Sirtuin 2-mediated deacetylation of cyclin-dependent kinase 9 promotes STAT1 signaling in type I interferon responses.

Type I interferons (IFNs) induce expression of multiple genes that control innate immune responses to invoke both antiviral and antineoplastic activities. Transcription of these interferon-stimulated genes (ISGs) occurs upon activation of the canonical Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways. Phosphorylation and acetylation are both events crucial to tightly regulate expression of ISGs. Here, using mouse embryonic fibroblasts and an array of biochemical methods including immunoblotting and kinase assays, we show that sirtuin 2 (SIRT2), a member of the NAD-dependent protein deacetylase family, is involved in type I IFN signaling. We found that SIRT2 deacetylates cyclin-dependent kinase 9 (CDK9) in a type I IFN-dependent manner and that the CDK9 deacetylation is essential for STAT1 phosphorylation at Ser-727. We also found that SIRT2 is subsequently required for the transcription of ISGs and for IFN-driven antiproliferative responses in both normal and malignant cells. These findings establish the existence of a previously unreported signaling pathway whose function is essential for the control of JAK-STAT signaling and the regulation of IFN responses. Our findings suggest that targeting sirtuin activities may offer an avenue in the development of therapies for managing immune-related diseases and cancer.

Differential Response of Glioma Stem Cells to Arsenic Trioxide Therapy Is Regulated by MNK1 and mRNA Translation.

Mesenchymal (MES) and proneural (PN) are two distinct glioma stem cell (GSC) populations that drive therapeutic resistance in glioblastoma (GBM). We screened a panel of 650 small molecules against patient-derived GBM cells to discover compounds targeting specific GBM subtypes. Arsenic trioxide (ATO), an FDA-approved drug that crosses the blood-brain barrier, was identified as a potent PN-specific compound in the initial screen and follow-up validation studies. Furthermore, MES and PN GSCs exhibited differential sensitivity to ATO. As ATO has been shown to activate the MAPK-interacting kinase 1 (MNK1)-eukaryotic translation initiation factor 4E (eIF4E) pathway and subsequent mRNA translation in a negative regulatory feedback manner, the mechanistic role of ATO resistance in MES GBM was explored. In GBM cells, ATO-activated translation initiation cellular events via the MNK1-eIF4E signaling axis. Furthermore, resistance to ATO in intracranial PDX tumors correlated with high eIF4E phosphorylation. Polysomal fractionation and microarray analysis of GBM cells were performed to identify ATO's effect on mRNA translation and enrichment of anti-apoptotic mRNAs in the ATO-induced translatome was found. Additionally, it was determined that MNK inhibition sensitized MES GSCs to ATO in neurosphere and apoptosis assays. Finally, examination of the effect of ATO on patients from a phase I/II clinical trial of ATO revealed that PN GBM patients responded better to ATO than other subtypes as demonstrated by longer overall and progression-free survival.Implications: These findings raise the possibility of a unique therapeutic approach for GBM, involving MNK1 targeting to sensitize MES GSCs to drugs like arsenic trioxide. Mol Cancer Res; 16(1); 32-46. ©2017 AACR.

Central Regulatory Role for SIN1 in Interferon γ (IFNγ) Signaling and Generation of Biological Responses.

The precise signaling mechanisms by which type II IFN receptors control expression of unique genes to induce biological responses remain to be established. We provide evidence that Sin1, a known element of the mammalian target of rapamycin complex 2 (mTORC2), is required for IFNγ-induced phosphorylation and activation of AKT and that such activation mediates downstream regulation of mTORC1 and its effectors. These events play important roles in the assembly of the eukaryotic translation initiation factor 4F (eIF4F) and mRNA translation of IFN-stimulated genes. Interestingly, IFNγ-induced tyrosine phosphorylation of STAT1 is reduced in cells with targeted disruption of Sin1, leading to decreased transcription of several IFNγ-inducible genes in an mTORC2-independent manner. Additionally, our studies establish that Sin1 is essential for generation of type II IFN-dependent antiviral effects and antiproliferative responses in normal and malignant hematopoiesis. Together, our findings establish an important role for Sin1 in both transcription and translation of IFN-stimulated genes and type II IFN-mediated biological responses, involving both mTORC2-dependent and -independent functions.

Merestinib blocks Mnk kinase activity in acute myeloid leukemia progenitors and exhibits antileukemic effects in vitro and in vivo.

