Efstratios Katsoulidis

Role of the p38 Mitogen-Activated Protein Kinase Pathway in the Generation of Arsenic Trioxide–Dependent Cellular Responses

Arsenic trioxide (As(2)O(3)) induces differentiation and apoptosis of leukemic cells in vitro and in vivo, but the precise mechanisms that mediate such effects are not known. In the present study, we provide evidence that the kinases MAPK kinase 3 (Mkk3) and Mkk6 are activated during treatment of leukemic cell lines with As(2)O(3) to regulate downstream engagement of the p38 mitogen-activated protein kinase. Using cells with targeted disruption of both the Mkk3 and Mkk6 genes, we show that As(2)O(3)-dependent activation of p38 is defective in the absence of Mkk3 and Mkk6, establishing that these kinases are essential for As(2)O(3)-dependent engagement of the p38 pathway. Pharmacologic inhibition of p38 enhances As(2)O(3)-dependent activation of the c-jun NH(2)-terminal kinase (JNK) and subsequent induction of apoptosis of chronic myelogenous leukemia (CML)- or acute promyelocytic leukemia (APL)-derived cell lines. In addition, in APL blasts, inhibition of p38 enhances myeloid cell differentiation in response to As(2)O(3), as well as suppression of Bcl-2 expression and loss of mitochondrial membrane potential. Similarly, induction of As(2)O(3)-dependent apoptosis is enhanced in mouse embryonic fibroblasts (MEF) with targeted disruption of both the Mkk3 and Mkk6 genes, establishing a key role for this pathway in the regulation of As(2)O(3)-induced apoptosis. In other studies, we show that the small-molecule p38 inhibitors SD-282 and SCIO-469 potentiate As(2)O(3)-mediated suppression of myeloid leukemic progenitor growth from CML patients, indicating a critical regulatory role for p38 in the induction of antileukemic responses. Altogether, our data indicate that the Mkk3/6-p38 signaling cascade is activated in a negative regulatory feedback manner to control induction of As(2)O(3)-mediated antileukemic effects.

Role of the p38 Mitogen-Activated Protein Kinase Pathway in Cytokine-Mediated Hematopoietic Suppression in Myelodysplastic Syndromes

The p38 mitogen-activated protein kinase (MAPK) pathway is activated by IFNs and other cytokines to mediate signals for important cellular functions, including transcriptional regulation and apoptosis. We examined the role of the p38 pathway in the generation of the effects of myelosuppressive cytokines on human hematopoiesis. Pharmacologic inhibition of p38 using BIX-01208 resulted in reversal of IFN-, tumor necrosis factor-alpha (TNF-alpha)-, and transforming growth factor-beta (TGF-beta)-mediated suppression of human erythroid (blast-forming unit-erythroid) and myeloid (granulocyte-macrophage colony-forming unit) colony formation, consistent with a key role for p38 in the generation of myelosuppressive signals by different cytokines. Similarly, the myelosuppressive effects of TNF-alpha and TGF-beta were reversed by small interfering RNAs targeting p38alpha expression, further establishing the requirement of this kinase in the induction of myelosuppressive responses. As TNF overproduction has been implicated in the pathophysiology of bone marrow failure states, we determined whether pharmacologic inhibition of p38 reverses the hematopoietic defects seen in bone marrows from patients with myelodysplastic syndromes (MDS) and the anemia of chronic disease. Addition of pharmacologic inhibitors of p38 on such bone marrows resulted in increased numbers of erythroid and myeloid progenitors. Similarly, inhibition of the activity of the downstream effectors of p38, MAPK activated protein kinase-2, and mitogen and stress activated kinase 1 partially restored the hematopoietic defect seen in these bone marrows. Taken altogether, our data implicate the p38 MAPK in the pathophysiology of myelodysplasias and suggest that p38 pharmacologic inhibitors may have therapeutic applications in the treatment of MDS.

Suppression of Programmed Cell Death 4 (PDCD4) Protein Expression by BCR-ABL-regulated Engagement of the mTOR/p70 S6 Kinase Pathway

There is accumulating evidence that mammalian target of rapamycin (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. Moreover, PDCD4 protein expression is up-regulated by inhibition of the BCR-ABL kinase in K562 cells and BaF3/BCR-ABL transfectants, suggesting a mechanism for the generation of the proapoptotic 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 suppression of PDCD4 expression.

