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Leukemic T-cell Precursors from T-lineage All Patients Characterized by Profound Ku80 Deficiency
7
1.3 Ku-dependent expression of
Ikaros
target genes
in primary human lymphocyte precursors from
pediatric ALL Patients
Lymphocyte precursors from mice with
IKZF1
null
mutation are characterized by >2-fold decreased
expression levels of 201 transcripts representing 137
IK
target genes, including 61 transcripts representing
45 genes harboring IK binding sites as confirmed by
chromatin immunoprecipitation followed by DNA
sequencing (Uckun et al., 2012). For 19 of these 45
genes, we identified 27 transcripts in the Affymetrix
platforms for human transcriptome that showed a
highly significant correlation with
IKZF1
gene
expression levels in primary human lymphocyte
precursors from ALL patients consistent with their
status as an
IK
target gene (Uckun et al., 2012). For
the expression level of each one of these 19 genes, a
striking correlation was also noted with Ku expression
levels (Table 1, Figure 3). Hierarchical cluster analysis
identified
PITRM1
(1.21 SD units, P = 1.2 × 10
-59
),
MDM1
(1.13 SD units, P = 2.2 × 10
-49
),
ITGA4
(1.19
SD units, P = 2.7 × 10
-49
),
KIF23
(1.13 SD units, P =
1.3 × 10
-46
) and
PREP
(1.06 SD units, P = 1.5 × 10
-43
)
as the most significantly upregulated genes in 314
patient samples with high
Ku
expression (Table 1).
These findings extend our earlier study that
demonstrated the ability of both Ku70-specific siRNA
and Ku80-specific siRNA to knock down the
expression of these validated IK target genes in human
293T cells (Ozer et al., 2013).
Figure 3 Correlation between transcript expression levels of
Ku
and
Ikaros
target genes
Note: The expression values of validated
IK
target genes in Standard Deviation units were compiled for the 5 studies and rank
ordered according to the mean expression of three highly correlated transcripts (208642_s_at (
XRCC5
), 208643_s_at (
XRCC5
),
200792_at (
XRCC6
). These samples were also rank ordered according to
IKZF1
expression level (205038_at, 205039_s_at,
216901_s_at, 227344_at and 227346_at; 3 of these were common in all Affymetrix platforms-205038_at, 205039_s_at, 216901_s_at).
T-tests were performed for the combined Standard Deviation units from the 5 datasets (2-sample, Unequal variance correction,
p-values<0.05 deemed significant) to reveal 27 transcripts representing 19 genes as significantly upregulated samples with
upregulated
Ku
and
Ikaros
express
We also examined the expression levels of 7 signaling
pathway genes (viz.:
IL4
,
IL5
,
IL10
,
IL13
,
STAT4
,
FGFR4
, and
VIPR1
) previously reported to be
regulated by native IK in human cells (35-40) for
Ku
-dependency.
Notably,
primary lymphocyte
precursors with high
Ku
expression levels (N=67)
exhibited significantly higher expression of the
IK1
target gene set than lymphocyte precursors with low
Ku
expression levels (N= 70) (Figure 4). Borderline
significant increases were observed with two
IK1
targets (IL13, P= 0.06) and (
VIPR1
, P=0.09) and
significant increases were exhibited with the other 7
IK1
targets. Two transcripts for
STAT4
and
FGFR4
showed highly significant (P<0.0001) overexpression
Molecular Medical Science, Int’l Journal of