Denise K Walters

Publications (14)130.98 Total impact

• Article: The antineoplastic antibiotic taurolidine promotes lung and liver metastasis in two syngeneic osteosarcoma mouse models and exhibits severe liver toxicity.

  Matthias J E Arlt, Denise K Walters, Ingo J Banke, Patrick Steinmann, Gabor J Puskas, Josefine Bertz, Katharina M Rentsch, Felix Ehrensperger, Walter Born, Bruno Fuchs
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  ABSTRACT: Osteosarcoma (OS) is the most frequent primary bone tumor. Despite multiagent neoadjuvant chemotherapy, patients with metastatic disease have a poor prognosis. Moreover, currently used chemotherapeutics have severe toxic side effects. Thus, novel agents with improved antimetastatic activity and reduced toxicity are needed. Taurolidine, a broad-spectrum antimicrobial, has recently been shown to have antineoplastic properties against a variety of tumors and low systemic toxicity. Consequently, we investigated in our study the antineoplastic potential of taurolidine against OS in two different mouse models. Although both OS cell lines, K7M2 and LM8, were sensitive for the compound in vitro, intraperitoneal application of taurolidine failed to inhibit primary tumor growth. Moreover, it enhanced the metastatic load in both models 1.7- to 20-fold and caused severe liver deformations and up to 40% mortality. Thus, systemic toxicity was further investigated in tumor-free mice histologically, by electron microscopy and by measurements of representative liver enzymes. Taurolidine dose-dependent fibrous thickening of the liver capsule and adhesions and atrophies of the liver lobes were comparable in healthy and tumor-bearing mice. Liver toxicity was further indicated by up to eightfold elevated levels of the liver enzymes alanine transaminase, aspartate transaminase and GLDH in the circulation. Ultrastructural analysis of affected liver tissue showed swollen mitochondria with cristolysis and numerous lipid vacuoles in the cytoplasm of hepatocytes. The findings of our study question the applicability of taurolidine for OS treatment and may suggest the need for caution regarding the widespread clinical use of taurolidine as an antineoplastic agent.
  International Journal of Cancer 11/2011; 131(5):E804-12. · 5.44 Impact Factor
• Source

  Available from: Roman Muff

  Article: Antimetastatic activity of honokiol in osteosarcoma.

  Patrick Steinmann, Denise K Walters, Matthias J E Arlt, Ingo J Banke, Urs Ziegler, Bettina Langsam, Jack Arbiser, Roman Muff, Walter Born, Bruno Fuchs
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  ABSTRACT: Metastasizing osteosarcoma has a mean 5-year survival rate of only 20% to 30%. Therefore, novel chemotherapeutics for more effective treatment of this disease are required. The antineoplastic activity of honokiol, which was demonstrated previously in numerous malignancies, was studied in vivo in C3H mice subcutaneously injected with syngeneic β-galactosidase bacterial gene (lacZ)-expressing LM8 osteosarcoma (LM8-lacZ) cells. In vitro cytotoxic effects of honokiol were investigated in 8 human and 2 murine osteosarcoma cell lines with different in vivo metastatic potential. Seven days after subcutaneous flank injection of LM8-lacZ cells, daily intraperitoneal treatment of mice with 150 mg/kg honokiol reduced the number of micrometastases in the lung by 41% and reduced the number of macrometastases in the lung and liver by 69% and 80%, respectively, compared with control. Primary tumor growth was not inhibited. In osteosarcoma cell lines, honokiol inhibited the metabolic activity with a half-maximal concentration (IC(50) ) between 8.0 μg/mL and 16 μg/mL. Cyclosporin A partially reversed the inhibition of metabolic activity in LM8-lacZ cells. Cell proliferation and wound healing migration of LM8-lacZ cells were inhibited by honokiol with an IC(50) between 5.0 μg/mL and 10 μg/mL. Higher concentrations caused rapid cell death, which was distinct from necrosis, apoptosis, or autophagy but was associated with swelling of the endoplasmic reticulum, cytoplasmic vacuolation, and morphologically altered mitochondria. Honokiol exhibited prominent antimetastatic activity in experimental osteosarcoma and caused rapid cell death in vitro that was unrelated to necrosis, apoptosis, or autophagy. The authors concluded that honokiol has considerable potential for the treatment of metastasizing osteosarcoma.
  Cancer 09/2011; 118(8):2117-27. · 4.77 Impact Factor
• Article: LacZ transgene expression in the subcutaneous Dunn/LM8 osteosarcoma mouse model allows for the identification of micrometastasis.

