Barbara Kroczynska

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. Clin Cancer Res; 17(13); 4378–88. ©2011 AACR.

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.

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 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.

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.

Regulatory Effects of Programmed Cell Death 4 (PDCD4) Protein in Interferon (IFN)-Stimulated Gene Expression and Generation of Type I IFN Responses

ABSTRACT 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 β-TRCP (β-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), p21WAF1/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.

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 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.

Critical Roles for Rictor/Sin1 Complexes in Interferon-dependent Gene Transcription and Generation of Antiproliferative Responses

Background: The precise roles for Rictor/Sin1 complexes in IFN signaling remain to be defined. Results: Targeted disruption Rictor/Sin1 results in defects in activation of elements of Stat pathways. These proteins are required for IFN antineoplastic effects on malignant erythroid precursors. Conclusion: Rictor/Sin1 play critical roles in IFN signaling. Significance: This study provides evidence for a key mechanism for gene regulation associated with generation of IFN antineoplastic 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

Background: The role of Slfn genes in antineoplastic responses is yet to be defined. Results: Murine Slfn genes are up-regulated in an IFN-dependent manner in melanoma, and RCC cells and members of this family differentially suppress proliferation and anchorage-independent malignant cell growth. Conclusion: Slfns play important roles in controlling malignant melanoma and RCC tumorigenesis. Significance: Modulation of Slfn expression may provide a novel approach for the treatment of malignancies. 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.