Pre-miRNA Hsa-Let-7a-2: a Novel Intracellular Partner of Angiotensin II Type 2 Receptor Negatively Regulating its Signals

G protein-coupled receptors (GPCRs) are the largest family of druggable targets, and their biological functions depend on different ligands and intracellular interactomes. Some microRNAs (miRNAs) bind as ligands to RNA-sensitive toll-like receptor 7 to regulate the inflammatory response, thereby contributing to the pathogenesis of cancer or neurodegeneration. It is unknown whether miRNAs bind to angiotensin II (Ang II) type 2 receptor (AGTR2), a critical protective GPCR in cardiovascular diseases, as ligands or intracellular interactomes. Here, screening for miRNAs that bind to AGTR2, we identified and confirmed that the pre-miRNA hsa-let-7a-2 non-competitively binds to the intracellular third loop of AGTR2. Functionally, intracellular hsa-let-7a-2 overexpression suppressed the Ang II-induced AGTR2 effects such as cAMP lowering, RhoA inhibition, and activation of Src homology 2 domain-containing protein-tyrosine phosphatase 1, whereas hsa-let-7a-2 knockdown enhanced these effects. Consistently, overexpressed hsa-let-7a-2 restrained the AGTR2-induced antiproliferation, antimigration, and proapoptosis of cells, and vasodilation of mesenteric arteries. Our findings demonstrated that hsa-let-7a-2 is a novel intracellular partner of AGTR2 that negatively regulates AGTR2-activated signals.

Proteomic approaches define rocaglates as translation remodelers with multiple protein targets

Intro: Deregulated protein synthesis is a common trait across solid and hematologic malignancies and an attractive target for cancer therapy. Rocaglates compounds that inhibit eukaryotic initiation factor 4A1 (eIF4A1), the essential DEAD-box RNA helicase that resolves mRNA 5'UTR secondary structures during cap-dependent translation initiation. Rocaglates' unique mechanism of action causes sequence-selective mRNA binding by eIF4A1, clamping the inactive helicase onto the transcript. This suppresses translation globally and affects many oncogenic and pro-survival transcripts in particular. Zotatifin, the first-in class synthetic rocaglate, is currently in Phase I clinical trials for the treatment of solid tumors and as an antiviral against SARS-CoV2. Currently, eIF4A1 and DDX3 are the only reported targets of rocaglate-mediated RNA clamping. Employing unbiased proteomic approaches, we have discovered that rocaglates, thought to act as pure eIF4A/translation inhibitors, extensively remodel the translation machinery and translatome. Additionally, mass-spec interrogation for proteins interacting with specific RNA sequences reveals novel targets of rocaglate-mediated, sequence-specific RNA clamping. Methods: We conducted original mass-spectrometry analyses of translational reprogramming by rocaglates. TMT-pSILAC assessed acute changes in protein production, while MATRIX, which captures high-resolution profiles of the translation machinery, revealed translation factors that drive reprogramming in response to rocaglate exposure. We validated results biochemically, in cellulo, and in vivo using patient-derived xenograft (PDX) mouse models. To probe existing and novel rocaglate RNA-clamping targets, we developed unbiased “clampome” assays - in cellulo protein-RNA-pull downs followed by mass-spec analysis of proteins with increased binding to RNA in the presence of rocaglates. Results: We find rocaglates, including zotatifin, have effects far more complex than simple “translational inhibition” as currently defined. Indeed, translatome analysis by TMT-pSILAC revealed myriad up-regulated proteins that drive hitherto unrecognized cytotoxic mechanisms. The GEF-H1 guanine exchange factor, for example, drives anti-survival RHOA/JNK activation, suggesting novel candidate biomarkers of rocaglate clinical outcomes. Translation-machinery analysis by MATRIX identifed rocaglate-induced dependence on specific translation factors including eEF1ϵ1 that drive remodeling. Novel rocaglate RNA-binding targets revealed by clampome studies remain under detailed evaluation as mediators of drug activities. Discussion: Our original proteome-level interrogation revealed that the complete cellular response to these historical “translation inhibitors” is mediated by comprehensive translational landscape remodeling. Effects on a broader suite of RNA binding proteins than eIF4A1 alone we suggest mediate the potent antitumor activities of these unique compounds, elucidation of which permits development of novel precision approaches to targeted translational deregulation in cancer.

