PKR Activation Research Articles

Abstract 17142: Deficiency of Smooth Muscle ADAR1 Exacerbates Vascular Remodeling and Pulmonary Hypertension

Background: Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes the conversion of adenosine (A) to inosine (I) in double-stranded RNA, which is critical to prevent auto-inflammatory responses mediated by activation of the type I interferon (IFN) signaling. Here, we define the role of ADAR1-dependent RNA editing in IFNβ activation and pulmonary artery smooth muscle cell (PASMC) remodeling in pulmonary arterial hypertension (PAH), a devastating disease leading to right heart failure and premature death. Methods: Total RNAs from IPAH PASMCs (N=3) and healthy control (N=3) were deep sequenced by RNA-seq and determined the fraction of reads with a ‘G’ nucleotide editing sites using RADAR database. A transgenic line ADAR1 SMC-KO was generated by knocking down ADAR1 selectively in αSMA+ cells, followed by 3 weeks of hypoxia. PKR inhibitor Imoxin was used to treat ADAR1 SMC-KO . Results: SMC layers of human IPAH tissues expressed reduced levels of ADAR1. Moreover, IPAH PASMCs displayed decreased mRNA and protein levels of ADAR1, along with reduced editing sites compared to healthy PASMCs (Fig.1A). The knockdown of ADAR1 in PASMCs resulted in the activation of the MDA5-PKR-IFNβ signaling pathway. Compared with controls in vivo , hypoxic ADAR1 SMC-KO mice developed severe PH, as evidenced by excessive vascular remodeling in distal arterioles (Fig.1B) and increased vascular leakage resulting in elevated right ventricular systolic pressure (37 vs 23 mmHg) and right ventricular hypertrophy by Fulton Index (46 vs. control 20%) (Fig.1C). The immunofluorescence staining showed higher expression of IFNβ and increased recruitment of inflammatory immune cells. Imoxin treatment prevented PH & RVH. Conclusions: The deficiency of ADAR1 leads to the activation of IFNβ thus contributing to the pathological processes observed in PAH. Inhibiting PKR activity could attenuate the aberrant IFNβ activation and potentially serve as a new therapeutic strategy to treat PAH.

Voxelotor and Red Blood Cell Pyruvate Kinase Activator Affect Clot Strength in Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is a common genetic red blood cell (RBC) disorder that results from a single substitution of valine for glutamic acid in the β-globin gene, resulting in sickle hemoglobin (HbSS). Under hypoxia, HbS polymerizes and forms fibers within the RBCs, thus making the cells rigid, deformed, and sickle-shaped [1]. Deformed RBCs influence the compactness and dynamics of blood clots [2]. In addition, people with SCD are susceptible to thrombophilia [2,3,4]. Recently, there has been a heightened interest in ameliorating SCD using RBCs pyruvate kinase (PKR) activators, which increase ATP and decrease 2,3 DPG, resulting in increased oxygen affinity and improved RBCs deformability [5]. Voxelotor is also an oxygen affinity-modifying drug, recently FDA-approved. Voxelotor delays deoxy-HbSS formation, preventing in vitro HbSS polymerization and RBC sickling, thus improving RBC deformability [6]. The in vitro impact of PKR activators and Voxelotor on blood clotting in clinical samples from people with SCD has not been examined. Here we use a microfluidic dielectric sensor, termed ClotChip ® [7,8], to investigate the effects of Voxelotor and a PKR activator on the clotting time and clot strength of samples from people with SCD. Methods: Venous blood samples were collected in sodium-citrate from subjects with homozygous HbSS and healthy volunteers (HbAA) under an IRB-approved protocol. ClotChip ® microsensors were fabricated as previously described [7]. Blood samples were centrifuged at 200 g for 10 min. Plasma and buffy coats were removed and stored at 4 °Cfor 6 hours. Isolated, washed RBCs were then re-suspended in PBS at 20% hematocrit and then mixed with 67 mg/mL of Voxelotor (GBT440; Selleckchem) to a final concentration of 600 µM (in DMSO) or a PKR activator (PKR activator 3; MedChem Express) at 10 mM (in DMSO) and incubated at 37 °Cfor 6 hours. For controls, HbSS- or HbAA-containing RBCs at 20% hematocrit were mixed with PBS containing 0.5% v/v DMSO and incubated at 37 °C for 6 hours. After incubation, HbSS+Vox, HbSS+PKR, and control samples were centrifuged at 500 g for 10 min to remove PBS. Each sample's plasma was used to reconstitute RBCs at 20% hematocrit. CaCl 2 was added to the samples to induce coagulation, and the samples were injected into the ClotChip ® microfluidic sensors. An Agilent 4294A impedance analyzer was used to obtain the ClotChip ® readout curve defined as the temporal variation of the normalized real part of blood dielectric permittivity at 1 MHz (see Figure 1A). Based on our previous studies [7,8], the time to reach a permittivity peak (T peak) parameter was taken to indicate the clotting time, whereas the maximum change in permittivity after the peak (ΔƐ r,max) parameter was taken to indicate the clot strength. Data are reported as mean ± standard deviation (SD). Results: We analyzed the ClotChip ® T peak and ΔƐ r,max readout parameters for HbSS (n=11), HbSS+Vox (n=11), HbSS+PKR (n=11), and HbAA control (n=8) samples ( Figure 1B). The clot strength was ~50% higher in HbAA than HbSS (HbAA vs. HbSS: p= 0.001, Mann-Whitney test). However, when the SCD samples were treated with Voxelotor, the clot strength was observed to increase by ~10% (HbSS vs. HbSS+Vox, p=0.01, Paired t-test). Treatment with a PKR activator increased the clot strength by ~30% (HbSS vs. HbSS+PKR, p=0.003, Figure 1B). A significant difference was observed in the clotting time between HbAA vs. HbSS (p=0.001) but treatment with Voxelotor or a PKR activator did not change the clotting time for HbSS (HbSS vs. HbSS+Vox, p=0.43; HbSS vs. HbSS+ PKR, p=0.29) ( Figure 1B). (p values were determined using a non-parametric Mann-Whitney test and paired t-test). Discussion : We found that treatment of HbS-containing RBCs with a PKR activator or voxelotor improves clot strength but not clotting time. PKR activators improved the strength of the clot significantly more than Voxelotor. Patients with SCD had a faster clotting time than healthy participants, which agrees with earlier researchers on hypercoagulability in SCD [4]. These data suggest that HbS polymerization and RBC health affect clot characteristics but not clot kinetics in SCD. Further investigations may be required to relate RBC deformability, which is well-known to impact SCD pathophysiology, to blood clotting and venous thromboembolism.