  Matthias J E Arlt, Ingo J Banke, Denise K Walters, Gabor J Puskas, Patrick Steinmann, Roman Muff, Walter Born, Bruno Fuchs
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  ABSTRACT: More effective treatment of patients with metastasizing osteosarcoma (OS) with a mean 5-year survival rate of <20% requires more detailed knowledge on the complex mechanisms of metastasis for the design of new drugs, which selectively target metastasizing cells. Moreover, novel diagnostic imaging technology for early detection of metastases is needed. Mouse models, which reproduce human metastasizing OS and allow visualization of single metastatic cells are instrumental for preclinical testing of new pharmaceuticals and diagnostic instruments. Here, the low metastatic Dunn cell line and its highly metastatic LM8 subline, both equipped with a constitutively expressed lacZ gene, were used to improve the well-established OS models in syngeneic C3H mice to achieve ex vivo visualization of single metastatic cells in affected organs by X-gal staining. These models, combined with a technique for in situ high quality lung tissue-maintaining perfusion revealed, as a novel finding, single metastasizing Dunn cells in lung and liver. Importantly, constitutive lacZ gene expression did not affect in vitro and in vivo tumorigenic and metastatic properties of Dunn and LM8 cells. Thus, these improved Dunn and LM8 OS mouse models will in the future serve as a benchmark for the development of new metastasis-targeting drugs and metastasis-imaging technology.
  Journal of Orthopaedic Research 01/2011; 29(6):938-46. · 2.81 Impact Factor
• Article: Cytotoxic effects of curcumin on osteosarcoma cell lines.

  Denise K Walters, Roman Muff, Bettina Langsam, Walter Born, Bruno Fuchs
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  ABSTRACT: Curcumin (diferuloylmethane), one of the main components of the Indian spice turmeric, is known to possess potent anti-inflammatory and anti-oxidant properties. In addition, curcumin has also been shown to have in vitro and in vivo efficacy against a variety of malignancies. In the current study we examined the cytotoxic effect of curcumin on seven osteosarcoma (OS) cell lines with varying degrees of in vivo metastatic potential. Curcumin inhibited the growth of all OS cell lines tested with half-maximal inhibitory concentration values ranging from 14.4 to 24.6 microM. Growth inhibition was associated with a dose dependent increase in the number of apoptotic cells and accumulation of cells in the G(2)/M phase of the cell cycle. Curcumin treatment also resulted in cleavage of caspase-3 and poly adenosine diphosphate-ribose polymerase. Moreover, curcumin treatment was associated with an increase in cellular levels of the apoptotic B-cell leukemia/lymphoma 2 (Bcl-2)-associated X protein and a decrease in cellular content of the anti-apoptotic protein Bcl-2. In addition, curcumin treatment also inhibited the migration of OS cell lines. These data indicate that the potent cytotoxic activity of curcumin on OS cell lines is mediated by induction of apoptotic processes. Thus, curcumin has potential to be a novel OS chemotherapeutic agent.
  Investigational New Drugs 09/2008; 26(4):289-97. · 3.36 Impact Factor
• Article: RNAi screening of the tyrosine kinome identifies therapeutic targets in acute myeloid leukemia.

