OAS-RNaasi L välittää DNA-metylaatiolääkkeiden sytotoksisuutta.

OAS-RNase L Innate Immune Pathway Mediates the Cytotoxicity of a DNA-demethylating Drug

Shuvojit Banerjee  ¹ , Elona Gusho

Affiliations

DOI: 10.1073/pnas.1815071116
Free PMC article

Abstract

Drugs that reverse epigenetic silencing, such as the DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (AZA), have profound effects on transcription and tumor cell survival. AZA is an approved drug for myelodysplastic syndromes and acute myeloid leukemia, and is under investigation for different solid malignant tumors. AZA treatment generates self, double-stranded RNA (dsRNA), transcribed from hypomethylated repetitive elements. Self dsRNA accumulation in DNMTi-treated cells leads to type I IFN production and IFN-stimulated gene expression. Here we report that cell death in response to AZA treatment occurs through the 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway.

  OASs are IFN-induced enzymes that synthesize the RNase L activator 2-5A in response to dsRNA. Cells deficient in RNase L or OAS1 to 3 are highly resistant to AZA, as are wild-type cells treated with a small-molecule inhibitor of RNase L. A small-molecule inhibitor of c-Jun NH₂-terminal kinases (JNKs) also antagonizes RNase L-dependent cell death in response to AZA, consistent with a role for JNK in RNase L-induced apoptosis.

 In contrast, the rates of AZA-induced and RNase L-dependent cell death were increased by transfection of 2-5A, by deficiencies in ADAR1 (which edits and destabilizes dsRNA), PDE12 or AKAP7 (which degrade 2-5A), or by ionizing radiation (which induces IFN-dependent signaling).
Finally, OAS1 expression correlates with AZA sensitivity in the NCI-60 set of tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. These studies may eventually lead to pharmacologic strategies for regulating the antitumor activity and toxicity of AZA and related drugs.

Keywords: 5-azacytidine; DNA methyltransferase inhibitor; OAS; RNase L; innate immunity.


2016 Feb 23;113(8):2241-6.

doi: 10.1073/pnas.1519657113. Epub 2016 Feb 8.

Activation of RNase L Is Dependent on OAS3 Expression During Infection With Diverse Human Viruses

Yize Li  ¹ , Shuvojit Banerjee et al. 

DOI: 10.1073/pnas.1519657113 

The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–RNase L system is an IFN-induced antiviral pathway. RNase L activity depends on 2-5A, synthesized by OAS. Although all three enzymatically active OAS proteins in humans—OAS1, OAS2, and OAS3—synthesize 2-5A upon binding dsRNA, it is unclear which are responsible for RNase L activation during viral infection. We used clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) technology to engineer human A549-derived cell lines in which each of the OAS genes or RNase L is knocked out. Upon transfection with poly(rI):poly(rC), a synthetic surrogate for viral dsRNA, or infection with each of four viruses from different groups (West Nile virus, Sindbis virus, influenza virus, or vaccinia virus), OAS1-KO and OAS2-KO cells synthesized amounts of 2-5A similar to those synthesized in parental wild-type cells, causing RNase L activation as assessed by rRNA degradation. In contrast, OAS3-KO cells synthesized minimal 2-5A, and rRNA remained intact, similar to infected RNase L-KO cells. All four viruses replicated to higher titers in OAS3-KO or RNase L-KO A549 cells than in parental, OAS1-KO, or OAS2-KO cells, demonstrating the antiviral effects of OAS3. OAS3 displayed a higher affinity for dsRNA in intact cells than either OAS1 or OAS2, consistent with its dominant role in RNase L activation. Finally, the requirement for OAS3 as the major OAS isoform responsible for RNase L activation was not restricted to A549 cells, because OAS3-KO cells derived from two other human cell lines also were deficient in RNase L activation. 

 

 

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Figure 6.

An active RNase L dimer preferentially localizes to the polysome to form a complex with Dom34. (A) HeLa cells were transfected with a combination of pCMV-5×Myc-Dom34 (lanes 1–6) and either pCMV-5×Flag (lanes 1 and 4), pCMV-5×Flag-RNase L (lanes 2 and 5) or pCMV-5×Flag-RNase L Y312A (lanes 3 and 6). The cells were lysed in buffer B. The immunoprecipitates (lanes 4–6) and inputs (lanes 1–3, 10% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. The leftmost five lanes, which analyzed 3-fold dilutions of total protein, show that the conditions used for western blotting are semi-quantitative. (B) The amount of immunoprecipitated 5×Myc-Dom34 protein in (A) was measured and normalized to the input 5×Myc-Dom34 protein, and either immunoprecipitated 5×Flag-RNase L wt or RNase L Y312A. Dom34 protein level in pCMV-5×Flag-RNase L wt-transfected cells (lane 5) was defined as 100% and Dom34 protein level in pCMV-5×Flag-RNase L Y312A-transfected cells (lane 6) was represented. (C) HeLa/5×Flag-RNase L cells were transfected with 2–5A using Neon™ Transfection System. At 6 h after 2–5A electroporation, the cells were harvested and lysed in buffer G. The cell lysate was fractionated by sucrose gradients. 5×Flag-RNase L in the polysomal fractions and the input (0.5% of the amount loaded onto sucrose gradients) was analyzed by western blotting with an anti-Flag antibody. (D) The amount of endogenous 5×Flag-tagged RNase L protein in (C) was measured and normalized to the input. RNase L protein levels in mock cells were set to 1 and fold-changes were indicated (mean ± SEM, n = 3). (E) Model for the Dom34-mediated decay of exogenous mRNA (see Discussion).