Biology Reference
In-Depth Information
focused on assessing the level of phosphorylation of the
direct Wee1 substrate Cdc2 at Tyr15. Importantly, both
p-CDC2Y15 and pHH3 (ser10 and ser28) have been
reported in clinical samples of tumors of various types,
further underlining the potential of these biomarkers to
be of clinical relevance in the trials of MK1775. Addi-
tionally, it has been shown that p-CDC2Y15 can be
detected in hair bulbs in skin, and that the level of
phoshorylation was modulated by MK1775 treatment
with good correlation to that observed in xenograft
samples, thus suggesting a surrogate tissue strategy
could potentially be applied,
signature suggests that it could be used as a quantitative
PD biomarker in either tumor or surrogate tissues such
as skin. This is particularly powerful given the difficulty
in obtaining serial biopsies from tumors that are not
readily accessible such as pancreas and lung.
Checkpoint Abrogation and Safety
There still remain many questions regarding the
safety and tolerability associated with the use of these
agents, such as the potential for side effects in normal
tissues, as well as the possibility of exaggerated toxicity
from the combination of these drugs with existing
chemotherapeutics. In terms of selective activity in
cancer cells, and avoiding the potential for normal
tissue toxicity, the ideal target for checkpoint abroga-
tion would be one that is solely associated with the
G2 checkpoint and not involved in other checkpoints,
or possessing a role in normal cellular function. This
is not the case for either Chk1 or Wee1. Chk1 plays
a broad role in cell cycle checkpoint control, and also
serves to monitor the integrity of the replication fork
during normal cell division, i.e. in the absence of exog-
enously induced DNA damage.
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It is also responsible
for the phosphorylation of a number of substrates
involved in normal cell cycle progression as well as
checkpoint signaling, DNA repair and modulation of
chromatin dynamics.
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Thus, Chk1 is important in
the maintenance of genomic integrity, and so chronic
exposure to Chk inhibitors may ultimately be associ-
ated with increased tumorigenesis. Several of the
agents discussed are also potent inhibitors of Chk2,
and it is known that while not spontaneously cancer
prone, Chk2 knockout mice are more sensitive to the
formation of skin cancer in response to treatment with
chemical carcinogens. The mice show an increase both
in overall tumor burden and rate at which tumors are
formed.
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Whether this is a result of an increase in
genomic stability or a relative resistance to stress-
induced apoptosis remains to be clarified. Similarly,
the phosphorylation of cdc2 at Tyr15 by Wee1 is
required for G2 progression in every cell cycle, thus
long term exposure to inhibitors could potentially
impair normal cell cycle progression at G2-M. In addi-
tion to the possibility of side effects from the inhibition
of these targets in normal tissues, there is also the possi-
bility of exaggerated toxicity from the combination of
these agents with existing chemotherapeutics. In this
regard, it should be borne in mind that, although less
selective than the newer agents, UCN01 in combination
with topotecan was found in a phase I trial to exhibit
myleosuppression at lower doses of topotecan than
are observed for topotecan used as a single agent
therapy, suggesting the potential for the exaggeration
of normal tissue toxicities. This again underscores the
reducing the need
for patient biopsies.
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More recently, a gene signature was identified in
response to gemcitabine and MK1775 that may lead to
the identification of additional PD markers for tumor
or surrogate tissue in future. The study took a non-
biased, genome-wide approach and used mRNA
expression profiling to determine which genes were
regulated in response to gemcitabine and MK1775 treat-
ment, both in cancer cell lines, WiDR xenograft samples
and rat skin. In cancer cells, of 39,558 probes, the expres-
sion of 55 genes was found to be significantly altered in
response to the combination of gemcitabine andMK1775
versus gemcitabine alone versus changes in the levels of
expression of 48 genes in rat skin. Interestingly, only five
genes overlapped in the two sets, providing a potential
gene signature to be used as a PD marker independent
of p53 status. This signature comprises changes in clas-
pin, mini-chromosome maintenance complex compone-
net 10 (MCM10), F-box protein 5 (FBXO5), cyclin E1, and
cyclin E2. All five genes have known functions that
relate to the S-G2 cell cycle, inferring a relationship
between Wee1 inhibition and the changes seen, consis-
tent with the mode of action of MK1775 as a G2 check-
point abrogating agent. It was also determined that
these changes in gene expression in xenograft samples
and rodent skin samples were dose-dependent, and
correlated with levels of pCdc2 and antitumor response.
To further validate the signature, siRNA to Wee1 was
used to recapitulate the gene signature obtained with
MK1775.
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Although promising, significant challenges remain to
be overcome before there is clinical validation of this
gene signature, such as its demonstration in human
tumor and surrogate tissue samples, and the differential
level of response in rapidly proliferating tumor versus
normal cells. The variability of the signature also needs
to be better understood. If the results do, however, trans-
late into the clinic, approaches of this type offer
significant advantages over more traditional immuno-
histochemical measurements. The measurement of
mRNA expression profiles requires only a very small
sample of biopsy tissue, and results are highly quantita-
tive, and in this case, the commonality of the gene
