Abstract B159: Exploring the functions of TAM kinases: assay development and characterization of potential therapeutics
Authors: Chakrabarti Alokta , Feger Daniel , Totzke Frank , Umber Sarah , Stus Orysya , Birkle Marianne , Siedentopf Oliver , Pathe Marcel , Weber Thomas , Kubbutat Michael H.G. , Ehlert Jan E. 
Published: August 2018

TAM kinases, a relatively recent group of RTKs, have gained interest as therapeutic targets in cancer, chronic inflammatory, and autoimmune diseases. Comprising Tyro-3, Axl, and MerTK, TAMs need an extracellular lipid-protein complex for their activation, which is unique to this family of RTKs. For example, they bind to phosphatidyl serine bound gamma-carboxylated proteins as Gas-6 on apoptotic cells as ligands. Normally, TAMs function as regulators of the immune system such as controlling innate immunity or differentiation of natural killer cells, and lately a significant role in hematopoiesis has been deciphered. Though not potent oncogenic drivers, deregulated TAM signaling is often strongly associated with cancer progression, metastasis, and resistance to targeted therapies. They promote tumor growth by activation of prosurvival signals in tumor cells and reduction of antitumor immune responses. Several TAM kinase inhibitors are under (pre-)clinical investigation, e.g., BGB324 (R428), an Axl inhibitor is currently in Phase 1 trials as mono- or combination therapy. While for dual MerTK-Flt3 inhibitors MRX-2843 has received FDA Investigational New Drug (IND) status, UNC2025 is restricted to certain cancers like acute myeloid leukemia since inhibition of Flt3 has been related to severe hematopoietic toxicity. With the rapid increase in TAM-selective targeting strategies, there is an unmet need of assay systems to identify and categorize specific inhibitors. Due to their unique activation mechanism, set-up of cellular assay systems especially poses a challenge. Here, we report on the progress of set-up of various cell-based assays for all TAM kinases. To this end, we successfully developed a MefToff system for Axl and full-length expression in one cell background (e.g. Rat1) for Axl and Tyro-3 to measure cellular kinase activity. Additionally, we compared the data and feasibility of our cellular systems with biochemical tests using recently published commercially available inhibitors including UNC2025. To identify off-targets, we also tested 320 kinases in our ProQinase biochemical wild-type profiler assays. Furthermore, phenotypic screens were run to substantiate these outcomes. Currently, we try to set up assays for MerTK using various transmembrane domain mutants and chimeric systems. Assays with primary as well as tumor cells are in progress that would allow us to validate results from the cell-based artificial systems in to a more physiologic context. We believe such assays will allow a detailed molecular characterization of TAM kinases and will open opportunities for development of more advanced molecules as well for many new areas of interest.

Citation Format: Alokta Chakrabarti, Daniel Feger, Frank Totzke, Sarah Umber, Orysya Stus, Marianne Birkle, Oliver Siedentopf, Marcel Pathe, Thomas Weber, Michael H.G. Kubbutat, Jan E. Ehlert. Exploring the functions of TAM kinases: assay development and characterization of potential therapeutics [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B159.