Analysis of Interaction Network Between Host Protein and M Protein of Swine Acute Diarrhea Syndrome Coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an enterovirus that can cause acute diarrhea and death in piglets and cause serious economic losses to the pig industry. SADS-CoV membrane (M) protein mainly plays a key role in biological processes, such as virus assembly, budding, and host innate immune regulation. Understanding the interaction between M protein and host proteins is very important to define the molecular mechanism of cells at the protein level and to understand specific cellular physiological pathways. In this study, 289 host proteins interacting with M protein were identified by glutathione-S-transferase (GST) pull-down combined with liquid chromatography-mass spectrometry (LC-MS/MS), and the protein-protein interaction (PPI) network was established by Gene Ontology (GO) terms and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways analysis. Results showed that SADS-CoV M protein was mainly associated with the host metabolism, signal transduction, and innate immunity. The Co-Immunoprecipitation (CO-IP) validation results of six randomly selected proteins, namely, Rab11b, voltage-dependent anion-selective channel 1 (VDAC1), Ribosomal Protein L18 (RPL18), RALY, Ras Homolog Family Member A (RHOA), and Annexin A2 (ANXA2), were consistent with LC-MS results. In addition, overexpression of RPL18 and PHOA significantly promoted SADS-CoV replication, while overexpression of RALY antagonized viral replication. This work will help to clarify the function of SADS-CoV M protein in the life cycle of SADS-CoV.

A Real World Experience of Combined Treatment with Romidepsin and Azacitidine in Patients with Peripheral T-Cell Lymphoma

