Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice.

Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.

Frequent somatic gene conversion as a mechanism for loss of heterozygosity in tumor suppressor genes.

The major processes in carcinogenesis include the inactivation of tumor-suppressor genes (TSGs). Although Knudson's two-hit model requires two independent inactivating mutations, perhaps more frequently, a TSG inactivation can occur through a loss of heterozygosity (LOH) of an inactivating mutation. Deletion and uniparental disomy (UPD) have been well documented as LOH mechanisms, but the role of gene conversion is poorly understood. Here, we developed a simple algorithm to detect somatic gene conversion from short-read sequencing data. We applied it to 6285 cancer patient samples, from which 4978 somatic mutations that underwent gene conversion to achieve LOH were found. This number accounted for 14.8% of the total LOH mutations. We further showed that LOH by gene conversion was enriched in TSGs compared with non-TSG genes, showing a significant contribution of gene conversion to carcinogenesis.

Roles of TGF-ß signals in tumor microenvironment via regulation of the formation and plasticity of vascular system.

Tumor cells evolve in tumor microenvironment composed of multiple cell types. Among these, endothelial cells (ECs) are the major players in tumor angiogenesis, which is a driver of tumor progression and metastasis. Increasing evidence suggests that ECs also contribute to tumor progression and metastasis as they modify their phenotypes to differentiate into mesenchymal cells through a process known as endothelial-mesenchymal transition (EndoMT). This plasticity of ECs is mediated by various cytokines, including transforming growth factor-ß (TGF-ß), and modulated by other stimuli depending on the cellular contexts. Recent lines of evidence have shown that EndoMT is involved in various steps of tumor progression, including tumor angiogenesis, intravasation and extravasation of cancer cells, formation of cancer-associated fibroblasts, and cancer therapy resistance. In this review, we summarize current updates on EndoMT, highlight the roles of EndoMT in tumor progression and metastasis, and underline targeting EndoMT as a potential therapeutic strategy.

Inhibition of transforming growth factor-ß signals suppresses tumor formation by regulation of tumor microenvironment networks.

The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-ß (TGF-ß) promotes tumor progression by inducing epithelial-mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-ß receptor containing both TGF-ß type I (TßRI) and type II (TßRII) receptors (TßRI-TßRII-Fc), which trapped all TGF-ß isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TßRI-TßRII-Fc protein effectively suppressed in vitro EMT of oral cancer cells and in vivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TßRI-TßRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TßRI-TßRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1ß (IL-1ß) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1ß and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-ß signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1ß/EREG pathways and that TßRI-TßRII-Fc protein is a promising tool for targeting the TME networks.

CD40 is expressed in the subsets of endothelial cells undergoing partial endothelial-mesenchymal transition in tumor microenvironment.

Tumor progression and metastasis are regulated by endothelial cells undergoing endothelial-mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells lose their properties and differentiate into mesenchymal cells. The cells undergoing EndoMT differentiate through a spectrum of intermediate phases, suggesting that some cells remain in a partial EndoMT state and exhibit an endothelial/mesenchymal phenotype. However, detailed analysis of partial EndoMT has been hampered by the lack of specific markers. Transforming growth factor-ß (TGF-ß) plays a central role in the induction of EndoMT. Here, we showed that inhibition of TGF-ß signaling suppressed EndoMT in a human oral cancer cell xenograft mouse model. By using genetic labeling of endothelial cell lineage, we also established a novel EndoMT reporter cell system, the EndoMT reporter endothelial cells (EMRECs), which allow visualization of sequential changes during TGF-ß-induced EndoMT. Using EMRECs, we characterized the gene profiles of multiple EndoMT stages and identified CD40 as a novel partial EndoMT-specific marker. CD40 expression was upregulated in the cells undergoing partial EndoMT, but decreased in the full EndoMT cells. Furthermore, single-cell RNA sequencing analysis of human tumors revealed that CD40 expression was enriched in the population of cells expressing both endothelial and mesenchymal cell markers. Moreover, decreased expression of CD40 in EMRECs enhanced TGF-ß-induced EndoMT, suggesting that CD40 expressed during partial EndoMT inhibits transition to full EndoMT. The present findings provide a better understanding of the mechanisms underlying TGF-ß-induced EndoMT and will facilitate the development of novel therapeutic strategies targeting EndoMT-driven cancer progression and metastasis.

