Imatinib Inhibits Proteasomal Activity in CML Cells.
Authors: Held Stefanie A.E. , Brauer Katharina M. , Rückrich Thomas , Werth Daniela , von Schwarzenberg Karin , Bringmann Anita , Kanz Lothar , Grünebach Frank , Driessen Christoph , Brossart Peter 
Published: October 2019

Abstract
Imatinib mesylate (Gleevec) is a specific tyrosine kinase inhibitor, which inhibits phosphorylation of downstream proteins involved in BCR-ABL signal transduction. In the treatment of CML it has become indispensable and shows few side effects. Nevertheless, imatinib has been described to influence the function and differentiation of APCs, inhibit the effector function of T lymphocytes and decrease the immunogenicity of CML cells. In the present study, we analyzed the possible effects of imatinib on proteasomal activity in CML cells. Proteasomal activity was determined using a biotinylated active site-directed probe which, depending on their activity, covalently binds and labels proteasomal subunits beta-1, beta-2 and beta-5. We show that Imatinib treatment leads to a decreased activity of the proteasome in BCR-ABL positive cells. Imatinib sensitive K-562 cells as well as HLA-A2 transfected K-562 cells displayed a profound downregulation of proteasome activity at sub-therapeutic concentrations of imatinib. This effect was not due to downregulation of the protein expression of proteasome subunits or the induction of apoptosis. In imatinib resistant K-562R cells a higher concentration of imatinib was required for a detectable inhibition of proteasomal activity corresponding to a higher expression of BCR-ABL in these cells. Interestingly, the use of specific siRNA against the BCR-ABL fusion site b3a2 to downregulate the expression of the protein in K-562 and K-562R cells did not influence the activity of the proteasome indicating that this effect is independent of the expression and activity of BCR-ABL. In line with these results transfection of BaF/3 cells with BCR-ABL had no effect on proteasomal activity. To further analyze the mechanisms involved in proteasome inhibition by imatinib we performed activity assays with isolated 20S and 26S proteasomes. Incubation of both proteasome subtypes with imatinib resulted in a reduced acitivity in a concentration dependent manner as shown by reduced proteolytic cleavage of fluorogenic substrates. As the reduction in activity was seen in the ATP-independent 20S proteasome as well as the ATP-dependent 26S proteasome, a competition of imatinib with ATP similar to the mechanism of BCR-ABL inhibition is not responsible for this effect. In order to expand these studies, we additionally examined HLA class I expression on the cell surface to determine the influence of imatinib and subsequent proteasome inhibition on antigen presentation. Correlating with the inhibition of proteasome activity, HLA class I expression was strongly reduced on HLA-A2 transfected K-562 cells after imatinib treatment. Taken together these findings show that imatinib directly affects proteasomal activity in vitro. This effect is more pronounced in BCR-ABL positive cells, which might be due to BCR-ABL mediated deregulation of cellular pathways, but seems to be, however, independent of BCR-ABL.