Biology subjects

Murai, J.

Publications and source records attributed to Murai, J..

8 recordsLinked to original sources

bioRxiv2026

BRCA2 loss triggers a downward spiral of genomic instability via ROS-dependent metabolic collapse

BRCA2 plays a central role in maintaining genome integrity through homologous recombination and replication-fork protection, yet the compensatory networks sustaining BRCA2-deficient cells remain unclear. Here we show BRCA2 enforces a homeostatic mechanism aligning mitochondrial respiration with DNA repair capacity in both cancer and non-cancer contexts. Genome-wide CRISPR screening identified glutathione metabolism and base-excision repair as the key compensatory networks sustaining BRCA2-deficient cells by detoxifying mitochondria-derived reactive oxygen species. BRCA2 loss provokes an acute mitochondrial ROS surge, causing 8-oxoguanine accumulation and a systemic metabolic crisis marked by NAD+ and glutathione depletion. PARP inhibitor targets DNA replication vulnerabilities, increasing the cellular requirement for BRCA2. The resulting oxidative burden primes cells for TP53-dependent apoptosis in G1 during olaparib treatment, which extends cytotoxicity beyond canonical S-phase stress. These findings indicate BRCA2 prevents metabolic flux from outpacing repair capacity, providing a rationale for combining PARP inhibition with redox modulation to enhance efficacy and overcome resistance. HighlightsO_LIAcute BRCA2 loss induces ROS and mitochondrial dysfunction creating a metabolic scar C_LIO_LIOxidative lesions drive PARP hyperactivation and precipitate a cellular NAD crisis C_LIO_LIPARP inhibitors provoke TP53-dependent apoptosis in G1 beyond replication stress in S phase C_LIO_LIGlutathione deficiency exacerbates bone marrow failure under BRCA2 depletion C_LIO_LIBRCA2 tightly couples mitochondrial redox homeostasis to genomic maintenance C_LI

cell biology↗

bioRxiv2026

Ubiquitin-dependent recruitment of SLFN11 to chromatin is regulated by deubiquitinase and RNF168

The molecular mechanisms driving SLFN11 chromatin recruitment remain partially elucidated. Using high-throughput imaging of 162 oncology-focused compounds in U2OS cells with inducible SLFN11 expression, we discovered that deubiquitinase (DUB) inhibitors drive massive SLFN11 recruitment to chromatin, preferentially at promoter regions while concurrently suppressing transcription. DUB inhibitors such as VLX-1570 promote ubiquitin-dependent enrichment of SLFN11 without detectable DNA damage, distinct from the camptothecin-induced RPA-associated SLFN11 foci formed at stressed replication forks. Yet, SLFN11 chromatin recruitment both by DUB inhibitors and DNA damage are suppressed by TAK243 demonstrating their ubiquitylation dependency. RNF168 is required for SLFN11 ubiquitylation and its subsequent chromatin association, and ubiquitylation within SLFN11s middle linker domain (lysines 390, 391, and 429) with K27-linked polyubiquitin chains is essential for the chromatin recruitment of SLFN11. These findings suggest the importance of SLFN11 ubiquitylation by RNF168 for SLFN11 chromatin recruitment and SLFN11 transcriptional regulatory role at promoter regions.

cell biology↗

bioRxiv2023

Clonal and Scalable Endothelial Progenitor Cell Lines from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) can be used as a renewable source of endothelial cells for treating cardiovascular disease and other ischemic conditions. Here, we present the derivation and characterization of a panel of distinct clonal embryonic endothelial progenitor cell (eEPC) lines that were differentiated from human embryonic stem cells (hESCs). The hESC line, ESI-017, was first partially differentiated to produce candidate cultures from which eEPC were cloned. Endothelial cell identity was assessed by transcriptomic analysis, cell surface marker expression, immunocytochemical marker analysis, and functional analysis using a vascular network forming assay. The transcriptome of the eEPC lines was compared to various adult endothelial lines as well as various non-endothelial cells including both adult and embryonic origins. This resulted in a variety of distinct cell lines with functional properties of endothelial cells and strong transcriptomic similarity to adult endothelial primary cell lines. The eEPC lines, however, were distinguished from adult endothelium by a novel pattern of embryonic gene expression. We demonstrated scalability of up to 80 population doublings and stable with long-term expansion over 50 passages and stable angiogenic properties at late passage in the EPC line. Taken together, these data support the finding that hESC-derived clonal eEPC lines are useful as a source of scalable therapeutic cells and cell products for treating cardiovascular disease. These eEPC lines offer a highly promising resource for preclinical studies and therapeutic interventions.

