Alternative splicing of BCR/ABL in chronic myelogenous leukemia

grantor: University of TorontoThe hallmark of chronic myelogenous leukemia (CML) is the presence of the Philadelphia chromosome and its resultant fusion message, 'BCR-ABL ', and fusion protein, p210. Patients with Philadelphia positive acute lymphoblastic leukemia (ALL) can have a smaller 'BCR-ABL' fusion transcript possessing only the first exon of 'BCR' fused to ' ABL'. This smaller transcript encodes a 190 kD protein that is more strongly transforming than the p210 protein derived from the larger CML-associated transcript. We examined the peripheral blood or marrow of 67 patients with CML and found that 35 of them expressed both transcripts while the remainder expressed the p210-encoding transcript exclusively. A comparison of patients co-expressing the p190- and p210-encoding transcripts with those patients who only expressed the p210-encoding transcript detected significantly higher white blood cell (WBC) counts and blast cell counts at time of testing as well as significantly higher white blood cell counts at diagnosis. We believe that the expression of the P190-encoding transcript in the chronic phase of CML is due to alternative splicing. 'BCR' and 'BCR'/'ABL' minigenes were constructed to study this phenomenon in more detail. These constructs were transfected into various cell types and splicing was assessed by RT-PCR. While the basic ' BCR' minigene expressed exon-inclusive transcripts only, insertion of genomic DNA spanning 'ABL' exon 2 induced exon-skipping but only when expressed in the CML cell lines K562 or EM3. In this study we localize the required sequence element to 'ABL' exon 2 itself. These results mimic the splicing phenotype displayed by most CML patients. RNA-binding assays using nuclear extracts from both CML and non-CML cells detected differential binding to 'ABL' exon 2. We propose a model where trans-factors present in some CML cells interact with ABL exon 2 pre-mRNA to promote skipping of upstream BCR exons.Ph.D