Discovery Of The Vulnerability Of Tumors Carrying BAP1 Mutations To BET Inhibitors And Study Of Involved Mechanisms

BackgroundUveal melanoma(UM)is the most common primary intraocular malignancy in adults.The main treatments for in situ UM are surgery and local radiotherapy.However,approximately half of UM patients develop metastasis,with about 90%metastasis in the liver.Once metastasized,there are no effective therapies at present,with an overall survival of 2 to 8 months.Inactivating somatic mutations of BAP 1 gene were identified in more than 80%metastasizing UM.Our study found that BAP1 knockout increases histone H2AK119 monoubiquitination,leading to an abnormal epigenetic status in knockout cells compared to their wild type counterparts.By Epigenetic Compound Library screening,we identified that BET inhibitors exhibited selective cytotoxicity to BAP1 knockout UM cells than compared wirh their wild type counterparts.As epigenetic readers,Bromodomain and extraterminal domain(BET)family proteins can recognize and bind acetylated histones and then regulate gene transcription.BET inhibitors target the bromodomain extra-terminal(BET)subfamily and mimic the acetyl-lysine moiety,preventing the interaction of BET bromodomains with acetylated histones.Since the first BET inhibitor JQ1 had been developed to display a promising anticancer effect in NUT midline carcinoma(NMC)preclinical study,accumulating BET inhibitors have been discovered,implying that they would be one of the most promising strategy in cancer.However,the vulnerability of BAP1 mutant tumors to BET inhibitors has not been reported,which is our novel findings.ObjectivesEncouraged by the discovery of the vulnerability of BAP1 knockout cells to BET inhibitors,we further explored the involved mechanism.Moreover,the in vivo anticancer activity of BET inhibitor was determined in xenografted mice models.This study aims to offer individualized treatment approaches for cancer patients carrying BAP1 mutations with a high risk of metastasis.Methods1.CRISPR/Cas9 was used to establish BAP1 KO isogenic UM cells.2.MTT method was used to screen the epigenetic compound library.3.The rescue experiments with retrovirus-mediated ectopic expression of wild type and mutant BAP1 proteins was used for phenotype study.4.mRNA-sequencing,MTT assay,QPCR,cell cycle and apoptosis analysis,CRISPR Cas9 technology,retrovirus-mediated ectopic expression and Western blot were used to explore the molecular mechanism underlying the vulnerability of BAP1 mutant tumors to BETi.5.Nude mice xenograft model was established to study the vulnerability of BAP1 mutant tumors to BETi in vivo.Results1.OCM1 and OMM2.3 BAP1 KO isogenic cell lines were established successfully.2.Through the screening and verification of epigenetic compound library,we found that BET inhibitors have selective killing effect on BAP1 KO UM cells.3.When exogenous wild type BAP1 was introduced,the sensitivity to BET inhibitors was reversed in various cancer cells,including BAP1 KO UM cells,renal clear cell carcinoma(ccRCC)and human mesothelioma cells carrying BAP 1 inactive mutants.4.Compared with WT UM cells,BET inhibitors significantly induced the G0/G1phase cell arrest and promoted apoptosis in BAP1 KO UM cells,which was accompanied with a significant reduction in a variety of proteins such as c-Myc protein(a classic target of BET inhibitors).5.Overexpression of c-Myc and the anti-apoptotic gene Bcl-2 in the BAP1 KO UM cells partially reversed cellular sensitivity to the BET inhibitor OTX015.6.Single and double knockout of PRC1 complex proteins RINGI and RING2 showed that RING1 and RING2 were antagonistic to BAP1 in UM cells in terms of H2A ubiquitination.And the cell proliferation was inhibited when double knokouted RING1/2.7.H2AK119 ubiquitination was increased by RING1 and RING2 in WT UM cells,leading to the enhanced sensitivity to OTX015.8.BET inhibitors exhibited cytotoxic action via inhibiting BRD2 and BRD4.9.BAP 1-mutated UM tumors were more sensitive to OTX015 in nude mice xenograft tumors.Conclusion1.BAP1 mutant tumors are vulnerable to BET inhibitors.2.BET inhibitors exhibit selective cytotoxicity to BAP1 mutant UM cells by inhibiting BRD2 and BRD4 via cell cycle arrest and apoptosis.3.High levels of H2AK119 ubiquitination may be a marker for the vulnerability to BET inhibitors in cancer cells.4.BET inhibitor OTX015 showed selective antitumor activity against BAP1 mutant tumors in vivo.