Targeting Deubiquitylating Enzyme USP1 in Multiple Myeloma

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-E_KL reporter assays, we found higher USP1 deubiquitylating activity in patient MM cells versus normal cells, suggesting a favorable therapeutic index for targeting USP1. Importantly, siRNA-knockdown of USP1 both promoted degradation of tumorigenic ID1 protein, and inhibited proliferation of bortezomib-resistant (ANBL-6.BR) MM cells, suggesting that novel agents targeting USP1 may overcome bortezomib resistance. We next examined the effects of USP1 inhibitor SJB3 on MM cell growth and survival in our models of MM. Analysis using Ub-Rhodamine, Ub-AMC, and Ub-E_KL reporter assays in a panel of MM cell lines showed that SJB is a potent, specific, and selective inhibitor of USP1 DUB activity (EC₅₀ = 50 ± 5.7 nM), which does not inhibit other DUBs (USP2/USP5/USP7/USP14) or other families of cysteine proteases (EC₅₀>100 μM). SJB blocks labeling of USP1 with HA-Ub-VS probe in a concentration-dependent manner, but did not alter labeling of other DUBs with HA-Ub-VS. SJB inhibits USP1-mediated cleavage of K48 linked polyubiquitin chains, but not that mediated by USP2 or USP7. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, Dox-40, ARP1, KMS11, U266, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreases their viability (IC₅₀ range 100nM to 500nM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for SJB. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Mechanistic studies show that SJB-triggered apoptosis is associated with degradation of USP1 and Id1 protein. Finally, combination of SJB with lenalidomide, pomalidomide, HDACi ACY-1215, or bortezomib both induces synergistic anti-MM activity and overcomes drug resistance.

Conclusion Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential MM therapy.