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

genesis of acute myeloid leukaemia (AML). Flt3-activating mutations
have been associated with poor prognosis and decreased overall survival
of AML patients. Among them, internal tandem duplications (ITD)
of Flt3 are found in a high proportion of cases with acute myeloid
leukemia (AML). These genetic aberrations may lead to the constitutive
activation of the receptor, thus providing the molecular basis for a persisting
growth stimulus. On the other hand, alterations of transcriptional
regulators are also key events in leukaemogenesis. Constitutive activation
of MAP/Akt/STAT/NF-κB signal transduction pathways have
been described in AML and they are commonly attributed to Flt3 activity.
With the aim of better understanding the molecular mechanisms
accompanying the acquisition of Flt3 activating mutations, a comparative
proteomic approach was undertaken on Flt3/ITD and wild-type Flt3
AML cells. Proteomic analyses of three AML samples showing Flt3/ITD
and three samples with wild-type Flt3 were carried out using 2-DE and
MALDI-TOF mass fingerprinting analysis. Proteins identified as more
significantly altered between the different AMLs analyzed belonged to
the group of suppressor genes, metabolic enzymes, antioxidants and signal
transduction mediators. Among them, four identified proteins were
found significantly deregulated in Flt3/ITD leukemic cells in comparison
to wild-type Flt3 AML cells, including two up-regulated proteins (peroxirredoxin
and catalase) and two down-regulated proteins (calreticulin
and RhoGDI). These proteins were widely involved in differentiation
and cell survival. Furthermore, several intracellular pathways and transcription
factors also implicated in differentiation and cell survival such
as ERK 1/2, p38 MAPK, Akt, STAT5 and NFkB were found constitutively
activated in the Flt3/ITD AML samples. Employing methods of cell
biology and proteome analysis tools, we examined the effects of an
inhibitor of Flt3 phosphorylation, AG1296 (AG), on the proliferation/
apoptosis characteristics of myelomonocitic leukemia-derived cells
MV411, which carried the Flt3/ITD mutation. AG induced apoptosis by
suppressing Flt3 activity, which was followed by p38 MAPK and Akt
inhibition. The proteomic analysis revealed several proteins that were
differentially expressed due to AG treatment namely, calreticulin, eIF-5a,
nucleophosmin, protein disulfide isomerase, glutamate dehydrogenase,
peroxirredoxin and RhoGDI, all of them involved in the regulation of cell
differentiation, proliferation and apoptosis. The further investigation of
the relationship between the altered protein expression found in
FLT3/ITD AML cells and the constitutive activation of those intracellular
pathways, will uncover new clues to understanding leukemogenic
effects of FLT3 activating mutations. This study shows the power of
proteomic profiling for the discovery of novel molecular targets and a
better understanding of the actions of Flt3/ITD at the molecular level in
AML cells. Supported by FIS 050910, FIS 041291, JA 0024/05, 0060/05
and FIJC-06/ESP
0470
LOW DOSE RAPAMYCIN IS NOT EFFLUXED BY P-GLYCOPROTEIN IN AML
M. Pallis, 1 S. Shang, 2 C.H. Seedhouse, 2 N.H. Russell2 1 Nottingham University Hospitals, NOTTINGHAM; 2 University of Nottingham,
NOTTINGHAM, United Kingdom
Background. A clinical trial of rapamycin in elderly patients with AML
is currently underway in Nottingham, UK. A potential confounding factor
to the clinical effectiveness of rapamycin is its reported interaction
with the drug efflux pump p-glycoprotein. Aims. We investigated the
effect of rapamycin on mTOR targets and on pgp in a single cohort of
AML cells in order to determine whether the mTOR target-modulating
dose of rapamycin is above or below the threshold for interaction with
pgp. Methods. Three pgp positive cell lines - KG1, KG1a and TF-1 were
used. MTOR targets p70 S6K(phosphothr389) and 4E-BP1 (phosphoser65)
were measured by Western blotting. Pgp function was measured
using the UIC2 shift assay and the rhodamine 123 accumulation assay
with cyclosporin A and vinblastine as positive controls. Results.10 nM
rapamycin induced a complete loss of phospho-p70 S6K in KG1 and
KG1a cells. TF-1 cells had undetectable basal levels. At 10 nM, rapamycin
also reduced phospho-4E-BP1 levels to 56% of control values in KG1a,
26% of control in KG1 and 39% of control in TF-1 cells. At this dose,
rapamycin failed to induce a UIC2 shift, indicating that there was no
interaction with pgp. 10 nM rapamycin also failed to increase rhodamine
123 accumulation, whether added concurrently or up to one hour before
the rapamycin, indicating that pgp has no significant interaction with 10
nM rapamycin. Higher doses of rapamycin did induce a small increase
in rhodamine 123 accumulation (by approximately 30% in all 3 cell lines
at 100 nM). Similarly, at 100 nM there was an approximately 20%
increase in UIC2 binding in KG1a and TF-1, but not KG1 cells. Many
drugs are documented to upregulate pgp within 48 hours. 72 hours’ incu-
12 th Congress of the European Hematology Association
bation with 10 nM rapamycin did not upregulate pgp protein expression
in the three cell lines. Conclusions. We conclude that pgp-mediated drug
resistance is unlikely to be a significant factor in the response of AML
cells to rapamycin.
