One of the key questions that arise during the study of leukemia-associated rearrangements and especially during the analysis of fusion genes is whether the fusion gene is capable of causing leukemia. The question whether a given (fusion) oncogene is actually able to transform hematopoietic cells is not easy to answer. The transforming potential of a fusion gene can be analyzed in cell line systems or in animal models.
The murine pro-B cell line Ba/F3 and fibroblast cell lines like NIH-3T3 or Rat-1 are some of the cellular systems used for the study of the oncogenic potential of fusion genes or mutated genes, especially in the case of constitutively activated tyrosine kinases. Ba/F3 cells are interleukin-3 (IL3) dependent and can only proliferate in the presence of IL3. The fibroblast cell lines (eg.
NIH3T3) are anchorage dependent and need to attach to the surface of the culture flask to proliferate. As an indication of its transformation potential the expression of a putative oncogene can induce IL3 independent proliferation of Ba/F3 cells or anchorage independent growth of the fibroblasts in soft agar.
As mentioned above, these two cellular systems are especially suitable to test the activity of activated tyrosine kinases. However, these cell line systems are not sufficient to accurately measure the transformation potential of all types of fusion genes or oncogenes, and they can not recapitulate the complexity of tumor development in the organism. To obtain a more complete
understanding of the true tumorogenic potential of a given oncogene whole animal models like bone marrow transplantation (BMT) models, transgenic animals and knock-in models are available. Bone marrow transplantation models, in which the oncogene is retrovirally transduced into primary bone marrow cells which are then transplanted into lethally irradiated syngeneic mice, are especially useful to assay the potential of an oncogene to induce tumors in the hematopoietic systems.
It should be kept in mind that in many cases the read outs obtained in the cell line systems do not match the read outs obtained from the animal model systems. For example, the transformation potentials of the BCR/ABL1 and the ETV6/ABL1 fusiongenes are more or less similar in the Ba/F3 cell line system, but in the bone marrow transplant (BMT) mouse models these two ABL1 fusions induce different diseases with different latency periods. The recipients (Balb-c mice) of BCR/ABL1 transduced bone marrow develop a CML-like disease, whereas the recipients of ETV6/ABL1 expressing bone marrow cells did not develop leukemia but rather a myeloproliferative syndrome or a small bowel syndrome (Golub et al., 1996; Million et al., 2002). These differences were also observed for the NUP214/ABL1 fusion. The NUP214/ABL1 fusion induces very weak IL3 independent growth of the Ba/F3 cells compared to the BCR/ABL1 fusion. However, in the BM transplanted mice a strong ALL phenotype is seen (De Keersmaecker et al., 2008).
Thus, for the study of the oncogenic potential of particular genetic lesion, animal models are more realistic. However, these model systems also have their limitations. For example, in BMT models, the gene, which is retrovirally transduced in the bone marrow cells, randomly integrates in the genome of these cells. These random integrations may disrupt or activate neighboring tumor suppressor gene or oncogene, respectively, thereby altering the phenotype. In addition, animal models are very expensive and time consuming to establish and to evaluate. This is especially true for the transgenic and knock-in models.
4.5.1 Behavior of SHIP1/ABL1 in Ba/F3 cells: SHIP1/ABL1 induces IL3 independent proliferation in a murine pro-B
cell line, which is inhibited by the tyrosine kinase inhibitor Imatinib
To obtain an initial cellular read out of the oncogenic potential of the SHIP1/ABL1 fusion, we used the IL3-dependent Ba/F3 cell line system. We expressed the SHIP1/ABL1 and the BCR/ABL1 fusion in IL3 dependent Ba/F3 cells. The expression of SHIP1/ABL1 induced IL3 independent proliferation of Ba/F3 cells, which was comparable with that induced by the BCR/ABL1 fusion. The IL3 independent growth induced by the SHIP1/ABL1 fusion and the fact that the SHIP1/ABL1 protein contains the same portion of ABL1 as the other ABL1 fusions, suggested that the ABL1 tyrosine kinase is constitutively activated in the SHIP1/ABL1 fusion protein. To assess this possibility, we analyzed the sensitivity of the SHIP1/ABL1 fusion to the tyrosine kinase inhibitor STI571 (Imatinib).
STI571, also known as Imatinib, is an ABL1 specific tyrosine kinase inhibitor as described in the introduction. All ABL1 fusions are sensitive to Imatinib. We assessed the sensitivity of SHIP1/ABL1 and BCR/ABL1 by proliferation assays using the Ba/F3 cell line. As expected, the IL3-independent proliferation of SHIP1/ABL1 expressing Ba/F3 cells could be inhibited by Imatinib treatment. Interestingly, SHIP1/ABL1 expressing Ba/F3 cells were significantly more sensitive to Imatinib than BCR/ABL1 expressing cells. The rate of proliferation was much lower in the SHIP1/ABL1 expressing Ba/F3 cells compared to that in the Ba/F3 cells expressing BCR/ABL1 at a particular concentration of Imatinib. The Imatinib concentrations required to inhibit the proliferation of SHIP1/ABL1 expressing Ba/F3 cells by 50% (inhibitory concentration 50, IC50) was less than one-fourth of that required in case of the BCR/ABL1-transduced Ba/F3 cells. The IC50 IMATNIB for SHIP1/ABL1 was 0.16 µM and that for BCR/ABL1 was 0.71 µM. These results clearly demonstrated that the ABL1 tyrosine kinase is constitutively active in the SHIP1/ABL1 fusion.
This higher sensitivity of SHIP1/ABL1 to Imatinib compared to that of BCR/ABL1 is surprising and difficult to explain. One explanation could be that, because of the strong tetramerization domain of BCR, the tyrosine activity of
the BCR/ABL1 fusion protein is more strongly activated than that of the SHIP1/ABL1 fusion protein.
To better understand these results and the leukemogenic mechanisms used by the SHIP1/ABL1 fusion, it might be helpful to look at the mechanism of ABL1 tyrosine kinase activation in the other ABL1 fusions and to examine more closely the protein domains in the SHIP1 portion of the SHIP1/ABL1 fusion.
4.6 THE N-TERMINAL PORTION OF SHIP1 CONTAINS TWO