FORTITUDE: A Phase 2 Open-Label Study in Progress to Evaluate Etavopivat for the Treatment of Anemia in Patients with Lower-Risk Myelodysplastic Syndromes

Background: Most patients with lower-risk myelodysplastic syndrome (LR-MDS) develop anemia and a high proportion of patients become dependent on red blood cell (RBC) transfusions. MDS-related anemia is associated with low hemoglobin (Hb) levels, iron overload and poor quality of life. Erythropoiesis-stimulating agents and other drugs, including lenalidomide, luspatercept and imetelstat (an investigational product), can lead to RBC transfusion independence and improve Hb levels in many patients; however, responses are generally transient and novel treatments are needed for this population. Etavopivat is an investigational, once-daily, selective erythrocyte pyruvate kinase (PKR) activator in development for the treatment of sickle cell disease (SCD) and anemia in patients with LR-MDS. In a phase 1 study in adult and adolescent patients with SCD by Saraf et al. HemaSphere. (2022), etavopivat 400 mg once daily was considered well tolerated and showed sustained Hb increases up to 12 weeks for most patients. Additional studies in healthy volunteers and patients with SCD support an acceptable safety profile together with improved RBC health and function during continuous treatment with etavopivat. In patients with LR-MDS, ineffective hematopoiesis may be due, in part, to insufficient ATP production, leading to increased erythroid lineage apoptosis. By activating PKR, etavopivat may improve RBC health and function by stimulating energy production (increasing ATP levels), increasing Hb levels (decreasing anemia), and decreasing erythroid lineage apoptosis. Aims:Describe the design of a multicenter, open-label, phase 2 study (FORTITUDE; NCT05568225) evaluating hematologic improvements in patients with very low-, low- and intermediate-risk MDS during treatment with etavopivat. Study design:Key eligibility criteria include age ≥18 years; very low-, low- and intermediate-risk MDS per the Revised International Prognostic Scoring System; and anemia defined by mean Hb <10.0 g/dL and <3 RBC transfusions <16 weeks prior to first dose for non-transfusion-dependent (NTD) patients or ≥3 units of RBCs for anemia <16 weeks prior to first dose for transfusion-dependent (TD) patients. Key exclusion criteria include MDS associated with del 5q cytogenetic abnormality and known TP53 abnormality, history of acute myeloid leukemia, and an absolute neutrophil count <0.5 × 10 9/L. Additional exclusion criteria include prior treatment with azacitidine; decitabine; erythropoietin, other hematopoietic growth factor treatment or lenalidomide within 30 days of Day 1 or anticipated to be required during the study; and luspatercept within 30 days of Day 1 for NTD patients and within 16 weeks of Day 1 for TD patients. Patients will receive etavopivat 400 mg once daily for at least 48 weeks. Treatment may be continued beyond 48 weeks in patients demonstrating clinical benefit. The primary objective of the study is to assess hematologic improvements based on an erythroid response over any continuous period ≥8 weeks within 24 weeks on etavopivat treatment in patients with LR-MDS. The primary endpoint will be based on the combined incidence of: ≥1.5 g/dL increase in Hb from baseline maintained for ≥8 consecutive weeks for NTD patients; absence of any transfusion for ≥8 consecutive weeks for low transfusion-burden patients; and reduction by ≥50% of RBC units for ≥8 consecutive weeks for high transfusion-burden patients. Safety, tolerability, pharmacokinetic, and pharmacodynamic properties of etavopivat will be evaluated. FORTITUDE is open for enrollment in Canada, France, Germany, and the USA. Planned enrollment includes approximately 15 patients in each of the three transfusion requirement-based groups (Figure). Statistical analysis: The primary endpoint (pooled incidence) will be tested with a one-sided Fisher's exact test at the significance level of 0.025 where 0.2 is the assumed null proportion. Summary:Etavopivat is a novel, investigational, once-daily, selective PKR activator with the potential to improve RBC health and lifespan. This phase 2 study will assess the safety of etavopivat and its impact on Hb levels and RBC transfusion burden in patients with LR-MDS and anemia.

