We did not detect the active form of ATF6 in cells treated with either b-AP15 or bortezomib (data not shown). b-AP15 with suberoylanilide hydroxamic acid, lenalidomide, or dexamethasone induces synergistic anti-MM activity. Our preclinical data showing efficacy of b-AP15 in MM disease models validates targeting DUBs in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance and provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient end result in MM. == Introduction == The ubiquitin proteasome pathway is usually a validated therapeutic target in multiple myeloma (MM), evidenced by the US Food and Drug Administration approval of bortezomib and carfilzomib.1,2Recent studies have focused on targeting enzymes that modulate protein ubiquitin conjugation/deconjugation upstream of the proteasome rather than the proteasome itself, with the goal of producing more specific, potent, and less harmful therapies targeting the ubiquitin proteasome pathway. Importantly, many human diseases are linked to dysfunction of ubiquitin ligases and/or deubiquitylating enzymes (DUBs), suggesting 5-Iodo-A-85380 2HCl that inhibitors of ubiquitylating or DUB enzymes represent a potential therapeutic strategy.3,4 The human genome encodes approximately 100 putative DUBs, which are classified into five families: USP (ubiquitin-specific processing protease), UCH (ubiquitin C-terminal hydrolase), OTU (ovarian tumor ubiquitin), MJD (Josephin domain), and 5-Iodo-A-85380 2HCl JAMM (Jab1/Mov34 metalloenzyme).5,6Note that this first 4 families are cysteine proteases, whereas the fifth family consists of metalloproteases; to date, USP and UCH are the best characterized families.7DUBs play a central role in regulating cellular processes, such as cell growth, proliferation, apoptosis, DNA repair, kinase activation, and transcription.8,9In mammalian cells, three DUBs are associated with the proteasome: USP14, UCHL5/Uch37, and Rpn11.6,10-12Both USP14 and UCHL5 reversibly associate with the 19S regulatory particle, whereas Rpn11 is an intrinsic subunit of the proteasome lid subcomplex of the 19S regulatory particle; therefore, modulating their functions may impact the proteasomal uptake of protein substrate for degradation. These DUBs have also been implicated in malignancy. Screening for genetic abnormalities by using retroviral expression libraries 5-Iodo-A-85380 2HCl and the 3T3 focus formation assay shows involvement of USP14 in ovarian carcinogenesis.13A recent study shows that USP14 is 5-Iodo-A-85380 2HCl highly expressed in colorectal malignancy and correlates with pathologic stage as well as liver and lymph node metastases.14Deubiquitylation of CXCR4 by USP14 is also crucial for both CXCR4 degradation and chemotaxis.15UCLH5, like USP14, plays an important role in regulating oncogenic signaling.16For example, transforming growth factor- (TGF-) is a critical regulator of cell proliferation, differentiation, and tumor pathogenesis; UCHL5 regulates TGF-/Smad signaling by binding to intracellular Smad transcription factors, thereby allowing for deubiquitylation and stabilization of the TGF receptor.17UCHL5/Rpn13 complex also regulates nuclear factor B signaling pathway via conversation with inhibitor of B.18Additionally, knockdown of UCHL5 induces apoptosis, whereas overexpression of UCHL5 promotes cell proliferation in A549 cells. Finally, the clinical significance of UCHL5 was exhibited in a recent analysis of tumor specimens from 111 patients with esophageal squamous cell carcinoma, showing a direct correlation between the elevated expression of UCHL5 and lymph node metastasis. 19 A recent study explained a small molecule inhibitor of 5-Iodo-A-85380 2HCl USP14 and UCHL5.20In contrast to the proteasome inhibitors, b-AP15 blocks the deubiquitylating activity of 19S regulatory particleassociated USP14 and UCHL5 without affecting proteolytic activities of the 20S core particle.20Interestingly, b-AP15 triggers cancer cell apoptosis, regardless of TP53 status and BCL2 expression level, and inhibits tumor growth and metastasis in vivo.21Given the therapeutic success of proteasome inhibition in MM, we attempted to examine whether b-AP15 blockade of USP14 and UCHL5 had anti-MM activity. Our in vitro and in vivo studies suggest that targeting DUBs can overcome proteasome inhibitor resistance, providing the basis for their clinical evaluation. == Materials and methods == == Cell culture and reagents == Human MM cell lines MM.1S, MM.1R, KMS-11, ARP-1, RPMI 8226, DOX40, LR5, ANBL-6 (ANBL6.WT), and ANBL-6-bortezomib-resistant (ANBL-6.BR), sound tumor cell lines COLO320, LS174, and SHSY5Y, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were cultured in RPMI-1640 medium supplemented with HBEGF complete medium (10% fetal bovine serum, 100 models/mL penicillin, 100 g/mL streptomycin, and 2 mMl-glutamine) at 37C and 5% CO2. Solid tumor cell lines DLD-1, 7860,.
