Michele S. Redell

Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor

Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%, despite aggressive chemotherapy. New therapies for AML are targeted at signal transduction pathways known to support blast survival, such as the Stat3 pathway. Aberrant activation of Stat3 has been demonstrated in many different malignancies, including AML, and this finding is frequently associated with more aggressive disease. The objectives of this study were: (1) to characterize Stat3 signaling patterns in AML cells lines and primary pediatric samples; and (2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We found that Stat3 was constitutively activated in 6 of 7 AML cell lines and 6 of 18 primary pediatric AML samples. Moreover, constitutively phosphorylated Stat3 was frequent in samples with normal karyotype but uncommon in samples with t(8;21). Most cell lines and primary samples responded to G-CSF stimulation, although the sensitivity and magnitude of the response varied dramatically. Our novel small-molecule Stat3 inhibitor, C188-9, inhibited G-CSF-induced Stat3 phosphorylation, induced apoptosis in AML cell lines and primary samples, and inhibited AML blast colony formation with potencies in the low micromolar range. Therefore, Stat3 inhibition may be a valuable strategy for targeted therapies for AML.

Targeting transcription factors for cancer therapy.

Advances in the molecular biology of oncogenesis have established a key role for transcription factors in malignant transformation. In some cases the activity of the transcription factor itself is altered by mutation. In many other cases, the activity of the transcription factor is affected by mutations in upstream signaling or regulatory proteins. This review highlights four transcription factors--Stat3, Stat5, NF-kappaB, and HIF-1--which are associated with cancer development. The evidence for the involvement of these factors in oncogenesis is reviewed. Further, we examine the efforts to specifically target these transcription factors for therapeutic intervention. Such strategies include using peptidomimetics, antisense oligonucleotides, small molecule inhibitors, and G-quartet oligonucleotides. Inhibition of transcription factor activity may occur at the level of activation, translocation, or DNA binding. Application of these approaches to in vitro and in vivo models of tumorigenesis is discussed.

Stat3 Isoforms, α and β, Demonstrate Distinct Intracellular Dynamics with Prolonged Nuclear Retention of Stat3β Mapping to Its Unique C-terminal End

Two isoforms of Stat3 (signal transducer and activator of transcription 3) are expressed in cells, α (p92) and β (p83), both derived from a single gene by alternative mRNA splicing. The 55-residue C-terminal transactivation domain of Stat3α is deleted in Stat3β and replaced by seven unique C-terminal residues (CT7) whose function remains uncertain. We subcloned the open reading frames of Stat3α and Stat3β into the C terminus of green fluorescent protein (GFP). Fluorescent microscopic analysis of HEK293T cells transiently transfected with GFP-Stat3α or GFP-Stat3β revealed similar kinetics and cytokine concentration dependence of nuclear accumulation; these findings were confirmed by high throughput microscope analysis of murine embryonic fibroblasts that lacked endogenous Stat3 but stably expressed either GFP-Stat3α or GFP-Stat3β. However, although time to half-maximal cytoplasmic reaccumulation after cytokine withdrawal was 15 min for GFP-Stat3α, it was >180 min for GFP-Stat3β. Furthermore, although the intranuclear mobility of GFP-Stat3α was rapid and increased with cytokine stimulation, the intranuclear mobility of GFP-Stat3β in unstimulated cells was slower than that of GFP-Stat3α in unstimulated cells and was slowed further following cytokine stimulation. Deletion of the unique CT7 domain from Stat3β eliminated prolonged nuclear retention but did not alter its intranuclear mobility. Thus, Stat3α and Stat3β have distinct intracellular dynamics, with Stat3β exhibiting prolonged nuclear retention and reduced intranuclear mobility especially following ligand stimulation. Prolonged nuclear retention, but not reduced intranuclear mobility, mapped to the CT7 domain of Stat3β.

DOT1L inhibition sensitizes MLL-rearranged AML to chemotherapy.

DOT1L, the only known histone H3-lysine 79 (H3K79) methyltransferase, has been shown to be essential for the survival and proliferation of mixed-linkage leukemia (MLL) gene rearranged leukemia cells, which are often resistant to conventional chemotherapeutic agents. To study the functions of DOT1L in MLL-rearranged leukemia, SYC-522, a potent inhibitor of DOT1L developed in our laboratory, was used to treat MLL-rearranged leukemia cell lines and patient samples. SYC-522 significantly inhibited methylation at H3K79, but not H3K4 or H3K27, and decreased the expression of two important leukemia-relevant genes, HOXA9 and MEIS1, by more than 50%. It also significantly reduced the expression of CCND1 and BCL2L1, which are important regulators of cell cycle and anti-apoptotic signaling pathways. Exposure of MLL-rearranged leukemia cells to this compound caused cell cycle arrest and promoted differentiation of those cells, both morphologically and by increased CD14 expression. SYC-522 did not induce apoptosis, even at 10 µM for as long as 6 days. However, treatment with this DOT1L inhibitor decreased the colony formation ability of primary MLL-rearranged AML cells by up to 50%, and promoted monocytic differentiation. Notably, SYC-522 treatment significantly increased the sensitivity of MLL-rearranged leukemia cells to chemotherapeutics, such as mitoxantrone, etoposide and cytarabine. A similar sensitization was seen with primary MLL-rearranged AML cells. SYC-522 did not affect chemotherapy-induced apoptosis in leukemia cells without MLL-rearrangement. Suppression of DOT1L activity inhibited the mitoxantrone-induced increase in the DNA damage response marker, γH2AX, and increased the level of cPARP, an intracellular marker of apoptosis. These results demonstrated that SYC-522 selectively inhibited DOT1L, and thereby altered gene expression, promoted differentiation, and increased chemosensitivity by preventing DNA damage response. Therefore, inhibition of DOT1L, in combination with DNA damaging chemotherapy, represents a promising approach to improving outcomes for MLL-rearranged leukemia.

