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A high-throughput screen of pharmacologically active compounds for inhibitors of UHRF1 reveals epigenetic activity of anthracycline derivative chemotherapeutic drugs.

ABSTRACT: DNA methylation can mediate epigenetic silencing of tumor suppressor and cancer protective genes. The protein ubiquitin-like containing PHD and ring finger domains 1 (UHRF1) is an essential component in cells for DNA methylation maintenance. The SET- and RING-associated (SRA) domain of UHRF1 can bind hemimethylated DNA, and mediate recruitment of DNA methyltransferases to copy the methylation pattern to the newly synthesized daughter strand. Loss of UHRF1 function can lead to demethylation and re-expression of epigenetically silenced tumor suppressor genes and can reduce cancer cell growth and survival. We created a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to screen for inhibitors capable of disrupting the interaction between the UHRF1-SRA domain and hemimethylated DNA. Using this assay (Z' factor of 0.74 in 384-well format) we screened the Library of Pharmacologically Active Compounds (LOPAC) for UHRF1-SRA inhibitors, and validated 7 hit compounds. These compounds included the anthracycline derivatives idarubicin and mitoxantrone, which are commonly used chemotherapeutic drugs known to mediate cytotoxicity by acting as class II topoisomerase (TOP2) poisons. In a panel of additional known topoisomerase poisons, only the anthracycline derivatives showed dose responsive inhibition of UHRF1-SRA. Additionally, mitoxantrone and doxorubicin showed dose-responsive global DNA demethylation and demonstrated a synergistic growth inhibition of multiple cancer cell lines when combined with the DNA methyltransferase (DNMT) inhibitor decitabine. These data validate a novel TR-FRET assay for identification of UHRF1 inhibitors, and revealed unexpected epigenetic properties of commonly used chemotherapeutic drugs that showed synergistic cytotoxicity of cancer cells when combined with a demethylating agent.

SUBMITTER: Giovinazzo H

PROVIDER: S-EPMC6508961 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UHRF1 (ubiquitin-like with PHD and ring finger domains 1) is highly expressed in various human cancers including retinoblastoma, and associated with tumor-promoting effects such as inhibition of apoptosis and high proliferation. However, the molecular mechanisms underlying tumor-promoting functions of UHRF1 in retinoblastoma still remain elusive. Here, we show that stable knockdown of UHRF1 renders retinoblastoma cells sensitized to conventional chemotherapeutic drugs such as etoposide and camptothecin, resulting in enhanced DNA damage and apoptotic cell death. We found that UHRF1-depleted retinoblastoma cells can recognize DNA damages normally but have markedly low expression of XRCC4 (X-ray repair cross complementing 4) among the components of nonhomologous end-joining (NHEJ) repair complex. Conversely, overexpression of UHRF1 increased the XRCC4 expression and stable knockdown of XRCC4 sensitized retinoblastoma cells to etoposide treatment, suggesting that XRCC4 is a key mediator for the drug sensitivity upon UHRF1 depletion in retinoblastoma cells. Consistent with the findings, chromatin association of DNA ligase IV in response to acute DNA damage was found to be significantly reduced in UHRF1-depleted retinoblastoma cells and functional complementation for XRCC4 in UHRF1-depleted cells attenuated the drug sensitivity, demonstrating that XRCC4 downregulation in UHRF1-depleted cells impaired DNA repair and consequently induced robust apoptosis upon genotoxic drug treatment. In human primary retinoblastoma, high expression of UHRF1 and XRCC4 could be detected, and elevated XRCC4 expression correlated with reduced apoptosis markers, implying that UHRF1-mediated XRCC4 upregulation under pathophysiological conditions triggered by RB1 gene inactivation may confer protection against endogenous DNA damages that arise during retinoblastoma development. Taken together, these results present a new mechanistic insight into how UHRF1 mediates its tumor-promoting functions in retinoblastoma, and also provide a basis for UHRF1 targeting to improve the efficacy of current chemotherapy for retinoblastoma treatment.

Project description:BackgroundPraziquantel represents the frontline chemotherapy used to treat schistosomiasis, a neglected tropical disease (NTD) caused by infection with macro-parasitic blood fluke schistosomes. While this drug is safe, its inability to kill all schistosome lifecycle stages within the human host often requires repeat treatments. This limitation, amongst others, has led to the search for novel anti-schistosome replacement or combinatorial chemotherapies. Here, we describe a repositioning strategy to assess the anthelmintic activity of epigenetic probes/inhibitors obtained from the Structural Genomics Consortium.Methodology/principle findingsThirty-seven epigenetic probes/inhibitors targeting histone readers, writers and erasers were initially screened against Schistosoma mansoni schistosomula using the high-throughput Roboworm platform. At 10 μM, 14 of these 37 compounds (38%) negatively affected schistosomula motility and phenotype after 72 hours of continuous co-incubation. Subsequent dose-response titrations against schistosomula and adult worms revealed epigenetic probes targeting one reader (NVS-CECR2-1), one writer (LLY-507 and BAY-598) and one eraser (GSK-J4) to be particularly active. As LLY-507/BAY-598 (SMYD2 histone methyltransferase inhibitors) and GSK-J4 (a JMJD3 histone demethylase inhibitor) regulate an epigenetic process (protein methylation) known to be critical for schistosome development, further characterisation of these compounds/putative targets was performed. RNA interference (RNAi) of one putative LLY-507/BAY-598 S. mansoni target (Smp_000700) in adult worms replicated the compound-mediated motility and egg production defects. Furthermore, H3K36me2, a known product catalysed by SMYD2 activity, was also reduced by LLY-507 (25%), BAY-598 (23%) and siSmp_000700 (15%) treatment of adult worms. Oviposition and packaging of vitelline cells into in vitro laid eggs was also significantly affected by GSK-J4 (putative cell permeable prodrug inhibitor of Smp_034000), but not by the related structural analogue GSK-J1 (cell impermeable inhibitor).Conclusion/significanceCollectively, these results provide further support for the development of next-generation drugs targeting schistosome epigenetic pathway components. In particular, the progression of histone methylation/demethylation modulators presents a tractable strategy for anti-schistosomal control.

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A high-throughput screen of pharmacologically active compounds for inhibitors of UHRF1 reveals epigenetic activity of anthracycline derivative chemotherapeutic drugs.

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