Christian Lutz

An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor

Activation of the aryl hydrocarbon receptor (AHR) by environmental xenobiotic toxic chemicals, for instance 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), has been implicated in a variety of cellular processes such as embryogenesis, transformation, tumorigenesis and inflammation. But the identity of an endogenous ligand activating the AHR under physiological conditions in the absence of environmental toxic chemicals is still unknown. Here we identify the tryptophan (Trp) catabolite kynurenine (Kyn) as an endogenous ligand of the human AHR that is constitutively generated by human tumour cells via tryptophan-2,3-dioxygenase (TDO), a liver- and neuron-derived Trp-degrading enzyme not yet implicated in cancer biology. TDO-derived Kyn suppresses antitumour immune responses and promotes tumour-cell survival and motility through the AHR in an autocrine/paracrine fashion. The TDO–AHR pathway is active in human brain tumours and is associated with malignant progression and poor survival. Because Kyn is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient for activating the human AHR, these results provide evidence for a previously unidentified pathophysiological function of the AHR with profound implications for cancer and immune biology.

Toll‐Like Receptor Engagement Enhances the Immunosuppressive Properties of Human Bone Marrow‐Derived Mesenchymal Stem Cells by Inducing Indoleamine‐2,3‐dioxygenase‐1 via Interferon‐β and Protein Kinase R

Mesenchymal stem cells (MSC) display unique suppressive properties on T‐cell immunity, thus representing an attractive vehicle for the treatment of conditions associated with harmful T‐cell responses such as organ‐specific autoimmunity and graft‐versus‐host disease. Toll‐like receptors (TLR) are primarily expressed on antigen‐presenting cells and recognize conserved pathogen‐derived components. Ligation of TLR activates multiple innate and adaptive immune response pathways to eliminate and protect against invading pathogens. In this work, we show that TLR expressed on human bone marrow‐derived MSC enhanced the immunosuppressive phenotype of MSC. Immunosuppression mediated by TLR was dependent on the production of immunosuppressive kynurenines by the tryptophan‐degrading enzyme indoleamine‐2,3‐dioxygenase‐1 (IDO1). Induction of IDO1 by TLR involved an autocrine interferon (IFN)‐β signaling loop, which was dependent on protein kinase R (PKR), but independent of IFN‐γ. These data define a new role for TLR in MSC immunobiology, which is to augment the immunosuppressive properties of MSC in the absence of IFN‐γ rather than inducing proinflammatory immune response pathways. PKR and IFN‐β play a central, previously unidentified role in orchestrating the production of immunosuppressive kynurenines by MSC. STEM CELLS 2009;27:909–919

The Indoleamine-2,3-Dioxygenase (IDO) Inhibitor 1-Methyl-D-tryptophan Upregulates IDO1 in Human Cancer Cells

1-methyl-D-tryptophan (1-D-MT) is currently being used in clinical trials in patients with relapsed or refractory solid tumors with the aim of inhibiting indoleamine-2,3-dioxygenase (IDO)-mediated tumor immune escape. IDO is expressed in tumors and tumor-draining lymph nodes and degrades tryptophan (trp) to create an immunsuppressive micromilieu both by depleting trp and by accumulating immunosuppressive metabolites of the kynurenine (kyn) pathway. Here we show that proliferation of alloreactive T-cells cocultured with IDO1-positive human cancer cells paradoxically was inhibited by 1-D-MT. Surprisingly incubation with 1-D-MT increased kyn production of human cancer cells. Cell-free assays revealed that 1-D-MT did not alter IDO1 enzymatic activity. Instead, 1-D-MT induced IDO1 mRNA and protein expression through pathways involving p38 MAPK and JNK signalling. Treatment of cancer patients with 1-D-MT has transcriptional effects that may promote rather than suppress anti-tumor immune escape by increasing IDO1 in the cancer cells. These off-target effects should be carefully analyzed in the ongoing clinical trials with 1-D-MT.

CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell–autonomous detoxification mechanism that must be overcome to prevent drug resistance.

Mouse mesenchymal stem cells suppress antigen-specific TH cell immunity independent of indoleamine 2,3-dioxygenase 1 (IDO1).