0471
SEPT2 IS A NEW FUSION PARTNER OF MLL IN ACUTE MYELOID LEUKEMIA WITH
T(2;11)(Q37;Q23)
N. Cerveira, C. Correia, S. Bizarro, C. Pinto, S. Lisboa, J.M. Mariz,
M. Marques, M.R. Teixeira
Portuguese Oncology Institute, PORTO, Portugal
Background. Abnormalities of 11q23 involving the MLL gene are found
in several hematological malignancies, including acute lymphoblastic
leukemia (ALL) and acute myeloid leukemia (AML), and also in a proportion
of patients with therapy-related leukemia after treatment with
topoisomerase II inhibitors. Aims/Methods. Cytogenetic, fluorescence in
situ hybridization, and molecular studies were used to identify a new
fusion partner of the MLL gene in a patient with a diagnosis of therapyrelated
acute myeloid leukemia with a t(2;11)(q37;q23). Results/Discussions.
Fluorescence in situ hybridization demonstrated a rearrangement
of the MLL gene with the chromosome band 2q37. RNA and DNA
analyses showed the existence of an in-frame fusion of MLL exon 7 with
SEPT2 exon 3, with the genomic breakpoints located in intron 7 and 2
of MLL and SEPT2, respectively. Search for DNA sequence motifs
revealed the existence of two sequences with 94.4% homology with
the topoisomerase II consensus cleavage site in MLL intron 7 and SEPT2
intron 2. SEPT2 is the fifth septin family gene fused with MLL, making
this gene family the most frequently involved in MLL-related AML
(about 10% of all known fusion partners). Conclusions. We have identified
a new MLL gene fusion partner in a patient with treatment-related
AML presenting a t(2;11)(q37;q23) as the only cytogenetic abnormality.
SEPT2 is the fifth septin that has been found fused with MLL in acute
leukemia, but the precise role played by this family of genes in this disease
remains incompletely known.
0472
PROGNOSTIC VALUE OF CYTOCHROME C RELATED ACTIVATION OF CASPASES IN
PEDIATRIC ACUTE MYELOID LEUKEMIA
H. Meyer, S.M. Eckhoff, M. Queudeville, K. Stahnke, K.-M. Debatin
University of Ulm, ULM, Germany
Background. Deficient activation of apoptosis signaling pathways has
been considered to be responsible for treatment failure in acute leukemia.
Studies have been performed to analyze the expression of apoptosis
molecules with respect to their prognostic value for treatment outcome
but could not establish a correlation with response to treatment and
prognosis. In a previous study we investigated functionally the importance
of intact apoptosis signaling for treatment response in childhood
lymphoblastic leukemia by analysis of two key apoptogenic events, caspase-3
activation and mitochondrial cytochrome c release. By combining
both parameters, we identified the novel indicator CRAC
(cytochrome c related activation of capase-3) which directly connects the
extent of caspase-3 activation to cytochrome c release in single cells in
an individual patient sample. Pediatric B-ALL patients with positive
CRAC values were found to be good responding to initial treatment and
showed a significantly higher probability of relapse free survival in contrast
to patients with negative CRAC values (Meyer et al., Blood, 2006,
107(11): 4524-31). Aims. Since the investigation of cytochrome c release
and caspase activation and the calculation of its relation as expressed by
the parameter CRAC is of prognostic value in childhood lymphoblastic
leukemia we were interested in the significance of intact apoptosis signaling
represented by this parameter in pediatric patients suffering from
acute myeloid leukemia. Methods. Myeloid leukemia cells identified by
surface staining in samples (cryopreserved bone marrow or peripheral
blood) obtained at diagnosis from pediatric AML patients were analyzed
for active caspase-3 and released cytochrome c flowcytometrically. The
relation between these two key apoptogenic events was analyzed upon
induction of apoptosis by factor withdrawal and the parameter CRAC
was calculated for each patient sample. Results. In the samples investigated
a heterogenous pattern of caspase activation and cytochrome c
release was observed. Especially insufficient caspase activation in the
presence of mitochondrial released cytochrome c was found in a number
of samples resulting in negative values for the CRAC parameter.
This indicates deficient apoptosis signaling in this subset of AML patient
samples. 16 of the 34 AML patients (47,1%) showed negative CRAC val-
haematologica/the hematology journal | 2007; 92(s1) | 175