The Imatinib and Nilotinib Induced Modulation of the Proteasomal Activity and Antigen Processing in Chronic Myeloid Leukemia Cells

Abstract 2748

The tyrosine kinase inhibitors (TKIs) Imatinib mesylate (IM, Gleevec, Glivec) and nilotinib (NI, Tasigna, AMN) are currently used in treatment of chronic myeloid leukaemia (CML). IM has been described to influence the function and differentiation of antigen presenting cells, to inhibit the effector function of T lymphocytes and to decrease the immunogenicity of CML cells by downregulation of tumor associated antigens. In the present study, we analyzed the effect of IM and NI on proteasomal activity in IM-sensitive or IM/NI- resistant CML cells as well as in patient samples using a biotinylated active site-directed probe, which, covalently binds and labels proteasomal subunits beta-1, beta-2 and beta-5 and their immunosubunit counterparts beta-1i, beta-2i and beta-5i, in an activity-dependent fashion. Incubation of CML cell lines and primary CML cells with IM or NI resulted in a concentration dependent inhibition of proteasomal activity that was independent of BCR-ABL, as these effects were observed in TKI-resistant and BCR-ABL negative cells. In addition, these effects were not due to a downregulation of the expression of proteasomal subunits as analyzed by Western blot and independent of apoptosis induction. To further analyze and confirm the direct effects of TKIs on proteasomal function, isolated h20S proteasome assays were performed. In line with the results of activity site labeling, IM or NI treated isolated h20S, showed a reduced concentration-dependent activity, as measured by fluorometric cleavage of the substrate suc-leu-leu-val-tyr-AMC. Furthermore, incubation of purified h20S proteasomes with serine or tyrosine specific phosphatases reduced the proteasomal activity as observed with the TKIs, indicating that serine and tyrosine phosphorylation plays an important role in the regulation of the proteasome function. In the next set of experiments we analyzed the effects of IM and NI on the proteasomal generation of antigenic peptides derived from BCR-ABL. In in vitro digestion experiments using purified proteasomes in the presence of NI or IM, we found that the treatment of immunoproteasome i20S with the TKIs almost completely abolished the generation of the long precursor peptides for the HLA-A3/A11 (KQSSKALQR) and the HLA-B8 (GFKQSSKAL) epitopes, while the cleavage of the short peptides significantly increased. Both epitopes have been shown to be naturally processed and presented in CML cells. However, we show that both epitopes can be generated as N-terminal elongated precursors in vitro by both constitutive and immuno-20S proteasomes. Interestingly, in all performed experiments NI was more effective as compared to IM, while other TKIs had no effect. Our results demonstrate that treatment with IM and NI can affect the immunogenicity of malignant cells by affecting proteasomal degradation of cytosolic antigens, thereby modulating the repertoire of presented antigens. These strong effects of the TKIs IM and NI on proteasomal activity might be a result of changes in the phosphorylation of proteasomal subunits akin to the recently showed endogenous phosphorylation sites of the mammalian 20S proteasome.