Synthesis, Activity and Metabolic Stability of Non-Ribose Containing Inhibitors of Histone Methyltransferase DOT1L

Histone methyltransferase DOT1L is a drug target for MLL leukemia. We report an efficient synthesis of a cyclopentane-containing compound that potently and selectively inhibits DOT1L (Ki = 1.1 nM) as well as H3K79 methylation (IC50 ~ 200 nM). Importantly, this compound exhibits a high stability in plasma and liver microsomes, suggesting it is a better drug candidate.

CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

Immunotherapy with CD123-specific T-cell engager proteins or with T cells expressing CD123-specific chimeric antigen receptors is actively being pursued for acute myeloid leukemia. T cells secreting bispecific engager molecules (ENG-T cells) may present a promising alternative to these approaches. To evaluate therapeutic potential, we generated T cells to secrete CD123/CD3-bispecific engager molecules. CD123-ENG T cells recognized primary acute myeloid leukemia (AML) cells and cell lines in an antigen-dependent manner as judged by cytokine production and/or tumor killing, and redirected bystander T cells to AML cells. Infusion of CD123-ENG T cells resulted in regression of AML in xenograft models conferring a significant survival advantage of treated mice in comparison to mice that received control T cells. At high effector to target ratios, CD123-ENG T cells recognized normal hematopoietic stem and progenitor cells (HSPCs) with preferential recognition of HSPCs from cord blood compared to bone marrow. We therefore introduced the CD20 suicide gene that can be targeted in vivo with rituximab into CD123-ENG T cells. The expression of CD20 did not diminish the anti-AML activity of CD123-ENG T cells, but allowed for rituximab-mediated ENG-T cell elimination. Thus, ENG-T cells coexpressing CD20 suicide and CD123 engager molecules may present a promising immunotherapeutic approach for AML.

FACS analysis of Stat3/5 signaling reveals sensitivity to G-CSF and IL-6 as a significant prognostic factor in pediatric AML: a Children's Oncology Group report

Signal transducer and activator of transcription 3 (Stat3) and Stat5 are critical signaling intermediates that promote survival in myeloid leukemias. We examined Stat3 and Stat5 activation patterns in resting and ligand-stimulated primary samples from pediatric patients with acute myeloid leukemia. Phosphorylated Stats were measured by FACS before and after stimulation with increasing doses of granulocyte-colony stimulating factor or IL-6. We also measured positive and negative regulators of Stat signaling, and we compared the variation in multiple parameters to identify biologic relationships. Levels of constitutively phosphorylated Stats were variable and did not correlate with survival. In terms of induced phospho-Stats, 15 of 139 specimens (11%) phosphorylated Stat3 in response to moderate doses of both granulocyte-colony stimulating factor and IL-6. Compared with groups that were resistant to 1 or both ligands, this pattern of dual sensitivity was associated with a superior outcome, with a 5-year event-free survival of 79% (P = .049) and 5-year overall survival of 100% (P = .006). This study provides important and novel insights into the biology of Stat3 and Stat5 signaling in acute myeloid leukemia. Patterns of ligand sensitivity may be valuable for improving risk identification, and for developing new agents for individualized therapy.

Conditional overexpression of Stat3α in differentiating myeloid cells results in neutrophil expansion and induces a distinct, antiapoptotic and pro-oncogenic gene expression pattern

Normal neutrophil development requires G‐CSF signaling, which includes activation of Stat3. Studies of G‐CSF‐mediated Stat3 signaling in cell culture and transgenic mice have yielded conflicting data regarding the role of Stat3 in myelopoiesis. The specific functions of Stat3 remain unclear, in part, because two isoforms, Stat3α and Stat3β, are expressed in myeloid cells. To understand the contribution of each Stat3 isoform to myelopoiesis, we conditionally overexpressed Stat3α or Stat3β in the murine myeloid cell line 32Dcl3 (32D) and examined the consequences of overexpression on cell survival and differentiation. 32D cells induced to overexpress Stat3α, but not Stat3β, generated a markedly higher number of neutrophils in response to G‐CSF. This effect was a result of decreased apoptosis but not of increased proliferation. Comparison of gene expression profiles of G‐CSF‐stimulated, Stat3α‐overexpressing 32D cells with those of cells with normal Stat3α expression revealed novel Stat3 gene targets, which may contribute to neutrophil expansion and improved survival, most notably Slc28a2, a purine nucleoside transporter, which is critical for maintenance of intracellular nucleotide levels and prevention of apoptosis, and Gpr65, an acid‐sensing, G protein‐coupled receptor with pro‐oncogenic and antiapoptotic functions.

Targeting transcription factors in cancer: Challenges and evolving strategies

As investigators uncover the molecular pathways leading to cancer, an abundance of potential molecular targets is accumulating. Among these targets, transcription factors are especially promising. This review discusses the reasons for focusing targeting efforts on transcription factors and highlights some salient examples. The advantages and problems with current methods for targeting are summarized. Finally, we discuss the drug delivery technologies in development that may one day make transcription factor targeting a therapeutic reality.:

Rhodium(II) Proximity‐Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti‐Leukemia Activity

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML.