Cancer is a highly complex, multigenic disease with tumor cells underlying constant transition. Single drug treatments against specific targets frequently result in only partial success because mutations and redundant pathways cause drug resistance. Therefore, drug combinations that effect different synergistically acting targets in the cancer cell in parallel have become a promising strategy to improve the success in many fields of cancer therapy. One example for such an approach is the co-treatment of B-Raf driven tumors with Raf and MEK1 inhibitors. In our study, we determined the combinatorial effect of the pan-Raf inhibitor AZ-628 and the MEK1 inhibitor AZD-6244 on the viability of a large panel of more than 100 tumor cell lines. Whittaker et al. (Mol.CancerTher, 2015) could already show that this drug combination has a significant synergistic effect in several melanoma and colon cancer cell lines. The authors showed evidence that synergistic activity was correlated with enhanced mutation-triggered RAS/RAF/MAPK signaling with a special role for C-Raf in resistance due to a negative feedback mechanism. We here applied the combination of AZD-6244 and AZ-628 in a broad checkerboard pattern to a multitude of cell lines from other entities beyond melanoma and colorectal cancer. Based on these results we correlated the observed synergistic and non-synergistic effects with gene expression profiles of these cell lines and analyzed the requirement of a MAPK activating signaling. Our approach revealed that synergistic activity is not confined to melanoma and colon cancer but is observed in tumors from other entities as well. This observation may expand the usefulness of MEK/Raf inhibitor co-treatment to a larger panel of cancer types. Citation Format: Alokta Chakrabarti, Daniel Feger, Sarah Umber, Orysya Stus, Marianne Birkle, Oliver Siedentopf, Jan E. Ehlert. Analysis of the combinatorial antiproliferative effect of pan RAF inhibitor AZ-628 and MEK1-inhibitor AZD-6244 on a large panel of tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1208. doi:10.1158/1538-7445.AM2017-1208
Anticipation of the in vivo activity of new investigational drugs is challenging. In the context of the living body, compounds are subjected to degradation, modification or binding to plasma proteins - mechanisms that may compromise the activity predicted from target-specific in vitro assays. As such assays are performed in artificial cell-culture media it was our aim to measure the activity directly in human plasma, as initially described by Levis et al.1. We hence established a method to analyze the activity of kinase inhibitors in human or rodent plasma (plasma inhibitory activity, PIA) in high throughput compatible ELISA-based cellular kinase assays for e.g. FLT-3 or BCR-Abl. Testing reference compounds such as Sunitinib or Sorafenib, we could confirm the expected correlation between the plasma binding of a compound and the loss of its activity in this assay setup compared to the activity in serum free medium. Furthermore we observed a donor dependent interference of the plasma with assay performance. This issue was solved by appropriate plasma heat-inactivation which made confirmation of compound stability to heat treatment mandatory before generating pharmacokinetic data using this methodology. Our aim is to establish the PIA assay for our panel of more than 30 kinases and analyze the activity of important reference kinase inhibitors spiked in human plasma. In the next step samples from mice treated with reference compounds shall be tested. This way, we want to compile reliable in vitro PIA assays to better predict the in vivo effect of kinase-directed drugs and to determine the amount of freely available active test substance in the blood of treated patients. 1 = Levis et al.; Blood, 2006; 108:3477 Citation Format: Kira E. Boehmer, Holger Weber, Daniel Feger, Marianne Birkle, Oliver Siedentopf, Melanie Mueller, Sarah Umber, Jan E. Ehlert. Determination of the plasma inhibitory activity of drugs using cellular kinase assays. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B139.
In anticancer drug discovery a common strategy is to start with a target-specific screen followed later by characterization of hit compounds in phenotypic cell-based assays. One important advantage of this sequential screening procedure is the feasibility of high throughput compound testing. However, a significant disadvantage of the approach is the fact that due to the sequential nature of the approach, compounds showing promising phenotypic activities, but addressing so far unknown targets will be lost as false-negative compounds. Here we describe a medium throughput screening strategy, where we characterized 360 natural compounds isolated from marine organisms in a parallel target- and phenotypic-driven screening approach. As target-specific read out, we analyzed biochemically the inhibitory potential of the compounds against 16 recombinant protein kinases, known to be relevant targets in different kinds of human tumors. In parallel we determined the impact of the compounds on cell transformation, migration and angiogenesis by analyzing their behavior in soft agar growth, wound healing and three-dimensional endothelial cell sprouting. Furthermore, unspecific toxicity of the compounds was tested via a viability assay with human peripheral mononuclear cells from healthy donors. Examples of identified compounds showing differential inhibitory profiles in the different assays applied will be presented. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C109. Citation Format: Daniel Feger, Holger Weber, Frank Totzke, Marianne Birkle, Oliver Siedentopf, Melanie Müller, Sarah Umber, Wenhan Lin, Peter Proksch, Jan E. Ehlert, Michael H.G. Kubbutat. Discovery of new natural anti-tumoral compounds using a parallel target- and phenotypic-driven screening approach. [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 C109.
Acute myeloid leukemia (AML) is the most common leukemia affecting adults. In 70-90% of all AML patients FLT3, a receptor tyrosine kinase which plays an important role in normal hematopoiesis, is highly expressed. Furthermore, in about 30% of all AML patients activating internal tandem duplications (ITDs) or point mutations within the FLT3 gene have been identified. FLT3, therefore, is one of the most promising drug targets for the treatment of AML. Most often novel FLT3 protein kinase inhibitors are tested in subcutaneous xenograft mouse models using various human AML cell lines. To generate more relevant, orthotopic AML in vivo models, we stably transduced the human AML cell lines MOLM-13 and MV4-11 with fire fly luciferase (MOLM-13-Luci and MV4-11-Luci) suitable for in vivo bioluminescence imaging. MOLM-13 carries a heterozygous and MV4-11 a homozygous FLT3 internal tandem duplication (ITD) mutation. In vivo growth of MOLM-13-Luci and MV4-11-Luci cells after subcutaneous implantation in NMRI nude mice proofed that stable expression of luciferase did not alter growth characteristics of these cells. To initiate orthotopic growth, both AML cell lines were implanted intravenously via the tail vein into NOD SCID mice after Cyclophosphamide treatment. Cyclophosphamide facilitates engraftment of the human cells by reducing the endogenous bone marrow cell population. Approximately two (MOLM-13-Luci), or three (MV4-11-Luci) weeks after implantation, luciferase activity was detectable in the pelvic region of the mice indicating growth of both tumor cell lines. Within the next weeks luciferase activity was also seen in additional parts of the mouse at different levels. After necropsy a set of different organs and tissues was analysed ex vivo for luciferase activity. The pattern of metastasising tumor cells was compared in both models. Furthermore, detection of human Ki-67 protein using appropriate human specific antibodies allowed verification of growth of MOLM-13-Luci and MV4-11-Luci cells in different tissues. Sutent, a small-molecule kinase inhibitor, totally abolished tumor growth of both orthotopically growing tumors, indicating that both in vivo models are highly suitable for the characterisation of novel drugs directed against AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1609. doi:10.1158/1538-7445.AM2011-1609
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