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

12 th Congress of the European Hematology Association
observed at a concentration as low as 10 nM, and almost complete inhibition
(99%) occurred after 24h at a concentration of 50nM (EC50=11
nM). In line with these results, we also observed reduced IL-2 production
in dasatinib treated purified T cells (EC50=1.3nM, measured by
ELISA), while 1nM staurosporine led to a 70% inhibition of IL-2 release,
and 10nM almost to a complete inhibition (99.9%). CD4 + T cells were
more sensitive than CD8 + T cells to the inhibitory effects of staurosporine
on activation and proliferation (89% proliferation inhibition
for CD4 + cells vs. 81% for CD8 + cells at 10 nM). The same held true for
dasatinib (activation: EC50 9 nM for CD4 + cells and 15 nM for CD8 + cells,
proliferation: EC50 5 nM for CD4 + cells and 14 nM for CD8 + cells). Since
CD8 + T cell-mediated immunity is essential for long-term control of persistent
DNA viruses, we evaluated dasatinib’s impact on antigen-specific
T-cell responses to CMV and EBV. Proliferation inhibition of CFSElabeled
CMV- and EBV-specific CD8 + T cells was observed, which might
explain the increased frequency of viral infections in dasatinib treated
patients besides the described induction of myelosuppression. Conclusions.
Close monitoring of patients under TK inhibitor treatment seems
warranted with respect to reactivation of persistent viral infections and
newly acquired opportunistic infections. However, these findings also
indicate dasatinib’s potential as an immunosuppressant in the fields of
transplantation and rheumatology.
0321
BOSUTINIB (SKI-606) EXHIBITS CLINICAL ACTIVITY IN PATIENTS WITH PHILADELPHIA
CHROMOSOME POSITIVE CML OR ALL WHO FAILED IMATINIB
T. Brummendorf, 1 J. Cortes, 2 H. Kantarjian, 2 G. Martinelli, 3 D. Liu, 4
T. Fischer, 5 B. Hewes, 6 A.D.G. Volkert, 6 R. Abbas, 6
C. Gambacorti-Passerini7 1 Universitaetsklinikum Hamburg, HAMBURG, Germany; 2 MD Anderson
Cancer Center, HOUSTON, USA; 3 University of Bologna, BOLOGNA, Italy;
4 New York Medical College, VALHALLA, USA; 5 Universitaetsklinikum Mainz,
MAINZ, Germany; 6 Wyeth Research, CAMBRIDGE, USA; 7 Opedale San
Gerardo-Monza, MONZA, Italy
Background. Bosutinib (SKI-606) is an orally available, dual Src/Abl
kinase inhibitor. Aims. To assess safety and preliminary clinical activity
of bosutinib, we conducted a phase 1/2 study in patients (pts) with
Philadelphia chromosome positive (Ph + ) chronic myelogenous leukemia
(CML) or acute lymphocytic leukemia (ALL) who were imatinib resistant/intolerant.
Methods. In part 1, 18 pts with imatinib-relapsed/refractory
chronic phase (CP) CML received bosutinib 400 mg/day (3 pts),
500 mg/day (3 pts), or 600 mg/day (12 pts). Part 2 was an expanded
cohort of 51 pts with all phases of Ph + CML and ALL dosed at 500 mg
daily. Timed blood samples were collected on days 1-3, 15 for PK analysis.
Results. Of 69 pts, median age was 59 yrs; 48 were CP; 90% imatinib
resistant. Drug-related grade 1/2 adverse events (AEs) occurring in 3 10%
of CP pts: diarrhea (69%), nausea (44%), vomiting (19%), abdominal
pain (13%), rash (13%). Grade 3/4 AEs occurring in 3 5% of CP pts: rash
(6%), thrombocytopenia (6%). 17 pts required dose reductions. In evaluable
imatinib-resistant CP-CML pts with no prior exposure to other Abl
inhibitors, 16/19 (84%) had complete hematologic response (CHR); 4/21
had partial and 7/21 had complete cytogenetic responses for major cytogenetic
response (MCyR) rate of 52%. Of 58 pts evaluable for mutations,
13 different imatinib-resistant mutations were found in 32 pts. 12/14 CP
pts with non-P-loop mutations and 3/3 with P-loop mutations achieved
CHR. 5/11 CP pts with non-P-loop mutations and 1/1 with P-loop mutation
achieved MCyR. 4/9 evaluable advanced leukemia pts had CHR, 2
had MCyR. After oral administration, steady state exposure of bosutinib
was nearly 2-fold higher than single-dose exposure. Mean elimination
half-life was approximately 22-27 hours, supporting a once-daily dosing
regimen. Conclusions. Bosutinib was well tolerated in pts with CML,
with primarily low-grade gastrointestinal and dermatologic AEs. Bosutinib
showed clinical activity in imatinib-resistant pts with cytogenetic
responses and CHR across a range of mutations. Durability of response
continues to be assessed.
