Biology Reference
In-Depth Information
Parp inhibition is synergistic in tumor cell lines with
genetic aberrations in the DNA damage response and
repair pathways, and future work will likely reveal
additional therapeutic hypotheses.
141
contrast to the activation events, levels of this marker
are reduced.
Phosphorylation of histone variant 2AX at Ser139
(known as
g
-H2AX) is a sensitive marker of DNA
double-strand breaks (DSBs).
143
e
145
This phosphoryla-
tion event (by ATM, ATR, and/or DNA-PK) is extremely
rapid and within seconds of the formation of a DNA
DSB thousands of H2AX molecules are phosphorylated,
leading to the formation of megabase chromatin
domains flanking the site of the strand break. This phos-
phorylation is now one of the most established chro-
matin modifications linked to DNA damage and
repair, and importantly can be quantified by immuno-
fluorescence-based assays or fluorescence microscopy
which allow visualization of the discrete foci formed in
response to a DSB. Each break has been shown to corre-
spond to one
PD Biomarkers
Preclinical studies have suggested a number of PD
biomarkers that may be valuable in assessing target
modulation for both checkpoint kinase and Wee1 inhib-
itors, although clearly this remains to be shown to have
clinical utility.
InthecaseoftheChkinhibitors,ithasnotbeen
possible to use direct Chk1 substrates as only a limited
number of Chk1 substrates have been identified,
limiting the options to find robust biomarkers. In addi-
tion, unfortunately, reagent quality (antibodies),
robustness of response and the complexity of combina-
tion with DNA damaging agent make many of these
impractical for use as reliable clinical tools
.
An
increased understanding of the impact of Chk inhibi-
tion, however, revealed possible alternatives for clini-
cally useful biomarkers such as phosphorylated Chk1,
histone H3, and histone 2AX that all have the potential
to be useful as PDmarkers. To date, most of the preclin-
ical studies seem to have focused on the assessment of
levels of pChk1 and
g
-H2AX and the demonstration
that changes in these markers correlate with target
modulation
in vivo
. The choice of these markers was
based on the observation that inhibition of Chk1, either
via small molecules or molecular tools, resulted in an
ATR-driven feedback loop. Thus, Chk1 inhibition
resultsinanincreaseinATRactivitywhichinturn
means that ATR substrates such as Chk1 and H2AX
are hyperphosphorylated. A major advantage associ-
ated with using these markers is that levels should
increase following combination treatment of a DNA-
damaging agent and a Chk inhibitor rather than
decreasing. This reduces the probability of obtaining
false-positive results. In addition, it should be easier
for the combination response to be distinguished from
baseline samples acquired from no treatment or DNA-
damaging agent alone.
There are three phosphorylation sites on Chk1 that
can potentially be used to provide a measure of PD
activity
-H2AX focus.
146,147
As well as being an
extremely sensitive measure, as
g
-H2AX is formed
de
novo
, it is a more accurate marker of DNA damage
than some other repair proteins which may be present
in the nucleus even if DNA damage is not present,
meaning that background levels may be problematic.
148
These factors combine to make
g
-H2AX a good pharma-
codynamic marker when investigating strategies to
improve the effectiveness of DNA damaging anticancer
therapies such as those described here, and indeed there
have now been several examples of the clinical assess-
ment of
g
-H2AX levels in blood (PBMCs), surrogate
tissues (skin) and tumor biopsies in cancer trials.
145
Preclinical work with the Chk inhibitors currently in
clinical trials has demonstrated that these markers do
indeed behave as predicted, and can be used to
understand PK/PD/efficacy relationships.
149,150
The
one exception is the finding that combination of
PF00477736 with gemcitabine results in a decrease in
pChk levels (both ser317 and ser345), although
increases in
g
2AX levels are still observed. The reason
for this is not fully understood, but could suggest that
PF00477736 in some way modulates the Chk1/protein
phosphatase 2A regulatory circuit that antagonizes
the phosphorylation of Chk1 by ATR.
151
Both
in vitro
and
in vivo
, preclinical studies with either
Chk inhibitors, or MK1775 reveal that pHH3 levels
increase following combination treatment with DNA
damaging agents, demonstrating abrogation of cell cycle
checkpoints and entry into mitosis. The premature entry
into mitosis is speculated to induce mitotic catastrophe,
which is a form of apoptosis that occurs during mitosis
and may result from deficient cell cycle checkpoints,
particularly DNA damage checkpoint(s) and the spindle
assembly checkpoint. Like the formation of
g
-H2AX,
phosphorylation events at Ser10 and Ser28 of histone
H3 are very well characterized, and robust and reliable
antibodies are readily available. In addition to pHH3
levels, biomarker strategies for MK1775 have also
g
activation phosphorylations (on Ser317 and
Ser345) and an autophosphorylation (on Ser296) that
triggers downstream signaling and checkpoint activa-
tion.
142
In response to the detection of DNA damage or
replication stress, ATR phosphorylates, and hence acti-
vates, Chk1 on the carboxy terminal residues Ser317
and Ser345 and as outlined above the extent of this phos-
phorylation increases in response to Chk1 inhibition.
The autophoshorylation on Ser296 is reduced, so in
e
