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

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may be quite complex, and may depend on the level of CIN reached in a given tissue in a given
genetic model. However, the fact that in currently available mouse models CIN is already present
during early development and in most cases cannot be induced in the adult mouse poses a
significant problem in interpreting its effects on tumor formation and inhibition. Therefore, we have
designed and tested a genetic strategy to inducibly reduce or ablate Mps1 activity in mice. In this
model, named CiMKi (Cre-inducible Mps1 Knock-in), timed and local induction of Cre-mediated
recombination using the Cre-ER T2 system will result in knock-in of two activity-reducing Mps1
point mutations in the endogenous Mps1 locus. This will allow us to control when and where and
to what extent Mps1 activity will be reduced, and therefore what level of CIN will be induced.
As expected from our previous work 172,299 and predicted from our experiments with the CiMKi T649A
strain, different activities of Mps1 will cause diverse levels of CIN. This makes CiMKi a suitable model to
test the influence of various levels of CIN on tumor initiation, progression and tumor growth inhibition
(Fig.1).
Results
To study the contributions of mild CIN to tumorigenesis as well as to assess whether inducing
excessive CIN is a feasible anti-cancer strategy, we have developed the CiMKi (Cre-inducible Mps1
Knock-in) model. Inspired by a recently published conditional V600E mutation in the BRAF locus 448 ,
CiMKi allows conditional mutation of the endogenous allele of Mps1 to one that encodes D637A
(equivalent of human D664; CiMKi D637A ) or T649A (equivalent of human T676; CiMKi T649A ). Both the
D664A and T676A mutation have been shown to reduce kinase activity of human Mps1 to 0% and
20% respectively. D664 is one of the catalytic aspartates, which is required for ATP positioning via Mg 2+
binding, and substitution of this residue renders the kinase inactive 143 . T676 is an autophosphorylation
site present in the activation loop of Mps1 and substitution for alanine reduces Mps1 kinase
56
Mps1
% kinase
activity
(expected)
Phenotype
(expected)
WT/WT WT/T649A WT/D637A T649A/T649A T649A/D637A D637A/D637A
100 60 50 20 10 0
No CIN ? ? Mild CIN Moderate
CIN
G1
M S
G2
Normal
cell cycle
progression
STOP
Tumorigenesis Cell cycle
arrest
Cell death
Severe
CIN
Cell death
Figure 1
Schematic overview of the CiMKi mouse models that will be generated. Expected residual Mps1 kinase activity of each mouse model
is indicated as well as the phenotype expected in the specific genotypes. The absence of CIN will produce two identical daughters
after each cell division and normal cell cycle progression. The presence of mild CIN will lead to mild chromosome missegregations
(~1-2 chromosomes/cell division), which will lead to intermediate levels of aneuploidy. This mild CIN could lead to cell death, cell
cycle arrest or tumorigenesis. Severe CIN will induce severe chromosome segregation errors upon mitotic exit (>3 chromosomes/
cell division) causing enhanced genomic instability, which is incompatible with cell viability.