Sonali Joshi

Role of the Akt pathway in mRNA translation of interferon-stimulated genes

Multiple signaling pathways are engaged by the type I and II IFN receptors, but their specific roles and possible coordination in the generation of IFN-mediated biological responses remain unknown. We provide evidence that activation of Akt kinases is required for IFN-inducible engagement of the mTOR/p70 S6 kinase pathway. Our data establish that Akt activity is essential for up-regulation of key IFN-α- and IFN-γ-inducible proteins, which have important functional consequences in the induction of IFN responses. Such effects of the Akt pathway are unrelated to regulatory activities on IFN-dependent STAT phosphorylation/activation or transcriptional regulation. By contrast, they reflect regulatory activities on mRNA translation via direct control of the mTOR pathway. In studies using Akt1 and Akt2 double knockout cells, we found that the absence of Akt kinases results in dramatic reduction in IFN-induced antiviral responses, establishing a critical role of the Akt pathway in IFN signaling. Thus, activation of the Akt pathway by the IFN receptors complements the function of IFN-activated JAK–STAT pathways, by allowing mRNA translation of IFN-stimulated genes and, ultimately, the induction of the biological effects of IFNs.

Type I interferon (IFN)-dependent activation of Mnk1 and its role in the generation of growth inhibitory responses

We provide evidence for the existence of an IFN-regulated cellular pathway involving the mitogen-activated protein kinase (MAPK)-integrating kinase (Mnk) 1. Our data demonstrate that type I (α, β) IFNs induce phosphorylation/activation of Mnk1, which, in turn, regulates phosphorylation of the eukaryotic initiation factor 4E (eIF4E) on Ser-209. Such Mnk activation depends on upstream engagement of Jak1, and requires downstream activation of the Mek/Erk MAPK pathway. In studies using double Mnk1−/−Mnk2−/− knockout mouse embryonic fibroblasts (MEFs), we found that engagement of Mnk kinases is essential for mRNA translation of the Isg15 and Isg54 genes, suggesting an important role for this pathway in mRNA translation of IFN-stimulated genes (ISGs). Importantly, our data demonstrate that pharmacological inhibition of Mnk kinases or siRNA-mediated knockdown of Mnk1 and Mnk2 results in partial reversal of the suppressive effects of IFNα on normal and leukemic hematopoietic progenitors, establishing a key role for this pathway in the generation of the growth inhibitory effects of type I IFNs. Together, our findings establish that the Mnk/eIF4E kinase pathway is activated in an IFN-inducible manner and plays important roles in mRNA translation for ISGs and generation of IFN-inducible anti-proliferative responses.

Mnk kinase pathway: Cellular functions and biological outcomes.

The mitogen-activated protein kinase (MAPK) interacting protein kinases 1 and 2 (Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs (p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E (eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4E. The role of Mnk kinases in inflammation and inflammation-induced malignancies is also discussed.

AMPK as a therapeutic target in renal cell carcinoma

AMPK is a cellular energy sensor that negatively regulates the mTOR signaling pathway. As mTOR plays critical roles in cell growth and tumorigenesis of renal cell carcinoma (RCC), we examined whether exogenous induction of AMPK activity exhibits inhibitory effects on growth and survival of renal cell carcinoma cells. Activation of AMPK by AICAR resulted in potent suppressive effects on RCC growth, while combinations of AICAR with statins were potent inducers of apoptosis in such cells. The effects of AICAR resulted from inhibition of mTOR and its effectors, resulting from induction of AMPK activity. Similar results on RCC cell growth were obtained when combinations of metformin with statins were examined. Importantly, studies to examine the effects of AICAR or metformin, alone or in combinations with statins, on anchorage-independent growth demonstrated potent suppressive effects on RCC tumorigenicity in vitro. Altogether, our studies demonstrate that AMPK plays critical regulatory roles in the regulation of growth of RCC cells and raise the prospect of future use of AMPK activators in the treatment of renal cell carcinoma in humans.

