Blockade of Deubiquitylating Enzyme USP1 Inhibits DNA Repair and Triggers Apoptosis in Multiple Myeloma Cells

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.

Figure 1. USP1 in MM

(A) USP1 expression in plasma cells from normal healthy donors, as well as tumor cells from patients with MGUS, SMM, and MM. Expression data: GSE5900 and GSE2658 from GEO (https://www.ncbi.nlm.nih.gov/geo/) Normal-MM and normal-SMM USP1 expression p values are 0.0005 and 0.002 respectively (B) Purified peripheral blood mononuclear cells (PBMCs), bone marrow MNCs from normal donors and MM cell lines were assessed for USP1 by immunoblotting with anti-USP1 and anti-GAPDH antibodies. (C) Kaplan-Meier plots of USP1 expression versus overall and event free survival of MM patients. Red line indicates patient group with higher USP1 expression, and blue line show patient cohort with lower USP1 expression. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39754 (D) MM.1S cells were transfected with genome control-siRNA/scr-siRNA or USP1-siRNA and cultured for 24h, followed by analysis of cell viability by WST assay. Percent cell viability was normalized against scr-siRNA control (mean ± SE; p < 0.005, n=3). Western blot shows USP1 expression in cells transfected with Scr-siRNA or USP1 siRNA.

Figure 2. Biochemical Characterization of USP1 inhibitor SJB3-019A (SJB)

(A) Chemical structure of SJB-019A: 2-(pyridin-3-yl) naphtho[2,3-d]oxazole-4,9-dione. (B) Schematic representation of Ub-AMC assay. DUB removes ubiquitin from its substrate Ub-AMC, and fluorescent AMC is measured. (C) Recombinant USP1/UAF1 complex or rUSP2 were incubated with DMSO control, USP1 inhibitor SJB, or USP7 inhibitor P5091 for 30 mins at 37°C, followed by the assessment of DUB activity using Ub-AMC assay (mean ± SE; p < 0.05 for USP1 activity in control verses SJB treated samples, n=3). (D) Indicated rDUBs were incubated with DMSO control or USP1 inhibitor SJB for 30 mins at 37°C, followed by assessment of DUB activity using Ub-AMC assay (mean ± SE; p < 0.05, n=3). (E) MM.1S cells were treated with DMSO control or SJB for 3h; protein lysates were subjected to immunoprecipitation with different DUBs (USP1, USP2, USP5, USP7 and USP14), followed by analysis of DUB expression and enzymatic activity. DUB immunoprecipitates were examined for DUB activity with Ub-AMC assay (mean ± SE; p < 0.05 for USP1 activity in control verses treated cell lysates, n=3). Immunoblot: DUB immunoprecipitates were subjected to immunoblotting with antibodies specific against USP1, USP2, USP5, USP7, or USP14.

Figure 3. SJB binds to USP1 and inhibits its deubiquitylating enzymatic activity

(A) MM.1S cells were treated with DMSO or SJB for 3h; protein lysates were incubated with HA-Ub-VS probe for 30 mins, followed by immunoblot analysis using antibodies against USP1, USP2, USP5 or USP7. (B) Ub-chain disassembly reactions of K48-linked Ub tetramers by purified recombinant USP1/UAF1 complex after 30 mins incubation with DMSO, USP1 inhibitor SJB, or proteasome inhibitor marizomib.

Figure 4. Cytotoxic activity of SJB against MM cells

(A) Indicated MM-cell lines were treated with DMSO control or SJB for 24h, followed by assessment for cell viability using WST-1 assay (mean ± SE; p < 0.05 for all cell lines; n=3). Cell viability data are presented in a Heatmap. (B) CD138+ patient MM cells were treated with DMSO or SJB for 24h, followed by assessment for cell viability of patient samples (Pt #1-Pt #5) using CellTiter-Glo assay (mean ± SE; p < 0.001, n=3), (C) Normal donor PBMCs were treated with SJB for 24h, and then analyzed for viability using WST-1 assay (mean ± SE of quadruplicate cultures). (D) MM.1S cells were cultured with or without BMSCs for 24h in the presence or absence of SJB, and cell growth was assessed using WST1 assay (mean ± SE of triplicate cultures; p < 0.05 for all samples). (E) MM.1S cells were cultured with or without patient plasmacytoid dendritic cells (pDCs) for 24h with or without SJB, and cell growth was assessed by WST1 assay (mean ± SE of triplicate cultures; p < 0.005 for all samples).

Figure 5. Mechanisms of action of SJB in MM cells

(A) MM.1S cells were treated with SJB for 15h, and analyzed for DNA content with propidium idodide (PI) staining and FACS. Percentage of cell populations in G2/M-, S-, or G1-phase of cell cycle is shown in bar graph (mean ± SD; n = 3; p < 0.001). Immunoblot: MM.1S cells were treated with indicated concentrations of SJB for 15h; protein lysates were then subjected to immunoblot analysis using anti-p21 and anti-β–actin Abs. (B) Bar graph: MM.1S cells were treated with SJB for 15h, and then analyzed for apoptosis using Annexin V/PI staining (mean ± SD; n = 3; p < 0.005). Immunoblot: MM.1S cells were treated with SJB for 12h; protein lysates were subjected to western blotting using antibodies directed against PARP, caspase-3, caspase-8, caspase-9 or β-actin. (C) MM.1S cells were treated with DMSO control or SJB for 12h; protein lysates were subjected to immunoblot analysis using antibodies specific against with FANCD2, FANCI, PCNA, or GAPDH. (D) MM.1S cells were treated with DMSO or SJB for 6h, and cells were stained with anti-RAD51 Abs. DAPI was used as counterstain for nuclei. Bar graph: Quantification of RAD51 Foci/nuclei using multiple fields. (E) MM.1S cells were treated with DMSO or SJB for 4h; cells were washed with plain medium to remove SJB, and then cultured in fresh complete medium for 24h, followed by cell viability cell viability analysis (mean ± SE; p < 0.05, n=3). In addition, cells were treated with SJB continuously for 24h, and then subjected to viability analysis (mean ± SE; p < 0.05, n=3).

Figure 6. SJB degrades USP1, ID proteins and alters MM cell morphology

(A) MM.1S cells were treated with DMSO control or SJB for 12h; protein lysates were subjected to immunoblot analysis using antibodies directed against USP1, ID1, ID2, ID3, ID4 or GAPDH. (B) MM.1S cells were treated with DMSO control or SJB for 6h; cells were seeded on the glass slides and stained with giemsa stain. (C) MM.1S cells were treated with DMSO control or SJB for 12h; protein lysates were subjected to immunoblot analysis using antibodies directed against SOX-4, SOX-2, NOTCH-2, NOTCH-1 or β-actin. (D) RPMI-8226 cells were treated with DMSO control or SJB for 12h; and subjected to dot plot analysis of MM side population (MM-SP) using FACS. MM-SP was sorted using staining and FACS. Hoechst 33342 accumulation was assessed in RPMI-8226 cells cultured with DMSO alone or in the presence of SJB (100 nM), Verapamil (100 μM) and reserpine (50 μM). Abscissa represents Hoechst red fluorescence intensity, and ordinate is Hoechst blue fluorescence intensity, with flow gate representing the MM-SP fraction of RPMI-8226 cells.