Transforming growth factor-ß signals promote progression of squamous cell carcinoma by inducing epithelial-mesenchymal transition and angiogenesis.

At present, the molecular mechanisms driving the progression and metastasis of oral squamous cell carcinoma (OSCC) remain largely uncharacterized. The activation of transforming growth factor-ß (TGF-ß) signaling in the tumor microenvironment has been observed in various types of cancer and has been implicated their progression by enhancing the migration and invasion of epithelial cancer cells. However, its specific roles in the oral cancer progression remain unexplored. In this study, we examined the effects of TGF-ß signaling on the murine squamous cell carcinoma, SCCVII cells in vitro and in vivo. The incubation of SCCVII cells with TGF-ß induced the activation of TGF-ß signals and epithelial-mesenchymal transition (EMT). Notably, the motility of SCCVII cells was increased upon the activation of the TGF-ß signaling. RNA sequencing revealed upregulation of genes related to EMT and angiogenesis. Consistent with these in vitro results, the inhibition of TGF-ß signals in SCCVII cell-derived primary tumors resulted in suppressed angiogenesis. Furthermore, we identified six candidate factors (ANKRD1, CCBE1, FSTL3, uPA, TSP-1 and integrin ß3), whose expression was induced by TGF-ß in SCCVII cells, and associated with poor prognosis for patients with head and neck squamous cell carcinoma. These results highlight the role of TGF-ß signals in the progression of OSCC via multiple mechanisms, including EMT and angiogenesis, and suggest novel therapeutic targets for the treatment of OSCC.

Impaired tumor immune response in metastatic tumors is a selective pressure for neutral evolution in CRC cases.

A Darwinian evolutionary shift occurs early in the neutral evolution of advanced colorectal carcinoma (CRC), and copy number aberrations (CNA) are essential in the transition from adenoma to carcinoma. In light of this primary evolution, we investigated the evolutionary principles of the genome that foster postoperative recurrence of CRC. CNA and neoantigens (NAG) were compared between early primary tumors with recurrence (CRCR) and early primary tumors without recurrence (precancerous and early; PCRC). We compared CNA, single nucleotide variance (SNV), RNA sequences, and T-cell receptor (TCR) repertoire between 9 primary and 10 metastatic sites from 10 CRCR cases. We found that NAG in primary sites were fewer in CRCR than in PCRC, while the arm level CNA were significantly higher in primary sites in CRCR than in PCRC. Further, a comparison of genomic aberrations of primary and metastatic conditions revealed no significant differences in CNA. The driver mutations in recurrence were the trunk of the evolutionary phylogenic tree from primary sites to recurrence sites. Notably, PD-1 and TIM3, T cell exhaustion-related molecules of the tumor immune response, were abundantly expressed in metastatic sites compared to primary sites along with the increased number of CD8 expressing cells. The postoperative recurrence-free survival period was only significantly associated with the NAG levels and TCR repertoire diversity in metastatic sites. Therefore, CNA with diminished NAG and diverse TCR repertoire in pre-metastatic sites may determine postoperative recurrence of CRC.

Subclonal accumulation of immune escape mechanisms in microsatellite instability-high colorectal cancers.

BACKGROUND: Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH. METHODS: We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed. RESULTS: Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment. CONCLUSIONS: Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.

Visible-light-driven enantioselective intermolecular [2 + 2] photocyclization utilizing bathochromic excitation mediated by a chiral phosphoric acid.

We report herein an enantioselective intermolecular [2 + 2] photocyclization of alkenyl 2-pyrrolyl ketones using the bathochromic shift mediated by a chiral phosphoric acid. This synthetic method provides access to cyclobutanes with up to 98% ee. According to the UV-Vis spectra, the bathochromic effect was observed by mixing alkenyl 2-pyrrolyl ketones and a chiral phosphoric acid. A non-linear correlation was observed between the ee of the catalyst and the ee of the cycloadduct, suggesting that both substrates bind to the chiral phosphoric acid and form a dimer complex before photocycloaddition.

