Breast cancer malignancy is governed by regulation of the macroH2A2/TM4SF1 axis, the AKT/NF-κB pathway, and elevated MMP13 expression.

The histone variant, macroH2A (mH2A) influences gene expression through epigenetic regulation. Tumor suppressive function of mH2A isoforms has been reported in various cancer types, but few studies have investigated the functional role of mH2A2 in breast cancer pathophysiology. This study aimed to determine the significance of mH2A2 in breast cancer development and progression by exploring its downstream regulatory mechanisms. Knockdown of mH2A2 facilitated the migration and invasion of breast cancer cells, whereas its overexpression exhibited the opposite effect. In vivo experiments revealed that augmenting mH2A2 expression reduced tumor growth and lung metastasis. Microarray analysis showed that TM4SF1 emerged as a likely target linked to mH2A2 owing to its significant suppression in breast cancer cell lines where mH2A2 was overexpressed among the genes that exhibited over twofold upregulation upon mH2A2 knockdown. Suppressing TM4SF1 reduced the migration, invasion, tumor growth, and metastasis of breast cancer cells in vitro and in vivo. TM4SF1 depletion reversed the increased aggressiveness triggered by mH2A2 knockdown, suggesting a close interplay between mH2A2 and TM4SF1. Our findings also highlight the role of the mH2A2/TM4SF1 axis in activating the AKT/NF-κB pathway. Consequently, activated NF-κB signaling leads to increased expression and secretion of MMP13, a potent promoter of metastasis. In summary, we propose that the orchestrated regulation of the mH2A2/TM4SF1 axis in conjunction with the AKT/NF-κB pathway and the subsequent elevation in MMP13 expression constitute pivotal factors governing the malignancy of breast cancer.

Characterization of 3D Organotypic Culture of Mouse Adipose-Derived Stem Cells.

Although stem cells are a promising avenue for harnessing the potential of adipose tissue, conventional two-dimensional (2D) culture methods have limitations. This study explored the use of three-dimensional (3D) cultures to preserve the regenerative potential of adipose-derived stem cells (ADSCs) and investigated their cellular properties. Flow cytometric analysis revealed significant variations in surface marker expressions between the two culture conditions. While 2D cultures showed robust surface marker expressions, 3D cultures exhibited reduced levels of CD44, CD90.2, and CD105. Adipogenic differentiation in 3D organotypic ADSCs faced challenges, with decreased organoid size and limited activation of adipogenesis-related genes. Key adipocyte markers, such as lipoprotein lipase (LPL) and adipoQ, were undetectable in 3D-cultured ADSCs, unlike positive controls in 2D-cultured mesenchymal stem cells (MSCs). Surprisingly, 3D-cultured ADSCs underwent mesenchymal-epithelial transition (MET), evidenced by increased E-cadherin and EpCAM expression and decreased mesenchymal markers. This study highlights successful ADSC organoid formation, notable MSC phenotype changes in 3D culture, adipogenic differentiation challenges, and a distinctive shift toward an epithelial-like state. These findings offer insights into the potential applications of 3D-cultured ADSCs in regenerative medicine, emphasizing the need for further exploration of underlying molecular mechanisms.

A cleavage-based surrogate reporter for the evaluation of CRISPR-Cas9 cleavage efficiency.

CRISPR-Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR-Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.

G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer.

Epigenetic abnormalities affect tumor progression, as well as gene expression and function. Among the diverse epigenetic modulators, the histone methyltransferase G9a has been focused on due to its role in accelerating tumorigenesis and metastasis. Although epigenetic dysregulation is closely related to tumor progression, reports regarding the relationship between G9a and its possible downstream factors regulating breast tumor growth are scarce. Therefore, we aimed to verify the role of G9a and its presumable downstream regulators during malignant progression of breast cancer. G9a-depleted MCF7 and T47D breast cancer cells exhibited suppressed motility, including migration and invasion, and an improved response to ionizing radiation. To identify the possible key factors underlying these effects, microarray analysis was performed, and a TGF-ß superfamily member, BMP5, was selected as a prominent target gene. It was found that BMP5 expression was markedly increased by G9a knockdown. Moreover, reduction in the migration/invasion ability of MCF7 and T47D breast cancer cells was induced by BMP5. Interestingly, a G9a-depletion-mediated increase in BMP5 expression induced the phosphorylation of Smad proteins, which are the intracellular signaling mediators of BMP5. Accordingly, we concluded that the observed antitumor effects may be based on the G9a-depletion-mediated increase in BMP5 expression and the consequent facilitation of Smad protein phosphorylation.

