Novel mechanism of inhibition of the proteasome by Bruton’s Tyrosine Kinase inhibitors

Abstract

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. This synergy was also observed in cells that do not express BTK and in cells that do express BTK, synergy was observed at concentrations much higher than the concentration at which BTK activity was completely inhibited; however, the mechanism responsible for synergy is not known. We report here that an off-target effect of Ibrutinib causes synergy and another BTK inhibitor, CGI-1746, showed even stronger synergy. We reveal through inhibition of the degradation of a model substrate by a purified 26S proteasome as well as inhibition of classical fluorescent proteasome substrates and inhibition of ATP hydrolysis by the 19S subunit of the proteasome, that CGI-1746 allosterically inhibits the proteasome’s ATPase and peptidase activities. We show that the inhibition of the proteasome by CGI-1746 is responsible for the synergy with proteasome inhibitors by comparing the cytotoxic mechanisms governing cell death due to CGI-1746 with FDA-approved proteasome inhibitors. We thereby demonstrate a conceptually novel mode of inhibition that may aid the development of more potent proteasome inhibitors and improve response in solid tumors clinically. We also demonstrate, using CRISPR mutated cell lines, that multi-site inhibition of the proteasome is more efficacious in reducing cancer cell viability compared to single-site inhibition and allows for significant dose reduction which may mitigate off-target adverse events. In addition, through knockdown of small nucleolar RNAs and proteomic analysis, we elucidate a previously unknown non-coding RNA mechanism that regulates proteostasis and modulates the response of cancer cells to proteasome inhibition