A globin-family protein, Cytoglobin 1, is involved in the development of neural crest-derived tissues and organs in zebrafish.

The zebrafish is an excellent model animal that is amenable to forward genetics approaches. To uncover unknown developmental regulatory mechanisms in vertebrates, we conducted chemical mutagenesis screening and identified a novel mutation, kanazutsi (kzt). This mutation is recessive, and its homozygotes are embryonic lethal. Mutant embryos suffered from a variety of morphological defects, such as head flattening, pericardial edema, circulation defects, disrupted patterns of melanophore distribution, dwarf eyes, a defective jaw, and extensive apoptosis in the head, which indicates that the main affected tissues are derived from neural crest cells (NCCs). The expression of tissue-specific markers in kzt mutants showed that the early specification of NCCs was normal, but their later differentiation was severely affected. The mutation was mapped to chromosome 3 by linkage analyses, near cytoglobin 1 (cygb1), the product of which is a globin-family respiratory protein. cygb1 expression was activated during somitogenesis in somites and cranial NCCs in wild-type embryos but was significantly downregulated in mutant embryos, despite the normal primary structure of the gene product. The kzt mutation was phenocopied by cygb1 knockdown with low-dose morpholino oligos and was partially rescued by cygb1 overexpression. Both severe knockdown and null mutation of cygb1, established by the CRISPR/Cas9 technique, resulted in far more severe defects at early stages. Thus, it is highly likely that the downregulation of cygb1 is responsible for many, if not all, of the phenotypes of the kzt mutation. These results reveal a requirement for globin family proteins in vertebrate embryos, particularly in the differentiation and subsequent development of NCCs.

Pathological complete remission of relapsed tumor by photo-activating antibody-mimetic drug conjugate treatment.

Antibody-mimetic drug conjugate is a novel noncovalent conjugate consisting of an antibody-mimetic recognizing a target molecule on the cancer cell surface and low-molecular-weight payloads that kill the cancer cells. In this study, the efficacy of a photo-activating antibody-mimetic drug conjugate targeting HER2-expressing tumors was evaluated in mice, by using the affibody that recognize HER2 (ZHER2:342 ) as a target molecule and an axially substituted silicon phthalocyanine (a novel potent photo-activating compound) as a payload. The first treatment with the photo-activating antibody-mimetic drug conjugates reduced the size of all HER2-expressing KPL-4 xenograft tumors macroscopically. However, during the observation period, relapsed tumors gradually appeared in approximately 50% of the animals. To evaluate the efficacy of repeated antibody-mimetic drug conjugate treatment, animals with relapsed tumors were treated again with the same regimen. After the second observation period, the mouse tissues were examined histopathologically. Unexpectedly, all relapsed tumors were eradicated, and all animals were diagnosed with pathological complete remission. After the second treatment, skin wounds healed rapidly, and no significant side effects were observed in other organs, except for occasional microscopic granulomatous tissues beneath the serosa of the liver in a few mice. Repeated treatments seemed to be well tolerated. These results indicate the promising efficacy of the repeated photo-activating antibody-mimetic drug conjugate treatment against HER2-expressing tumors.

Antibody mimetic drug conjugate manufactured by high-yield Escherichia coli expression and non-covalent binding system.

Antibody-drug conjugates (ADCs) are a major therapeutic tool for the treatment of advanced cancer. Malignant cells in advanced cancer often display multiple genetic mutations and become resistant to monotherapy. Therefore, a therapeutic regimen that simultaneously targets multiple molecules with multiple payloads is desirable. However, the development of ADCs is hampered by issues in biopharmaceutical manufacturing and the complexity of the conjugation process of low-molecular-weight payloads to biologicals. Here, we report antibody mimetic-drug conjugates (AMDCs) developed by exploiting the non-covalent binding property of payloads based on high-affinity binding of mutated streptavidin and modified iminobiotin. Miniprotein antibodies were fused to a low immunogenic streptavidin variant, which was then expressed in Escherichia coli inclusion bodies, solubilized, and refolded into functional tetramers. The AMDC developed against human epidermal growth factor receptor 2 (HER2) effectively killed cultured cancer cells using bis-iminobiotin conjugated to photo-activating silicon phthalocyanine. The HER2-targeting AMDC was also effective in vivo against a mouse KPL-4 xenograft model. This AMDC platform provides rapid, stable, and high-yield therapeutics against multiple targets.

