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

0872
MN1 INDUCES LEUKEMIA IN MICE AND PREDICTS ATRA RESISTANCE AND SENSITIVITY
IN AML PATIENTS
M.H. Heuser, 1 B. Argiropoulos, 1 F. Kuchenbauer, 1 E. Yung, 1 J. Piper, 1
S. Fung, 1 R.F. Schlenk, 2 K. Dohner, 2 T. Hinrichsen, 3 C. Rudolph, 3 A.
Schambach, 3 C. Baum, 3 B. Schlegelberger, 3 H. Dohner, 2 A. Ganser, 3
R.K. Humphries 1
1 British Columbia Cancer Agency, VANCOUVER, Canada; 2 University Hospital
of Ulm, ULM, Germany; 3 Hannover Medical School, HANNOVER, Germany
Recently, we found that overexpression of wild-type MN1 is a negative
prognostic factor in acute myeloid leukemia (AML) patients with
normal cytogenetics. We evaluated whether MN1 plays a functional role
in leukemogenesis. Strikingly, we now demonstrate using retroviral gene
transfer and bone marrow transplantation that MN1 overexpression is
sufficient to induce a rapidly lethal AML in mice (disease latency 35 days).
Insertional mutagenesis and chromosomal instability were ruled out as
secondary aberrations. MN1 dramatically increased resistance to all-trans
retinoic acid (ATRA)-induced cell-cycle arrest and differentiation (by
>3,000-fold) in an in vitro differentiation model. We show by chromatinimmunoprecipitation
that MN1 directly interferes with RARα signaling
through binding to regulatory sequences and transcriptional repression of
the RARα-target genes Rarbeta, C/ebpepsilon, and PU.1. The morphologically
and functionally evident block in differentiation of MN1-transduced
cells thus appears to be mediated by repression of RARα signaling. This
could be confirmed functionally by fusion of a transcriptional activator
(VP16) to MN1 in order to mask the potential repressive activity of MN1.
MN1VP16-transduced bone marrow cells gave rise to immortalized cell
lines, however, these cells differentiated to mature granulocytes and eventually
went into cell cycle arrest upon treatment with ATRA. We then
evaluated whether MN1 expression levels in AML patients (excluding
the M3 subtype) correlated with resistance to ATRA treatment in a cohort
of patients older than 60 years uniformly treated within treatment protocol
AML HD98-B. Of a total of 83 patients 40 were treated with standard
chemotherapy (idarubicin, cytarabin, and etoposide), and 43 patients
were treated with standard chemotherapy plus ATRA. MN1 expression
levels at diagnosis were measured by quantitative RT-PCR. Patient characteristics
were equally distributed between ATRA versus no-ATRA treated
patients including event-free survival (EFS) and overall survival (OS).
Patients with MN1 expression above the median expression level did not
differ in EFS or OS whether they were treated with ATRA or not. Strikingly,
patients with low MN1 expression who received ATRA had a significantly
prolonged EFS (p=0.008) and OS (p=0.04) compared to the
remaining patients. Our data show that MN1 is a unique oncogene in
hematopoiesis that both promotes proliferation/ self-renewal and blocks
differentiation. Such findings provide tools and impetus to unravel the
mechanisms of MN1 function. Of immediate consequence, MN1 expression
levels allow to identify a subset of AML patients that appear to benefit
from treatment with ATRA and thus may become useful as a predictive
marker to guide the treatment in AML patients.
12 th Congress of the European Hematology Association
Acute lymphoblastic leukemia - Biology
0873
DIFFERENTIAL CHROMOSOMAL BREAKPOINTS IMPACT LEVEL OF LMO2 EXPRESSION
IN T-ALL
A.W. Langerak1 , W.A. Dik1 , B. Nadel2 , G.K. Przybylski3 , V. Asnafi4 ,
P. Grabarczyk5 , J.M. Navarro2 , B. Verhaaf1 , C.A. Schmidt5 ,
E.A. Macintyre4 , J.J.M. Van Dongen1 1 Erasmus MC, ROTTERDAM, The Netherlands; 2 CIML INSERM-CNRS,
MARSEILLE, France; 3 Institute of Human Genetics, POZNAN, Poland; 4 Hôpital
Necker-Enfants-Malades, PARIS, France; 5 Universität Greifswald, GREIF-
SWALD, Germany
Background. Human T-cell acute lymphoblastic leukemias (T-ALLs) are
associated with chromosomal translocations involving TCR genes and
proto-oncogenes. The t(11;14)(p14;q11), which is considered the paradigm
for TCR-associated translocations, is presumed to arise from erroneous
TCRD V(D)J recombination and to result in ectopic LMO2 activation.
