Heath L. Bradley

Increased numbers of committed myeloid progenitors but not primitive hematopoietic stem/progenitors in mice lacking STAT6 expression.

Signal transducer and activator of transcription‐6 (STAT6) plays important roles in cytokine signaling via interleukin‐4 and ‐13 receptors (IL‐4R and IL‐13R). Mice in which STAT6 has been disrupted by homologous recombination show defects in T helper cell type 2 (Th2) lymphocyte production, resulting in an accumulation of Th1 cells. In addition to defects in differentiation and proliferation of T lymphocytes, STAT6‐deficient mice show increased cell‐cycle activation and frequency of myeloid progenitors. Although this has been shown to be mediated through Oncostatin M production by T cells, IL‐4Rα and STAT6 have also recently been found to be enriched for expression in primitive hematopoietic stem cells (HSCs) in gene expression‐profiling studies. Therefore, we have investigated whether defects in hematopoietic function in mice lacking STAT6 expression extended into the primitive hematopoietic compartments of the bone marrow. Here, we report that STAT6 deficiency increased bone marrow‐committed myeloid progenitors but did not alter the number of cells enriched for HSC/multipotent progenitors, primitive cobblestone area‐forming cells assayed in vitro, or bone marrow short‐term or long‐term repopulating cells assayed in vivo. Therefore, the requirement for STAT6 activation during hematopoiesis is limited, and primitive hematopoietic cell types are insulated against possible effects of cytokine stimulation by Th1 cells.

Tissue inhibitor of matrix metalloproteinase-1 overexpression in M1 myeloblasts impairs IL-6-induced differentiation

The balance between matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) is important for extracellular matrix interactions of hematopoietic cells. MMP-independent growth modulating activity for TIMP-1 on B lymphocytes and erythroid progenitors has also been described, but a role for TIMP-1 in myelomonocytic differentiation has not been previously reported. In this study, we demonstrate that TIMP-1 overexpression impairs differentiation of the myeloblastic M1 cell line following interleukin (IL)-6 stimulation. We generated retroviral vectors coexpressing human TIMP-1 and the green fluorescent protein (GFP) and stably transduced murine M1 myeloid cells. TIMP-1 expressing cells showed a large reduction in IL-6-induced macrophage differentiation in vitro that was reversible with a specific monoclonal antibody. The differentiation delay in M1/TIMP-1 cells was also specifically reversible by pharmacologic phosphatidylinositol-3 kinase (PI3-K) inhibition. Additionally, overexpression of a TIMP-1/GFP fusion protein also impaired M1 differentiation and this protein was localized to the cell surface, consistent with an autocrine receptor-mediated mechanism. Surprisingly, TIMP-1 transduced cells had a selective advantage for growth in IL-6, indicating that functional effects on growth and differentiation of M1 cells were primarily through an autocrine mechanism. Intrinsic TIMP-1 expression in myeloid leukemia cells might thus impact upon survival or differentiation.

Capillary nano-immunoassay for Akt 1/2/3 and 4EBP1 phosphorylation in acute myeloid leukemia

BackgroundOverall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse being a major cause of mortality. The PI3K-Akt-mTOR kinase pathway plays a vital role in pro-survival signals within leukemic cells and inhibition of this pathway is being investigated to improve patient outcomes. Tracking activation of multiple signaling proteins simultaneously in patient samples can be challenging especially with limiting cell numbers within rare sub-populations.MethodsThe NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have utilized this nano-immunoassay to examine activation of Akt 1/2/3 and downstream mTOR target - eukaryotic initiation factor 4E-Binding Protein 1 (4EBP1).ResultsAssays for Akt 1/2/3 and 4EBP1 were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. Target inhibition was studied using mTOR 1/2 inhibitor AZD-8055 and results were corroborated by Western blotting. The assay was able to quantify nanogram amounts of 4EBP1 and Akt 1/2/3 in AML cell lines and primary pediatric AML samples and results were quantifiable, consistent and reproducible.ConclusionOur data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this nano-immunoassay prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy.

Synergistic cell death in FLT3-ITD positive acute myeloid leukemia by combined treatment with metformin and 6-benzylthioinosine

Current therapy for acute myeloid leukemia (AML) primarily includes high-dose cytotoxic chemotherapy with or without allogeneic stem cell transplantation. Targeting unique cellular metabolism of cancer cells is a potentially less toxic approach. Monotherapy with mitochondrial inhibitors like metformin have met with limited success since escape mechanisms such as increased glycolytic ATP production, especially in hyperglycemia, can overcome the metabolic blockade. As an alternative strategy for metformin therapy, we hypothesized that the combination of 6-benzylthioinosine (6-BT), a broad-spectrum metabolic inhibitor, and metformin could block this drug resistance mechanism. Metformin treatment alone resulted in significant suppression of ROS and mitochondrial respiration with increased glycolysis accompanied by modest cytotoxicity (10-25%). In contrast, 6-BT monotherapy resulted in inhibition of glucose uptake, decreased glycolysis, and decreased ATP with minimal changes in ROS and mitochondrial respiration. The combination of 6-BT with metformin resulted in significant cytotoxicity (60-70%) in monocytic AML cell lines and was associated with inhibition of FLT3-ITD activated STAT5 and reduced c-Myc and GLUT-1 expression. Therefore, although the anti-tumor and metabolic effects of metformin have been limited by the metabolic reprogramming within cells, the novel combination of 6-BT and metformin targets this bypass mechanism resulting in reduced glycolysis, STAT5 inhibition, and increased cell death.

