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XRN1 deletion induces PKR-dependent cell lethality in interferon-activated cancer cells

ABSTRACT: Emerging data suggest that induction of viral mimicry responses through activation of double-stranded RNA (dsRNA) sensors in cancer cells is a promising therapeutic strategy. One approach to induce viral mimicry is to target molecular regulators of dsRNA sensing pathways. Here, we show that the exoribonuclease XRN1 is a negative regulator of the dsRNA sensor protein kinase R (PKR) in cancer cells with high interferon-stimulated gene (ISG) expression. XRN1 deletion causes PKR pathway activation and consequent cancer cell lethality. Disruption of interferon signaling with the JAK1/2 inhibitor ruxolitinib can decrease cellular PKR levels and rescue sensitivity to XRN1 deletion. Conversely, interferon-b stimulation can increase PKR levels and induce sensitivity to XRN1 inactivation. Lastly, XRN1 deletion causes accumulation of endogenous complementary sense/anti-sense RNAs, which may represent candidate PKR ligands. Our data demonstrate how XRN1 regulates PKR, and how this interaction creates a vulnerability in cancer cells with an activated interferon cell state.

ORGANISM(S): Homo sapiens

PROVIDER: GSE248036 | GEO | 2024/04/17

REPOSITORIES: GEO

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ADAR1 utilizes independent mechanisms to suppress MDA5 and PKR activation by cellular double-stranded RNA

Project description:The antiviral defense in vertebrates requires the innate immune system to sense foreign “non-self” nucleic acids while avoiding “self” nucleic acids, which is accomplished by an intricate system. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA editing enzyme ADAR1 to prevent their dsRNA structure pattern from being recognized as viral dsRNA. Lack of RNA editing by ADAR1 enables activation of MDA5, a cytosolic dsRNA sensor, by cellular dsRNA. Additional RNA editing- independent functions of ADAR1 have been proposed, but the specific mechanism remains elusive. Here we demonstrate that RNA binding by ADAR1, independent of its editing activity, restricts the activation of PKR, another cytosolic dsRNA sensor, by cellular dsRNA. Mechanistically, the loss of ADAR1 editing caused MDA5 activation to induce interferon signaling, while a lack of ADAR1 protein or its dsRNA binding ability led to PKR activation, with subsequent stress granule formation and proliferation arrest. Based on these findings we rescued the Adar1−/− mice from embryonic lethality to adulthood by deleting both MDA5 and PKR, in contrast to the limited rescue of Adar1−/− mice by removing MDA5 or PKR alone. Our findings reveal a multifaceted contribution of ADAR1 in regulating the immunogenicity of “self” dsRNAs. Furthermore, ADAR1 is an immuno-oncology target for drug development, and the separation of ADAR1’s RNA editing and binding functions provides mechanistic insights for such developments.

2023-10-03 | GSE198386 | GEO

Expressed dsRNA provokes selective sequence-independent PKR response in transient co-transfection experiments

Project description:In mammals, double-stranded RNA (dsRNA) plays roles in sequence-specific RNA interference, sequence-independent interferon response, and RNA editing by adenosine deaminases. We have previously shown that long hairpin dsRNA expression in cultured cells does not activate the interferon response, it is poorly processed into siRNAs, and it is partially edited. Here, we demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits expression of co-transfected reporter plasmids but not expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent and independent of a cell type, transfection method, or dsRNA sequence. The inhibition occurs at the level of translation and is mediated by protein kinase R (PKR). PKR binds the expressed dsRNA, becomes phosphorylated and changes its distribution along polysome fractions. In conclusion, we demonstrate that expression from plasmids is selectively repressed if one of co transfected plasmids produces dsRNA. Our results highlight the importance of proper controls and careful interpretation of co-transfection experiments. HEK293 cells (human origin) were transiently transfected with hairpin dsRNA-expressing (MosIR) and control (pCag) plasmids.

2013-12-27 | E-GEOD-46959 | biostudies-arrayexpress

Expressed dsRNA provokes selective sequence-independent PKR response in transient co-transfection experiments

2013-12-27 | GSE46959 | GEO

PKR activity modulation by phosphomimetic mutations of serine residues located three aminoacids upstream of double-stranded RNA binding motifs

Project description:Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), better known as PKR plays a key role in the response to viral infections and cellular homeostasis by regulating mRNA translation. Upon binding dsRNA, PKR is activated through homodimerization and subsequent autophosphorylation on Thr446 and Thr451 residues. In this study, we identified a novel PKR phosphorylation site, Ser6, located 3 aminoacids upstream the first double-stranded RNA binding domain (DRBM1). Another Ser residue occurs in PKR at position 97, the very same position relative to the DRBM2. Ser or Thr residues also occur 3 amino acids upstream DRBMs of other proteins such as ADAR1 or DICER. Phosphoinhibiting mutations (Ser-to-Ala) introduced at Ser6 and Ser97 spontaneously activated PKR. In contrast, phosphomimetic mutations (Ser-to-Asp) inhibited PKR activation following either poly (I:C) transfection or virus infection. These mutations moderately affected dsRNA binding, suggesting a model where negative charges occuring at position 6 and 97 tighten the interaction of DRBMs with the kinase domain, thus keeping PKR in an inactive, closed conformation even in the presence of dsRNA. This study provides new insights on PKR regulation mechanisms and identifies Ser6 and Ser97 as potential targets to modulate PKR activity for therapeutic purposes

2021-05-10 | PXD022564 | Pride

Synthesis of immunogenicity-free RNA circles as potent PKR inhibitors

Project description:Exon(s) back-splicing-generated circular RNAs as a group can suppress the double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing circular RNAs as potent PKR inhibitors. Here, we report that circular RNAs synthesized by permuted td introns from T4 bacteriophage and by Anabeana pre-tRNA group I intron induce an immune response. Mechanistically, autocatalytic splicing introduces extra ~74 nt and ~186 nt fragments (E1+E2+spacer) that form additional stable loop structures that likely provoke innate immune responses in circular RNA products. In contrast, circular RNAs produced by T4 RNA ligase without unwanted fragment exhibit little immunogenicity. Importantly, directly-ligated circular RNAs form short dsRNA-regions exhibit 103~106 fold higher efficiency than the reported chemical compounds C16 and 2-AP in suppressing PKR activation, highlighting the use of circular RNAs as novel inhibitors for PKR over-reaction diseases.

2021-12-23 | GSE183941 | GEO

Transcription profiling of human NIH 3T3 inducible cell line to study the role of PKR in regulating gene expression

Project description:PKR is an interferon induced serine/threonine protein kinase, that is activated by double stranded RNA. PKR plays an important role in the antiviral defense by interferon. In addition to its role in translation, PKR participates in several signaling pathways to transcription. The goal of this experiment is to study the role of PKR in regulating gene expression in our NIH 3T3 inducible cell line, which could overexpress PKR wt protein after the removal of tetracycline (Donze O, Dostie J, Sonenberg N. (1999) Virology 256: 322-9).

2004-01-30 | E-MEXP-50 | biostudies-arrayexpress

XRN1 knockdown stimulate innate immune reponse in murine melanoma cells

Project description:Despite the remarkable achievement of immune checkpoint blockade (ICB) therapy, the response rate is relatively low and only a subset of patients can benefit from the treatment. We hypothesize that targeting RNA decay machinery may lead to accumulation of aberrantRNA, triggering interferon (IFN) signaling and sensitizing tumor cells to immunotherapy. With this in mind, we identified an RNA exoribonuclease, XRN1 as a potential target. Silencing of XRN1 suppressed tumor growth in syngeneic immunocompetent mice and potentiated immunotherapy, while silencing of XRN1 alone did not affect tumor growth in immune deficient mice. Mechanistically, XRN1 depletion activated interferon signaling and viral defense pathway; both pathways play determinant roles in regulating immune evasion. We identified aberrant-RNA sensing signaling proteins (RIG-I/MAVS and PKR) in mediating the expression of IFN genes, as depletion of each of them blunted the elevation of anti-viral/IFN signaling in Xrn1 silenced cells. Analysis of pan-cancer CRISPR screening data indicated that IFN signaling triggered by Xrn1 silencing is a common phenomenon, suggesting that the effect of Xrn1 silencing may be extend to multiple types of cancers.

2023-02-21 | GSE199744 | GEO

XRN1 knockdown in tumor cells stimulates immune cell infiltration

Project description:Despite the remarkable achievement of immune checkpoint blockade (ICB) therapy, the response rate is relatively low and only a subset of patients can benefit from the treatment. We hypothesize that targeting RNA decay machinery may lead to accumulation of aberrantRNA, triggering interferon (IFN) signaling and sensitizing tumor cells to immunotherapy. With this in mind, we identified an RNA exoribonuclease, XRN1 as a potential target. Silencing of XRN1 suppressed tumor growth in syngeneic immunocompetent mice and potentiated immunotherapy, while silencing of XRN1 alone did not affect tumor growth in immune deficient mice. Mechanistically, XRN1 depletion activated interferon signaling and viral defense pathway; both pathways play determinant roles in regulating immune evasion. In murine tumors engrafted on immmunocompetent mice, XRN1 depletion significantly enhanced immune cell infiltration in solid tumors especially in combinatory with PD-1 blockade. We identified aberrant-RNA sensing signaling proteins (RIG-I/MAVS and PKR) in mediating the expression of IFN genes, as depletion of each of them blunted the elevation of anti-viral/IFN signaling in Xrn1 silenced cells. Analysis of pan-cancer CRISPR screening data indicated that IFN signaling triggered by Xrn1 silencing is a common phenomenon, suggesting that the effect of Xrn1 silencing may be extend to multiple types of cancers.

2023-02-21 | GSE202587 | GEO

PKR overexpression

Project description:Global analysis of gene expression in NIH3T3 cells over-expressing RNA-dependent serine/threonine protein kinase (PKR). Keywords: other

2004-09-22 | GSE682 | GEO

Long non-coding RNA DARS-AS1 promotes tumor progression by suppressing PACT-mediated PKR activation

Project description:Cancer cells evolve various mechanisms to overcome cellular stresses and maintain progression. Protein kinase R (PKR) and its protein activator (PACT) are the initial responders in monitoring diverse stress signals and lead to cell proliferation inhibition and cell apoptosis in consequence. However, the regulation of PACT-PKR pathway in cancer cells is largely unknown. Herein, we identify that the long non-coding RNA (lncRNA) aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) is directly involved in the inhibition of the PACT-PKR pathway and promotes cancer cell proliferation. Using large-scale CRISPRi functional screening of 971 cancer-associated lncRNAs, we find that DARS-AS1 is associated with significantly enhanced cancer cell proliferation. Accordingly, knocking down DARS-AS1 inhibits cell proliferation of multiple cancer cell lines and promotes cancer cell apoptosis in vitro and significantly reduces tumor growth in vivo. Mechanistically, DARS-AS1 directly binds to the activator domain of PACT and prevents PACT-PKR interaction, thereby decreasing PKR activation, eIF2α phosphorylation and inhibiting apoptotic cell death. Clinically, DARS-AS1 is broadly expressed across multiple cancers and the increased expression of this lncRNA indicates poor prognosis. This study elucidates the lncRNA DARS-AS1 directed cancer-specific modulation of the PACT-PKR pathway and provides a novel target for cancer prognosis and therapeutic treatment.

2022-05-15 | GSE197980 | GEO

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