USP22 controls necroptosis by regulating receptor-interacting protein kinase 3 ubiquitination.

Dynamic control of ubiquitination by deubiquitinating enzymes is essential for almost all biological processes. Ubiquitin-specific peptidase 22 (USP22) is part of the SAGA complex and catalyzes the removal of mono-ubiquitination from histones H2A and H2B, thereby regulating gene transcription. However, novel roles for USP22 have emerged recently, such as tumor development and cell death. Apart from apoptosis, the relevance of USP22 in other programmed cell death pathways still remains unclear. Here, we describe a novel role for USP22 in controlling necroptotic cell death in human tumor cell lines. Loss of USP22 expression significantly delays TNFα/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFα-mediated NF-κB activation or extrinsic apoptosis. Ubiquitin remnant profiling identified receptor-interacting protein kinase 3 (RIPK3) lysines 42, 351, and 518 as novel, USP22-regulated ubiquitination sites during necroptosis. Importantly, mutation of RIPK3 K518 reduced necroptosis-associated RIPK3 ubiquitination and amplified necrosome formation and necroptotic cell death. In conclusion, we identify a novel role of USP22 in necroptosis and further elucidate the relevance of RIPK3 ubiquitination as crucial regulator of necroptotic cell death.

Comprehensive multiplexed autoantibody profiling of patients with advanced urothelial cancer.

BACKGROUND: Comprehensive profiling of autoantibodies (AAbs) in metastatic urothelial cancer (mUC) has not been performed to date. This may aid in diagnosis of UC, uncover novel therapeutic targets in this disease as well as identify associations between AAbs and response and toxicity to systemic therapies. METHODS: We used serum from patients with mUC collected prior to and after systemic therapy (immune checkpoint inhibitor (ICI) or platinum-based chemotherapy (PBC)) at Dana-Farber Cancer Institute. 38 age-matched and sex-matched healthy controls (HCs) from healthy blood donors were also evaluated. The SeroTag immuno-oncology discovery array (Oncimmune) was used, with quantification of the AAb reactivity toward 1132 antigens. Bound AAbs were detected using an anti-immunoglobulin G-specific detection antibody conjugated to the fluorescent reporter dye phycoerythrin. The AAb reactivity was reported as the median fluorescence intensity for each color and sample using a Luminex FlexMAP3D analyzer. Clinical outcomes of interest included radiographic response and development of immune-related adverse events (irAEs). Significance analysis of microarray was used to compare mUC versus HC and radiographic response. Associations with irAE were evaluated using a logistic regression model. P<0.05 was considered statistically significant. RESULTS: 66 patients were included with a median age of 68 years; 54 patients (82%) received ICI and 12 patients (18%) received PBC. Compared with HCs, AAbs against the cancer/testis antigens (CTAG1B, CTAG2, MAGEB18), HSPA1A, TP53, KRAS, and FGFR3 were significantly elevated in patients with mUC. AAbs against BRCA2, TP53, and CTNBB1 were associated with response, and those against BICD2 and UACA were associated with resistance to ICI therapy. AAbs against MITF, CDH3, and KDM4A were associated with development of irAEs in patient who received an ICI. A higher variance in pre-to-post treatment fold change in AAb levels was seen in patients treated with ICI versus PBC and was associated with response to ICI. CONCLUSIONS: This is the first report of comprehensive AAb profiling of patients with mUC and identified key AAbs that were elevated in patients with mUC versus HCs as well as AAbs associated with therapeutic response to ICI. These findings are hypothesis generating and further mechanistic studies evaluating humoral immunity in UC are required.

USP9X stabilizes XIAP to regulate mitotic cell death and chemoresistance in aggressive B-cell lymphoma.

The mitotic spindle assembly checkpoint (SAC) maintains genome stability and marks an important target for antineoplastic therapies. However, it has remained unclear how cells execute cell fate decisions under conditions of SAC-induced mitotic arrest. Here, we identify USP9X as the mitotic deubiquitinase of the X-linked inhibitor of apoptosis protein (XIAP) and demonstrate that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons. We find that primary human aggressive B-cell lymphoma samples exhibit high USP9X expression that correlate with XIAP overexpression. We show that high USP9X/XIAP expression is associated with shorter event-free survival in patients treated with spindle poison-containing chemotherapy. Accordingly, aggressive B-cell lymphoma lines with USP9X and associated XIAP overexpression exhibit increased chemoresistance, reversed by specific inhibition of either USP9X or XIAP. Moreover, knockdown of USP9X or XIAP significantly delays lymphoma development and increases sensitivity to spindle poisons in a murine Eµ-Myc lymphoma model. Together, we specify the USP9X-XIAP axis as a regulator of the mitotic cell fate decision and propose that USP9X and XIAP are potential prognostic biomarkers and therapeutic targets in aggressive B-cell lymphoma.