Due to their immunosuppressive properties, human mesenchymal stem cells (hMSC) represent a promising tool for cell-based therapies of autoimmune diseases such as multiple sclerosis (MS). Mouse MSC (mMSC) have been used extensively to characterize and optimize route of administration, motility, cellular targets, and immunosuppressive mechanisms in mouse models of autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE). Tryptophan (trp) catabolism by indolamine-2,3-dioxygenase 1 (IDO1) is a chief endogenous metabolic pathway that tightly regulates unwanted immune responses through depletion of trp and generation of immunosuppressive kynurenines (kyn). IDO1 activity contributes to the immunosuppressive phenotype of hMSC. Here, we demonstrate that although IDO1 is inducible in bone marrow-derived mMSC by proinflammatory stimuli such as interferon-g (IFN-g) and ligands of toll-like receptors (TLR), it does not lead to catabolism of trp in vitro. This failure to catabolize trp is not due to defective TLR signaling as demonstrated by induction of interleukin 6 (IL-6) by TLR activation. While mMSC suppressed the activation of antigen-specific myelin oligodendrocyte glycoprotein (MOG)-reactive T-cell receptor (TCR) transgenic T-helper (TH) cells in co-culture, neither pharmacologic inhibition nor genetic ablation of IDO1 reversed this suppressive effect. Finally, systemic administration of both, IDO1-proficient and phenotypically identical IDO1-deficient mMSC, equally resulted in amelioration of EAE. mMSC, unlike hMSC, do not display IDO1-mediated suppression of antigen-specific T-cell responses.

A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity

BackgroundMany studies of the eukaryotic transcription mechanism and its regulation rely on in vitro assays. Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purification and gel electrophoresis is laborious and not always quantitative.ResultsHerein, we describe a new, non-radioactive, robust and reproducible eukaryotic in vitro transcription assay that has been established in our laboratory. Upon transcription, the newly synthesized RNA is directly detected and quantified using the QuantiGene assay. Alternatively, the RNA can be purified and a primer extension followed by PCR detection or qPCR quantification can be performed. When applied to assess the activity of RNA polymerase II inhibitors, this new method allowed an accurate estimation of their relative potency.ConclusionsOur novel assay provides a non-radioactive alternative to a standard in vitro transcription assay that allows for sensitive detection and precise quantification of the newly transcribed, unlabelled RNA and is particularly useful for quantification of strong transcriptional inhibitors like α-amanitin. Moreover, the method can be easily adapted to quantify the reaction yield and the transcription efficiency of other eukaryotic in vitro systems, thus providing a complementary tool for the field of transcriptional research.

Abstract 77: Preclinical evaluation of HDP-101, an anti-BCMA antibody-drug conjugate

Background: ATACs (antibody-targeted Amanitin conjugates) comprise a new class of antibody-drug conjugates using amanitin as toxic payload. Amanitin binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription process at very low concentrations. In the current study, in vitro and in vivo data of new ATACs targeting BCMA (B Cell Maturation Antigen, also known as CD269) are presented. BCMA is selectively expressed on malignant plasma cells like in multiple myeloma (MM) and hence considered an ideal target for Amanitin based ADCs. Material and methods: MM cell lines: NCI-H929, MM.1S Luc (stable luciferase transfected) and CCRF-CEM (BCMA negative). Antibody: anti-BCMA Thiomab (Max Delbruck Centrum, Berlin; derivatization and production at Heidelberg Pharma). Synthesis of HDP-101: Maleimide amanitin compounds were conjugated to substituted cysteine residues of the anti-BCMA Thiomab. Cell proliferation assay: Quantitative determination of cytotoxicity was performed by CellTiter Glo 2.0 assay (Promega) or WST.1 assay (Roche). Animal models: Subcutaneous and metastatic mouse xenograft tumor models with MM cell lines NCI-H929 and MM.1S Luc were performed in single-dose and multiple-dosing experiments. Tolerability was assessed in mice and nonhuman primates (NHP). Results: HDP-101 showed in vitro cytotoxicity on BCMA+ cell lines in picomolar range, whereas no cytotoxic activity on BCMA- cells was observed. In mouse xenograft models, HDP-101 caused dose-dependent tumor regression and complete remission after a single i.v. dose of 2.0 mg/kg and 4.0 mg/kg in s.c. xenografts and after single i.v. doses from 0.1 mg/kg to 2.0 mg/kg in disseminating xenografts. Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index after sequentially applied doses of 0.3, 1.0, and multiple dose application of 4 x 3.0 mg/kg. Hematology and clinical chemistry parameters were unaffected except liver enzymes and LDH: A mild to moderate and transient increase was observed. The half-life of the ADC in serum was 7-9 days; the free toxin was detectable at levels close to the lower limit of quantification only (LLOQ = 1.2nM). Conclusions: Targeted cytotoxic drug delivery to BCMA positive MM cell lines was achieved by using HDP-101, an anti-BCMA-ATAC. The mode of action of the payload Amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in NHP studies. Using ADCs in the therapy of multiple myeloma is a promising approach, especially by using a cytotoxic agent whose mode of action differs from other commonly used toxins, like ATACs. First-in-human trial is expected to start in 2018. Citation Format: Torsten Hechler, Aniko Palfi, Christoph Muller, Christian Lutz, Andreas Pahl, Michael Kulke. Preclinical evaluation of HDP-101, an anti-BCMA antibody-drug conjugate [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 77. doi:10.1158/1538-7445.AM2017-77