Smek promotes corticogenesis through regulating Mbd3's stability and Mbd3/NuRD complex recruitment to genes associated with neurogenesis.

The fate of neural progenitor cells (NPCs) during corticogenesis is determined by a complex interplay of genetic or epigenetic components, but the underlying mechanism is incompletely understood. Here, we demonstrate that Suppressor of Mek null (Smek) interact with methyl-CpG-binding domain 3 (Mbd3) and the complex plays a critical role in self-renewal and neuronal differentiation of NPCs. We found that Smek promotes Mbd3 polyubiquitylation and degradation, blocking recruitment of the repressive Mbd3/nucleosome remodeling and deacetylase (NuRD) complex at the neurogenesis-associated gene loci, and, as a consequence, increasing acetyl histone H3 activity and cortical neurogenesis. Furthermore, overexpression of Mbd3 significantly blocked neuronal differentiation of NPCs, and Mbd3 depletion rescued neurogenesis defects seen in Smek1/2 knockout mice. These results reveal a novel molecular mechanism underlying Smek/Mbd3/NuRD axis-mediated control of NPCs' self-renewal and neuronal differentiation during mammalian corticogenesis.

Smek1/2 is a nuclear chaperone and cofactor for cleaved Wnt receptor Ryk, regulating cortical neurogenesis.

The receptor-like tyrosine kinase (Ryk), a Wnt receptor, is important for cell fate determination during corticogenesis. During neuronal differentiation, the Ryk intracellular domain (ICD) is cleaved. Cleavage of Ryk and nuclear translocation of Ryk-ICD are required for neuronal differentiation. However, the mechanism of translocation and how it regulates neuronal differentiation remain unclear. Here, we identified Smek1 and Smek2 as Ryk-ICD partners that regulate its nuclear localization and function together with Ryk-ICD in the nucleus through chromatin recruitment and gene transcription regulation. Smek1/2 double knockout mice displayed pronounced defects in the production of cortical neurons, especially interneurons, while the neural stem cell population increased. In addition, both Smek and Ryk-ICD bound to the Dlx1/2 intergenic regulator element and were involved in its transcriptional regulation. These findings demonstrate a mechanism of the Ryk signaling pathway in which Smek1/2 and Ryk-ICD work together to mediate neural cell fate during corticogenesis.

DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes.

Ectopic expression of the double homeodomain transcription factor DUX4 causes facioscapulohumeral muscular dystrophy (FSHD). Mechanisms of action of DUX4 are currently unknown. Using immortalized human myoblasts with a titratable DUX4 transgene, we identify by mass spectrometry an interaction between the DUX4 C-terminus and the histone acetyltransferases p300/CBP. Chromatin immunoprecipitation shows that DUX4 recruits p300 to its target gene, ZSCAN4, displaces histone H3 from the center of its binding site, and induces H3K27Ac in its vicinity, but C-terminal deleted DUX4 does not. We show that a DUX4 minigene, bearing only the homeodomains and C-terminus, is transcriptionally functional and cytotoxic, and that overexpression of a nuclear targeted C-terminus impairs the ability of WT DUX4 to interact with p300 and to regulate target genes. Genomic profiling of DUX4, histone H3, and H3 modifications reveals that DUX4 binds two classes of loci: DNase accessible H3K27Ac-rich chromatin and inaccessible H3K27Ac-depleted MaLR-enriched chromatin. At this latter class, it acts as a pioneer factor, recruiting H3K27 acetyltransferase activity and opening the locus for transcription. In concert with local increased H3K27Ac, the strong H3K27Ac peaks at distant sites are significantly depleted of H3K27Ac, thus DUX4 uses its C-terminus to induce a global reorganization of H3K27 acetylation.

The Wnt receptor Ryk reduces neuronal and cell survival capacity by repressing FOXO activity during the early phases of mutant huntingtin pathogenicity.

The Wnt receptor Ryk is an evolutionary-conserved protein important during neuronal differentiation through several mechanisms, including γ-secretase cleavage and nuclear translocation of its intracellular domain (Ryk-ICD). Although the Wnt pathway may be neuroprotective, the role of Ryk in neurodegenerative disease remains unknown. We found that Ryk is up-regulated in neurons expressing mutant huntingtin (HTT) in several models of Huntington's disease (HD). Further investigation in Caenorhabditis elegans and mouse striatal cell models of HD provided a model in which the early-stage increase of Ryk promotes neuronal dysfunction by repressing the neuroprotective activity of the longevity-promoting factor FOXO through a noncanonical mechanism that implicates the Ryk-ICD fragment and its binding to the FOXO co-factor ß-catenin. The Ryk-ICD fragment suppressed neuroprotection by lin-18/Ryk loss-of-function in expanded-polyQ nematodes, repressed FOXO transcriptional activity, and abolished ß-catenin protection of mutant htt striatal cells against cell death vulnerability. Additionally, Ryk-ICD was increased in the nucleus of mutant htt cells, and reducing γ-secretase PS1 levels compensated for the cytotoxicity of full-length Ryk in these cells. These findings reveal that the Ryk-ICD pathway may impair FOXO protective activity in mutant polyglutamine neurons, suggesting that neurons are unable to efficiently maintain function and resist disease from the earliest phases of the pathogenic process in HD.