ADAR1p150 prevents MDA5 and PKR activation via distinct mechanisms to avert fatal autoinflammation

Effective immunity requires the innate immune system to distinguish foreign nucleic acids from cellular ones. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA-editing enzyme ADAR1 to evade being recognized as viral dsRNA by cytoplasmic dsRNA sensors, including MDA5 and PKR. The loss of ADAR1-mediated RNA editing of cellular dsRNA activates MDA5. Additional RNA-editing-independent functions of ADAR1 have been proposed, but a specific mechanism has not been delineated. We now demonstrate that the loss of ADAR1-mediated RNA editing specifically activates MDA5, whereas loss of the cytoplasmic ADAR1p150 isoform or its dsRNA-binding activity enabled PKR activation. Deleting both MDA5 and PKR resulted in complete rescue of the embryonic lethality of Adar1p150-/- mice to adulthood, contrasting with the limited or no rescue by removing MDA5 or PKR alone. Our findings demonstrate that MDA5 and PKR are the primary invivo effectors of fatal autoinflammation following the loss of ADAR1p150.

PKR activation in mitochondrial unfolded protein response-mitochondrial dsRNA might do the trick.

PKR activation in mitochondrial unfolded protein response-mitochondrial dsRNA might do the trick.

A co-formulation of interferons alpha2b and gamma distinctively targets cell cycle in the glioblastoma-derived cell line U-87MG

BackgroundHeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell carcinomas. In glioblastoma (GBM), HeberFERON has displayed promising preclinical and clinical results. This led us to design a microarray experiment aimed at identifying the molecular mechanisms involved in the distinctive effect of HeberFERON compared to the individual interferons in U-87MG model.MethodsTranscriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive transcription patterns of HeberFERON. Validation of most representative genes was performed by qPCR. For the cell cycle analysis of cells treated with HeberFERON for 24 h, 48 and 72 h we used flow cytometry.ResultsThe three treatments show different behavior based on the gene expression profiles. The enrichment analysis identified several mitotic cell cycle related events, in particular from prometaphase to anaphase, which are exclusively targeted by HeberFERON. The FOXM1 transcription factor network that is involved in several cell cycle phases and is highly expressed in GBMs, is significantly down regulated. Flow cytometry experiments corroborated the action of HeberFERON on the cell cycle in a dose and time dependent manner with a clear cellular arrest as of 24 h post-treatment. Despite the fact that p53 was not down-regulated, several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relationship of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain this distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes, as well as its reduced MDM2 mediated ubiquitination and export from the nucleus to the cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON on the proliferation of U-87MG.ConclusionsWe proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to the treatments with the individual interferons in U-87MG model, where cell cycle related events were highly relevant.

SARS-CoV-2 nucleocapsid protein inhibits the PKR-mediated integrated stress response through RNA-binding domain N2b.

The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding-prevented PKR activation, inhibited β-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Similar observations were made for the N protein of human coronavirus 229E, suggesting that this may be a general trait conserved among members of other orthocoronavirus (sub)genera.

Directed natural evolution generates a next-generation oncolytic virus with a high potency and safety profile

Oncolytic viruses (OVs) represent a type of encouraging multi-mechanistic drug for the treatment of cancer. However, attenuation of virulence, which is generally required for the development of OVs based on pathogenic viral backbones, is frequently accompanied by a compromised killing effect on tumor cells. By exploiting the property of viruses to evolve and adapt in cancer cells, we perform directed natural evolution on refractory colorectal cancer cell HCT-116 and generate a next-generation oncolytic virus M1 (NGOVM) with an increase in the oncolytic effect of up to 9690-fold. The NGOVM has a broader antitumor spectrum and a more robust oncolytic effect in a range of solid tumors. Mechanistically, two critical mutations are identified in the E2 and nsP3 genes, which accelerate the entry of M1 virus by increasing its binding to the Mxra8 receptor and antagonize antiviral responses by inhibiting the activation of PKR and STAT1 in tumor cells, respectively. Importantly, the NGOVM is well tolerated in both rodents and nonhuman primates. This study implies that directed natural evolution is a generalizable approach for developing next-generation OVs with an expanded scope of application and high safety.

Double-stranded RNA-dependent protein kinase (PKR) in antiviral defence in fish and mammals

The interferon-inducible double-stranded RNA-dependent protein kinase (PKR) is one of the key antiviral arms of the innate immune system. Upon binding of viral double stranded RNA, a viral Pattern Associated Molecular Pattern (PAMP), PKR gets activated and phosphorylates the eukaryotic translation initiation factor 2α (eIF2α) resulting in a protein shut-down that limits viral replication. Since its discovery in the mid-seventies, PKR has been shown to be involved in multiple important cellular processes including apoptosis, proinflammatory and innate immune responses. Viral subversion mechanisms of PKR underline its importance in the antiviral response of the host. PKR activation pathways and its mechanisms of action were previously identified and characterised mostly in mammalian models. However, fish Pkr and fish-specific paralogue Z-DNA-dependent protein kinase (Pkz) also play key role in antiviral defence. This review gives an update on the current knowledge on fish Pkr/Pkz, their conditions of activation and their implication in the immune responses to viruses, in comparison to their mammalian counterparts.

