The prognostic value of 19S ATPase proteasome subunits in acute myeloid leukemia and other forms of cancer.

Introduction: The ubiquitin-proteasome system (UPS) is an intracellular organelle responsible for targeted protein degradation, which represents a standard therapeutic target for many different human malignancies. Bortezomib, a reversible inhibitor of chymotrypsin-like proteasome activity, was first approved by the FDA in 2003 to treat multiple myeloma and is now used to treat a number of different cancers, including relapsed mantle cell lymphoma, diffuse large B-cell lymphoma, colorectal cancer, and thyroid carcinoma. Despite the success, bortezomib and other proteasome inhibitors are subject to severe side effects, and ultimately, drug resistance. We recently reported an oncogenic role for non-ATPase members of the 19S proteasome in chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and several different solid tumors. In the present study, we hypothesized that ATPase members of the 19S proteasome would also serve as biomarkers and putative therapeutic targets in AML and multiple other cancers. Methods: We used data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) available at UALCAN and/or GEPIA2 to assess the expression and prognostic value of proteasome 26S subunit, ATPases 1-6 (PSMC1-6) of the 19S proteasome in cancer. UALCAN was also used to associate PSMC1-6 mRNA expression with distinct clinicopathological features. Finally, cBioPortal was employed to assess genomic alterations of PSMC genes across different cancer types. Results: The mRNA and protein expression of PSMC1-6 of the 19S proteasome were elevated in several cancers compared with normal controls, which often correlated with worse overall survival. In contrast, AML patients demonstrated reduced expression of these proteasome subunits compared with normal mononuclear cells. However, AML patients with high expression of PSMC2-5 had worse outcomes. Discussion: Altogether, our data suggest that components of the 19S proteasome could serve as prognostic biomarkers and novel therapeutic targets in AML and several other human malignancies.

Phase 1 study of the protein deubiquitinase inhibitor VLX1570 in patients with relapsed and/or refractory multiple myeloma.

This phase 1 study sought to characterize the safety, tolerability, and pharmacokinetic behavior of VLX1570, a small molecule inhibitor of the deubiquitinases (DUBs) that remove sterically bulky ubiquitin chains from proteins during processing in the19S regulatory subunit of the proteasome, in patients with relapsed and refractory multiple myeloma (MM). Fourteen patients were treated with escalating doses of VLX1570 ranging from 0.05 to 1.2 mg/kg as a brief intravenous (IV) infusion on Days 1, 2, 8, 9, 15, and 16 of a 28-day cycle. Due to its poor aqueous solubility, VLX1570 was formulated in polyethylene glycol, polyoxyethylated castor oil, and polysorbate 80 and administered as a brief intravenous (IV) infusion via a central venous catheter. Anti-myeloma effects were noted at doses at or above 0.6 mg/kg, however, two patients treated at the 1.2 mg/kg dose level experienced severe, abrupt, and progressive respiratory insufficiency, which was associated with diffuse pulmonary infiltrates on imaging studies, similar to those rarely noted with bortezomib and other inhibitors of the 20S proteasome, culminating in death. Although the contribution of VLX1570's formulation to the pulmonary toxicity could not be ruled out, the severity and precipitous nature of the toxicity and the steep relationship between dose and toxicity, the study was discontinued. Despite the severe pulmonary toxicity noted with VLX1570, efforts directed at identifying DUB inhibitors with greater therapeutic indices appear warranted based on the unique mechanism of action, robustness of preclinical antitumor activity, and activity of the DUB inhibitors in MM resistant to PIs targeting the 20S proteasome subunit.

TOR Facilitates the Targeting of the 19S Proteasome Subcomplex To Enhance Transcription Complex Assembly at the Promoters of the Ribosomal Protein Genes.