FACT-mediated exchange of histone variant H2AX regulated by phosphorylation of H2AX and ADP-ribosylation of Spt16.

The phosphorylation of histone variant H2AX at DNA double-strand breaks is believed to be critical for recognition and repair of DNA damage. However, little is known about the molecular mechanism regulating the exchange of variant H2AX with conventional H2A in the context of the nucleosome. Here, we isolate the H2AX-associated factors, which include FACT (Spt16/SSRP1), DNA-PK, and PARP1 from a human cell line. Our analyses demonstrate that the H2AX-associated factors efficiently promote both integration and dissociation of H2AX and this exchange reaction is mainly catalyzed by FACT among the purified factors. The phosphorylation of H2AX by DNA-PK facilitates the exchange of nucleosomal H2AX by inducing conformational changes of the nucleosome. In contrast, poly-ADP-ribosylation of Spt16 by PARP1 significantly inhibits FACT activities for H2AX exchange. Thus, these data establish FACT as the major regulator involved in H2AX exchange process that is modulated by H2AX phosphorylation and Spt16 ADP-ribosylation.

DNA methylation of Runx1 regulatory regions correlates with transition from primitive to definitive hematopoietic potential in vitro and in vivo.

The transcription factor Runx1 (AML1) is a central regulator of hematopoiesis and is required for the formation of definitive hematopoietic stem cells (HSCs). Runx1 is alternatively expressed from two promoters: the proximal (P2) prevails during primitive hematopoiesis, while the distal (P1) dominates in definitive HSCs. Although some transcription factor binding sites and cis-regulatory elements have been identified, a mechanistic explanation for the alternative promoter usage remains elusive. We investigated DNA methylation of known Runx1 cis-elements at stages of hematopoietic development in vivo and during differentiation of murine embryonic stem cells (ESCs) in vitro. In vivo, we find loss of methylation correlated with the primitive to definitive transition at the P1 promoter. In vitro, hypomethylation, acquisition of active chromatin modifications, and increased transcriptional activity at P1 are promoted by direct interaction with HOXB4, a transcription factor that confers definitive repopulation status on primitive hematopoietic progenitors. These data demonstrate a novel role for DNA methylation in the alternative promoter usage at the Runx1 locus and identify HOXB4 as a direct activator of the P1 promoter. This epigenetic signature should serve as a novel biomarker of HSC potential in vivo, and during ESC differentiation in vitro.

Inducing positive inotropy in human iPSC-derived cardiac muscle by gene editing-based activation of the cardiac α-myosin heavy chain.

Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure-function. This includes studies centered on disease etiology, drug development, and for potential clinical applications in heart regeneration/repair. Ultimately, for these applications to achieve success, a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state, and this represents a major hurdle for the field. To address this roadblock, we have developed a hiPSC-CMs (ß-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere, the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile, the molecular motor of the heart, which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of α-MyHC in ß-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis, we used gene editing with an inducible α-MyHC expression cassette into isogeneic hiPSC-CMs, and separately by gene transfer, and then investigated the direct effects of increased α-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with α-MyHC. Positive inotropy and relaxation was evident in comparison to ß-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening, as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration, repair and re-muscularization applications.

Regulatory role of Echinochrome A in cancer-associated fibroblast-mediated lung cancer cell migration.

