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

12 th Congress of the European Hematology Association
(24%). The frequency of molecular response was significantly higher
in patients without baseline mutations (54% versus 18%), p=0.005.
During therapy, 18/68 patients (26%) developed 7 different mutations
that were not detectable at baseline (Table 1) (5/18 did not have any
baseline mutation). These seven mutations were predicted from the in
vitro screening studies to be resistant to nilotinib. Progression was documented
in 21 patients (4/4 Ph + ALL, 8/15 BC [53%]; 2/6 AP [33%];
7/43 CP [16%]). There was no significant difference in the frequency
of progression for those with baseline mutations (36%) and those without
(26%). Of the 21 patients with progression, 12 developed mutations
during therapy; 5 had mutations at baseline that were identified in the
resistance screens (3, F359V; 1, F359I; 1, Q252H) but had no further
mutation at progression; and 4 did not have a mutation at any time. Of
the 20 patients with molecular response who did not have any of the
mutations that were identified in the in vitro resistance screens, none has
lost molecular response whereas 3/5 who developed mutations during
therapy lost response, p=0.004. At 90% of assessment timepoints the
patients with mutations identified in the resistance screens received at
least 400mg BID (9% 600mg BID). Summary/Conclusions. Progression
was associated with the detection of mutations that were previously
identified in nilotinib resistance screens. Rather than the predominant
emergence of T315I with resistance, as predicted by the in vitro studies,
the mutations that developed most frequently were Y253H, E255K and
E255V. Among the 7 mutations that developed, only T315I has an IC50
value >1000 nM and is the only one predicted to be insensitive to nilotinib
concentrations of 400 mg BID. This might be due to intracellular
concentrations of nilotinib being lower than the plasma concentrations
predicted from pharmacokinetic studies.
Table 1. Mutations that developed during nilotinib therapy.
0905
NOVEL PATHWAY IN BCR-ABL SIGNAL TRANSDUCTION INVOLVES AKT-INDEPENDENT
ACTIVATION OF PLC-GAMMA/MTOR/P70-S6 KINASE
B. Markova, 1 F. Breitenbuecher, 2 J.V. Melo, 3 J. Duyster, 4 C. Huber, 2
T. Fischer2 1 University Hospital, MAINZ, Germany; 2 Hematology, University Hospital,
MAINZ, Germany; 3 Imperial College, Hammersmith Hospital, LONDON,
United Kingdom; 4 Internal Medicine 3 Technical University, MUNICH, Germany
Background. In CML, defining new, additional therapeutic targets in
the pathways, activated by BCR-ABL is critical for the development of
new treatment strategies, especially for patients resistant or refractory
to imatinib or other tyrosine kinase inhibitors. While studying the
involvement of PI3K/Akt/mTOR signaling pathway in the development
of such resistance we have uncovered the existence of additional,
Akt-independent mechanism of activation of mTOR/p70-S6 Kinase
pathway. Aims. In this work, we characterize a new, PhosphoLipaseCgamma
(PLCγ) dependent pathway for activation of mTOR/p70-
S6Kinase and its importance for the control of proliferation and apoptosis
of BCR-ABL-positive cells. Methods. BCR – ABL + cell lines LAMA84,
AR320, KCL22, K562, Ba/F3-BCR-ABL were treated with various
338 | haematologica/the hematology journal | 2007; 92(s1)
inhibitors and their effect on cell signaling was analyzed by Western
blotting using phosphorylation-specific antibodies. In addition to the
chemical inhibition, siRNA-mediated knock down of PLCγ was utilized
to better understand the necessity/sufficiency of PLCγ for full
activation of mTOR/p70-S6K pathway in the BCR - ABL + cells. The
function of PLCγ/mTOR/p70-S6K pathway for the BCR-ABL driven
cells was also analyzed in proliferation (MTS) and apoptosis assays
where cells were treated with imatinib alone or in combination with
U73122, BAPTA-AM, RAD001 or PKC412 inhibitors. Results. Short
term treatment with imatinib (1 µM, 4h) of BCR - ABL + cell lines caused
downregulation of phosphorylation of p70-S6K and of S6 ribosomal
protein without decreasing phosphorylation levels of Akt, as detected
by Western blotting using the respective phosphorylation-specific antibodies
p-p70-S6K (Thr389), p-S6 (Ser240/244) and p-Akt (Ser473). Inhibition
of Akt by the specific inhibitor SH-6 (10 µM, 4h) did not affect
the phosphorylation of p70-S6K and S6. These results were consistent
in all analyzed cell lines, and led us to consider alternative mechanism
for mTOR/p70-S6K pathway activation. One such mechanism, recently
described in FGF9 signaling is a PLCγ-controlled Calcium signaling
pathway involving Ca/Calmodulin (CaM) and Ca/Calmodulindependent
Kinase (CaM-K). In all BCR - ABL + cell lines analyzed, we
detected strong PLC-γ activation (examined by p-PLCγ-Tyr783 antibody),
which was effectively suppressed by imatinib (1 µM, 4h). Incubation
of the cells for 30 min with 10 µM U73122, a specific PLC
inhibitor, in contrast to the inactive analog U73343, significantly
blocked p70-S6K and S6 phosphorylation. siRNA mediated suppression
of PLCγ also reduced S6 phosphorylation. In general, activation
of PLCγ leads to activation of various PKC isoform and increased Cadependent
signaling. By employing inhibitors of the Ca-signaling (BAP-
TA-AM, EDTA), CaM-K (KN-93) and the PKC inhibitor PKC412 we
studied the participation of these molecules in the pathway. Inhibition
of PLCγ led to suppression of cell proliferation and enhanced apoptosis.