Targeting all transforming growth factor-ß isoforms with an Fc chimeric receptor impairs tumor growth and angiogenesis of oral squamous cell cancer.

Tumor progression is governed by various growth factors and cytokines in the tumor microenvironment (TME). Among these, transforming growth factor-ß (TGF-ß) is secreted by various cell types residing in the TME and promotes tumor progression by inducing the epithelial-to-mesenchymal transition (EMT) of cancer cells and tumor angiogenesis. TGF-ß comprises three isoforms, TGF-ß1, -ß2, and -ß3, and transduces intracellular signals via TGF-ß type I receptor (TßRI) and TGF-ß type II receptor (TßRII). For the purpose of designing ligand traps that reduce oncogenic signaling in the TME, chimeric proteins comprising the ligand-interacting ectodomains of receptors fused with the Fc portion of immunoglobulin are often used. For example, chimeric soluble TßRII (TßRII-Fc) has been developed as an effective therapeutic strategy for targeting TGF-ß ligands, but several lines of evidence indicate that TßRII-Fc more effectively traps TGF-ß1 and TGF-ß3 than TGF-ß2, whose expression is elevated in multiple cancer types. In the present study, we developed a chimeric TGF-ß receptor containing both TßRI and TßRII (TßRI-TßRII-Fc) and found that TßRI-TßRII-Fc trapped all TGF-ß isoforms, leading to inhibition of both the TGF-ß signal and TGF-ß-induced EMT of oral cancer cells, whereas TßRII-Fc failed to trap TGF-ß2. Furthermore, we found that TßRI-TßRII-Fc suppresses tumor growth and angiogenesis more effectively than TßRII-Fc in a subcutaneous xenograft model of oral cancer cells with high TGF-ß expression. These results suggest that TßRI-TßRII-Fc may be a promising tool for targeting all TGF-ß isoforms in the TME.

Activation of ß2-adrenergic receptor signals suppresses mesenchymal phenotypes of oral squamous cell carcinoma cells.

Metastasis is a primary reason related to the mortality of oral squamous cell carcinoma (OSCC) patients. A program called epithelial-mesenchymal transition (EMT) has been shown to play a critical role in promoting metastasis in epithelium-derived carcinoma. During EMT, epithelial cancer cells acquire motile mesenchymal phenotypes and detach from primary tumors. Recent lines of evidence have suggested that EMT confers cancer cells with tumor-initiating ability. Therefore, selective targeting of EMT would lead to the development of effective therapeutic agents. In this study, using a chemical biology approach, we identified isoxsuprine, a ß2-adrenergic receptor (ß2-AR) agonist as a low-molecular-weight compound that interferes with the acquisition of mesenchymal phenotypes of oral cancer cells. Treatment of multiple types of oral cancer cells with isoxsuprine led to the downregulation of mesenchymal cell markers that was accompanied by reduced cell motility. Similar inhibitory effects were also observed for isoprenaline, a non-selective ß-adrenergic receptor (ß-AR) agonist. In addition, inhibition of cell migration upon treatment with isoxsuprine was reverted by a non-selective ß-AR antagonist, propranolol, and the CRISPR/Cas9 system-mediated deletion of the ß2-AR gene, suggesting that the effects exerted by isoxsuprine involved signals mediated by ß2-AR. In addition, in a subcutaneous xenograft model of oral cancer cells, the administration of isoxsuprine effectively suppressed primary tumor growth, suggesting ß2-AR signals to be a promising cancer therapeutic target for treatment of OSCC.

Independently programmable frequency-multiplexed phase-sensitive optical parametric amplification in the optical telecommunication band.

We experimentally demonstrate programmable multimode phase-sensitive amplification multiplexed in the frequency domain for flexible control of parallelly generated squeezed states. We utilize the unique phase-matching condition of a type-II periodically poled potassium titanyl phosphate (PPKTP) crystal and pulse shaping technique to fully control the frequency-domain parallel generation of squeezed states in the optical telecommunication band. We experimentally verify that the independent programmability of phase-sensitive optical parametric amplification (OPA) for the modes corresponding to different frequency bands can be achieved by shaping the pump laser pulse from optical parametric gain measurements using a coherent probe light generated by a degenerate synchronously pumped optical parametric oscillator.

