The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor, also referred to as IRF8. ICSBP acts as a suppressor of myeloid leukemia, although few target genes explaining this effect have been identified. In the current studies, we identified the gene encoding growth arrest specific 2 (GAS2) as an ICSBP target gene relevant to leukemia suppression. We find that ICSBP, Tel, and histone deacetylase 3 (HDAC3) bind to a cis element in the GAS2 promoter and repress transcription in myeloid progenitor cells. Gas2 inhibits calpain protease activity, and beta-catenin is a calpain substrate in these cells. Consistent with this, ICSBP decreases beta-catenin protein and activity in a Gas2- and calpain-dependent manner. Conversely, decreased ICSBP expression increases beta-catenin protein and activity by the same mechanism. This is of interest, because decreased ICSBP expression and increased beta-catenin activity are associated with poor prognosis and blast crisis in chronic myeloid leukemia (CML). We find that the expression of Bcr/abl (the CML oncoprotein) increases Gas2 expression in an ICSBP-dependent manner. This results in decreased calpain activity and a consequent increase in beta-catenin activity in Bcr/abl-positive (Bcr/abl(+)) cells. Therefore, these studies have identified a Gas2/calpain-dependent mechanism by which ICSBP influences beta-catenin activity in myeloid leukemia.

The precise Stat-regulated gene targets that inhibit cell growth and generate the antitumor effects of Type I IFNs remain unknown. We provide evidence that Type I IFNs regulate expression of schlafens (SLFNs), a group of genes involved in the control of cell cycle progression and growth inhibitory responses. Using cells with targeted disruption of different Stat proteins and/or the p38 Map kinase, we demonstrate that the IFN-dependent expression of distinct schlafen genes is differentially regulated by Stat-complexes and the p38 Map kinase pathway. We also provide evidence for a key functional role of a member of the SLFN family, SLFN2, in the induction of the growth suppressive effects of IFNs. This is shown in studies demonstrating that knockdown of SLFN2 enhances hematopoietic progenitor colony formation and reverses the growth suppressive effects of IFNalpha on normal hematopoietic progenitors. Importantly, NIH3T3 or L929 cells with stable knockdown of SLFN2 form less colonies in soft agar, implicating this protein in the regulation of anchorage-independent growth. Altogether, our data implicate SLFN2 as a negative regulator of the metastatic and growth potential of malignant cells and strongly suggest a role for the SLFN family of proteins in the generation of the antiproliferative effects of Type I IFNs.

Although the roles of Jak-Stat pathways in Type I and II 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 the eukaryotic translation initiation factor 4B (eIF4B). Our data show that eIF4B is phosphorylated on Ser422 during treatment of sensitive cells with IFNalpha or IFNgamma. Such phosphorylation is regulated, in a cell-type-specific manner, by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6 kinase (RSK) and results in enhanced interaction of the protein with the eukaryotic translation initiation factor 3a (p170/eIF3A) and increased associated ATPase activity. Our data also demonstrate that IFN-inducible eIF4B activity and ISG15 or CXCL10 protein expression are diminished in double S6k1/S6k2 knockout mouse embryonic fibroblasts (MEFs). In addition, IFNalpha-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, generation of IFN-dependent growth inhibitory effects on primitive leukemic progenitors, are dependent on activation of the S6K/eIF4B or RSK/eIF4B pathways. Taken altogether, 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, acting as common mediator and integrator of IFN-generated signals from these kinases.

