Cancer Research in Switzerland - Krebsliga Schweiz

Peter Matthias | Regulation of genome stability by
Rtt101p/cullin4-based E3-ubiquitin ligases in yeast
and mammalian cells (OCS 02189­02­2008)
Cullin 4 (Cul4)­based ubiquitin ligases have emerged as
critical regulators of DNA replication and repair. Over 50
Cul4­specific adaptors (DCAFs) have been identified and
are thought to assemble functionally distinct Cul4 complexes.
Using a live­cell imaging­based RNAi screen, we
analyzed the function of DCAFs and Cul4­linked proteins
and identified specific subsets required for progression
through G1 and S­phase. We discovered C6orf167/
Mms22L as a putative human orthologue of budding
yeast Mms22, which together with the cullin Rtt101 regulates
genome stability by promoting DNA replication
through natural pause sites and damaged templates. Loss
of Mms22L function in human cells results in S­phase­dependent
genomic instability characterized by spontaneous
double strand breaks and DNA damage checkpoint
activation. Unlike yeast Mms22, human Mms22L does
not stably bind to Cul4 but is degraded in a Cul4­dependent
manner and upon replication stress. Mms22L physically
and functionally interacts with the scaffold­like protein
Nfkbil2 that co­purifies with histones, several
chromatin remodelling and DNA replication/repair factors.
Together, our results strongly suggest that the
Mms22L­Nfkbil2 complex contributes to genome stability
by regulating the chromatin state at stalled replication
forks.
Project coordinator
Prof. Dr. Matthias Peter
Institut für Biochemie
ETH Zürich
HPM G8
Schafmattstrasse 18
CH­8093 Zürich
Phone +41 (0)44 633 65 86
Fax +41 (0)44 632 12 98
matthias.peter@bc.biol.ethz.ch
Plückthun Andreas | Tumor targeting of ErbB2 with
designed ankyrin repeat proteins
(OCS 02128­08­2007)
In the last ten years, targeted tumour therapy has made a
major leap forward, mainly due to improvements in the
design and production of antibodies. However, despite the
technological advances, targeting molecules such as antibodies,
in particular as fusion proteins with toxic moieties,
often suffer from unfavourable biophysical properties, expensive
production and limited efficacy if used as monotherapy.
Therefore, complementary molecular scaffolds
are in high demand and the subject of intense research.
We have developed a new class of binding molecules,
termed “designed ankyrin repeat proteins” (DARPins) that
display high affinities for their targets, outstanding biophysical
properties and a wide range of targets, and they
can be manufactured in bacteria in large amounts at low
cost.
We chose the ErbB2 receptor as a target tumour antigen.
Overexpression of ErbB2 occurs in a broad range of human
cancers, including up to 30 % of breast carcinoma,
and high ErbB2 levels have been correlated with an aggressive
metastatic tumour phenotype. Consequently,
high expression of ErbB2 may be employed for tumour
targeting, where the receptor is used as a cellular gate to
convey therapeutic payloads into the tumour cells. Notably,
the inhibition of tumour growth may be induced
solely by the binding to the receptor in as much as ErbB2
is required for the proliferation of tumour cells. In order to
achieve high treatment efficiency, we aimed to construct
ErbB2­targeting molecules combining several favourable
attributes.
In the course of this project, we established an array of
powerful methods for selection and maturation of the
DARPin binders. By this means, a number of ErbB2­directed
binders were selected and profiled for their efficiency
to inhibit tumour growth in cell culture models.
Several binders were further engineered by methods of
molecular biology and computational molecular design,
and the resulting molecules reached tumouristatic activity
that surpassed the efficacy of the anti­ErbB2 antibodies
currently used in the clinic. The high anti­tumour activity
of these novel binders has been related to a potent induction
of cell death (apoptosis) and growth arrest selectively
on the tumour cells expressing ErbB2. Importantly, due to
the absence of any cytotoxic additives or moieties, these
DARPin variants are expected to be devoid of adverse side
effects. This property may translate into high therapeutic
benefit in the clinical treatment.
Finally, we conducted initial studies in animal models
aimed at determining the cytotoxicity, biodistribution and
pharmacokinetics of DARPins. The DARPins appeared to
be well tolerated and localized to the tumour sites with
very high specificities. We are therefore confident that
DARPins can be used as effective vehicles for the ErbB2mediated
tumour targeting. Based on their high tumouricidal
activity, specificity of targeting and low­cost production,
they can be potentially envisaged as candidates
to complement or even substitute antibodies in a number
of therapeutic applications.
Project coordinator
Prof. Dr. Andreas Plückthun
Biochemisches Institut
Universität Zürich
Winterthurerstrasse 190
8057 Zürich
Phone +41 (0)44 635 55 70
Fax +41 (0)44 635 57 12
plueckthun@bioc.uzh.ch
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