12th Congress of the European Hematology ... - Haematologica

12 th Congress of the European Hematology Association
period of 7 days to investigate CCN3 growth regulation. K562 cells were
transfected with vector alone, vector containing CCN3 construct or with
treated with Imatinib (1 micromolar) for 24h prior to plating in methyl
cellulose. Primary human CML CD34 + cells were isolated from peripheral
blood and treated with CCN3 (1 nanomolar) or imatinib (1 micromolar)
for 24h before plating into culture. Results. K562 cells that had
been transfected with vector alone or vector containing full-length
CCN3 were compared to cells treated with Imatinib. Increased CCN3
expression significantly reduced colony formation by 65.4%±18.8 when
compared to cells transfected with vector alone (p=0.027, n=3). Treatment
with Imatinib also reduced colony formation (75%±8.2; p=0.001,
n=3) compared to untreated cells. Full-length CCN3 comprises 5
domains. To further characterise the functional component within
CCN3, partial length constructs encoding domains 2-5 (NH25), 3-5
(NH35) and 4-5 (NH45) were transfected into K562 cells using nucleofector
technology. Expression of NH25 and NH35 significantly
reduced colony formation capacity by 20% (p=0.001) and 25% (p=0.036)
respectively, whilst expression of the NH45 construct did not alter
colony formation. We next assessed the clonogenic effects of CCN3 and
Imatinib on primary human CD34+ progenitor cells derived from CML
peripheral blood samples at diagnosis (n=3). Cells pre-treated with
exogenous addition of full-length CCN3 protein for 24h prior to plating
in methyl cellulose cultures reduced clonogenic capacity by 25.5±3.9%
(p=0.011). Pre- treatment with Imatinib reduced colony formation by
37.9%±19.9 (p=0.010). Conclusions. This study demonstrates that CCN3
expression regulates the colony formation capacity of BCR-ABL + cells.
Domains 2 and 3 comprising an insulin-like growth factor binding and
Von Willebrand type C domain appear to be significant contributory
components in mediating negative growth regulation. Loss of CCN3
expression mediated by BCR-ABL disrupts cell growth regulation confering
growth advantage to CML cells.
0531
TRAIL MEDIATED ANTI-LEUKEMIC EFFECTS OF INTERFERON-ALPHA COMBINED WITH
G-CSF ON CHRONIC MYELOGENOUS LEUKEMIA DURING IMATINIB THERAPY
S. Ota, 1 M. Musashi, 1 N. Toyoshima, 1 J. Tanaka, 1 M. Kasai, 2
M. Imamura, 1 M. Asaka1 1 2 Hokkaido University, SAPPORO; Sapporo Hokuyu Hospital, SAPPORO,
Japan
Aims. In the treatment of patients with chronic myelogenous leukemia
(CML), a major goal of therapy is the eradication of BCR-ABL-positive
leukemic clone. Simultaneous exposure of BCR-ABL-positive leukemia
cells to imatinib and interferon-α (IFN) produced synergistic anti-proliferative
effects. As G-CSF augments IFN-induced release of soluble TNFrelated
apoptosis- inducing ligand (sTRAIL) from leukocytes, if G-CSF
has a proliferative effect on normal clone but not malignant clone, a
combination therapy of imatinib, IFN, and G-CSF is expected to eradicate
malignant clone. In this in vitro study using bone marrow and peripheral
blood mononuclear cells (BMC, PMC) from patients with CML and
healthy volunteer donors (HVD), we evaluated whether imatinib in
combination with IFN and G-CSF enhanced the anti-leukemic effects via
TRAIL/TRAIL receptor (DR4 & DR5) system. Methods. Colony assay
was performed by a methylcellulose method. Patient’s BMC and
leukemic cell lines were incubated with imatinib, IFN, G-CSF or recombinant
human sTRAIL (rhsTRAIL). Ratio of BCR-ABL- positive cells to
-negative cells in pooled colonies was evaluated by RT-PCR with BCR-
ABL mRNA. To evaluate indirect cytotoxic effects of IFN-stimulated
BMC, PMC, and neutrophils from HVD, their co-culture with K562 was
performed using transwell chamber. Apoptosis was detected by Annexin
V staining. Concentration of sTRAIL was measured by ELISA. Expression
of DR4 and DR5 on leukemic cell lines was examined by FACScan.
