Inhibition of RNase L and RNA-dependent Protein Kinase (PKR) by Sunitinib Impairs Antiviral Innate Immunity

Abstract

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC 50 values of 1.4 and 0.3 M, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-␤ levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.RNase L and PKR 2 are host enzymes of higher vertebrates that participate in innate immunity against viral infections (1-4). Activation of both RNase L and PKR is triggered by the viral pathogen-associated molecular pattern, double-stranded RNA (dsRNA). However, whereas dsRNA directly binds to and activates PKR, in the case of RNase L activation is indirect. Interferon (IFN) treatment of cells induces PKR that, upon binding to dsRNA, phosphorylates first itself and then EIF2␣ thus blocking protein synthesis among other effects. RNase L degrades single-stranded RNA resulting in pleiotropic antiviral effects (5). Short 5Ј-triphosphorylated, 2Ј,5Ј-oligoadenylates (2-5A) are produced from ATP when viral dsRNA stimulates IFN-inducible oligoadenylate synthetases. 2-5A binds ankyrin repeats 2 and 4 in the N-terminal region of RNase L causing its dimerization and activation (6). RNase L is also pseudokinase with amino acid sequence homology to the PKR kinase domains (7).IRE1, a kinase and endoribonuclease involved in the unfolded protein response, is another relative of RNase L (8). IRE1 spans the endoplasmic reticulum (ER) membrane. The intralumenal domains of IRE1 directly or indirectly sense unfolded proteins in the ER leading to autophosphorylation and ribonuclease activities in the cytoplasmic domains. IRE1 excises an intron from pre-mRNA for a transcription factor (HAC1 in yeast and XBP1 in mammals) leading to splicing and translation (9). HAC1/XBP1 drives expression of ER chaperones and protein folding enzymes that re-establish ER function. The kinase-extension-nuclease (KEN) domains of RNase L and IRE1 ar...

The Multiple Functions of TRBP, at the Hub of Cell Responses to Viruses, Stress, and Cancer

“…PKR is therefore implicated in the integration and transmission of signals to the translational machinery as well as to factors such as STAT, IFN regulatory factor 1, p53, Jun Nterminal protein kinase (JNK), p38, ATF-3, and NF-B (48,121,125). PKR overexpression decreases-whereas its inhibition increases-the expression levels of several viruses (34,72,106,109,117). The cellular function of PKR on cell growth appears to be more complicated, because its upregulation or overexpression can inhibit the growth of some tumor cells, whereas an upregulation of PKR has been identified in some malignancies (13,104,141).…”

RNase L Attenuates Mitogen-stimulated Gene Expression via Transcriptional and Post-transcriptional Mechanisms to Limit the Proliferative Response

“…Interestingly, the RNase L pseudokinase domain is critical for coordinating 2-5A binding (39,40) and homodimerization (33,34) that occur upon its activation. Furthermore, the ATP competitive kinase inhibitor sunitinib was recently shown to bind the ATP pocket within this domain and inhibit RNase L activation (93). Together, these studies establish a central role for the pseudokinase domain in RNase L activation and suggest that heterologous proteins that interact in this domain may positively or negatively impact RNase L activity as a novel mechanism to modulate its biologic functions.…”

Oxidative Stress-Associated Protein Tyrosine Kinases and Phosphatases in Fanconi Anemia

“…It has been shown that inhibition of PKR represents an interesting strategy for neuroprotection, and preventing the cell death induced by ER stress in cultured human neuroblastoma cells (16,36,48,49,51,106,139). PKR was constitutively activated in FA-C cells, which contributed to the hypersensitivity of bone marrow progenitor cells to growth repression mediated by the inhibitory cytokines IFN-c and FIG.…”