Suppressive effects of statins on acute promyelocytic leukemia cells.

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.

Interferon-Dependent Engagement of Eukaryotic Initiation Factor 4B via S6 Kinase (S6K)- and Ribosomal Protein S6K-Mediated Signals

ABSTRACT 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-α) or IFN-γ. 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-γ-inducible chemokine CXCL-10 protein expression are diminished in S6k1/S6k2 double-knockout mouse embryonic fibroblasts. In addition, IFN-α-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.

Activation of the Mitogen- and Stress-activated Kinase 1 by Arsenic Trioxide

Arsenic trioxide (As2O3) is a potent inducer of apoptosis of leukemic 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 activation of the mitogen- and stress-activated kinase 1 (MSK1) and downstream phosphorylation of its substrate, histone H3, in leukemia cell lines. Such activation requires upstream engagement of p38 MAPK, as demonstrated by experiments using pharmacological inhibitors of p38 or p38α knock-out cells. Arsenic-induced apoptosis was enhanced in cells in which MSK1 expression was decreased using small interfering RNA and in Msk1 knock-out mouse embryonic fibroblasts, suggesting that this kinase is activated in a negative feedback regulatory manner to regulate As2O3 responses. Consistent with this, pharmacological inhibition of MSK1 enhanced the suppressive effects of As2O3 on the growth of primary leukemic progenitors from chronic myelogenous leukemia patients. Altogether, these findings indicate an important role for MSK1 downstream of p38 in the regulation of As2O3 responses.

Role of Schlafen 2 (SLFN2) in the Generation of Interferon α-induced Growth Inhibitory Responses

The precise STAT-regulated gene targets that inhibit cell growth and generate the antitumor effects of Type I interferons (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 IFNα on normal hematopoietic progenitors. Importantly, NIH3T3 or L929 cells with stable knockdown of SLFN2 form more 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.

Role of Interferon α (IFNα)-inducible Schlafen-5 in Regulation of Anchorage-independent Growth and Invasion of Malignant Melanoma Cells

IFNα exerts potent inhibitory activities against malignant melanoma cells in vitro and in vivo, but the mechanisms by which it generates its antitumor effects remain unknown. We examined the effects of interferon α (IFNα) on the expression of human members of the Schlafen (SLFN) family of genes, a group of cell cycle regulators that mediate growth-inhibitory responses. Using quantitative RT-real time PCR, we found detectable basal expression of all the different human SLFN genes examined (SLFN5, SLFN11, SLFN12, SLFN13, and SLFN14), in malignant melanoma cells and primary normal human melanocytes, but SLFN5 basal expression was suppressed in all analyzed melanoma cell lines. Treatment of melanoma cells with IFNα resulted in induction of expression of SLFN5 in malignant cells, suggesting a potential involvement of this gene in the antitumor effects of IFNα. Importantly, stable knockdown of SLFN5 in malignant melanoma cells resulted in increased anchorage-independent growth, as evidenced by enhanced colony formation in soft agar assays. Moreover, SLFN5 knockdown also resulted in increased invasion in three-dimensional collagen, suggesting a dual role for SLFN5 in the regulation of invasion and anchorage-independent growth of melanoma cells. Altogether, our findings suggest an important role for the SLFN family of proteins in the generation of the anti-melanoma effects of IFNα and for the first time directly implicate a member of the human SLFN family in the regulation of cell invasion.

Regulatory effects of mammalian target of rapamycin-mediated signals in the generation of arsenic trioxide responses

Arsenic 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 mammalian target of rapamycin (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 knock-out 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 acute myeloid leukemia, we found that pharmacological inhibition of mTOR enhances the suppressive effects of arsenic trioxide on leukemic progenitor colony formation. Moreover, short interfering RNA-mediated inhibition of expression of the negative downstream effector, translational repressor 4E-BP1, partially reverses the effects of As2O3. Altogether, 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 anti-leukemic properties of As2O3.

Regulation of Arsenic Trioxide-induced Cellular Responses by Mnk1 and Mnk2

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 MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38α MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1–/– or Mnk2–/–, or double Mnk1–/–Mnk2–/– knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. 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.