Echinochrome A (Ech A), a marine biosubstance isolated from sea urchins, is a strong antioxidant, and its clinical form, histochrome, is being used to treat several diseases, such as ophthalmic, cardiovascular, and metabolic diseases. Cancer-associated fibroblasts (CAFs) are a component of the tumor stroma and induce phenotypes related to tumor malignancy, including epithelial-mesenchymal transition (EMT) and cancer stemness, through reciprocal interactions with cancer cells. Here, we investigated whether Ech A modulates the properties of CAFs and alleviates CAF-induced lung cancer cell migration. First, we observed that the expression levels of CAF markers, Vimentin and fibroblast-activating protein (FAP), were decreased in Ech A-treated CAF-like MRC5 cells. The mRNA transcriptome analysis revealed that in MRC5 cells, the expression of genes associated with cell migration was largely modulated after Ech A treatment. In particular, the expression and secretion of cytokine and chemokine, such as IL6 and CCL2, stimulating cancer cell metastasis was reduced through the inactivation of STAT3 and Akt in MRC5 cells treated with Ech A compared to untreated MRC5 cells. Moreover, while conditioned medium from MRC5 cells enhanced the migration of non-small cell lung cancer cells, conditioned medium from MRC5 cells treated with Ech A suppressed cancer cell migration. In conclusion, we suggest that Ech A might be a potent adjuvant that increases the efficacy of cancer treatments to mitigate lung cancer progression.

Quantitative and qualitative mutational impact of ionizing radiation on normal cells.

The comprehensive genomic impact of ionizing radiation (IR), a carcinogen, on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells, we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs), including balanced inversions, translocations, composite SVs (deletion-insertion, deletion-inversion, and deletion-translocation composites), and complex genomic rearrangements (CGRs), including chromoplexy, chromothripsis, and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome, comprising 2.15 indels, 0.17 SVs, and 0.01 CGRs, despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose, regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall, our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes.

Identification of preferential target sites for human DNA methyltransferases.

DNA methyltransferases (DNMTs) play an important role in establishing and maintaining DNA methylation. Aberrant expression of DNMTs and their isoforms has been found in many types of cancer, and their contribution to aberrant DNA methylation has been proposed. Here, we generated HEK 293T cells stably transfected with each of 13 different DNMTs (DNMT1, two DNMT3A isoforms, nine DNMT3B isoforms and DNMT3L) and assessed the DNA methylation changes induced by each DNMT. We obtained DNA methylation profiles of DNA repetitive elements and 1505 CpG sites from 808 cancer-related genes. We found that DNMTs have specific and overlapping target sites and their DNA methylation target profiles are a reflection of the DNMT domains. By examining H3K4me3 and H3K27me3 modifications in the 808 gene promoter regions using promoter ChIP-on-chip analysis, we found that specific de novo DNA methylation target sites of DNMT3A1 are associated with H3K4me3 modification that are transcriptionally active, whereas the specific target sites of DNMT3B1 are associated with H3K27me3 modification that are transcriptionally inactive. Our data suggest that different DNMT domains are responsible for targeting DNA methylation to specific regions of the genome, and this targeting might be associated with histone modifications.

Downregulation of complement factor H attenuates the stemness of MDA­MB­231 breast cancer cells via modulation of the ERK and p38 signaling pathways.

The complement system is a powerful innate immune system deployed in the immediate response to pathogens and cancer cells. Complement factor H (CFH), one of the regulators involved in the complement cascade, can interrupt the death of target cells. Certain types of cancer, such as breast cancer, can adopt an aggressive phenotype, such as breast cancer stem cells (BCSCs), through enhancement of the defense system against complement attack by amplifying various complement regulators. However, little is known about the association between CFH and BCSCs. In the present study, the roles of CFH in the CSC characteristics and radioresistance of MDA-MB-231 human breast cancer cells were investigated. CFH knockdown in MDA-MB-231 cells decreased the viability of the cells upon complement cascade activation. Notably, CFH knockdown also decreased cell survival and suppressed mammosphere formation, cell migration and cell invasion by attenuating radioresistance. Additionally, CFH knockdown further enhanced irradiation-induced apoptosis through G2/M cell cycle arrest. It was also discovered that CFH knockdown attenuated the aggressive phenotypes of cancer cells by regulating CSC-associated gene expression. Finally, by microarray analysis, it was found that the expression of erythrocyte membrane protein band 4.1-like 3 (EPB41L3) was markedly increased following CFH knockdown. EPB41L3 inhibited ERK and activated the p38 MAPK signaling pathway. Taken together, these results indicated that CFH knockdown attenuated CSC properties and radioresistance in human breast cancer cells via controlling MAPK signaling and through upregulation of the tumor suppressor, EPB41L3.

Evaluation of Radiation Sensitivity Differences in Mouse Liver Tumor Organoids Using CRISPR/Cas9-Mediated Gene Mutation.

BACKGROUND: To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1. METHODS: The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay. RESULTS: The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations. CONCLUSIONS: This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.

Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, namely, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR.

Genomic targets and selective inhibition of DNA methyltransferase isoforms.