Structural study of novel vaccinia virus E3L and dsRNA-dependent protein kinase complex

E3L (RNA-binding protein E3) is one of the key IFN resistance genes encoded by VV and consists of 190 amino acids with a highly conserved carboxy-terminal double-stranded RNA-binding domain (dsRBD). PKR (dsRNA-dependent protein kinase) is an IFN-induced protein involved in anti-cell and antiviral activity. PKR inhibits the initiation of translation through alpha subunit of the initiation factor eIF2 (eIF2α) and mediates several transcription factors such as NF-κB, p53 or STATs. Activated PKR also induces apoptosis in vaccinia virus infection. E3L is required for viral IFN resistance and directly binds to PKR to block activation of PKR. In this work, we determined the three-dimensional complex structure of E3L and PKR using cryo-EM and determined the important residues involved in the interaction. In addition, PKR peptide binds to E3L and can increase protein levels of phosphorus-PKR and phosphorus-eIF2α-induced cell apoptosis through upregulation of phosphorus-PKR in HEK293 cells. Taken together, structural insights into E3L and PKR will provide a new optimization and development of vaccinia virus drugs.

Luteolin protects DYT-PRKRA cells from apoptosis by suppressing PKR activation.

DYT-PRKRA is a movement disorder caused by mutations in the PRKRA gene, which encodes for PACT, the protein activator of interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR. PACT brings about PKR's catalytic activation by a direct binding in response to stress signals and activated PKR phosphorylates the translation initiation factor eIF2α. Phosphorylation of eIF2α is the central regulatory event that is part of the integrated stress response (ISR), an evolutionarily conserved intracellular signaling network essential for adapting to environmental stresses to maintain healthy cells. A dysregulation of either the level or the duration of eIF2α phosphorylation in response to stress signals causes the normally pro-survival ISR to become pro-apoptotic. Our research has established that the PRKRA mutations reported to cause DYT-PRKRA lead to enhanced PACT-PKR interactions causing a dysregulation of ISR and an increased sensitivity to apoptosis. We have previously identified luteolin, a plant flavonoid, as an inhibitor of the PACT-PKR interaction using high-throughput screening of chemical libraries. Our results presented in this study indicate that luteolin is markedly effective in disrupting the pathological PACT-PKR interactions to protect DYT-PRKRA cells against apoptosis, thus suggesting a therapeutic option for using luteolin to treat DYT-PRKRA and possibly other diseases resulting from enhanced PACT-PKR interactions.

HIV co-opts a cellular antiviral mechanism, activation of stress kinase PKR by its RNA, to enable splicing of rev/tat mRNA

BackgroundActivation of RNA-dependent stress kinase PKR, especially by viral double-stranded RNA, induces eukaryotic initiation factor 2 α-chain (eIF2α) phosphorylation, attenuating thereby translation. We report that this RNA-mediated negative control mechanism, considered a cornerstone of the cell’s antiviral response, positively regulates splicing of a viral mRNA.ResultsExcision of the large human immunodeficiency virus (HIV) rev/tat intron depends strictly on activation of PKR by the viral RNA and on eIF2α phosphorylation. Rev/tat mRNA splicing was blocked by viral PKR antagonists Vaccinia E3L and Ebola VP35, as well as by a trans-dominant negative mutant of PKR, yet enhanced by overexpressing PKR. Expression of non-phosphorylatable mutant eIF2αS51A, but not of wild type eIF2α, abrogated efficient splicing of rev/tat mRNA. By contrast, expression of eIF2αS51D, a phosphomimetic mutant of eIF2α, left rev/tat mRNA splicing intact. Unlike eIF2αS51A, eIF2αS51D does not inhibit eIF2α phosphorylation by activated PKR. All HIV mRNA species contain terminal trans-activation response (TAR) stem-loop sequences that potentially could activate PKR, yet even upon TAR deletion, HIV mRNA production remained sensitive to inhibitors of PKR activation. Bioinformatic and mutational analyses revealed a compact RNA pseudoknot upstream of 3′-terminal TAR that promotes splicing by activating PKR. Supporting its essential role in control of splicing, this pseudoknot is conserved among diverse HIV and nonhuman primate SIVcpz isolates. The pseudoknot and 3′-terminal TAR collaborate in mediating PKR-regulated splicing of rev/tat intron, the pseudoknot being dominant.ConclusionsOur results on HIV provide the first example of a virus co-opting activation of PKR by its RNA, a cellular antiviral mechanism, to promote splicing. They raise the question whether other viruses may use local activation of host kinase PKR through RNA elements within their genome to achieve efficient splicing of their mRNA. Our experiments reveal an indispensable role for eIF2α phosphorylation in HIV rev/tat mRNA splicing that accounts for the need for PKR activation.

PKR induces TGF-β and limits oncolytic immune therapy

BackgroundMammalian cells have developed multiple intracellular mechanisms to defend against viral infections. These include RNA-activated protein kinase (PKR), cyclic GMP-AMP synthase and stimulation of interferon genes (cGAS-STING) and toll-like receptor-myeloid...

