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

12 th Congress of the European Hematology Association
0745
IN VITRO ACTIVITY OF TYROSINE KINASE, FARNESYL TRANSFERASE AND AKT KINASE
INHIBITORS ON C-KIT POSITIVE/NEGATIVE AND MDR-PGP POSITIVE/NEGATIVE
LEUKAEMIA TUMOR CELL LINES
M. Malagola, 1 D. Damiani, 2 A. Michelutti, 2 G. Martinelli, 3
E. Ottaviani, 3 S. Paolini, 3 P.P. Piccaluga, 3 C. Skert, 4 A. Roccaro, 4 A. Peli, 4
E. Capuzzi, 4 C. Filì, 4 C. Bergonzi, 4 R. Fanin, 2 M. Baccarani, 3 D. Russo 4
1 Chair of Haematology, Brescia, BRESCIA; 2 Clinic of Haematology, UNIVER-
SITY OF UDINE; 3 Institute of Haematology Seràgnoli, UNIVERSITY OF
BOLOGNA; 4 Chair of Haematology, UNIVERSITY OF BRESCIA, Italy
Imatinib is a target inhibitor of the tyrosin kinase (TK) activity associated
to P210/190-BCR-ABL chimeric protein and to platelet-derived
growth factor receptor (PDGF-R) α and β or c-kit receptor. Actually, few
haematologic responses were observed in the c-kit positive acute
myeloid leukaemias (AMLs) patents treated with Imatinib. The sensitivity
of Imatinib to several proteins involved in the mechanisms of multidrug
resistance (MDR), such as the over-expression of P-glycoprotein
(Pgp), has been suggested as one the causes of failure of Imatinib in ckit
positive AML. In this study, we evaluated the effects of MDR-Pgp
expression on the in vitro activity of Imatinib and other TK inhibitors
such as Nilotinib (AMN107, Novartis Pharma) and Dasatinib (BMS-
354825, Bristol-Myers Squibb) on c-kit positive (HL60/HLA60-DNR )
and c-kit negative (CCRF-CEM/CEM-VLB) human leukaemic cell lines.
Secondly, we tested the in vitro activity of farnesyl-transferase inhibitors
(FTIs) Tipifarnib (R115777, Zarnestra, Johnson & Johnson) and Lonafarnib
(SCH66336, Sarasar, Schering-Plough) and of AKT inhibitor (A-
443654, Abbott) either alone or in combination with Imatinib on c-kit
positive/MDR-Pgp positive cell line (HLA60-DNR) to investigate alternative
strategies against c-kit positive AML blasts. By using the MTTmicrocultured
tetrazolium colorimetric assay, we measured a consistent
growth inhibitory effect of TK inhibitors in MK1/BCR-ABL positive cell
line and only in the HL60 DNR/c-kit positive cell line, even though this
latter was over-expressing MDR-Pgp. Both in MK1/BCR-ABL positive
and HL60 DNR/c-kit positive cell lines, Nilotinib and Dasatinib resulted
10 and 100 folds more potent than Imatinib, respectively. In all of the
leukaemic cell lines, Tipifarnib (ID50 range = 0.0065 to 0.21 µM) was
more toxic than Lonafarnib (ID50 range = 0.3 to 6.5 µM), while AKT
inhibitor (A-443654) showed a very low growth inhibitory effect (ID50
range = 0.1 and 0.35 microM). Neither FTIs nor AKT cytotoxic activity
was influenced by the MDR-Pgp over-expression. When we tested the
combinations of Imatinib with Tipifarnib or other inhibitors on the
HL60 DNR/c-kit positive/MDR-Pgp positive or on the other leukaemic
cell lines we did not measure any synergistic effects, but only additive
effects. The results of our in vitro study indicate that the MDR-Pgp overexpression
doesn’t seem to reduce the sensitivity of c-kit positive cells
to Imatinib or to other TK, FTIs or AKT inhibitors. To improve the
inhibitory effects on c-kit positive cells may be more useful to employ
Nilotib or Dasatinib. As we did not observe any synergistic effects, the
use in vivo of combinations with different inhibitors could be more toxic
than useful, because the costs in terms of haematological and extrahaematological
toxicities could be much higher than the benefits in
terms of antitumor activity.
This work was supported in part by FIRB (protocol number:
RBAU01RLNB005 - 2004; D. Russo), progetto 60% 2005 (D.Russo) and
COFIN 60% 2006.
