Rolle von ICSBP in der Genetik BCR-ABL-induzierter Transformation und bei der Resistenzentstehung von BCR-ABL-transformierten Zellen gegenüber Imatinib

The reciprocal gene translocation t(9;22)(q34;q11) creates the Philadelphia chromosome (Ph+) carrying the BCR-ABL fusion gene. It is the causative genetic aberration of chronic myelogenous leukaemia (CML) and encodes a constitutively active protein tyrosine kinase. CML is characterized by three clinical stages including the chronic phase, the accelerated phase and the blast crisis. After a median of 3-4 years rather indolent chronic phase accelerates to blast crisis, an acute leukaemia. Although the underlying mechanisms are not fully understood, the appearance of additional genetic and/or epigenetic abnormalities in the blast phase strongly suggests that superimposed genetic alterations account for disease progression. There is mounting evidence that the family of interferon regulatory factors (IRFs), among them particularly IRF-8 (ICSBP) and IRF-4, is involved in the pathogenesis of CML. In the peripheral blood of CML patients in chronic phase the ICSBP-mRNA expression is very low or absent compared with blood from healthy donors. INF-α therapy leads to an increase of ICSBP transcripts in the treated patients. Furthermore there is a correlation between good response to INF-α and high ICSBP levels. These data suggest a role for ICSBP as a tumour suppressor and regulator of apoptosis in diseases of the myeloid system. In an attempt to address whether there is genetic evidence for the cooperation between BCR-ABL and the loss of ICSBP in the oncogenic transformation of haematopoiesis we performed a replating assay. This experiment was based on the observation that BCR ABL transduced mononuclear bone marrow cells from ICSBP-/- mice could be replated more often than the corresponding cells from ICSBP+/+ mice. Flow-sorted mononuclear bone marrow cells from ICSBP+/+ and ICSBP+/- mice were transduced with BCR-ABL. We then looked for their replating capacities in methylcellulose culture. The replating pattern did not differ significantly. Both cells from ICSBP+/+ and from ICSBP+/- mice did not form any CFUs beyond the first round of replating. These results indicate that the genetic selection pressure in the course of the replating assay is not sufficient to lead to LOH. Thus, genetic evidence for the cooperation between BCR-ABL and loss of ICSBP in transformation could not be found. In the second part of the thesis we investigated whether ICSBP has an impact on resistance to imatinib based on the observation that 32D/BA ICSBP cells are significantly more sensitive to apoptosis in the presence of imatinib than 32D/BA cells. These data suggest a role for ICSBP as a tumour suppressor and regulator of apoptosis. In an ENU based mutagenesis assay we compared the resistence pattern of 32D/BA and 32D/BA-ICSBP cells and found that ICSBP seems to increase the rate of resistance. Because ENU causes point mutations in multiple proteins, we hypothesized that in our assay, resistance to imatinib may be multifactorial. However in western blot it became obvious that the resistant clones had an active tyrosine kinase suggesting that point mutations in the kinase domain of BCR-ABL may be responsible for resistance to imatinib. Whether treatment with ENU has an impact on the increased resistance in 32D/BA ICSBP cells and by which mechanisms ICSBP leads to increased resistance to imatinib will be part of further investigation. Taken together, genetic evidence for the cooperation between BCR-ABL and ICSBP in the oncogenic transformation could not be found, and ICSBP increases the resistance rate in BCR ABL-transduced 32D cells in the presence of imatinib.