BACKGROUND Patients with peripheral T-cell lymphoma (PTCL) lack good treatment options, particularly in the relapsed and refractory setting (Mak V et al. J Clin Oncol 2013). The development of the targeted therapies in PTCL has been lagging behind those developed for B cell lymphomas. Our work suggested that combinations of epigenetic therapies can be a safe and effective approach for patients with PTCL, particularly those with T-cell lymphomas with a follicular helper phenotype (Marchi E et al. Br. J Haematol 2015;O'Connor O.A. et al;Blood 2019;Falchi L et al. Blood 2020). While the reason for this is not clear, it is thought recurrent mutations in epigenetic factors, including Ten-Eleven Translocation-2 (TET2), DNA methyl transferase-3A (DNMT3A) and isocitrate dehydrogenase-2 (IDH2) may contribute for their increased vulnerability (Couronné L. et al. N Eng J Med 2012;Lemonnier F et al. Blood 2012). Despite these presumptions, a direct explanation for the sensitivity to epigenetic based treatment remains to be established. OBJECTIVES To evaluate the merits of romidepsin plus subcutaneous azacitidine in patients with PTCL when administered in a ‘real-world’ scenario. METHODS We retrospectively identified PTCL patients that were treated with azacitidine and romidepsin outside of a clinical trial based upon queries regarding off study use. The study was reviewed and approved by each Medical Center Institutional Review Board. We have identified 13 patients world-wide whose pretreatment characteristics are shown in Table 1. These patients were treated using 3 different schedules: Schedule A: azacitidine 75mg/m2 s.c. on days 1-7, romidepsin 14 mg/m2 on day 1, 8 and 15 of a 28 day cycle (total of 6 patients);Schedule B: azacitidine 75mg/m2 s.c. on days 1-5, romidepsin 14 mg/m2 on day 8, 15 and 22 of a 35 day cycle;and Schedule C (total of 2 patients): azacitidine 75mg/m2 s.c. on days 1-7, romidepsin 12-14 mg/m2 on day 8, 15 and 22 of a 28 day cycle (total of 5 patients). RESULTS We retrospectively identified 13 patients that were treated with romidepsin and azacitidine off study. Ten patients had angioimmunoblastic lymphoma (AITL), 2 had adult T-cell leukemia/lymphoma (ATLL) and 1 had PTCL-NOS. Eight of the 13 patients had next generation sequencing performed. Most common mutations found were those of TET2 (5 pts), RHOA (4pts), IDH2 (3pts) and DNMT3A (1 pt). One ATLL patient had mutations in TRAF3, FAT1 and MED12. Among these 13 patients, overall response rate (ORR) was 84% and the complete response rate (CR) was 61%. Median number of cycles was 3 (range 1-12). Treatment was well tolerated but notable adverse effects included nausea, fatigue, rash, neutropenia and thrombocytopenia. One patient experienced febrile neutropenia while another had pulmonary infiltrates (differential diagnosis included drug toxicity versus infection). Thrombocytopenia was the most common reason for dose reduction of romidepsin (to 12mg/m2) or its omission on day 8, 15 or 22. In 3 patients, azacitidine and romidepsin were used to achieve remission prior to allogeneic transplant (range of cycles 1-3), with all 3 patients were in CR at their last disease assessment. One patient died of transplant related mortality 8 months after his allogeneic stem cell transplant. There was 1 patient with AITL (treatment naïve) noted to have progression of disease at first imaging following 2 cycles of romidepsin and azacitidine. On the day of her PET/CT, she was however diagnosed with symptomatic Covid19 infection and was hospitalized. A repeat PET/CT 6 weeks later (without any additional lymphoma treatment) revealed PR. CONCLUSIONS Subcutaneous azacitidine and romidepsin administered in a ‘real-world’ situation is highly effective in patients with relapsed PTCL with tolerable toxicity, and can be used to successfully bridge patients to stem cell transplant. Notably, the efficacy was similar to the one reported on a clinical study with oral azacitidine and romidepsin. [Formula presented] Disclosures: Kalac: Astra Zeneca: Consultancy;Kyowa Kirin Consultancy;Gilead: Consultancy;Johnson and Johnson: Research Funding;Guidepoint: Consultancy;GLG: Consultancy. Tam: Beigene: Research Funding;Janssen: Research Funding;Abbvie: Research Funding;Loxo: Honoraria;Beigene: Honoraria;Janssen: Honoraria;Abbvie: Honoraria. Montanari: Seattle Genetics: Research Funding. O'Connor: Servier: Research Funding;Mundipharma: Honoraria;Myeloid Therapeutics: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees;Kymera: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees;Astex: Research Funding;BMS: Research Funding;Merck: Research Funding;TG Therapeutics: Current Employment, Current equity holder in publicly-traded company. Marchi: BMS: Research Funding;Astex: Research Funding;Merck: Research Funding;Myeloid Therapeutics: Honoraria;Kyowa Kirin: Honoraria;Kymera Therapeutics: Other: Scientific Advisor.

Matrix stiffness regulates lipid nanoparticle-mRNA delivery in cell-laden hydrogels.

mRNA therapeutics have increased in popularity, largely due to the transient and fast nature of protein expression and the low risk of off-target effects. This has increased drastically with the remarkable success of mRNA-based vaccines for COVID-19. Despite advances in lipid nanoparticle (LNP)-based delivery, the mechanisms that regulate efficient endocytic trafficking and translation of mRNA remain poorly understood. Although it is widely acknowledged that the extracellular matrix (ECM) regulates uptake and expression of exogenous nano-complexed genetic material, its specific effects on mRNA delivery and expression have not yet been examined. Here, we demonstrate a critical role for matrix stiffness in modulating both mRNA transfection and expression and uncover distinct mechano-regulatory mechanisms for endocytosis of mRNA through RhoA mediated mTOR signaling and cytoskeletal dynamics. Our findings have implications for effective delivery of therapeutic mRNA to targeted tissues that may be differentially affected by tissue and matrix stiffness.

