Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

[摘要] Multiple Myeloma (MM) is an incurable plasma cell malignancy and, although novel treatment regimes in the past decade have improved patient outcome, long-term treatment leads to relapse and refractory disease. The centrosomal kinase NEK2 is found overexpressed in MM and promotes chromosomal instability, drug resistance and increased proliferation. Although much research shows NEK2 having a detrimental effect in cancer, much of its mechanisms of overexpression and drug resistance has not been studied in detail. In this work we expand our understanding of NEK2 in MM. Using Tandem Affinity Purification coupled with Mass Spectrometry, we show that NEK2 directly interacts with the de-ubiquitinase USP7. We confirm this interaction in cell lines of MM and lung cancer. Since USP7 has been shown to have important cancer-promoting roles we tested if USP7 was necessary for NEK2-driven bortezomib resistance. We found that USP7 shRNA was sufficient to sensitize the bortezomib resistant NEK2 overexpressing cells to bortezomib. Surprisingly, we found that USP7 inhibition with shRNA or by treatment with the small molecule USP7 inhibitor P5091 led to depletion of NEK2 protein in every cell line tested. Previous research shows USP7's main function is a de-ubiquitinase and, since NEK2 is a target of the ubiquitin-proteasome system, we hypothesized USP7 may be de-ubiquitinating NEK2. Through western blots and immunoprecipitations, we show the NEK2-USP7 interaction promotes the de-ubiquitination and subsequent stabilization of NEK2, presenting USP7 as the first discovered de-ubiquitinating enzyme of NEK2. To understand how NEK2 promotes drug resistance in cancer we studied a previously published list of NEK2-regulated genes and, using the UCSC genome browser (Track Name:GM12878+TNFa RELA) ChIP-seq data, we found approximately half of these genes have the NF-κB transcription factor p65 bound throughout the gene sequence. We also produced a signaling score using an average of 11 known targets of NF-κB and patients with high NEK2 showed a significantly increased score of NF-κB signaling. Additionally, through western blots and immunofluorescence, we found that patients with high NEK2 protein levels consisitently had activation higher signal of p65 protein and phosphorylated p65 at Serine 536, indicative of increased activity. We then causally show NEK2 activates canonical NF-κB by performing western blots and a dual-luciferase reporter assay on control and NEK2 overexpressing cells. Using AKT and PP1α inhibitors, we found that NEK2 drives NF-κB by phosphorylating and inactivating PP1α, leading to hyperactive AKT. Using this model of NEK2-NF-κB activation, we aimed at targeting NEK2 directly with the

[发布机构] University of Iowa