cell biology↗

bioRxiv2023

Synergistic effects of PARP inhibitors by Schlafen 11 and BRCA2-deficiency through accumulation of single-strand DNA gaps behind a fork

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) induce synthetic lethality in breast cancer gene (BRCA)-deficient tumors. Besides the original model proposed by accumulation of double-strand DNA breaks due to the impaired homologous recombination, accumulation of single-strand DNA (ssDNA) gaps due to impaired BRCA-mediated Okazaki fragment processing has emerged as an alternative mechanism of synthetic lethality. Accordingly, PARPis induce ssDNA gaps behind a replication fork in BRCA-deficient cells. Schlafen 11 (SLFN11), a member of the SLFN family, binds replication protein A (RPA)-coated ssDNA gaps and sensitizes cancer cells to DNA-damaging anticancer agents. These facts motivated us to examine the combinational effects of SLFN11 and BRCA-deficiency on PARPis sensitivity. Here, we show that SLFN11 and BRCA2-deficiency synergistically increased sensitivity to PARPis (talazoparib, niraparib, olaparib, and veliparib) at specific concentrations, where SLFN11 alone showed marginal effects. Using chromatin-bound proteins and alkaline BrdU comet assays in human cancer cells, we revealed that ssDNA gaps induced by PARPis were increased by SLFN11 or BRCA2-deficiency and that the combination of the two had the greatest effect. SLFN11 was recruited to and colocalized with chromatin-bound RPA2 under PARPis. SLFN11 recruited around a fork under DNA damage blocked replication, whereas SLFN11 recruited behind a fork under PARPis did not. Chromatin recruitment of SLFN11 and RPA2 was attenuated by the MRE11 inhibitor mirin. Hence, our studies showed that BRCA2-deficiency increased ssDNA gaps behind a fork under PARPis treatment, where SLFN11 bound and further increased the gaps. Our findings provide a mechanistic understanding of favorable responses to PARPis in SLFN11-proficient and BRCA-deficient tumors. SignificanceThis study reveals how SLFN11 enhances the antitumor effects of PARP inhibitors in BRCA2-deficient cancer cells and highlights the importance of analyzing SLFN11 expression in addition to BRCA analysis in clinical practice.

cancer biology↗

bioRxiv2022

Metabolic clogging of mannose triggers genomic instability via dNTP loss in human cancer cells

Mannose has anti-cancer activity that inhibits cell proliferation and enhances the efficacy of chemotherapy. How mannose exerts its anti-cancer activity, however, remains poorly understood. Here, using genetically engineered human cancer cells that permit the precise control of mannose metabolic flux, we demonstrate that the large influx of mannose exceeding its metabolic capacity induced metabolic remodeling, leading to the generation of slow-cycling cells with limited deoxyribonucleoside triphosphates (dNTPs). This metabolic remodeling impaired dormant origin firing required to rescue stalled forks by cisplatin, thus exacerbating replication stress. Importantly, pharmacological inhibition of de novo dNTP biosynthesis was sufficient to retard cell cycle progression, sensitize cells to cisplatin, and inhibit dormant origin firing, suggesting dNTP loss-induced genomic instability as a central mechanism for the anti-cancer activity of mannose.

cancer biology↗

bioRxiv2022

The germline factor DDX4 contributes to the chemoresistance of small cell lung cancer cells

