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

12 th Congress of the European Hematology Association
0525
CD74 IS A NOVEL SURVIVAL RECEPTOR IN CHRONIC LYMPHOCYTIC LEUKEMIA
M.Z.H. Haran, 1 I. Binsky, 2 D. Starlets, 2 N. Harpaz, 3 L. Shvidel, 3
A. Berrebi, 3 I. Shachar 2
1 Kaplan Medical Center, REHOVOT; 2 Immunology, Weizmann Institue of science,
REHOVOT; 3 Hematology Inst., Kaplan Medical Center, REHOVOT,
Israel
Background. Previous studies have shown that chronic lymphocytic
leukemia (CLL) lymphocytes express relatively large amounts of CD74
mRNA compared to normal B cells. We have recently demonstrated in
a murine model that CD74 stimulation with anti-CD74 antibody leads
to an induction of a signaling cascade resulting in NF-κB activation, entry
of the stimulated cells into the S phase, elevation of DNA synthesis, cell
division, and augmented expression of BCL-XL. These findings therefore
demonstrated that surface CD74 functions as a survival receptor. Aims.
In the current study we aimed to determine whether activation of cell
surface CD74 in B-CLL cells leads to induction of a signaling cascade
resulting in cell survival.
Figure 1. A) CD74 stimulation induces IL-8 and Bcl-2 expression in CLL B
cells. Cells were incubated in the presence or absence of anti-CD74 antibody,
Id2, a control antibody or MIF for 18 h. RNA was purified and levels of IL-8,
Bcl-2 and actin mRNA were analyzed. The results presented are representative
of 12 CLL patients. B) The conditioning medium of the cells was collected,
and the level of IL-8 was assessed by ELISA. The results are representative
of 3 independent experiments. C) IL-8 secreted following CD74
stimulation regulates B-CLL cell survival. CLL cells were incubated in the
presence or absence of an agonistic anti-CD74 antibody, anti-IL-8 or a control
antibody (c-jun) for 48 h. Cells were stained with annexin V and analyzed
by FACS. The results presented are representative of 7 CLL patients.
Materials and Methods. B cells were purified from the peripheral blood
of CLL patients in different stages, in accordance with the IRB of our hospital.
CD74 stimulation was achieved using an agonistic anti-CD74antibody
or MIF (a natural occurring ligand of CD74). IL-8 expression and
function was determined by RT-PCR, western blot, ELISA and Annexin
V staining. Results. In all the cells obtained from CLL patients there was
a significantly increased expression of cell surface CD74, as compared
to normal B cells. Activation of cell surface CD74 initiated a signaling
cascade that resulted in increased expression and secretion of Interleukin
8, as well as over-expression of bcl-2 (see panels A, B in Figure 1). Stimulation
of CD74 led to increased cell survival, as seen by annexin staining,
whereas blocking of IL-8 led to increased apoptosis (panel C in Figure
1). Conclusions. Our data show that over-expression of CD74 in CLL
is an important survival mechanism, operational from the very early
stages of the disease, and inherent in all further stages. This survival
mechanism thus appears to be an early and significant event in the
pathogenesis of the disease. Our findings open prospects for novel therapeutic
strategies aimed at interruption of this survival pathway.
196 | haematologica/the hematology journal | 2007; 92(s1)
Chronic myeloid leukemia - Biology
0526
OVER EXPRESSION OF THE ABC-TRANSPORTER BCRP, MRP1 AND MDR1 IN CML
PATIENTS AT DIAGNOSIS, WHO RESPOND AND WITH ACQUIRED RESISTANCE TO
IMATINIB MESYLATE
I. Bendit, 1 L. Nardinelli, 2 M. Bendit, 2 M.C. Mello, 2 M.N.Y. Novaes, 2
W.M. Lima, 2 R.R. Giorgi, 2 J.E.B. Neto, 2 D.A.F. Chamone, 2
P. Llacer-Dorliac2 1 2 University of Sao Paulo, SAO PAULO; Medical School at USP, SAO
PAULO, Brazil
IM is widely used for the treatment of CML inhibiting the BCR-ABL
tyrosine kinase activity. Although, 90% of the patients respond to IM,
resistance to this drug has been seen mainly due mutations in ABL tyrosine
kinase domain. Recently, it was reported that IM is a substrate for
the ABC-transporters BCRP (breast cancer resistance protein), MRP1
(multidrug resistant protein 1) and MDR1 (P-Glycoprotein). We studied
29 patients (9 CP, 17 AP, and 3 BC) who initiated treatment with IM after
49 mo (±10 mo) from diagnosis and develop resistance 31 mo (± 4mo)
after beginning of therapy. To compare the expression of the resistant
group, 34 CML patients at diagnosis (23 CP, 5 AP, and 2 BC) and 29
good responders to IM (22 CP, 5 AP, and 2 BC) with a mean time from
diagnosis to initiation of IM of 29 mo (±6,6 mo). To detect the expression
of those ABC-transporters RNA was extracted and cDNA was synthesized
and amplified through real time PCR applying SYBR Green as
a dye and reported as the 2- Ct where it is assumed that the amplification
efficiency of the target gene (BCRP, MRP1 or MDR1) and the internal
control gene (ABL) are the same. Over expression of BCRP was
observed in 65.5% of the resistant group (4 CP, 12 AP, and 3 BC) when
compared to 11,8% of patients at diagnosis (2 CP, 1 AP, and 1 BC), and
to 10,3% of patients that respond to IM (3 CP) (Kruskal-Wallis test,
p