Mitoxantrone Targets Human Ubiquitin-Specific Peptidase 11 (USP11) and Is a Potent Inhibitor of Pancreatic Cancer Cell Survival

Abstract

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related death in the United States, with a 95% five-year mortality rate. For over a decade, gemcitabine (GEM) has been the established first-line treatment for this disease despite suboptimal response rates. The development of PARP inhibitors that target the DNA damage repair (DDR) system in PDA cells has generated encouraging results. Ubiquitinspecific peptidase 11 (USP11), an enzyme that interacts with the DDR protein BRCA2, was recently discovered to play a key role in DNA double-strand break repair and may be a novel therapeutic target. A systematic high-throughput approach was used to biochemically screen 2,000 U.S. Food and Drug Administration (FDA)-approved compounds for inhibition of USP11 enzymatic activity. Six pharmacologically active small molecules that inhibit USP11 enzymatic activity were identified. An in vitro drug sensitivity assay demonstrated that one of these USP11 inhibitors, mitoxantrone, impacted PDA cell survival with an IC 50 of less than 10 nM. Importantly, across six different PDA cell lines, two with defects in the Fanconi anemia/ BRCA2 pathway (Hs766T and Capan-1), mitoxantrone is 40-to 20,000-fold more potent than GEM, with increased endogenous USP11 mRNA levels associated with increased sensitivity to mitoxantrone. Interestingly, USP11 silencing in PDA cells also enhanced sensitivity to GEM. These findings establish a preclinical model for the rapid discovery of FDA-approved compounds and identify USP11 as a target of mitoxantrone in PDA.Implications: This high-throughput approach provides a strong rationale to study mitoxantrone in an early-phase clinical setting for the treatment of PDA. Mol Cancer Res; 11(8); 901-11. Ó2013 AACR. IntroductionPancreatic ductal adenocarcinoma remains the fourth leading cause of cancer-related death in the United States.

Structure and Catalytic Regulatory Function of Ubiquitin Specific Protease 11 N-Terminal and Ubiquitin-like Domains

“…We observed a K m of 0.55 μM which is consistent with K m values reported ranging from 0.12–0.77 μM. 1,16 Comparison of FL-USP11 to the construct lacking the catalytic domain UBL2 containing insert, FLΔUBL2, showed that these constructs display similar kinetic parameters as summarized in Figure 4C. Furthermore, the kinetic parameters for FLΔUBL2 and a construct additionally missing the N-terminal DU domains, CatΔUBL2, were comparable (Figure 4C).…”

“…12,13 Importantly, USP11 has been shown to exhibit altered expression levels in several types of cancers such as lung and breast cancer, 14,15 and the enzymatic activity of USP11 is inhibited by mitoxantrone which affects pancreatic cancer cell survival. 16 There is currently no structure for USP11 domains or information on how its catalytic activity is regulated and substrate is recognized.…”

USP11-dependent selective cIAP2 deubiquitylation and stabilization determine sensitivity to Smac mimetics

“…Recently, mitoxantrone was identified as a USP11 inhibitor. 34 Consistently, USP11 inhibition by mitoxantrone induced cIAP2 destabilization, which was reversed with MG132 treatment (Figures 4h-i). USP11 depletion by siRNA abrogated mitoxantrone-induced cIAP2 degradation, suggesting that mitoxantrone regulates cIAP2 degradation via USP11 inhibition (Figure 4j).…”

A selective USP1–UAF1 inhibitor links deubiquitination to DNA damage responses

“…USP1, USP1-UAF1, USP46-UAF1 and USP11 were generated as previously described 21,48 . Ub-PCNA was prepared as previously reported 49 .…”