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

No major toxicity, neither hematological nor non-hematological, was
observed in the animals. Conclusions. The tyrosine kinase inhibitor BVT
I has significant in vitro and in vivo effect in AML. Lack of toxicity in the
hollow fiber study, suggests that BVT I is well tolerated in mice. Further
preclinical studies in AML are ongoing.
0318
FACTORS DETERMINING CLINICAL ACTIVITY OF THE HISTONE DEACETYLASE INHIBITOR
SODIUM VALPROATE IN PATIENTS WITH HIGH RISK ACUTE MYELOID LEUKAEMIA
C. Craddock, 1 F. Khanim, 2 J. Arrazi, 2 M. Griffiths, 3 B. Turner, 2
C. Bunce2 1 Queen Elizabeth Hospital, BIRMINGHAM; 2 University of Birmingham,
BIRMINGHAM; 3 Birmingham Women's Hospital, BIRMINGHAM, United
Kingdom
Background. Histone deacetylases inhibitors (HDIs) represent an important
new class of drugs in the treatment of acute myeloid leukaemia
(AML). However the mechanism by which they induce tumour specific
apoptosis in vivo and the factors regulating tumour cell sensitivity are
not known. Aims; In order to define factors determining clinical sensitivity
to the HDI valproic acid (VPA) we have correlated the impact of
VPA exposure on gene expression in vitro and in vivo with clinical responses
in patients with high risk AML. Methods; A panel of 14 haemato-lymphoid
cell lines were tested for their sensitivity (IC50) to apoptotic cell
killing by VPA. Gene expression array analyses using HGMP chip 6500
gene arrays were performed on the same cell lines prior to HDI exposure.
A bioinformatics approach combined the array and IC50 data to
generate a score for each gene identifying those whose elevated expression
correlated with resistance, or sensitivity to VPA in vitro. In a concurrent
Phase I/II clinical trial 24 patients with high risk AML (relapsed
n=11, newly diagnosed n=6, primary refractory n=3) with a median age
of 64 yrs (41 -83 yrs) received combination treatment with VPA, all transretinoic
acid (ATRA) and theophylline. in vivo histone acetylation and
expression of HDI responsive genes was measured in leukaemic blasts
before and after commencement of VPA therapy. Expression of genes
associated with VPA sensitivity in vitro were re-analysed with respect to
their expression in pre-treatment blasts from non-responding and
responding trial patients. Results. By combining LC50 values to VPA and
microarray data generated from pre-treatment RNA in 14 haematolymphoid
cell lines we were able to identify candidate genes mediating sensitivity
to VPA killing in vitro. These studies identified potential VPA-signalling
networks containing sensitivity genes including IL-1β and genes
associated with VPA resistance including PLOD2, cyclin B1 (CCNB1)
and ACVR2A. In the clinical trial 5 patients, all with relapsed AML,
achieved clinical responses (complete remission n=1, partial remission
n=4). Treatment with VPA and ATRA for 28 days resulted in increased
histone acetylation in peripheral blood mononuclear cells and increased
expression of the pro-apoptotic genes p15, p16, p21 and TRAIL in patient
myeloblasts. Combined upregulation of p21 and TRAIL was only
observed in myeloblasts from the patient whom achieved a CR. We
then specifically compared the pattern of gene expression, as defined by
microarray studies, in responding and non-responding patients with the
previously identified in vitro VPA signalling networks. The pattern of
expression of VPA sensitivity and resistance genes in pre-treatment
myeloblasts from 4 patients identified similar networks to those defined
in vitro and appeared to be correlated with clinical response. Conclusions.
This study demonstrates induction of pro-apoptotic gene expression by
VPA in a clinical context and identifies potential mechanisms through
which its anti-leukaemic effect may be mediated in vivo. Furthermore we
have identified potential VPA-signalling networks containing novel sensitivity
and resistance genes whose expression correlates with VPAmediated
tumour cell killing in vitro and may predict clinical activity.