Sprouty Proteins Are Negative Regulators of Interferon (IFN) Signaling and IFN-inducible Biological Responses

Background: The potential involvement of Spry proteins in IFN signaling is unknown. Results: Type I IFN treatment results in up-regulation of Spry proteins, which negatively control generation of IFN responses. Conclusion: Spry proteins play important regulatory roles in IFN signaling and the generation of the biological effects of IFNs. Significance: This study provides evidence for the existence of a key signaling pathway that controls IFN responses. Interferons (IFNs) have important antiviral and antineoplastic properties, but the precise mechanisms required for generation of these responses remain to be defined. We provide evidence that during engagement of the Type I IFN receptor (IFNR), there is up-regulation of expression of Sprouty (Spry) proteins 1, 2, and 4. Our studies demonstrate that IFN-inducible up-regulation of Spry proteins is Mnk kinase-dependent and results in suppressive effects on the IFN-activated p38 MAP kinase (MAPK), the function of which is required for transcription of interferon-stimulated genes (ISGs). Our data establish that ISG15 mRNA expression and IFN-dependent antiviral responses are enhanced in Spry1,2,4 triple knock-out mouse embryonic fibroblasts, consistent with negative feedback regulatory roles for Spry proteins in IFN-mediated signaling. In other studies, we found that siRNA-mediated knockdown of Spry1, Spry2, or Spry4 promotes IFN-inducible antileukemic effects in vitro and results in enhanced suppressive effects on malignant hematopoietic progenitors from patients with polycythemia vera. Altogether, our findings demonstrate that Spry proteins are potent regulators of Type I IFN signaling and negatively control induction of Type I IFN-mediated biological responses.

Essential Role for Mnk Kinases in Type II Interferon (IFNγ) Signaling and Its Suppressive Effects on Normal Hematopoiesis

IFNγ exhibits potent antitumor effects and plays important roles in the innate immunity against cancer. However, the mechanisms accounting for the antiproliferative effects of IFNγ still remain to be elucidated. We examined the role of Mnk1 (MAPK-interacting protein kinase 1) in IFNγ signaling. Our data demonstrate that IFNγ treatment of sensitive cells results in engagement of Mnk1, activation of its kinase domain, and downstream phosphorylation of the cap-binding protein eIF4E on Ser-209. Such engagement of Mnk1 plays an important role in IFNγ-induced IRF-1 (IFN regulatory factor 1) gene mRNA translation/protein expression and is essential for generation of antiproliferative responses. In studies aimed to determine the role of Mnk1 in the induction of the suppressive effects of IFNs on primitive hematopoietic progenitors, we found that siRNA-mediated Mnk1/2 knockdown results in partial reversal of the suppressive effects of IFNγ on human CD34+-derived myeloid (CFU-GM) and erythroid (BFU-E) progenitors. These findings establish a key role for the Mnk/eIF4E pathway in the regulatory effects of IFNγ on normal hematopoiesis and identify Mnk kinases as important elements in the control of IFNγ-inducible ISG mRNA translation.

Negative regulatory effects of Mnk kinases in the generation of chemotherapy-induced antileukemic responses.

Mnk kinases are downstream effectors of mitogen-activated protein kinase pathways and mediate phosphorylation of the eukaryotic initiation factor (eIF4E), a protein that plays a key role in the regulation of mRNA translation and is up-regulated in acute myeloid leukemia (AML). We determined the effects of chemotherapy (cytarabine) on the activation status of Mnk in AML cells and its role in the generation of antileukemic responses. A variety of experimental approaches were used, including immunoblotting, apoptosis assays, small interfering RNA (siRNA)-mediated knockdown of proteins, and clonogenic hematopoietic progenitor assays in methylcellulose. Cytarabine induced phosphorylation/activation of Mnk and Mnk-mediated phosphorylation of eIF4E on Ser209, as evidenced by studies involving pharmacological inhibition of Mnk or experiments using cells with targeted disruption of Mnk1 and Mnk2 genes. To assess the functional relevance of cytarabine-inducible engagement of Mnk/eIF4E pathway, the effects of pharmacological inhibition of Mnk on cytarabine-mediated suppression of primitive leukemic progenitors [leukemic colony forming unit (CFU-L)] were examined. Concomitant treatment of cells with a pharmacological inhibitor of Mnk or siRNA-mediated knockdown of Mnk1/2 strongly enhanced the suppressive effects of low cytarabine concentrations on CFU-L. It is noteworthy that the mammalian target of rapamycin (mTOR) inhibitor rapamycin also induced phosphorylation of eIF4E in a Mnk-dependent manner, whereas inhibition strongly enhanced its antileukemic effects. These data demonstrate that Mnk kinases are activated in a negative-feedback regulatory manner in response to chemotherapy and impair the generation of antileukemic responses. They also identify this pathway as a novel target for the design of new approaches to enhance the antileukemic effects of chemotherapy or mTOR inhibitors in AML.

