TRIP13 Modulates Protein Deubiquitination to Promote Myeloma Progression

Thyroid Hormone Receptor 13 (TRIP13) is an exciting emerging epitope in numerous cancers and is related to genomic integrity. In multiple myeloma (MM), TRIP13 is the third most upregulated gene in the 70-gene high-risk signature and is one of the top 10 genes correlated with drug resistance and disease relapse. Expression of TRIP13 is elevated in MM cells, when compared to normal, smoldering MM, and MGUS plasma cells. MM patients with elevated TRIP13 expression had inferior event-free and overall survival. Here we introduce a novel role for TRIP13 in the modulation of ubiquitin homeostasis and in its contribution to cancer progression.

Because TRIP13 has been shown to affect MAD2 stability in a proteasome-dependent fashion, we hypothesized that TRIP13 may modulate protein ubiquitination. The overexpression of TRIP13 in MM cell lines resulted in decreased cellular ubiquitination and similar decreases in ubiquitin were found in vivo upon the creation of a TRIP13 transgenic mouse (TRIP13^TG). Conversely, targeted knockdown of TRIP13 through the expression of shRNA resulted in elevated cellular ubiquitin. While these results indicate that TRIP13 promotes deubiquitination, TRIP13 is known to have ATPase activity but not protease activity. Using in vitro deubiquitination assays we found that TRIP13 lacked endogenous deubiquitination capacity. However, TRIP13 did increase the efficiency of ubiquitin cleavage by USP7.

To investigate the phenotypic outcome of TRIP13 overexpression, we crossed TRIP13^TG mice with the Em-MYC mice, which spontaneously develop diffuse large B cell lymphoma (DLBCL) and/or Burkitts' lymphoma. Kaplan-Meier analyses demonstrated that expression of TRIP13 ^TG/ Em-MYC ^TG decreased overall survival by greater than 25% compared to TRIP13 ^WT/ Em-MYC ^TG mice. Treatment of TRIP13 ^TG/ Em-MYC ^TG mice with a USP7 inhibitor, P5091, rescued this phenotype, restored overall survival to nearly that of TRIP13 ^WT/ Em-MYC ^TG mice. These data indicate that USP7 plays a key role in TRIP13-induced tumor progression. We further investigated whether TRIP13 regulates USP7 important substrates, such as p53 and PTEN. RNA-sequencing analysis was performed on splenic premalignant B cells derived from both TRIP13 ^TG/ Em-MYC ^TG and TRIP13 ^WT/ Em-MYC ^TG mice. GSEA identified that p53 and PTEN signaling pathways were significant inhibited in TRIP13 ^TG/ Em-MYC ^TG mice compared to TRIP13 ^WT/ Em-MYC ^TG mice. Western blots confirmed that p53 protein was decreased in TRIP13 ^TG/ Em-MYC ^TG mouse tissues. Using cellular fractionation and immunoblotting, we found that PTEN was excluded from the nucleus in TRIP13 ^TG/ Em-MYC ^TG mice and TRIP13-overexpressing MM cell lines. Treatment with the USP7 inhibitor, P5091, restored p53 stabilization and nuclear PTEN localization in TRIP13 overexpressing MM cells, demonstrating that USP7 signaling pathways are involved in TRIP13-induced MM progression.

These data describe an exciting new role for TRIP13 in the promotion of deubiquitination through USP7 and highlight TRIP13 as a target in MM. The elevated expression of TRIP13 has been associated with inferior outcomes in many forms of cancers suggesting that these results may have relevance beyond MM.