BACKGROUND: DNA methylation in the human genome is established and maintained by DNA methyltransferases (DNMTs). DNMT isoforms show differential expression by cell lineage and during development, but much remains to be elucidated about their shared and unique genomic targets. RESULTS: We examined changes in the epigenome following overexpression of 13 DNMT isoforms in HEK293T cells. We observed increased methylation (Δß > 0.2) at 43,405 CpG sites, with expression of DNMT3A2, DNMTΔ3B4 and DNMTΔ3B2 associated with the greatest impact. De novo methylation occurred primarily within open sea regions and at loci with intermediate methylation levels (ß: 0.2-0.6). 53% of differentially methylated loci showed specificity towards a single DNMT subfamily, primarily DNMTΔ3B and DNMT3A and 39% towards a single isoform. These loci were significantly enriched for pathways related to neuronal development (DNMTΔ3B4), calcium homeostasis (DNMTΔ3B3) and ion transport (DNMT3L). Repetitive elements did not display differential sensitivity to overexpressed DNMTs, but hypermethylation of Alu elements was associated with their evolutionary age following overexpression of DNMT3A2, DNMT3B1, DNMT3B2 and DNMT3L. Differential methylation (Δß > 0.1) was observed at 121 of the 353 loci associated with the Horvath 'epigenetic clock' model of ageing, with 51 showing isoform specificity, and was associated with reduction of epigenetic age by 5-15 years following overexpression of seven isoforms. Finally, we demonstrate the potential for dietary constituents to modify epigenetic marks through isoform-specific inhibition of methylation activity. CONCLUSIONS: Our results provide insight into regions of the genome methylated uniquely by specific DNMT isoforms and demonstrate the potential for dietary intervention to modify the epigenome.

Early epigenetic changes and DNA damage do not predict clinical response in an overlapping schedule of 5-azacytidine and entinostat in patients with myeloid malignancies.

Sequential administration of DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors has demonstrated clinical efficacy in patients with hematologic malignancies. However, the mechanism behind their clinical efficacy remains controversial. In this study, the methylation dynamics of 4 TSGs (p15(INK4B), CDH-1, DAPK-1, and SOCS-1) were studied in sequential bone marrow samples from 30 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) who completed a minimum of 4 cycles of therapy with 5-azacytidine and entinostat. Reversal of promoter methylation after therapy was observed in both clinical responders and nonresponders across all genes. There was no association between clinical response and either baseline methylation or methylation reversal in the bone marrow or purified CD34(+) population, nor was there an association with change in gene expression. Transient global hypomethylation was observed in samples after treatment but was not associated with clinical response. Induction of histone H3/H4 acetylation and the DNA damage-associated variant histone gamma-H2AX was observed in peripheral blood samples across all dose cohorts. In conclusion, methylation reversal of candidate TSGs during cycle 1 of therapy was not predictive of clinical response to combination "epigenetic" therapy. This trial is registered with http://www.clinicaltrials.gov under NCT00101179.

Longitudinal Imaging of Liver Cancer Using MicroCT and Nanoparticle Contrast Agents in CRISPR/Cas9-Induced Liver Cancer Mouse Model.

INTRODUCTION: Micro-computed tomography with nanoparticle contrast agents may be a suitable tool for monitoring the time course of the development and progression of tumors. Here, we suggest a practical and convenient experimental method for generating and longitudinally imaging murine liver cancer models. METHODS: Liver cancer was induced in 6 experimental mice by injecting clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 plasmids causing mutations in genes expressed by hepatocytes. Nanoparticle agents are captured by Kupffer cells and detected by micro-computed tomography, thereby enabling longitudinal imaging. A total of 9 mice were used for the experiment. Six mice were injected with both plasmids and contrast, 2 injected with contrast alone, and one not injected with either agent. Micro-computed tomography images were acquired every 2- up to 14-weeks after cancer induction. RESULTS: Liver cancer was first detected by micro-computed tomography at 8 weeks. The mean value of hepatic parenchymal attenuation remained almost unchanged over time, although the standard deviation of attenuation, reflecting heterogeneous contrast enhancement of the hepatic parenchyma, increased slowly over time in all mice. Histopathologically, heterogeneous distribution and aggregation of Kupffer cells was more prominent in the experimental group than in the control group. Heterogeneous enhancement of hepatic parenchyma, which could cause image quality deterioration and image misinterpretation, was observed and could be due to variation in Kupffer cells distribution. CONCLUSION: Micro-computed tomography with nanoparticle contrast is useful in evaluating the induction and characteristics of liver cancer, determining appropriate size of liver cancer for testing, and confirming therapeutic response.