Combined treatment with imatinib and U73122 drastically
increased the apoptosis rate of the cells. In addition, this combination
was able to suppress proliferation and induce apoptosis in imatinib
resistant cells. Combination of these two inhibitors was more efficient
in Ba/F3 p185-F317V cells exhibiting moderate imatinib resistance
as compared to Ba/F3 p185 E255K and T315I cells exhibiting strong
resistance to imatinib. Conclusions. In summary, we demonstrate the
existence of additional, Akt-independent, PLCγ -dependent mode of
activation of mTOR/p70-S6K which operates in Bcr-Abl-positive cells.
This alternative pathway may prove novel therapeutic targets for CML
treatment.
0906
DELETIONS OF THE DERIVATIVE CHROMOSOME 9 DO NOT INFLUENCE RESPONSE
TO IMATINIB IN EARLY CHRONIC PHASE CHRONIC MYELOID LEUKEMIA PATIENTS
(A GIMEMA WORKING PARTY ANALYSIS)
F. Castagnetti, 1 F. Palandri, 1 G Marzocchi, 1 S. Luatti, 1 N. Testoni, 1
M. Amabile, 1 S. Soverini, 1 S. Kerim, 2 E. Giugliano, 3 G. Specchia, 4
A. Cuneo, 5 G. Martinelli, 1 G. Alimena, 6 M. Baccarani, 1 G Rosti1 1 Institute of Hematology Seràgnoli, BOLOGNA; 2 Patologia Medica, Orbassano,
TORINO; 3 CEINGE, Università di Napoli, NAPOLI; 4 Chair of Hematology,
Università di Bari BARI; 5 Chair of Hematology. Università di Ferrara,
FERRARA; 6 Chair of Hematology, Università La Sapienza, ROMA, Italy
Background. BACKGROUND Extensive submicroscopic deletions
adjacent to the breakpoint on derivative chromosome 9 [der(9)] have
been reported in a subset of Chronic Myeloyd Leukemia (CML)
patients and have been associated with an adverse outcome with conventional
drugs and α-interferon (α-IFN). Huntly et al (Blood. 2003;
102.2205-12) reported 275 CML pts who were treated with imatinib
in CP, suggesting that der(9) deletions were associated with lower
response rates and a shorter time to progression. Different data were
reported by Quintas-Cardama et al (Blood. 2005; 105:2281-6), who
did not find any difference related with der(9) deletions in other 320
patients treated with imatinib. In these 2 studies, some patients began
imatinib in early CP (51 and 152, respectively) while many patients
(224 and 168, respectively) were treated in late CP. Aims. To establish
the relationship of der(9) deletions with the response to imatinib in early
CP patients,we performed a sub-analysis within 3 simultaneously
running trials of the GIMEMA (Gruppo Italiano Malattie Ematologiche
dell'Adulto) CML WP (n.CML/021, phase II - ima 800 in intermediate
Sokal risk; CML/022, phase III- ima 400 vs 800 mg in high Sokal risk,
n . CML/023, observational - ima 400 mg). Patients and Methods. 442
evaluable CML patients in early CP have been enrolled from January,