Abstract IA012: Reducing disparities in breast cancer mortality through the identification of novel targets in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) remains a challenge to clinicians, laboratory investigators, and patients due to its disproportionate number of breast cancer deaths and its lack of an established therapeutic target. Numerous studies have identified potential novel mutational gene targets in TNBC, but single-agent therapeutics have lacked substantial impact in TNBC. Novel effective therapies are desperately needed. Alongside these therapies, we need accurate biomarkers that can be used to predict which patients will respond. We find that the ARF tumor suppressor expression is lost alongside p53 mutation in 60% of TNBC. ARF is the key player in sensing cellular oncogene-induced stress, such as signals emanating from oncogenic Ras and Myc alleles. ARF suppresses tumor formation in both p53-dependent and -independent mechanisms by interacting with proteins within the cell nucleolus. Our findings have advanced the premise that the p53-indendent functions of ARF are critical to preventing epithelial-based cancers. Our data show that mammary epithelial cells rapidly induce ARF expression in vitro and in vivo upon loss of functional p53. This induced ARF suppresses mammary tumor formation in vivo, wherein loss of both p53 and ARF dramatically accelerates tumorigenesis. This is consistent with our evidence that ARF and p53 function are concomitantly lost in TNBC. Potentially stemming from the dual loss of ARF and p53, we have observed that type I IFN signaling is elevated in TNBC. Our laboratory and others have shown that cancer cell lines, including those derived from TNBC, with elevated IFN signaling are dependent on the RNA deaminase ADAR1. Loss of ADAR1 in these cells leads to hyperactivation of the type I IFN pathway and cell death. We hypothesized that loss of ARF and p53 results in elevated type I IFN signaling and sensitizes cells to ADAR1 depletion. Beyond the potential genetic interaction between ARF-loss and ADAR1-dependency in TNBC, recent work from our lab has revealed that ARF and ADAR1 physically interact in cells lacking functional p53. We find that ARF can fully titrate much of the cellular ADAR1 into ARF complexes. Additionally, TNBC cells, but not non-TNBC cells, are sensitive to ADAR1 knockdown. Sensitivity to ADAR1 depletion appears to require type I IFN signaling and subsequent PKR activation. Analysis of primary human TNBC showed a marked separation of IFN signaling and ADAR1 localization/function between African American and Caucasian TNBC patients, suggesting that this pathway may serve as a functional marker of African American TNBC. Citation Format: Kyle Cottrell, Pat Kung, Sua Ryu, Leonard Maggi, Catherine Kuzmicki, Raleigh Kladney, Ling Yiu, Graham Colditz, Jason D. Weber. Reducing disparities in breast cancer mortality through the identification of novel targets in triple-negative breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA012.

PKR deficiency delays vascular aging via inhibiting GSDMD-mediated endothelial cell hyperactivation

Vascular aging is an independent risk factor for cardiovascular diseases, but the regulatory mechanism is not clearly understood. In this study, we found that endothelial PKR activity is elevated in aging aorta tissues, which is accompanied with increased endothelial cell hyperactivation, IL-1β and HMGB1 release and vascular smooth muscle cell (VSMC) phenotype transforming. Global knockout of PKR exhibits significantly delayed vascular aging compared to wild-type mice at the same age. Invitro, using PKR siRNA or the cell hyperactivation inhibitor glycine or disulfiram can effectively inhibit H2O2 or palmitic acid-induced endothelial cell hyperactivation, IL-1β and HMGB1 release and co-cultured VSMC phenotype transforming. These results demonstrate that endothelial PKR activation induces GSDMD-mediated endothelial cell hyperactivation torelease HMGB1 and IL-1β, which promotes the phenotype transforming of VSMC and subsequent accelerates the process of vascular aging. These discoveries will help to explore the new drug target to inhibit vascular aging.

ADAR1p110 promotes Enterovirus D68 replication through its deaminase domain and inhibition of PKR pathway

BackgroundSevere respiratory and neurological diseases caused by human enterovirus D68 (EV-D68) pose a serious threat to public health, and there are currently no effective drugs and vaccines. Adenosine deaminase acting on RNA1 (ADAR1) has diverse biological functions in various viral infections, but its role in EV-D68 infections remains undetermined.MethodsRhabdomyosarcoma (RD) and human embryonic kidney 293 T (293 T) cells, and HeLa cells were used to evaluate the expression level of ADAR1 upon EV-D68 (Fermon strain) and human parainfluenza virus type 3 (HPIV3; NIH47885) infection, respectively. Knockdown through silencing RNA (siRNA) and overexpression of either ADAR1p110 or ADAR1p150 in cells were used to determine the function of the two proteins after viral infection. ADAR1p110 double-stranded RNA binding domains (dsRBDs) deletion mutation was generated using a seamless clone kit. The expression of ADAR1, EV-D68 VP1, and HPIV3 hemagglutinin–neuraminidase (HN) proteins was identified using western blotting. The median tissue culture infectious dose (TCID50) was applied to detect viral titers. The transcription level of EV-D68 mRNA was analyzed using reverse transcription-quantitative PCR (RT-qPCR) and the viral 5′-untranslated region (5′-UTR)-mediated translation was analyzed using a dual luciferase reporter system.ConclusionWe found that the transcription and expression of ADAR1 was inhibited upon EV-D68 infection. RNA interference of endogenous ADAR1 decreased VP1 protein expression and viral titers, while overexpression of ADAR1p110, but not ADAR1p150, facilitated viral replication. Immunofluorescence assays showed that ADAR1p110 migrated from the nucleus to the cytoplasm after EV-D68 infection. Further, ADAR1p110 lost its pro-viral ability after mutations of the active sites in the deaminase domain, and 5′-UTR sequencing of the viral genome revealed that ADAR1p110 likely plays a role in EV-D68 RNA editing. In addition, after ADAR1 knockdown, the levels of both phosphorylated double-stranded RNA dependent protein kinase (p-PKR) and phosphorylated eukaryotic initiation factor 2α (p-eIF2α) increased. Attenuated translation activity of the viral genome 5′-UTR was also observed in the dual-luciferase reporter assay. Lastly, the deletion of ADAR1p110 dsRBDs increased the level of p-PKR, which correlated with a decreased VP1 expression, indicating that the promotion of EV-D68 replication by ADAR1p110 is also related to the inhibition of PKR activation by its dsRBDs. Our study illustrates that ADAR1p110 is a novel pro-viral factor of EV-D68 replication and provides a theoretical basis for EV-D68 antiviral research.

