ZNF397 Deficiency Triggers TET2-driven Lineage Plasticity and AR-Targeted Therapy Resistance in Prostate Cancer.

Cancer cells exhibit phenotypical plasticity and epigenetic reprogramming, which allows them to evade lineage-dependent targeted treatments by adopting lineage plasticity. The underlying mechanisms by which cancer cells exploit the epigenetic regulatory machinery to acquire lineage plasticity and therapy resistance remain poorly understood. We identified Zinc Finger Protein 397 (ZNF397) as a bona fide coactivator of the androgen receptor (AR), essential for the transcriptional program governing AR-driven luminal lineage. ZNF397 deficiency facilitates the transition of cancer cell from an AR-driven luminal lineage to a Ten-Eleven Translocation 2 (TET2)-driven lineage plastic state, ultimately promoting resistance to therapies inhibiting AR signaling. Intriguingly, our findings indicate that a TET2 inhibitor can eliminate the resistance to AR targeted therapies in ZNF397-deficient tumors. These insights uncover a novel mechanism through which prostate cancer acquires lineage plasticity via epigenetic rewiring and offer promising implications for clinical interventions designed to overcome therapy resistance dictated by lineage plasticity.

Faslpr gene dosage tunes the extent of lymphoproliferation and T cell differentiation in lupus.

Sle1 and Faslpr are two lupus susceptibility loci that lead to manifestations of systemic lupus erythematosus. To evaluate the dosage effects of Faslpr in determining cellular and serological phenotypes associated with lupus, we developed a new C57BL/6 (B6) congenic lupus strain, B6.Sle1/Sle1.Faslpr/+ (Sle1homo.lprhet) and compared it with B6.Faslpr/lpr (lprhomo), B6.Sle1/Sle1 (Sle1homo), and B6.Sle1/Sle1.Faslpr/lpr (Sle1homo.lprhomo) strains. Whereas Sle1homo.lprhomo mice exhibited profound lymphoproliferation and early mortality, Sle1homo.lprhet mice had a lifespan comparable to B6 mice, with no evidence of splenomegaly or lymphadenopathy. Compared to B6 monogenic lupus strains, Sle1homo.lprhet mice exhibited significantly elevated serum ANA antibodies and increased proteinuria. Additionally, Sle1homo.lprhet T cells had an increased propensity to differentiate into Th1 cells. Gene dose effects of Faslpr were noted in upregulating serum IL-1⍺, IL-2, and IL-27. Taken together, Sle1homo.lprhet strain is a new C57BL/6-based model of lupus, ideal for genetic studies, autoantibody repertoire investigation, and for exploring Th1 effector cell skewing without early-age lymphoproliferative autoimmunity.

The anti-myotoxic effects and mechanisms of Sinonatrix annularis serum and a novel plasma metalloproteinase inhibitor towards Deinagkistrodon acutus envenomation.

Non-venomous snakes commonly evolve natural resistance to venom to escape predators. Sinonatrix annularis serum has been shown to inhibit Deinagkistrodon acutus venom-induced hemorrhage and upregulation of serum CK, CK-MB, LDH, AST and ALT levels. Using TMT-labeled proteomics analysis, 168 proteins were found to be altered significantly in the envenomed gastrocnemius muscle and categorized into pathways such as complement and coagulation cascades, leukocyte transendothelial migration, and JAK/STAT signaling. These alterations were mitigated by S. annularis serum. Subsequently, a novel metalloproteinase inhibitor, SaMPI, was isolated from S. annularis serum by two-step chromatography. It showed strong antidotal effects against D. acutus envenomation, including inhibition of subcutaneous bleeding caused by crude venom and DaMP (a metalloproteinase derived from D. acutus) activity in a 1:1 ratio. Histology and immunoblotting analyses demonstrated that SaMPI mitigated myonecrosis, reduced neutrophil infiltration and local inflammatory factor release, and retarded JAK/STAT and MAPK signaling activation. Analysis of the SaMPI gene cloned by 5'-RACE revealed a shared sequence identity of 58-79% with other SVMP inhibitors. These findings demonstrate the protective effects of SaMPI and indicate its potential value as a candidate for viper bite adjuvant therapy.

