Advances in Bioscience and Biotechnology, 2013, 4, 129-135 ABB
http://dx.doi.org/10.4236/abb.2013.41A019 Published Online January 2013 (http://www.scirp.org/journal/abb/)
Polycomb response element-binding sites in the MDR of
CLL: Potential tumor suppressor regulation
Christine E. Cutucache1, Javeed Iqbal2, Philip J. Bierman3, Robert Gregory Bociek3,
Dennis D. Weisenburger2, Shantaram S. Joshi4
1Department of Biology, University of Nebraska at Omaha, Omaha, USA
2Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, USA
3Department of Internal Medicine Oncology/Hematology, University of Nebraska Medical Center, Omaha, USA
4Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, USA
Received 1 November 2012; revised 5 December 2012; accepted 10 January 2013
ABSTRACT
Chronic lymphocytic leukemia [CLL] is the most com-
mon adult leukemia and is heterogeneous in clinical
presentation. CLL cases present with various chro-
mosomal aberrations, including 11q23, 14q32, 17p,
and trisomy 12, with the most common abnormality
being deletion of 13q14 [1]. Although monoallelic de-
letion of 13q14 is common, there is a subset of pa-
tients who have complete nullisomy at 13q14, a locus
that has been hypothesized to contribute to CLL pa-
thogenesis [2] due to loss of tumor suppressors [ DLEU
and miR-15a/16-1]. We hypothesized that deletion of
both copies of 13q14 would lead to uncontrollable
proliferation of CLL cells and a poor prognosis. We
examined our 13q14 nullisomy for survival, treat-
ment-free survival, lymphocyte doubling time, and
the presence of lymphadenopathy. Furthermore, we
compared the gene expression profiles between pa-
tients with 13q14 monosomy, nullisomy, or normal
karyotype. Our results suggest that patients with 13q
nullisomy have a higher incidence of bulky lympha-
denopathy [16.6% compared to 10% of monosomy
patients], a higher frequency of lymphocyte doubling
time [27.7% compared to 7.4% of monosomy pa-
tients], and a higher rate of needing treatment [50%
compared to 18.5% of monosomy patients]. We ob-
served deletion of DLEU1 and HTR2A, consistent
with a gene dosage effect, and observed PRE-binding
sites on DLEU1. Patients with homozygous deletion of
13q14 had a worse prognosis compared to heterozy-
gotes. Lastly, the DLEU1 locus is a possible “second
hit” loss for CLL progression.
Keywords: Chronic Lymphocytic Leukemia; Gene
Expression; 13q14; Nullisomy; DLEU; Tolerogenic
1. INTRODUCTION
Chronic lymphocytic leukemia [CLL] is the most com-
mon adult leukemia and is heterogenous in clinical pres-
entation. Previous studies have focused on identifying
prognostic markers such as CD38− and ZAP70-expres-
sion, immunoglobulin mutational status, lymphocyte dou-
bling time, presence of lymphadenopathy, and chromo-
somal abnormalities to better group patients for therapy
[1,2]. Patients with CLL may have none, one, or several
of the following chromosomal abnormalities: del [11q23],
del [13q14], del [17p], trisomy 12, and, less frequently,
del [14q32]. However, there exists a cohort of patients
with loss of both alleles at 13q14 [13q null] that is dis-
tinct of patients with monosomy at this region [13q mono]
and wild type [WT]. The 13q14 locus was hypothesized
to contribute to CLL pathogenesis due to the loss of tu-
mor suppressors, DLEU and miR-15a/16-1 [3-6]. We
hypothesized that deletion of both copies of 13q14 would
lead to uncontrollable proliferation of CLL cells and a
poor prognosis. To test this hypothesis, we performed
retrospective analyses with the data we accumulated
from CLL patients cared for at the University of Ne-
braska Medical Center [UNMC].
2. PATIENTS AND METHODS
2.1. Patient Samples
All peripheral blood CLL [PB-CLL] samples were ob-
tained by venipuncture, and the CLL cells from bone
marrow [BM-CLL] and lymph node [LN-CLL] were
collected from the excess tissue samples. All protocols
were approved by the Institutional Review Board [IRB]
and Scientific Review Committee [SRC] at UNMC. PB-
CLL and BM-CLL cells were separated by using mag-
netic bead cell separation [Miltenyi Biotec, Auburn, CA,
USA], and samples with 95% or greater CD5+CD19+
cells were used for analyses. To obtain LN-CLL cells,

C. E. Cutucache et al. / Advances in Bioscience and Biotechnology 4 (2013) 129-135 130
serial sections of frozen lymph node biopsies were ob-
tained from the UNMC tissue bank. Immunohistochem-
istry was used to stain each section for CD5 and CD19 to
identify areas of >90% CLL cells. The CLL cells were
microdissected out for the analyses.
