Project description:Liver cancer claims over 800,000 human deaths each year. Liver cancer is notoriously refractory to conventional therapeutics. Further insight into the etiology carries promise for innovative diagnostics and therapeutics. Tumor progression is governed by interplay between tumor promoting genes and suppressor genes. BRD4, an acetyl-lysine binding protein, plays a critical role in development and human diseases. In many cancer types, BRD4 is overexpressed and promotes activation of a pro-tumor gene network. But the underlying mechanism for BRD4 overexpression remains elusive. As BRD4 has risen as a promising therapeutic target, to understand the mechanism regulating BRD4 protein level will shed insight into BRD4-targeting therapeutics. In this study, we find BRD4 protein level in liver cancer is significantly regulated by P53, the most frequently dysregulated tumor suppressor. We identify a strong negative correlation between protein levels of P53 and BRD4 in liver cancer. We then show P53 promotes BRD4 protein degradation. Mechanistically, P53 represses the transcription of USP1, a deubiquitinase, through P21-RB. We show USP1 is a deubiquitinase of BRD4, which increases its stability. We show the pro-tumor role of USP1 is partially mediated by BRD4 and the USP1-BRD4 axis upholds expression of a group of cancer-related genes. In summary, we identify a functional P53-P21-RB-USP1-BRD4 axis in liver cancer.
Project description:CRISPR Cas9-based functional genomics screening is a powerful approach for identifying and characterizing novel oncology drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacological inhibition of USP1 leads to decreased DNA synthesis concomitant with the induction of S-phase-specific DNA damage. Genome-wide CRISPR-Cas9 screens identified RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as downstream mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition was associated with a reduction in total PCNA protein levels. Ectopic expression of WT and ubiquitin-dead K164R PCNA reversed USP1 inhibitor sensitivity. Our results demonstrate, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to a novel subset of BRCA1/2 WT cancer enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant cell lines and xenograft models. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to post-translational modifications of PCNA. Taken together, USP1 inhibition may represent a unique therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.
Project description:Ewing sarcoma (EWS) is a malignant pediatric bone cancer. Most Ewing sarcomas are driven by EWS-FLI1 oncogenic transcription factor that plays roles in transcriptional regulation, DNA damage response, cell cycle checkpoint control, and alternative splicing. USP1, a deubiquitylase which regulates DNA damage and replication stress responses, is overexpressed at both the mRNA and protein levels in EWS cell lines compared to human mesenchymal stem cells, the EWS cell of origin. The functional significance of high USP1 expression in Ewing sarcoma is not known. Here, we identify USP1 as a transcriptional target of EWS-FLI1 and a key regulator of EWS cell survival. We show that EWS-FLI1 knockdown decreases USP1 mRNA and protein levels. ChIP and ChIP-seq analyses show EWS-FLI1 occupancy on the USP1 promoter. Importantly, USP1 knockdown or inhibition arrests EWS cell growth and induces cell death by apoptosis. We observe destabilization of Survivin (also known as BIRC5 or IAP4) and activation of caspases-3 and -7 following USP1 knockdown or inhibition in the absence of external DNA damage stimuli. Notably, EWS cells display hypersensitivity to combinatorial treatment of doxorubicin or etoposide, EWS standard of care drugs, and USP1 inhibitor compared to single agents alone. Together, our study demonstrates that USP1 is regulated by EWS-FLI1, the USP1-Survivin axis promotes EWS cell survival, and USP1 inhibition sensitizes EWS cells to standard of care chemotherapy.
Project description:Efforts to therapeutically target EZH2 have generally focused on inhibition of its methyltransferase activity, although it remains less clear whether this is the central mechanism whereby EZH2 promotes cancer. We demonstrate that EZH2 directly interacts with both MYC family oncoproteins, MYC and MYCN, and promotes their stabilization in a methyltransferase-independent manner. By competing against the SCFFBW7 ubiquitin ligase to bind MYC and MYCN, EZH2 counteracted FBW7-mediated MYC(N) polyubiquitination and proteasomal degradation. Depletion, but not enzymatic inhibition, of EZH2 induced robust MYC(N) degradation and inhibited tumor cell growth in MYC(N) driven neuroblastoma and small cell lung cancer. These findings unveil the MYC family proteins as global EZH2 oncogenic effectors and EZH2 pharmacologic degraders as potential MYC(N) targeted cancer therapeutics, pointing out that MYC(N) driven cancers may develop inherent resistance to the canonical EZH2 enzymatic inhibitors currently in clinical development.
Project description:Identification of critical survival determinants of PDGF-driven proneural glioma. Results provided information about the genes and pathways that are regulated by PDGF signaling in PDGF-driven proneural glioma and led to the assessment of the importance of the USP1-ID2 axis in proneural glioma.
Project description:Identification of critical survival determinants of PDGF-driven proneural glioma. Results provided information about the genes and pathways that are regulated by PDGF signaling in PDGF-driven proneural glioma and led to the assessment of the importance of the USP1-ID2 axis in proneural glioma.
Project description:Identification of critical survival determinants of PDGF-driven proneural glioma. Results provided information about the genes and pathways that are regulated by PDGF signaling in PDGF-driven proneural glioma and led to the assessment of the importance of the USP1-ID2 axis in proneural glioma. Total RNA obtained from PDGF-driven glioma spheroid cells (PDGF-GSC) and primary tumors arising in the Gfap-tTa/Tre-PDGFB mouse model used in our study was analyzed to determine to which subtype of GBM these specimens belonged.