Genetics of AML

Internal tandem duplication (ITD)

Internal tandem duplication (ITD)

FLT3

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Activation loop

Fig. 4.1 FLT3-ITD and activation loop mutations in AML

Twenty to twenty-five percent of cases of AML have internal tandem duplications (ITD) in the juxtamembrane domain of FLT3. These can range from several to more than 50 amino acids in length, and result in ligand-independent activation of FLT3 tyrosine kinase activity. The mechanism of activation is not clear, but may be the disruption of a negative autoregulatory module in the juxtamembrane domain, with subsequent ligand-independent activation. Point mutations in the 'activation loop' occur in about 5-10% of cases of AML and result in constitutive activation of the FLT3 tyrosine kinase.

Activation loop

Fig. 4.1 FLT3-ITD and activation loop mutations in AML

Twenty to twenty-five percent of cases of AML have internal tandem duplications (ITD) in the juxtamembrane domain of FLT3. These can range from several to more than 50 amino acids in length, and result in ligand-independent activation of FLT3 tyrosine kinase activity. The mechanism of activation is not clear, but may be the disruption of a negative autoregulatory module in the juxtamembrane domain, with subsequent ligand-independent activation. Point mutations in the 'activation loop' occur in about 5-10% of cases of AML and result in constitutive activation of the FLT3 tyrosine kinase.

roviras containing a FLT3-ITD, followed by transplantation into lethally irradiated syngeneic recipient mice by tail vein injection. When this experiment is performed with various FLT3-ITDs, mice develop a myeloproliferative disease and die with a median latency of about 45 days. However, these animals never develop AML, but rather a leukocytosis with normal maturation and differentiation of myeloid lineage cells, and splenomegaly due to extramedullary hematopoiesis. The disease is not transplantable into secondary recipient mice. These data indicate that FLT3-ITDs alone are not sufficient to induce an AML phenotype in primary murine hematopoietic progenitors.

The FLT3-ITD phenotype is similar to that reported in the murine bone marrow transplantation assay for other constitutively activated tyrosine kinases associated with myeloproliferative phenotypes in humans, including BCR-ABL, TEL-PDGFßR, TEL-ABL and TEL-JAK2. Taken together, these data indicate that constitutive activation of tyrosine ki-nases is sufficient to induce a myeloproliferative phenotype, but not AML.

region, with subsequent kinase activation. In addition, mutations may also occur in the so-called activation loop of FLT3 in about 5-10% of AMLs. These also result in constitutive kinase activation. Although the structure of FLT3 is not yet available, these mutations, in the context of other tyrosine kinases, result in folding out of the activation loop, providing access of the catalytic site to ATP and substrate. There are rare examples of both ITD and activation loop mutations in the same allele of FLT3, suggesting that the combination of mutations may hyperactivate the kinase and provide added proliferative advantage to cells that harbor both mutations.

FLT3 mutant AML constitutes an important subset of AMLs that has a poor prognosis in most studies of children and adults as an independent prognostic indicator. FLT3 has not been reported as a poor prognostic indicator in adults with AML over the age of 65, which may reflect the overall worse prognosis of this group compared with younger individuals.

As one surrogate of transformation, FLT3-ITD confers in-terleukin (IL)-3-independent growth on the murine hematopoietic cell line Ba/F3, which is normally dependent on IL-3 for growth and survival. FLT3-ITDs activate several signal transduction pathways in Ba/F3 cells that are known to confer proliferative and/or survival advantage, including the RAS/ MAPK, STAT and PI3K/AKT pathways. FLT3-ITD also induces a myeloproliferative disease in primary hematopoietic progenitors in a murine bone marrow transplantation assay. In this assay, mice are treated with 5-fluorouracil to induce transient pancytopenia. Cytopenias induce hematopoietic progenitors into the cell cycle and render them susceptible to transduction with a murine ecotropic retrovirus. Bone marrow is harvested from donor animals, and transduced with ret-

Mutations associated with AML that affect hematopoietic differentiation

Core binding factor in acute leukemias

In contrast with chromosomal translocations in chronic myeloid leukemias, which almost invariably involve constitutively activated tyrosine kinases, the cloning of recurring chromosomal translocations associated with AML usually identifies fusion genes involving transcription factors or transcriptional co-activators that are important for normal hematopoietic development. Multiple translocations target the core binding factor (CBF) in acute leukemias. Of these, the most extensively studied are the AML1-ETO, CBFP-SMMHC and TEL-AML1 fusions. CBF is a heterodimeric transcription factor comprising AML1 (also known as RUNX1) and CBFP sub-units. Homozygous loss of function of either AML1 or CBFP in genetically engineered mice results in a complete lack of definitive hematopoiesis, indicating that both components of CBF are necessary for normal hematopoietic development.