Pharmacokinetics of CuGTSM, a Novel Drug Candidate, in a Mouse Model of Menkes Disease.

PURPOSE: Menkes disease is a rare hereditary disease in which systemic deficiency of copper due to mutation of the ATP7A gene causes severe neurodegenerative disorders. The present parenteral drugs have limited efficacy, so there is a need for an efficacious drug that can be administered orally. This study focused on glyoxal-bis (N(4)-methylthiosemicarbazonato)-copper(II (CuGTSM), which has shown efficacy in macular mice, a murine model of Menkes disease, and examined its pharmacokinetics. In addition, nanosized CuGTSM (nCuGTSM) was prepared, and the effects of nanosizing on CuGTSM pharmacokinetics were investigated. METHODS: CuGTSM or nCuGTSM (10 mg/kg) was administered orally to male macular mice or C3H/HeNCrl mice (control), and plasma was obtained by serial blood sampling. Plasma concentrations of CuGTSM and GTSM were measured by LC-MS/MS and pharmacokinetic parameters were calculated. RESULTS: When CuGTSM was administered orally, CuGTSM and GTSM were both detected in the plasma of both mouse strains. When nCuGTSM was administered, the Cmax was markedly higher, and the mean residence time was longer than when CuGTSM was administered for both CuGTSM and GTSM in both mouse strains. With macular mice, the AUC ratio (GTSM/CuGTSM) was markedly higher and the plasma CuGTSM concentration was lower than with C3H/HeNCrl mice when either CuGTSM or nCuGTSM was administered. CONCLUSION: Absorption of orally administered CuGTSM was confirmed in macular mice, and the nano-formulation improved the absorption and retention of CuGTSM in the body. However, the plasma concentration of CuGTSM was lower in macular mice than in control mice, suggesting easier dissociation of CuGTSM.

Non-competitive fluorescence polarization immunoassay for detection of H5 avian influenza virus using a portable analyzer.

Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 µL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.

TNF-α enhances TGF-ß-induced endothelial-to-mesenchymal transition via TGF-ß signal augmentation.

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer-associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF-ß, which induces endothelial-to-mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor-infiltrating inflammatory cells secrete various cytokines, including TNF-α. However, the role of TNF-α in TGF-ß-induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF-α on TGF-ß-induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF-ß and TNF-α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF-ß and TNF-α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF-ß type I receptor, TGF-ß2, activin A, and integrin αv, suggesting that TNF-α enhanced TGF-ß-induced EndMT by augmenting TGF-ß family signals. Furthermore, oral squamous cell carcinoma-derived cells underwent epithelial-to-mesenchymal transition (EMT) in response to humoral factors produced by TGF-ß and TNF-α-cultured ECs. This EndMT-driven EMT was blocked by inhibiting the action of TGF-ßs. Collectively, our findings suggest that TNF-α enhances TGF-ß-dependent EndMT, which contributes to tumor progression.

Replication stress induces accumulation of FANCD2 at central region of large fragile genes.

During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.

A Stable and Cleavable O-Linked Spacer for Drug Delivery Systems.

Anti-cancer chemotherapy with good efficacy and fewer side effects is highly desirable. A drug delivery system comprising a cancer-targeting module and a cytotoxic agent connected with a cleavable linker is promising for reducing side effects. The development of a cleavable linker satisfying the requirements of both stability and cleavability, however, is difficult, especially when a carbonate moiety is used for conjugating the linker to a hydroxy group in a drug of interest. We herein report a new stable linker comprising carbamate and ester spacers, which can be introduced on a hydroxy group of a drug. This linker is more stable in aqueous neutral buffer than a corresponding carbonate-type linker, and releases a payload anti-cancer drug, SN-38, through a two-step sequence upon cathepsin B treatment. This linker may have potential use in other drug delivery systems to lower side effects by selectively transporting cytotoxic drugs to tumor cells.