Key Points Deubiquitinases Usp9x and Usp24 regulate Mcl-1 and myeloma cell survival. Small-molecule–mediated Usp9x/Usp24 inhibition induces apoptosis and blocks myeloma tumor growth in vivo.

Gene products that modify the apoptotic susceptibility of cancer cells may offer novel drug response markers or therapeutic targets. In this study, we probed the contribution of 53 different isopeptidases to apoptosis triggered by bortezomib and etoposide. USP18, a type I IFN-induced protein that deconjugates the ubiquitinlike modifier ISG15 from target proteins, was found to limit apoptotic susceptibility to IFN-α or bortezomib. Ablating USP18 in cells treated with IFN-α increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) production; upregulated expression of transcription factors IFN-regulatory factor (IRF)-1, IRF-7, and IRF-9; and promoted the extrinsic pathway of apoptosis. The proapoptotic effects of ablating USP18 were abrogated by FLIP overexpression or TRAIL silencing. However, in bortezomib-treated cells, weak spontaneous signaling from type I IFNs was implicated in the proapoptotic effect of USP18 ablation. Ectopic USP18 repressed apoptotic signaling by IFN-α, TRAIL, or bortezomib. Similar effects were produced by a catalytically inactive USP18 mutant, indicating that the antiapoptotic function of USP18 is independent of its catalytic activity. These findings suggest that USP18 may significantly limit operation of the extrinsic apoptotic pathway triggered by type I IFN and drugs. Cancer Res; 70(2); 655-65. ©2010 AACR.

ETS transcription factors are commonly deregulated in cancer by chromosomal translocation, overexpression or post-translational modification to induce gene expression programs essential in tumorigenicity. Targeted destruction of these proteins may have therapeutic impact. Here we report that Ets-1 destruction is regulated by the deubiquitinating enzyme, Usp9x, and has major impact on the tumorigenic program of metastatic melanoma. Ets-1 deubiquitination blocks its proteasomal destruction and enhances tumorigenicity, which could be reversed by Usp9x knockdown or inhibition. Usp9x and Ets-1 levels are coincidently elevated in melanoma with highest levels detected in metastatic tumours versus normal skin or benign skin lesions. Notably, Ets-1 is induced by BRAF or MEK kinase inhibition, resulting in increased NRAS expression, which could be blocked by inactivation of Usp9x and therapeutic combination of Usp9x and MEK inhibitor fully suppressed melanoma growth. Thus, Usp9x modulates the Ets-1/NRAS regulatory network and may have biologic and therapeutic implications.

Usp5 is a deubiquitinase (DUB) previously shown to regulate unanchored polyubiquitin (Ub) chains, p53 transcriptional activity and double-strand DNA repair. In BRAF mutant melanoma cells, Usp5 activity was suppressed by BRAF inhibitor (vemurafenib) in sensitive but not in acquired or intrinsically resistant cells. Usp5 knockdown overcame acquired vemurafenib resistance and sensitized BRAF and NRAS mutant melanoma cells to apoptosis initiated by MEK inhibitor, cytokines or DNA-damaging agents. Knockdown and overexpression studies demonstrated that Usp5 regulates p53 (and p73) levels and alters cell growth and cell cycle distribution associated with p21 induction. Usp5 also regulates the intrinsic apoptotic pathway by modulating p53-dependent FAS expression. A small molecule DUB inhibitor (EOAI3402143) phenocopied the FAS induction and apoptotic sensitization of Usp5 knockdown and fully blocked melanoma tumor growth in mice. Overall, our results demonstrate that BRAF activates Usp5 to suppress cell cycle checkpoint control and apoptosis by blocking p53 and FAS induction; all of which can be restored by small molecule-mediated Usp5 inhibition. These results suggest that Usp5 inhibition can provide an alternate approach in recovery of diminished p53 (or p73) function in melanoma and can add to the targeted therapies already used in the treatment of melanoma.

Inhibitors of the ubiquitin-proteasome system (UPSIs) promote apoptosis of cancer cells and show encouraging anti-tumor activities in vivo. In this study, we evaluated the death activities of two different UPSIs: bortezomib and the isopeptidase inhibitor G5. To unveil whether these compounds elicit different types of death, we compared their effect both on apoptosis-proficient wild type mouse embryo fibroblasts and on cells defective for apoptosis (double-deficient Bax/Bak mouse embryo fibroblasts) (double knockout; DKO). We have discovered that (i) both inhibitors induce apoptosis in a Bax and Bak-dependent manner, (ii) both inhibitors elicit autophagy in WT and DKO cells, and (iii) only G5 can kill apoptosis-resistant DKO cells by activating a necrotic response. The induction of necrosis was confirmed by different experimental approaches, including time lapse analysis, HMGB1 release, and electron microscopy studies. Neither treatment with antinecrotic agents, such as antioxidants, poly(ADP-ribose) polymerase and JNK inhibitors, necrostatin, and the intracellular Ca 2؉ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester, nor overexpression of Bcl-2 and Bcl-xL prevented necrosis induced by G5. This necrotic death is characterized by the absence of protein oxidation and by the rapid cyclosporin A-independent dissipation of the mitochondrial membrane potential. Notably, a peculiar feature of the G5-induced necrosis is an early and dramatic reorganization of the actin cytoskeleton, coupled to an alteration of cell adhesion. The importance of cell adhesion impairment in the G5-induced necrotic death of DKO cells was confirmed by the antagonist effect of the extracellular matrixadhesive components, collagen and fibronectin.