Mitogen-activated protein kinase interacting protein kinases (Mnks) play important roles in the development and progression of acute myeloid leukemia (AML) by regulating eukaryotic translation initiation factor 4E (eIF4E) activation. Inhibiting Mnk1/2-induced phosphorylation of eIF4E may represent a unique approach for the treatment of AML. We provide evidence for antileukemic effects of merestinib, an orally bioavailable multikinase inhibitor with suppressive effects on Mnk activity. Our studies show that merestinib effectively blocks eIF4E phosphorylation in AML cells and suppresses primitive leukemic progenitors from AML patients in vitro and in an AML xenograft model in vivo. Our findings provide evidence for potent preclinical antileukemic properties of merestinib and support its clinical development for the treatment of patients with AML.

Interferon γ (IFNγ) Signaling via Mechanistic Target of Rapamycin Complex 2 (mTORC2) and Regulatory Effects in the Generation of Type II Interferon Biological Responses.

We provide evidence for a unique pathway engaged by the type II IFN receptor, involving mTORC2/AKT-mediated downstream regulation of mTORC1 and effectors. These events are required for formation of the eukaryotic translation initiation factor 4F complex (eIF4F) and initiation of mRNA translation of type II interferon-stimulated genes. Our studies establish that Rictor is essential for the generation of type II IFN-dependent antiviral and antiproliferative responses and that it controls the generation of type II IFN-suppressive effects on normal and malignant hematopoiesis. Together, our findings establish a central role for mTORC2 in IFNγ signaling and type II IFN responses.

Human Schlafen 5 (SLFN5) Is a Regulator of Motility and Invasiveness of Renal Cell Carcinoma Cells.

We provide evidence that human SLFN5, an interferon (IFN)-inducible member of the Schlafen (SLFN) family of proteins, exhibits key roles in controlling motility and invasiveness of renal cell carcinoma (RCC) cells. Our studies define the mechanism by which this occurs, demonstrating that SLFN5 negatively controls expression of the matrix metalloproteinase 1 gene (MMP-1), MMP-13, and several other genes involved in the control of malignant cell motility. Importantly, our data establish that SLFN5 expression correlates with a better overall survival in a large cohort of patients with RCC. The inverse relationship between SLFN5 expression and RCC aggressiveness raises the possibility of developing unique therapeutic approaches in the treatment of RCC, by modulating SLFN5 expression.

Central role of ULK1 in type I interferon signaling.

We provide evidence that the Unc-51-like kinase 1 (ULK1) is activated during engagement of the type I interferon (IFN) receptor (IFNR). Our studies demonstrate that the function of ULK1 is required for gene transcription mediated via IFN-stimulated response elements (ISRE) and IFNγ activation site (GAS) elements and controls expression of key IFN-stimulated genes (ISGs). We identify ULK1 as an upstream regulator of p38α mitogen-activated protein kinase (MAPK) and establish that the regulatory effects of ULK1 on ISG expression are mediated possibly by engagement of the p38 MAPK pathway. Importantly, we demonstrate that ULK1 is essential for antiproliferative responses and type I IFN-induced antineoplastic effects against malignant erythroid precursors from patients with myeloproliferative neoplasms. Together, these data reveal a role for ULK1 as a key mediator of type I IFNR-generated signals that control gene transcription and induction of antineoplastic responses.

Regulatory effects of SKAR in interferon α signaling and its role in the generation of type I IFN responses.

We provide evidence that S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) is engaged in IFN-α signaling and plays a key role in the generation of IFN responses. Our data demonstrate that IFN-α induces phosphorylation of SKAR, which is mediated by either the p90 ribosomal protein S6 kinase (RSK) or p70 S6 kinase (S6K1), in a cell type-specific manner. This type I IFN-inducible phosphorylation of SKAR results in enhanced interaction with the eukaryotic initiation factor (eIF)4G and recruitment of activated RSK1 to 5' cap mRNA. Our studies also establish that SKAR is present in cap-binding CBP80 immune complexes and that this interaction is mediated by eIF4G. We demonstrate that inducible protein expression of key IFN-α-regulated protein products such as ISG15 and p21(WAF1/CIP1) requires SKAR activity. Importantly, our studies define a requirement for SKAR in the generation of IFN-α-dependent inhibitory effects on malignant hematopoietic progenitors from patients with chronic myeloid leukemia or myeloproliferative neoplasms. Taken altogether, these findings establish critical and essential roles for SKAR in the regulation of mRNA translation of IFN-sensitive genes and induction of IFN-α biological responses.

