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

0927
MK-0457, A NOVEL MULTIKINASE INHIBITOR, IS ACTIVE IN PATIENTS WITH CHRONIC
MYELOID LEUKEMIA (CML) AND ACUTE LYMPHOCYTIC LEUKEMIA (ALL) WITH THE T315I
BCR-ABL RESISTANCE MUTATION AND PATIENTS WITH REFRACTORY JAK-2 POSITIVE
MYELOPROLIFERATIVE DISEASES (MPD)
J. Giles, 1 J. Cortes, 1 D.A. Bergstrom, 2 A. Xiao, 2 D. Jones, 1
S. Verstovsek, 1 D. Thomas, 1 D. Rizzieri, 3 S.J. Freedman, 2 H. Kantarjian1 1 M.D. Anderson Cancer Center, HOUSTON, USA; 2 Merck & Co., Inc.,
UPPER GWYNEDD, USA; 3 Duke University, DURHAM, USA
Background. MK-0457 (VX-680) is a small molecule inhibitor of aurora
kinases A, B, and C, FLT3, and JAK-2 with nanomolar level broad
spectrum pre-clinical anti-tumor activity. The T315I BCR-ABL mutation
mediates high level resistance to imatinib, dasatinib and nilotinib. MK-
0457 has in vitro activity against cells expressing wild-type or mutated
BCR-ABL, including the T315I BCR-ABL mutation. Aims. The aim of
this study was to determine the tolerability of MK-0457 in patients with
refractory hematological malignancies. Methods. After IRB approval,
fifty-one consenting patients with either refractory AML, CML, ph+
ALL or MPD were enrolled onto the study. The study regimen is a 5-day
continuous IV regimen given every 2 to 3 weeks. To date, dose levels of
8 (N=4), 12 (N=5), 16 (N=3), 20 (N=6), 24 (N=15), 28 (N=4), 32 (N=3) and
40 (N=11) mg/m 2 /hr have been investigated. Results. MK-0457 is a well
tolerated agent in the treatment of hematological malignancies. At the
40 mg/m 2 /hr dose level, grades 2 and 3 asymptomatic lipase elevations
were observed and in one instance, it was determined to be a DLT. Additional
toxicities possibly related to MK-0457 include grade 1 amylase elevation,
nausea, alopecia, myalgias, althralgias, and cough, and grades 1
and 2 mucositis. Dose-proportional PK has been observed across dose
levels. Myelosuppression is consistently seen with MK-0457 at all dose
levels studied to date and appears to be dose-related. Of the 51 patients
on part 1 of the study to date, 16 had CML, 5 had ALL, 21 had AML and
9 had rapidly progressive or transforming MPD. A nested PCR strategy
followed by direct DNA sequencing using the dideoxy chain termination
method was used to detect and monitor mutations in codons 221
to 500 of the BCR-ABL kinase domain in CML and ALL patients. Of 15
evaluable patients with CML, 9 had a T315I BCR-ABL mutation as did
3 Ph + ALL patients. Of 14 currently evaluable patients with CML, 11
had an objective (hematologic, cytogenetic, and/or molecular) response,
including all 9 patients with the T315I mutation. Two patients with
T315I-mutant ALL had an objective response, one achieving a complete
hematologic and cytogenetic response as well as resolution of 2 out of
3 mutations present prior to treatment on study. Six of 8 currently evaluable
patients with JAK-2 positive refractory MPD have achieved an
objective response. Downregulation of BCR-ABL and CrkL phosphorylation
in leukemia cells from CML and ALL patients treated on study has
been documented - the possible role of aurora kinase inhibition in these
clinical responses requires further investigation. Accrual to the study is
ongoing at the 40 mg/m 2 /hr dose level as well as utilizing both single 24hour
and single 6-hour infusions to sequentially determine the maximum-tolerated
dose of MK-0457 using shorter infusion durations. Conclusions.
The currently reported cases are the first observed clinical activity
of a kinase inhibitor against the T315I positive CML/ALL and JAK-2
positive MPD. The observation of responses in these patients to doses
of MK-0457 associated with no significant toxicity warrants further
study.