  Jeffrey W Tyner, Denise K Walters, Stephanie G Willis, Mary Luttropp, Jason Oost, Marc Loriaux, Heidi Erickson, Amie S Corbin, Thomas O'Hare, Michael C Heinrich, Michael W Deininger, Brian J Druker
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  ABSTRACT: Despite vast improvements in our understanding of cancer genetics, a large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of numerous types of cancer, but identification and validation of tyrosine kinase targets in cancer can be a time-consuming process. We report the establishment of an efficient, functional screening assay using RNAi technology to directly assess and compare the effect of individually targeting each member of the tyrosine kinase family. We demonstrate that siRNA screening can identify tyrosine kinase targets containing activating mutations in Janus kinase (JAK) 3 (A572V) in CMK cells and c-KIT (V560G) in HMC1.1 cells. In addition, this assay identifies targets that do not contain mutations, such as JAK1 and the focal adhesion kinases (FAK), that are crucial to the survival of the cancer cells. This technique, with additional development, might eventually offer the potential to match specific therapies with individual patients based on a functional assay.
  Blood 03/2008; 111(4):2238-45. · 9.90 Impact Factor
• Article: Taurolidine: a novel anti-neoplastic agent induces apoptosis of osteosarcoma cell lines.

  Denise K Walters, Roman Muff, Bettina Langsam, Philipp Gruber, Walter Born, Bruno Fuchs
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  ABSTRACT: Taurolidine, the active agent of Taurolin, is a broad spectrum anti-biotic that has been used for over 15 years for the treatment of severe surgical infections. Recently, taurolidine has been shown to possess anti-neoplastic properties in vitro and in vivo against a variety of cancers including ovarian, colon and prostate. In this study we assessed the cytotoxic activity of taurolidine against human osteosarcoma (OS) cell lines and normal human bone cells. Treatment with taurolidine inhibited the growth of all ten osteosarcoma cell lines tested and taurolidine was equally potent against cell lines with and without distinct genetic defects (i.e. p53, Rb). Moreover, taurolidine-induced growth inhibition was found to be associated with a dose dependent increase in the number of apoptotic cells and apoptosis was shown to be caspase-dependent. Taurolidine treatment was also found to inhibit adhesion of OS cell lines. Compared to OS cell lines, normal bone cells in primary culture were found to be less sensitive to the cytotoxic and anti-adhesive effects of taurolidine. These data indicate that taurolidine possesses potent anti-neoplastic activity against osteosarcoma cell lines and may have potential as a novel OS chemotherapeutic agent.
  Investigational New Drugs 09/2007; 25(4):305-12. · 3.36 Impact Factor
• Article: A novel fusion of RBM6 to CSF1R in acute megakaryoblastic leukemia.

  Ting-lei Gu, Thomas Mercher, Jeffrey W Tyner, Valerie L Goss, Denise K Walters, Melanie G Cornejo, Cynthia Reeves, Lana Popova, Kimberly Lee, Michael C Heinrich, John Rush, Masanori Daibata, Isao Miyoshi, D Gary Gilliland, Brian J Druker, Roberto D Polakiewicz
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  ABSTRACT: Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles.
  Blood 08/2007; 110(1):323-33. · 9.90 Impact Factor
• Article: Phosphotyrosine profiling identifies the KG-1 cell line as a model for the study of FGFR1 fusions in acute myeloid leukemia.

  Ting-Lei Gu, Valerie L Goss, Cynthia Reeves, Lana Popova, Julie Nardone, Joan Macneill, Denise K Walters, Yi Wang, John Rush, Michael J Comb, Brian J Druker, Roberto D Polakiewicz
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  ABSTRACT: The 8p11 myeloproliferative syndrome (EMS) is associated with translocations that disrupt the FGFR1 gene. To date, 8 fusion partners of FGFR1 have been identified. However, no primary leukemia cell lines were identified that contain any of these fusions. Here, we screened more than 40 acute myeloid leukemia cell lines for constitutive phosphorylation of STAT5 and applied an immunoaffinity profiling strategy to identify tyrosine-phosphorylated proteins in the KG-1 cell line. Mass spectrometry analysis of KG-1 cells revealed aberrant tyrosine phosphorylation of FGFR1. Subsequent analysis led to the identification of a fusion of the FGFR1OP2 gene to the FGFR1 gene. Small interfering RNA (siRNA) against FGFR1 specifically inhibited the growth and induced apoptosis of KG-1 cells. Thus, the KG-1 cell line provides an in vitro model for the study of FGFR1 fusions associated with leukemia and for the analysis of small molecule inhibitors against FGFR1 fusions.
  Blood 01/2007; 108(13):4202-4. · 9.90 Impact Factor
• Article: Phosphoproteomic analysis of AML cell lines identifies leukemic oncogenes.