Deletion of type I IFN genes and resistance to apoptosis induced by type I IFNs are common in glioblastoma. Here we have investigated the importance of the constitutive weak IFN-signaling in the apoptotic response to IFN-α in glioblastoma cells. U87MG cells hold a deletion of type I IFN genes, whereas in T98G cells the spontaneous IFN signaling is intact. In response to IFN-α U87MG cells produce much less TRAIL, while other IFN-inducible genes were efficiently up-regulated. Alterations in TRAIL promoter sequence and activity were not observed. DNA methylation can influence TRAIL transcription but without overt differences between the two cell lines. We also discovered that TRAIL mRNA stability is influenced by IFN-α, but again no differences can be appreciated between the two cell lines. By silencing IFNAR1 we provide evidences that the spontaneous IFN signaling loop is required to sustain elevated levels of TRAIL expression, possibly through the regulation of IRF-1. Despite the presence/absence of the constitutive IFN signaling, both cell lines were resistant to IFN-α induced apoptosis. Targeting the deisgylase USP18 can overcome resistance to IFN-induced apoptosis only in T98G cells. Alterations in elements of the extrinsic apoptotic pathway, such as Bid and c-FLIP contribute to apoptotic resistance of U87MG cells. Down-regulation of USP18 expression together with the induction of ER-stress efficiently restored apoptosis in U87MG cells. Finally, we demonstrated that the BH3-only protein Noxa provides an important contribution in the apoptotic response to ER-stress in USP18 silenced cells.

Usp9x has emerged as a potential therapeutic target in some hematologic malignancies and a broad range of solid tumors including brain, breast, and prostate. To examine Usp9x tumorigenicity and consequence of Usp9x inhibition in human pancreatic tumor models, we carried out gain- and loss-of-function studies using established human pancreatic tumor cell lines (PANC1 and MIAPACA2) and four spontaneously immortalized human pancreatic patient-derived tumor (PDX) cell lines. The effect of Usp9x activity inhibition by small molecule deubiquitinase inhibitor G9 was assessed in 2D and 3D culture, and its efficacy was tested in human tumor xenografts. Overexpression of Usp9x increased 3D growth and invasion in PANC1 cells and up-regulated the expression of known Usp9x substrates Mcl-1 and ITCH. Usp9x inhibition by shRNA-knockdown or by G9 treatment reduced 3D colony formation in PANC1 and PDX cell lines, induced rapid apoptosis in MIAPACA2 cells, and associated with reduced Mcl-1 and ITCH protein levels. Although G9 treatment reduced human MIAPACA2 tumor burden in vivo, in mouse pancreatic cancer cell lines established from constitutive (8041) and doxycycline-inducible (4668) KrasG12D/Tp53R172H mouse pancreatic tumors, Usp9x inhibition increased and sustained the 3D colony growth and showed no significant effect on tumor growth in 8041-xenografts. Thus, Usp9x inhibition may be therapeutically active in human PDAC, but this activity was not predicted from studies of genetically engineered mouse pancreatic tumor models.

Melanoma tumors driven by BRAF mutations often do not respond to BRAF/MEK/ERK pathway inhibitors currently used in treatment. One documented mechanism of resistance is upregulation of SOX2, a transcription factor that is essential for tumor growth and expansion, particularly in melanoma tumors with BRAF mutations. Targeting transcription factors pharmacologically has been elusive for drug developers, limiting treatment options. Here we show that ubiquitin-specific peptidase 9, X-linked (Usp9x), a deubiquitinase (DUB) enzyme controls SOX2 levels in melanoma. Usp9x knockdown in melanoma increased SOX2 ubiquitination, leading to its depletion, and enhanced apoptotic effects of BRAF inhibitor and MEK inhibitors. Primary metastatic melanoma samples demonstrated moderately elevated Usp9x and SOX2 protein expression compared to tumors without metastatic potential. Usp9x knockdown, as well as inhibition with DUB inhibitor, G9, blocked SOX2 expression, suppressed in vitro colony growth, and induced apoptosis of BRAF-mutant melanoma cells. Combined treatment with Usp9x and mutant BRAF inhibitors fully suppressed melanoma growth in vivo . Our data demonstrate a novel mechanism for targeting the transcription factor SOX2, leveraging Usp9x inhibition. Thus, development of DUB inhibitors may add to the limited repertoire of current melanoma treatments.