Bortezomib-Induced Down-Regulation of KIT Is Mediated by Inhibition of Sp1 and NF-kB in AML1/ETO-Positive Cells.

Activating mutations of KIT, encoding a type III receptor tyrosine kinase, are frequently detected in core binding factor AML (i.e., AML1/ETO and CBFB/MYH11 AML), promote cell survival and proliferation of leukemic cells and predict poor outcome. Kinase inhibitors (e.g., imatinib or PKC-412) have been shown to block constitutively activated KIT. However, novel therapeutic approaches that target mutated KIT are necessary, since resistance to these agents can be predicted in a substantial proportion of patients in these subgroups of AML. We observed that expression levels of KIT in AML1/ETO-positive Kasumi-1 cells are more than 25 fold higher than in AML1/ETO-negative AML cell lines (i.e., THP-1, K562, and MV4–11). We also found that in Kasumi-1 cells, bortezomib (Velcade), a proteasome inhibitor already used in the clinic, induces time- (60nM for 0, 1, 3, 6 12 and 24hr) and dose- (0, 1, 6, 20, 60 and 100nM for 24hr) dependent down-regulation (>90%) of total KIT expression. Noticeably, dephosphorylation of KIT occurred 6hr before reduction of the total KIT level after bortezomib exposure. We also found >50% down-regulation of KIT expression in patients’ primary blasts treated with 60nM bortezomib for 24hr and in Kasumi-1 cells treated with the proteasome inhibitor MG132 (300nM for 24hr). Down-regulation of KIT appeared to be associated with inhibition of NF-kB and Sp1, which is necessary for regulation of the KIT promoter activity by the SCL complex. In fact, treatment with bortezomib inactivated NF-kB and decreased transcription of Sp1 in Kasumi-1 cells. Furthermore, exposure to the NF-kB or Sp1 inhibitors parthenolide (30μM for 24hr) and mithramycin (100ng/ml for 24hr), respectively, resulted in a dose-dependent decrease in KIT expression in Kasumi-1 cells. When cells were treated with bortezomib (20nM) in combination with mithramycin (30ng/ml for 24hr), we observed synergy in down-regulation of KIT RNA and KIT protein. This correlated with growth arrest and increased cell death. Interestingly, the magnitude of these effects was higher when Kasumi-1 cells were pretreated with mithramycin for 24hr before being exposed to bortezomib. Taken all together, our data suggest that bortezomib downregulates KIT expression and might also inhibit KIT phosphorylation and should be considered in future therapeutic strategies targeting AML subgroups harboring mutated KIT.