Regulation of interferon-dependent mRNA translation of target genes.

Interferons (IFNs) are released by cells on exposure to various stimuli, including viruses, double-stranded RNA, and other cytokines and various polypeptides. These IFNs play important physiological and pathophysiological roles in humans. Many clinical studies have established activity for these cytokines in the treatment of several malignancies, viral syndromes, and autoimmune disorders. In this review, the regulatory effects of type I and II IFN receptors on the translation-initiation process mediated by mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways and the known mechanisms of control of mRNA translation of IFN-stimulated genes are summarized and discussed.

Critical roles for Rictor/Sin1 complexes in interferon-dependent gene transcription and generation of antiproliferative responses.

We provide evidence that type I IFN-induced STAT activation is diminished in cells with targeted disruption of the Rictor gene, whose protein product is a key element of mTOR complex 2. Our studies show that transient or stable knockdown of Rictor or Sin1 results in defects in activation of elements of the STAT pathway and reduced STAT-DNA binding complexes. This leads to decreased expression of several IFN-inducible genes that mediate important biological functions. Our studies also demonstrate that Rictor and Sin1 play essential roles in the generation of the suppressive effects of IFNα on malignant erythroid precursors from patients with myeloproliferative neoplasms. Altogether, these findings provide evidence for critical functions for Rictor/Sin1 complexes in type I IFN signaling and the generation of type I IFN antineoplastic responses.

Expression and regulatory effects of murine Schlafen (Slfn) genes in malignant melanoma and renal cell carcinoma.

There is emerging evidence that the IFN-inducible family of Slfn genes and proteins play important roles in cell cycle progression and control of cellular proliferation, but the precise functional roles of different Slfn members in the regulation of tumorigenesis remain unclear. In the present study, we undertook a systematic analysis on the expression and functional relevance of different mouse Slfn genes in malignant melanoma and renal cell carcinoma cells. Our studies demonstrate that several mouse Slfn genes are up-regulated in response to IFN treatment of mouse melanoma and renal cell carcinoma cells, including Slfn1, Slfn2, Slfn4, Slfn5, and Slfn8. Our data show that Slfn2 and Slfn3 play essential roles in the control of mouse malignant melanoma cell proliferation and/or anchorage-independent growth, suggesting key and non-overlapping roles for these genes in the control of malignant melanoma tumorigenesis. In renal cell carcinoma cells, in addition to Slfn2 and Slfn3, Slfn5 also exhibits important antineoplastic effects. Altogether, our findings indicate important functions for distinct mouse Slfn genes in the control of tumorigenesis and provide evidence for differential involvement of distinct members of this gene family in controlling tumorigenesis. They also raise the potential of future therapeutic approaches involving modulation of expression of members of this family of genes in malignant melanoma and renal cell carcinoma.

Essential role for the Mnk pathway in the inhibitory effects of type I interferons on myeloproliferative neoplasm (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.

Inhibition of Mnk kinase activity by cercosporamide and suppressive effects on acute myeloid leukemia precursors.

Mnk kinases regulate the phosphorylation and activation of the eukaryotic initiation factor 4E (eIF4E), a protein that plays key roles in the initiation of messenger RNA translation and whose activity is critical for various cellular functions. eIF4E is deregulated in acute myeloid leukemia (AML), and its aberrant activity contributes to leukemogenesis. We determined whether cercosporamide, an antifungal agent that was recently shown to act as a unique Mnk inhibitor, exhibits antileukemic properties. Treatment of AML cells with cercosporamide resulted in a dose-dependent suppression of eIF4E phosphorylation. Such suppression of Mnk kinase activity and eIF4E phosphorylation by cercosporamide resulted in dose-dependent suppressive effects on primitive leukemic progenitors (CFU-L) from AML patients and enhanced the antileukemic properties of cytarabine (Ara-C) or mammalian target of rapamycin (mTOR) complex 1 inhibition. Similarly, the combination of cercosporamide with cytarabine resulted in enhanced antileukemic responses in a xenograft mouse model in vivo. Altogether, this work demonstrates that the unique Mnk inhibitor cercosporamide suppresses phosphorylation of eIF4E and exhibits antileukemic effects, in support of future clinical-translational efforts involving combinations of Mnk inhibitors with cytarabine and/or mTOR inhibitors for the treatment of AML.