  Denise K Walters, Valerie L Goss, Eric P Stoffregen, Ting-Lei Gu, Kimberly Lee, Julie Nardone, Laura McGreevey, Michael C Heinrich, Michael W Deininger, Roberto Polakiewicz, Brian J Druker
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  ABSTRACT: STAT5 is constitutively phosphorylated in leukemic cells in approximately 70% of acute myeloid leukemia (AML) patients. To identify kinase candidates potentially responsible for STAT5 phosphorylation, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) mass spectrometry to detect phosphoproteins in AML cell lines. We established TEL-ARG and BCR-ABL fusion proteins as the mechanism underlying STAT5 phosphorylation in HT-93 and KBM-3 cells, respectively. In addition, we identified a JAK2 pseudokinase domain mutation in HEL cells and using siRNA downregulation, established JAK2 as the kinase responsible for phosphorylating STAT5. This study illustrates the benefit of LC-MS/MS mass spectrometry and siRNA for the identification of novel targets and mutations.
  Leukemia Research 10/2006; 30(9):1097-104. · 2.92 Impact Factor
• Source

  Available from: Michael C Heinrich

  Article: Activating alleles of JAK3 in acute megakaryoblastic leukemia.

  Denise K Walters, Thomas Mercher, Ting-Lei Gu, Thomas O'Hare, Jeffrey W Tyner, Marc Loriaux, Valerie L Goss, Kimberly A Lee, Christopher A Eide, Matthew J Wong, [......], Laura McGreevey, Julie Nardone, Sandra A Moore, John Crispino, Titus J Boggon, Michael C Heinrich, Michael W Deininger, Roberto D Polakiewicz, D Gary Gilliland, Brian J Druker
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  ABSTRACT: Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3(A572V), JAK3(V722I), and JAK3(P132T) each transform Ba/F3 cells to factor-independent growth, and JAK3(A572V) confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations.
  Cancer Cell 08/2006; 10(1):65-75. · 26.57 Impact Factor
• Article: Combined Abl inhibitor therapy for minimizing drug resistance in chronic myeloid leukemia: Src/Abl inhibitors are compatible with imatinib.

  Thomas O'Hare, Denise K Walters, Eric P Stoffregen, Daniel W Sherbenou, Michael C Heinrich, Michael W N Deininger, Brian J Druker
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  ABSTRACT: Chronic myeloid leukemia (CML) is effectively treated with imatinib. However, reactivation of Bcr-Abl via kinase domain mutations that reduce sensitivity to imatinib can cause relapse. As combination therapy is frequently used to prevent emergence of resistance, the combination of imatinib with an inhibitor of imatinib-resistant Bcr-Abl mutants (e.g., Src/Abl inhibitors AP23848 and BMS-354825) was investigated. To test this approach, cellular proliferation and Bcr-Abl tyrosine phosphorylation assays were done on Ba/F3 cells expressing wild-type (WT) Bcr-Abl and four common imatinib-resistant mutants (Y253F, E255K, T315I, and M351T). Colony-forming assays with primary CML cells were also done. Both Src/Abl inhibitors retained full inhibitory capacity when coadministered with imatinib at concentrations above typical clinical levels. For cells expressing WT Bcr-Abl or the marginally imatinib-resistant mutant M351T, inclusion of imatinib at therapeutic levels enhanced the effects of the Src/Abl inhibitors. By comparison, for the highly imatinib-resistant mutants Y253F and E255K, inclusion of imatinib at clinical levels resulted in only a slight enhancement beyond the effects of the Src/Abl inhibitors. None of the inhibitors affected Bcr-Abl T315I cells. Colony-forming assays with primary CML cells yielded analogous results. Our results indicate that Src/Abl inhibitors are compatible with imatinib and suggest that combined Abl inhibitor therapy is a feasible treatment strategy for patients with CML.
  Clinical Cancer Research 11/2005; 11(19 Pt 1):6987-93. · 7.74 Impact Factor
• Article: In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants.