Human cancers often re-express germline factors, yet their mechanistic role in oncogenesis and cancer progression remains unknown. Here we demonstrate that DDX4, a germline factor and RNA helicase conserved in all multicellular organisms, contributes to epithelial mesenchyme transition (EMT)-like features and cisplatin resistance in small cell lung cancer (SCLC) cells. DDX4 depletion in H69AR and SHP77 cell lines decreased motility and resistance to cisplatin, whereas its overexpression increased these features. Proteomic analysis suggests that DDX4 upregulates metabolic protein expression related to DNA repair and immune/inflammatory response, suggesting its fundamental function may be in regulating cellular metabolism. Consistent with these trends in cell lines, DDX4 depletion compromised in vivo tumor development while its overexpression enhanced tumor growth even after cisplatin treatment in nude mice. Although the DDX4 expression level in somatic tumors is generally low compared to that in the germline, the relatively higher DDX4 expression in SCLC patients correlates with decreased survival and shows increased expression of EMT and cisplatin resistance markers. Taken together, we conclude that DDX4 influences the survival of SCLC patients by altering cellular metabolism in response to environmental cues such as drug treatments. This fundamental function of DDX4 as a germline factor might be applicable in other cancer types that express DDX4 and may serve as a key to combat specific tumors that are highly resistant to treatments. HighlightsO_LIDDX4 contributes to cellular motility and drug resistance in SCLC cells. C_LIO_LIDDX4-overexpression globally alters the proteome and suppresses cytokine production. C_LIO_LIDDX4 promotes tumorigenesis and drug resistance in vitro and in vivo. C_LIO_LIDDX4 expression correlates with survival in SCLC patients and with immune/inflammatory response both in cell lines and patient samples. C_LI

cancer biology↗

bioRxiv2020

Epigenetic suppression of SLFN11 in germinal center B cells in the process of the dynamic expression change during B-cell development

BackgroundSLFN11 enhances cellular toxicity of genotoxic anti-cancer agents, and its important role under physiological conditions has not been appreciated yet. Somatic hypermutations and class switch recombination that can cause physiological genotoxic stress arise in germinal center B cells (GCBs). GCBs are a major origin of B-cell lymphomas that are frequently treated by cytosine arabinoside, a genotoxic anti-cancer agent. ObjectiveTo clarify the expression profile of SLFN11 in different stages of B cells and B-cell lymphomas. MethodsWe analyzed the expression of SLFN11 by mining publicly available databases for different stages of normal B cells and various types of B-cell lymphoma lines and also by performing immunohistochemical staining of human lymph nodes. We investigated the effects of two epigenetic modifiers, an EZH2 inhibitor, tazemetostat (EPZ6438) and a histone deacetylase inhibitor, panobinostat (LBH589) on SLFN11 expression in B-cell lymphoma lines and examined the therapeutic efficacy of these epigenetic modifiers in the combination with cytosine arabinoside. ResultsSLFN11 expression was specifically low in GCBs compared to non-GCBs, which was consolidated by the immunohistochemical staining for SLFN11 with human lymph nodes. SLFN11 expression levels in B-cell lymphoma lines largely correlated to those of their normal counterparts. The epigenetic modifiers upregulated SLFN11 expression in GCB-derived lymphomas and made the lymphomas further susceptible to cytosine arabinoside. ConclusionsThe expression of SLFN11 may be epigenetically suppressed in GCBs and GCB-derived lymphomas. GCB-derived lymphomas with low SLFN11 expression can be treated by the combination of epigenetic modifiers and cytosine arabinoside.

molecular biology↗

bioRxiv2019

BAMscale: quantification of DNA sequencing peaks and generation of scaled coverage tracks

BAMscale is a one-step tool that processes DNA sequencing datasets from chromatin binding (ChIP-seq) and chromatin state changes (ATAC-seq, END-seq) experiments to DNA replication data (OK-seq, NS-seq and replication timing). The outputs include normalized peak scores in text format and scaled coverage tracks (BigWig) which are directly accessible to data visualization programs. BAMscale (available at https://github.com/ncbi/BAMscale) effectively processes large sequencing datasets (~100Gb size) in minutes, outperforming currently available tools.

bioinformatics↗