Faculty Summaries
Stephen M. Sykes, PhD
Stephen M. Sykes, PhD
Assistant Professor
Office Phone: 215-728-3539
Lab Phone: 215-728-3563
Office: W364
  • Identify and Delineate the Molecular Pathways That Allow Leukemia Cells to Tolerate FOXO Inhibition
    Effects of FOXO3 inhibition in human AML cells
    Effects of FOXO3 inhibition in human AML cells

    Our previous work indicates that AML segregates into two broadly defined groups based on the activities of the AKT/FOXO and JNK/c-JUN signaling pathways (Figure 1). We are now interested in delineating the pathways that promote toleration of reduced FOXO activity in leukemia as well as to understand the downstream modulators that FOXOs utilize to maintain leukemia.

    1. Investigate the role of JNK substrates in suppressing the anti-leukemic effects of FOXO inhibition. Activation of the JNK pathway allows leukemia cells to tolerate the growth-inhibitory effects of diminished FOXO activity. JNKs are a family of stress-activated kinases that phosphorylate numerous downstream substrates including c-JUN, ATF2, p53, Smad4, BCL-2 and BCL-XL. To delineate the pathogenic relevant JNK substrate we are using shRNA-mediated approaches to examine how inhibition of each known JNK substrate affects the growth and survival of human AML cells where FOXO3 has been depleted. JNK substrates that protect AML cells against FOXO inhibition will be further validated in vivo using our MLL-AF9-induced mouse model of AML. We are also characterizing the inverse correlation of the expression and activity of c-JUN and FOXOs in AML in collaboration with Dr. Sekhar Reddy at the University of Illinois – Chicago.
    2. Assess the relationship between FOXOs and ß-catenin in AML. The ß-catenin signaling pathway is activated in a wide spectrum of human cancers including myeloid leukemias. However, the precise molecular mechanisms by which ß-catenin supports leukemia remain unclear. FOXOs and ß-catenin have previously been shown to compete with one another to regulate the transcription of numerous genes. Our preliminary studies have shown that inhibition of FOXOs results in activation of both the JNK signaling pathway and c-JUN transcription suggesting that FOXOs inhibit these processes. On the contrary, ß-catenin has been shown to activate the Jnk signaling pathway and directly induce c-Jun transcription in colon cancer. Therefore, we are investigating whether ß-catenin contributes to FOXO-resistance in AML.
  • Identify the Molecular Components Utilized by FOXOs to Maintain the Differentiation Blockade
    AML blasts in the peripheral blood of a leukemic mouse.
    B. AML blasts in the peripheral blood of a leukemic mouse.

    We have observed that inhibition of FOXOs causes AML cells to overcome the differentiation blockade and subsequently undergo apoptosis. FOXOs are transcription factors that regulate the transcription of numerous genes. We now aim to identify and characterize the downstream modulators that FOXOs regulate in both normal HSPCs and leukemic progenitors. We are using deep sequencing technology to assess how deletion of FoxO1/3/4 affects the global gene expression pattern in both normal and malignant hematopoietic stem and progenitor cells (HSPCs). Concurrently, we are examining the global chromatin-binding pattern of different FOXO family members in normal and malignant HSPCs to identify putative FOXO target genes. To determine which FoxO target genes are functionally required for FoxOs to maintain the differentiation blockade, we are developing candidate-based shRNA (for genes up-regulated upon FoxO deletion) and cDNA (for genes down-regulated upon FoxO deletion) screen platforms.

  • Validate the Therapeutic Potential of Targeting the Aforementioned Pathways Using Pre-Clinical Animal Models of Leukemia
    FOXO3 is localized in the nucleus of leukemia cells but cytoplasmic in normal myeloid cells.
    C. FOXO3 is localized in the nucleus of leukemia cells. (Left)
    FOXO3 is localized in the nucleus of leukemia cells but cytoplasmic in normal myeloid cells.
    C. FOXO3 is localized in the nucleus of leukemia cells. (Right)
    1. Assess the therapeutic potential of combining JNK inhibitors with FOXO deletion in treating AML. Combined inhibition of FOXOs and JNK in vitro significantly decreases AML cell survival in comparison to inhibiting each alone. We are now evaluating how the administration of the JNK inhibitor SP600125 affects leukemic growth in vivo using our MLL-AF9-induced mouse model of AML. Moreover, we want to evaluate whether the status of FoxO1/3/4 expression impacts the feasibility of SP600125 treatment in vivo using our mouse models of AML.
    2. Evaluate the effectiveness of combining JNK inhibitors with rapamycin in the treatment of AKT+ AMLs. FOXOs are directly inactivated by the oncogenic kinase AKT and therefore AMLs that display elevated AKT activity may be preferentially sensitive to JNK inhibitors. However, AKT regulates numerous signaling pathways in addition to FOXOs to promote tumorigenesis. Specifically, AKT activates the mTOR pathway, which supports malignant growth through the regulation of protein translation and metabolism (Figure 2). mTOR inhibitors such as rapamycin or its derivatives have been relatively unsuccessful in the treatment of AML. We are examining the efficacy of simultaneously targeting the mTOR and JNK pathways in AKT+ AMLs. Our previous work has shown that MLL-AF9 expressing leukemia cells from our AML mouse model display low AKT activity (AKT-). To generate AKT+ AML cells, we have engineered our MLL-AF9-leukemia cell to express a constitutively active form of AKT (myrAkt) and we are using this pre-clinical system to assess how the co-administration of JNK inhibitors and rapamycin derivatives affect both AKT-dependent and –independent AML in vivo.
    3. Determine whether JNK inhibitors increase the effectiveness of conventional chemotherapies. Although conventional chemotherapeutic agents are effective in reducing the bulk leukemia burden a small fraction of cells, referred to as LICs, are intrinsically resistant to these therapies. Given that FoxOs are required to maintain LIC function, we want to examine whether targeting the FoxO pathway can enhance the effectiveness of traditional anti-AML therapies. We will do this by evaluating how the FoxO1/3/4 deletion impacts the efficacy of standard drugs, such as cytarabine and daunorubicin in our pre-clinical mouse model of AML.

    To execute studies and others we maintain several ongoing collaborations with a number of laboratories including:

    Dr. Franco Aversa, University of Parma, Parma, Italy
    Dr. Lars Bullinger, University Hospital of Ulm, Ulm, Germany
    Dr. Stefan Fröhling and Claudia Scholl, University of Heidelberg, Heidelberg, Germany
    Dr. Giuliana Gobbi, University of Parma, Parma, Italy
    Dr. Steven Lane, Queensland Medical Institute of Research, Brisbane, Australia
    Dr. Sekhar Reddy, University of Chicago – Illinois