The Molecular Pathology of Pediatric Acute Leukemia
St. Jude Children's Hospital
A major goal of leukemia research has been to gain a better understanding of the genetic changes
responsible for the establishment of the leukemic clone. This pursuit is fueled by the hope that the
information obtained will not only help us to understand the differences in clinical response that are
observed among leukemic patients, but will also lead to the identification of rational molecular targets
against which novel therapeutic agents can be developed. One of the major insights into the underlying
pathogenesis of acute leukemia has come from the careful analysis of chromosomal changes within the
leukemic cells. These studies have revealed the presence of clonal chromosomal translocations and
inversions in greater than 50% of cases. Efforts to determine the targets of these abnormalities have
identified a series of genes that play critical roles in the normal development of the hematopoietic
The genes encoding the AML1/CBFb transcription factor complex are among the most frequent targets of
genetic alterations in pediatric and adult acute leukemia
. AML1 is
targeted by the t(8;21) and t(16;21) in acute myeloid leukemia (AML), the t(3;21) in myelodysplatic
syndrome, and the t(12;21) in pediatric acute lymphoblastic leukemia (ALL), where as CBFb is the target of the AML-associated chromosomal rearrangements, inv(16) and
t(16;16). In addition, germ-line mutations of AML1 are the underlying
abnormality in a familial platelet disorder in which the patients have a high predisposition to develop
AML, and somatic mutations in AML1 occur in rare cases of sporadic AML.
Collectively these leukemias are referred to as core binding factor (CBF) leukemias.
Work performed in my laboratory, as well as others, demonstrated that AML1/CBFb functions as a master
regulatory switch that establishes a transcriptional cascade necessary for the development of the
definitive hematopoietic stem cell (HSC) . To explore the leukemia potential of translocation-encoded
AML1 chimeric genes, we generated mice with an AML1-ETO fusion gene by knocking ETO into the AML1 genomic locus . Expression of AML1-ETO induced an embryonic lethal
phenotype similar to that resulting from the loss of AML1/CBFb. However, although AML1/CBFb-deficient
embryos lacked detectable hematopoietic progenitors, fetal livers from AML1-ETO expressing embryos
contained dysplastic hematopoietic progenitors that had a high self-renewal capacity. These cells,
however, failed to result in overt leukemias either within the developing embryos, or when transplanted
into recipient mice. Thus, expression of AML1-ETO was insufficient to induce leukemia, but instead
altered the self-renewal capacity of HSC resulting in a preleukemic population.
To further define the leukemic potential of AML1-ETO, we generated mice that contain a conditional
AML1-ETO knock-in allele, in which a strong transcriptional stop cassette
bracketed by loxP sites was placed 5' to the AML1-ETO fusion . To induce the activation of the allele in vivo, the mice were crossed with a murine line that contains an
interferon-inducible Mx1-Cre transgene. Induction of Cre expression in
double transgenic mice resulted in the efficient expression of AML1-ETO within HSC and their mature
progeny. Interestingly, expression of AML1-ETO in these adult mice resulted in only minor perturbation
within the hematopoietic system. Moreover, no overt leukemia developed during the first year. However,
when mice were mutagenized with ENU at a dose that induced only rare T-cell leukemias in control animals,
35% of the AML1-ETO expressing mice developed an AML that closely resembled human t(8;21)-containing AML.
An important insight into the nature of the secondary mutation that cooperated with AML1-ETO in this
experimental system arose from the observation that the non-leukemic AML1-ETO expressing cell lines were
cytokine-dependent, where as cell lines derived from the murine AML1-ETO expressing leukemias were
factor-independent. These data strongly suggest that one signaling pathway that collaborates with
AML1-ETO is cytokine- or growth factor-mediated proliferation or survival. To further assess this
possibility, we analyzed 31 human CBF leukemias and a similar number of control non-CBF leukemias for
evidence of activating mutations in N-RAS, K-RAS, and c-KIT. Remarkably, 48% of the CBF
leukemias contained activating mutations in N-RAS or K-RAS, and an additional 13% had c-KIT activating mutations, where as the
frequency of these mutations in the non-CBF leukemias were 13% and 0%, respectively (unpublished
observation, Sun and Downing). Thus, activating mutations in these growth factor signaling molecules
appears to be a frequent even in CBF leukemias. These data suggest that inhibitors of either the
AML1/CBFb transcriptional cascade or RAS-mediated signaling pathways may be an effective means to inhibit
the growth of leukemic cells containing these genetic lesions. These hypotheses are being tested in
murine models engineered to contain these specific genetic lesions. In addition, the conditional
AML1-ETO mouse is being used in genetic screens to determine the range of mutations that can cooperate
with the expression of AML1-ETO to induce leukemia. Any lesions identified through these murine screens
will be assessed in human AML1-ETO leukemias to see if they contribute to the molecular pathology of this
A second area of investigation that will provide important insights into the molecular mechanisms of
leukemogenesis is the analysis of the expression profiles of primary human leukemia samples
studies performed in my laboratory, expression profiles have been obtained from a large number of
and AMLs. Specifically, three human leukemia gene expression datasets have been
generated: (i) An ALL expression database containing the expression profiles of over 327 diagnostic bone
marrow samples using the Affymetrix U95Av2 microarray (http://www.stjuderesearch.org/data/ALL1), (ii) An ALL
expression database containing the expression profiles of 132 diagnostic bone marrow samples obtained
using the Affymetrix U133A and B microarrays (http://www.stjuderesearch.org/data/ALL3), and (iii) an
AML expression database containing the expression profiles from 130 pediatric and 20 adult diagnostic
bone marrow samples obtained using the Affymetrix U133A microarray (in press). The analysis of these
databases have demonstrate specific gene expression profiles for each of the known prognostically and
therapeutically relevant subgroups of childhood ALL and AML. Distinct gene expression profiles were
identified for ALL blasts with T-lineage, hyperdiploid >50 chromosomes, BCR-ABL, E2A-PBX1, TEL-AML1, and MLL gene rearrangement, where as in
AML, distinct expression profiles were identified for AML blasts with t(15;17)[PML-RARa], t(8;21)[AML1-ETO], inv(16)[CBFb-MYH11], and MLL gene rearrangement. Moreover, using a variety of
computer-assisted supervised learning algorithms, the single platform of expression profiling was shown
to accurately diagnosis the various leukemia subtypes with an overall accuracy of 96%. Remarkably, this
level of accuracy exceeds that typically achieved using a combination of contemporary diagnostic
approaches, suggesting that microarray–based gene expression profiling may provide a viable approach for
the front-line diagnostic work up of patients with acute leukemia.
The identified expression profiles also suggested new insights into the pathogenesis of these
diseases. However, sorting through this data to identify those genes whose altered expression is
mechanistically involved in disease pathogenesis will be a significant challenge. This analysis is
likely to benefit from cross-species comparisons between the human leukemias and murine models of the
same disease. The ability to specifically program into mice the identical genetic lesions found in the
human disease, coupled with our ability to follow the murine disease from a preleukemic phase to overt
leukemia, should help to identify the alterations in gene expression that are involved in disease
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