, is to administer a single-dose prior to HMA. Ven can depolarize mitochondrial membranes;mitochondrial membrane-potential is essential to function of the mitochondrial enzyme DHODH that produces cytidine/deoxycytidine that competes with HMA in cells. Thus, Ven prior to HMA dosing temporarily inhibits de novo pyrimidine synthesis, to counter a major mechanism of resistance to HMA in MDS/AML, without suppressing normal myelopoiesis (https://doi.org/10.1182/blood-2020-143200). We conducted a retrospective analysis of all pts with TP53mut MDS or AML treated with weekly Ven and low-dose subcutaneous Dec at our institution. We analyzed the characteristics of these pts, response to therapy, and outcomes using standard descriptive statistics. Mutational testing was performed using a commercial next-generation sequencing (NGS) panel. Five pts, 3 male and 2 female, with TP53mut MDS or AML were treated with weekly Ven 400 mg on D1 and subcutaneous Dec 0.2 mg/kg on D2, administered weekly in 28 day cycles. Two pts had MDS (1 de novo, 1 treatment related) and 3 pts had AML (1 de novo, 2 secondary from prior MDS). Four pts (80%) received the treatment in frontline, all with poor performance status (PS), and 1 pt (20%) had R/R disease. Median age at diagnosis was 79 years [41-82]. The only young pt had prolonged severe cytopenias after 1 cycle Dec standard dosing during the peak of COVID-19 pandemic so was switched to this regimen. Of the 4 frontline treated pts, 2 pts had high-risk MDS, and 2 pts had adverse risk AML. The R/R pt had high-risk MDS transformed to AML that was refractory to 2 prior lines of therapy: standard Aza/Ven x5 cycles, then standard Vyxeos. Disease cytogenetics were complex in all pts. 60% (3/5) pts had sole TP53mut on NGS, with median variant allelic frequency (VAF) 48% [28-79]. 80% (4/5) pts were transfusion dependent prior to treatment. Median time to initiating therapy was 7 days from initial or refractory diagnosis [3-59] and median follow-up was 7.8 months (mo) [2.9-11.4]. The overall response rate (ORR) was 100%: 4/4 frontline pts had complete remissions (CR), and the 1 R/R pt achieved morphologic leukemia-free state (MLFS). Median time to best response was 2.9 mo. 50% (2/4) pts became transfusion independent. 40% (2/5) pts lost their TP53mut at best response, and another 40% (2/5) pts had significant reductions (83% and 38%) in TP53 mut VAF. The regimen was well tolerated with no pts stopping therapy due to adverse effects (AE). AE included G3/G4 neutropenia (80%), G1 thrombocytopenia (40%), nausea (20%), fatigue (20%), lower extremity edema (20%), pneumonia (60%), and neutropenic fever (20%) with a median of 1 unplanned hospitalization per pt during follow-up. 60% (3/5) pts remain in CR on continued therapy for a median of 7.8 mo [7.2-9.4] thus far. One pt underwent allogeneic stem cell transplantation, however, died 11.4 mo after conditioning due to transplant related mortality. The R/R pt died after being lost to follow-up 2.9 mo after therapy initiation. No pt had measurable relapse during follow-up. Combination weekly Ven with subcutaneous low-dose Dec is well tolerated yielding igh rates of clinical and molecular response in pts with TP53mut MDS/AML. Although small, this case-series extends previous clinical trial proof-of-activity of non-cytotoxic DNMT1-targeting to a high-risk, poor PS, historically chemorefractory patient population. The regimen allowed frequent, sustained exposure to therapy often not possible with standard HMA/Ven regimens. [Formula presented] Disclosures: Shastri: Kymera Therapeutics: Research Funding;Guidepoint: Consultancy;GLC: Consultancy;Onclive: Honoraria. Gritsman: iOnctura: Research Funding. Feldman: Glycomimetics: Current Employment, Current holder of stock options in a privately-held company. Verma: Celgene: Consultancy;Acceleron: Consultancy;Novartis: Consultancy;Stelexis: Consultancy, Current equity holder in publicly-traded company;Eli Lilly: Research Funding;Curis: Research Funding;Medpacto: Research Funding;Incyte: Research Funding;GSK: Research Funding;BMS: Research Funding;Stelexis: Current equity holder in publicly-traded company;Throws Exception: Current equity holder in publicly-traded company. Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Coronavirus Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Interacts with p53 To Induce Cell Cycle Arrest in S-Phase and Promotes Viral Replication.

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S71NWHFYYLGTGPHADLRYRT90 and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S171RGNSQNRGNNQGRGASQNRGGNN194 (NS171-N194), in which G183RG185 are core residues. NS171-N194 and G183RG185 were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N194 domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication (P < 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. IMPORTANCE Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.