Proteasomal adaptations underlying carfilzomib-resistance in human bone marrow plasma cells.

Donor-specific antibodies (DSAs) have a deleterious effect on allografts and remain a major immunologic barrier in transplantation. Current therapies to eliminate DSAs are ineffective in highly HLA-sensitized patients. Proteasome inhibitors have been employed as a strategy to target bone marrow plasma cells (BMPCs), the source of long-term antibody production; however, their efficacy has been limited by poorly defined drug-resistance mechanisms. Here, we performed transcriptomic profiling of CD138+ BMPCs that survived in vivo desensitization therapy with the proteasome inhibitor carfilzomib to identify mechanisms of drug resistance. The results revealed a genomic signature that included increased expression of the immunoproteasome, a highly specialized proteasomal variant. Western blotting and functional studies demonstrated that catalytically active immunoproteasomes and the immunoproteasome activator PA28 were upregulated in carfilzomib-resistant BMPCs. Carfilzomib-resistant BMPCs displayed reduced sensitivity to the proteasome inhibitors carfilzomib, bortezomib, and ixazomib, but enhanced sensitivity to an immunoproteasome-specific inhibitor ONX-0914. Finally, in vitro carfilzomib treatment of BMPCs from HLA-sensitized patients increased levels of the immunoproteasome ß5i (PSMB8) catalytic subunit suggesting that carfilzomib therapy directly induces an adaptive immunoproteasome response. Taken together, our results indicate that carfilzomib induces structural changes in proteasomes and immunoproteasome formation.

MUC5AC and MUC5B enhance the characterization of mucinous adenocarcinomas of the lung and predict poor prognosis.

AIMS: From the viewpoint of histogenesis, lung adenocarcinoma can be subdivided into two groups: terminal respiratory unit (TRU) and non-TRU types. We recently reported a non-TRU type adenocarcinoma designated as ciliated adenocarcinoma (we now prefer central type adenocarcinoma). We suggest reasons that mucinous adenocarcinoma should encompass central type adenocarcinoma to represent its biological characteristics as non-TRU type adenocarcinoma. METHODS AND RESULTS: Mucin (MUC)5AC and MUC5B were expressed more significantly in non-TRU type adenocarcinoma (P < 0.01). Thirty-five (76.1%) and 45 cases (97.8%) of 46 non-TRU type adenocarcinoma showed positivity for MUC5AC and MUC5B. Twelve (7.6%) and eight (5.1%) cases of 157 TRU type adenocarainoma showed positivity for MUC5B and MUC5AC. NKX2-1 gene expression was measured with quantitative reverse transcription-polymerase chain reaction (qRT-PCR). ΔΔCt of NKX2-1 gene expression was 6.79 for TRU type adenocarcinoma and 0.6 for non-TRU type adenocarcinoma. Overall survival and disease-free survival were poorer in non-TRU type adenocarcinoma (P = 0.02 and P = 0.03). A multivariate test also showed that non-TRU type adenocarcinoma is an independent prognostic factor (P = 0.04). CONCLUSION: MUC5AC and MUC5B were specific makers for non-TRU adenocarcinoma, including both central type adenocarcinoma and mucinous adenocarcinoma. We suggest that non-TRU type adenocarcinoma presents a poorer prognosis, so it should be regarded separately from TRU type adenocarcinoma.

Development of withaferin A analogs as probes of angiogenesis.

The natural product withaferin A (WFA) is a potent angiogenesis inhibitor and it targets the ubiquitin-proteasome pathway in vascular endothelial cells. We generated a biotinylated affinity analog WFA-LC(2)B for use as a probe to study angiogenesis. WFA-LC(2)B inhibits angiogenic sprouting in vitro and it causes levels of ubiquitinated proteins to increase in tumor necrosis factor-alpha-treated human umbilical vein endothelial cells, confirming the retention of WFA's biological activity. We show that WFA-LC(2)B forms protein adducts in endothelial cells which are competed by free WFA in vivo. This WFA-LC(2)B analog will be useful to isolate the biological target of WFA.

Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation.

The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCF(beta-TRCP)-binding phosphopeptide derived from IkappaBalpha linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the alpha isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer.