Recent evidence suggests that several deubiquitinases (DUB) are overexpressed or activated in tumor cells and many contribute to the transformed phenotype. Agents with DUB inhibitory activity may therefore have therapeutic value. In this study, we describe the mechanism of action of WP1130, a small molecule derived from a compound with Janus-activated kinase 2 (JAK2) kinase inhibitory activity. WP1130 induces rapid accumulation of polyubiquitinated (K48/K63-linked) proteins into juxtanuclear aggresomes, without affecting 20S proteasome activity. WP1130 acts as a partly selective DUB inhibitor, directly inhibiting DUB activity of USP9x, USP5, USP14, and UCH37, which are known to regulate survival protein stability and 26S proteasome function. WP1130-mediated inhibition of tumor-activated DUBs results in downregulation of antiapoptotic and upregulation of proapoptotic proteins, such as MCL-1 and p53. Our results show that chemical modification of a previously described JAK2 inhibitor results in the unexpected discovery of a novel DUB inhibitor with a unique antitumor mechanism. Cancer Res; 70(22); 9265-76. ©2010 AACR.

Host Cell Factor-1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression, undergoes proteolytic maturation in which any of six repeated sequences is cleaved by the nutrient-responsive glycosyltransferase, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). We report that the tetratricopeptide-repeat domain of O-GlcNAc transferase binds the C-terminal portion of an HCF-1 proteolytic repeat such that the cleavage region lies in the glycosyltransferase active site above UDP-GlcNAc. The conformation is similar to that of a glycosylation-competent peptide substrate. Cleavage occurs between cysteine and glutamate residues and results in a pyroglutamate product. Conversion of the cleavage site glutamate into serine converts an HCF-1 proteolytic repeat into a glycosylation substrate. Thus, protein glycosylation and HCF-1 cleavage occur in the same active site.

IntroductionChronic myelogenous leukemia (CML) is associated with a chromosomal abnormality in the hematopoietic stem cell that results in the expression of Bcr-Abl with unregulated tyrosine kinase activity. 1 These observations supported the development and clinical testing of the first Bcr-Abl kinase inhibitor, imatinib, which demonstrated remarkable clinical efficacy in CML patients. Imatinib is the frontline therapy for CML and other Bcr-Ablexpressing leukemias, and most patients treated with imatinib in the chronic phase achieve a complete cytogenetic response. 2,3 However, molecular studies of imatinib-treated patients in remission demonstrated that Bcr-Abl expression is still detectable in most cases, and discontinuation of imatinib therapy often results in disease relapse. 4,5 Limited duration of imatinib response is also common in advanced CML patients, and imatinib resistance can occur at any stage of the disease. 5,6 Acquired imatinib resistance and disease progression are frequently characterized by Bcr-Abl mutations and posttranslational modifications that affect imatinib binding and kinase inhibition. 5,6 Some of the molecular changes in imatinib-resistant disease can be overcome with second-generation tyrosine kinase inhibitors, which bind Bcr-Abl with higher affinity or inhibit imatinib-insensitive kinases associated with resistance. [7][8][9][10][11] However, the activity of these inhibitors can also be limited by mutations and other mechanisms. [12][13][14] These observations suggest that additional approaches and targeting strategies may be needed to provide long-term effective treatment for CML patients.Kinase inhibition by small molecules that bind the ATP or the switch pocket region of Bcr-Abl are effective inhibitors but require continuous treatment because Bcr-Abl protein levels themselves are not directly regulated through kinase inhibition. Some evidence suggests that Bcr-Abl can function as a protein scaffold to organize signaling complexes that are not fully dependent on kinase activity. 15,16 These observations suggest that compounds that modulate Bcr-Abl protein levels may be more effective and appropriate for CML therapy in some settings. In light of that possibility and as a novel approach to overcoming kinase inhibitor resistance, several compounds have been described that affect Bcr-Abl function through mechanisms other than direct kinase inhibition. These include heat shock protein 90 (Hsp90) inhibitors, arsenic trioxide, homoharringtonine, histone deacetylase inhibitors, proteasome inhibitors, PP2A activators, and others. 5,10 All are reported to lead to CML cell death through down-regulation of Bcr-Abl expression or a loss of Bcr-Abl stability. However, most of these compounds reduce Bcr-Abl levels only after extended incubation intervals and display only limited selectivity for Bcr-Abl, which may increase their toxicity and decrease their clinical utility. [17][18][19] Additional compounds that induce rapid changes in Bcr-Abl levels with limited impact on other proteins are ...

IntroductionChronic myelogenous leukemia (CML) is a consequence of reciprocal translocation between chromosomes 9 and 22 resulting in the so-called Philadelphia chromosome, 1,2 in which the first exon of the c-Abl gene is replaced with sequences of the breakpoint cluster region (bcr) gene 3,4 to create the Bcr/Abl oncogene. The constitutively active kinase activity of Bcr/Abl in the cytosol contributes to its transforming function 5 and drug resistance through activation of several key survival pathways, including the mitogen-activated protein kinase/extracellular signal-regulating kinase cascade, nuclear factor B, and the signal transducer and activator of transcription (Stat) family. [6][7][8] Previous studies showed that reducing intracellular levels of Bcr/Abl mRNA or protein led to inhibition of proliferation and clonogenic survival of Bcr/Abl-expressing leukemia cells. 9,10 The introduction of imatinib mesylate (Gleevec; STI-571; CGP57148B; Novartis, East Hanover, NJ) revolutionized the treatment of CML, because it selectively inhibits the kinase activity of Bcr/Abl 11 without adversely affecting normal cells. Imatinib mesylate is currently used as frontline therapy for CML and is effective in most cases. However, although imatinib mesylate produces treatment responses at both the hematologic and cytogenetic levels, a growing number of patients in blast crisis eventually experience relapse despite continued treatment with imatinib mesylate. [12][13][14] Mutations within the kinase domain of Abl that interfere with the binding of the drug constitute a primary cause of resistance, [15][16][17][18][19][20][21][22] although other mechanisms have been proposed. Many different approaches to overcome clinical resistance to imatinib mesylate have been described. Farnesyltransferase inhibitors such as SCH66336 and the proteasome inhibitor bortezomib (Velcade) were shown to have growth inhibitory effects on certain imatinib mesylate-resistant leukemias. 23 The pyrido-pyrimidine-type kinase inhibitors PD166326 and SKI-606 24 are active against common kinase-domain mutants of Bcr/Abl that cause resistance to imatinib mesylate. However, these agents do not affect the kinase activity of the T315I mutant, which sterically reduces drug/kinase affinity and prevents direct contact of these agents with the Bcr/Abl protein. Other kinase inhibitors such as PD180970 and CGP76030 show similar restrictions in affinity for the T315I mutant. 25 Recently, second-generation compounds such as nilotinib (AMN107), with higher affinity for abl, or dasatinib (BMS-354825), with high affinity for both abl and src kinases, have been tested in phase 1 and 2 trials. Despite their effectiveness against many Bcr/Abl mutants in imatinib mesylate-resistant disease, these agents cannot suppress the kinase activity of T315 mutants, suggesting that the emergence of the T315 mutation in patients with CML will severely reduce the benefit of these kinase inhibitors. 26,27 The novel compound ONO12380 was recently reported to inhibit Bcr/Abl kinase activi...