2.2. Fluorescent in Situ Hybridization [FISH]
and Microarray Analyses
Fluorescent in situ hybridization [FISH] was used to de-
termine chromosomal abnormalities in CLL cells as pre-
viously described [7]. The annotated patient data was
examined to determine the association between time to
treatment, treatment-free survival, lymphocyte doubling
time, and the presence of lymphadenopathy. Microarray
analyses were used to determine differential expression
of genes in a region of 13q suggested to be deleted in
CLL [8,9].
Previously, a tolerogenic signature was associated with
poor prognosis in CLL [9-12]. Genes from the tolero-
genic signature were examined among patients with 13q
null, 13q mono, and WT cases. Next, the genes within
the 13q locus that were included in our array were clus-
tered by CLL patients WT at this locus compared to 13q
mono and 13q null. These results would describe whe-
ther there was a gene copy effect between CLL cases
with losses at this region, which would identify a candi-
date tumor suppressor gene[s].
2.3. Examination of Regulatory Elements
The sequence for DLEU1 was examined using Vista
[pipeline.lbl.gov/cgi-bin/gateway2] and the University of
California, Santa Cruz [UCSC] Genome Bioinformatics
[genome.ucsc.edu] Programs. The entire coding and non-
coding sequence of DLEU1 was blasted for the poly-
comb response elements [PRE], including: GAF, G10,
PHO, and Z binding sites.
3. RESULTS AND DISCUSSION
3.1. Prognosis for CLL Patients with 13q
Nullisomy
Our cohort of 13q null patients [n = 34] had divergent
clinical characteristics from that of 13q mono CLL cases
[n = 27]. 13q null patients had higher incidences of bulky
lymphadenopathy [16.6% null compared to 0% mono],
shorter lymphocyte doubling time [27.7% null compared
to 7.4% mono], and greater occurrences of needing treat-
ment (50% null, 18.5% mono; Table 1). CLL patients
were broadly defined as having a poor prognosis if there
was lymph node-involvement, a short lymphocyte dou-
bling time [defined as less than a year for doubling], and
if they required treatment. Although CLL patients with
13q deletion are generally classified as having a favor-
able outcome, we suggest a divergence in prognosis be-
tween patients with a heterozygous, compared to a ho-
mozygous, deletion at this locus. Accordingly, patients
with homozygous deletion of 13q14 might benefit from
earlier treatment.
We next examined the percentage of CLL cells with
13q null to determine their susceptibility to treatment.
The FISH results from pre-treatment and post-treatment
for CLL patients showed a decrease in the number of
CLL cells positive for 13q14 nullisomy from 18% pre-
treatment to only 5% patients after treatment (p = 0.015;
Figure 1(a)). This 73% decline preliminarily suggests
that early treatment of patients with 13q null could be
beneficial.
3.2. Gene Expression Profiles [GEP] of Patients
with 13q Nullisomy
In order to determine whether there was a divergence in
GEP between WT, 13q null, and 13q mono cases, mi-
croarray data were examined. Previously, we reported
that a tolerogenic signature is indicative of patients with
an unfavorable outcome compared to those with a more
indolent disease [8]. Furthermore, based on clinical pro-
gnostic indicators, our results suggested that CLL pa-
tients with 13q14 null had a less favorable prognosis
compared to those mono or WT. As a consequence, we
examined the tolerogenic GEP between CLL cases WT,
mono, or null at 13q14 to determine if this expression
signature correlated with clinical outcome. We hypothe-
sized that patients with 13q14 null would have higher
expression of the tolerogenic signature compared to 13q
mono or WT cases.
Changes in the GEP were compared between 13 null,
13q mono, and patients WT at the 13q14 locus (Figure
1(b)). There was an increase in the immunosuppressive
Table 1. Characteristics of CLL patients with 13q14 nullisomy compared to 13q14 heterozygous deletion.
Chromosomal
Aberration
Avg Age at Diagnosis
Diagnosis (Range) N (male) % Patients with BLA% Patients with <1 yr LDT
% Patients ever
Treated
Avg Months to
Treatment (Range)
13q14 wildtype 63.15 years (45 - 85) 34 (16) 23.5%* 17.6% 56.3% 40.75 (1 - 245)
13q14 monosomy 62.03 years (46 - 79) 27 (13) 0% 7.4% 18.5% 57.1 (1 - 185)
13q14 nulilsomy 63.1 years (48 - 84) 18 (11) 16.6% 27.7% 50% 39.56 (1 - 96)
*Indicates patients with favorable chromosomal abnormality, but with a high percentage of CLL cells expressing CD38; BLA: Bulky lymphadenopathy.

