Biology Reference
In-Depth Information
of both DNA-damaging cytotoxics and antimetabolites.
85
Early studies revealed synergistic growth inhibition with
UCN-01 in combination with mytomycin C, 5-fluoro-
uracil or methotrexate, both
in vitro
in cell lines, and
in
vivo
inmouse xenograft or syngenic models.
82
This syner-
gism was later related to the ability of UCN-01 to abro-
gate the G2 checkpoint. It was subsequently discovered
that UCN-01 could also abrogate the S-phase arrest
induced by cisplatin or camptothecin. Of particular
importance was the finding that the potentiating effect
of UCN-01 was greater in p53-deficient cells, and the
belief that the biological effects seen in these combination
studies were primarily driven through the inhibition of
Chk1, and hence, abrogation of cell cycle arrest.
41,86,87
Despite its lack of specificity, UCN-01 was the first
agent that potently inhibits Chk1 (IC
50
10 nM) to enter
clinical trials, but its pan-kinase activity means that the
clinical effects are not solely due to the drug's ability
to inhibit Chk1. To date, UCN-01 has been tested in
22 clinical trials, three of which are still ongoing.
88
e
92
The drug has been used in a variety of settings, across
numerous tumor types, and, because of its unique
profile of activity, is in trials both as a single agent and
in combination with cytotoxic agents. In general, combi-
nation treatment with UCN-01 has been well tolerated,
the exception being with cisplatin where targeted thera-
peutic levels could not be achieved owing to toxicity.
91
While some evidence of stable disease was reported in
combination with carboplatin in patients with plat-
inum-resistant disease in advanced solid tumors, many
other trials failed to demonstrate increased efficacy.
Thus, while these observations sparked an interest in
checkpoint abrogators as potentially efficacious anti-
cancer agents when used in combination with existing
therapies, more selective compounds were needed in
order to build a more sophisticated understanding of
the DDR networks. The identification of one of the
targets of UCN-01 as Chk1 increased interest in Chk1
as a useful therapeutic target that could lead to enhanced
cytotoxicity in tumor cells, and initiated many efforts to
design and develop novel, more selective small molecule
inhibitors. The current generation of more selective
checkpoint kinase andWee1 inhibitors that have reached
clinical trials are described in more detail below.
clinical trials (September 2005) as a single agent for the
treatment of CLL. The purpose of the study was to
assess the safety and tolerability of different doses of
XL-844 when given orally to patients with recurrent or
refractory CLL or small lymphocytic lymphoma. This
trial, however, was terminated in March 2007 due to
slow patient enrolment. In May 2007, a phase I trial
was initiated with XL-844 in combination with gemcita-
bine in patients with solid tumors or lymphoma.
XL-844 (the structure of which has not been publicly
released) is a potent, orally available, adenosine
triphosphate (ATP) competitive, aminopyrazine inhib-
itor of the checkpoint kinases Chk1 and Chk2, having
K
i
values of 2.2 nM and 0.07 nM, respectively.
93
In
a selectivity profile versus 86 additional kinases, only
four other kinases were inhibited to a significant degree
(IC
50
<
20 nM), these being Flt3, Flt4, KDR, and PDGF.
In vitro
cellular assays in PANC-1 cells have demon-
strated that XL-844 dramatically increases gemcita-
bine-induced Chk1 phosphorylation at the Ser
317
activation site. This suggests that Chk1 inhibition leads
to increased DNA damage and hence increased
upstream signaling from ATR. Importantly, this
increase in Chk1 phosphorylation does not result in
downstream degradation of Cdc25a, and thus the
S-phase checkpoint is not activated. As with p-Chk1,
increases are also seen in the levels of
g
-H2AX
(a marker of double-stranded DNA damage). Together
these markers indicate a greatly increased level of DNA
damage resulting from the combination treatment
versus treatment with gemcitabine alone.
Further
in vitro
studies also demonstrated that XL-844
is able to release the late G1/early S phase cell cycle
arrest induced by gemcitabine. Gemcitabine treatment
arrested PANC-1 cells and prevented them from
entering mitosis, as measured by a decrease in phos-
pho-histone H3 (p-hH3) levels. In the presence of
XL-844, p-hH3 increased, and cells had
4N DNA
content, indicating abrogation of arrest, in turn resulting
in premature entry into mitosis. Control experiments
confirmed that XL-844 alone had no impact on the cell
cycle in the absence of gemcitabine. Clonogenic assays
were also carried out in order to demonstrate that the
ability of XL-844 to significantly sensitize cells to the
cytotoxicity of gemcitabine was general across a number
of cell lines derived from diverse solid tumor types.
In
vitro
studies also showed that inhibition of replication
checkpoints by XL-844, in the presence of DNA-
damaging agents, induces cell death through mitotic
catastrophe in chronic lymphocytic leukemia (CLL)
leukemic lines, with greatest emphasis on p53-deficient
lines.
94,95
Increased efficacy has also been demonstrated
in vivo
in a PANC-1 ectopic xenograft model.
93
In this study,
gemcitabine used alone resulted in a tumor growth
<
CHECKPOINT ABROGATING AGENTS AS
POTENTIATING AGENTS
SELECTIVE
CHECKPOINT KINASE AND WEE1
INHI
BITORS IN CLINICAL TR
IALS
e
XL-844 (EXEL-844)
XL-844 (Exelixis) was the first of the current genera-
tion of more selective Chk inhibitors to enter Phase I
