The Transcription Factors RUNX1/AML1 and RUNX3/AML2 Protect Bcr-Abl-Transformed B-Cells from Imatinib Induced Apoptosis.

The Abl-tyrosine kinase inhibitor Imatinib efficiently targets the Bcr-Abl kinase and produces major cytogenetic responses in most patients with chronic phase CML. In contrast, patients with advanced stage CML or Ph+ ALL frequently become refractory to Imatinib treatment. Resistance arises predominantly from point mutations in the Abl-kinase region, Bcr-Abl amplification or clonal evolution due to secondary genetic aberrations. To screen for genes contributing to clonal evolution, we have employed retroviral insertional mutagenesis in a murine CML/ALL model to identify potential candidate genes leading to Imatinib resistance. We found proviral insertions near the RUNX3/AML2 promoter in Imatinib resistant leukemic clones, leading to upregulation of RUNX3 mRNA expression. To analyze the effects of high RUNX3 levels on Imatinib response, we expressed RUNX3 in a Bcr-Abl-transformed murine pre-B-cell line. Significantly, whereas there was no effect on Imatinib-mediated proliferation inhibition, the cells displayed a marked reduction of apoptosis. A RUNX3R193A mutant carrying a mutation in the DNA-binding domain of RUNX3 did not protect from apoptosis, indicating that transcriptional regulation by RUNX3 was required to induce the anti-apoptotic effects. To allow for a controlled activation of RUNX transcriptional activity and to extend our analysis to other members of the RUNX family of transcription factors, we constructed 4-OH-tamoxifen (TAM) inducible RUNX3/AML2- and RUNX1/AML1-Estrogen receptor (ER) fusion proteins. These fusion proteins readily translocated from the cytoplasm into the nucleus and activated a RUNX-dependent TCRß-luciferase construct upon addition of TAM. Using these constructs, we could demonstrate that activation of RUNX3 as well as RUNX1 protected Bcr-Abl-transformed Ba/F3 cells from Imatinib-induced apoptosis. Furthermore, we found that RUNX1 mRNA levels were significantly upregulated in patients with Ph+ ALL upon resistance development. Taken together, our data indicate that elevated RUNX3 or RUNX1 levels may contribute to Imatinib resistance in Bcr-Abl expressing leukemic cells.