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Summary Long noncoding RNAs (lncRNAs) modulate various biological processes, but their role in host antiviral responses is largely unknown. Here we identify a lncRNA as a key regulator of antiviral innate immunity. Following from the observation that a lncRNA that we call negative regulator of antiviral response (NRAV) was dramatically downregulated during infection with several viruses, we ectopically expressed NRAV in human cells or transgenic mice and found that it significantly promotes influenza A virus (IAV) replication and virulence. Conversely, silencing NRAV suppressed IAV replication and virus production, suggesting that reduction of NRAV is part of the host antiviral innate immune response to virus infection. NRAV negatively regulates the initial transcription of multiple critical interferon-stimulated genes (ISGs), including IFITM3 and MxA, by affecting histone modification of these genes. Our results provide evidence for a lncRNA in modulating the antiviral interferon response., Graphical Abstract, Highlights • Genome-wide lncRNA microarray analysis of influenza virus-infected cells identifies NRAV • NRAV overexpression promotes and silencing suppresses influenza virus infection • NRAV suppresses interferon-stimulated genes via regulating histone modification of ISGs • NRAV downregulation is suggested an innate antiviral host defense mechanism, Ouyang et al. identify NRAV, a functional long noncoding RNA that negatively regulates the initial transcription of multiple critical interferon-stimulated antiviral genes, and can promote influenza virus replication and virulence. Host cells dramatically reduce NRAV levels during viral infection. These findings position lncRNAs as modulators of antiviral innate immunity.

Innate cytokine response provides the first line of defense against influenza virus infection. However, excessive production of cytokines appears to be critical in the pathogenesis of influenza virus. Interferon lambdas (IFN-λ) have been shown to be overproduced during influenza virus infection, but the precise pathogenic processes of IFN-λ production have yet to be characterized. In this report, we observed that influenza virus induced robust expression of IFN-λ in alveolar epithelial cells (A549) mainly through a RIG-I-dependent pathway, but IFN-λ-induced phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was dramatically inhibited in the infected cells. Remarkably, influenza virus infection induced robust expression of suppressor of cytokine signaling-1 (SOCS-1), leading to inhibition of STAT1 activation. Interestingly, the virus-induced SOCS-1 expression was cytokine-independent at early stage of infection both in vitro and in vivo. Using transgenic mouse model and distinct approaches altering the expression of SOCS-1 or activation of STAT signaling, we demonstrated that disruption of the SOCS-1 expression or expression of constitutively active STAT1 significantly reduced the production of IFN-λ during influenza virus infection. Furthermore, we revealed that disruption of IFN-λ signaling pathway by increased SOCS-1 protein resulted in the activation of NF-κB and thereby enhanced the IFN-λ expression. Together, these data imply that suppression of IFN-λ signaling by virus-induced SOCS-1 causes an adaptive increase in IFN-λ expression by host to protect cells against the viral infection, as a consequence, leading to excessive production of IFN-λ with impaired antiviral response., Author Summary Influenza virus infection triggers innate immune responses. However, aberrant host immune responses such as excessive production of cytokines contribute to the pathogenesis of influenza virus. Type III interferons (IFN-λ) constitute the major innate immune response to influenza virus infection, but the precise pathogenic processes of IFN-λ production and mechanistic underpinnings are not well understood. In this study, we report that influenza virus induces robust IFN-λ expression mainly through a RIG-I-dependent pathway, but signaling activated by IFN-λ was dramatically inhibited by virus-induced SOCS-1. Importantly, we found that disruption of the SOCS-1 expression or forced activation of STAT1 significantly reduced the expression of IFN-λ in vitro and in vivo, suggesting that suppression of IFN-λ signaling by SOCS-1 results in their excessive production during influenza virus infection. Furthermore, our experiments revealed that disruption of IFN-λ signaling pathway resulted in the activation of NF-κB that governs the IFN-λ expression. Together these findings, we propose that impaired antiviral response of IFN-λ due to the inhibitory effect of SOCS-1 causes an adaptive increase in IFN-λ expression by host to protect cells against the viral infection. This is a novel mechanism that may be critical in the pathogenesis of the influenza virus strains that induce hypercytokinemia.

Dysregulation of non-coding RNA H19 has been observed in various tumors. However, it remains unknown whether H19 is involved in Bcr-Abl-induced leukemia. Here, we demonstrate a critical requirement for H19 in Bcr-Abl-mediated tumorigenesis. H19 was highly expressed in Bcr-Abl-transformed cell lines and primary cells derived from patients in a Bcr-Abl kinase-dependent manner. Silencing H19 expression sensitized leukemic cells to undergo imatinib-induced apoptosis and inhibited Bcr-Abl-induced tumor growth. Furthermore, H19 was shown to be regulated by c-Myc in Bcr-Abl-expressing cells. These results reveal an important role H19 plays in Bcr-Abl-mediated transformation and provide novel insights into complex mechanisms underlying Bcr-Abl-induced cancers.

CDKN3 (cyclin-dependent kinase inhibitor 3), a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs) and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML) remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

It is well documented that malignant transformation of myeloid and lymphoid cells by Bcr-Abl involves the dysregulation or mutation of a variety of protein-coding genes that are normally involved in regulating the proliferation and survival of hematopoietic cells. However, the functional relevance of long noncoding RNAs (lncRNAs) and their in vivo regulation during Bcr-Abl-mediated tumorigenesis remain unknown, although recent studies have shown that aberrant expression of lncRNAs is associated with various human cancers. In this study, we performed a comprehensive analysis of lncRNAs in human CML cells using an lncRNA cDNA microarray, and identified an lncRNA termed lncRNA-BGL3 that acted as a key regulator of Bcr-Abl-mediated cellular transformation. Notably, we observed that lncRNA-BGL3 was highly induced in response to disruption of Bcr-Abl expression or by inhibiting Bcr-Abl kinase activity in K562 cells and leukemic cells derived from CML patients. Ectopic expression of lncRNA-BGL3 sensitized leukemic cells to undergo apoptosis and inhibited Bcr-Abl-induced tumorigenesis. Furthermore, transgenic mice expressing lncRNA-BGL3 were generated. We found that transgenic expression of lncRNA-BGL3 alone in mice was sufficient to impair primary bone marrow transformation by Bcr-Abl. Interestingly, we identified that lncRNA-BGL3 was a target of miR-17, miR-93, miR-20a, miR-20b, miR-106a and miR-106b, microRNAs that repress mRNA of PTEN. Further experiments demonstrated that lncRNA-BGL3 functioned as a competitive endogenous RNA (ceRNA) for binding these microRNAs to cross-regulate PTEN expression. Additionally, our experiments have begun to address the mechanism of how lncRNA-BGL3 is regulated in the leukemic cells and showed that Bcr-Abl repressed lncRNA-BGL3 expression through c-Myc-dependent DNA methylation. Taken together, these results reveal that Bcr-Abl-mediated cellular transformation critically requires silence of tumor suppressor lncRNA-BGL3 and suggest a potential strategy for the treatment of Bcr-Abl-positive leukemia. Citation Format: Guijie Guo, Qingzheng Kang, Qinghuang Chen, Ouyang Jing, Jilong Chen. A long noncoding RNA plays a critical role in Bcr-Abl-mediated cellular transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3543. doi:10.1158/1538-7445.AM2014-3543