12 th Congress of the European Hematology Association
Epigenetics, transcription and signalling
0928
TRANSCRIPTIONAL REGULATION OF HEMO-OXIGANASE-1 AND A SECRETED PROTEIN
ACIDIC AND RICH IN CYSTEINE (SPARC) IN BORTEZOMIB-TREATED ADULT T-CELL
LEUKEMIA CELLS
H. Ryoko, J.H. Ohyashiki, C. Kobayashi, A. Hirota, Y. Zhang,
T. Takaku, K. Ohyashiki
Tokyo Medical University, TOKYO, Japan
Background and Aims. Adult T-cell leukemia (ATL) is a fatal neoplasia
derived from HTLV-1 infected T lymphocytes frequently exhibiting
nuclear factor-kappaB (NF-κB) activation. Despite the development of
various treatment regimens, the prognosis of ATL is poor, and new treatment
strategies need to be determined. The aim of this study is to investigate
the effect and the molecular mechanism of a proteasome inhibitor,
bortezomib, in ATL cells. Methods. An IL-2 dependent ATL cell line, TaY,
two IL-2 independent ATL cell lines, MT-2, and MT-4, and an HTLV-1
negative T cell line, Jurkat, were used. After obtaining informed consent,
peripheral blood mononuclear cells (PBMCs) isolated from a patient with
ATL were also used. We explored gene expression profiling as well as
gene network-based analysis using a pathway-focused oligonucleotide
assay (GPL 3837). Quantitative RT-PCR and Western blotting was done
to confirm the microarray results. Results. We found bortezomib-induced
cell death in ATL cell lines with decreased activity of NF-κB. Differential
gene expression analysis of an ATL cell line, TaY, revealed up-regulation
of oxygen-dependent gene, hemo-oxigenase-1(HMOX-1) which
is known as a target gene of hypoxia-inducible gene -1 α (HIF-1 α).
Induction of HMOX-1 by cobalt protoporphyrin (CoPP) increased apoptosis
of TaY cells in a pharmacologically effective dose of bortezomib,
while CoPP did not affect the cell growth in the absence of bortezomib.
Gene network analysis by a Bayesian statistical framework extracted a
secreted protein acidic and rich in cysteine (SPARC), a tumor-invasiveness
related gene. Inhibition of SPARC by siRNA enhanced the apoptotic
effect of bortezomib on TaY cells. Conclusions. Targeting SPRC may be
challenging to treat ATL patients exhibiting extra-nodal lesions including
skin invasion. The enhanced sensitivity of TaY cells to bortezomib
by CoPP is also intriguing and suggests that HMOX-1 may be an attractive
target for treating ATL patients. Applying network-based analysis
in addition to gene expression profiling may be new option to provide
a novel insight into proteasome inhibitor, bortezomib in ATL.
0929
LACK OF MHC-II MOLECULE EXPRESSION IN T-LEUKAEMIA IS ASSOCIATED
WITH DISTINCT EPIGENETIC DNA AND HISTONE MODIFICATIONS AT PROMOTER III
CHROMATIN OF MHC2TA
P.J. van den Elsen, T.M. Holling, M.C.J.A. Van Eggermond
Leiden University Medical Center, LEIDEN, Netherlands
Previously we have shown that, unlike normal peripheral T cells, in vitro
stimulation of T-leukaemia cell lines with well-known T-cell activation
agents did not result in the induction of class II transactivator (CIITA) and
MHC-II molecule expression. The co-activator CIITA, encoded by the
MHC2TA gene, is essential for transcriptional activation of all MHC-II
genes. Because other T-cell activation markers like IFNγ, IL-4, CD69, and
CD45RO were readily induced in the stimulated T-leukaemia cells, we
eliminated the possibility that general T-cell activation pathways were
corrupted. Additionally, we showed in a transient promoter-reporter assay
that promoter III of MHC2TA (CIITA-PIII), which is the principal T-cell
employed MHC2TA promoter, was readily activated in CIITA-deficient
T-leukaemia cells to levels similar as observed in MHC-II expressing Tlymphoma
cells. These observations reveal that all essential transcription
factors for CIITA-PIII activation are expressed in the leukaemia T-cells.
However, in vivo genomic footprint analysis revealed lack of transcription
factor binding to CIITA-PIII and hyper-methylation at CpG of CIITA-PIII
in the CIITA-deficient T-leukaemia cells. Subsequent inhibition of DNA
methyltransferase activity with 5AZA-2’-deoxycytidine resulted in reexpression
of the CIITA-PIII isoform in these leukaemia T-cells. Moreover,
we also found hyper-methylation of CIITA-PIII in HLA-DR-deficient
primary leukaemia T-cells. Therefore, the defect in CIITA expression
in leukaemia T-cells correlates with hypermethylation of promoter DNA,
which blocks factor assembly on CIITA-PIII resulting in impairment of its
activation. Since epigenetic DNA and histone modifications work in concert
in promoting accessibility of the transcriptional machinery to regulatory
elements of genes, we have extended our research towards the epi-
haematologica/the hematology journal | 2007; 92(s1) | 347