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SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition.

ABSTRACT: Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.

SUBMITTER: Ma J

PROVIDER: S-EPMC8486843 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeAberrant epigenetic changes, like DNA methylation, histone modifications, or ubiquitination, could trigger metabolic disorders in human cancer cells. This study planed to uncover the biological roles of epigenetic SPOP/CHAF1A axis in modulating tumor autophagy during Diffuse large B-cell lymphoma (DLBCL) tumorigenesis.Materials and methodsThe Immunohistochemistry (IHC) was performed to assess the CHAF1A expressions. The expression data of CHAF1A was derived from The Cancer Genome Atlas (TCGA), GSE32918 and GSE83632 datasets. Bioinformatic assays contain differential analysis, functional enrichment analysis and Kaplan-Meier survival curve analysis. The colony generation assay, Transwell assay and CCK-8 assays were conducted for the in vitro assays. The in vivo ubiquitination assays were used to assess regulations of SPOP on CHAF1A. The Chromatin immunoprecipitation (ChIP) assays were used to uncover epigenetic regulations of CHAF1A on TFEB. The relevant DLBCL cells were subcutaneously injected to SCID beige mice to establish the xenograft models.ResultsBioinformatic results revealed that CHAF1A expressed highly in DLBCL that were validated in patients samples. Patients with high CHAF1A suffered from inferior prognosis with shorter survival months relative to those with low CHAF1A. High CHAF1A enhanced DLBCL aggressiveness, including cell proliferation, migration and in vivo growth. Mechanistically, E3 ubiquitin ligase SPOP binds to and induces the degradative ubiquitination of CHAF1A via recognizing a consensus SPOP-binding motif in CHAF1A. SPOP is down-regulated in DLBCL and habours two DLBCL-associated mutations. Deficient SPOP leads to accumulated CHAF1A proteins that promote malignant features of DLBCL. Subsequently, ChIP-qPCR assay revealed that CHAF1A directly binds to TFEB promoters to activate the expressions. High CHAF1A could enhance the transcriptional activity of TFEB and downstream genes. The SPOP/CHAF1A axis modulates TFEB-dependent transactivation to regulate the lysosomal biogenesis and autophagy. The in vivo models suggested that TFEB inhibition is effective to suppress growth of SPOP-deficient DLBCLs.ConclusionsCHAF1A is aberrantly elevated in SPOP-deficient DLBCL. The in-depth mechanism understanding of SPOP/CHAF1A/TFEB axis endows novel targets for DLBCL treatment.

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SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition.

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