One might therefore predict that an acquired gene rearrangement or mutation that resulted in CBF loss of function might impair hematopoietic differentiation. Indeed, several lines of evidence indicate that the leukemia-associated fusion genes result in loss of CBF function through dominant negative activity mediated by aberrant recruitment of the nuclear co-repressor complex. For example, the use of homologous recombination strategies to express AML1-ETO or CBFP-SMMHC from their endogenous promoters in mice results in a phenotype nearly identical to that of the AML1 or CBFP knock-outs, namely a loss of definitive hematopoiesis.

Additional evidence that implicates loss of function of CBF in the pathogenesis of leukemias has come from the analysis of pedigrees with an inherited predisposition to develop leukemia. For example, the familial platelet disorder with propensity to develop acute myeloid leukemia (FPD/AML syndrome) is an autosomal dominant disorder that is caused by haploin-sufficiency of the AML1 gene. Furthermore, in sporadic AML there are loss-of-function point mutations involving AML1 in about 3-5% of cases, most of which impair AML1 DNA binding activity. In many AML patients there is loss of function of both alleles of AML1, suggesting that homozygous loss may contribute to disease progression or severity of disease.

Although mutations and gene rearrangements affecting CBF function are clearly important in the pathogenesis of AML, in part through the disruption of normal hematopoi-etic differentiation programs, it is equally clear that they are not sufficient to cause AML. For example, conditional alleles of AML1-ETO expressed in adult hematopoietic progenitors are not sufficient to cause AML. It is necessary to treat animals with chemical mutagens such as ethyl-nitrosourea to induce AML. However, AML1-ETO expression does confer an immortalization phenotype, in that AMLl-ETO-expressing progenitors can be propagated in serial transfer assays in vitro. It is not clear whether AML1-ETO expression itself induces a transcriptional program that confers the immortalization phenotype, or whether the phenotype simply reflects a block in differentiation at the level of the hematopoietic stem cell that has self-renewal capacity.

Mutations involving retinoic acid receptor-alpha (RARa)

As with CBF, there are also multiple chromosomal translocations that involve the RARa locus. Each of these is associated with an acute promyelocytic leukemia (APL) characterized by a block in differentiation at the promyelocyte stage of hematopoietic development. The most extensively studied, and the most common, is the PML-RARa fusion associated with t(15;17). PML-RARa expression is associated with a block in differentiation due to aberrant recruitment of the nuclear co-repressor complex, similar to observations in the context of the AML1-ETO, CBFP-MYH11 and TEL-AML1 fusions. All-trans-retinoic acid (ATRA), a ligand for RARa, has proved to be an effective therapy for APL, especially when given in combination with conventional induction chemotherapy with anthracyclines and cytosine arabinoside. The efficacy of ATRA in the treatment of APL appears to be related to the ability of ATRA to bind to the fusion protein, with resultant dissociation of the nuclear co-repressor complex. Promyelocytes are then able to engage normal hematopoietic differentiation programs that ultimately result in apoptotic cell death. The efficacy of agents that induce normal differentiation, such as ATRA, has suggested that inhibitors of histone deacetylase, a key component of the nuclear co-repressor complex, might have therapeutic efficacy not only in APL, but also in other leukemias characterized by aberrant recruitment of the nuclear co-repressor complex, such as AML1-ETO and CBFP-MHY11.

Expression of PML-RARa and/or its reciprocal is not sufficient to induce AML. There are several lines of evidence that support this assertion. These include transgenic murine models of PML-RARa-induced AML. Expression of PML-RARa has been directed to the promyelocyte compartment using promyelocyte-specific promoters, including the cathepsin G promoter, and the MRP8 promoter. However, although the fusion gene is present in the germline and is expressed during embryonic and adult development, these animals do not develop AML until 3-6 months after birth, and even then with a modest penetrance of only 15-30%, and often with acquisition of secondary cytogenetic abnormalities. Although co-expression of the reciprocal RARa-PML and PML-RARa under the control of the cathepsin G promoter in double transgenic mice increases penetrance to about 60%, double transgenic mice do not have shortened latency of disease. These data indicate that second mutations are necessary in the pathogenesis of APL in this murine model system.

A second line of reasoning that supports a need for more than one mutation in the pathogenesis of APL is derived from genotyping. At least 30% of APL patients harbor activating mutations in FLT3-ITD mutations in addition to t(15;17), which gives rise to the PML-RARa fusion. These mutations are not observed in normal individuals; thus, their concordance in this context indicates that both are required for pathogenesis of APL in at least a subset of patients.

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