116 | haematologica/the hematology journal | 2007; 92(s1)
0322
SULINDAC SULFIDE REVERTS THE ACTIVATION OF THE WNT-SIGNALING AND ABERRANT
SELF RENEWAL INDUCED BY THE AML-ASSOCIATED FUSION PROTEINS PML/RAR AND
PLZF/RAR
M. Ruthardt, 1 E. Puccetti, 1 G. Steinert, 1 G. Steinert, 2 M. Ruthardt, 2
E.Puccetti2 1 2 Klinikum der J. W. Goethe Universität, FRANKFURT, Germany; Laboratory
for Tumor Stem Cell Biology, Department of Hematology, J. W. Goethe University
Frankfurt, Germany
Background. More than 60% of acute myeloid leukemias (AML) harbor
specific chromosomal aberrations mainly translocations. Translocations
such as t(15;17), t(11;17), or t(8;21) lead to the formation of
chimeric genes encoding for fusion proteins such as PML/RAR,
PLZF/RAR and AML-1/ETO (leukemia associated fusion proteins -
LAFP). LAFP are able to induce and to maintain the leukemic phenotype
by blocking terminal differentiation of early hematopoietic progenitors
and by increasing the self renewal potential of the leukemic stem cells
(LSC). The key mechanims by which LAFP increase LSC self renewal is
the activation of the Wnt-signaling pathway. PML/RAR, PLZF/RAR or
AML-1/ETO activate Wnt-signaling by up-regulating γ-catenin and βcatenin
at a transcriptional level. Activation of the Wnt signaling augments
self renewal of normal HSC and LSC. We and others have recently
shown that the aberrant activation of Wnt-signaling by the LAFP decisively
contributes to the pathogenesis of AML. Aim. To disclose whether
a leukemic stem cell therapy is effective we targeted the Wnt-signaling by
Sulindac Sulfid (SuSu) in PML/RAR- or PLZF/RAR- (X-RAR) positive
stem cell models. SuSu represents the active metabolite of Sulindac, a
nonsteroidal anti-inflammatory drug (NSAID), known to inactivate the
Wnt-signaling. Methods. SuSu was used at a concentration of 50-100 µM
which is achievable in patients at a dosage of 0.2-0.4g. As leukemia
models we used U937 cells stably expressing PML/RAR or PLZF/RAR
and the PML/RAR-positive cell line NB4. As stem cell models we used
i.) the CD34 + /CD38 – fraction of the KG-1 cells stably expressing
PML/RAR or PLZF/RAR under serum free culture conditions; ii.) Sca-
1+/lin- murine HSC retrovirally transduced with PML/RAR or
PLZF/RAR and plated in methyl cellulose containing mIL-3, mSCF and
mIL-6. The amount of total γ-catenin and β-catenin and activated βcatenin
was determined by immunoblotting. Results. Here we report
that SuSu i.) down-regulated not only β-catenin but also γ-catenin in X-
RAR expressing U937 and KG-1 cells; ii.) reduced the active form of βcatenin
in the presence of X-RAR; iii.) induced a high apoptosis rate in
PML/RAR-positive NB4 cells; iv.) reduced the CD34 + /CD38 – stem cell fraction
of KG-1 cells expressing X-RAR but not of mock transfected controls;
iv.) reduced the self renewal potential of X-RAR-positive HSC as
revealed by a significantly reduced replating efficiency. Conclusions. Here
we provide first evidence that it is possible to the exposure to therapeutically
achievable dosages of a NSAID revert the aberrant activation of
the Wnt-signaling by LAFP. The significant reduction of the aberrant
self renewal potential of HSC in the presence of X-RAR further support
that the inhibition of the aberrantly activated Wnt signaling in AML
might be a valid molecular therapy approach which has to further validated
in in vivo leukemia models and in a clinical setting.AML-1/ETO
activate Wnt-signaling by up-regulating γ-catenin and β-catenin at a transcriptional
level. Activation of the Wnt signaling augments self renewal
of normal HSC and LSC. We and others have recently shown that the
aberrant activation of Wnt-signaling by the LAFP decisively contributes
to the pathogenesis of AML. Aim. To disclose whether a leukemic stem cell
therapy is effective we targeted the Wnt-signaling by Sulindac Sulfid
(SuSu) in PML/RAR- or PLZF/RAR- (X-RAR) positive stem cell models.
SuSu represents the active metabolite of Sulindac, a nonsteroidal antiinflammatory
drug (NSAID), known to inactivate the Wnt-signaling.
Methods. SuSu was used at a concentration of 50-100 µM which is
achievable in patients at a dosage of 0.2-0.4g. As leukemia models we
used U937 cells stably expressing PML/RAR or PLZF/RAR and the
PML/RAR-positive cell line NB4. As stem cell models we used i.) the
CD34 + /CD38 – fraction of the KG-1 cells stably expressing PML/RAR or
PLZF/RAR under serum free culture conditions; ii.) Sca-1+/lin- murine
HSC retrovirally transduced with PML/RAR or PLZF/RAR and plated in
methyl cellulose containing mIL-3, mSCF and mIL-6. The amount of
total γ-catenin and β-catenin and activated β-catenin was determined by
immunoblotting. Results. Here we report that SuSu i.) down-regulated
not only β-catenin but also γ-catenin in X-RAR expressing U937 and
KG-1 cells; ii.) reduced the active form of β-catenin in the presence of
X-RAR; iii.) induced a high apoptosis rate in PML/RAR-positive NB4
cells; iv.) reduced the CD34 + /CD38 – stem cell fraction of KG-1 cells expressing
X-RAR but not of mock transfected controls; iv.) reduced the self