0319
GOLD COMPOUND AURANOFIN INDUCES APOPTOSIS IN MYELOMA CELLS VIA
TARGETING IL-6 SIGNALING PATHWAY
A. Nakaya, M. Sagawa, A. Muto, H. Uchida, Y. Ikeda, M. Kizaki
Keio University School of Medicine, TOKYO, Japan
[Background] Multiple myeloma is an incurable B-cell malignancy,
requiring new therapeutic strategies. Recent understanding of the biology
in multiple myeloma has led to the development of various novel
agents, which target the myeloma cells and its microenvironment. These
new agents have shown remarkable activity against refractory myeloma
in early clinical trials, but prolonged drug exposure may result in the
development of de novo drug resistance. In addition, unexpected pul-
12 th Congress of the European Hematology Association
monary complications by bortezomib, a promising agent for multiple
myeloma, have been reported. Therefore, identification and validation
of novel agents with less toxicity to overcome drug resistance and to
improve clinical outcome of multiple myeloma are necessary. Aims. Auranofin
(AF) is a coordinated gold compound, which has been widely used
for the treatment of rheumatoid arthritis. Recent studies have shown that
AF induces apoptosis in leukemia cells. Therefore, assuming that AF has
a potency to induce apoptosis in myeloma cells as well, it may become
a candidate of a novel targeted therapeutic agent. Methods. To address
our hypothesis, the effects of AF on inducing apoptosis of various myeloma
cells were examined. Further, the molecular mechanism of AFinduced
apoptosis in myeloma cells was investigated. Results. AF inhibited
the growth of U266 myeloma cells in a time (0-24 h)-dependent
manner with IC50 of 50 nM. AF significantly induced cell cycle arrest at
G1 phase and subsequent apoptosis of U266 cells. AF upregulated the
expression of cdk inhibitor p21 and caused dephosphorylation of Rb
protein. Furthermore, AF-induced apoptosis in myeloma cells involved
the activation of caspase-8 and the cleavage of Bid, a mediator that is
known to connect the death receptor to the mitochondrial apoptosis
pathway. We also found that AF induced the cleavage of Mcl-1 protein,
but had no effect on the expression of Bax or Bcl-2 proteins. To clarify
the importance of Mcl-1 in AF-induced apoptosis, the Mcl-1 expression
vector was introduced into U266 cells. Induction of apoptosis by AF was
almost abrogated in Mcl-1-overexpressed U266 cells. AF also inhibited
IL-6-induced activation of JAK2 and phosphorylation of STAT3 in U266
cells, suggesting that AF could inhibit the IL-6-mediated signaling pathway
in myeloma cells. Previous reports have demonstrated that STAT3
binds to the Mcl-1 gene promoter and activates its transcription, resulting
in the upregulation of Mcl-1 expression. Therefore, we then examined
the effect of AF on the DNA binding activity of STAT3. Electrophoretic
mobility shift assays using U266 nuclear extracts demonstrated
that IL-6-induced STAT3 binding activity was inhibited by the
presence of AF. Conclusion. We report here for the first time that AF
induced apoptosis in human multiple myeloma cells. Down-regulation
of Mcl-1 with modulation of IL-6-mediated signaling pathway plays an
important role in AF-induced apoptosis in myeloma cells. Low pharmacological
concentration (50 nM) of AF is widely employed for the management
of rheumatoid arthritis without any side effects, and may be
used for treating myeloma without the risk of severe toxicity. We conclude
that AF is one of the promising candidates for the new therapeutic
agent as a signal transduction therapy of myeloma.
0320
DASATINIB INHIBITS T CELL ACTIVATION AND PROLIFERATION IN A DOSE-DEPENDENT
MANNER
R. Weichsel, 1 C. Dix, 1 J. Zezula, 2 E. Greiner, 2 D.A. Price, 3 H. Einsele, 1
R. Seggewiss1 1 University of Wuerzburg, WUERZBURG, Germany; 2 Laboratory of Medicinal
Chemistry, NIDDK, BETHESDA, USA; 3 Human Immunology Section, VRC,
NIH, BETHESDA, USA
Background. Dasatinib (Sprycel ® , BMS-354825, Bristol-Myers Squibb)
is a novel inhibitor of BCR-ABL, SRC kinases, c-kit, PDGF receptor and
other kinases. It has been demonstrated to be safe and more effective in
CML patients than Imatinib (Glivec ® , STI571, Novartis) and has been
approved in 2006. An increased infection rate, which has been described
in patients undergoing dasatinib treatment, might be due to inhibitory
effects on LCK and FYN, since dasatinib targets tyrosine kinases (TK)
that play an important role in T cell development and function. A recent
case report also demonstrated dasatinib’s efficacy in the treatment of a
patient with thymoma. Aims. We have now evaluated dasatinib’s effects
on human purified T cells from healthy blood donors and compared
them with the effects of the promiscuous TK inhibitor staurosporine,
which has been the base for several SRC kinase inhibitors in clinical
development. Methods and Results. All assays described herein were
performed at clinically relevant doses between 1nM and 100nM. CFSE
labeled T cells were stimulated with the murine monoclonal CD3 antibody
OKT-3 for four days. A dose-dependent inhibition of T cell proliferation
was detected (EC50=7 nM) and almost complete inhibition (96%
inhibition) occurred at a concentration of 20 nM. T cells incubated with
dasatinib for 24h, and then removed from dasatinib, proliferated as well
as untreated T cells (p=0.118). This argues for a reversible blockade of T
cell proliferation, in contrast to staurosporine that led to a dose-dependent
but irreversible inhibition of proliferation at a dose of 10 nM (on
average 90% inhibition). We further investigated the functional effects
of dasatinib on T cells. A statistically significant inhibition of OKT-3
induced up-regulation of the T cell activation marker CD69 was
haematologica/the hematology journal | 2007; 92(s1) | 115