SARS-CoV-2 Spike Protein Disrupts Blood-Brain Barrier Integrity via RhoA Activation.

The SARS-CoV-2 spike protein has been shown to disrupt blood-brain barrier (BBB) function, but its pathogenic mechanism of action is unknown. Whether angiotensin converting enzyme 2 (ACE2), the viral binding site for SARS-CoV-2, contributes to the spike protein-induced barrier disruption also remains unclear. Here, a 3D-BBB microfluidic model was used to interrogate mechanisms by which the spike protein may facilitate barrier dysfunction. The spike protein upregulated the expression of ACE2 in response to laminar shear stress. Moreover, interrogating the role of ACE2 showed that knock-down affected endothelial barrier properties. These results identify a possible role of ACE2 in barrier homeostasis. Analysis of RhoA, a key molecule in regulating endothelial cytoskeleton and tight junction complex dynamics, reveals that the spike protein triggers RhoA activation. Inhibition of RhoA with C3 transferase rescues its effect on tight junction disassembly. Overall, these results indicate a possible means by which the engagement of SARS-CoV-2 with ACE2 facilitates disruption of the BBB via RhoA activation. Understanding how SARS-CoV-2 dysregulates the BBB may lead to strategies to prevent the neurological deficits seen in COVID-19 patients.

Proteomics reveal cap-dependent translation inhibitors remodel the translation machinery and translatome.

Tactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple "translational inhibition" as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical "translation inhibitors" is mediated by comprehensive translational landscape remodeling.

Effects of statins on myocarditis: A review of underlying molecular mechanisms.

Myocarditis refers to the clinical and histological characteristics of a diverse range of inflammatory cellular pathophysiological conditions which result in cardiac dysfunction. Myocarditis is a major cause of mortality in individuals less than 40 years of age and accounts for approximately 20% of cardiovascular disease (CVD) events. Myocarditis contributes to dilated cardiomyopathy in 30% of patients and can progress to cardiac arrest, which has a poor prognosis of <40% survival over 10 years. Myocarditis has also been documented after infection with SARS-CoV-2. The most commonly used lipid-lowering therapies, HMG-CoA reductase inhibitors (statins), decrease CVD-related morbidity and mortality. In addition to their lipid-lowering effects, increasing evidence supports the existence of several additional beneficial, 'pleiotropic' effects of statins. Recently, several studies have indicated that statins may attenuate myocarditis. Statins modify the lipid oxidation, inflammation, immunomodulation, and endothelial activity of the pathophysiology and have been recommended as adjuvant treatment. In this review, we focus on the mechanisms of action of statins and their effects on myocarditis, SARS-CoV-2 and CVD.

Computationally predicted SARS-COV-2 encoded microRNAs target NFKB, JAK/STAT and TGFB signaling pathways.

Recently an outbreak that emerged in Wuhan, China in December 2019, spread to the whole world in a short time and killed >1,410,000 people. It was determined that a new type of beta coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) was causative agent of this outbreak and the disease caused by the virus was named as coronavirus disease 19 (COVID19). Despite the information obtained from the viral genome structure, many aspects of the virus-host interactions during infection is still unknown. In this study we aimed to identify SARS-CoV-2 encoded microRNAs and their cellular targets. We applied a computational method to predict miRNAs encoded by SARS-CoV-2 along with their putative targets in humans. Targets of predicted miRNAs were clustered into groups based on their biological processes, molecular function, and cellular compartments using GO and PANTHER. By using KEGG pathway enrichment analysis top pathways were identified. Finally, we have constructed an integrative pathway network analysis with target genes. We identified 40 SARS-CoV-2 miRNAs and their regulated targets. Our analysis showed that targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C, JARID2 play important roles in NFKB, JAK/STAT and TGFB signaling pathways as well as cells' epigenetic regulation pathways. Our results may help to understand virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. As there is no current drug and effective treatment available for COVID19, it may also help to develop new treatment strategies.