343 Endogenous double-stranded RNA is a potential target for psoriasis therapy

The activation of endogenous double-stranded RNA (endo-dsRNA) signalling pathways is linked to a variety of autoimmune diseases. Here, we investigated the role of endogenous dsRNA in psoriasis by quantifying dsRNA binding protein (DRBP) mRNA and mitochondrial dsRNA formation in response to narrow-band UVB (NB-UVB) treatment. RNAseq data from 34 psoriatic patients treated with NB-UVB were analysed. Biopsies were obtained from four sites 24h after NB-UVB irradiation (lesional unirradiated, nonlesional unirradiated lesional irradiated with one minimal erythemal dose (MED), lesional irradiated with three MEDs. Psoriatic lesional skin had a significantly higher expression of all examined DRBP (RIG, MDA5, LGP2, PKR, OAS1-3, OASL) as compared to nonlesional skin (increase between 1.5-21.7 fold, p <0.001 for all eight genes). NB-UVB treatment had no effect on the DRBPs after 24h of one or three MEDs of NB-UVB. Additionally, a significant reduction (about 20%) of dsRNA from mitochondrial transcripts was detected in response to both NB-UVB doses(p-value 0.012 and 0.001, respectively). These results indicate that increased levels of dsRNA activate RIG, MDA5, and LGP2 in lesional compared to nonlesional psoriasis skin. RIG, MDA5, and LGP2 in turn upregulate type1 IFN via recruiting TBK1, and IRF3/7, all of which were significantly upregulated in the lesional unirradiated skin. PKR binds endo-dsRNA, and activation of PKR enhances IFN-α/β expression via IRF3. Moreover, OASs trigger an antiviral response via a RIG induced IFN pathway. Once activated, IFN strongly enhances the transcription of DRBP thus creating a vicious cycle that exacerbates the immune response. In conclusion, upregulation of all DRBP genes in psoriatic lesional skin indicates that endo-dsRNA may play a crucial role in psoriasis pathogenesis, and the early reduction of the mitochondrial dsRNA after NB-UVB treatment suggests that dsRNA is an important target for psoriasis therapy.

Mitapivat Improves Iron Overload in Patients with Pyruvate Kinase Deficiency

Background: Pyruvate kinase (PK) deficiency is a rare hereditary disease resulting in chronic hemolytic anemia. Iron overload is highly prevalent in patients (pts) with PK deficiency regardless of transfusion requirements, suggesting dyserythropoietic features. Iron overload can lead to serious complications including liver cirrhosis, cardiomyopathy, arrhythmia, sudden cardiac death, and endocrine dysfunction. In pts with PK deficiency, ineffective erythropoiesis combined with chronic hemolysis contributes to the manifestation of iron overload. Increased erythropoietic drive leading to erythroferrone-induced hepcidin suppression results in increased iron absorption and abnormal iron metabolism in PK deficiency. Furthermore, transfusions increase the existing burden of iron overload in PK deficiency. Mitapivat (AG-348) is a first-in-class, oral, allosteric activator of the red blood cell wildtype and mutant PK enzyme (PKR) that is approved by the US Food and Drug Administration for the treatment of hemolytic anemia in adults with PK deficiency, which has been shown to improve ineffective erythropoiesis and hemolysis. Previously reported data from ACTIVATE (NCT03548220) and its long-term extension (LTE) study (NCT03853798) also showed that treatment with mitapivat improved markers of iron homeostasis in adults with PK deficiency (up to 72 weeks [wks]). Here, we provide longer-term data from the LTE study on the impact of mitapivat on iron homeostasis and iron overload, and also assess liver iron concentration (LIC) changes in pts with iron overload at baseline (BL). Methods: In ACTIVATE (double-blind, placebo [PBO]-controlled study), pts with PK deficiency who were not regularly transfused were randomized 1:1 to receive mitapivat or PBO. Pts who had demonstrated clinical benefit from mitapivat on completion of the fixed-dose period for ACTIVATE (24 wks), or who were assigned to the PBO arm in ACTIVATE, were eligible to continue in the LTE. All pts enrolled in the LTE received mitapivat. Pts from ACTIVATE who continued in the LTE were categorized into the mitapivat-to-mitapivat arm (M/M) or PBO-to-mitapivat arm (P/M). The ACTIVATE/LTE analysis assessed changes from BL over time (up to 96 wks), for both study arms, in markers related to iron homeostasis and overload, including erythroferrone, soluble transferrin receptor (sTfR), hepcidin, and LIC by magnetic resonance imaging. LIC changes over time in pts with evidence of iron overload at BL, were also assessed; pts were considered to have iron overload at BL if they met at least 1 of 3 criteria: BL ferritin >1000 mg/L, BL average LIC >3 mg Fe/g dry weight (dw), or chelation therapy within the last year before study start. For this calculation, BL was defined as the last assessment before start of treatment with mitapivat, and LIC data from pts in the M/M arm were pooled together with data from pts in the P/M arm and summarized over the time they were treated with mitapivat. Results: 80 pts were included in the ACTIVATE/LTE analysis (M/M=40; P/M=40). Meaningful improvements in hepcidin, erythroferrone, sTfR, and LIC values were observed with mitapivat treatment previously; here, we confirm these meaningful improvements are continued up to 96 wks (Table). These measurements were improved early-on, within 24 wks of starting treatment in the M/M arm, but remained relatively unchanged in the P/M arm while on PBO. Similar improvements to the M/M arm were seen in the P/M arm after switching to mitapivat in the LTE. Among pts treated with mitapivat (N=78), 55.1% (n=43) met the criteria for iron overload at BL. These pts showed clinically meaningful and continued improvements in iron overload over time as measured by LIC (median [Q1, Q3] decrease from BL to Wk 96 of mitapivat treatment of -1.95 [-4.85, -0.70] mg Fe/g dw) (Figure). Conclusions: Activation of PKR with mitapivat was associated with meaningful long-term improvements in iron homeostasis and overload. Mitapivat is the first disease-modifying pharmacotherapy shown to have beneficial effects on iron overload in pts with PK deficiency. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Pyruvate Kinase Activator Mitapivat Improves Red Blood Cell Deformability and Sickling Kinetics in Adult Patients with Sickle Cell Disease