0746
RADIOIMMUNOTHERAPY AS A PROMISING THERAPEUTIC OPTION IN PATIENTS WITH
RELAPSED OR REFRACTORY CD20 + NON-HODGKINS LYMPHOMA
A. Rohn, C. Leitner, R.I. Brezinschek, S. Sormann, W. Linkesch,
D. Strunk
Medical University of Graz, GRAZ, Austria
90Y ibritumomab tiuxetan is a murine anti-CD20 monoclonal antibody
developed for radioimmunotherapeutic targeting of CD20+ lymphoma
cells. As it emits therapeutic β radiation a wide ranging crossfire
effect can be achieved. Our aim was to evaluate the effectiveness of
90Y ibritumomab tiuxetan (Zevalin ® ) as a treatment option for patients
with CD20 + NHL, especially for those with follicular lymphoma (FL)
and diffuse large B-cell lymphoma (DLCL). Forty patients with CD20 +
NHL were treated with Zevalin ® between March 2004 and June 2006 in
this cohort. Twenty-two patients had relapsed or refractory FL, 11
patients had relapsed or refractory DLCL, 2 patients suffered from
Immunocytoma, one patient had mantle cell lymphoma and one suf-
278 | haematologica/the hematology journal | 2007; 92(s1)
fered from Burkitts lymphoma. One of the patients had a post-transplantation
B-lymphoproliferative disorder after 9 years of immunosuppressive
therapy after heart transplantation. All of them were extensively
pretreated with a median of 3 prior regimen of polychemotherapy (range
1 to 6). Two patients with FL and one patient with DLCL received
Zevalin ® as first-line therapy. The median age was 61 in FL (range 41-
79) and 69 in DLCL (range 36-86). The Zevalin ® treatment regimen consisted
of pre-treatment with Rituximab (250 mg/m 2 intravenously on
days 1 and 8) to deplete peripheral blood B-cells followed by Zevalin ®
infusion on day 8. A mean of 1084 MBq (range 680-2040) 90Y ibritumomab
tiuxetan (Yatrics-Zevalin, Schering AG) was administered.
Remission state was evaluated in DLCL three months after therapy.
Additionally we calculated overall survival and disease-free survival for
patients with FL. For FL overall survival was 87,5% (21/24) after a median
follow-up of 16 months (range 2-30) and disease-free survival was
79% (19/24) after a median follow-up of 15 months (range 3-30). Out
of eleven patients with DLCL, six developed progressive disease, three
achieved complete remission, one partial remission and one patient
remained in stable disease. One patient in CR died due to myocardial
infarction. Thus 73% (8/11) DLCL patients did not respond to this therapeutic
strategy. All of them were extensively pretreated. The main side
effects were thrombocytopenia followed by mild leukocytopenia and
anemia. Treatment associated myelodysplastic syndrome was diagnosed
in one patient twenty-nine months after therapy. Our results confirm
that Zevalin ® is a good treatment option in patients with relapsed or
refractory FL whereas extensively pretreated DLCL patients show hardly
any response to this therapeutic strategy. Possibly these patients
would benefit in an earlier use of Zevalin ® in the course of the disease.
0747
TARGETING THE RAF/MEK/ERK SIGNALING PATHWAY BY THE NOVEL MEK INHIBITOR
PD0325901: MOLECULAR AND FUNCTIONAL EFFECTS IN PRE-CLINICAL LEUKEMIA
MODELS
M.R. Ricciardi, 1 M. Milella, 2 M.C. Scerpa, 1 C. Gregorj, 1 F. De Cave, 1 S.
Abrams, 3 L. Steelman, 3 S. Chiaretti, 1 M. Konopleva, 4 M.T. Petrucci, 1
F. Cognetti, 5 R. Foà, 1 M. Andreeff, 4 J. McCubrey, 3 A. Tafuri1 1 University La Sapienza of Rome, ROME, Italy; 2 Regina Elena National Cancer
Institute, ROME, Italy; 3 Microbiology & Immunology, East Carolina,
GREENVILLE, USA; 4 University of Texas MDACC, HOUSTON, TX, USA;
5 Medical Oncology A, Regina Elena Nation, ROME, Italy
Background. The Raf/MEK/ERK pathway plays a pivotal role in the
regulation of cell proliferation, survival and differentiation. As reported
by our and other groups, this pathway is frequently deregulated in
hematological malignancies, particularly in AML, representing an attractive
target for therapeutic interventions. Aims. Here we investigated the
effects of PD0325901, a novel MEK inhibitor, on phospho-protein
expression, gene expression profile, cell proliferation, and apoptosis in
AML, ALL and multiple myeloma (MM) cell lines, in oncogene-transformed
hematopoietic murine myeloid cell lines, as well as in ex vivo-cultured
primary AML blasts. Results. AML cell lines (OCI-AML2, OCI-
AML3, HL-60) showed a high sensitivity to PD0325901 (IC50: 5-19 nM),
NB4 (APL) and U266 (MM) showed an intermediate sensitivity (IC50:
822 and 724 nM), while all the lymphoid cell lines tested and the
myeloid cell lines U937 and KG1 were resistant (IC50 >1000 nM). Cell
cycle analysis and Annexin V assay demonstrated that cell growth inhibition
was caused by cell cycle arrest and apoptosis induction. The
effects of PD0325901 were also examined on primary cells from 18 AML
patients. A statistically significant reduction in the percentage of S-phase
cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019)
was also observed in primary AML samples, in vitro exposed to 100 nM
of the MEK inhibitor. Next, PD0325901 effects were examined in a panel
of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to
grow in response to 11 different oncogenes in the absence of IL-3. Fms,
Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R- and STAT5a-transformed FDC-P1
cells were very sensitive to PD0325901 (IC50: ~ nM), while A-Raf-, BCR-
ABL-, EGFR- or Src-transformed cells were 10- to 100-fold less sensitive
(IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had
an IC50 >1000 nM. Semi-quantitative analysis of the phosphorylation
status of 18 different target proteins on OCI-AML3 cells after treatment
with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2 and
a 2-fold reduction in JNK and STAT3 phosphorylation. Conversely,
increased phosphorylation in response to PD0325901 was observed for
Raf-1 (2.5-fold), MEK1/2 (2.4-fold), AKT (2-fold) and p70S6K (2-fold).
The gene expression profile of the sensitive cell line OCI-AML3 was also
profoundly altered from PD0325901 (10 nM) treatment: 96 genes were