Abstract Inhibition of the spindle assembly checkpoint kinase TTK causes chromosome mis-segregation and tumor cell death. High levels of TTK correlate with chromosomal instability (CIN), which can lead to aneuploidy. To investigate the potential relationship of CIN and sensitivity to TTK inhibition, we performed CIN analysis in human cancer cell lines from different tumor tissue origins and with different relative sensitivity to the selective TTK inhibitor NTRC 0066-0 [1]. By time lapse microscopy we observed that treatment with TTK inhibitor resulted in overriding of the mitotic checkpoint, irrespective of cell line sensitivity. Aneuploid but chromosomal stable cell lines were more sensitive than cell lines that already displayed high levels of CIN. In sensitive cell lines, treatment with TTK inhibitor induced acute chromosome mis-segregration. On the contrary, in populations of cells that already showed high levels of CIN, there was only a small additional fraction of cells mis-segregating their chromosomes. The resistant cell lines were all hypo-triploid instable cell lines, which are thought to be derived from cells with a double diploid genome (tetraploid cells) that have lost certain chromosomes. Next, we studied the effect of NTRC 0066-0 on three cell lines evolved via tetraploidization of the colorectal adenocarcinoma cell line HCT 116 referred as tetraploids. The tetraploids have low levels of CIN and were recently shown to display low level multidrug resistance against various cytotoxic agents and several targeted drugs. Proliferation of the tetraploids was inhibited with the same potency as the diploid parental cell line by three TTK inhibitors. Parental and tetraploids were also equally sensitive to reversine, a compound that is often used as a TTK reference inhibitor but whose selectivity has been disputed. In surface plasmon resonance binding experiments we measured a 800 times more potent binding of reversine to TTK over Aurora B. In a comparative cancer cell panel profiling study with 122 different anti-cancer agents [2], reversine clustered together with TTK inhibitors, demonstrating that in cells reversine acts as a TTK inhibitor. Finally, we show that NTRC 0066-0 inhibits the proliferation of primary human patient-derived colorectal cancer organoids with potencies similar to that of immortalized cancer cell lines. In contrast, treatment with TTK inhibitor did not reduce the viability T cell acute lymphoblastic leukemia cell samples, which are non-proliferating cells and very sensitive to classic chemotherapeutic agents, such as daunorubicin. These data is consistent with the function of TTK as a spindle assembly checkpoint kinase that is only active in proliferating cells. Consequently, TTK inhibitor therapy is expected to spare non-dividing cells, whereas it may be used to target stable aneuploid tumors. [1] Maia et al. (2015) Annals of Oncology; [2] Uitdehaag et al. (2016) Molecular Cancer Therapeutics. Citation Format: Marion A.A. Libouban, Jeroen A.D.M. de Roos, Joost C.M. Uitdehaag, Nicole Willemsen-Seegers, Sara Mainardi, Jelle Dylus, Jos de Man, Bastiaan Tops, Jules P.P. Meijerink, Zuzana Storchová, Rogier C. Buijsman, René H. Medema, Guido J.R. Zaman. Stable aneuploid cells are more sensitive to TTK inhibition than chromosome instable 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 3457. doi:10.1158/1538-7445.AM2017-3457