Type I interferons (IFNs) are cytokines with diverse biological properties, including antiviral, growth inhibitory, and immunomodulatory effects. Although several signaling pathways are activated during engagement of the Type I IFN receptor and participate in the induction of IFN responses, the mechanisms of generation of specific signals for distinct biological effects remain to be elucidated. We provide evidence that a novel member of the PKC family of proteins is rapidly phosphorylated and activated during engagement of the Type I IFN receptor. In contrast to other members of the PKC family that are also regulated by IFN receptors, PKC eta does not regulate IFN-inducible transcription of ISGs or generation of antiviral responses. However, its function promotes cell cycle arrest and is essential for the generation of the suppressive effects of IFN-alfa on normal and leukemic human myeloid (CFU-GM) bone marrow progenitors. Altogether, our studies establish PKC eta as a unique element in IFN- signaling that plays a key and essential role in the generation of the regulatory effects of Type I IFNs on normal and leukemic hematopoiesis.

The important antiviral and antitumor properties of interferons (IFNs) in vitro and in vivo have triggered extensive investigations over the years to understand the signals that control such responses in normal and malignant cells. The discovery of IFN-regulated Jak-Stat pathways and various ancillary cascades has led to the definition and establishment of models by which early signals at the IFN receptor level ultimately induce transcription of IFN-controlled genes to generate antiviral and antitumor responses. An important outstanding issue in the field has been the identification of the mechanisms responsible for regulation of mRNA translation of IFN-sensitive genes. There is emerging evidence suggesting that mTOR and its effectors play key and essential roles in the generation of such responses. Moreover, recent studies point towards Akt as a common and central integrator for such responses in Type I and II IFN signaling, via its regulatory effects on mTOR. Here, we review the accumulating evidence on the importance of Akt in IFN-signaling, with particular emphasis on its role in mRNA translation of IFN-sensitive genes. The implications of such studies on the overall perception of the Akt pathway are also discussed.

Arsenic trioxide (As2O3) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As2O3 induces phosphorylation/activation of the mitogen- activated protein kinase signal-integrating kinases (Mnk) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38 Map kinase gene, indicating that it requires upstream engagement of the p38 Map kinase pathway. Studies using Mnk1-/- or Mnk2 -/-, or double Mnk1-/-Mnk2-/- knockout cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E (eIF4E) at Ser209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide-responses. Consistent with this, pharmacological inhibition of Mnk-activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.

The interferons (IFNs) are cytokines that play key roles in host defense against viral infections and immune surveillance against cancer. We report that BCR-ABL transformation of hematopoietic cells results in suppression of IFN-dependent responses, including transcription of IFN-inducible genes and generation of IFN-mediated antiviral effects. BCR-ABL trans-formation suppresses expression of several IFN-regulated genes containing ISRE or GAS elements in their promoters, including Isg15, Irf1, Irf9 and Ifit2. Suppressed suppression of trans-cription of ISRE-containing genes is also seen in cells expressing various BCR-ABL kinase domain mutants, including T315I, H396P, Y253F and E255K, but not kinase-defective BCR-ABL. Such effects are associated with impaired IFN-dependent phosphorylation of Stat1 on Tyr701 and Stat3 on Tyr705 and defective binding of Stat-complexes to ISRE or GAS elements. Beyond suppression of Stat-activities, BCR-ABL inhibits IFN-inducible phosphorylation/ activation of the p38 Map kinase, suggesting a dual mechanism by which this abnormal fusion protein blocks IFN-transcriptional responses. The inhibitory activities of BCR-ABL ultimately result in impaired IFN-mediated protection against encephalomyocarditis virus (EMCV) infection and reversal of IFN-dependent growth suppression. Altogether, our data provide evidence for a novel mechanism by which BCR-ABL impairs host-defenses and promotes malignant transformation, involving dual suppression of IFN-activated signaling pathways.

