An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered “off-target” effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo.
The process of bone remodeling is governed by mechanical stresses and strains. Studies on the effects of mechanical stimulation on cell response are often difficult to compare as the nature of the stimuli and differences in parameters applied vary greatly. Experimental systems for the investigation of mechanical stimuli are mostly limited in throughput or flexibility and often the sum of several stimuli is applied. In this work, a flexible system that allows the investigation of cell response to isolated intermittent cyclic hydrostatic pressure (icHP) on a high throughput level is shown. Human bone derived cells were cultivated with or without mechanical stimulus in the presence or absence of chemical cues triggering osteogenesis for 7-10 days. Cell proliferation and osteogenic differentiation were evaluated by cell counting and immunohistochemical staining for bone alkaline phosphatase as well as collagen 1, respectively. In either medium, both cell proliferation and level of differentiation were increased when the cultures were mechanically stimulated. These initial results therefore qualify the present system for studies on the effects of isolated icHP on cell fate and encourage further investigations on the details behind the observed effects.
Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) catalyze the oxidation of L-Tryptophan (TRP) leading to the formation of immunosuppressive kynurenine (KYN) pathway metabolites. According to TCGA, a substantial fraction of different tumor types express both IDO1 and TDO2. In 82 tumor samples (GBM, hepatocellular carcinoma, NSCLC, and ovarian cancer) analyzed for IDO1/TDO2 RNA expression (RNA sequencing) and TRP metabolite content (LC-MS/MS), we found TDO2 highly expressed in hepatocellular carcinoma and to lower levels in NSCLC and ovarian cancer. IDO1 was found to be expressed in NSCLC and ovarian cancer but at higher levels and its expression was correlating with KYN levels. Immunosuppressive properties of KYN and downstream metabolites are mediated through T cell apoptosis and regulatory T cell (Treg) induction. In our hands, KYN at concentrations ≥ 50uM was shown to be immunosuppressive, notably inducing T cell apoptosis. According to our and published data, such high KYN concentrations (≥ 50uM) can be found in a subset of human tumors. As outlined, both IDO1 and TDO2 can contribute to high KYN levels in tumors. Hence, dual IDO1/TDO2 inhibition should be superior in decreasing tumor KYN levels and levering immunosuppression as compared to IDO1 selective inhibition. In this regard, we have generated potent dual IDO1/TDO2 inhibitors inhibiting IDO1 and TDO2 enzyme activity in cellular assays in vitro. To proof the concept of superiority in vivo, we established a dual IDO1/TDO2 expressing human tumor model (LOVO+IFNg) in the mouse and showed that the oral administration of a dual IDO1/TDO2 inhibitor as compared to an IDO1 selective inhibitor showed enhanced KYN/TRP modulation in the tumor. Lead compounds with favorable drug-like properties are currently investigated for pre-clinical development. Citation Format: Carina Lotz-Jenne, Sylvaine Cren, Christoph Joesch, Sabine Ackerknecht, Jennifer Brandes, Claire Moebs, Dominik Hartl, Felix Hartrampf, Philippe Guerry, Julien Pothier, Alexia Chavanton-Arpel. Superiority of dual IDO1/TDO2 inhibition versus IDO1 selective inhibition in reducing immunosuppressive KYN levels in tumors co-expressing IDO1 and TDO2 [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B66.
Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) catalyze the oxidation of L-Tryptophan (TRP) leading to the formation of immunosuppressive kynurenine (KYN) pathway metabolites. According to TCGA, a substantial fraction of different tumor types expresses both IDO1 and TDO2. In 82 tumor samples (GBM, hepatocellular carcinoma, NSCLC, and ovarian cancer) analyzed for IDO1/TDO2 RNA expression (RNA sequencing) and TRP metabolite content (LC-MS/MS), we found TDO2 highly expressed in hepatocellular carcinoma and to lower levels in NSCLC and ovarian cancer. IDO1 was found to be expressed in NSCLC and ovarian cancer but at higher levels and its expression was correlating with KYN levels. Immunosuppressive properties of KYN and downstream metabolites are mediated through T cell apoptosis and regulatory T cell (Treg) induction. In our hands, KYN at concentrations ≥ 50uM was shown to be immunosuppressive, notably inducing T cell apoptosis. According to our and published data, such high KYN concentrations (≥ 50uM) can be found in a subset of human tumors. As outlined, both IDO1 and TDO2 can contribute to high KYN levels in tumors. Hence, dual IDO1/TDO2 inhibition should be superior in decreasing tumor KYN levels and levering immunosuppression as compared to IDO1 selective inhibition. In this regard, we have generated potent dual IDO1/TDO2 inhibitors inhibiting IDO1 and TDO2 enzyme activity in cellular assays in vitro. To prove the concept of superiority in vivo, we established a dual IDO1/TDO2 expressing human tumor model (LOVO+IFNg) in the mouse and showed that the oral administration of a dual IDO1/TDO2 inhibitor as compared to an IDO1 selective inhibitor showed enhanced KYN/TRP modulation in the tumor. Lead compounds with favorable drug-like properties are currently investigated for pre-clinical development. Citation Format: Carina Lotz-Jenne, Sylvaine Cren, Christoph Joesch, Sabine Ackerknecht, Jennifer Brandes, Claire Moebs, Dominik Hartl, Felix Hartrampf, Philippe Guerry, Julien Pothier, Alexia Chavanton-Arpel. Superiority of dual IDO1/TDO2 inhibition versus IDO1 selective inhibition in reducing immunosuppressive KYN levels in tumors co-expressing IDO1 and TDO2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1849.
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