Background: A hallmark of sickle cell disease (SCD) is compromised red blood cell (RBC) deformability. Polymerization of hemoglobin S (HbS) underlies this "sickling" which results in altered biomechanical properties that impact rheology and hemodynamics (Barabino et al, 2010). There have been exciting clinical developments in anti-sickling therapies - including enhancement of red cell pyruvate kinase (PKR) modality. Importantly, deformability measurements of RBCs via ektacytometry are increasingly used to aid the assessment of their therapeutic efficacy. Here we employed the Laser assisted Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics), which permits the evaluation of RBC deformability as a function of shear stress, osmotic pressure, and oxygen tension, to evaluate the changes in RBC deformability in subjects undergoing treatment with mitapivat (AG-348), a PKR activator under clinical development for sickle cell disease. We analyzed changes in deformability measurements of RBCs in HbSS subjects after 4 weeks and 12 weeks of mitapivat therapy and compared them to t50 - a single parameter measure of sickling kinetics (Dunkelberger et al, 2018). Methods: We studied subjects currently enrolled under IRB approved clinical trial NCT04610866 that evaluates the long-term safety and tolerability of mitapivat in SCD patients. Fifteen subjects (aged 25 - 57 years; 5 females, 10 males; African/African-American) were evaluated, though not all endpoints were available for every patient across all assays. Fresh whole blood (collected in EDTA tubes) was obtained at baseline (prior to drug initiation), after 4 weeks and after 12 weeks of therapy. Cells were counted and diluted in polyvinylpyrrolidone (PVP) solution before undergoing same-day LORRCA assays. The Elongation Index (EI) is a measure of the cells' ability to undergo deformation at different physiologically relevant conditions. EI was assessed at increasing shear stresses of .3 Pascals (Pa), .95 Pa, 3 Pa, 9.49 Pa, and 30 Pa (elongation assay). Parameters derived under continuous osmotic changes (0-600mOsm/kg - osmoscan) include O min which informs on RBC fragility, O Hyper which informs on hydration state and corresponds to 50% of the maximum deformability in the hypertonic region, EI max (osmo) which reveals maximum deformability and informs on RBC cytoskeletal mechanics, and O Max which gives the osmolality at which EI Max is achieved. Point of Sickling (POS) gives the oxygen tension at which the cells start to sickle during nitrogen facilitated deoxygenation of the sample (oxyscan). These parameters of deformability are generated by LORRCA software. For all assays, we analyzed the % change from baseline after 4 weeks and 12 weeks of therapy (Wilcoxon signed rank test, R v1.4.1106) and compared these changes to those in t50 (Spearman correlation test, Prism v9) (Figure 1). Results: Mitapivat therapy resulted in increased elongation indices across all shear stress levels, with significance reached at shears of .3 Pa, .95 Pa, and 3 Pa (respective mean % changes of 78.5, 181.8, and 23.0 (4 weeks) and 101.2, 212.9, and 30.1 (12 weeks)) suggesting improved deformability of RBCs. Likewise, significant increases in O hyper (mean % changes of 2.8 and 3.4) and osmo EI Max (mean % changes 7.9 and 9.5) respectively, indicate improvements in RBC hydration and cytoskeletal dynamics in response to changing osmotic pressures. Furthermore, a significant decrease in the average point of sickling was noted at both 4 weeks and 12 weeks (-20.15% and -16.8%, respectively), suggesting RBCs were susceptible to sickling at partial pressures of oxygen lower than those observed before therapy. Conclusion: Altogether, these findings indicate that mitapivat therapy results in improved ability of RBCs to withstand deformational changes in response to shear stress, osmotic pressure, and decreasing oxygen tension, likely via bolstered membrane integrity. Interestingly, when compared to t50 values, significant correlation was observed at all levels of shear stress and at the maximal deformability (EI max) achieved during both the osmoscan and oxyscan assays. Acknowledgement: This study is part of a Cooperative Research and Development Agreement (CRADA) with AGIOS Pharmaceuticals, Inc., Cambridge, MA, United States Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Activating Pyruvate Kinase Improves Red Blood Cell Integrity By Reducing Band3 Tyrosine Phosphorylation