However, the molecular mechanisms underlying formation of this
translocation and the resulting LMO2 activation are poorly defined. Aims
.We aimed to investigate how t(11;14)(p13;q11) aberrations lead to LMO2
activation in T-ALL. Methods. Following genomic breakpoint analysis in
9 T-ALL cases, breakpoints were cloned and tested in an ex vivo recombination
substrate assay and in silico using a RIC algorithm. LMO2 levels
in T-ALL were determined by real-time quantitative PCR. Occurrence
of TCRD-LMO2 translocations was evaluated in normal thymocytes by
nested PCR. Results. In our T-ALL cases we identified two t(11;14)
(p13;q11) translocation mechanisms: 1) RAG mis-targeting of LMO2
through cryptic recombination signal sequences (cRSSs), and 2) V(D)J
post cleavage synaptic complex repair mistakes. Furthermore, contrary to
the common concept of TCR-enhancer driven activation, we provide
compelling evidence that removal of a T-cell specific negative regulatory
element (NRE) upstream of LMO2 is the main determinant for LMO2
activation in the majority of t(11;14)(p13;q11) cases. Activation levels are
determined by the exact LMO2 breakpoint position and the configuration
of the derivative chromosome. Summary/Conclusions. Our combined
in vivo, ex vivo, and in silico analyses on nine new t(11:14)(p13;q11) positive
T-ALL cases as well as normal thymocytes illustrate the critical role
of the exact LMO2 breakpoint location in influencing the level of LMO2
activation and the consequent pressure for pre-leukemic selection and T-
ALL development.
0874
EVIDENCE FOR POSTNATAL ORIGIN OF CHILDHOOD T-LINEAGE ALL
S. Fischer, 1 G. Mann, 2 M. Metzler, 3 G. Ebetsberger, 4 N. Jones, 5 B. Nadel, 6
O. Bodamer, 7 O.A. Haas, 2 K. Schmitt, 8 H. Gadner, 2R. Panzer-Grümayer1 1 Children's Cancer Research Institute, VIENNA, Austria; 2 St. Anna Kinderspital,
VIENNA, Austria; 3 University Erlangen-Nürnberg, ERLANGEN, Germany;
4 Kinderklinik, LINZ, Austria; 5 University of Slazburg, SALZBURG, Austria;
6 Université de la Méditerranée, MARSEILLE, France; 7 Universitiy of Vienna,
VIENNA, Austria; 8 University of Linz, LINZ, Austria
Background. T ALL is an aggressive hematological malignancy that comprises
about 15% of childhood ALL. One hallmark of T ALL is the activation
of oncogenic transcription factors that results from deregulation,
deletion or, rarely though, chromosomal translocations. The latter are
considered early events in leukemogenesis that require further complementary
mutations for the clinical manifestation of the disease. Such
genetic alterations as well as the clone-specific T cell receptor (TCR)
rearrangements characterize the leukemic clone and thus can be used to
scrutinize neonatal blood spots. If they can be detected at birth, this
would indicate that the leukemia originates already in utero. A fetal origin
has been suggested for the majority of genetic subgroups of childhood
B cell precursor ALLs. Aims. The aim of this study was to assess whether
childhood T ALL also originated in utero by backtracking leukemia-associated
molecular markers back to birth. Patients and Methods. Neonatal
blood spots from 18 children, 1,5 - 11,2 years old at the time of diagnosis
of T ALL, were included into this study. Molecular markers were identified
and used for highly sensitive and specific PCR (RQ-PCR or nested
PCR) approaches. A sensitivity to detect one cell with the particular marker
among 100.000 normal cells was required for inclusion into this study.
The targets comprised TCRD-LMO2 breakpoint regions (n=2), TAL1
deletions (n=3), Notch1 mutations (n=1), and TCRD or TCRG rearrangements
(n=15). Between one third and half of a neonatal blood spot was
haematologica/the hematology journal | 2007; 92(s1) | 325