Allosteric inhibition of proteolytic and ATPase activities of the proteasome by the BTK inhibitor CGI-1746
Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib has been shown to synergize in vitro with proteasome inhibitors (PIs) in reducing the viability of cells derived from B-cell malignancies, but the mechanism is not known. We report here that an off-target effect of Ibrutinib causes synergy because not all BTK inhibitors exhibited the synergistic effect, and those that synergized did so even in cells that do not express BTK. The allosteric BTK inhibitor CGI-1746 showed the strongest synergy. Co-treatment of cells with CGI-1746 increased PI-induced expression of heat shock proteins and accumulation of ubiquitin conjugates. The CGI-1746 inhibited the degradation of a model substrate by a purified 26S proteasome. CGI-1746, but not other BTK inhibitors, allosterically inhibited ATPase activity and ?2 and ?1 peptidase activities of the 26S proteasomes. Although the effect is unlikely to contribute to the anti-neoplastic activity of BTK inhibitors in multiple myeloma and mantle cell lymphoma, it demonstrates a conceptually novel mode of inhibition that may aid the development of more potent proteasome inhibitors and improve response in solid tumors clinically. Overall design: MDA-MB-231 cells were treated for four hours with CGI-1746, LU-102, a combination of the two, or bortezomib as a control for pathways changed by proteasome inhibition. RNA was extracted using the Qiagen RNeasy Mini Kit according to the manufacturer's protocol, frozen, and shipped on dry ice to LCSciences (Houston, TX) for total RNA sequencing (150 bp PE, 40 million reads per sample).