Background: The scientific rationale for mitapivat (AG-348) as an anti-sickling therapy is that it enhances red cell pyruvate kinase (PKR) activity and glycolytic flux, leading to a reduction of intracellular 2,3-diphosphoglycerate (2,3-DPG) and a concomitant increase in adenosine triphosphate (ATP). Reducing 2,3-DPG destabilizes hemoglobin S (HbS) fibers, while increasing ATP improves red blood cell (RBC) integrity and cellular hydration. Mitapivat increased ATP and decreased 2,3-DPG levels in a dose-dependent manner in this Phase 1 study (Xu et al, 2022). The Hb increase was sustained after stopping mitapivat until end of study (EOS) at 4 weeks, suggesting an improvement in RBC survival. However, pathways modulated by mitapivat that may contribute to sickle RBC survival remain largely unknown. Mitapivat has been shown to improve oxidative stress by increasing GSH/GSSG ratio (Matte et al 2021). In SCD, elevated oxidative stress inhibits RBC tyrosine (Tyr) phosphatases that normally maintain the RBC Band3 (Bd3) tyrosines in an unphosphorylated state. Tyr-phosphorylation of Bd3 destabilizes membrane integrity and reduces RBC deformability (Noomuna et al, 2020). We hypothesize that PKR activators, such as mitapivat and AG-946 (a next-generation activator of PKR), improve the membrane integrity via regulating Tyr-phosphorylation of Bd3. Methods: Of the 16 patients in the Phase1 study, frozen whole blood samples were evaluable in 15, from which RBC ghosts (membranes) at the different timepoints (baseline and following 2 weeks of treatment at each dose level) were isolated. Tyr-phosphorylation of Bd3 was analyzed by Western blotting using a specific phospho-Tyr Bd3 antibody. Ex-vivo experiments were conducted by treating RBCs from different HbSS patients with different concentrations (1 µM, 3 µM, 10 µM, and 30 µM) of mitapivat or AG-946, for 4 hours at 37°C. To investigate the effect of mitapivat on kinases and phosphatases, HbSS RBCs were treated with different concentrations of mitapivat in a combination or without Syk kinase inhibitor-fostamatinib (MedChemExpress). We measured the effect of mitapivat on ATP production in-vitro by treating HbSS RBCs with 30 µM mitapivat for 2h, 4h, 6h, and 8h. The ATP level was detected using CellTiter-Glo® 2.0 Cell Viability Assay (Promega). Meanwhile, the same samples were used to explore the effect of mitapivat on the expression of intact protein tyrosine phosphatase 1B (PTP1B) on the RBC membrane. Results: A dose-dependent decrease in mean Bd3 Tyr-phosphorylation of 20%, 34%, 42%, and 42%, from baseline was observed in the 15 subjects, corresponding to ascending BID dose levels of 2 weeks mitapivat 5 mg, 20 mg, 50 mg, 100 mg, respectively; followed by a return to near baseline by EOS (Figure 1A). Consistent with the in-vivo data, ex-vivo treated HbSS RBCs also showed a dose-dependent decrease in Bd3 Tyr-phosphorylation after either mitapivat or AG-946 treatment (Figure 1B, C). Interestingly, combination of mitapivat with fostamatinib showed an enhanced effect on the reduction of Bd3 Tyr-phosphorylation compared with single drug treatment, which indicated that mitapivat may affect the activity of PTP1B (Figure 1D). Since the activity of PTP1B could be regulated by ATP, we then investigated the effect of mitapivat on ATP level. The ATP level was elevated after 6 hours' mitapivat treatment, even after correction for cell number (Figure 1E). Concomitantly, the intact PTP1B associated with cell membrane also increased after 6 hours of mitapivat treatment (Figure 1F), which might explain the de-phosphorylation of Bd3. Conclusion: A recent study showed that PTP1B is the major phosphatase that maintains Bd3 tyrosine in an unphosphorylated state (Noomuna et al, 2022). PTP1B could be cleaved into an inactive form by calpain, which needs to be activated by calcium. The balance of calcium in RBC relies on the activity of the plasma membrane Ca2+ ATPase (PCMA) pump, which is fueled by ATP. Our results suggest that in enhancing PKR activity, mitapivat leads to an increase in intracellular ATP which increases activity of the PCMA pump, and an increased efflux of intracellular calcium. A reduction in intracellular Ca2+ reduces the activity of calpain, and thereby suppresses calpain's cleavage of phosphatase-PTP1B, resulting in reduction of Bd3 Tyr-phosphorylation. ATP increase is thus a key mechanism of mitapivat's beneficial effect on the hemoglobin increase in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal