Identification of Deubiquitinase OTUD1 As a Novel Player in Resistance of Multiple Myeloma to Bortezomib

Introduction

Multiple myeloma (MM) being one of the most widely spread haematological malignancies remains an incurable disease. As malignant plasma cells produce abnormally large amounts of immunoglobulins they particularly rely on the ubiquitin-proteasome system (UPS) to avoid aberrant protein overload. This unique feature is targeted by proteasome inhibitors (PI) that induce MM cell death especially by increasing levels of reactive oxygen species (ROS).

Despite the high efficacy of PI most of the MM patients eventually relapse and expansion of drug resistant clones makes the treatment ineffective. Therefore, uncovering new molecular mechanisms of drug resistance is a crucial task. UPS is a very complex system that involves hundreds of proteins. While the roles of the proteasome and E3 ligases in PI resistance are well established, the third UPS component, deubiquitinating enzymes (DUBs), is much less explored. In this work, we performed a comprehensive search for DUBs with impact on MM pathogenesis and PI resistance, and further investigated the underlying molecular mechanisms.

Methods

Gene expression and survival

For analysis of DUB genes (n = 101) expression in blood cells, expression dataset Gds3997, DICE database and data from (Jourdan et al., J Immunol. 2011 Oct 15;187(8):3931-41., Jourdan et al., Blood. 2009 Dec 10;114(25):5173-81.) submitted to http://www.genomicscape.com were used. For survival analysis MM patients were divided into two groups by median of gene expression for each DUB (datasets GSE2658, GSE4581 and GSE9782).

Cell line models

RPMI8226 and HEK293 cells were used as model cell lines. Cells with OTUD1 knockdowns and overexpression were generated by lentiviral infection using vectors containing doxycycline-inducible shRNA's and different versions of OTUD1 gene, respectively.

Proliferation and cell death

MTT assay was used for the analysis of cell proliferation and viability. Cell death was also evaluated by flow cytometry by staining with Annexin V and 7-AAD.

Cell migration

Cells were labelled with calcein-AM and placed into the upper chamber of transwell insert with 8-µM pores. After 16 hours of migration towards SDF-1α gradient, cells were counted by fluorescence detection.

ROS analysis

ROS was detected by labelling cells with 2′,7′-dichlorodihydrofluorescein diacetate and measuring fluorescence intensity using flow cytometry.

Immunoprecipitation

HEK293 cells were co-transfected with HA-OTUD1 and FLAG-KEAP1, and reciprocal co-immunoprecipitation and western blot analyses were performed.

Results

We analysed the expression of all human DUBs in different blood cell types and identified OTUD1 as the most differentially expressed DUB between B-cell lineage and other haematopoietic cells. During B-cell maturation OTUD1 expression reaches the maximum in the bone marrow plasma cells.

MM patients with low OTUD1 expression had significantly worse prognosis in OS based on three large datasets (p value= 0,035; 0,008; 2.4e−06. HR=0,55; 0,19; 0,41).

Expression of shRNA targeting OTUD1 in MM cell line RPMI8226 did not affect cell proliferation and migration but dramatically increased survival under oxidative stress (high ROS) conditions induced by bortezomib.

Treatment with bortezomib promoted expression of OTUD1 in the wild type MM cells in a ROS-dependent manner.

Additionally, we identified oxidative stress regulator, the E3 ligase KEAP1 as a novel direct interaction partner of OTUD1 that regulates OTUD1 stability under high ROS conditions.

Conclusion

Based on the gene expression analysis, OTUD1 was identified as a novel, potentially important player in MM pathogenesis. Low levels of OTUD1 expression in MM patients correlate with significantly worse OS. Knocking down OTUD1 in MM cells causes resistance to bortezomib. Mechanistically, bortezomib-induced ROS promotes transcription of OTUD1 mRNA and further induces stabilization of OTUD1 on protein level via disruption of OTUD1-KEAP1 complex. Our collective data suggest on a crucial role of OTUD1 in bortezomib-mediated MM cytotoxity. Further mechanistic studies delineating the role of OTUD1 in MM pathogenesis and PI resistance are ongoing.