Nanoproteomic Assays on Hematopoietic Stem Cells

Dysregulation of cytokine signaling pathways is associated with benign and malignant hematologic disorders. Improvements in therapy rely on understanding the biology of the pathways and the proteins involved. Studying these pathways in patient samples is challenging as samples are difficult to obtain, contain fewer cells, and are heterogeneous in nature. To address some of these difficulties, we have utilized the technique of microcapillary electrophoresis. Using the NanoPro 1000 system (ProteinSimple) which is built on an automated, capillary-based immunoassay platform, we have developed rapid and quantitative assays for specific proteins from relatively small sample sizes. The NanoPro provides precise and quantitative data of the phosphorylation states of a specific protein of interest. We describe our experience with NanoPro assay development and optimization with specific application toward understanding aberrant cytokine signaling in human leukemia cells.

Imipramine blue sensitively and selectively targets FLT3-ITD positive acute myeloid leukemia cells

Aberrant cytokine signaling initiated from mutant receptor tyrosine kinases (RTKs) provides critical growth and survival signals in high risk acute myeloid leukemia (AML). Inhibitors to FLT3 have already been tested in clinical trials, however, drug resistance limits clinical efficacy. Mutant receptor tyrosine kinases are mislocalized in the endoplasmic reticulum (ER) of AML and play an important role in the non-canonical activation of signal transducer and activator of transcription 5 (STAT5). Here, we have tested a potent new drug called imipramine blue (IB), which is a chimeric molecule with a dual mechanism of action. At 200–300 nM concentrations, IB is a potent inhibitor of STAT5 through liberation of endogenous phosphatase activity following NADPH oxidase (NOX) inhibition. However, at 75–150 nM concentrations, IB was highly effective at killing mutant FLT3-driven AML cells through a similar mechanism as thapsigargin (TG), involving increased cytosolic calcium. IB also potently inhibited survival of primary human FLT3/ITD+ AML cells compared to FLT3/ITDneg cells and spared normal umbilical cord blood cells. Therefore, IB functions through a mechanism involving vulnerability to dysregulated calcium metabolism and the combination of fusing a lipophilic amine to a NOX inhibiting dye shows promise for further pre-clinical development for targeting high risk AML.

Abstract C200: Rapid nano-immunoassay for evaluation of mTOR pathway activation in acute myeloid leukemia.

Overall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse in AML being a major cause of mortality. Risk stratification by cytogenetic and molecular aberrations has changed the prognosis for some patients, but for a majority of the patients (especially with normal cytogenetics) improvement in therapy is needed. Understanding key differences in survival pathways such as the mTOR pathway within leukemic cells could be potentially exploited to develop new drug therapy and design relapse therapy. Tracking pathway activation in patient samples is challenging in AML since samples are difficult to obtain, contain fewer cells, and are heterogeneous in nature. The NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have used this novel nano-technology based system to study the mTOR pathway in AML samples (n=8) obtained from the Children9s Oncology Group (COG) Myeloid Disease Reference Laboratory. Assays for downstream mTOR target protein-eukaryotic initiation factor 4E binding protein 1 (4EBP1) were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. The assay was able to detect as little as 80 ng of protein which corresponded to 1000 cells per assay. MV4-11 cells were treated with the dual mTOR 1/2 inhibitor AZD-8055 for 24 hours and reduced phosphorylation of 4EBP1 was confirmed by Western Blotting and corroborated by the NanoPro assay. This nano-immunoassay was able to reliably quantify amounts of 4EBP1 (total and phosphorylated forms) in primary pediatric AML samples (n=8) thawed following cryopreservation. The relative luminescence units (measure of chemiluminescence and protein signal) were plotted against exposure time and the linearity of the graph provided evidence that the signal is sensitive over a range of three logs. Further, these samples were treated with AZD-8055 for 24 hours followed by analysis of changes in signaling profiles for total and phosphorylated Ser65 and Thr37/46. As expected, complete reduction of phosphorylation was noted in the treated samples (n=4). The assays obtained were quantifiable, consistent, and reproducible and enabled us to delineate activation patterns of the mTOR pathway in primary AML samples and evaluate drug efficacy for targeted agents. Our data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this novel nano-immunoassay based platform prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C200. Citation Format: Himalee Sabnis, Heath Bradley, Todd Cooper, Kevin Bunting. Rapid nano-immunoassay for evaluation of mTOR pathway activation in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C200.