Chromosome segregation errors: a double-edged sword - TI Pharma

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these cells with E1A virus 584 . Strikingly, the transformed BJ-Tert cell line that had undergone all
three events was more sensitive to Mps1 inhibition (Fig.5C). While wild-type BJ-Tert cells did not
show any decrease in colony formation capacity following treatment with 30nM compound-5, the
viability of fully transformed BJ-Tert cells (p53KD, HRASV12 and E1A) was clearly affected (Fig.5C).
These data indeed indicate that transformed cells are much more affected by partial Mps1 inhibition
compared to healthy diploid cells. These differences are most likely due to more severe effects of partial
Mps1 inhibition on mitotic timing and chromosome congression in tumor cells, which eventually lead
to chromosome segregation errors, DNA damage and cell death.
High doses of compound 5 lead to deleterious effects on intestinal structures in mice.
Since these and published data suggest that Mps1 inhibition is a possible interesting candidate in
specific anti-cancer treatment 176,299,578,585 we plan to test the effect of Mps1 inhibition in a mouse
model for spontaneous tumor formation 586 . We tested the bio-availability in Balb/c mice and the
maximum tolerated dose (MTD) of compound-5 in wild-type FVB female mice (Fig.6). Pharmacokinetic
studies of intravenous and orally delivered compound-5 revealed a long latency time of the inhibitor
in the blood of more than 24 hours with a half-time of ~6 hours (Fig.6A). Initial MTD studies (50, 100
and 150 mg kg -1 compound-5) resulted in lethality in a very short time-span (~4-5 days). Haematoxylin
and Eosin (H&E) staining of the intestines of compound-5 treated mice revealed deleterious effects of
the inhibitor on the intestinal structure, as is evident by small villae and unstructured crypts (Fig.6B).
Moreover, actively dividing cells were almost completely absent in the intestinal crypts of mice treated
with compound-5 as determined with staining for phosphoS10-Histone H3, a mitotic marker, and Ki67,
a proliferation marker (Fig.6B). These effects on the intestine indicate that the cell cycle progression
of crypt cells in vivo has been affected. In future studies we wish to find well tolerated doses of
compound-5 and test the activity in mouse models for spontaneous tumor formation.
Discussion
In this study, we have validated Mps1 as a promising anti-cancer target using a small
molecule inhibitor. The inhibitor, referred to as compound-5, binds with subnanomolar
affinity to Mps1 and is more than 60-fold selective over a panel of 281 other kinases. Its
selectivity entropy, which is a novel quantitative measure of quantifying selectivity from panel
screening data 580 , is 0.13 and this low value signifies it is an excellently selective inhibitor.
In fact there are only three kinase targets on which more selective reference inhibitors were
reported so far 587 . Furthermore, the compound has a long retention time on Mps1 (~46 h) (data
not shown). The specificity and long latency could make compound-5 useful for in vivo tumor
studies, since one single administration could inhibit Mps1 molecules for a prolonged period
of time, which could decrease the need for multiple rounds of compound-5 administration.
Nanomolar concentrations of compound-5 specifically reduced the mitotic timing and affected
chromosome alignment of tumor cells, whereas immortalized, diploid fibroblasts were less affected
(Fig. 5). This suggests that healthy cells can cope with slight defects in Mps1 activity in contrast to tumor
cells, which die at relatively low doses of Mps1 inhibitor (Fig.4A). One explanation for this difference
could be that healthy, untransformed cells need less time to align their diploid content of chromosomes
and might therefore be less affected by checkpoint inhibition induced by partial Mps1 inhibition when
compared to tumor cells that have to cope with the presence of extra chromosomes. This hypothesis is
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