Assays and models for in vitro and in vivo evaluation of small molecule inhibitors of tryptophan dioxygenase

Abstract

Assays and models for in vitro and in vivo evaluation of small molecule inhibitors of tryptophan dioxygenase Ching, Lai-Ming1, Tomek, Petr1, Palmer, Brian1, Tijono, Sofian1, Henare, Kimiora1, Braun, Lukas1, De Leeuw, Matthew1 1Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand Background: Cancers co-opt indoleamine 2,3-dioxygenase 1 (IDO1) as a mechanism for evading the immune system, and IDO1 inhibitors have emerged as a promising new approach for the treatment cancer through their potential to restore anti-tumour immunity and to synergise with existing therapies. As part of our investigations into novel small molecule inhibitors of IDO1, we screened a number of human and murine tumour lines for expression of tryptophan dioxygenases. All the human and murine tumour lines that we tested were found not to express tryptophan dioxygenases unless induced with IFN-gamma. Our survey included testing 50 primary human melanoma lines developed from tumour samples obtained New Zealand Melanoma patients (NZMel lines) for expression of IDO1, IDO2 and tryprophan dioxygenase (TDO). After co-culture with IFN-gamma, 86% of the NZMel lines were IDO1+. IDO2 and TDO was not detected in any of the NZMel lines before or after IFN-gamma exposure. To overcome the need for IFN-gamma induction for our cell based assays of IDO1 inhibitors, we transfected the wild-type murine Lewis Lung carcinoma line (LLTC) to constitutively express murine IDO1 (LLTC-mIDO1) or human IDO1 (LLTC-hIDO1), and used these engineered lines to assay for potential species selectivity of our inhibitors. Results: IDO1 expression in the lines was consistent and stable, but when LLTC-hIDO1 cells were implanted subcutaneously into syngeneic mice for in vivo testing of inhibitors, we found considerable heterogeneity in IDO1 expression between tumours. Approximately half of the tumours from the same batch of implants were completely negative for IDO1 expression by Western blot analysis. More intriguingly, when fragments of an IDO1+ or an IDO1- donor tumour were implanted into new recipients, the same degree of inter-tumoural heterogeneity in IDO1 expression was seen in the subsequent generation of tumours. In contrast, subcutaneous tumours developing from murine GL261 glioma cells similarly engineered to over-express hIDO1 (GL261-hIDO1) were homogeneous in their expression of hIDO1, and were used for subsequent evaluation of inhibitors in vivo. GL261 lines constitutively expressing TDO have also been developed and are being used to select for TDO-selective and TDO/IDO1 dual inhibitors.