Multistep pathogenesis of AML

These data suggest that at least two mutations are required for the development of AML. Genotypic analysis of known leukemia oncogenes indicates that there are at least two broad complementation groups of mutations. One class of mutations, exemplified by FLT3-ITD or oncogenic RAS mutations, confer a proliferative and/or survival advantage on hematopoietic progenitors, but have minimal effects on differentiation programs in hematopoietic progenitors. These mutations are each relatively frequent in AML, but only very rarely are observed together in the same patient. In contrast, mutations resulting in loss of function of hematopoietic transcription factors result in a block in differentiation at a specific stage in hematopoietic development, as exemplified by the PML-RARa fusion, which is associated with a block in differentiation at the promyelocyte stage. Again, although CBF mutations and PML-RARa mutations occur in a significant proportion of AML patients, they are never observed together in the same patient, suggesting that they also constitute a complementation group. These mutations may also confer an immortalization phenotype, but are not sufficient to cause acute myeloid leukemia. On the basis of these observations, and genotype data, a model emerges for the pathogenesis of AML in which there are at least two broad classes or complementation groups of mutations. When mutations that confer proliferative and/or survival advantage are expressed alone, they result in a myeloproliferative disease with leuko-cytosis and normal differentiation. When mutations such as AML1-ETO are expressed alone, they impair differentiation and confer an immortalization phenotype, reminiscent of the behavior of myelodysplastic cells. In this model, co-expression of a mutant that confers a proliferative and/or survival advantage, such as an FLT3-ITD, and a mutation than impairs hematopoietic differentiation, such as PML-RARa, results in AML (Figure 4.2).

To test this hypothesis, bone marrow from transgenic C3H/C57BL6 mice expressing the PML-RARa fusion under

Proliferation and survival Mutations that impair mutations differentiation the control of the cathepsin G promoter was harvested and transduced with retrovirus containing an FLT3-ITD mutant. In control experiments, PML-RARa transgenic bone marrow transduced with an empty vector control resulted in an APL-like disease in secondary recipient mice, with a latency of approximately 6 months and a penetrance of about 15-30%, in agreement with previous reports. In an additional control experiment, FLT3-ITD retrovirus transduced into the C3H/ C57BL6 wild-type background resulted in T-cell lymphomas with a latency of 3-6 months (not shown).

PML-RARa bone marrow transduced with FLT3-ITD resulted in a shortened latency, with 100% penetrance of disease (Figure 4.3). This murine model of cooperativity not only provides experimental evidence for cooperation between these two types of mutations, but also provides a model in which FLT3 inhibitors can be tested alone and in combination with ATRA in the treatment of APL. Indeed, initial data in a similar system using MRP8-PML-RARa transgenic mice and FLT3-ITD indicate that small-molecule inhibitors of FLT3 have at least additive effects with ATRA in treating APL in this model system. Additional experimentation will be

Rx Rx with

FLT3 inhibitors ATRA, HDAC

prenylation inhibitors inhibitors

Fig. 4.2 Therapeutic insights from a multistep model of pathogenesis of AML

AML appears to be due to cooperation between at least two broa d classes of mutations. The first class, exemplified by activating mutations in FLT3 or oncogenic RAS, confers a proliferative and/or survival advantage on hematopoietic progenitors but does not affect differentiation. A corollary of the hypothesis, supported by murine models of disease, is that expression of FLT3-ITD alone would result in a myeloproliferative phenotype. A second class of mutations, exemplified by loss-of-function mutations in hematopoietic transcription factors such as core binding factor (CBF) and the PML-RARa fusion, results in impaired hematopoietic differentiation and may confer an immortalization phenotype due to the inability to undergo terminal differentiation and apoptosis. These mutations alone are not sufficient to cause AML, but appear to confer a phenotype most similar to myelodysplastic syndrome. Together, these two mutations would result in the acute myeloid leukemia phenotype, characterized by a proliferative and/or survival advantage of hematopoietic progenitors and by impaired hematopoietic differentiation. The hypothesis has important clinical therapeutic implications. For example, targeting the proliferative and survival pathways with FLT3 inhibitors or with inhibitors of RAS, such as farnesyltransferase inhibitors (FTI), might have therapeutic benefit. Alternatively, agents that relieve the block in differentiation in AML, as ATRA does in APL, may have therapeutic benefit.

100 80 60 40 20 0



empty vector n =20

0 25 50 75 100 125 150 175 200 225 Days

Fig. 4.3 Cooperativity between FLT3-ITD and PML-RARa in the induction of an APL-like disease in a murine model

About 30-40% of human APL patients have both the t(15;17) mutation, which gives rise to the PML-RARa fusion, and the FLT3-ITD mutation. Expression of PML-RARa in the promyelocyte compartment under the control of the cathepsin G promoter results in the development of leukemia with a long latency (3-6 months) and incomplete penetrance (15-30%), strongly indicating the need for a second mutation in disease pathogenesis. Retroviral transduction of FLT3-ITD into cathepsin G-PML-RARa-transgenic bone marrow results in shortened latency and 100% penetrance, indicating cooperativity between FLT3-ITD and PML-RARa in the induction of APL. These data indicate that FLT3-ITD and PML-RARa may both be targets for molecularly targeted therapy with FLT3-specific small-molecule inhibitors and ATRA, respectively. APL patients with FLT3-ITD appear to have a worse prognosis than FLT3-negative patients, suggesting that introduction of FLT3-ITD inhibitors could have therapeutic benefit in this subgroup of patients.

required to assess the transforming properties of oncogenic RAS expressed from its endogenous reporter, and co-expression of oncogenic RAS with potential cooperating mutations. Overall, these data support the multistep model of disease described in Figure 4.2.

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