C. E. Cutucache et al. / Advances in Bioscience and Biotechnology 4 (2013) 129-135 131
Figure 1. Clinical and molecular characteristics of CLL patients with 13q14 nullisomy: (a) The
change in the percentage of CLL cells positive for nullisomy at 13q14 pre- and post-treatment.
The percentage of CLL cells positive for deletion at 13q14 as analyzed by FISH before and af-
ter therapy. N = 5, P = 0.015; (b) Tolerogenic gene expression profiles between CLL patients
WT at the 13q14 locus, heterozygous (13q mono), or bearing a homozygous deletion (13q null).
The genes in our microarray within this region were compared between patients who are WT,
have a heterozygous deletion, or with a homozygous deletion at this region. Cluster analysis
was performed using Cluster 3.0 and Treeview software; (c) GEP of genes at the 13q14 locus
among CLL patients who are WT, have a heterozygous deletion, or with a homozygous dele-
tion at this region. The genes in our microarray within this region were compared between pa-
tients who are WT, have a heterozygous deletion, or with a homozygous deletion at this region.
Cluster analysis was performed using Cluster 3.0 and Treeview software; (d) Genes on 13q14
that were on our microarray chip (not an exhaustive list). Of the genes in the 13q14 region
commonly deleted in CLL, the genes listed above were on our microarray. We were able to de-
termine the gene expression profile for these select genes for CLL patients with both copies of
13q14 compared to those that were missing either one or both copies at this locus.
molecules APC and TG Fβ1 in 13q null CLL cases when
compared to 13q mono cases. APC, a member of the Wnt
signaling pathway, was shown to regulate cell migration
and adhesion and colorectal cancer is associated with
mutations in this gene [13,14]. TGFβ1 has been impli-
cated in immunosuppression, in addition to cellular pro-
liferation, differentiation, adhesion, and migration [15-
20].
3.3. Gene Expression in CLL Cases Nullisomy,
Monosomy, or WT at 13q14
After determining a prognostic significance for complete
loss of 13q14, the next objective was to identify whether
a tolerogenic/immunosuppressive signature was differen-
tially expressed in these cells. Supervised cluster analy-
ses were performed on the genes included in our mi-
croarray platform that were within the 13q14 locus [8;
MWG Biotech, Germany, Human 10K oligo set A; Fig-
ure 1(c)]. The expected result was to observe a dosage
effect of genes within the deleted region at 13q14. To
determine critical genes in the region of interest, we ex-
pected patients WT at the locus to have high expression
[red], those missing one copy [+/−] to have a lower ex-
pression [lighter red or green], and individuals null at
this region to have no expression of critical genes [black].
We anticipated the results of these analyses would de-
termine a region that was commonly deleted in CLL that
we could compare to other reports in the literature.
The expression of two genes was consistent with the
predicted expression pattern; these were HTR2A and DL-
EU1 (Figure 1(c)). The location of HTR2A and DLEU1
is depicted in Figure 1(d). 5-hydroxytryptamine [sero-
tonin] receptor 2A [HTR2A], G-protein-coupled receptor,
has never been reported as having a role in CLL. Fur-
thermore, as serotonin functions as a neurotransmitter, its
association with cancer pathophysiology is an inter-
esting observation. The gene deleted in lymphocytic

C. E. Cutucache et al. / Advances in Bioscience and Biotechnology 4 (2013) 129-135 132
leukemia 1 [DLEU1] is a long non-coding RNA [lnRNA]
previously reported to participate in CLL [21-25]. Al-
though there are only 15 published papers describing
DLEU1 to date, early reports and observations show
promise that the regulation of this lnRNA appears to
have a great influence on CLL progression.
Although the role of DLEU1 in CLL is still elusive,
we searched the sequence of this region to gain greater
insight. A polycomb response element [PRE] conserved
sequence was identified, known to serve as an antagonist
for epigenetic regulation of gene expression [26] within
this region (Figure 2(a)). This region is a regulatory
switchable element that influences the architecture of
chromatin and the expression of nearby genes [27,28]. In
this regard, DLEU1 is potentially a docking site for the
polyhomeotic [PHO] protein. This evolutionarily con-
served, regulatory system was identified in Drosophila.
