Mechanism of blastic transformation in CML

Chronic myeloid leukemia (CML) is caused by BCR/ABL, a constitutively active tyrosine kinase that is the result of the t(9;22)(q34;q11) translocation. BCR-ABL activates multiple signaling pathways, leading to increased proliferation, reduced apoptosis, and genetic instability. The clinical course of CML usually is two- or three- phased. During the chronic phase (CP), the myeloid cell compartment is expanded, but differentiation is maintained. After a variable length of time the disease progresses to blast crisis (BC), resembling an acute leukemia with myeloid or lymphoid phenotype that is often poorly responsive to therapy. Progression to BC may occur suddenly or through an intermediate stage termed accelerated phase (AP). Although imatinib has dramatically improved the therapy of CML, there is a certain rate of progression to BC even in newly diagnosed CP patients. Additionally, most patients continue to harbor minimal residual disease, and several of them relapse after a variable latency period. Thus, further improvement of therapy is warranted to eliminate mortality from CML. Although important clues have emerged, a fundamental mechanistic explanation of disease progression to BC has yet to be established. The hallmark of BC is loss of differentiation capacity, the molecular basis of which is not well understood. We hypothesize that disruption of the function of a critical transcription factor or transcriptional co-activator underlies the loss of differentiation capacity at transformation of CP CML to BC. The major aim of the current project is to define the molecular mechanisms underlying disease progression in CML. In a first step, we will combine an inducible model of CP CML with insertional mutagenesis (IM) to identify genetic lesions capable of cooperating with BCR-ABL to induce BC. Given that cooperating lesions in AML have been detected by IM, we expect to identify such lesions with our system. Through this approach, we anticipate identifying a number of candidate genes relevant for the differentiation block that contributes to BC. Priority would be given to recurrent integration sites and genes with a function that might explain the differentiation block, particularly transcription factors (TFs) or proteins that regulate transcription (transcriptional inhibitors or transcriptional co-activators). In a second step, we will validate respective candidate genes in a murine leukemia model. In case of genes with increased expression, we will clone the gene of interest into a retroviral expression vector. Bone marrow from trangenic mice and controls will be infected with this retrovirus and transplanted into lethally irradiated recipients. After stable engraftment, the mice will be monitored for leukemia. Further experiments will be performed to test the expression and function of identified genes in primary CML cells. For this purpose, samples from patients with CP, AP, and BC CML will be compared for expression of candidate genes. The functional significance of expression or loss of function of such molecules will be determined in approprite bioassays employing specific siRNA and/or antisense oligonucleotides (ASO), and in case of tumor suppressors, by transducing respective genes into leukemic cells by lentiviral-mediated gene transfer. The long term aim will be to identify pharmacologic inhibitors/modulators targeting gene products responsible for transformation to BC. It can be expected that the results obtained in this project will enhance our understanding of the mechanisms that underly transformation of CP to BC, which may be used as a rational basis for the development of novel treatment strategies.