ABSTRACT
Purpose: The ubiquitin proteasome pathway is a validated therapeutic target in multiple myeloma. Deubiquitylating enzyme USP1 participates in DNA damage response and cellular differentiation pathways. To date, the role of USP1 in multiple myeloma biology is not defined. In the present study, we investigated the functional significance of USP1 in multiple myeloma using genetic and biochemical approaches.Experimental Design: To investigate the role of USP1 in myeloma, we utilized USP1 inhibitor SJB3-019A (SJB) for studies in myeloma cell lines and patient multiple myeloma cells.Results: USP1-siRNA knockdown decreases multiple myeloma cell viability. USP1 inhibitor SJB selectively blocks USP1 enzymatic activity without blocking other DUBs. SJB also decreases the viability of multiple myeloma cell lines and patient tumor cells, inhibits bone marrow plasmacytoid dendritic cell-induced multiple myeloma cell growth, and overcomes bortezomib resistance. SJB triggers apoptosis in multiple myeloma cells via activation of caspase-3, caspase-8, and caspase-9. Moreover, SJB degrades USP1 and downstream inhibitor of DNA-binding proteins as well as inhibits DNA repair via blockade of Fanconi anemia pathway and homologous recombination. SJB also downregulates multiple myeloma stem cell renewal/survival-associated proteins Notch-1, Notch-2, SOX-4, and SOX-2. Moreover, SJB induced generation of more mature and differentiated plasma cells. Combination of SJB and HDACi ACY-1215, bortezomib, lenalidomide, or pomalidomide triggers synergistic cytotoxicity.Conclusions: Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential novel multiple myeloma therapy. Clin Cancer Res; 23(15); 4280-9. ©2017 AACR.
Subject(s)
DNA-Binding Proteins/genetics , Drug Synergism , Multiple Myeloma/drug therapy , Neoplasm Proteins/genetics , Ubiquitin-Specific Proteases/genetics , Apoptosis/drug effects , Bortezomib/administration & dosage , Cell Line, Tumor , Cell Survival/drug effects , DNA Damage/drug effects , DNA Repair/drug effects , DNA-Binding Proteins/drug effects , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lenalidomide , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasm Proteins/drug effects , Thalidomide/administration & dosage , Thalidomide/analogs & derivatives , Ubiquitin-Specific Proteases/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
Testicular Sertoli cells (Sc) are main somatic component of seminiferous tubules that govern the differentiation of germ cells (Gc) and provide them physical support. Sc are the target of follicle stimulating hormone (FSH) and testosterone (T) which are known to regulate spermatogenesis. FSH and T levels in human and sub-human male primates remain high during infancy (4-6 months post birth), similar to those during puberty. Subsequently, juvenile phase is marked with low levels of these hormones. In spite of prolonged hormonal exposure, spermatogenesis is not discerned during infancy unlike that during puberty. Situation during infancy is similar to certain idiopathic male infertility, where prolonged hormone supplementation fails to initiate spermatogenesis. In our quest to determine non hormonal causes of idiopathic infertility which may reside within the Sc, we investigated the association between spermatogenesis and Sc specific gene(s) expressed differentially during puberty and infancy. Although products of several genes may be necessary for quantitatively normal spermatogenesis, one needs to investigate their roles one by one. Differential display and real time PCR analysis revealed higher expression of a known tumor suppressor, Dickkopf homolog 3 (DKK3), by pubertal monkey Sc as compared to infant Sc. To evaluate role of DKK3 in spermatogenesis, we generated DKK3 knock down mice (DKDM) using shRNA construct targeted to DKK3. In testis of adult DKDM, expression of DKK3 mRNA and protein were significantly (p<0.05) low and was associated with elevated WNT-4/ß-CATENIN activity. Elevated ß-CATENIN activity is known to restrict Sc maturation. Abundant expression of infant Sc marker, Mullerian inhibiting substance (MIS), in the testes of adult DKDM confirmed lack of Sc maturation in DKDM. Gc differentiation and fertility was severely compromised in DKDM. This is the first report of role of DKK3 in the testis and DKK3 mediated regulation of spermatogenesis via WNT-4/ß-CATENIN modulation.
Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , Sertoli Cells/metabolism , Spermatogenesis , Wnt Signaling Pathway , Adaptor Proteins, Signal Transducing , Animals , Apoptosis , Cell Differentiation , Female , Fertility , Gene Expression Profiling , Gene Knockdown Techniques , Germ Cells/cytology , Germ Cells/metabolism , Haplorhini , Humans , Intercellular Signaling Peptides and Proteins/genetics , Male , Mice , RNA, Small Interfering/metabolism , Seminiferous Tubules/cytology , Seminiferous Tubules/metabolism , Sexual Maturation , Spermatogenesis/geneticsABSTRACT
Apoptosis-inducing factor (Aif) is a mitochondrial flavoprotein that regulates cell metabolism and survival in many tissues. We report that aif-hypomorphic harlequin (Hq) mice show thymic hypocellularity and a cell-autonomous thymocyte developmental block associated with apoptosis at the ß-selection stage, independent of T cell receptor ß recombination. No abnormalities are observed in the B cell lineage. Transgenes encoding wild-type or DNA-binding-deficient mutant Aif rectify the thymic defect, but a transgene encoding oxidoreductase activity-deficient mutant Aif does not. The Hq thymic block is reversed in vivo by antioxidant treatment, and Hq T but not B lineage cells show enhanced oxidative stress. Thus, Aif, a ubiquitous protein, serves a lineage-specific nonredundant antiapoptotic role in the T cell lineage by regulating reactive oxygen species during thymic ß-selection.
