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.

The t(8;21)(q22;q22) translocation is one of the most common genetic abnormalities in acute myeloid leukemia (AML), identified in 15% of all cases of AML, including 40-50% of FAB M2 subtype and rare cases of M0, M1 and M4 subtypes. The most commonly known AML1-ETO fusion protein (full-length AML1-ETO) from this translocation has 752 amino acids and contains the N-terminal portion of RUNX1 (also known as AML1, CBFalpha2 or PEBP2alphaB), including its DNA binding domain, and almost the entire RUNX1T1 (also known as MTG8 or ETO) protein. Although alterations of gene expression and hematopoietic cell proliferation have been reported in the presence of AML1-ETO, its expression does not lead to the development of leukemia. Here, we report the identification of a previously unknown alternatively spliced isoform of the AML1-ETO transcript, AML1-ETO9a, that includes an extra exon, exon 9a, of the ETO gene. AML1-ETO9a encodes a C-terminally truncated AML1-ETO protein of 575 amino acids. Expression of AML1-ETO9a leads to rapid development of leukemia in a mouse retroviral transduction-transplantation model. More importantly, coexpression of AML1-ETO and AML1-ETO9a results in the substantially earlier onset of AML and blocks myeloid cell differentiation at a more immature stage. These results indicate that fusion proteins from alternatively spliced isoforms of a chromosomal translocation may work together to induce cancer development.

A common chromosomal translocation in acute myeloid leukemia (AML) involves the AML1 (acute myeloid leukemia 1, also called RUNX1, core binding factor protein (CBFa), and PEBP2aB) gene on chromosome 21 and the ETO (eight-twenty one, also called MTG8) gene on chromosome 8. This translocation generates an AML1-ETO fusion protein. t(8;21) is associated with 12% of de novo AML cases and up to 40% in the AML subtype M2 of the French-American-British classification. Furthermore, it is also reported in a small portion of M0, M1, and M4 AML samples. Despite numerous studies on the function of AML1-ETO, the precise mechanism by which the fusion protein is involved in leukemia development is still not fully understood. In this review, we will discuss structural aspects of the fusion protein and the accumulated knowledge from in vitro analyses on AML1-ETO functions, and outline putative mechanisms of its leukemogenic potential.

ISG15 is one of the most strongly induced genes upon viral infection, type I interferon (IFN) stimulation, and lipopolysaccharide (LPS) stimulation. Here we report that mice lacking UBP43, a protease that removes ISG15 from ISGylated proteins, are hypersensitive to type I IFN. Most importantly, in UBP43-deficient cells, IFN-β induces a prolonged Stat1 tyrosine phosphorylation, DNA binding, and IFN-mediated gene activation. Furthermore, restoration of ISG15 conjugation in protein ISGylation-defective K562 cells increases IFN-stimulated promoter activity. These findings identify UBP43 as a novel negative regulator of IFN signaling and suggest the involvement of protein ISGylation in the regulation of the JAK-STAT pathway.

They have also served as consultants for Kura Oncology, have equity ownership in the company, and are coinventors (along with SK, TW, LS, and PR) on patent applications covering MI-3454 (PCT/US2017/022535). PR is an employee of Kura Oncology, Inc. and has a significant ownership interest in the parent of Wellspring Biosciences, Inc. FB is an employee of Kura Oncology, Inc. Kura Oncology, Inc. and the University of Michigan have filed patent applications covering MI-3454 and they hold intellectual property rights on this compound. OAW has served as a consultant for H3B Biomedicine, Foundation Medicine Inc, Merck, and Janssen, and has received prior research funding from H3B Biomedicine unrelated to the current manuscript. MG receives research support from Cellectis and serves as a consultant in SeqRx.

Normal blood-cell differentiation is controlled by regulated gene expression and signal transduction. Transcription deregulation due to chromosomal translocation is a common theme in hematopoietic neoplasms. AML1-ETO, which is a fusion protein generated by the 8;21 translocation that is commonly associated with the development of acute myeloid leukemia, fuses the AML1 runx family DNA-binding transcription factor to the ETO corepressor that associates with histone deacetylase complexes. Analyses have demonstrated that AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. Here, we report that the loss of the molecular events of AML1-ETO C-terminal NCoR͞SMRT-interacting domain transforms AML1-ETO into a potent leukemogenic protein. Contrary to full-length AML1-ETO, the truncated form promotes in vitro growth and does not obstruct the cell-cycle machinery. These observations suggest a previously uncharacterized mechanism of tumorigenesis, in which secondary mutation(s) in molecular events disrupting the function of a domain of the oncogene promote the development of malignancy.t(8;21) ͉ truncated protein ͉ hematopoietic malignancy ͉ mouse model ͉ cell cycle C hromosomal translocations involving transcriptional regulators and͞or signaling molecules are a common theme associated with dysregulation of hematopoiesis. The transcription factor AML1 (Acute Myeloid Leukemia 1, also called RUNX1, CBF␣, and PEBP2␣B) and its heterodimerization partner CBF␤ are frequent targets of genetic alterations or chromosomal translocations in leukemia (1-3). A common AML1 involved chromosomal translocation, t(8;21)(q22;q22), is found in 12% of all acute myeloid leukemia (AML) cases, including 40-50% of AML M2 subtype and a small portion of M0, M1, and M4 subtypes [French-American-British (FAB) classification] (4-7). The translocation brings together the DNA sequence encoding the N terminus of the AML1 with nearly all of ETO (eighttwenty-one; also called MTG8 and CBFA2T1) to generate the fusion protein AML1-ETO (8-11). AML1-ETO-involved leukemogenesis provides a challenging and excellent model to study cell proliferation and differentiation.A critical step toward understanding many human pathological processes is to produce the human disease in animal models. Several of the mouse models of AML1-ETO expression that have been developed over the years have shown limited success in reproducing and dissecting molecular mechanisms of t(8;21)-associated AML development. Heterozygous AML1-ETO knockin mice (12, 13) died at the embryonic stage of embryonic day 12.5 (E12.5)-E13.5, failing to establish definitive hematopoiesis. Various subsequent transgenic mouse models that circumvented embryonic lethality did not develop any malignancies (14-16), indicating that AML1-ETO alone was not sufficient for leukemia development. Furthermore, by using retroviralmediated AML1-ETO expression and bone marrow transplantation, AML1-ETO alone did not induce AML (17-19). However, treatment of MRP8-AML1-ETO transgenic mice (15)...

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 ...

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.