Regulatory effects of programmed cell death 4 (PDCD4) protein in interferon (IFN)-stimulated gene expression and generation of type I IFN responses.

The precise mechanisms by which the activation of interferon (IFN) receptors (IFNRs) ultimately controls mRNA translation of specific target genes to induce IFN-dependent biological responses remain ill defined. We provide evidence that IFN-α induces phosphorylation of programmed cell death 4 (PDCD4) protein on Ser67. This IFN-α-dependent phosphorylation is mediated by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) in a cell-type-specific manner. IFN-dependent phosphorylation of PDCD4 results in downregulation of PDCD4 protein levels as the phosphorylated form of PDCD4 interacts with the ubiquitin ligase ß-TRCP (ß-transducin repeat-containing protein) and undergoes degradation. This process facilitates IFN-induced eukaryotic translation initiation factor 4A (eIF4A) activity and binding to translation initiation factor eIF4G to promote mRNA translation. Our data establish that PDCD4 degradation ultimately facilitates expression of several ISG protein products that play important roles in the generation of IFN responses, including IFN-stimulated gene 15 (ISG15), p21(WAF1/CIP1), and Schlafen 5 (SLFN5). Moreover, engagement of the RSK/PDCD4 pathway by the type I IFNR is required for the suppressive effects of IFN-α on normal CD34(+) hematopoietic precursors and for antileukemic effects in vitro. Altogether, these findings provide evidence for a unique function of PDCD4 in the type I IFN system and indicate a key regulatory role for this protein in mRNA translation of ISGs and control of IFN responses.

Dual mTORC2/mTORC1 targeting results in potent suppressive effects on acute myeloid leukemia (AML) progenitors.

PURPOSE: To determine whether mTORC2 and rapamycin-insensitive (RI)-mTORC1 complexes are present in acute myeloid leukemia (AML) cells and to examine the effects of dual mTORC2/mTORC1 inhibition on primitive AML leukemic progenitors. EXPERIMENTAL DESIGN: Combinations of different experimental approaches were used, including immunoblotting to detect phosphorylated/activated forms of elements of the mTOR pathway in leukemic cell lines and primary AML blasts; cell-proliferation assays; direct assessment of mRNA translation in polysomal fractions of leukemic cells; and clonogenic assays in methylcellulose to evaluate leukemic progenitor-colony formation. RESULTS: mTORC2 complexes are active in AML cells and play critical roles in leukemogenesis. RI-mTORC1 complexes are also formed and regulate the activity of the translational repressor 4E-BP1 in AML cells. OSI-027 blocks mTORC1 and mTORC2 activities and suppresses mRNA translation of cyclin D1 and other genes that mediate proliferative responses in AML cells. Moreover, OSI-027 acts as a potent suppressor of primitive leukemic precursors from AML patients and is much more effective than rapamycin in eliciting antileukemic effects in vitro. CONCLUSIONS: Dual targeting of mTORC2 and mTORC1 results in potent suppressive effects on primitive leukemic progenitors from AML patients. Inhibition of the mTOR catalytic site with OSI-027 results in suppression of both mTORC2 and RI-mTORC1 complexes and elicits much more potent antileukemic responses than selective mTORC1 targeting with rapamycin.

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 p21(WAF1/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.

Mechanisms of mRNA translation of interferon stimulated genes.

Over the last two decades, a lot of research work has been focused on the interferon (IFN)-regulated JAK-STAT pathway and understanding the mechanisms governing the transcription of interferon stimulated genes (ISGs). Evidence suggests that the JAK-STAT pathway alone does not account in its entirety for mediating cellular responses to IFNs. There is emerging evidence that non-Stat pathways play important roles in mediating signals for the generation of IFN-responses. Various studies have underscored the importance of mitogen activated protein kinases (MAPKs), especially p38 and ERK1/2, as well as the PI 3'K/AKT pathway in transmitting signals that are of critical importance for the biological effects of IFNs. Besides regulating the transcription of ISGs in some cases, engagement of these signaling pathways by the IFN-receptor (IFNR) associated complexes also plays an important role in mediating the translation of ISGs. The mechanisms regulating mRNA translation of ISGs is an area of ongoing active research and a lot more efforts will be required to complete our understanding of the various cellular elements involved in this process. In this review we highlight the mechanisms regulating translation of ISGs. We focus on the proteins regulated by the PI 3'K/AKT pathway, their role in mediating mRNA translation of ISGs and the functional consequences of this regulation. In addition, MAPKs are known to regulate the phosphorylation of various eukaryotic initiation factors and we summarize the roles of eIF4B and eIF4E phosphorylations on the translation of ISGs. The emerging roles of microRNAs in mRNA translation of ISGs are also discussed.

