A Reversible and Highly Selective Inhibitor of the Proteasomal Ubiquitin Receptor Rpn13 Is Toxic to Multiple Myeloma Cells

Trader, Darci J.; Simanski, Scott; Kodadek, Thomas

doi:10.1021/jacs.5b02069

Cited by 74 publications

(93 citation statements)

References 49 publications

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“…Two independently discovered hRpn13-targeting molecules are reported to restrict multiple myeloma cells (9,10). We have found that one such molecule, RA190, interferes with DNA replication and G 2 progression in HeLa cells (Fig.…”

Section: Discussionmentioning

confidence: 93%

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The Proteasome Ubiquitin Receptor hRpn13 and Its Interacting Deubiquitinating Enzyme Uch37 Are Required for Proper Cell Cycle Progression

Randles

Anchoori

Roden

et al. 2016

Journal of Biological Chemistry

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Recently, we reported that bisbenzylidine piperidone RA190 adducts to Cys-88 of the proteasome ubiquitin receptor hRpn13, triggering accumulation of ubiquitinated proteins and endoplasmic reticulum stress-related apoptosis in various cancer cell lines. hRpn13 contains an N-terminal pleckstrin-like receptor for ubiquitin domain that binds ubiquitin and docks it into the proteasome as well as a C-terminal deubiquitinase adaptor (DEUBAD) domain that binds the deubiquitinating enzyme Uch37. Here we report that hRpn13 and Uch37 are required for proper cell cycle progression and that their protein knockdown leads to stalling at G 0 /G 1 . Moreover, serum-starved cells display reduced hRpn13 and Uch37 protein levels with hallmarks of G 0 /G 1 stalling and recovery to their steady-state protein levels following release from nutrient deprivation. Interestingly, loss of hRpn13 correlates with a small but statistically significant reduction in Uch37 protein levels, suggesting that hRpn13 interaction may stabilize this deubiquitinating enzyme in human cells. We also find that RA190 treatment leads to a loss of S phase, suggesting a block of DNA replication, and G 2 arrest by using fluorescence-activated cell sorting. Uch37 deprivation further indicated a reduction of DNA replication and G 0 /G 1 stalling. Overall, this work implicates hRpn13 and Uch37 in cell cycle progression, providing a rationale for their function in cellular proliferation and for the apoptotic effect of the hRpn13-targeting molecule RA190.

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Section: Discussionmentioning

confidence: 93%

“…We recently discovered a bisbenzylidine piperidone class of molecules that restricts mouse xenograft models of multiple myeloma and ovarian cancer and covalently attaches to the proteasome ubiquitin receptor hRpn13 (9). Another group independently also identified an hRpn13-targeting molecule to restrict multiple myeloma cells (10).…”

mentioning

confidence: 99%

The Proteasome Ubiquitin Receptor hRpn13 and Its Interacting Deubiquitinating Enzyme Uch37 Are Required for Proper Cell Cycle Progression

Randles

Anchoori

Roden

et al. 2016

Journal of Biological Chemistry

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“…In HCT116 cells, RA190 can also bind with UCH37, a deubiquitinating enzyme interacting with ADRM1, to suppress proteasome function [35]. Meanwhile, it was reported that KDT-11 could also suppress multiple myeloma by targeting ADRM1 [36]. In ICC cells the specificity of RA190 on ADRM1 inhibition was confirmed by : (1) the accumulation of higher-molecular weight polyubiquitylated proteins and p-IκBα in RA190-treated ICC cells; and (2) reduced RA190 sensitivity following knockdown of ADRM1 in ICC cells [14].…”

Section: Discussionmentioning

confidence: 99%

RA190, a Proteasome Subunit ADRM1 Inhibitor, Suppresses Intrahepatic Cholangiocarcinoma by Inducing NF-KB-Mediated Cell Apoptosis

Wang

Zheng

et al. 2018

Cell Physiol Biochem

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Background/Aims: Effective drug treatment for intrahepatic cholangiocarcinoma (ICC) is currently lacking. Therefore, there is an urgent need for new targets and new drugs that can prolong patient survival. Recently targeting the ubiquitin proteasome pathway has become an attractive anti-cancer strategy. In this study, we aimed to evaluate the therapeutic effect of and identify the potential mechanisms involved in targeting the proteasome subunit ADRM1 for ICC. Methods: The expression of ADRM1 and its prognostic value in ICC was analyzed using GEO and TCGA datasets, tumor tissues, and tumor tissue arrays. The effects of RA190 on the proliferation and survival of both established ICC cell lines and primary ICC cells were examined in vitro. Annexin V/propidium iodide staining, western blotting and immunohistochemical staining were performed. The in vivo anti-tumor effect of RA190 on ICC was validated in subcutaneous xenograft and patient-derived xenograft (PDX) models. Results: ADRM1 levels were significantly higher in ICC tissues than in normal bile duct tissues. ICC patients with high ADRM1 levels had worse overall survival (hazard ratio [HR] = 2.383, 95% confidence interval [CI] =1.357 to 4.188) and recurrence-free survival (HR = 1.710, 95% CI =1.045 to 2.796). ADRM1 knockdown significantly inhibited ICC growth in vitro and in vivo. The specific inhibitor RA190 targeting ADRM1 suppressed proliferation and reduced cell vitality of ICC cell lines and primary ICC cells significantly in vitro. Furthermore, RA190 significantly inhibited the proteasome by inactivating ADRM1, and the consequent accumulation of ADRM1 substrates decreased the activating levels of NF-κB to aggravate cell apoptosis. The therapeutic benefits of RA190 treatment were further demonstrated in both subcutaneous implantation and PDX models. Conclusions: Our findings indicate that up-regulated ADRM1 was involved in ICC progression and suggest the potential clinical application of ADRM1 inhibitors (e.g., RA190 and KDT-11) for ICC treatment.

