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Leukemic T-cell Precursors from T-lineage All Patients Characterized by Profound Ku80 Deficiency
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alternative NHEJ pathway (Fattah et al., 2010), the
development of a T-cell precursor hyperplasia in
haplodeficient mice (Ozer et al., 2013) cannot be
explained by DNA repair deficits and provides
compelling evidence that the interaction of Ku
components with IK likely contributes to the
anti-leukemic effects of IK as a tumor suppressor.
Thus, Ku deficiency may in part be responsible for the
IK malfunction in proliferating T-cell precursors in
mice, which is a hallmark of pediatric high-risk
T-lineage ALL (Sun et al., 1999).
The purpose of the present study was to explore if the
IK-regulatory function of Ku might play a role in the
biology of pediatric T-lineage ALL. We examined the
expression levels of validated IK target genes that
harbored IK binding sites (Uckun et al., 2012) in
primary leukemic cells from 1104 pediatric ALL
patients in relationship to Ku expression levels.
Hierarchical cluster analysis of cellular gene
expression profiles revealed a highly significant
correlation between Ku expression level and
expression levels of validated IK target genes.
Leukemic T-cell precursors from primary bone
marrow specimens of children with T-lineage ALL
were characterized by profoundly diminished Ku80
transcript levels as well as functional IK deficiency
with very low IK target gene expression levels. These
results uniquely implicate Ku-deficiency as a
contributor to the functional IK-deficiency in pediatric
T-lineage ALL.
1 Results and Discussion
1.1 Ikaros-Ku Molecular Complex
By using multiple assay platforms, including
coimmunoprecipitations,
pull-down assays, and
EMSAs with supershift assays, we recently
demonstrated that native as well as recombinant Ku
and IK proteins form heterodimers capable of binding
IK target DNA sequences with greater affinity that IK
homodimers (Ozer et al., 2013). In order to gain
further insights into the physical interactions between
Ku and IK proteins, we examined the ability of
MBP-tagged purified recombinant Ikaros 1 (IK1) and
Ikaros 5 (IK5) proteins in solution phase to bind to
immobilized recombinant
Ku70,
Ku80,
and
Ku70/Ku80 heterodimer by surface plasmon
resonance (SPR), which permits direct measurements
of the association and dissociation kinetics of binding
interactions. Reflective of a high biological affinity,
the SPR-based dissociation constant values for the
IK1-Ku binding interactions were in the low
nanomolar range. MBP-IK1 (but not MBP-IK5)
showed high affinity binding to Ku70 (Figure 1A.1),
Ku80 (Figure 1A.2) and Ku70/Ku80 heterodimer
(Figure 1A.3) with nanomolar KD values. The KD
values for the IK1-Ku70,
IK1-Ku80,
and
IK1-Ku70/Ku80 binding interactions were 15.2 nM,
14.1 nM, and 15.3 nM, respectively.
We constructed a molecular model of the Ku-IK1
hetero-complex focusing on the contribution of the
N-terminal zinc finger structures, ZF (1-4) of IK
(110-256) to the experimentally documented
differential binding of Ikaros isoforms to Ku
components. Our refined molecular model of the IK1
protein complexed with the Ku70/80 heterodimer is
depicted in Figure 1B1-B3. According to our model,
Ku80 subunit of Ku protein forms an excellent
docking site for IK1 near the entrance of the “ring”
along the minor groove that fits well with the contours
of the ZF (2-4) domains of IK1 approaching along
with the major groove and extending almost a
complete turn of DNA duplex. As a result, IK1 and
Ku80 form an interface with at least 970 Å2 buried
surface. There are 40% polar and 60% non-polar
residues involved in the interaction. The following
residues of Ku80 are located in the interaction region:
E49, K51, R242, H246, P248, R250, R260, Y264,
K273-4, T275, T277, K282, T283, L284, K285, K286,
E287. E329, K334, S335,E336, K338, A367, R368,
D369, D370 and K399. Ku70 is in the middle of the
DNA major groove and there is only 194 Å2 contact
area for Ku70 from this orientation. However, IK1-ZF
(2-4) can readily interact with Ku70 assuming Ku70 is
flexible and the segment of the βO-to-α12 turn in
Ku70 is capable of adopting a more open
conformation. Our model illustrates the importance of
IK1 ZF domains for its interactions with Ku and
readily explains why IK1 exhibited better binding to
Ku components (Ku70 and Ku80) than IK5 lacking
ZF (2-4) in SPR assays.
IK1 protein contains four Cys2His2 ZF motifs near its
Molecular Medical Science, Int’l Journal of