Analysis of the Leukemogenic Potential of the CALM/AF10 Fusion Gene in Patients, Transgenic Mice and Cell Culture Models

The t(10;11)(p13;q14) is a recurring translocation resulting in the
fusion of the CALM and AF10 genes. The leukemogenic CALM/AF10
fusion genes codes for a 1595 amino acids protein. This
translocation was first identified in a patient with hystiocytic
lymphoma and was subsequently found in patients with AML, T-ALL and
malignant lymphoma. This translocation is found in younger patients
and is associated with a poor prognosis. The CALM/AF10-associated
leukemias can exhibit myeloid, lymphoid or mixed lymphoid-meyloid
features, indicating a stem cell or an early commited progenitor as
the target cell of leukemic transformation. At the present time the
target cells in CALM/AF10-associated leukemogenesis are unknown. It
is also not known which target genes are up or downregulated by the
presence of the CALM/AF10 fusion protein. To answer these
questions, the following experiments were performed: 1) Five
transgenic mouse lines, two expressing CALM/AF10 under the control
of the immunoglobulin heavy chain enhancer promoter and three under
the control of the murine proximal Lck promoter were generated.
Although the CALM/AF10 expression was confirmed to be present and
specific to the cells targeted by the promoters used (B- and T-cell
progenitors for IgH and Lck promoters, respectively), the
transgenic animals did not show a phenotype that could be detected
after meticulous clinical, haematological, immunological, flow
cytometrical and immunohistopatological analysis . 2) We performed
molecular characterization of several CALM/AF10 patient samples: A
group of 13 patients with different types of leukemia: case 1 (AML
M2), case 2 (Acute Biphetnotypic leukemia), case 3 (Pre T-ALL),
case 4 (Acute Undifferentiated Leukemia), case 5 (PreT-ALL), cases
6 and 7 (ProT-ALL), case 8 (T-ALL), case 9 (AML), case 14 (T-ALL),
case 15, 16 and 17 (AML) with a t(10;11) translocation detected by
cytogenetic analysis suggesting a CALM/AF10-rearrangement. The
samples were analyzed for the presence of the CALM/AF10 and
AF10/CALM fusion transcripts by RT-PCR and sequence analysis. All
these patients were found to be positive for the CALM/AF10 fusion.
In addition, we analyzed a series of twenty-nine patients with
T-ALL with T-cell receptor ≥¥ rearrangement. Among these patients,
four (case 10 to 13) were positive for the CALM/AF10 fusion
transcript, indicating a high incidence of CALM/AF10 fusions in
this group of leukemia. Three different breakpoints in CALM at
nucleotide 1926, 2091 and a new exon, with 106 bases inserted after
nt 2064 of CALM in patient 4 were found. In AF10 four breakpoints
were identified: at nucleotide position 424, 589, 883 and 979. In
patient 16 we found an extra exon before nt 424 of AF10. In seven
patients it was also possible to amplify the reciprocal AF10/CALM
fusion transcript (case 1, 3, 4, 8, 9, 10 and 14). There was no
correlation between disease phenotype and breakpoint location. Ten
CALM/AF10 positive patients were analyzed using oligonucleotide
microarrays representing 33,000 different genes (U133 set,
Affymetrix). Analysis of microarray gene expression signatures of
these patients revealed high expression levels of the polycomb
group gene BMI1, the homeobox gene MEIS1 and the HOXA cluster genes
HOXA1, HOXA4, HOXA5, HOXA7, HOXA9, and HOXA10. The overexpression
of HOX genes seen in these CALM/AF10 positive leukemias is
reminiscent to the pattern seen in leukemias with rearrangements of
the MLL gene, normal karyotypes and complex aberrant karyotypes
suggesting a common effector pathway (i.e. HOX gene deregulation)
for these diverse leukemias. In addition, the general pattern of
gene expression of CALM/AF10 patients when compared to other
leukemia subtypes and to normal bone marrow was dominated by a
global downregulation of genes some of them with function
identified as related to important molecular mechanisms, such as
membrane trafficking, cell growth regulation, proliferation,
differentiation and tumor suppression. 3) We cloned CALM/AF10
fusion gene into a vector that allowed us to induce the expression
of CALM/AF10 using doxycycline in transiently and
stably-transfected NIH3T3 and HEK293 cells. This system will be an
important tool to identify direct CALM/AF10 target genes and to
answer the question whether continued CALM/AF10 expression is
necessary to maintain the CALM/AF10-associated expression pattern.