The mammalian homolog of PHO in Drosophila is the
(a)
(b)
Figure 2. Polycomb response element binding site: (a) Putative PRE
binding sites on the long noncoding RNA, DLEU1 at 13q14. The bind-
ing sites for the GAGA factor (GAF; 29), G10, extended GAGA site
(G10; 30), PHO consensus (31), and Zeste binding site (Z; 30) are high-
lighted in the selected portion of the sequence as indicated by the legend.
Due to the length of DLEU1, an abbreviated portion of the sequence is
provided in this figure; (b) Hypothesized mechanism of regulation by
DLEU1. DLEU1, a long non-coding RNA (lnRNA) deleted in CLL with
unknown function. We have identified PRE binding sites on DLEU1
and describe a hypothesized mechanism of DLEU1 regulating transcrip-
tion to maintain homeostasis, but when DLEU1 is lost as in the case of
13q null, then transcription is dysregulated and multiple “hits” to the

C. E. Cutucache et al. / Advances in Bioscience and Biotechnology 4 (2013) 129-135 133
genome likely contribute to CLL progression.
transcription factor YY 1 [29]. Recently, a lnRNA was the
link between copy number variation and a polycomb/
trithorax epigenetic switch in muscular dystrophy [26],
and we suspect a similar switch may be occurring in
CLL following the loss of regulation of the region con-
taining DLEU1 (Figure 2(b)). This finding suggests a
potential negative feedback loop with the binding of YY1
to DLEU1 thereby regulating gene expression. Therefore,
this finding would explain the over decade-long conun-
drum of observing a deletion in this region, but being
unable to identify a clear point mutation or tumor sup-
pressor gene.
In summary, this report describes an unfavorable
prognosis for patients with biallelic deletion of 13q14,
compared to 13q mono patients. However, the percent-
age of CLL cells with 13q null decreased after treatment,
suggesting that chemotherapy was effective at killing
these malignant cells. GEP of patients with 13q null,
compared to 13q mono, identified the overexpression
APC and TGFβ1. Therefore, the upregulation of these
immunoregulatory molecules in 13q null patients might
lead to a greater immunosuppression in these cases.
The limitation of this study is the number of cases
examined. A larger cohort is needed to conclusively de-
termine the biological relevance of DLEU1 in CLL cases
with 13q14 null, but hopefully this study will serve as a
basis for additional in-depth analyses. Previously, a
similar study with a large sample size [n = 323] showed
that a higher percentage of 13q null cells were associated
with a significantly shorter time to treatment [6]. Simi-
larly, we suggest that CLL patients with 13q null have a
worse prognosis than patients that are mono or WT at
13q14. Contrastingly, these data contradict those de-
scribed earlier this year [30]. Garg et al. observed that
the baseline characteristics between CLL cases with
mono- or bi-allelic deletion of 13q differed only by
ZAP70-expression and albumin levels [31]. This study
differs from ours in that solely fluorescent in situ hy-
bridization [FISH] was used to assess the genetic abnor-
malities in CLL cells. Patients included in our study were
first screened using both FISH and gene expression pro-
filing. Therefore, this study presents a comprehensive
picture as to the genetic abnormalities present within this
frequently deleted region.
It will be difficult to prove a role for the consensus
sequence of DLEU1 in CLL without engineering a dele-
tion at that region both in CLL samples and an in vivo
animal model. While this manuscript was in preparation,
Lia et al. (2012) published a report regarding this precise
question [31]. Transgenic mice engineered to have loss
of 13q14 had a shorter life expectancy, similar to our
data from patients described in this report [31; Tabl e 1].
The data presented herein suggest that, first, CLL cases
show a heterozygous deletion at 13q14, and then a “sec-
ond hit” or loss of the second allele [13q null] is neces-
sary to take CLL from an indolent to an aggressive stage.
Prospective studies are necessary to conclusively deter-
mine this switch causing CLL cells to drive a more ag-
gressive disease.
4. SUMMARY
The findings presented herein are significant in that they
suggest a different way of classifying CLL cases with
13q null. However, it is important to note the limitations
with the sample size described in this study. Based on
this small sample size, we suggest that patients with the
deletion of both copies of 13q14 potentially need therapy
initiated earlier than their heterozygous counterparts and,
thus, might require more frequent monitoring of lym-
phocyte counts. The influence of DLEU1 in CLL was
reported previously, and the microRNAs in this region
have been studied extensively [2-5]. However, this is the
first report suggesting that regulatory elements, specifi-
cally PRE-sequences, of DLEU1 might contribute as a
“second hit” to lead to a more aggressive disease pro-
gression for patients with CLL.
5. ACKNOWLEDGEMENTS
Thanks to the CLL patients who so willingly donated cells for our
study. Also, thank you to the clinicians and nurses at the University of
Nebraska Medical Center for collecting these samples.
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