There is accumulating evidence that mTOR-activated pathways play important roles in cell growth and survival of BCR-ABL-transformed cells. We have previously shown that the mTOR/p70 S6 kinase (p70 S6K) pathway is constitutively activated in BCR-ABL transformed cells and that inhibition of BCR-ABL kinase activity by imatinib mesylate abrogates such activation. We now provide evidence for the existence of a novel regulatory mechanism by which BCR-ABL promotes cell proliferation, involving p70 S6K-mediated suppression of expression of programmed cell death 4 (PDCD4), a tumor suppressor protein that acts as an inhibitor of cap-dependent translation by blocking the translation initiation factor eIF4A. Our data also establish that second generation BCR-ABL kinase inhibitors block activation of p70 S6K and downstream engagement of the S6 ribosomal protein in BCR-ABL transformed cells. More-over, PDCD4 protein expression is upregulated by inhibition of the BCR-ABL kinase in K562 cells and BaF3/BCR-ABL transfectants, sug-gesting a mechanism for the generation of the pro-apoptotic effects of such inhibitors. Knockdown of PDCD4 expression results in reversal of the suppressive effects of nilotinib and imatinib mesylate on leukemic progenitor colony formation, suggesting an important role for this protein in the generation of antileukemic responses. Altogether, our studies identify a novel mechanism by which BCR-ABL may promote leukemic cell growth, involving sequential engagement of the mTOR/p70 S6K pathway and downstream suppres-sion of PDCD4 expression.

AArsenic trioxide (As2O3) is a potent inducer of apoptosis of leukemic cells in vitro and in vivo, but the mechanisms that mediate such effects are not well understood. We provide evidence that the Akt kinase is phosphorylated/ activated during treatment of leukemia cells with As2O3, to regulate downstream engagement of mTOR and its effectors. Using cells with targeted disruption of both the Akt1 and Akt2 genes, we found that induction of arsenic trioxide-dependent apoptosis is strongly enhanced in the absence of these kinases, suggesting that Akt1/Akt2 are activated in a negative feedback regulatory manner, to control generation of As2O3-responses. Consistent with this, As2O3-dependent pro-apoptotic effects are enhanced in double knockout cells for both isoforms of the p70 S6 kinase (S6k1/S6k2), a downstream effector of Akt and mTOR. On the other hand, As2O3-dependent induction of apoptosis is diminished in cells with targeted disruption of TSC2, a negative upstream effector of mTOR. In studies using primary hematopoietic progenitors from patients with AML we found that pharmacological inhibition of mTOR enhances the suppressive effects of arsenic trioxide on leukemic progenitor colony formation. Moreover, siRNA mediated inhibition of expression of the negative downstream effector, translational repressor 4E-BP1, partially reverses the effects of As2O3. Altoget-her, these data provide evidence for a key regulatory role of the Akt/mTOR pathway in the generation of the effects of As2O3, and suggest that targeting this signaling cascade may provide a novel therapeutic approach to enhance the antileukemic properties of As2O3.

The family of statins includes pharmacologic inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase that are potent regulators of cholesterol biosynthesis. In addition to their cholesterol-lowering effects, statins inhibit cell proliferation and promote apoptosis of malignant cells in vitro, but their potential therapeutic roles in the treatment of malignancies remain to be defined. We examined the effects of statins on the growth and differentiation of acute myeloid leukemia (AML) cells. Atorvastatin and fluvastatin were found to be potent inducers of cell differentiation and apoptosis of the NB4 acute promyelocytic leukemia (APL) cell line. Such effects correlated with activation of the small G-proteins Rac1/Cdc42 and downstream engagement of the c-Jun NH(2)-terminal kinase kinase pathway, whose function was found to be essential for the generation of proapoptotic responses. Importantly, different statins were found to enhance all-trans-retinoic acid (ATRA)-dependent differentiation of APL blasts and reverse resistance to the antileukemic effects of ATRA. In addition, fluvastatin exhibited growth-inhibitory properties on primary bone marrow-derived leukemic progenitors from patients with AML and enhanced the suppressive effects of ATRA on leukemic progenitor colony formation. Altogether, these studies establish that statins exhibit potent antileukemic properties in vitro and raise the possibility that combinations of statins with ATRA may be an effective approach to overcome the development of ATRA resistance by the leukemic cells.[Cancer Res 2007;67(9):4524-32].