Loss of SYNCRIP unleashes APOBEC-driven mutagenesis, tumor heterogeneity, and AR-targeted therapy resistance in prostate cancer.

Tumor mutational burden and heterogeneity has been suggested to fuel resistance to many targeted therapies. The cytosine deaminase APOBEC proteins have been implicated in the mutational signatures of more than 70% of human cancers. However, the mechanism underlying how cancer cells hijack the APOBEC mediated mutagenesis machinery to promote tumor heterogeneity, and thereby foster therapy resistance remains unclear. We identify SYNCRIP as an endogenous molecular brake which suppresses APOBEC-driven mutagenesis in prostate cancer (PCa). Overactivated APOBEC3B, in SYNCRIP-deficient PCa cells, is a key mutator, representing the molecular source of driver mutations in some frequently mutated genes in PCa, including FOXA1, EP300. Functional screening identifies eight crucial drivers for androgen receptor (AR)-targeted therapy resistance in PCa that are mutated by APOBEC3B: BRD7, CBX8, EP300, FOXA1, HDAC5, HSF4, STAT3, and AR. These results uncover a cell-intrinsic mechanism that unleashes APOBEC-driven mutagenesis, which plays a significant role in conferring AR-targeted therapy resistance in PCa.

CircIFNGR2 enhances proliferation and migration of CRC and induces cetuximab resistance by indirectly targeting KRAS via sponging to MiR-30b.

Currently the clinical efficacy of colorectal cancer (CRC) which is the most common malignant tumors over the world has not reached an ideal level. Cetuximab, the monoclonal antibody targeting the extracellular domain of EGFR, has shown its great efficacy in the promotion of apoptosis and the inhibition of tumor cells-like characteristics in numerous cancers. However certain KRAS wild-type CRC patients unexpectedly show cetuximab resistance and the specific mechanism remains unclear. Circular RNAs (circRNAs) as the promising novel type of biomarkers in the cancer diagnosis and therapy, have been reported to be related with the drug resistance. In this study, with wondering the mechanism of cetuximab resistance in KRAS wild-type CRC patients, we evaluate the impact of circIFNGR2 on CRC and detect the association among circIFNGR2, miR-30b and KRAS via various experiments such as RT-qPCR, immunohistochemistry, luciferase assays, cell functional experiments and xenograft model. We conclude that circIFNGR2 induces cetuximab resistance in colorectal cancer cells by indirectly regulating target gene KRAS by sponging miR-30b at the post-transcriptional level. It is thus suggested that inhibition of circIFNGR2 can be a promising therapeutic strategy for malignant CRC patients with cetuximab resistance.

Dopamine/DOPAC-assisted immobilization of bone morphogenetic protein-2 loaded Heparin/PEI nanogels onto three-dimentional printed calcium phosphate ceramics for enhanced osteoinductivity and osteogenicity.

