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

gest diverse sequences of NPM mutant NES motifs function differently.
Hereby, we provide evidence of how C-terminus alterations functionally
cooperate to delocalize NPM mutants to cytoplasm. We first investigated
whether different NPM leukemic mutants differ in their ability to
be exported in the cytoplasm. NIH 3T3 cells were transfected with eGFPtagged
NPM mutants A and E. After incubation with low doses of Leptomycin
B (a specific inhibitor of Crm1, the protein responsible for NESmediated
nuclear export), NPM mutant A was almost completely nuclear
whilst NPM mutant E was still markedly cytoplasmic. This demonstrate
that NPM mutant E is less sensitive to Crm1 inhibition than NPM mutant
A. To directly measure the export efficiency of each of the six different
NPM C-terminal NESs so far identified, we isolated and cloned them into
a pREV(1.4)-eGFP plasmid expressing a mutagenized REV protein lacking
its NES but retaining its Nuclear Localization Signal, an assay that
allows to measure the export efficiency of various NES sequences (Henderson,
Exp Cell Res 256:213, 2000). The REV(1.4) fusion protein containing
the most common NPM mutant NES LxxxVxxVxL (never found with
W288) was nuclear in the majority of transfected cells (indicating a functional
NES with weak activity), whilst all variant NESs were mostly cytoplasmic
(indicating a stronger activity). These findings prove that NPM
mutants carry NES motifs with different nuclear export efficiency. NPM
subcellular localization is dictated by opposing balance of forces (tryptophans
and NES), and no NPM mutant from AML leukemic patients has
ever been found to contain the weak LxxxVxxVxL NES in the presence
of W288. We therefore investigated the consequence of artificially combining
these two features on NPM subcellular traffic in NIH 3T3 cells.
Notably, these artificial NPM mutants were not exported efficiently into
cytoplasm, since the force (W288) driving mutants towards the nucleolus
overwhelmed the force (NES motif) exporting them into cytoplasm.
These findings show that NPM leukemic mutants must carry a strong
NES motif if W288 is retained in order to ensure efficient cytoplasmic
accumulation. This reveals a mutational selective pressure toward efficient
NPM nuclear export and points to this event as critical for leukemogenesis
and therefore as a potential therapeutic target.
Table 1. NESs from NPM leukemic mutants: types, incidence upon NPM
mutants, and correlation with Tryptophan loss.
12 th Congress of the European Hematology Association
Chronic myeloid leukemia - Biology
0903
BMS-214662 TARGETS AN EARLY PROGENITOR POPULATION IN PRIMARY CML AND
INDUCES APOPTOSIS IN THE QUIESCENT FRACTION AFTER SENSITIZATION BY THE MEK
1/2 INHIBITOR U0126
F. Pellicano, M. Copland, H. Jorgensen, T. Holyoake
University of Glasgow, GLASGOW, United Kingdom
Background. Chronic myeloid leukemia (CML) is a hematopoietic
stem cell disorder that cannot be eradicated by the targeted Abl tyrosine
kinase inhibitor (TKI), imatinib mesylate (IM; Gleevec, Gilvec) or
dasatinib (Sprycel; a more potent multi-targeted TKI) as these drugs
reversibly arrest proliferation of CML stem/progenitor cells but do not
induce apoptosis or kill the most primitive quiescent cells. Farnesyltransferase
inhibitors (FTI) inhibit farnesylation of oncogenic RAS as
well as of other intracellular proteins involved in hematological malignancies.
BMS-214662, a cytotoxic FTI has been shown to preferentially
kill non-proliferating cells, to induce potent tumour regression, and
has anti-leukemic activity in acute myeloid leukemia. Aims. We tested
BMS-214662 for ability to target CML stem/progenitor cells. Methods.
Primitive CD34 + 38 – cells, derived from CML patients in chronic phase
and normal donors, were treated with BMS-214662 alone or in combination
with a pharmacologic inhibitor, U0126, and analysed for caspase-3
activation by flow cytometry. Western blotting analysis was
used to investigate BMS-214662 mechanism of action. Results. BMS-
214662 significantly and selectively increased caspase-3 activity in CML
versus normal cells (27.7 and 6.4%, respectively, after 48 hours of treatment).
Remarkably, since CD34 + 38 – cells are almost exclusively quiescent,
this highlights for the first time the effectiveness of BMS-214662
against the more quiescent primary CML stem cell population. Moreover,
neither IM, nor dasatinib, nor lonafarnib (Sarasar), nor nilotinib
(Tasigna) showed similar apoptotic activity to BMS-214662. In CML
CD34 + progenitor cells, BMS-214662 potently blocked the pro-survival
MAPK pathway by inhibiting phosphorylation of RAF-1 and ERK. In
addition, the inhibition of MEK with U0126, together with BMS-214662
resulted in a dramatic synergistic enhancement of apoptosis. In an
attempt to understand how BMS-214662 induces apoptosis, we
analysed expression level of the Bcl-2 family proteins. For both CML cell
lines and primary CD34 + cells Mcl-1, Bcl-2 and BimXL levels were
unchanged after treatment with BMS-214662. However, BMS-214662
decreased the level of IAP-1, a known suppressor of apoptosis. Conclusions.
Our group is the first to report that BMS-214662 selectively kills
quiescent cancer stem cells and therefore offers potential for eradication
of CML in chronic phase.
0904
THE P-LOOP MUTATIONS Y253H AND E255K/V MAY DEVELOP MORE FREQUENTLY THAN
T315I DURING NILOTINIB THERAPY AFTER IMATINIB FAILURE AND ARE ASSOCIATED
WITH PROGRESSION IN PATIENTS WITH PH-POSITIVE LEUKEMIA
S. Branford, 1 Y. Shou, 2 R. Lawrence, 1 Z. Rudzki, 1 T. Hughes1 1 Institute of Medical &Veterinary Science, ADELAIDE, Australia; 2 Novartis
Pharmaceuticals, NEW JERSEY, USA
Background. In vitro data suggest a central role for BCR-ABL mutations
in clinical resistance to nilotinib for patients with Philadelphia-positive
(Ph + ) leukemia. Fifteen nilotinib resistant mutations were identified in
resistance screens (Blood, 2006;108:1328-1333, Blood, 2006;108:2332-
2338). With the exception of T315I, all mutations were effectively suppressed
with nilotinib concentrations of 2000 nM, which falls within the
peak-trough plasma levels (3600-1700 nM) measured in patients treated
with 400mg BID. Aims. We aimed to determine the effect of mutations
in vivo on nilotinib resistance and molecular response of patients
treated with 400mg nilotinib BID after imatinib failure. Methods. Sixtyeight
patients were treated in a Phase II trial (Ph + ALL, n=4; CML-blast
crisis [BC], n=15; CML-accelerated phase [AP], n=6; CML-chronic phase
[CP], n=43). Patients were followed by RQ-PCR and mutation analysis
while receiving nilotinib therapy for a median of 6 (range 2-15) months.
The results were correlated with disease status. Molecular response
was defined as the reduction of BCR-ABL to ≤ 1% (minor) and ≤ 0.10%
(major) on the international scale. Loss of molecular response was
defined as >2-fold rise of BCR-ABL and loss of a minor molecular
response. Results. Prior to nilotinib (baseline), 22 different mutations
were detected in 33/68 patients (49%). Molecular response occurred in
25/68 patients (37%). A major molecular response occurred in 16/68
haematologica/the hematology journal | 2007; 92(s1) | 337