Results. Sequential colony assay of the patients showed that the colony
formation decreased paralleled with reduction of Ph clone after imatinib
administration, and recovered after achieving a cytogenetic remission.
In untreated cases, colonies were effectively suppressed by an addition
of imatinib to cultures, while that were more strongly suppressed
by IFN than imatinib in patients undergoing imatinib. Although there
were no significant differences in BCR-ABL mRNA ratio of pooled
colonies between mono-agent treatment and the combinations, IFN
with or without G-CSF suppressed BCR-ABL mRNA expression level
compared to the control. IFN increased sTRAIL concentration in culture
of BMC from patients with CML, also induced sTRAIL from BMC,
PMC, or neutrophils obtained from HVD, in a cell number-dependent
manner. To clarify the effects of sTRAIL minutely, we examined
sTRAIL-induced apoptosis in leukemic cell lines. DR4 and DR5 were
highly expressed on K562, KU812, and HL60. rhsTRAIL induced dose-
198 | haematologica/the hematology journal | 2007; 92(s1)
and time-dependent apoptosis in the cell lines, and the effect was completely
abolished by anti-TRAIL antibody. Apoptosis of K562 was not
induced by a co-culture with IFN, but was induced by IFN-stimulated
BMC, PMC, or neutrophils from HVD in the membrane-separated transwell
chamber. There were no significant differences in incidence of
apoptosis between IFN alone and IFN plus G-CSF treatment. Conclusions.
We suggest that IFN, adding to its direct effects, induces sTRAIL
from BMC, PBC, and neutrophils, which may contribute to reduce
leukemic progenitor cells in patients with CML. G-CSF dose not abrogate
the cytotoxic effects of IFN, but increases sTRAIL release from
blood cells. From these observations, we suggest that G-CSF can
enhance anti-leukemic effect of IFN on BCR-ABL-expressing cells via
sTRAIL during imatinib therapy.
0532
THE LEVEL OF CIRCULATING T REGULATORY CELLS CORRELATES WITH THE DISEASE
BURDEN IN CHRONIC MYELOID LEUKAEMIA
J.-M. Rojas, S. Owen, L.H. Wang, K. Knight, R.E. Clark
University of Liverpool, LIVERPOOL, United Kingdom
Chronic myeloid Leukaemia (CML) is characterised by the BCR-ABL
gene recombination. We and others have demonstrated that T cells capable
of recognising BCR-ABL junctional peptides are present in the circulation
of CML patients. We also demonstrated an inverse correlation
between the level of these BCR-ABL-specific T cells and the disease burden,
as assessed by the BCR-ABL/ABL transcript ratio. However, in spite
of the presence of these BCR-ABL-specific T cells, the CML cells persist
in the patients. The CD4 + CD25 + naturally-occurring T regulatory cell
(Treg) population has immunosuppressive activity involved in limiting
immune responses. Studies have also shown that Treg cells can inhibit
antitumour immune responses. Thus, we hypothesised that Treg cells
may be involved in limiting anti-CML immune responses. In the present
study, we carried out an analysis of the amount of Treg cells detected
in the peripheral blood of 22 CML patients at multiple time points.
All patients were in first chronic phase and currently receiving or having
recently received imatinib 400-600 mg daily. The Treg cell numbers
were evaluated using flow cytometry for the Treg cell markers CD25
and FOXP3. We correlated these data with BCR-ABL/ABL transcript
ratios measured by real-time RT-PCR for each time point. We observed
that the Treg population was consistently elevated in patients with high
BCR-ABL/ABL transcript ratio (ratio>10%) as compared to patients with
low BCR-ABL/ABL transcript ratio (ratio