Amanitins and their development as a payload for antibody-drug conjugates

Amanitin-based ADCs represent a new class of ADCs using a novel mode of action. This payload introduces a novel mode of action into oncology therapy, the inhibition of RNA Polymerase II. The high potency of the toxin leads to highly efficacious ADCs. The development of the technology around this toxin will be described. These developments support the clinical development of amanitin-based ADCs by using a toxin with a new mode of action and with a favorable therapeutic index. HDP-101 is an Amanitin based ADC directed against BCMA and will be advancing to the clinical phase in 2019.

Abstract A69: A novel mechanism mediates drug resistance in the exocrine-like pancreatic ductal adenocarcinoma (PDAC) subtype

PDAC is a highly aggressive disease with dismal prognosis [1, 2]. Despite extensive research and the discovery of several drug candidates, little progress has been reported since the approval of gemcitabine and erlotinib [1]. Moreover, recent trials with targeted therapies have shown only limited or no benefit [1, 2]. For a number of other carcinomas, tumor subclasses have been uncovered that allow the use of targeted therapies. The mutational landscape of PDAC is complex and heterogeneous, raising the question whether subclasses also exist in PDAC [3]. Collisson et al. described three PDAC subtypes that were identified based on their gene-expression profiles: The classical, the quasi-mesenchymal and the exocrine-like subtype [4]. However, not all subtypes could be identified in the previously available model systems. We have established a novel patient-derived model system that allows the analysis of these three human PDAC subtypes in vitro and in vivo. Hence, we provide a systematic workflow to propagate human PDAC in orthotopic xenografts and to derive tumor-initiating primary cell lines of all three PDAC subtypes. HNF-1 and Keratin 81 were identified as markers for subtype stratification by immunohistochemistry. Application of this two-marker set on a 258 large patient cohort confirmed a predominantly non-overlapping staining and revealed a significant difference in overall survival across the three subtypes. Furthermore, a drug screen uncovered subtype-specific drug sensitivities towards a number of drugs, including gemcitabine and dasatinib. Notably, the exocrine-like subtype was resistant towards all compounds tested. Thus, we aimed to identify the underlying cause of the observed drug resistance. Molecular analysis including gene set enrichment analysis (GSEA) allowed us to identify a putative novel mechanism of drug resistance. Analysis by qRT-PCR and Western blot demonstrated the enhanced expression of several genes mediating this mechanism particularly in the exocrine-like subtype in vitro and in vivo. These findings led to the identification of a novel protein target central to this mechanism. Additionally, retrospective immunohistochemical analysis of a large patient cohort confirmed that this target is predominantly found in those patient tumors classified as exocrine-like. Hence, we hypothesized that the observed strong activation of this mechanism in the exocrine-like PDAC subtype could be responsible for the drug resistance observed in this subclass. In line with this, functional inhibition of this mechanism resulted in increased drug sensitivity in the exocrine-like subtype. Hence, our findings may ultimately advance personalized treatment by applying novel marker-based patient selection strategies in combination with tailored drug use, a strategy which will be presented in more detail at the conference. [1] Hidalgo, M. Pancreatic cancer. The New England journal of medicine. 362, 1605-1617, doi:10.1056/NEJMra0901557 (2010). [2] Vincent, A., Herman, J., Schulick, R., Hruban, R. H. & Goggins, M. Pancreatic cancer. Lancet. 378, 607-620, doi:10.1016/S0140-6736(10)62307-0 (2011). [3] Jones, S. et al. Core signalling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 321, 1801-1806, doi:10.1126/science.1164368 (2008). [4] Collisson, E. A. et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nature medicine. 17, 500-503, doi:10.1038/nm.2344 (2011). Citation Format: Elisa M. Noll, Christian Eisen, Elisa Espinet, Vanessa Vogel, Corinna Klein, Albrecht Stenzinger, Franziska Zickgraf, Peter Neuhaus, Marcus Bahra, Bruno V. Sinn, Christian Lutz, Michael Kulke, Andreas Pahl, Nathalia A. Giese, Oliver Strobel, Jens Werner, Wilko Weichert, Andreas Trumpp, Martin R. Sprick. A novel mechanism mediates drug resistance in the exocrine-like pancreatic ductal adenocarcinoma (PDAC) subtype. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A69.