Biology Reference
In-Depth Information
with deletions in chromosome 11q involving
ATM
did
not respond well to conventional therapy and had
more rapid disease progression.
256
Table 8.2
provides
examples of more recent analyses of potential NHEJ
markers of patients with different cancers. Some
investigators looked for association between SNPs and
clinical outcome or overall survival, while others made
use of reverse transcriptase-polymerase chain reaction
(RT
e
PCR) to measure mRNA levels to gauge gene
expression or assessed protein expression directly by
immunohistochemistry.
An unanticipated finding, apparent in several of the
studies to identify potential NHEJ-based biomarkers
shown in
Table 8.2
, is the better prognosis or response
to treatment of individuals with tumors with higher
expression of the NHEJ genes. For example, S¨derlund
et al.
257
found a superior response to radiotherapy for
breast cancer patients with higher levels of the MRN
complex. Similarly, head and neck cancer patients
responded better to treatment if their tumors expressed
higher levels of
Ku70
,
Ku80
, and
DNA-PKcs
, especially
Ku70.
258
These outcomes are surprising because the
expectation would be that tumors with higher levels of
NHEJ repair proteins would be more resistant to
therapy with the corollary that low expression of the
repair proteins would render tumors more sensitive to
radiation and chemotherapy. There are currently no
proven explanations for these observations, but it has
been speculated that impaired expression of either the
MRN or DNA-PK complexes might fail to induce cell
cycle arrest and apoptosis.
257,258
Ku70 and Ku80 have
been shown to form a complex with clusterin in irradi-
ated breast cancer cells that acts as a death signal for
severely damaged cells.
259
In addition to tumor response, it is important to take
into consideration treatment-related normal tissue
complications when treating patients, since these
complications are often the limiting factor to therapy. It
would thus be beneficial to have biomarkers that could
identify individuals who should not receive a particular
therapeutic regimen due to the likelihood of severe treat-
ment-related complications. The early deleterious effects
of radiation occurring within 90 days are primarily
fatigue and dry and peeling skin. The more persistent
and troublesome late effects of radiation include fibrosis
due to cytokine release that stimulates collagen deposi-
tion, chronic inflammation, which is particularly
dangerous in the lung (pneumonitis), and necrosis,
which can be fatal in organs such as the brain. Several
genomic studies have compared SNPs, including those
in NHEJ genes, of radiosensitive patients to those of
radiotolerant patients. SNPs in
ATM
have been associ-
ated with late fibrosis in breast cancer patients,
260,261
while SNPs in
LIG4
have been associated with radiosen-
sitive prostate cancer patients,
262,263
and a SNP in
Ku70
was associated with serious dysphagia (difficulty in
swallowing) following irradiation of a population of
patients with squamous cell carcinoma of head and
neck.
264
Inhibition of NHEJ Proteins as
a Therapeutic Approach
Given the importance of the NHEJ pathway in the
repair of IR and chemotherapeutic agent-induced DSBs
it is not surprising that this pathway has gained atten-
tion as a possible therapeutic target. Here we will review
some of the approaches currently being used to target
the NHEJ pathway for therapeutic advantage.
Small Molecule Inhibitors of DNA-PK
and ATM Protein Kinases
The protein kinase activities of DNA-PKcs and ATM
are required for their cellular functions in DSB repair
and the DNA damage response, making them attractive
therapeutic targets. Both DNA-PKcs and ATM are
inhibited
in vitro
by the phosphatidylinositol 3-kinase
(PI3K) inhibitor, wortmannin which alkylates a specific
lysine in the PI3K active site
265
as well as by the methyl
xanthine, caffeine.
266
A series of more specific small
molecule inhibitors of DNA-PKcs that includes
NU7026 and NU7441 has recently been derived from
the PI3K inhibitor LY294002.
267
NU7441 (2-N-morpho-
lino-8-dibenzothiophenyl-chromen-4-one) is a potent
and ATP-competitive inhibitor of DNA-PKcs kinase
activity with an
in vitro
IC
50
of 14 nM that sensitizes
HeLa cells to IR and etoposide.
268
A similar strategy
generated KU55933 (2-morpholin-4-yl-6-thianthren-1-
yl-pyran-4-one), a potent and specific inhibitor of
ATM protein kinase (IC
50
13 nM) that sensitizes cells
to IR, etoposide, camptothecin and other DNA
damaging drugs.
269
Screening of small molecule
libraries against
in vitro
ATM kinase activity has yielded
CP466722 [2-(6,7-dimethoxyquinazolin-4-yl)-5-(pyri-
din-2-yl)-2H-1,2,4-triazol-3-amine], which, like KU55933,
inhibits ATM dependent signaling and cell cycle check-
point activation in irradiated cells, and sensitizes cells to
IR.
270
Other classes of DNA-PK inhibitors have been
generated such as vanillin
271
but
they appear less
specific than the NU compounds.
Although these compounds have proven to be
extremely useful in the research laboratory, allowing
the roles of ATM and DNA-PKcs to be distinguished,
whether derivatives of these compounds will prove to
be efficacious in the clinic remains to be determined.
Also, although the KU and NU inhibitors were highly
selective for their targets in
in vitro
assays against a panel
of protein kinases, it is possible that they will be less
specific
in vivo
since they both act as ATP competitive
inhibitors. Moreover, since ATM and DNA-PKcs are