  Thomas O'Hare, Denise K Walters, Eric P Stoffregen, Taiping Jia, Paul W Manley, Jürgen Mestan, Sandra W Cowan-Jacob, Francis Y Lee, Michael C Heinrich, Michael W N Deininger, Brian J Druker
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  ABSTRACT: Imatinib, a Bcr-Abl tyrosine kinase inhibitor, is a highly effective therapy for patients with chronic myelogenous leukemia (CML). Despite durable responses in most chronic phase patients, relapses have been observed and are much more prevalent in patients with advanced disease. The most common mechanism of acquired imatinib resistance has been traced to Bcr-Abl kinase domain mutations with decreased imatinib sensitivity. Thus, alternate Bcr-Abl kinase inhibitors that have activity against imatinib-resistant mutants would be useful for patients who relapse on imatinib therapy. Two such Bcr-Abl inhibitors are currently being evaluated in clinical trials: the improved potency, selective Abl inhibitor AMN107 and the highly potent dual Src/Abl inhibitor BMS-354825. In the current article, we compared imatinib, AMN107, and BMS-354825 in cellular and biochemical assays against a panel of 16 kinase domain mutants representing >90% of clinical isolates. We report that AMN107 and BMS-354825 are 20-fold and 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl and that similar improvements are maintained for all imatinib-resistant mutants tested, with the exception of T315I. Thus, both inhibitors hold promise for treating imatinib-refractory CML.
  Cancer Research 06/2005; 65(11):4500-5. · 7.86 Impact Factor
• Article: RNAi-induced down-regulation of FLT3 expression in AML cell lines increases sensitivity to MLN518.

  Denise K Walters, Eric P Stoffregen, Michael C Heinrich, Michael W Deininger, Brian J Druker
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  ABSTRACT: FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in approximately 30% of acute myelogenous leukemia (AML) patients and appears to confer an adverse prognosis. Thus, development of inhibitors and/or antibodies that specifically target FLT3 has been of substantial interest. In this regard, phase 1 and 2 trials involving FLT3 inhibitors have recently reported FLT3 inhibition and leukemic blast reduction in some patients. Despite this, issues such as specificity and resistance need to be addressed. Consequently, the development of alternative approaches for targeting FLT3 would be of great consequence. In the present report, we demonstrate that FLT3 siRNA effectively down-regulates FLT3 expression in Ba/F3 cells transfected with FLT3 containing an activating internal tandem duplication (ITD) in the juxtamembrane domain and FLT3-ITD-positive Molm-14 human leukemia cells. Treatment with the FLT3 siRNA results in growth inhibition and apoptosis of these cells. Furthermore, siRNA-induced down-regulation of FLT3 increased the sensitivity of both cell lines to treatment with the FLT3 inhibitor MLN518. This illustrates the potential benefit of combined therapeutic approaches.
  Blood 05/2005; 105(7):2952-4. · 9.90 Impact Factor
• Article: AMN107: tightening the grip of imatinib.

  Thomas O'Hare, Denise K Walters, Michael W N Deininger, Brian J Druker
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  ABSTRACT: The Abl inhibitor imatinib is a highly effective therapy for patients with chronic myeloid leukemia. Relapses are relatively uncommon in newly diagnosed patients, but are the rule in patients with more advanced disease. Mutations in the BCR-ABL gene are the most common cause of relapse. Working from the imatinib chemical structure, a higher-affinity family member, AMN107, was designed. AMN107 is approximately 20-fold more potent than imatinib, and this translates into improved inhibitory activity against most of the common BCR-ABL mutations. The implications of these results, and the potential role this and other novel ABL inhibitors may have in treating patients with CML, are discussed.
  Cancer Cell 03/2005; 7(2):117-9. · 26.57 Impact Factor