Nowadays, the three-dimensional (3D) printed calcium phosphate (CaP) ceramics have well-designed geometric structure, but suffer from relative weak osteoinductivity. Surface modification by incorporating bone morphogenetic protein-2 (BMP2) onto scaffolds is considered as an efficient approach to improve their bioactivity. However, high dose and uncontrolled burst release of BMP2 may cause undesired side effect. In the present study, porous BCP ceramics with inverse face-centred cube structure prepared by digital light processing (DLP)-based 3D printing technique were used as the substrates. BMP2 proteins were loaded in the self-assembled Heparin/PEI nanogels (NP/BMP2), and then immobilized onto BCP substrates through the intermediate mussel-derived bioactive dopamine and dihydroxyphenylacetic acid (DA/DOPAC) coating layers to construct functional BCP/layer/NP/BMP2 scaffolds. Our results showed that Heparin/PEI nanogel was a potent delivery system for BMP2, and BCP/layer/NP/BMP2 scaffolds exhibited the high loading capacity, controlled release rate, and sustained local delivery of BMP2. In vitro cell experiments with bone marrow stromal cells (BMSCs) found that BCP/layer/NP/BMP2 could promote cell proliferation, facilitate cell spreading, accelerate cell migration, up-regulate expression of osteogenic genes, and improve synthesis of osteoblast-related proteins. Moreover, the murine intramuscular implantation model suggested that BCP/layer/NP/BMP2 had a superior osteoinductive capacity, and the rat femoral condyle defect repair model showed that BCP/layer/NP/BMP2 could enhance in situ bone repair and regeneration. These findings demonstrate that the incorporation of BMP2 loaded Heparin/PEI nanogels to 3D printed scaffolds holds great promise in fabricating bone graft with a superior biological performance for orthopedic application.

SIK2 inhibition enhances PARP inhibitor activity synergistically in ovarian and triple-negative breast cancers.

Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7-associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2, EXO1, and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.

Fabrication and biological evaluation of 3D-printed calcium phosphate ceramic scaffolds with distinct macroporous geometries through digital light processing technology.

Digital light processing (DLP)-based 3D printing technique holds promise in fabricating scaffolds with high precision. Here raw calcium phosphate (CaP) powders were modified by 5.5% monoalcohol ethoxylate phosphate (MAEP) to ensure high solid loading and low viscosity. The rheological tests found that photocurable slurries composed of 50 wt% modified CaP powders and 2 wt% toners were suitable for DLP printing. Based on geometric models designed by computer-aided design (CAD) system, three printed CaP ceramics with distinct macroporous structures were prepared, including simple cube, octet-truss and inverse face-centered cube (fcc), which presented the similar phase composition and microstructure, but the different macropore geometries. Inverse fcc group showed the highest porosity and compressive strength. The in vitro and in vivo biological evaluations were performed to compare the bioactivity of three printed CaP ceramics, and the traditional foamed ceramic was used as control. It suggested that all CaP ceramics exhibited good biocompatibility, as evidence by an even bone-like apatite layer formation on the surface, and the good cell proliferation and spreading. A mouse intramuscular implantation model found that all of CaP ceramics could induce ectopic bone formation, and foam group had the strongest osteoinduction, followed by inverse fcc, while cube and octet-truss had the weakest one. It indicated that macropore geometry was of great importance to affect the osteoinductivity of scaffolds, and spherical, concave macropores facilitated osteogenesis. These findings provide a strategy to design and fabricate high-performance orthopedic grafts with proper pore geometry and desired biological performance via DLP-based 3D printing technique.

The role of micro-vibration parameters in inflammatory responses of macrophages cultured on biphasic calcium phosphate ceramics and the resultant influence on osteogenic differentiation of mesenchymal stem cells.

