Targeting Deubiquitylating Enzyme USP1 in Multiple Myeloma

• Published in: Blood 2015-12, Vol.126 (23), p.1813-1813
• Main Authors: Das, Deepika Sharma, Song, Yan, Ray, Arghya, Richardson, Paul, Chauhan, Dharminder, Anderson, Kenneth C
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V126.23.1813.1813

Introduction Proteasome inhibitor Bortezomib is effective therapy of relapsed/refractory and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Importantly, the ability of bortezomib to overcome resistance to conventional therapies has validated therapeutically targeting the Ubiquitin Proteasome System (UPS), and suggested potential utility of inhibitors of other components of the UPS including deubiquitylating enzymes (DUBs). Therapeutic strategies directed against DUBs may allow for more specific targeting of the UPS, and therefore be less likely to have off-target activities with associated toxicities. Our prior studies have identified a role of USP7, USP14, and UCHL5 in MM pathogenesis, and provided the rationale for targeting these DUBs in MM (Chauhan et al., Cancer Cell 2012, 11:345-358; Tian et al., Blood 2014, 123:706-716). Among DUBs, USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner UAF1, and through its deubiquitylation of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. Here we examined the role of USP1 DUB in MM using both biochemical and RNA interference strategies. Methods We utilized MM cell lines, patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST and CellTiter-Glo assays. MM.1S MM cells were transiently transfected with control short interfering RNA (siRNA), USP1 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. A biochemical inhibitor of USP1 SJB3-019A (SJB) was purchased from Medchem Express, USA. In vitro DUB enzymatic activity was assessed using Ubiquitin-AMC and Ubiquitin-Rhodamine assay kits, as well as Ub-CHOP-reporter and K48-linked Ubiquitin tetramers. Competitive Ub-VS probe labeling was performed, as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Signal transduction pathways were evaluated using immunoblotting. Statistical significance of data was determined using a Student's t test. Results Immunoblot analyses show higher USP1 levels in MM cell lines and patient cells than normal cells.USP1-siRNA inhibited MM cell proliferation, which was rescued by transfection of USP1 (WT). Using Ub-Rhodamine, Ub-AMC, and Ub-EKL reporter assays, we found higher USP1 deubiquitylating activity in patient MM cells versus normal cells, suggesting a favorable therapeutic index