Arsenic trioxide-dependent activation of thousand-and-one amino acid kinase 2 and transforming growth factor-beta-activated kinase 1.

Arsenic trioxide (As(2)O(3)) has potent antileukemic properties in vitro and in vivo, but the mechanisms by which it generates its effects on target leukemic cells are not well understood. Understanding cellular mechanisms and pathways that are activated in leukemic cells to control the generation of As(2)O(3) responses should have important implications in the development of novel approaches using As(2)O(3) for the treatment of leukemias. In this study, we used immunoblotting and immune complex kinase assays to provide evidence that the kinases thousand-and-one amino acid kinase 2 (TAO2) and transforming growth factor-beta-activated kinase 1 (TAK1) are rapidly activated in response to treatment of acute leukemia cells with As(2)O(3). Such activation occurs after the generation of reactive oxygen species and regulates downstream engagement of the p38 mitogen-activated protein kinase. Our studies demonstrate that siRNA-mediated knockdown of TAO2 or TAK1 or pharmacological inhibition of TAK1 enhances the suppressive effects of As(2)O(3) on KT-1-derived leukemic progenitor colony formation and on primary leukemic progenitors from patients with acute myelogenous leukemia. These results indicate key negative-feedback regulatory roles for these kinases in the generation of the antileukemic effects of As(2)O(3). Thus, molecular or pharmacological targeting of these kinases may provide a novel approach to enhance the generation of arsenic-dependent antileukemic responses.

Growth suppressive cytokines and the AKT/mTOR pathway.

The mTOR signaling pathway plays a very important role in the transmission of signals for initiation of mRNA translation and protein expression in mammalian cells. mTOR activates various downstream effectors to promote initiation of cap-dependent mRNA translation and mediate pro-mitogenic and pro-survival signals. Recent evidence has implicated effectors of this signaling cascade in mRNA translation for interferon stimulated genes (ISGs). In addition, it was recently shown that AKT/mTOR-mediated signals play important roles in the generation of IFN-dependent antiviral and growth inhibitory responses, suggesting that mTOR and its effectors can mediate diverse biological responses, depending on the cellular context and the triggering stimuli. In this review, the regulatory effects of various growth suppressive cytokines on the mTOR pathway are summarized and the emerging new functions of mTOR are discussed.

Interferon-dependent engagement of eukaryotic initiation factor 4B via S6 kinase (S6K)- and ribosomal protein S6K-mediated signals.

Although the roles of Jak-Stat pathways in type I and II interferon (IFN)-dependent transcriptional regulation are well established, the precise mechanisms of mRNA translation for IFN-sensitive genes remain to be defined. We examined the effects of IFNs on the phosphorylation/activation of eukaryotic translation initiation factor 4B (eIF4B). Our data show that eIF4B is phosphorylated on Ser422 during treatment of sensitive cells with alpha IFN (IFN-alpha) or IFN-gamma. Such phosphorylation is regulated, in a cell type-specific manner, by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) and results in enhanced interaction of the protein with eIF3A (p170/eIF3A) and increased associated ATPase activity. Our data also demonstrate that IFN-inducible eIF4B activity and IFN-stimulated gene 15 protein (ISG15) or IFN-gamma-inducible chemokine CXCL-10 protein expression are diminished in S6k1/S6k2 double-knockout mouse embryonic fibroblasts. In addition, IFN-alpha-inducible ISG15 protein expression is blocked by eIF4B or eIF3A knockdown, establishing a requirement for these proteins in mRNA translation/protein expression by IFNs. Importantly, the generation of IFN-dependent growth inhibitory effects on primitive leukemic progenitors is dependent on activation of the S6K/eIF4B or RSK/eIF4B pathway. Taken together, our findings establish critical roles for S6K and RSK in the induction of IFN-dependent biological effects and define a key regulatory role for eIF4B as a common mediator and integrator of IFN-generated signals from these kinases.