Although in vitro studies have shown that biomaterials and mechanical stimuli can mediate inflammatory responses or regulate osteogenesis of MSCs, the underlying behaviour of the inflammatory response of macrophages on biomaterials mediated by mechanical stimuli, which regulates osteogenesis, is relatively unknown. Thus, it is imperative to explore the role of bionic mechanical stimulation in the biomaterial-mediated inflammatory response of macrophages. In this study, we used osteoinductive biphasic calcium phosphate (BCP) ceramics as the model biomaterial and chose micro-vibration stimulation (MVs) with three variable parameters (frequency, magnitude, and time). Based on orthogonal experiments, nine combinations of MVs parameters were generated, and their effects on the BCP-mediated macrophage inflammatory response were investigated. MVs significantly affected the gene expression and cytokine secretion of macrophages grown on BCP ceramics and further influenced the behaviour of bone marrow mesenchymal stem cells (BMMSCs) in a paracrine manner. Moreover, frequency seemed to be the most dominant factor (compared with magnitude and time) in regulating the inflammatory response of macrophages. The optimal combination of MVs parameters (frequency 10 Hz, magnitude 0.45 g, and time 60 min) could induce a healing-associated M2 phenotype, as evidenced by the downregulated pro-inflammatory gene (Il-1ß, and Tnf-α) expression, the upregulated anti-inflammatory gene (Il10) expression, and the inhibited pro-inflammatory cytokine (Il-1ß and Tnf-α) secretion of macrophages grown on BCP ceramics, and its conditioned medium (CM) could further promote osteogenic differentiation of BMMSCs. These findings provide valuable insights into the mechanical stimulus-mediated macrophage inflammatory response and osteogenesis in the presence of osteoinductive BCP ceramics and allow accurate evaluation of the biological performance of biomaterials in vitro, in order to optimize bone substitute materials to achieve the desired clinical performance.

GuidePro: a multi-source ensemble predictor for prioritizing sgRNAs in CRISPR/Cas9 protein knockouts.

MOTIVATION: The efficiency of CRISPR/Cas9-mediated protein knockout is determined by three factors: sequence-specific sgRNA activity, frameshift probability and the characteristics of targeted amino acids. A number of computational methods have been developed for predicting sgRNA efficiency from different perspectives. However, an integrative method that combines all three factors for rational sgRNA selection is still lacking. RESULTS: We developed GuidePro, a two-layer ensemble predictor that enables the integration of multiple factors for the prioritization of sgRNAs in protein knockouts. Tested on independent datasets, GuidePro outperforms existing methods and demonstrates consistent superior performance in predicting phenotypes caused by protein loss-of-function, suggesting its robustness for prioritizing sgRNAs in various applications of CRISPR/Cas9 knockouts. AVAILABILITY AND IMPLEMENTATION: GuidePro is available at https://github.com/MDhewei/GuidePro. A web application for prioritizing sgRNAs that target protein-coding genes in human, monkey and mouse genomes is available at https://bioinformatics.mdanderson.org/apps/GuidePro. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.

Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers.

BACKGROUND: Nucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells. RESULTS: We generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs' tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs. CONCLUSIONS: The re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.

H2A.Z nucleosome positioning has no impact on genetic variation in Drosophila genome.

Nucleosome occupancy results in complex sequence variation rate heterogeneity by either increasing mutation rate or inhibiting DNA repair in yeast, fish, and human. H2A.Z nucleosome is extensively involved in gene transcription activation and regulation. To test whether H2A.Z nucleosome has the similar impact on sequence variability in the Drosophila genome, we profiled the H2A.Z nucleosome occupancy and sequence variation rate at gene ends and splicing sites. Consistent with previous studies, H2A.Z nucleosome positioning helps to demarcate the borders of exons. Nucleosome occupancy is anticorrelated with sequence divergence rate in the regions flanking transcription start sites and splicing sites. However, there is no rate heterogeneity between the linker DNA and H2A.Z nucleosomal DNA regardless of nucleosome occupancy, fuzziness, positioning in promoter, coding, and intergenic regions, young or old genes. But the rate at intergenic nucleosomes and the flanking linker regions is higher than that at the genic counterparts. Further analyses found that the high sequence divergence rate in the promoter regions that are usually nucleosome depleted regions may be likely resulted from the high mutation rate in the enriched tandem repeats. Interestingly, within nucleosomes spanning splicing sites, sequence variability of nucleosomal DNA significantly increases from the end within exons to the other end protruding into introns. The relaxed functional constraint in introns contributes to the high rate of nucleosomal DNA residing in introns while the strict functional constraint in exons maintains the low rate of nucleosomal DNA residing in exons. Taken together, H2A.Z nucleosome occupancy has no effect on sequence variability of Drosophila genome, which is likely determined by local sequence composition and the concomitant selection pressure.