TOR (target of rapamycin) has been previously implicated in transcriptional stimulation of the ribosomal protein (RP) genes via enhanced recruitment of NuA4 (nucleosome acetyltransferase of H4) to the promoters. However, it is not clearly understood how TOR enhances NuA4 recruitment to the promoters of the RP genes. Here we show that TOR facilitates the recruitment of the 19S proteasome subcomplex to the activator to enhance the targeting of NuA4 to the promoters of the RP genes. NuA4, in turn, promotes the recruitment of TFIID (transcription factor IID, composed of TATA box-binding protein [TBP] and a set of TBP-associated factors [TAFs]) and RNA polymerase II to the promoters of the RP genes to enhance transcriptional initiation. Therefore, our results demonstrate that TOR facilitates the recruitment of the 19S proteasome subcomplex to the promoters of the RP genes to promote the targeting of NuA4 for enhanced preinitiation complex (PIC) formation and consequently transcriptional initiation, hence illuminating TOR regulation of RP gene activation. Further, our results reveal that TOR differentially regulates PIC formation (and hence transcription) at the non-RP genes, thus demonstrating a complex regulation of gene activation by TOR.

The role of 19S proteasome associated deubiquitinases in activity-dependent hippocampal synaptic plasticity.

Posttranslational modification and degradation of proteins by the ubiquitin-proteasome system (UPS) is crucial to synaptic transmission. It is well established that 19S proteasome associated deubiquitinases (DUBs) reverse the process of ubiquitination by removing ubiquitin from their substrates. However, their potential contribution to hippocampal synaptic plasticity has not been addressed in detail. Here, we report that inhibition of the 19S proteasome associated DUBs, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14) by b-AP15 results in an accumulation of polyubiquitinated proteins and a reduction of monomeric ubiquitin without overt effects on 26S proteasome activity. b-AP15 led to a suppression of mTOR-p70S6K signaling and an increase in levels of p-p38 MAPK, two pathways essentially involved in establishing various forms of activity-dependent plasticity. Additionally, b-AP15 impaired the induction of late-phase long-term potentiation (L-LTP), induced the transformation of mGluR-mediated protein synthesis-independent long-term depression (early-LTD) to L-LTD and promoted heterosynaptic stabilization through synaptic tagging/capture (STC) in the hippocampal CA1 region of mice. The activity of 19S proteasome associated DUBs was also required for the enhancement of short-term potentiation (STP) induced by brain-derived neurotrophic factor (BDNF). Altogether, these results indicate an essential role of 19S proteasome associated DUBs in regulating activity-dependent hippocampal synaptic plasticity.

26S and PA28-20S Proteasome Activity in Cytosolic Extracts from Embryonic Stem Cells.

The proteasome is a complex multisubunit protease that plays a major role in the degradation of proteins in eukaryotic cells. Proteasome function is one of the key players regulating the proteome and it is vital for many cellular processes. The method described here makes it possible to assay the proteolytic capacities of proteasome complexes separately in crude cytosolic extracts from ES cells. The method is based on hydrolysis of a fluorogenic peptide substrate in lysates prepared under conditions that favor the interactions of the 20S proteasomal catalytical core with either the 19S or the PA28αß proteasome regulator.

Changes in the Proteasome Pool during Malignant Transformation of Mouse Liver Cells.

Multiple forms of proteasomes regulate cellular processes by destroying proteins or forming the peptides involved in those processes. Various pathologies, including carcinogenesis, are related to changes in the functioning of the proteasome forms. In this study, we looked at the changes in the pool of liver proteasomes during nodular regenerative hyperplasia and formation of adenoma and hepatocellular carcinoma in mice treated with Dipin, followed by partial liver resection. The relative content of various proteasome forms was determined using Western blot analysis. The chymotrypsin-like activity of proteasomes was assessed from the hydrolysis of the commercial Suc-LLVY-AMC substrate. It was found that changes in the proteasome pool appeared already during the formation of diffuse nodules, the changes being the increased expression of the X(ß5) constitutive subunit and the LMP7(ß5i) and LMP2(ß1i) immune subunits, accompanied by the increase of the total proteasome pool and the decrease in the chymotrypsin-like activity. These changes were more pronounced in hepatocellular carcinoma. The content of the total proteasome pool and the LMP2(ß1i) immune subunit and the chymotrypsin-like activity in adenoma were intermediate compared to those in the samples of liver with diffuse nodules and carcinoma. In addition, the level of the Rpt6 subunit present in the 19S proteasome activator was increased in carcinoma. Our results indicate that nodular regenerative hyperplasia and adenomatosis may be stages preceding carcinogenesis. We also conclude that there is a need to find signalling pathways that change the expression of various proteasome subunits during carcinogenesis. The 19S proteasome activator, which is overexpressed in malignant tumours, can be a promising target for the development of new anticancer drugs.