Mnk kinases in cytokine signaling and regulation of cytokine responses.

Abstract The kinases Mnk1 and Mnk2 are activated downstream of the p38 MAPK and MEK/ERK signaling pathways. Extensive work over the years has shown that these kinases control phosphorylation of the eukaryotic initiation factor 4E and regulate engagement of other effector elements, including hnRNPA1 and PSF. Mnk kinases are ubiquitously expressed and play critical roles in signaling for various cytokine receptors, and there is emerging evidence that they have important functions as mediators of proinflammatory cytokine production. In this review, the mechanisms of activation of MNK pathways by cytokine receptors are addressed and their roles in diverse cytokine-dependent biological processes are reviewed. The clinical-translational implications of such work and the relevance of future development of specific MNK inhibitors for the treatment of malignancies and autoimmune disorders are discussed.

Regulatory Effects of Ribosomal S6 Kinase 1 (RSK1) in IFNλ Signaling

Although the mechanisms of generation of signals that control transcriptional activation of Type III IFN (IFNλ)-regulated genes have been identified, very little is known about the mechanisms by which the IFNλ receptor generates signals for mRNA translation of IFNλ-activated genes. We provide evidence that IFNλ activates the p90 ribosomal protein S6 kinase 1 (RSK1) and its downstream effector, initiation factor eIF4B. Prior to its engagement by the IFNλ receptor, the non-active form of RSK1 is present in a complex with the translational repressor 4E-BP1 in IFNλ-sensitive cells. IFNλ-inducible phosphorylation/activation of RSK1 results in its dissociation from 4E-BP1 at the same time that 4E-BP1 dissociates from eIF4E to allow formation of eIF4F and initiation of cap-dependent translation. Our studies demonstrate that such IFNλ-dependent engagement of RSK1 is essential for up-regulation of p21WAF1/CIP1 expression, suggesting a mechanism for generation of growth-inhibitory responses. Altogether, our data provide evidence for a critical role for the activated RSK1 in IFNλ signaling.

Essential role for the Mnk-pathway in the inhibitory effects of Type I interferons on myeloproliferative neoplasm (MPN) precursors

Background: The mechanisms by which IFNs generate antineoplastic responses remain to be defined. Results: Type I IFN treatment results in activation of the Mnk/eIF4E pathway in Jak2V617F-transformed cells, and this activation is required for the antineoplastic effect. Conclusion: Mnk kinases are essential for the antineoplastic effects of IFN. Significance: This study provides evidence for a key mechanism mediating the effects of IFNs in malignant MPN precursors. The mechanisms of generation of the antineoplastic effects of interferons (IFNs) in malignant hematopoietic cells remain to be precisely defined. We examined the activation of type I IFN-dependent signaling pathways in malignant cells transformed by Jak2V617F, a critical pathogenic mutation in myeloproliferative neoplasms (MPNs). Our studies demonstrate that during engagement of the type I IFN receptor (IFNAR), there is activation of Jak-Stat pathways and also engagement of Mnk kinases. Activation of Mnk kinases is regulated by the Mek/Erk pathway and is required for the generation of IFN-induced growth inhibitory responses, but Mnk kinase activation does not modulate IFN-regulated Jak-Stat signals. We demonstrate that for type I IFNs to exert suppressive effects in malignant hematopoietic progenitors from patients with polycythemia vera, induction of Mnk kinase activity is required, as evidenced by studies involving pharmacological inhibition of Mnk or siRNA-mediated Mnk knockdown. Altogether, these findings provide evidence for key and essential roles of the Mnk kinase pathway in the generation of the antineoplastic effects of type I IFNs in Jak2V617F-dependent MPNs.