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“…For example, the human Rpn13/ADRM1 gene is overexpressed in lung, ovarian, colon, liver, kidney, bladder, and stomach cancers (18), and ADRM1 amplification in ovarian cancers correlates significantly with shorter time to recurrence and death (19). The Rpn13/ADRM1 inhibitor RA190 inhibits proteasome functions, triggers apoptosis, and restricts cancer growth in mice xenografts (20), and similar effects were observed with an Rpn13/ADRM1-targeting peptoid inhibitor (21). hHR23B has been identified as a candidate cancer biomarker that governs the response and sensitivity of tumor cells to HDAC inhibitors (22,23).…”

mentioning

confidence: 89%

Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation

Yang

Wang

et al. 2016

Molecular & Cellular Proteomics

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Proteasomes are protein degradation machines that exist in cells as heterogeneous and dynamic populations. A group of proteins function as ubiquitin receptors (UbRs) that can recognize and deliver ubiquitinated substrates to proteasome complexes for degradation. Defining composition of proteasome complexes engaged with UbRs is critical to understand proteasome function. However, because of the dynamic nature of UbR interactions with the proteasome, it remains technically challenging to capture and isolate UbR-proteasome subcomplexes using conventional purification strategies. As a result, distinguishing the molecular differences among these subcomplexes remains elusive. We have developed a novel affinity purification strategy, in vivo cross-linking (X) assisted bimolecular tandem affinity purification strategy (XBAP), to effectively isolate dynamic UbR-proteasome subcomplexes and define their subunit compositions using label-free quantitative mass spectrometry. In this work, we have analyzed seven distinctive UbR-proteasome complexes and found that all of them contain the same type of the 26S holocomplex. However, selected UbRs interact with a group of proteasome interacting proteins that may link each UbR to specific cellular pathways. The compositional similarities and differences among the seven UbRproteasome subcomplexes have provided new insights on functional entities of proteasomal degradation machineries. The strategy described here represents a gen- Proteasomes are multisubunit protein complexes that are responsible for the degradation of ubiquitinated substrates to maintain cell viability and homeostasis. The 26S proteasome is composed of at least 33 subunits (1-3), which can be divided into two subcomplexes: the 20S catalytic core particle (CP) 1 and the 19S regulatory particle (RP). The 20S CP is responsible for various proteolytic activities, and has a highly conserved "barrel"-like structure consisting of two copies each of 14 nonidentical subunits (␣1-7, ␤1-7), which are arranged into four heptameric rings stacked in the order of ␣ 7 ␤ 7 ␤ 7 ␣ 7 (4, 5). The 19S RP intimately interacts with the 20S CP, regulating its activity. In addition, the 19S RP carries diverse functions including substrate recognition and deubiquitination, protein unfolding, and substrate translocation to the 20S CP for degradation (2, 3, 6 -8). In contrast to the highly ordered and stable structure of the 20S CP, the 19S RP appears to be much more flexible and dynamic (3, 9 -11). Current structural analyses have revealed that six Rpt subunits of the 19S RP form a hexameric AAA-ATPase ring to associate with the cylinder ends of the 20S CP, and are surrounded by a shell of Rpn subunits (9 -11). Apart from the

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A Reversible and Highly Selective Inhibitor of the Proteasomal Ubiquitin Receptor Rpn13 Is Toxic to Multiple Myeloma Cells

Cited by 74 publications

References 49 publications

The Proteasome Ubiquitin Receptor hRpn13 and Its Interacting Deubiquitinating Enzyme Uch37 Are Required for Proper Cell Cycle Progression

The Proteasome Ubiquitin Receptor hRpn13 and Its Interacting Deubiquitinating Enzyme Uch37 Are Required for Proper Cell Cycle Progression

RA190, a Proteasome Subunit ADRM1 Inhibitor, Suppresses Intrahepatic Cholangiocarcinoma by Inducing NF-KB-Mediated Cell Apoptosis

Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation

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